Noémie Cresto

Camille Gardier

Marie-Claude Gaillard

Francesco Gubinelli

Pauline Roost

Daniel Molina

Charlène Josephine

Gwenaëlle Auregan

Martine Guillermier

Suéva Bernier

Caroline Jan

Pauline Gipchtein

Philippe Hantraye

Marie-Christine Chartier-Harlin

Gilles Bonvento

Nadja Van Camp

Jean-Marc Taymans

Karine Cambon

Géraldine Liot

Alexis-Pierre Bemelmans

Emmanuel Brouillet

. Statistical analysis

Keywords: Parkinson's disease, Leucine-rich repeat kinase 2, -synuclein, AAVs, cell-autonomous mechanisms

Background. Alpha-synuclein (α-syn) and leucine-rich repeat kinase 2 (LRRK2) likely play crucial roles both in sporadic and familial forms of Parkinson's disease (PD). The most prevalent mutation in LRRK2 is the G2019S substitution, which induces neurotoxicity through increased kinase activity. There is likely an interplay between LRRK2 and α-syn involved in the neurodegeneration of dopaminergic (DA) neurons in the substantia nigra (SNc) in PD. However, the mechanisms underlying this interplay are ill-defined. Here, we investigated whether LRRK2 G2019S can increase the neurotoxicity induced by a mutant form of α-syn (A53T mutation) in DA neurons in vivo.

We used a co-transduction approach with AAV2/6 vectors encoding human a-syn A53T and the C-terminal portion of LRRK2 (ΔLRRK2), which contains the kinase domain, with either the G2019S mutation (ΔLRRK2 G2019S ) alone or the D1994A mutation (ΔLRRK2 G2019S/D1994A ), which inactivates the kinase activity of LRRK2. The AAVs were co-injected into the rat SNc and histological evaluation was performed at 6-and 15-weeks post-injection (PI).

Results: Most SNc neurons co-expressed ΔLRRK2 and human α-syn A53T after transduction. ΔLRRK2 G2019S alone produced no cell loss at 15-weeks PI, whereas as expected, transduction with AAV-a-syn A53T mixed with a control AAV coding for GFP produced significant loss of DA neurons. Co-injection of AAV-ΔLRRK2 G2019S and AAV-α-syn A53T induced a loss of DA neurons slightly but significantly greater than that produced by co-injection of AAV-α-syn A53T and AAVGFP. We also studied the inactive form, ΔLRRK2 G2019S/D1994A at 6 weeks PI. Results showed that ΔLRRK2 G2019S/D1994A did not alter the early toxicity of α-syn A53T , in contrast to the active form ΔLRRK2 G2019S that produced a moderate but significant increase in α-syn A53T toxicity.

Conclusion. Thus, these results show that mutant LRRK2 may selectively facilitate α-syn toxicity in DA neurons through a cell-autonomous mechanism involving its kinase activity.

A p p e n d i x 2 -S u b m i t t e d a r t i c l e | 149 However, considering that the effect of ΔLRRK2 G2019S upon human α-syn A53T is moderate in our paradigm where pathological proteins are overexpressed, the study supports the hypothesis that the interplay between LRRK2 and α-syn also implicates non-cell-autonomous mechanisms such as those involved in neuroinflammation.

There are multiple people that I would like to thank for their involvement in the process of my PhD research.

First of all, I would like to thank Nadja, who, as my direct supervisor, stood by me during this whole journey. I am very thankful for all the help and guidance you provided me with during the past three years, not only on a scientific level, but also personal. In short, I am very glad to have had you as my supervisor.

Next, I would like to thank everybody in MIRCen that directly or indirectly was involved in my research projects or my social life: Merci tout le monde! I would particularly like to thank: *Philippe and Emmanuel, who gave me the upportunity to be involved in the BrainMatTrain project, and who gave import scientific insights on my projects. *Marie-Laure and Cecile, for answering all my questions asked in bad French, big or small. *Martine and Mylène, for all their help with the animal experimentation. *Leopold, for all the hours we spent locked up together in the PET zone. *Didier, there is not many people who are happy when you tell them things are broken/missing (and you want a replacement 3D-printed), and for helping with all the IT problems. *Caroline and Pauline G., for answering all my question regarding histology, troubleshooting, and even helping with some of the experiments. *Marie-Claude, for sharing all your knowledge about RT-qPCR with me, but also for all scientific insights and discussions. *Anastasie, Camille, Miriam, and Mathilde, for all the great times we had talking in the office, about science and life in general. *Francesco, for surviving the last three years together, both in the lab and at home. It was hard and challenging at times, but we have both made it. I am very happy we have taken this journey together. **To all of you I say: I could not have done this without you! MERCI! I would also like to thank all people involved in the BrainMatTrain program: ESRs, supervisors, and managers. We had great scientific interactions, but also spent great social time together all over Europe.

Next, there is some people outside MIRCEN I would like to thank: Thomas and Henriette, for trying to teach me French. Didier, Fabian, Nadya, and Lucia, for all the fun we had at dance class. YRLS boards 2019 & 2020, for showing me how international Paris can be, and how fun it is to organise big events.

Daarnaast zijn er natuurlijk ook nog een hele hoop mensen in Nederland die ik graag wil bedanken. Allereerst mijn ouders die mijn keuze om naar het buitenland te gaan voor mijn werk en opleiding altijd gesteund hebben. Bedankt voor al jullie morele steun, vooral tijdens de laatste periode van schrijven, en natuurlijk alle fysieke hulp iedere keer als ik weer ging verhuizen. Ook de rest van de familie wil ik graag bedanken voor de morele steun, maar ook dat jullie gezellig op bezoek zijn gekomen in Parijs om in mijn nieuwe leven te delen.

Lieve Mieke en Leonie, ook jullie steun heb ik hard nodig gehad de afgelopen jaren. Ik ben jullie dankbaar dat jullie nog niet hebben opgegeven en dat we nog altijd vriendinnen zijn ondanks de lange afstand. Ik ben blij met alle keren dat jullie me zijn komen opvrolijken in Parijs en alle uren die we samen achter de laptop doorgebracht hebben om te videobellen.

Als laatste: Marco. De afgelopen drie jaar ben je mijn steun en toeverlaat geweest. Ik ben heel erg blij met alle tijd die we samen hebben mogen doorbrengen in Parijs, maar in het vervolg blijf ik toch maar wat dichter in de buurt. Je t'aime. BEDANKT, THANK YOU, GRAZIE, DANKE, TACK, OBRIGADA, GRACIAS, MULTUMESC, BEDANKTJ, MERCI! N o n -s c i e n t i f i c s u m m a r i e s | 3

Niet-wetenschappelijke samenvatting

De ziekte van Parkinson (PD) is de op één na meest voorkomende ziekte waarbij hersencellen vergaan. PD wordt gekenmerkt door opeenhoping van het α-synucleïne eiwit, een progressief verlies van de dopamine-producerende neuronen en de aanwezigheid van ontstekingen in de neurale cellen.

In dit proefschrift heb ik het effect geëvalueerd van α-synucleïne aggregatie en milde neurale ontsteking op dopamine-neuronen in diermodellen van PD. Hiervoor heb ik een nucleaire beeldvormingtechniek gebruikt (positron emissie tomografie (PET)), en post-mortem analyses om de genen en eiwitten te evalueren in deze modellen.

Ik heb aangetoond dat gemuteerd α-synucleïne een gematigde maar progressieve dood van dopamine-neuronen veroorzaakt, gepaard gaande met een lichte ontstekingsreactie. Anderzijds heb ik aangetoond dat milde ontsteking van neurale cellen niet tot verlies van dopamine-neuronen leidt. PET beeldvorming visualiseerde het functionele gebrek van de dopamine neuronen, wat niet mogelijk was met de post-mortem analyses. Dit laat het belang van PET beeldvorming zien bij het bepalen van neuronaal verlies in diermodellen van PD.

Non-scientific summary

Parkinson's disease (PD) is characterised by the aggregation of α-synuclein, a progressive loss of the dopamine-producing neurons, and the presence of neuroinflammation During my PhD studies, I evaluated the impact of α-synuclein aggregation and moderate neuroinflammation on dopaminergic neurons in animal models of PD. To this end, I have used a nuclear imaging method, positron emission tomography (PET), and post-mortem analyses to assess the expression of different genes and proteins in these models.

I have demonstrated that mutated αsynuclein induces mild progressive death of dopamine neurons, coupled with a weak inflammatory response. Conversely, mild neuroinflammation did not lead to loss of dopamine neurons. PET imaging showed functional damage to the dopamine neurons, which was not detectable by post-mortem analyses. This highlights the crucial interest PET imaging to better characterise neurodegeneration in animals models of PD.

Résumé non scientifique

La maladie de Parkinson (MP) est caractérisée par l'agrégation de l'α-synucléine, la perte des neurones qui synthétisent la dopamine (neurone DA), et une inflammation cérébrale.

Pendant ma thèse, j'ai évalué les effets de l'α-synucléine et de l'inflammation modérée sur les neurones DA dans des modèles animaux de la MP. J'ai utilisé une méthode d'imagerie nucléaire, la tomographie d'émission de positons (TEP) et des analyses post mortem évaluant l'expression de différents gènes et protéines dans ces modèles.

J'ai démontré que l'α-synucléine mutée induit une mort des neurones DA, et une faible réponse inflammatoire. À l'inverse, l'inflammation modérée n'entraîne pas la mort de ces neurones. Dans les deux cas, la TEP montre de plus des atteintes fonctionnelles des neurones DA, qui ne sont pas détectées par les analyse post mortem, soulignant l'intérêt crucial de cette méthode non invasive pour mieux caractériser la dégénérescence dans les modèles animaux de la MP.

Table of Contents

Characteristics of PD

Parkinson's disease (PD) affects 2-3% of the population over 65 years of age, resulting in over 10 million patients worldwide, which makes it the second most common neurodegenerative disorder [START_REF] Poewe | Parkinson disease[END_REF]. The mean age of onset of PD is 55 years of age, and the prevalence increases with age [START_REF] Dauer | Parkinson's Disease: Mechanisms and Models[END_REF]. PD was named after James Parkinson, who was the first to describe the "paralysis agitans" in his essay from 1817 [START_REF] Parkinson | An essay on the shaking palsy. 1817[END_REF].

PD is a progressive neurological disorder that mainly affects the motor system. PD is characterised by tremor at rest, freezing, impaired balance, rigidity (passive stiffness), bradykinesia (slowness of movement), hypokinesia (reduction in amplitude of movement), akinesia (absence of unconscious movements), and stooped figure [START_REF] Parkinson | An essay on the shaking palsy. 1817[END_REF][START_REF] Dauer | Parkinson's Disease: Mechanisms and Models[END_REF][START_REF] Cooper | Modeling Parkinson's disease in C. elegans[END_REF]. These are displayed as hypomimia (reduction in facial expressions), hypophonia (reduced voice volume), dysarthria (speech problems), dysphagia (difficulty swallowing), micrographia (small handwriting) and slow handwriting, drooling, and smaller stride length [START_REF] Dauer | Parkinson's Disease: Mechanisms and Models[END_REF][START_REF] Broadfoot | Research-based Updates in Swallowing and Communication Dysfunction in Parkinson Disease: Implications for Evaluation and Management[END_REF]. Apart from motor symptoms, PD often also affects mental state; e.g. passiveness (lack of initiative), apathy, depression, anxiety, bradyphrenia (slowed cognitive processes), and dementia [START_REF] Dauer | Parkinson's Disease: Mechanisms and Models[END_REF][START_REF] Cooper | Modeling Parkinson's disease in C. elegans[END_REF]. Other symptoms include hyposmia (reduced smell), incontinence, constipation, and rapid eye movement sleep disorder [START_REF] Cooper | Modeling Parkinson's disease in C. elegans[END_REF][START_REF] Kouli | Parkinson's Disease: Etiology, Neuropathology, and Pathogenesis, Parkinson's Disease: Pathogenesis and Clinical Aspects[END_REF]. These non-motor symptoms can already start as early as 12-14 years before diagnosis, which defines the prodromal phase [START_REF] Postuma | Identifying prodromal Parkinson's disease: Pre-Motor disorders in Parkinson's disease[END_REF]. With such a diversity of symptoms, PD is a very heterogeneous disease; many symptoms can be part of PD. A clinical diagnosis is based on bradykinesia in combination with a resting tremor or rigidity. Additionally, an asymmetric onset and responsiveness to levodopa differentiates PD from other parkinsonian syndroms [START_REF] Kouli | Parkinson's Disease: Etiology, Neuropathology, and Pathogenesis, Parkinson's Disease: Pathogenesis and Clinical Aspects[END_REF].

Nowadays, PD is described as a disease with slow progressive neurodegeneration which starts years before diagnosis. PD implicates multiple neuro-anatomical areas. It results from a combination of genetic and environmental factors, and manifests with a broad range of symptoms (Kalia and Lang, 2015).

The prodromal phase of PD is particularly interesting from a therapeutic standpoint. During the prodromal phase non-motor symptoms present themselves, but no clinical diagnosis can currently be made (Figure 1), diagnosis is only made when quantifiable motor-symptoms appear. Better detection and characterisation may help to start appropriate neuroprotective therapies at this early stage.

It is believed that at time of diagnosis already between 30% [START_REF] Rizek | An update on the diagnosis and treatment of Parkinson disease[END_REF]) and 50% [START_REF] Fearnley | Ageing and Parkinson's disease: substantia nigra regional selectivity[END_REF], and even up to 80% [START_REF] Demaagd | Parkinson's Disease and Its Management: Part 1: Disease Entity, Risk Factors, Pathophysiology, Clinical Presentation, and Diagnosis[END_REF][START_REF] Sveinbjornsdottir | The clinical symptoms of Parkinson's disease[END_REF], of the dopaminergic neurons in the nigro-striatal pathway have perished, combined with 80% reduction of striatal dopamine levels [START_REF] Marsden | Parkinson's disease[END_REF][START_REF] Fearnley | Ageing and Parkinson's disease: substantia nigra regional selectivity[END_REF]. Clinical diagnosis of PD is especially difficult in the early stages [START_REF] De Lau | Epidemiology of Parkinson's disease[END_REF], this can delay a diagnosis for several years until symptoms of functional disability develop [START_REF] Abbasi Gharibkandi | Radiotracers for imaging of Parkinson's disease[END_REF].

Dopaminergic signalling 1.Basal ganglia circuitry

The basal ganglia are best known for their role in motor functions [START_REF] Bartels | Parkinson's disease: the syndrome, the pathogenesis and pathophysiology[END_REF], although they also play a role in goal-directed behaviours [START_REF] Delong | Changing views of basal ganglia circuits and circuit disorders[END_REF][START_REF] Haber | The place of dopamine in the cortico-basal ganglia circuit[END_REF].

The basal ganglia are a collection of interconnected subcortical nuclei. They include the striatum, substantia nigra, globus pallidus, and subthalamic nucleus (Figure 2). The substantia nigra has two separate nuclei; the GABAergic pars reticulate (SNr), and the dopaminergic pars compacta (SNpc). The dopaminergic neurons of the substantia nigra pars compacta project primarily to the striatum. The striatum is the main input structure of the basal ganglia and receives projections from the cerebral cortex, brainstem, and thalamus. The subthalamic nucleus (STN) is glutamatergic and is located between the two parts of the globus pallidus (external (GPe) and internal segments (GPi)). The subthalamic nucleus is the secondary input structure, receiving input from the cerebral cortex. The activity at the corticostriatal synapses is regulated by dopamine release from the terminals of the substantia nigra pars compacta [START_REF] Delong | Changing views of basal ganglia circuits and circuit disorders[END_REF][START_REF] Haber | The place of dopamine in the cortico-basal ganglia circuit[END_REF].

There are two main types of dopamine receptors in the striatum; D 1 -like and D 2 -like receptors, their activation causing opposing effects on the basal ganglionic output. Activation of the D 1 -like receptors by dopamine results in increased activity of the direct pathway (striatum  Gpi/SNr), leading to inhibition of GPi/SNr output neurons, and consequently to increased thalamocortical neuronal activity. Activation of the D 2 -like receptors leads to activation of the indirect pathway; striatum  GPe  STN  thalamus, leading to disinhibition of GPi/SNr output neurons, and ultimately to greater inhibition of thalamocortical neuronal activity (DeLong and Wichmann, 2010).

Dopamine physiology

Dopamine synthesis starts in the liver, directly from tyrosine (non-essential amino acid) or indirectly from phenylalanine (essential amino acid). L-phenylalanine is converted into L-tyrosine by phenylalanine hydroxylase (PH), and tyrosine is subsequently transported into the dopaminergic neurons in the brain via an active transport mechanism. In the brain, L-tyrosine is converted into L-3,4-dihydroxyphenylalanine (L-DOPA or levodopa) through hydroxylation at the phenol ring by tyrosine hydroxylase (TH). L-DOPA is subsequently converted into 3,4-dihydroxyphenethylamine (dopamine) through decarboxylation by aromatic-L-amino acid decarboxylase (AADC; also known as DOPA decarboxylase) in the pre-synaptic terminal [START_REF] Musacchio | Enzymes Involved in the Biosynthesis and Degradation of Catecholamines[END_REF][START_REF] Zahoor | Pharmacological Treatment of Parkinson's Disease, Parkinson's Disease: Pathogenesis and Clinical Aspects[END_REF].

Dopamine is metabolised after reuptake into dopaminergic neurons or glial cells. Dopamine undergoes oxidative deamination by monoamine oxidase (MAO) to produce reactive aldehyde 3,4dihydroxyphenylacetaldehyde (DOPAL). DOPAL, in turn, is converted into 3,4-dihydroxyphenylacetic acid (DOPAC) by aldehyde dehydrogenase (ALDH) [START_REF] Eisenhofer | Catecholamine metabolism: a contemporary view with implications for physiology and medicine[END_REF]. DOPAC is then degraded by the catechol-O-methyl transferase (COMT) into biologically inactive homovanillic acid (HVA) [START_REF] Zahoor | Pharmacological Treatment of Parkinson's Disease, Parkinson's Disease: Pathogenesis and Clinical Aspects[END_REF].

Hallmarks of PD and Physiopathology

As mentioned above, Parkinson's disease is a progressive neurodegenerative disease with early prominent death of the dopaminergic neurons in the substantia nigra pars compacta (Kalia and Lang, 2015). Nevertheless, other main hallmarks of PD are the presence of Lewy bodies and inflammation.

Dopaminergic neurodegeneration

The loss of dopaminergic neurons in the substantia nigra pars compacta is the most prominent neuropathological hallmark of PD [START_REF] Pienaar | Parkinson's disease: Insights from non-traditional model organisms[END_REF]. The nigrostriatal dopaminergic pathway is more affected than any other [START_REF] Cadet | Free radicals and the pathobiology of brain dopamine systems[END_REF], which is believed to stem from the sensitivity to oxidative stress [START_REF] Guo | Damage to dopaminergic neurons by oxidative stress in Parkinson's disease (Review)[END_REF] and high density of microglial cells [START_REF] Kim | Regional difference in susceptibility to lipopolysaccharide-induced neurotoxicity in the rat brain: role of microglia[END_REF]. The neuronal loss within the substantia nigra pars compacta decreases the dopamine signalling in the striatum, which leads to the motor symptoms of PD [START_REF] Cooper | Modeling Parkinson's disease in C. elegans[END_REF]. However, in later stages of the disease, neurodegeneration also occurs in many other brain regions, including locus ceruleus, raphe nucleus, amygdala, and hypothalamus [START_REF] Ikeda | Idiopathic parkinsonism with lewy-type inclusions in cerebral cortex. A case report[END_REF][START_REF] Dickson | Parkinson's disease and parkinsonism: neuropathology[END_REF], affecting cholinergic, serotonergic, and noradrenergic neurotransmitter systems [START_REF] Pienaar | Parkinson's disease: Insights from non-traditional model organisms[END_REF].

C h a p t e r 1 -I n t r o d u c t i o n | 16

Lewy pathology

In 1912 Friedrich Lewy discovered protein aggregates in brain regions outside the substantia nigra in PD patients. Later, in 1919, Konstantin Tretiakoff found similar aggregates in the substantia nigra and named them after Lewy [START_REF] Trétiakoff | Contribution a l'etude l'anatomie pathologique du locus Niger de soemmering: avec quelques déductions relatives à la pathogénie des troubles du tonus musculaire et de la maladie de Parkinson[END_REF]; Lewy bodies when in the cell body, and Lewy neurites when in the processes of neurons. However, Lewy pathology is not solely restricted to the brain, but can also affect the spinal cord and peripheral nervous system [START_REF] Beach | Multi-organ distribution of phosphorylated alpha-synuclein histopathology in subjects with Lewy body disorders[END_REF]. It took until 1997 before it was discovered that the main component of Lewy bodies is α-synuclein (see 1.3.1, page 17) [START_REF] Spillantini | a-Synuclein in Lewy bodies[END_REF]; in a misfolded state α-synuclein becomes insoluble and aggregates (Kalia and Lang, 2015).

Lewy aggregates also contain various other proteins (including tau), amongst which some are linked to familiar forms of PD; i.e. leucine-rich repeat kinase 2 (LRRK2), parkin, ubiquitin, and UCH-L1 [START_REF] Wakabayashi | The Lewy body in Parkinson's disease: molecules implicated in the formation and degradation of alpha-synuclein aggregates[END_REF]. Interestingly, Lewy pathology is not always found in the brains of patients with rare genetic forms of PD, such as parkin-related PD, and non-G2019S mutated LRRK2-related PD [START_REF] Poulopoulos | The neuropathology of genetic Parkinson's disease[END_REF].

In PD patients, Lewy bodies are commonly found in brain regions susceptible to neurodegeneration, and although generally accepted that they cause this neurodegeneration, there is also a movement suggesting that they are protective of neurodegeneration [START_REF] Olanow | Lewy-body formation is an aggresome-related process: A hypothesis[END_REF]. It is thought that toxic α-synuclein aggregates are separated in Lewy bodies, and that they promote their degradation [START_REF] Olanow | Lewy-body formation is an aggresome-related process: A hypothesis[END_REF][START_REF] Tanaka | Aggresomes formed by alpha-synuclein and synphilin-1 are cytoprotective[END_REF]. In support of this theory, in vitro studies have shown that cell death can precede Lewy body formation [START_REF] Tompkins | Contribution of somal Lewy bodies to neuronal death[END_REF][START_REF] Saha | Induction of neuronal death by alpha-synuclein[END_REF]. Moreover, neurons have been described as having a healthier morphology when containing Lewy bodies [START_REF] Terry | Do neuronal inclusions kill the cell?[END_REF], and no evidence was found to support association between either Lewy body distribution or density and dopaminergic cell loss [START_REF] Parkkinen | Disentangling the Relationship between Lewy bodies and nigral neuronal loss in Parkinson's disease[END_REF].

Inflammation

Another hallmark of PD is the presence of neuroinflammation; i.e. microglial activation, increased astrocyte and lymphocyte infiltration, and the presence of pro-inflammatory cytokines [START_REF] Glass | Mechanisms Underlying Inflammation in Neurodegeneration[END_REF].

Microglia are the resident innate immune cells of the brain, and play a major role in inflammatory processes. In a healthy brain they reside in a resting state that is characterized by a ramified morphology, while continuously monitoring the brain environment [START_REF] Nimmerjahn | Resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo[END_REF][START_REF] Glass | Mechanisms Underlying Inflammation in Neurodegeneration[END_REF]. In response to inflammation, microglia are activated and adopt an amoeboid morphology [START_REF] Nimmerjahn | Resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo[END_REF][START_REF] Cho | Pathological dynamics of activated microglia following medial forebrain bundle transection[END_REF].

Astrocytes are abundantly present in the brain and play a role in homeostasis, provide trophic support neurons, and regalute synapses. Activated microglia can induce reactive astrocytes via secretion of pro-inflammatory cytokines (i.e. interleukin (IL)-1α, tumor necrosis factor α (TNFα), and complement component 1, q subcomponent (C1q). Reactive astrocytes lose their beneficial role in brain homestasis and instead can induce neurodegeneration [START_REF] Liddelow | Neurotoxic reactive astrocytes are induced by activated microglia[END_REF].

In 1988, McGeer and colleagues described for the first time the presence reactive microglia in the brains of PD patients [START_REF] Mcgeer | Reactive microglia are positive for HLA-DR in the substantia nigra of Parkinson's and Alzheimer's disease brains[END_REF]. Damiar and colleagues reported an increase in glutathione peroxidase-containing cells (a subset of glial cells) surrounding the surviving dopaminergic neurons, which was inversely correlated with the amount of neuronal loss (Damier et C h a p t e r 1 -I n t r o d u c t i o n | 17 al., 1993). Nagatsu and colleagues (2000) measured increased levels of various cytokines in the striatum (e.g. TNFα, IL-1β) [START_REF] Nagatsu | Cytokines in Parkinson's disease[END_REF]. Finally, also activated astrocytes have been reported to accompany the Lewy pathology [START_REF] Braak | Development of alpha-synuclein immunoreactive astrocytes in the forebrain parallels stages of intraneuronal pathology in sporadic Parkinson's disease[END_REF].

Today, it is generally believed that the immune system plays an active role, and that the neuroinflammation in PD might not only be a consequence of the ongoing neurodegeneration as initially hypothesised [START_REF] Tansey | Neuroinflammation in Parkinson's disease: Its role in neuronal death and implications for therapeutic intervention[END_REF][START_REF] Cabezudo | Multiple-Hit Hypothesis in Parkinson's Disease: LRRK2 and Inflammation[END_REF]. However, the interplay between cytokines, neurodegeneration, and protein aggregation as cause or consequence remains largely unknown [START_REF] De Lau | Epidemiology of Parkinson's disease[END_REF][START_REF] Glass | Mechanisms Underlying Inflammation in Neurodegeneration[END_REF][START_REF] Tansey | Neuroinflammation in Parkinson's disease: Its role in neuronal death and implications for therapeutic intervention[END_REF]. One possibility is that neuronal death and the subsequent release of protein aggregates induces microglial activation. Indeed, α-synuclein can cause microglial activation, and eventually leed to a positive feedback-loop of neurodegeneration and neuroinflammation (reviewed by [START_REF] Roodveldt | Immunological features of α-synuclein in Parkinson's disease[END_REF]. On the other hand, the multiple hit hypothesis suggests an interaction between multiple genetic and/or environmental risk factors, including an inflammation component, is needed to trigger PD pathology [START_REF] Cabezudo | Multiple-Hit Hypothesis in Parkinson's Disease: LRRK2 and Inflammation[END_REF].

It is also under debate if presence of inflammatory cells is protective or deleterious. On one hand, astrocytes can protect against entry by inflammatory cells through borders or scars; on the other hand, astrocytes may also have strong pro-inflammatory potential (reviewed by [START_REF] Sofroniew | Astrocyte barriers to neurotoxic inflammation[END_REF].

Causes of PD / Aetiology

Parkinson's disease is a multifactorial disease [START_REF] Semchuk | Parkinson's disease: A test of the multifactorial etiologic hypothesis[END_REF], meaning that both genetic and environmental factors play a role. The biggest risk factor for PD is age [START_REF] Van Den Eeden | Incidence of Parkinson's disease: variation by age, gender, and race/ethnicity[END_REF]Kalia and Lang, 2015;[START_REF] Kouli | Parkinson's Disease: Etiology, Neuropathology, and Pathogenesis, Parkinson's Disease: Pathogenesis and Clinical Aspects[END_REF], with a mean age of onset of 55 years [START_REF] Dauer | Parkinson's Disease: Mechanisms and Models[END_REF]. PD seems to be more prevalent in men than in women [START_REF] Van Den Eeden | Incidence of Parkinson's disease: variation by age, gender, and race/ethnicity[END_REF][START_REF] De Lau | Epidemiology of Parkinson's disease[END_REF]. Additionally, PD is more prevalent in Europe and the Americas, as compared to Africa, Asia, and Arabia [START_REF] Van Den Eeden | Incidence of Parkinson's disease: variation by age, gender, and race/ethnicity[END_REF]Kalia and Lang, 2015). Apart from a genetic predisposition, also environmental or occupational exposure to certain substances, as well as life style choices, can affect the risk for PD (Kalia and Lang, 2015).

Genetic

About 15% of the parkinsonian cases have a family history of PD [START_REF] Deng | The genetics of Parkinson disease[END_REF]. To date 23 loci have been linked to PD, termed PARK1 to PARK23. There are both autosomal dominant and recessive mutations, while some mutations are considered risk factors rather than causative [START_REF] Deng | The genetics of Parkinson disease[END_REF]. Even though these monogenic forms of PD are less common, the same cellular pathways are involved as in multifactorial cases [START_REF] Cooper | Modeling Parkinson's disease in C. elegans[END_REF].

Dominant mutations in SNCA/PARK1/PARK4

The first gene mutation (A53T) to cause monogenic PD was discovered in 1997 in the SNCA/PARK1/PARK4 gene, which encodes α-synuclein [START_REF] Polymeropoulos | Mutation in the α-Synuclein Gene Identified in Families with Parkinson's Disease[END_REF]. Multiple other point mutations (A53T, A53E, A30P, E46K, H50Q, G51D) [START_REF] Krüger | AlaSOPro mutation in the gene encoding α-synuclein in Parkinson's disease[END_REF][START_REF] Zarranz | The new mutation, E46K, of α-synuclein causes parkinson and Lewy body dementia[END_REF][START_REF] Appel-Cresswell | Alpha-synuclein p.H50Q, a novel pathogenic mutation for Parkinson's disease[END_REF][START_REF] Kiely | α-Synucleinopathy associated with G51D SNCA mutation: a link between Parkinson's disease and multiple system atrophy?[END_REF][START_REF] Proukakis | A novel a-synuclein missense mutation in Parkinson disease[END_REF][START_REF] Pasanen | Novel α-synuclein mutation A53E associated with atypical multiple system atrophy and Parkinson's disease-type pathology[END_REF] and even C h a p t e r 1 -I n t r o d u c t i o n | 18 duplication and triplication [START_REF] Singleton | alpha-Synuclein locus triplication causes Parkinson's disease[END_REF][START_REF] Chartier-Harlin | alpha-Synuclein locus duplication as a cause of familial Parkinson's disease[END_REF] have now been linked to autosomal dominant PD. These mutations or increased protein expression make α-synuclein more prone to aggregation (Kalia and Lang, 2015). An overview of the use of SNCA (mutations) to model PD is discussed later on page 26. SNCA-related PD is relatively rare (Kalia and Lang, 2015), nonetheless, it led to the discovery of α-synuclein as a major component of Lewy bodies and Lewy neurites [START_REF] Spillantini | a-Synuclein in Lewy bodies[END_REF]. Even so, the exact function of α-synuclein remains unknown.

The primate (including human) α-synuclein sequence differs from other vertebrates by a substitution of Alanine for a Threonine at position 53 (A53T) [START_REF] Hamilton | alpha-Synuclein A53T substitution associated with Parkinson disease also marks the divergence of Old World and New World primates[END_REF]. Human α-synuclein has 140 amino acids, encoded by six exons, and has three main domains; an N-terminal domain which adopts an α-helical structure upon binding to cellular membranes, a median domain called non-amyloid component (NAC) which is normally folded as an α-helix, and an acidic non-folded Cterminal domain [START_REF] Snead | Alpha-synuclein function and dysfunction on cellular membranes[END_REF][START_REF] Bobela | Alpha-synuclein as a mediator in the interplay between aging and Parkinson's disease[END_REF]. All the domains have their own roles and are essential for the structure of the protein [START_REF] Snead | Alpha-synuclein function and dysfunction on cellular membranes[END_REF]. α-Synuclein is abundantly present presynaptically [START_REF] Maroteaux | Synuclein: a neuron-specific protein localized to the nucleus and presynaptic nerve terminal[END_REF], and is thought to modulate synaptic vesicle function [START_REF] Kahle | Structure/function of alpha-synuclein in health and disease: rational development of animal models for Parkinson's and related diseases[END_REF].

Mutations in LRRK2/PARK8

The most frequent cause of monogenic PD is a mutation in the LRRK2/PARK8 gene, encoding for leucine-rich repeat kinase 2 (LRRK2). There are at least eight different pathogenic mutations in the LRRK2 gene (Kalia and Lang, 2015); of which the G2019S substitution is the most common [START_REF] Healy | Phenotype, genotype, and worldwide genetic penetrance of LRRK2-associated Parkinson's disease: a case-control study[END_REF]. LRRK2 is normally localised in membranes [START_REF] Jackson-Lewis | Animal models of Parkinson's disease[END_REF], and while its normal function is not clear, there seems to be a link with mitochondria (reviewed in [START_REF] Winklhofer | Mitochondrial dysfunction in Parkinson's disease[END_REF]. Additionally, it is thought to be involved in neurite outgrowth (Kalia and Lang, 2015), synaptic morphogenesis [START_REF] Lee | The synaptic function of LRRK2[END_REF], vesicle trafficking [START_REF] Sanna | LRRK2 and vesicle trafficking[END_REF], autophagy (Kalia and Lang, 2015), and protein synthesis (Kalia and Lang, 2015). Moreover, LRRK2 is highly expressed in immune cells and has been functionally linked to pathways pertinent to immune cell function, such as cytokine release, autophagy and phagocytosis [START_REF] Dzamko | An emerging role for LRRK2 in the immune system[END_REF][START_REF] Wallings | LRRK2 regulation of immune-pathways and inflammatory disease[END_REF]. The LRRK2 gene codes for a large protein (2527 amino acids) in the ROCO family; formed by a core signalling region (Ras of complex (ROC) and C-terminal of ROC (COR) domains) and protein-protein interaction domains (e.g. leucine-rich repeats, and WD40 domains) [START_REF] Li | The role of the LRRK2 gene in Parkinsonism[END_REF]. Interestingly, most of the disease causing mutations are localised within the catalytic domains (ROC/COR/Kinase) of LRRK2 [START_REF] Healy | Phenotype, genotype, and worldwide genetic penetrance of LRRK2-associated Parkinson's disease: a case-control study[END_REF]. The G2019S mutation leads to increased kinase activity (Kalia and Lang, 2015). The use of LRRK2 mutations to model PD will be discussed later in this chapter (page 27). For a detailed review of the putative interplay between LRRK2 and α-synuclein, the reader is referred to Cresto and colleagues (2018).

Autosomal recessive genes

The autosomal recessive forms of PD typically have an earlier onset than classical PD [START_REF] Schrag | Epidemiological, clinical, and genetic characteristics of early-onset parkinsonism[END_REF], and most are linked to mitochondrial homeostasis [START_REF] Mccoy | Mitochondrial quality control and dynamics in Parkinson's disease[END_REF]. Both homozygous and compound heterozygous mutations in these genes might result in autosomal recessive PD (Kalia and Lang, 2015).

Mutations in the PRKN/PARK2/Parkin gene [START_REF] Kitada | Mutations in the parkin gene cause autosomal recessive juvenile parkinsonism[END_REF] account for almost half of the early-onset familial cases of PD [START_REF] Lücking | Association between Early-Onset Parkinson's Disease and Mutations in the Parkin Gene[END_REF][START_REF] Jackson-Lewis | Animal models of Parkinson's disease[END_REF], and there are over 100 mutations known for the Parkin gene [START_REF] Lücking | Association between Early-Onset Parkinson's Disease and Mutations in the Parkin Gene[END_REF], most of which are C h a p t e r 1 -I n t r o d u c t i o n | 19 loss-of-function [START_REF] Konnova | Animal Models of Parkinson's Disease, Parkinson's Disease: Pathogenesis and Clinical Aspects[END_REF]. Parkin is involved in the ubiquitin proteasome system as an E3 ubiquitin ligase [START_REF] Shimura | Familial Parkinson disease gene product, parkin, is a ubiquitin-protein ligase[END_REF][START_REF] Zhang | Parkin functions as an E2-dependent ubiquitin-protein ligase and promotes the degradation of the synaptic vesicle-associated protein, CDCrel-1[END_REF].

The gene for phosphatase and tensin (PTEN) homolog-induced novel kinase 1 (PINK1/PARK6) is the second most commonly mutated in early-onset PD [START_REF] Konnova | Animal Models of Parkinson's Disease, Parkinson's Disease: Pathogenesis and Clinical Aspects[END_REF]. Similar to Parkin, PINK1 mutations are recessive, and lead to loss-of-function [START_REF] Dawson | Genetic Animal Models of Parkinson's Disease[END_REF]. PINK1 is normally localised in the mitochondria [START_REF] Silvestri | Mitochondrial import and enzymatic activity of PINK1 mutants associated to recessive parkinsonism[END_REF], where it recruits Parkin and increases its ubiquination activity and induces Parkin-mediated mitophagy [START_REF] Lazarou | PINK1 drives Parkin self-association and HECT-like E3 activity upstream of mitochondrial binding[END_REF], which is a process to eliminate damaged mitochondria (Kalia and Lang, 2015).

DJ-1 is a redox sensitive molecular chaperone [START_REF] Moore | Lessons from Drosophila models of DJ-1 deficiency[END_REF], whose mutations play a role in early-onset PD [START_REF] Bonifati | Mutations in the DJ-1 gene associated with autosomal recessive early-onset parkinsonism[END_REF]; causing loss of function [START_REF] Macedo | The DJ-1L166P mutant protein associated with early onset Parkinson's disease is unstable and forms higher-order protein complexes[END_REF][START_REF] Moore | A missense mutation (L166P) in DJ-1, linked to familial Parkinson's disease, confers reduced protein stability and impairs homo-oligomerization[END_REF]. DJ-1 plays a role in the inhibition of α-synuclein aggregation [START_REF] Shendelman | DJ-1 is a redox-dependent molecular chaperone that inhibits alphasynuclein aggregate formation[END_REF]. It is expressed widely throughout the whole body, and localised in the cytosol and mitochondria [START_REF] Zhang | Mitochondrial localization of the Parkinson's disease related protein DJ-1: implications for pathogenesis[END_REF].

Genetic risk factors

The biggest genetic risk factor for PD is a mutation in the GBA1 gene [START_REF] Migdalska-Richards | The relationship between glucocerebrosidase mutations and Parkinson disease[END_REF]. GBA1 mutations have reduced penetrance, thus do not cause a Mendelian form of PD, however, they can increase the risk for PD 20-30 fold [START_REF] Sidransky | Multicenter analysis of glucocerebrosidase mutations in Parkinson's disease[END_REF][START_REF] Migdalska-Richards | The relationship between glucocerebrosidase mutations and Parkinson disease[END_REF]. About 30% of GBA1 mutation carriers develop PD before the age of 80 [START_REF] Migdalska-Richards | The relationship between glucocerebrosidase mutations and Parkinson disease[END_REF].

GBA1 encodes for β-glucocerebrosidase, a lysosomal enzyme [START_REF] Beutler | Gaucher disease: new molecular approaches to diagnosis and treatment[END_REF]. Bi-allelic mutations that results in greatly reduced glucocerebrosidase enzyme activity lead to Gaucher disease, an autosomal recessive lysosomal storage disorder [START_REF] Hruska | Gaucher disease: Mutation and polymorphism spectrum in the glucocerebrosidase gene (GBA)[END_REF][START_REF] Stoker | Pathological Mechanisms and Clinical Aspects of GBA1 Mutation-Associated Parkinson's Disease[END_REF]. Roughly 10% of patients with type 1 Gaucher disease develop PD before the age of 80 [START_REF] Rosenbloom | The incidence of Parkinsonism in patients with type 1 Gaucher disease: Data from the ICGG Gaucher Registry[END_REF], compared to 3-4% in the normal population [START_REF] Stoker | Pathological Mechanisms and Clinical Aspects of GBA1 Mutation-Associated Parkinson's Disease[END_REF].

In a genome-wide association study (GWAS) in PD patients, the SNCA and microtubuleassociated protein tau (MAPT) locus were identified as major risk factors for PD [START_REF] Simon-Sanchez | Genome-Wide Association Study reveals genetic risk underlying R e f e r e n c e s | 211 Parkinson's disease HHS Public Access Author manuscript[END_REF][START_REF] Bandres-Ciga | Genetics of Parkinson's disease: An introspection of its journey towards precision medicine[END_REF]. The MAPT locus has two major haplotypes, of which the MAPTH1 haplotype is associated with an increased risk for PD. This could be due to haplotypespecific differences in expression or alternative splicing of MAPT transcripts, which can affect cellular functions at different levels, and ultimately increase PD susceptibility [START_REF] Billingsley | Genetic risk factors in Parkinson's disease[END_REF].

In a recent GWAS around 37,700 PD patients and 1.4 million million controls were used to identify 90 independent risk factors associated with PD [START_REF] Nalls | Identification of novel risk loci, causal insights, and heritable risk for Parkinson's disease: a meta-analysis of genome-wide association studies[END_REF][START_REF] Bandres-Ciga | Genetics of Parkinson's disease: An introspection of its journey towards precision medicine[END_REF].

Environmental contributors & Lifestyle

In 1983 [START_REF] Chartier-Harlin | alpha-Synuclein locus duplication as a cause of familial Parkinson's disease[END_REF]3, was described to be the cause of a parkinsonian-like state of a small group of patients after injection as by-product of a designer drug (also see paragraph 2.2, page 24) [START_REF] Langston | Chronic Parkinsonism in humans due to a product of meperidine-analog synthesis[END_REF][START_REF] Langston | The MPTP Story[END_REF]. Since it was shown that parkinsonism can be caused by a toxin, the idea arose that nigral dopaminergic neurodegeneration could also be caused by other environmental toxins [START_REF] Kouli | Parkinson's Disease: Etiology, Neuropathology, and Pathogenesis, Parkinson's Disease: Pathogenesis and Clinical Aspects[END_REF].

C h a p t e r 1 -I n t r o d u c t i o n | 20

Pesticides

Investigation of a French cohort revealed a positive association between PD and the overall use of professional pesticides, with a dose effect for the number of years used. They observed a particularly strong relation between PD and the use of organochlorine insecticides [START_REF] Elbaz | Professional exposure to pesticides and Parkinson disease[END_REF]. One example of an organochlorine is dieldrin, which has been shown to deplete brain dopamine levels in several animal species and increase dopamine transporter (DAT) binding in the striatum [START_REF] Kanthasamy | Dieldrin-induced neurotoxicity: Relevance to Parkinson's disease pathogenesis[END_REF]. Additionally, a positive relationship has been shown between PD and the professional use of rotenone (mitochondrial complex I inhibitor), paraquat (inducer of oxidative stress), and other herbicides [START_REF] Semchuk | Parkinson's disease: A test of the multifactorial etiologic hypothesis[END_REF][START_REF] Tanner | Rotenone, paraquat, and Parkinson's disease[END_REF].

Heavy metals

The implication of heavy metals in PD etiology is highly debated, with several studies demonstrating relationships [START_REF] Cheng | Dietary intake of iron, zinc, copper, and risk of Parkinson's disease: a metaanalysis[END_REF], while others do not [START_REF] Jiménez-Jiménez | Serum levels of zinc and copper in patients with Parkinson's disease[END_REF][START_REF] Semchuk | Parkinson's disease: A test of the multifactorial etiologic hypothesis[END_REF][START_REF] Meamar | Assessing of plasma levels of iron, zinc and copper in Iranian Parkinson′s disease[END_REF]. Of the heavy metals, iron and copper are thought to induce oxidative stress, which might underlie neuronal death [START_REF] Ball | Parkinson's Disease and the Environment[END_REF]. As such, both iron and copper have been demonstrated to accelerate α-synuclein aggregation [START_REF] Uversky | Metal-triggered Structural Transformations, Aggregation, and Fibrillation of Human α-Synuclein[END_REF]. Lead, which can cross the blood-brain barrier by mimicking calcium channels, can cause neuronal loss [START_REF] Caudle | Industrial toxicants and Parkinson's disease[END_REF]. Lastly, mercury is a neurotoxin that can cause neuronal death, however, its association with PD is still debated [START_REF] Ball | Parkinson's Disease and the Environment[END_REF].

The dietary intake of these heavy metals was found to be unrelated to PD prevalence [START_REF] Mariani | Fe and Cu do not differ in Parkinson's disease: A replication study plus metaanalysis[END_REF]. In contrast, occupational exposure to these heavy metal shows increased associated with PD [START_REF] Gorell | Occupational exposures to metals as risk factors for Parkinson's disease[END_REF][START_REF] Coon | Whole-Body Lifetime Occupational Lead Exposure and Risk of Parkinson's Disease[END_REF][START_REF] Lin | Dose-Response Relationship Between Cumulative Mercury Exposure Index and Specific Uptake Ratio in the Striatum on Tc-99m TRODAT SPECT[END_REF] Lifestyle Both cigarette smoking [START_REF] Hernán | A meta-analysis of coffee drinking, cigarette smoking, and the risk of Parkinson's disease[END_REF][START_REF] Ritz | Pooled Analysis of Tobacco Use and Risk of Parkinson Disease[END_REF][START_REF] Noyce | Meta-analysis of early nonmotor features and risk factors for Parkinson disease[END_REF][START_REF] Breckenridge | Association between Parkinson's disease and cigarette smoking, rural living, well-water consumption, farming and pesticide use: Systematic review and metaanalysis[END_REF] and coffee drinking [START_REF] Ascherio | Prospective study of caffeine consumption and risk of Parkinson's disease in men and women[END_REF][START_REF] Hernán | A meta-analysis of coffee drinking, cigarette smoking, and the risk of Parkinson's disease[END_REF][START_REF] Noyce | Meta-analysis of early nonmotor features and risk factors for Parkinson disease[END_REF] have been linked to reduced incidences of PD.

It is still unclear why cigarette smoking is associated with a reduced risk of PD. One explanation is that the activation by nicotine of nicotinic acetylcholine receptors on the dopaminergic neurons are neuroprotective [START_REF] Kouli | Parkinson's Disease: Etiology, Neuropathology, and Pathogenesis, Parkinson's Disease: Pathogenesis and Clinical Aspects[END_REF], which has been shown in animal models [START_REF] Bordia | The α7 nicotinic receptor agonist ABT-107 protects against nigrostriatal damage in rats with unilateral 6-hydroxydopamine lesions[END_REF]. However, in a clinical trial there was no indication that nicotine could relieve PD symptoms [START_REF] Wood | Parkinson disease: Caffeine and nicotine do not provide symptomatic relief in Parkinson disease[END_REF]. Alternatively, it could be that PD itself reduces smoking behaviour [START_REF] Ritz | Parkinson disease and smoking revisited: Ease of quitting is an early sign of the disease[END_REF][START_REF] Kouli | Parkinson's Disease: Etiology, Neuropathology, and Pathogenesis, Parkinson's Disease: Pathogenesis and Clinical Aspects[END_REF]; the reduction of dopamine levels in PD patients could make them less susceptible for addictive behaviours [START_REF] Ritz | Parkinson disease and smoking revisited: Ease of quitting is an early sign of the disease[END_REF][START_REF] Kouli | Parkinson's Disease: Etiology, Neuropathology, and Pathogenesis, Parkinson's Disease: Pathogenesis and Clinical Aspects[END_REF]. This hypothesis is supported by research showing that prodromal and PD patients had greater ease in giving up smoking compared to controls [START_REF] Ritz | Parkinson disease and smoking revisited: Ease of quitting is an early sign of the disease[END_REF]. Interestingly, a significant decrease of nicotinic acetylcholine receptors was observed in the striatum and substantia nigra of PD patients that was not correlated with disease severity [START_REF] Kas | Decrease of nicotinic receptors in the nigrostriatal system in Parkinson's R e f e r e n c e s | 194 disease[END_REF].

For caffeine, as for nicotine, the exact pathway and causative roll of caffeine still need to be shown [START_REF] Kouli | Parkinson's Disease: Etiology, Neuropathology, and Pathogenesis, Parkinson's Disease: Pathogenesis and Clinical Aspects[END_REF]. Also caffeine did not relieve clinical symptoms in PD patients in a clinical trial [START_REF] Wood | Parkinson disease: Caffeine and nicotine do not provide symptomatic relief in Parkinson disease[END_REF]. Furthermore, there seemed to be marked differences between the effects in men and women; women show a weaker inverse relation between coffee and PD. This could be explained by the fact that oestrogen competitively inhibits caffeine metabolism [START_REF] Ascherio | Coffee consumption, gender, and Parkinson's disease mortality in the Cancer Prevention Study II cohort: The modifying effects of estrogen[END_REF][START_REF] Kouli | Parkinson's Disease: Etiology, Neuropathology, and Pathogenesis, Parkinson's Disease: Pathogenesis and Clinical Aspects[END_REF].

C h a p t e r 1 -I n t r o d u c t i o n | 21

Disease progression and α-synuclein spreading

Dual hit-theory Braak, Del Tredici and colleagues (2003) published on the temporal and spatial spreading of Lewy pathology in PD and recognized six different stages (Figure 3). In stage 1 the peripheral nervous system, olfactory system, and medulla are affected, followed by the pons and spinal cord grey matter in stage 2. Next, in stage 3, the midbrain -including the SN pars compacta-, basal forebrain, and amygdala display Lewy pathology, which spreads further to the rest of the limbic system, thalamus, and temporal cortex in stage 4. In stage 5 and 6 multiple cortical regions show Lewy pathology [START_REF] Braak | Staging of brain pathology related to sporadic Parkinson's disease[END_REF]Kalia and Lang, 2015). These brain pathological stages seem to correspond to the clinical symptoms of PD: in stages 1 and 2 pre-motor symptoms occur, in stage 3 motor features appear due to nigrostriatal dopamine deficiency, and stages 4-6 correspond to the non-motor symptoms of advanced PD (Kalia and Lang, 2015). While the Braak theory initially proposes a viral pathogen as initiator of PD pathology, it has been shown that peripherally administered rotenone (an environmental toxin, also see page 19 and page 141) can induce sequential α-synuclein accumulation, inflammation, and motor dysfunction [START_REF] Pan-Montojo | Progression of Parkinson's disease pathology is reproduced by intragastric administration of rotenone in mice[END_REF]. Braak, Rüb and colleagues (2003) have subsequently suggested that the stereotypic pattern of PD primarily affects cell types with a disposition for Lewy pathology; specifically, projection neurons with long, thin, incomplete or poorly myelinated axons (but not long sturdily myelinated or shortaxoned nerve cells) [START_REF] Braak | Idiopathic Parkinson's disease: Possible routes by which vulnerable neuronal types may be subject to neuroinvasion by an unknown pathogen[END_REF][START_REF] Visanji | The prion hypothesis in Parkinson's disease: Braak to the future[END_REF]. It is not known why these specific neuronal types are more susceptible, and why neighbouring regions with similar cell types remain unaffected [START_REF] Visanji | The prion hypothesis in Parkinson's disease: Braak to the future[END_REF].

In a subsequent paper these authors hypothesized that PD might originate in the gut, and from there spread towards the brain [START_REF] Braak | Idiopathic Parkinson's disease: Possible routes by which vulnerable neuronal types may be subject to neuroinvasion by an unknown pathogen[END_REF]. A few years later, the dual hit-theory emerged, which states that the neurotrophic pathogen enters the brain via two routes; 1) nasal, with anterograde progression into the temporal lobe, and 2) gastric, secondary to swallowing of nasal secretions in saliva [START_REF] Hawkes | Parkinson's disease: The dual hit theory revisited[END_REF]. The nasal route would explain the early involvement of olfactory structures and the olfactory dysfunction that is often seen in early PD [START_REF] Hawkes | Parkinson's disease: The dual hit theory revisited[END_REF]. Lewy pathology has been observed in several olfactory structures, as well as projection neurons connected to the olfactory epithelium [START_REF] Daniel | Preliminary diagnosis of Parkinson's disease by olfactory bulb pathology[END_REF][START_REF] Braak | Staging of brain pathology related to sporadic Parkinson's disease[END_REF]. A revised theory by [START_REF] Lerner | Olfactory pathogenesis of idiopathic parkinson disease revisited[END_REF] suggests not only anterograde, but also retrograde spreading of PD [START_REF] Lerner | Olfactory pathogenesis of idiopathic parkinson disease revisited[END_REF].

C h a p t e r 1 -I n t r o d u c t i o n | 22

The gut-brain axis

In support of the theory that the gut is one of two places of origin of PD, Lewy pathology has been found throughout the gastrointestinal tract of patients [START_REF] Wakabayashi | Parkinson's disease: the presence of Lewy bodies in Auerbach's and Meissner's plexuses[END_REF][START_REF] Beach | Multi-organ distribution of phosphorylated alpha-synuclein histopathology in subjects with Lewy body disorders[END_REF][START_REF] Shannon | Alpha-synuclein in colonic submucosa in early untreated Parkinson's disease[END_REF]. Moreover, PD patients often display non-motor symptoms related to the gastrointestinal tract [START_REF] Visanji | The prion hypothesis in Parkinson's disease: Braak to the future[END_REF]; gastrointestinal dysfunction is common already in the early stages of PD [START_REF] Pfeiffer | Gastrointestinal dysfunction in Parkinson's disease[END_REF], or even in the prodromal stages [START_REF] Abbott | Frequency of bowel movements and the future risk of Parkinson's disease[END_REF]. The nerve cells of the gastrointestinal tract share the same vulnerabilities to Lewy pathology as the central nervous system, and could thus propagate Lewy pathology to the central nervous system via the vagal preganglionic fibres [START_REF] Visanji | The prion hypothesis in Parkinson's disease: Braak to the future[END_REF]. In support of this, suggestive evidence was found for a potential protective effect of severing the vagal nerve against PD in humans [START_REF] Svensson | Vagotomy and subsequent risk of Parkinson's disease[END_REF][START_REF] Liu | Vagotomy and Parkinson disease[END_REF].

Additionally, dysbiosis of the gut microbiome is linked to the prodromal phase of PD. The gut microbiome is crucial for function, metabolism, and energy cycles, but it can have a neurological effect through metabolite production or the gut-brain axis. However, the specific link between the gut microbiome and PD is not clear yet [START_REF] Scheperjans | The gut and Parkinson's disease: Hype or hope?[END_REF][START_REF] Dutta | Parkinson's disease: The emerging role of gut dysbiosis, antibiotics, probiotics, and fecal microbiota transplantation[END_REF][START_REF] Yang | The Role of the Gut Microbiota in the Pathogenesis of Parkinson's Disease[END_REF]. Diet robustly impacts the gut microbiome; the Western diet is associated with an increased risk for PD, while the Mediterranean diet is associated with a reduced risk [START_REF] Jackson | Diet in Parkinson's Disease: Critical Role for the Microbiome[END_REF]. Interestingly, the PD microbiome is associated with increased levels of lipopolysaccharide (paragraph 2.3, page 25) [START_REF] Jackson | Diet in Parkinson's Disease: Critical Role for the Microbiome[END_REF]. It is hypothesised that the observed gastrointestinal permeability in PD patients could lead to increased levels of LPS in the blood, which in turn could cause neuroinflammation and ultimately neurodegeneration [START_REF] Brown | The endotoxin hypothesis of neurodegeneration[END_REF].

Prion hypothesis

Lewy body-like inclusions have occasionally been found within grafted neurons after transplantation of embryonic mesencephalic neurons into the putamen of PD patients (Kordower, C h a p t e r 1 -I n t r o d u c t i o n | 23 Chu, Hauser, Freeman, et al., 2008;[START_REF] Kordower | Transplanted dopaminergic neurons develop PD pathologic changes: A second case report[END_REF][START_REF] Li | Lewy bodies in grafted neurons in subjects with Parkinson's disease suggest host-to-graft disease propagation[END_REF]. These findings suggested that Lewy body pathology can propagate from host to graft cells [START_REF] Li | Lewy bodies in grafted neurons in subjects with Parkinson's disease suggest host-to-graft disease propagation[END_REF]. Indeed, several groups have shown that injection of brain homogenates containing αsynuclein aggregates or synthetic α-synuclein fibrils into the brain of wild-type model animals led to α-synuclein pathology [START_REF] Luk | Pathological α-synuclein transmission initiates Parkinson-like neurodegeneration in nontransgenic mice[END_REF][START_REF] Recasens | Lewy body extracts from Parkinson disease brains trigger α-synuclein pathology and neurodegeneration in mice and monkeys[END_REF][START_REF] Shimozawa | Propagation of pathological α-synuclein in marmoset brain[END_REF][START_REF] Melki | Alpha-synuclein and the prion hypothesis in Parkinson's disease[END_REF]. Based on these and other observations it is now widely believed that aggregated α-synuclein propagates and amplifies in a prion-like manner [START_REF] Brundin | Prion-like transmission of protein aggregates in neurodegenerative diseases[END_REF][START_REF] Chen | Prion-like propagation of α-synuclein in the gut-brain axis[END_REF][START_REF] Melki | Alpha-synuclein and the prion hypothesis in Parkinson's disease[END_REF].

Like in prion diseases, there is mulitple separate diseases characterised by pathological αsynuclein deposits, e.g. multiple system atrophy (MSA), dementia with Lewy Bodies (DLB) and PD. Experiments have shown differences in seed characteristics and seeding activity between patient brain extracts, which supports the hypothesis that MSA, DLB, and PD are caused by different αsynuclein strains (Figure 4), which have different functional properties, including tropism for different cell types or toxicity [START_REF] Melki | Alpha-synuclein and the prion hypothesis in Parkinson's disease[END_REF][START_REF] Jaunmuktane | Invited Review: The role of prion-like mechanisms in neurodegenerative diseases[END_REF]. Functional threshold theory Zaccai and colleagues (2008) suggest that α-synuclein pathology does not follow Braak staging in around half of all PD cases [START_REF] Zaccai | Patterns and stages of α-synucleinopathy[END_REF], indicating that Braak staging is not the only pattern of spreading for α-synuclein pathology. In fact, more recently, a different theory has emerged; the functional threshold theory [START_REF] Engelender | The Threshold Theory for Parkinson's Disease[END_REF]. This theory accounts better for the neurobiology of PD symptoms since it is based on the evidence of simultaneous degeneration of the central and peripheral nervous systems [START_REF] Engelender | The Threshold Theory for Parkinson's Disease[END_REF].

C h a p t e r 1 -I n t r o d u c t i o n | 24
This theory revolves around different vulnerabilities of brain regions, each having an individual threshold for PD pathology (e.g. α-synuclein abundance); more sensitive areas will show earlier signs of dysfunction as compared to more resistant areas. Some areas have a larger functional reserve and can compensate better for the pathology, while others will succumb and present Lewy pathology and ultimately neurodegeneration [START_REF] Engelender | The Threshold Theory for Parkinson's Disease[END_REF].

PD models

Even though, as described above, some important discoveries have been done on human samples, PD autopsy material is rare and precious. Animal models allow for study of different aspects of the disease, and explore both disease progression and evaluate possible treatment strategies. The biggest advantage however, is the possibility to study the prodromal phase with the use of slowly progressive models. At the moment, there is still no perfect model available that mimics all pathological aspects of PD. The current available models can be broadly divided in four types; 1) pharmacological models, 2) toxin-based models, 3) PD hallmark-based models, and 4) genetic-based models. A more extensive review on different animal models in PD, ranging from non-mammalian to non-human primates, can be found in the Additional Readings -Animal models of PD (page 135).

6-hydroxydopamine

The most-used toxin-based PD model is the 6-hydroxydopmine (6-OHDA) model, which is widely used in rodents [START_REF] Ungerstedt | 6-hydroxy-dopamine induced degeneration of central monoamine neurons[END_REF][START_REF] Thiele | Development of a unilaterally-lesioned 6-OHDA mouse R e f e r e n c e s | 213 model of Parkinson's disease[END_REF][START_REF] Stott | Time course of dopamine neuron loss and glial response in the 6-OHDA striatal mouse model of Parkinson's disease[END_REF], pigs [START_REF] Christensen | Pirouetting pigs: A large non-primate animal model based on unilateral 6-hydroxydopamine lesioning of the nigrostriatal pathway[END_REF] (Table 1). 6-OHDA is an analogue of dopamine and norepinephrine [START_REF] Dauer | Parkinson's Disease: Mechanisms and Models[END_REF][START_REF] Konnova | Animal Models of Parkinson's Disease, Parkinson's Disease: Pathogenesis and Clinical Aspects[END_REF] and is taken up preferentially by dopaminergic and noradrenergic transporters, resulting in relative cell selective toxicity [START_REF] Javoy | Specificity of dopaminergic neuronal degeneration induced by intracerebral injection of 6-hydroxydopamine in the nigrostriatal dopamine system[END_REF][START_REF] Jonsson | Chemical Neurotoxins as Denervation Tools in Neurobiology[END_REF]. 6-ODHA-induced toxicity is caused by its accumulation in the cytosol, where it generates reactive oxygen species (ROS), inhibits mitochondrial complex I, and inactivates biological macromolecules [START_REF] Cadet | Free radicals and the pathobiology of brain dopamine systems[END_REF][START_REF] Konnova | Animal Models of Parkinson's Disease, Parkinson's Disease: Pathogenesis and Clinical Aspects[END_REF]. A major down-side of 6-OHDA is its hydrophilic state, which makes it unable to cross the blood brain barrier, and thus requires direct injection into the brain [START_REF] Dauer | Parkinson's Disease: Mechanisms and Models[END_REF][START_REF] Jackson-Lewis | Animal models of Parkinson's disease[END_REF][START_REF] Jagmag | Evaluation of Models of Parkinson's Disease[END_REF].

6-OHDA injections in the substantia nigra (pars compacta) and medial forebrain bundle (MFB) lead to rapid and complete dopaminergic neurodegeneration [START_REF] Jeon | 6-Hydroxydopamine Lesion of the Rat Substantia Nigra: Time Course and Morphology of Cell Death[END_REF][START_REF] Cadet | Free radicals and the pathobiology of brain dopamine systems[END_REF][START_REF] Dauer | Parkinson's Disease: Mechanisms and Models[END_REF][START_REF] Jagmag | Evaluation of Models of Parkinson's Disease[END_REF]. Striatal injections induce progressive retrograde neuronal death in the substantia nigra and ventral tegmental area [START_REF] Berger | Retrograde degeneration of nigrostriatal neurons induced by intrastriatal 6-hydroxydopamine injection in rats[END_REF][START_REF] Sauer | Progressive degeneration of nigrostriatal dopamine neurons following intrastriatal terminal lesions with 6-hydroxydopamine: A combined retrograde tracing and immunocytochemical study in the rat[END_REF]. These dopaminergic lesions are dosedependent [START_REF] Kyono | Use of [18F]FDOPA-PET for in vivo evaluation of dopaminergic dysfunction in unilaterally 6-OHDA-lesioned rats[END_REF]. 6-OHDA is generally injected unilaterally, since bilateral injections carry a high mortality rate [START_REF] Jackson-Lewis | Animal models of Parkinson's disease[END_REF][START_REF] Konnova | Animal Models of Parkinson's Disease, Parkinson's Disease: Pathogenesis and Clinical Aspects[END_REF]. Unilateral models allow the use of the contralateral side to serve as an internal control.

MPTP

The neurodegenerative properties of [START_REF] Chartier-Harlin | alpha-Synuclein locus duplication as a cause of familial Parkinson's disease[END_REF]3, were first discovered in 1983 as a by-product of a designer drug (1-methyl-4-

phenyl-4- C h a p t e r 1 -I n t r o d u c t i o n | 25
propionoxypiperidine (MPPP)) by Dr. Langston (although a single case was reported previously [START_REF] Davis | Chronic parkinsonism secondary to intravenous injection of meperidine analogues[END_REF]). The patients intoxicated by MPTP presented many parkinsonian motor symptoms, including rigidity, slowness of movement, postural instability, and freezing, and several non-motor symptoms, like facial seborrhoea and mild cognitive deficits. Furthermore, they all reacted to levodopa and some developed levodopa-induced dyskinesia (LID) [START_REF] Davis | Chronic parkinsonism secondary to intravenous injection of meperidine analogues[END_REF][START_REF] Langston | Chronic Parkinsonism in humans due to a product of meperidine-analog synthesis[END_REF][START_REF] Dauer | Parkinson's Disease: Mechanisms and Models[END_REF][START_REF] Langston | The MPTP Story[END_REF]. PET imaging using a L-DOPA analogue (Fluorodopa, also see 4.4.2, page 40) suggested damage to the nigrostriatal pathway [START_REF] Calne | Positron emission tomography after MPTP: observations relating to the cause of Parkinson's disease[END_REF]. Post-mortem analyses showed a destruction of the substantia nigra in all cases, and a Lewy body-like inclusion in the first case only [START_REF] Davis | Chronic parkinsonism secondary to intravenous injection of meperidine analogues[END_REF][START_REF] Langston | Evidence of active nerve cell degeneration in the substantia nigra of humans years after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine exposure[END_REF].

MPTP is a lipophilic compound, which can cross the blood-brain barrier. In the brain it is taken up by astrocytes [START_REF] Ransom | Astrocytes convert the parkinsonism inducing neurotoxin, MPTP, to its active metabolite, MPP+[END_REF], where monoamine oxidase-B (MAO-B) [START_REF] Heikkila | Protection against the dopaminergic neurotoxicity of 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine by monoamine oxidase inhibitors[END_REF] converts it to 1-methyl-4-phenyl-2,3-dihydropyridinium (MPDP+) [START_REF] Dauer | Parkinson's Disease: Mechanisms and Models[END_REF][START_REF] Jagmag | Evaluation of Models of Parkinson's Disease[END_REF][START_REF] Konnova | Animal Models of Parkinson's Disease, Parkinson's Disease: Pathogenesis and Clinical Aspects[END_REF], followed by spontaneous oxidation into highly toxic 1-methyl-4-phenylpyridinium (MPP+) [START_REF] Dauer | Parkinson's Disease: Mechanisms and Models[END_REF]. Once released by the astrocytes through organic cation transporter 3 [START_REF] Cui | The organic cation transporter-3 is a pivotal modulator of neurodegeneration in the nigrostriatal dopaminergic pathway[END_REF], MPP+ is selectively taken up by dopaminergic cells through dopamine transporter (DAT) [START_REF] Javitch | Parkinsonism-inducing neurotoxin, N-methyl-4-phenyl-1,2,3,6tetrahydropyridine: uptake of the metabolite N-methyl-4-phenylpyridine by dopamine neurons R e f e r e n c e s | 193 explains selective toxicity[END_REF], where it accumulates in the cytoplasm, binds to vesicular monoamine transporter 2 (VMAT2), and accumulates in the mitochondria [START_REF] Dauer | Parkinson's Disease: Mechanisms and Models[END_REF]. Once inside the mitochondria, MPP+ blocks mitochondrial complex I [START_REF] Nicklas | Inhibition of NADH-linked oxidation in brain mitochondria by 1-methyl-4-phenyl-pyridine, a metabolite of the neurotoxin, 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine[END_REF], leading to reduced ATP production, increased oxidative stress, neuro-inflammation, and ultimately cell death [START_REF] Konnova | Animal Models of Parkinson's Disease, Parkinson's Disease: Pathogenesis and Clinical Aspects[END_REF].

MPTP is a powerful tool to model PD in both mice [START_REF] Mori | Immunohistochemical evaluation of the neurotoxic effects of 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine (MPTP) on dopaminergic nigrostriatal neurons of young adult mice using dopamine and tyrosine hydroxylase antibodies[END_REF] and non-human primates [START_REF] Burns | The neurotoxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in the monkey and man[END_REF][START_REF] Langston | MPTP-induced parkinsonism in human and nonhuman primates--clinical and experimental aspects[END_REF] (Table 1). Rats are resistant to MPTP toxicity [START_REF] Giovanni | Studies on species sensitivity to the dopaminergic neurotoxin 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine. Part 1: Systemic administration[END_REF][START_REF] Pienaar | Parkinson's disease: Insights from non-traditional model organisms[END_REF][START_REF] Jagmag | Evaluation of Models of Parkinson's Disease[END_REF][START_REF] Konnova | Animal Models of Parkinson's Disease, Parkinson's Disease: Pathogenesis and Clinical Aspects[END_REF], as the conversion from MPTP to MPP+ occurs at the blood brain barrier in rats, preventing great influx into the brain [START_REF] Riachi | Entry of 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine into the rat brain[END_REF][START_REF] Langston | The MPTP Story[END_REF]. The MPTP model is the gold-standard in PD non-human primate research [START_REF] Jackson-Lewis | Animal models of Parkinson's disease[END_REF][START_REF] Konnova | Animal Models of Parkinson's Disease, Parkinson's Disease: Pathogenesis and Clinical Aspects[END_REF]; it shows a striking loss of dopaminergic neurons in the substantia nigra pars compacta [START_REF] Burns | The neurotoxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in the monkey and man[END_REF], though not the ventral tegmental area (VTA) [START_REF] Jacobowitz | N-methyl-4-phenyl-1,2,3,6-tetra-hydropyridine (MPTP) causes destruction of the nigrostriatal but not the mesolimbic dopamine system in the monkey[END_REF]. Additionally, relocation of α-synuclein into aggregates in the cell body has been observed in non-human primates [START_REF] Forno | Locus ceruleus lesions and eosinophilic inclusions in MPTP-treated monkeys[END_REF][START_REF] Kowall | MPTP induces alpha-synuclein aggregation in the substantia nigra of baboons[END_REF]but not mice: Jagmag et al., 2016).

Lipopolysaccharide

As described above a hallmark of PD is the presence of neuro-inflammation (alinea 1.2.3, page 16). Lipopolysaccharide (LPS) is found on the outer membrane of gram-negative bacteria, being released physiologically as outer membrane vesicles and upon destruction of the bacteria [START_REF] Brown | The endotoxin hypothesis of neurodegeneration[END_REF]. LPS is a potent inducer of inflammation both in peripheral tissues as well as the central nervous system via the activation of toll-like receptor 4 (TLR4), which results in NF-κB transcriptional activation of hundreds of inflammatory genes, including pro-inflammatory cytokines such as TNFα, IL-6 and pro-IL-1β [START_REF] Kempuraj | Neuroinflammation Induces Neurodegeneration[END_REF][START_REF] Brown | The endotoxin hypothesis of neurodegeneration[END_REF]. Intracerebral administration of LPS locally activates pro-inflammatory microglia and astrocytes; microglia are activated directly through interaction with TLR4, while astrocytes can also be induced indirectly through brain proinflammatory factors expressed by microglia [START_REF] Liddelow | Neurotoxic reactive astrocytes are induced by activated microglia[END_REF].

C h a p t e r 1 -I n t r o d u c t i o n | 26
Prolonged neuroinflammation can lead to loss of dopaminergic neurons, which has been shown both in vitro and in vivo [START_REF] Choi | Striatal neuroinflammation promotes parkinsonism in rats[END_REF][START_REF] Khan | Dietary Supplementation of the Antioxidant Curcumin Halts Systemic LPS-Induced Neuroinflammation-Associated Neurodegeneration and Memory/Synaptic Impairment via the JNK/NF-κ B/Akt Signaling Pathway in Adult Rats[END_REF][START_REF] Wang | Locus coeruleus neurons are most sensitive to chronic neuroinflammationinduced neurodegeneration[END_REF]. However, the dose and target region of LPS are critical factors for the outcome of the neuro-inflammatory response, thus introducing a great heterogeneity in applied experimental protocols (Table 1) [START_REF] Lopes | LPS and neuroinflammation: a matter of timing[END_REF][START_REF] Batista | Lipopolysaccharide-induced neuroinflammation as a bridge to understand neurodegeneration[END_REF]. Generally, LPS injection is performed unilaterally in the striatum or in the substantia nigra inducing an acute and localised neuroinflammation within 24 hours that lasts up to 4 weeks [START_REF] Choi | Striatal neuroinflammation promotes parkinsonism in rats[END_REF][START_REF] Flores-Martinez | Acute neuroinflammatory response in the substantia nigra pars compacta of rats after a local injection of lipopolysaccharide[END_REF]. Intracerebral LPS doses vary from 1µg [START_REF] Pottier | PET imaging of cannabinoid type 2 receptors with[END_REF][START_REF] Sridharan | Comparative Evaluation of Three TSPO PET Radiotracers in a LPS-Induced Model of Mild Neuroinflammation in Rats[END_REF] to 5-10 µg [START_REF] Choi | Striatal neuroinflammation promotes parkinsonism in rats[END_REF][START_REF] Flores-Martinez | Acute neuroinflammatory response in the substantia nigra pars compacta of rats after a local injection of lipopolysaccharide[END_REF], and even up to 50µg [START_REF] Ory | PET imaging of TSPO in a rat model of local neuroinflammation induced by intracerebral injection of lipopolysaccharide[END_REF][START_REF] Ory | Quantification of TSPO overexpression in a rat model of local neuroinflammation induced by intracerebral injection of LPS by the use of [18F]DPA-714 PET[END_REF]. Reports range from no observed dopaminergic neuronal loss [START_REF] Hoban | Further characterisation of the LPS model of Parkinson's disease: A comparison of intra-nigral and intra-striatal lipopolysaccharide administration on motor function, microgliosis and nigrostriatal neurodegeneration in the rat[END_REF] to progressive nigrostriatal degeneration [START_REF] Choi | Striatal neuroinflammation promotes parkinsonism in rats[END_REF][START_REF] Deng | Lipopolysaccharide Animal Models of Parkinson's Disease: Recent Progress and Relevance to Clinical Disease[END_REF].

Intranigral LPS injection eventually leads to dopaminergic cell loss in the substantia nigra, albeit that different time courses have been described [START_REF] Sharma | Characterization of the lipopolysaccharide induced model of Parkinson's disease: Role of oxidative stress and neuroinflammation[END_REF][START_REF] Flores-Martinez | Acute neuroinflammatory response in the substantia nigra pars compacta of rats after a local injection of lipopolysaccharide[END_REF][START_REF] Deng | Lipopolysaccharide Animal Models of Parkinson's Disease: Recent Progress and Relevance to Clinical Disease[END_REF]. The LPS-induced inflammatory reaction in the substantia nigra is stronger than in the striatum [START_REF] Herrera | The single intranigral injection of LPS as a new model for studying the selective effects of inflammatory reactions on dopaminergic system[END_REF][START_REF] Hoban | Further characterisation of the LPS model of Parkinson's disease: A comparison of intra-nigral and intra-striatal lipopolysaccharide administration on motor function, microgliosis and nigrostriatal neurodegeneration in the rat[END_REF], which is partly attributed to the fact that the substantia nigra has the highest concentration of microglia in the brain [START_REF] Lawson | Heterogeneity in the distribution and morphology of microglia in the normal adult mouse brain[END_REF]. A study found that activated microglia express dopaminergic receptors (D 1 -D 4 ) [START_REF] Mastroeni | Microglial responses to dopamine in a cell culture model of Parkinson's disease[END_REF], while others found that dopamine itself can modulate cellular functions of both restingstate and activated microglial cells [START_REF] Fan | Differential Regulation of Adhesion and Phagocytosis of Resting and Activated Microglia by Dopamine[END_REF], which could explain the selective vulnerability of dopaminergic neurons in PD. A reduction of extracellular dopamine concentration, following TH inhibition, was indeed shown to prevent intranigral LPS-induced activation of microglial, and loss of TH immunostaining [START_REF] De Pablos | Dopamine-dependent neurotoxicity of lipopolysaccharide in substantia nigra[END_REF]. Additionally, human macrophages were found to express mRNA for all subtypes of dopaminergic receptors (D 1 -D 5 ), and cytokine production in macrophages is modulated by dopamine [START_REF] Gaskill | Characterization and function of the human macrophage dopaminergic system: Implications for CNS disease and drug abuse[END_REF]. [START_REF] Ungerstedt | 6-hydroxy-dopamine induced degeneration of central monoamine neurons[END_REF][START_REF] Berger | Retrograde degeneration of nigrostriatal neurons induced by intrastriatal 6-hydroxydopamine injection in rats[END_REF][START_REF] Jeon | 6-Hydroxydopamine Lesion of the Rat Substantia Nigra: Time Course and Morphology of Cell Death[END_REF][START_REF] Maia | Longitudinal and parallel monitoring of neuroinflammation and neurodegeneration in a 6-hydroxydopamine rat model of Parkinson's disease[END_REF][START_REF] Thiele | Development of a unilaterally-lesioned 6-OHDA mouse R e f e r e n c e s | 213 model of Parkinson's disease[END_REF]. (2) [START_REF] Burns | The neurotoxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in the monkey and man[END_REF][START_REF] Langston | MPTP-induced parkinsonism in human and nonhuman primates--clinical and experimental aspects[END_REF][START_REF] Mori | Immunohistochemical evaluation of the neurotoxic effects of 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine (MPTP) on dopaminergic nigrostriatal neurons of young adult mice using dopamine and tyrosine hydroxylase antibodies[END_REF][START_REF] Belloli | Early upregulation of 18-kDa translocator protein in response to acute neurodegenerative damage in TREM2-deficient mice[END_REF]. (3) [START_REF] Hoban | Further characterisation of the LPS model of Parkinson's disease: A comparison of intra-nigral and intra-striatal lipopolysaccharide administration on motor function, microgliosis and nigrostriatal neurodegeneration in the rat[END_REF][START_REF] Ory | PET imaging of TSPO in a rat model of local neuroinflammation induced by intracerebral injection of lipopolysaccharide[END_REF][START_REF] Lopes | LPS and neuroinflammation: a matter of timing[END_REF][START_REF] Flores-Martinez | Acute neuroinflammatory response in the substantia nigra pars compacta of rats after a local injection of lipopolysaccharide[END_REF][START_REF] Batista | Lipopolysaccharide-induced neuroinflammation as a bridge to understand neurodegeneration[END_REF].

Animal species Location Pathology Repreducability

Model

Mouse

Rat NHP SN MFB STR Systemic Neuro- degeneration Neuro- inflammation 6-ODHA    ++ ++ +  ++ ±  (1) MPTP        + +  (2) LPS    ++  +  ± ++  (3)

α-synuclein-based models

Alongside the A53T mutation in the SNCA gene (coding for α-synuclein) [START_REF] Polymeropoulos | Mutation in the α-Synuclein Gene Identified in Families with Parkinson's Disease[END_REF], multiple other mutations have been identified [START_REF] Krüger | AlaSOPro mutation in the gene encoding α-synuclein in Parkinson's disease[END_REF][START_REF] Zarranz | The new mutation, E46K, of α-synuclein causes parkinson and Lewy body dementia[END_REF]; Appel-C h a p t e r 1 -I n t r o d u c t i o n | 27 [START_REF] Appel-Cresswell | Alpha-synuclein p.H50Q, a novel pathogenic mutation for Parkinson's disease[END_REF][START_REF] Kiely | α-Synucleinopathy associated with G51D SNCA mutation: a link between Parkinson's disease and multiple system atrophy?[END_REF][START_REF] Proukakis | A novel a-synuclein missense mutation in Parkinson disease[END_REF]. While many models have been created using α-synuclein, its exact function is still unknown. α-synuclein is an abundantly presynaptic protein [START_REF] Maroteaux | Synuclein: a neuron-specific protein localized to the nucleus and presynaptic nerve terminal[END_REF], that is thought to modulate synaptic vesicle function [START_REF] Kahle | Structure/function of alpha-synuclein in health and disease: rational development of animal models for Parkinson's and related diseases[END_REF]. [START_REF] Feany | A Drosophila model of Parkinson's disease[END_REF] have shown that expression of wild-type or mutant (A30P or A53T) αsynuclein in Drosophila leads to loss of the dopaminergic neurons in adult flies, as well as motor dysfunction (reversible by levodopa [START_REF] Pendleton | Effects of Pharmacological Agents upon a Transgenic Model of Parkinson's Disease in Drosophila melanogaster[END_REF]), and Lewy Body-like inclusions [START_REF] Feany | A Drosophila model of Parkinson's disease[END_REF]. In mice, a knockout of α-synuclein resulted in behavioural deficits as well as decreased dopamine levels in the striatum, however there was no evidence of dopaminergic cell, fibre, or synapse loss [START_REF] Abeliovich | Mice Lacking α-Synuclein Display Functional Deficits in the Nigrostriatal Dopamine System[END_REF]. Interestingly, α-synuclein knockout mice are resistant to MPTP [START_REF] Dauer | Resistance of alpha -synuclein null mice to the parkinsonian neurotoxin MPTP[END_REF].

Apart from SNCA mutations, SNCA duplication and triplication have been linked to autosomal dominant PD [START_REF] Singleton | alpha-Synuclein locus triplication causes Parkinson's disease[END_REF][START_REF] Chartier-Harlin | alpha-Synuclein locus duplication as a cause of familial Parkinson's disease[END_REF]. Overexpression of α-synuclein resulted in variable phenotypes in mice, and only a few lines were reported to have alterations in the nigrostriatal pathway (reviewd by [START_REF] Fleming | Genetic mouse models of Parkinsonism: Strengths and limitations[END_REF]. Interestingly, mice overexpressing α-synuclein seem more sensitive to a mixture of toxins (paraquat and maneb; 1.3.4, page 141) [START_REF] Norris | Pesticide exposure exacerbates alpha-synucleinopathy in an A53T transgenic mouse model[END_REF].

Viral vector overexpression

In addition to transgenic lines, gene overexpression can be induced using viral vectors (Table 2). This has the advantage, unlike in transgenic lines, that the nigrostriatal system can be specifically targeted, expression of the vector can be dose-dependent, and is induced at a specific age. Both recombinant adeno-associated virus (rAAV) and lentivirus-based vectors have been used to overexpress SNCA and other genes in rodents [START_REF] Konnova | Animal Models of Parkinson's Disease, Parkinson's Disease: Pathogenesis and Clinical Aspects[END_REF], with or without PDrelated mutations (reviewed in [START_REF] Volpicelli-Daley | S-LRRK2 Expression Augments α-Synuclein Sequestration into Inclusions in Neurons[END_REF]. Unfortunately, high expression levelsfour to fivefold above normal -of α-synuclein are needed to obtain neurodegeneration [START_REF] Decressac | Comparison of the behavioural and histological characteristics of the 6-OHDA and α-synuclein rat models of Parkinson's disease[END_REF], while the inflammatory response is short-lived and modest [START_REF] Chung | Dynamic Changes in Presynaptic and Axonal Transport Proteins Combined with Striatal Neuroinflammation Precede Dopaminergic Neuronal Loss in a Rat Model of AAV -Synucleinopathy[END_REF].

Preformed a-synuclein fibrils

In contrast to the overexpression models of α-synuclein, preformed fibrils of α-synuclein can directly be injected into the rodent brain. These models allow the investigation of the impact and propagation of α-synuclein on the neuronal function [START_REF] Volpicelli-Daley | S-LRRK2 Expression Augments α-Synuclein Sequestration into Inclusions in Neurons[END_REF]. In both mice and rats, it has been shown that injection of α-synuclein fibrils leads to α-synuclein containing inclusions and neurodegeneration, as well as motor deficits [START_REF] Luk | Pathological α-synuclein transmission initiates Parkinson-like neurodegeneration in nontransgenic mice[END_REF][START_REF] Paumier | Intrastriatal injection of pre-formed mouse α-synuclein fibrils into rats triggers α-synuclein pathology and bilateral nigrostriatal degeneration[END_REF][START_REF] Rey | Widespread transneuronal propagation of α-synucleinopathy triggered in olfactory bulb mimics prodromal Parkinson's disease[END_REF][START_REF] Abdelmotilib | α-Synuclein fibril-induced inclusion spread in rats and mice correlates with dopaminergic Neurodegeneration[END_REF][START_REF] Blumenstock | Seeding and transgenic overexpression of alpha-synuclein triggers dendritic spine pathology in the neocortex[END_REF][START_REF] Patterson | Time course and magnitude of alpha-synuclein inclusion formation and nigrostriatal degeneration in the rat model of synucleinopathy triggered by intrastriatal α-synuclein preformed fibrils[END_REF][START_REF] Yabuki | Fatty Acid Binding Protein 3 Enhances the Spreading and Toxicity of α-Synuclein in Mouse Brain[END_REF].

Moreover, some groups have used a combination of α-synuclein overexpression and preformed fibrils. This lead to Lewy body-like aggregates, more progressive dopaminergic neurodegeneration, reduced dopaminergic nerve terminal volume, aggravated motor symptoms, and long-lasting inflammation [START_REF] Peelaerts | α-Synuclein strains cause distinct synucleinopathies after local and systemic administration[END_REF][START_REF] Thakur | Modeling Parkinson's disease pathology by combination of fibril seeds and αsynuclein overexpression in the rat brain[END_REF][START_REF] Espa | Seeding of protein aggregation causes cognitive impairment in rat model of cortical synucleinopathy[END_REF].

LRRK2-based models

Mutations in leucine rich repeat kinase 2 (LRRK2) were first discovered to cause autosomal dominant PD in 2004 (Zimprich et al., 2004). Although LRRK2 is normally localised in membranes C h a p t e r 1 -I n t r o d u c t i o n | 28 [START_REF] Jackson-Lewis | Animal models of Parkinson's disease[END_REF], its function is not clear, but there seems to be a link with mitochondria, autophagy, endocytosis, and many other cellular processes (reviewed by [START_REF] Winklhofer | Mitochondrial dysfunction in Parkinson's disease[END_REF][START_REF] Berwick | LRRK2 Biology from structure to dysfunction: research progresses, but the themes remain the same[END_REF]. The most common PD-related mutations in the LRRK2 gene are G2019S [START_REF] Kachergus | Identification of a novel LRRK2 mutation linked to autosomal dominant parkinsonism: evidence of a common founder across European populations[END_REF] and R1441C/G [START_REF] Dusonchet | A Rat Model of Progressive Nigral Neurodegeneration Induced by the Parkinson's Disease-Associated G2019S Mutation in LRRK2[END_REF][START_REF] Konnova | Animal Models of Parkinson's Disease, Parkinson's Disease: Pathogenesis and Clinical Aspects[END_REF]. These pathogenic mutations lead to hyperactivation of the LRRK2 kinase domain [START_REF] Alessi | LRRK2 kinase in Parkinson's disease[END_REF].

Transgenic Drosophila with a non-functional truncated version of LRRK2 showed no abnormalities in dopaminergic neurons [START_REF] Wang | Dispensable role of Drosophila ortholog of LRRK2 kinase activity in survival of dopaminergic neurons[END_REF], while over-expression leads to dopaminergic neurodegeneration and levodopa-reversible motor deficits [START_REF] Liu | A Drosophila model for LRRK2-linked parkinsonism[END_REF]. Overexpression of G2019S mutant LRRK2 caused a more severe phenotype [START_REF] Liu | A Drosophila model for LRRK2-linked parkinsonism[END_REF]. Knockout mice have abnormal accumulation and aggregation of proteins, including α-synuclein [START_REF] Jagmag | Evaluation of Models of Parkinson's Disease[END_REF]. Knock-out rats, unlike mice, show no significant dopaminergic cell loss, inhibited proinflammatory response, and inhibited rAAV-mediated α-synuclein expression [START_REF] Ness | Leucine-Rich Repeat Kinase 2 (LRRK2)-Deficient Rats Exhibit Renal Tubule Injury and Perturbations in Metabolic and Immunological Homeostasis[END_REF][START_REF] Daher | Abrogation of α-synuclein-mediated dopaminergic neurodegeneration in LRRK2-deficient rats[END_REF]. Similarly, LRRK2 transgenic rats (human wild-type LRRK2 or human mutated LRRK2: G2019S, R1441C or R1441G) did not show neurodegeneration in the substantia nigra, however, in many cases they did show behavioural deficits [START_REF] Walker | Behavioral deficits and striatal DA signaling in LRRK2 p.G2019S transgenic rats: a multimodal investigation including PET neuroimaging[END_REF][START_REF] Lee | Behavioral, neurochemical, and pathologic alterations in bacterial artificial chromosome transgenic G2019S leucine-rich repeated kinase 2 rats[END_REF][START_REF] Shaikh | Transgenic LRRK2R1441G rats-a model for Parkinson disease?[END_REF][START_REF] Sloan | LRRK2 BAC transgenic rats develop progressive, L-DOPA-responsive motor impairment, and deficits in dopamine circuit function[END_REF]. Conversely, overexpression of human G2019S LRRK2 in adulthood did generate significant dopaminergic neuronal loss [START_REF] Dusonchet | A Rat Model of Progressive Nigral Neurodegeneration Induced by the Parkinson's Disease-Associated G2019S Mutation in LRRK2[END_REF][START_REF] Zhou | Temporal expression of mutant LRRK2 in adult rats impairs dopamine reuptake[END_REF]. Recently, it has was shown by our lab that rAAV-mediated overexpression of truncated mutant (G2019S) LRRK2 in the rat substantia nigra caused dopaminergic neurodegeneration, suggesting the mutant kinase domain is sufficient to induce neuronal loss [START_REF] Cresto | The C-terminal domain of LRRK2 with the G2019S mutation is sufficient to produce neurodegeneration of dopaminergic neurons in vivo[END_REF].

Combination strategies (lrrk2&a-syn)

Overexpression of mutant LRRK2 in A53T mutated α-synuclein transgenic mice accelerated the PD neuropathology and promoted α-synuclein aggregation. Conversely, downregulation of LRRK2 was shown to suppress α-synuclein aggregation and slowed the neuropathology [START_REF] Lin | Leucine-rich repeat kinase 2 regulates the progression of neuropathology induced by Parkinson's-disease-related mutant alpha-synuclein[END_REF]. [START_REF] Cresto | The Unlikely Partnership Between LRRK2 and α-Synuclein in Parkinson's Disease[END_REF] have recently written an extensive review on the growing evidence for an interaction between LRRK2 and α-synuclein. While the underlying mechanisms for interaction between LRRK2 and α-synuclein are not clear, there is compelling evidence that the two proteins may act in concert to trigger dopaminergic neurodegeneration. As a consequence, therapeutic targeting of both proteins could slow down the disease process in patients with LRRK2 mutations but also in those with sporadic forms of PD [START_REF] Cresto | The Unlikely Partnership Between LRRK2 and α-Synuclein in Parkinson's Disease[END_REF].

Towards a cure

Even though PD has first been described over 200 years ago, there is still no curative treatment to this day (Kalia and Lang, 2015;[START_REF] Fahn | The 200-year journey of Parkinson disease: Reflecting on the past and looking towards the future[END_REF]. There are drugs available that help improve motor function, even though they have problematic side-effects in time [START_REF] Stoker | Stem Cell Treatments for Parkinson's Disease, Parkinson's Disease: Pathogenesis and Clinical Aspects[END_REF][START_REF] Zahoor | Pharmacological Treatment of Parkinson's Disease, Parkinson's Disease: Pathogenesis and Clinical Aspects[END_REF]. In tandem with drugs, there is the possibility of invasive deep brain stimulation (DBS), which can reduce the need for medication and reduce motor symptoms [START_REF] Dallapiazza | Considerations for Patient and Target Selection in Deep Brain Stimulation Surgery for Parkinson's Disease, Parkinson's Disease: Pathogenesis and Clinical Aspects[END_REF]. Lastly, to restore dopamine delivery in the brain in a more physiological way, gene therapy and cell grafting strategies have gained interest. Even though clinical trials with regenerative medicine have run into a number of difficulties, e.g. lack of efficacy, and ethical barriers [START_REF] Stoker | Stem Cell Treatments for Parkinson's Disease, Parkinson's Disease: Pathogenesis and Clinical Aspects[END_REF], one of the aims of the BrainMatTrain program 1,2 was to improve cell graft survival by employing hydrogels as a delivery support.

As previously described, PD is characterised by a selective loss of the dopaminergic neurons in the substantia nigra pars compacta, which leads to a dopamine deficiency in the striatum [START_REF] Dauer | Parkinson's Disease: Mechanisms and Models[END_REF]. Most treatment strategies aim to supplement dopamine in the striatum [START_REF] Zahoor | Pharmacological Treatment of Parkinson's Disease, Parkinson's Disease: Pathogenesis and Clinical Aspects[END_REF]. However, dopamine cannot cross the BBB, and must therefore be synthesised within the central nervous system [START_REF] Zahoor | Pharmacological Treatment of Parkinson's Disease, Parkinson's Disease: Pathogenesis and Clinical Aspects[END_REF].

Pharmacological dopamine increase

Levodopa

Currently, the most used treatments are based on levodopa [START_REF] Zahoor | Pharmacological Treatment of Parkinson's Disease, Parkinson's Disease: Pathogenesis and Clinical Aspects[END_REF], the dopamine precursor that can cross the BBB, in contrast to dopamine [START_REF] Samii | Parkinson's disease[END_REF][START_REF] Zahoor | Pharmacological Treatment of Parkinson's Disease, Parkinson's Disease: Pathogenesis and Clinical Aspects[END_REF]. In the brain, levodopa is converted into dopamine by AADC [START_REF] Musacchio | Enzymes Involved in the Biosynthesis and Degradation of Catecholamines[END_REF].

In 1957, Carlsson and colleagues showed that levodopa (L-dopa), a naturally occurring Lisomer of the amino acid D,L-dihydroxyphenylalanine, was able to restore the levels of dopamine. For this experiment rabbits were treated with reserpine to deplete all three catecholamines; interestingly L-dopa restored only dopamine levels, leaving the levels of adrenaline and noradrenaline almost untouched, while a precursor of serotonin did not elevated any catecholamine's level [START_REF] Carlsson | 3,4-Dihydroxyphenylalanine and 5-Hydroxytryptophan as Reserpine Antagonists[END_REF]. The important discoveries of Carlsson were rewarded with the Nobel Prize in Physiology or Medicine in 2000, together with Paul Greengard and Eric R. Kandel, for effectively proving the link between loss of dopamine and Parkinsonism. Carlsson suggested that dopamine was involved in PD and that L-dopa could restore levels of dopamine in the PD brain. In 1960, Ehringer and Hornykiewicz confirmed there was a neostriatal dopamine deficiency in post-mortem PD brains (translation [START_REF] Ehringer | Distribution of noradrenaline and dopamine (3hydroxytyramine) in the human brain and their behavior in diseases of the extrapyramidal system[END_REF]. Thanks to Hornykiewicz and Birkmayer, Carlsson's findings were rapidly translated into human testing, with the aim of restoring the missing dopamine. From 1961, levodopa was partially administered to PD patients but many reports were fairly sceptical about its therapeutic effect [START_REF] Tolosa | History of levodopa and dopamine agonists in Parkinson's disease treatment[END_REF]. Additionally various concerns were raised due to the gastrointestinal side effects caused by administration of even tiny doses. A solution to the problems was given by George Cotzias in 1969: he developed an oral levodopa intake based on high dosage together with a very slow release over time [START_REF] Cotzias | Modification of Parkinsonism -Chronic Treatment with L-Dopa[END_REF]. Since then, levodopa has become the major therapeutic against PD's motor symptoms.

Generally, levodopa administration rapidly shows a clinical effect, which can lasts for several hours. As the disease advances, the clinical effect becomes less prominent and the effects wear off faster, necessitating increased dosage and frequency of administration. Increasing the dosage leads to occurrence of adverse side-effects [START_REF] Fahn | The 200-year journey of Parkinson disease: Reflecting on the past and looking towards the future[END_REF][START_REF] Zahoor | Pharmacological Treatment of Parkinson's Disease, Parkinson's Disease: Pathogenesis and Clinical Aspects[END_REF]. Some of these sideeffects stem from the peripheral conversion of levodopa into dopamine, which can be counteracted by combined administration of levodopa and peripheral AADC blockers, like benserazide and carbidopa. These cannot cross the BBB, but selectively prevent the peripheral conversion of levodopa to dopamine, and thus reduce the peripheral side effects [START_REF] Silva | Increased neostriatal dopamine activity after intraperitoneal or intranasal administration of L-DOPA: on the role of benserazide pretreatment[END_REF]. Other side-effects result in significant motor complications such as levodopa-induced dyskinesia (LID) and severe on-off motor fluctuations. These can be handled by reducing the levodopa dose, making the right dosage of levodopa a fine balance to find [START_REF] Zahoor | Pharmacological Treatment of Parkinson's Disease, Parkinson's Disease: Pathogenesis and Clinical Aspects[END_REF]. Alternatively, research projects are aimed to identify molecules that reduce LID (e.g. Aron [START_REF] Badin | IRC-082451, a Novel Multitargeting Molecule, Reduces L-DOPA-Induced Dyskinesias in MPTP Parkinsonian Primates[END_REF]. Non-motor side-effects of levodopa include gastrointestinal disturbances (like nausea and vomiting), and anxiety and hallucinations, due to extra-nigral effects of dopamine (Kalia and Lang, 2015;[START_REF] Fahn | The 200-year journey of Parkinson disease: Reflecting on the past and looking towards the future[END_REF][START_REF] Zahoor | Pharmacological Treatment of Parkinson's Disease, Parkinson's Disease: Pathogenesis and Clinical Aspects[END_REF].

Dopamine degradation inhibitors

Monoamine Oxidase B inhibitors

Monoamine Oxidase B (MAO-B) inhibitors work through inhibition of dopamine metabolism, in order to increase the level of endogenous dopamine in the striatum [START_REF] Zahoor | Pharmacological Treatment of Parkinson's Disease, Parkinson's Disease: Pathogenesis and Clinical Aspects[END_REF], and to relieve motor symptoms [START_REF] Goldenberg | Medical management of Parkinson's disease[END_REF]. To delay the onset of complications with levodopa use, levodopa-sparing initial therapy can be considered, using MAO-B inhibitors or dopamine agonists [START_REF] Goldenberg | Medical management of Parkinson's disease[END_REF]Kalia and Lang, 2015). However, the effects of these are debatable [START_REF] Lang | Initiating dopaminergic treatment in Parkinson's disease[END_REF]PD Med Collaborative Group et al., 2014). MAO-B inhibitors are generally well tolerated, but can cause gastrointestinal side-effects by itself, and a myriad of other less-common adverse effects [START_REF] Goldenberg | Medical management of Parkinson's disease[END_REF].

Catechol-O-methyl transferase inhibitors

Catechol-O-methyl transferase (COMT) inhibitors are another way to limit dopamine degradation and preserving endogenous dopamine [START_REF] Goldenberg | Medical management of Parkinson's disease[END_REF]. They are mostly used in combination with levodopa, since they have a limited effect on PD symptoms on their own. COMT inhibitors are especially effective in patients with wearing-off problems of levodopa, since COMT inhibitors increase the half-life and brain delivery of levodopa [START_REF] Zahoor | Pharmacological Treatment of Parkinson's Disease, Parkinson's Disease: Pathogenesis and Clinical Aspects[END_REF]

. Since C h a p t e r 1 -I n t r o d u c t i o n | 32
COMT inhibitors can lead to amplification of levodopa-induced side-effects, they often necessitate a reduction in levodopa dose. Additionally, COMT inhibitors come with their own side-effects [START_REF] Zahoor | Pharmacological Treatment of Parkinson's Disease, Parkinson's Disease: Pathogenesis and Clinical Aspects[END_REF].

Dopamine agonists

Dopamine agonists stimulate the dopaminergic system by binding to the dopamine receptors, and thus do not need to be converted into dopamine [START_REF] Samii | Parkinson's disease[END_REF]. While dopamine agonists are less effective in controlling motor symptoms of PD, they can be useful in patients with minor symptoms, patients who do not tolerate levodopa, or in combination with levodopa therapy [START_REF] Zahoor | Pharmacological Treatment of Parkinson's Disease, Parkinson's Disease: Pathogenesis and Clinical Aspects[END_REF]. Commonly used are ropinirole, pramipexole, and rotigotine. Apomorphine is less commonly used but can be very useful as additional medication in patients with severe motor fluctuations [START_REF] Goldenberg | Medical management of Parkinson's disease[END_REF][START_REF] Zahoor | Pharmacological Treatment of Parkinson's Disease, Parkinson's Disease: Pathogenesis and Clinical Aspects[END_REF]. Some studies suggest that dopamine agonists additionally have a mild neuroprotective effect (reviewed by [START_REF] Olanow | Dopamine agonists and neuroprotection in R e f e r e n c e s | 204 Parkinson's disease[END_REF]. However, dopamine agonist treatment comes with severe sideeffects, including nausea and vomiting, insomnia, constipation, fainting, hallucinations, and sleepiness [START_REF] Samii | Parkinson's disease[END_REF][START_REF] Goldenberg | Medical management of Parkinson's disease[END_REF]. Another important side-effect is the development of impulse control disorder, which occurs in 15-20% PD patients taking dopamine agonists [START_REF] Weintraub | Impulse Control Disorders in Parkinson Disease: A Cross-Sectional Study of 3090 Patients[END_REF][START_REF] Poletti | A single-center, cross-sectional prevalence study of impulse control disorders in Parkinson disease: association with dopaminergic drugs[END_REF][START_REF] Zahoor | Pharmacological Treatment of Parkinson's Disease, Parkinson's Disease: Pathogenesis and Clinical Aspects[END_REF]. Impulse control disorder entails a wide range of behaviours, including hypersexuality, gambling, binge eating, compulsive shopping, and compulsive writing [START_REF] Weintraub | Impulse Control and Related Disorders in Parkinson's Disease[END_REF]. On the other hand, stopping with or reducing the dose of dopamine agonists can lead to dopamine agonist withdrawal syndrome [START_REF] Weintraub | Impulse Control and Related Disorders in Parkinson's Disease[END_REF], which also has psychological consequences [START_REF] Zahoor | Pharmacological Treatment of Parkinson's Disease, Parkinson's Disease: Pathogenesis and Clinical Aspects[END_REF].

Non-dopaminergic mechanisms

Besides pharmacological drugs that act on the dopaminergic system, as described above, there are also PD drugs that act through non-dopaminergic mechanisms, for example anticholinergics. The loss of dopaminergic neurons disturbs the normal balance between dopamine and acetylcholine in the brain. Anticholinergic drugs act as antagonist for cholinergic receptors and thereby reduce the activity of acetylcholine, this can lead to restoration of the balance [START_REF] Zahoor | Pharmacological Treatment of Parkinson's Disease, Parkinson's Disease: Pathogenesis and Clinical Aspects[END_REF]. Anticholinergics were the first pharmacological agents used as treatment for PD, but since the discovery of levodopa they are little used [START_REF] Brocks | Anticholinergic drugs used in Parkinson's disease: An overlooked class of drugs from a pharmacokinetic perspective[END_REF]. Anticholinergics are most useful to relief tremors and muscle stiffness, and can even be used as monotherapy in the early stages of tremordominant PD [START_REF] Zahoor | Pharmacological Treatment of Parkinson's Disease, Parkinson's Disease: Pathogenesis and Clinical Aspects[END_REF]. They can cause many side-effects, one of which is dry mouth, which is actually beneficial for patients who experience drooling [START_REF] Goldenberg | Medical management of Parkinson's disease[END_REF].

Conversely, nicotine has been shown to protect against nigrostriatal damage [START_REF] Ryan | Dose-related neuroprotective effects of chronic nicotine in 6hydroxydopamine treated rats, and loss of neuroprotection in alpha4 nicotinic receptor subunit knockout mice[END_REF][START_REF] Huang | Nicotine is neuroprotective when administered before but not after nigrostriatal damage in rats and monkeys[END_REF] and improve LID in PD models [START_REF] Huang | Nicotinic receptor agonists decrease L-dopa-induced dyskinesias most effectively in partially lesioned parkinsonian rats[END_REF][START_REF] Quik | Nicotine reduces established levodopa-induced dyskinesias in a monkey model of Parkinson's disease[END_REF]. Nicotine binds to nicotinic acetyncholine receptors, and AQW051 is a partial agonist for the nicotinic acetyncholine receptors. Even though AQW051 was shown to significantly reduce LID in MPTPtreated monkeys without compromising the beneficial effects of levodopa [START_REF] Di Paolo | AQW051, a novel and selective nicotinic acetylcholine receptor α7 partial agonist, reduces l-Dopa-induced dyskinesias and extends the duration of l-Dopa effects in parkinsonian monkeys[END_REF], AQW051 did not significantly reduce dyskinesia or parkinsonian severity in PD patients [START_REF] Trenkwalder | A Placebo-Controlled Trial of AQW051 in Patients With Moderate to Severe Levodopa-Induced Dyskinesia[END_REF].

C h a p t e r 1 -I n t r o d u c t i o n | 33
Other targets of interest are serotonin receptors and glutamate receptors. Sarizotan is a 5-HT1A agonist with additional affinity for D 3 and D 4 receptors, which was shown to inhibit levodopainduced turning in 6-OHDA treated rats in a dose-dependent manner [START_REF] Gerlach | Role of dopamine D 3 and serotonin 5-HT 1A receptors in l-DOPA-induced dyskinesias and effects of sarizotan in the 6-hydroxydopamine-lesioned rat model of Parkinson's disease[END_REF]. Mavoglurant (AFQ056) is a glutamate receptor antagonist with debatable outcome. It showed reduced LID without affecting levodopa therapy in two studies [START_REF] Berg | AFQ056 treatment of levodopa-induced dyskinesias: results of 2 randomized controlled trials[END_REF][START_REF] Stocchi | AFQ056 in Parkinson patients with levodopa-induced dyskinesia: 13-week, randomized, dose-finding study[END_REF], but failed to reduce LID in a third study [START_REF] Trenkwalder | Mavoglurant in Parkinson's patients with l-Dopa-induced dyskinesias: Two randomized phase 2 studies[END_REF].

Conclusion

The drugs that have been discussed above are used to control the motor symptoms of PD, but long-term use of dopaminergic therapies results in significant adverse effects [START_REF] Zahoor | Pharmacological Treatment of Parkinson's Disease, Parkinson's Disease: Pathogenesis and Clinical Aspects[END_REF][START_REF] Moriarty | Encapsulation of young donor age dopaminergic grafts in a GDNF-loaded collagen hydrogel further increases their survival, reinnervation, and functional efficacy after R e f e r e n c e s | 202 intrastriatal transplantation in hemi-Parkinsonian rats[END_REF]. Thus there is a requirement to develop better means of restoring striatal dopamine [START_REF] Moriarty | Encapsulation of young donor age dopaminergic grafts in a GDNF-loaded collagen hydrogel further increases their survival, reinnervation, and functional efficacy after R e f e r e n c e s | 202 intrastriatal transplantation in hemi-Parkinsonian rats[END_REF]. Additionally, the dopaminergic treatments do not alter the course of the disease, thus there is a need for treatments that are able to slow down progression of the disease [START_REF] Zahoor | Pharmacological Treatment of Parkinson's Disease, Parkinson's Disease: Pathogenesis and Clinical Aspects[END_REF][START_REF] Moriarty | Encapsulation of young donor age dopaminergic grafts in a GDNF-loaded collagen hydrogel further increases their survival, reinnervation, and functional efficacy after R e f e r e n c e s | 202 intrastriatal transplantation in hemi-Parkinsonian rats[END_REF].

Deep brain stimulation

As described above, levodopa can be accompanied by severe dyskinesias and other sideeffects after long-term use. In these patients deep brain stimulation might be considered, which may allow increased control of the motor symptoms and a reduction of levodopa dose [START_REF] Zahoor | Pharmacological Treatment of Parkinson's Disease, Parkinson's Disease: Pathogenesis and Clinical Aspects[END_REF]. Deep brain stimulation is a surgical technique in which electrodes are implanted in a target brain region. An impulse generator gives tiny electrical stimuli to the brain to disrupt or modulate neural signalling [START_REF] Okun | Deep-brain stimulation for Parkinson's disease[END_REF]. The positive therapeutic outcome has been shown by several clinical trials; demonstrating motor improvements, long-lasting effects, and general improvement of life quality [START_REF] Groiss | Deep brain stimulation in Parkinson-s disease[END_REF].

Deep brain stimulation has been in used for over 70 years, but has also greatly evolved over time. The first deep brain stimulation electrode was implanted in 1948 in the caudate nucleus to treat depression and anorexia in a PD patients [START_REF] Malek | Deep Brain Stimulation in Parkinson's Disease[END_REF]. However, [START_REF] Benabid | Combined (thalamotomy and stimulation) stereotactic surgery of the VIM thalamic nucleus for bilateral Parkinson disease[END_REF] has performed pioneering work for modern day deep brain stimulation [START_REF] Malek | Deep Brain Stimulation in Parkinson's Disease[END_REF].

Gene therapy

Apart from the drugs described above, that aim to restore dopamine levels in the brain, gene therapy is being tested, generally to achieve endogenous dopaminergic restoration in a more continuous fashion [START_REF] Zahoor | Pharmacological Treatment of Parkinson's Disease, Parkinson's Disease: Pathogenesis and Clinical Aspects[END_REF]. Gene therapy uses viral vectors, not for the creation of PD models as described above (2.4, page 26), but to introduce a beneficial gene [START_REF] Bjorklund | Repairing the brain: Gene therapy[END_REF][START_REF] Hitti | Human gene therapy approaches for the treatment of Parkinson's disease: An overview of current and completed clinical trials[END_REF].

Adeno-associated viral vectors (AAVs) are non-integrating vectors, which limits the risks of insertional mutagenesis that can occur during viral vector integration [START_REF] Mukherjee | Gene therapy for PIDs: Progress, pitfalls and prospects[END_REF]. Their main drawback is the relatively limited size of the genetic cargo, meaning that only one gene of the dopamine synthesis pathway can be incorporated [START_REF] Moriarty | Encapsulation of young donor age dopaminergic grafts in a GDNF-loaded collagen hydrogel further increases their survival, reinnervation, and functional efficacy after R e f e r e n c e s | 202 intrastriatal transplantation in hemi-Parkinsonian rats[END_REF]. An AAV coding for AADC has been shown to successfully express AADC in MPTP-lesioned non-human primates, leading to long-term conversion of levodopa to dopamine. In combination with levodopa administration this resulted in long-term improvement of clinical rating scores, lower levodopa requirements, and improved [ 18 F]FMT uptake [START_REF] Bankiewicz | Long-Term Clinical Improvement in MPTP-Lesioned Primates after Gene Therapy with AAV-hAADC[END_REF]. Subsequent phase I trials also C h a p t e r 1 -I n t r o d u c t i o n | 34 found improved clinical parameters and increased [ 18 F]FMT PET uptake [START_REF] Christine | Safety and tolerability of putaminal AADC gene therapy for Parkinson disease[END_REF][START_REF] Muramatsu | A phase i study of aromatic l-amino acid decarboxylase gene therapy for parkinson's disease[END_REF].

In contrast to AAVs, lentiviral vectors are integrating vectors, carrying a much larger capacity for genetic cargo [START_REF] Moriarty | Encapsulation of young donor age dopaminergic grafts in a GDNF-loaded collagen hydrogel further increases their survival, reinnervation, and functional efficacy after R e f e r e n c e s | 202 intrastriatal transplantation in hemi-Parkinsonian rats[END_REF]. This characteristic is exploited to restore endogenous dopamine production through medial spiny neurons by providing all the required building blocks [START_REF] Bjorklund | Repairing the brain: Gene therapy[END_REF][START_REF] Badin | Gene Therapy for Parkinson's Disease: Preclinical Evaluation of Optimally Configured TH:CH1 Fusion for Maximal Dopamine Synthesis[END_REF][START_REF] Hitti | Human gene therapy approaches for the treatment of Parkinson's disease: An overview of current and completed clinical trials[END_REF]; i.e. genes encoding the rate-limiting enzymes in dopamine synthesis: AADC, TH, and guanosine triphosphate cyclohydrolase-1 (GTPCH1) [START_REF] Palfi | Long-term safety and tolerability of ProSavin, a lentiviral vector-based gene therapy for Parkinson's disease: a dose escalation, open-label, phase 1/2 trial[END_REF]. Striatal injection of this triple-gene lentiviral vector was shown to results in functional improvements in both 6-OHDA-lesioned rats [START_REF] Azzouz | Multicistronic lentiviral vector-mediated striatal gene transfer of aromatic Lamino acid decarboxylase, tyrosine hydroxylase, and GTP cyclohydrolase I induces sustained transgene expression, dopamine production, and functional improvement in a rat model[END_REF] and MPTP-lesioned nonhuman primates [START_REF] Jarraya | Dopamine gene therapy for Parkinson's disease in a nonhuman primate without associated dyskinesia[END_REF]. This was translated into ProSavin (OXB-101; Oxford Biomedia, UK), which was found well tolerated and improved motor behaviour in PD patients in a phase I/II trial [START_REF] Palfi | Long-term safety and tolerability of ProSavin, a lentiviral vector-based gene therapy for Parkinson's disease: a dose escalation, open-label, phase 1/2 trial[END_REF][START_REF] Palfi | Long-Term Follow-Up of a Phase I/II Study of ProSavin, a Lentiviral Vector Gene Therapy for Parkinson's Disease[END_REF]. More recently, this lentiviral vector has been optimised [START_REF] Stewart | Optimizing Transgene Configuration and Protein Fusions to Maximize Dopamine Production for the Gene Therapy of Parkinson's Disease[END_REF], and subsequently tested in MPTP-lesioned non-human primates (Aron [START_REF] Badin | Gene Therapy for Parkinson's Disease: Preclinical Evaluation of Optimally Configured TH:CH1 Fusion for Maximal Dopamine Synthesis[END_REF]. This OXB-102 is currently in clinical trials under the name AXO-Lenti-PD [START_REF] Lewis | Study of OXB-102 (AXO-Lenti-PD) in Patients With Bilateral, Idiopathic Parkinson's Disease (SUNRISE-PD)[END_REF].

Alternatively, gene therapy is also being explored as a delivery system for neuroprotection. Viral vectors coding for glial-cell-derived neurotrophic factor (GDNF) or neurturin (its naturally occurring analogue) have been tested to slow dopaminergic neurodegeneration [START_REF] Wang | Delayed delivery of AAV-GDNF prevents nigral neurodegeneration and promotes functional recovery in a rat model of Parkinson's disease[END_REF][START_REF] Marks | Safety and tolerability of intraputaminal delivery of CERE-120 (adenoassociated virus serotype 2-neurturin) to patients with idiopathic Parkinson's disease: an open-label, phase I trial[END_REF][START_REF] Marks | Gene delivery of AAV2-neurturin for Parkinson's disease: A double-blind, randomised, controlled trial[END_REF][START_REF] Olanow | Gene delivery of neurturin to putamen and substantia nigra in Parkinson disease: A double-blind, randomized, controlled trial[END_REF].

Cell grafting approaches

Another way to ensure endogenous dopamine production is by introducing exogenous dopaminergic cells into the striatum. In this context, cell grafting approaches have great potential as regenerative treatment [START_REF] Stoker | Stem Cell Treatments for Parkinson's Disease, Parkinson's Disease: Pathogenesis and Clinical Aspects[END_REF]. Several strategies have been proposed and are in various stages of preclinical and clinical testing [START_REF] Moriarty | Encapsulation of young donor age dopaminergic grafts in a GDNF-loaded collagen hydrogel further increases their survival, reinnervation, and functional efficacy after R e f e r e n c e s | 202 intrastriatal transplantation in hemi-Parkinsonian rats[END_REF].

The first prove of concept studies showing the potential of brain cell grafting in the context of PD appeared in the late 70's [START_REF] Perlow | Brain grafts reduce motor abnormalities produced by destruction of nigrostriatal dopamine system[END_REF][START_REF] Björklund | Functional reactivation of the deafferented neostriatum by nigral transplants[END_REF]. Björklund and colleagues (1981) grafted fetal ventral mesencephalon (fVM) tissue in 6-ODHA-lesioned rats and observed improved motor symptoms [START_REF] Björklund | Functional reactivation of the deafferented neostriatum by nigral transplants[END_REF]. The first human trials with human fVM cells started in the late 1980s and showed promising results [START_REF] Lindvall | Grafts of fetal dopamine neurons survive and improve motor function in Parkinson's disease[END_REF]reviewed in Barker, Drouin-Ouellet and Parmar, 2015). Even though fVM cells were shown the be effective in treating the critical features of PD, there are many ethical and logistical barriers for widespread use, e.g. unpredictable and inadequate supply of fetal tissue [START_REF] Barker | Cell-based therapies for Parkinson diseasepast insights and future potential[END_REF][START_REF] Stoker | Stem Cell Treatments for Parkinson's Disease, Parkinson's Disease: Pathogenesis and Clinical Aspects[END_REF][START_REF] Moriarty | Encapsulation of young donor age dopaminergic grafts in a GDNF-loaded collagen hydrogel further increases their survival, reinnervation, and functional efficacy after R e f e r e n c e s | 202 intrastriatal transplantation in hemi-Parkinsonian rats[END_REF]. These initial studies have spurred the search for renewable sources of dopamine-producing cells [START_REF] Stoker | Stem Cell Treatments for Parkinson's Disease, Parkinson's Disease: Pathogenesis and Clinical Aspects[END_REF][START_REF] Moriarty | Encapsulation of young donor age dopaminergic grafts in a GDNF-loaded collagen hydrogel further increases their survival, reinnervation, and functional efficacy after R e f e r e n c e s | 202 intrastriatal transplantation in hemi-Parkinsonian rats[END_REF].

Embryonic stem cell (ESC) lines are isolated from the early blastocyst, and can be generated from surplus human embryos derived from in vitro fertilisation procedures [START_REF] Thomson | Embryonic stem cell lines derived from human blastocysts[END_REF]. This is thought to lead to smaller ethical barriers as compared to the use of fetal tissue [START_REF] Barker | Scientific and ethical issues related to stem cell research and interventions in neurodegenerative disorders of the brain[END_REF]. ESCs can be reprogrammed to midbrain dopaminergic neuronal progenitor cells with high efficiency, which have been shown to improve motor deficits in 6-OHDA-lesioned rats [START_REF] Grealish | Human ESC-derived dopamine neurons show similar preclinical efficacy and potency to fetal neurons when grafted in a rat model of Parkinson's disease[END_REF]. The first clinical trials with ESC-derived dopaminergic neuron progenitors are currently ongoing (NeuroStemCellRepair, TRANSEURO, and NYSTEM-PD) (reviewed by [START_REF] Barker | Human Trials of Stem Cell-Derived Dopamine Neurons for Parkinson's Disease: Dawn of a New Era[END_REF].

Induced pluripotent stem cells (iPSCs) are derived through reprogramming of somatic cells, such as fibroblasts, into pluripotent cells, which can then be converted into dopaminergic neuron C h a p t e r 1 -I n t r o d u c t i o n | 35 progenitor cells [START_REF] Takahashi | Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors[END_REF]. Grafting of human iPSC-derived dopaminergic neuron progenitors into MPTP-lesioned non-human primates showed graft cell survival and functional integration, hence, spontaneous movement recovery [START_REF] Kikuchi | Human iPS cell-derived dopaminergic neurons function in a primate Parkinson's disease model[END_REF]. These preclinical studies lead to clinical trials using iPSC-derived dopaminergic neuron progenitors (CiRA trial and Summit for PD trial) (reviewed by [START_REF] Barker | Human Trials of Stem Cell-Derived Dopamine Neurons for Parkinson's Disease: Dawn of a New Era[END_REF]. A major disadvantage of using autologous iPSCs is that any genetic defect of the host, will also be present in the grafted cells [START_REF] Moriarty | Encapsulation of young donor age dopaminergic grafts in a GDNF-loaded collagen hydrogel further increases their survival, reinnervation, and functional efficacy after R e f e r e n c e s | 202 intrastriatal transplantation in hemi-Parkinsonian rats[END_REF].

Finally, bone marrow-derived mesenchymal stem cells (MSCs) are multipotent cells that can be differentiated into TH-positive cells. Grafting of MSCs was shown to improve motor symptoms in 6-ODHA-lesioned mice [START_REF] Offen | Intrastriatal transplantation of mouse bone marrow-derived stem cells improves motor behavior in a mouse model of Parkinson's disease[END_REF]. Differentiation into dopaminergic neurons has proven to be challenging [START_REF] Moriarty | Encapsulation of young donor age dopaminergic grafts in a GDNF-loaded collagen hydrogel further increases their survival, reinnervation, and functional efficacy after R e f e r e n c e s | 202 intrastriatal transplantation in hemi-Parkinsonian rats[END_REF], but not impossible [START_REF] Trzaska | Specification of a Dopaminergic R e f e r e n c e s | 214 Phenotype from Adult Human Mesenchymal Stem Cells[END_REF]. It has been suggested that MSCs themselves have anti-inflammatory and paracrine activity, resulting in a neuroprotective effect. This could make MSC grafts useful even without differentiation into dopaminergic neurons [START_REF] Kim | Neuroprotective effects of human mesenchymal stem cells on dopaminergic neurons through anti-inflammatory action[END_REF]. A small open-label clinical trial using MSC grafts in PD patients has shown short-term safety and hinted at improved motor scores [START_REF] Venkataramana | Open-labeled study of unilateral autologous bone-marrowderived mesenchymal stem cell transplantation in Parkinson's disease[END_REF].

Positron Emission Tomography Imaging

Positron emission tomography (PET) imaging is used for clinical diagnosis, monitoring disease progression, evaluation of (neuroprotective) therapies, and drug occupancy studies both in patients [START_REF] Thobois | Contributions of PET and SPECT to the understanding of the pathophysiology of Parkinson's disease[END_REF], and preclinical settings [START_REF] Sahin | Differential dopamine receptor occupancy underlies L-DOPA-induced dyskinesia in a rat model of Parkinson's disease[END_REF].

PET imaging relies on the use of radiolabelled molecules, termed radiotracers, that diffuse freely through the body and selectively bind to specific biological targets [START_REF] Van Camp | Quantification of Brain Function and Neurotransmission System In Vivo by Positron Emission Tomography: A Review of Technical Aspects and Practical Considerations in Preclinical Research[END_REF]. PET can be used to monitor receptors [START_REF] Ishida | Alteration of striatal [11C]raclopride and 6-[18F]fluoro-L-3,4dihydroxyphenylalanine uptake precedes development of methamphetamine-induced rotation following unilateral 6-hydroxydopamine lesions of medial forebrain bundle in rats[END_REF], transporters [START_REF] Sérrière | In vivo PET quantification of the dopamine transporter in rat brain with [18F]LBT-999[END_REF], neurotransmitter synthesis and enzymes [START_REF] Becker | Comparative assessment of 6-[18F]fluoro-L-m-tyrosine and 6-[18F]fluoro-Ldopa to evaluate dopaminergic presynaptic integrity in a Parkinson's disease rat model[END_REF], intracellular proteins [START_REF] Ory | PET imaging of TSPO in a rat model of local neuroinflammation induced by intracerebral injection of lipopolysaccharide[END_REF], and aggregated proteins [START_REF] Franzmeier | Functional brain architecture is associated with the rate of tau accumulation in Alzheimer's disease[END_REF] in vivo (Figure 5) [START_REF] Van Camp | Quantification of Brain Function and Neurotransmission System In Vivo by Positron Emission Tomography: A Review of Technical Aspects and Practical Considerations in Preclinical Research[END_REF][START_REF] Abbasi Gharibkandi | Radiotracers for imaging of Parkinson's disease[END_REF][START_REF] Herfert | Quantitative Rodent Brain Receptor Imaging[END_REF]. Of all modalities to image the brain in vivo PET imaging has the highest sensitivity and allows quantitative evaluation [START_REF] Herfert | Quantitative Rodent Brain Receptor Imaging[END_REF], thus providing valuable information.

A big advantages of PET is its translational potential; it can be used on a wide spectrum of species, from humans to rodents [START_REF] Zheng | A study of non-invasive Patlak quantification for whole-body dynamic FDG-PET studies of mice[END_REF][START_REF] Ma | Reproducibility of a Parkinsonism-related metabolic brain network in non-human primates: A descriptive pilot study with FDG PET[END_REF][START_REF] Parkinson | The Effect of Endogenous Adenosine on Neuronal Activity in Rats: An FDG PET Study[END_REF][START_REF] Matthews | FDG PET Parkinson's disease-related pattern as a biomarker for clinical trials in early stage disease[END_REF], and even down to insects [START_REF] Currie | Biodistribution of 18F-FDG after oral administration to a honeybee: PET/CT proof of concept[END_REF]. Clinical and preclinical imaging can learn from each other due to the high translational value of PET imaging. Preclinical imaging has the possibility to compare with post-mortem studies, which is more complicated in human studies (but possible [START_REF] Sabri | Florbetaben PET imaging to detect amyloid beta plaques in Alzheimer's disease: phase 3 study[END_REF]). The main advantage of preclinical imaging, however, is the possibility to study the prodromal phase of diseases like PD.

C h a p t e r 1 -I n t r o d u c t i o n | 36

Principles of PET imaging

PET imaging utilises a positron-emitting radioisotope linked to a molecule, together forming the radiotracer [START_REF] Van Camp | Quantification of Brain Function and Neurotransmission System In Vivo by Positron Emission Tomography: A Review of Technical Aspects and Practical Considerations in Preclinical Research[END_REF]. This radioisotope undergoes radioactive decay in the form of positron emission, which subsequently travels a short distance, dependent on the initial emission energy and the medium through which it is travelling. Upon interaction with an electron there is an annihilation event, which gives rise to the emission of two 511keV gamma ray photons that are emitted in almost exactly opposite directions, with a directional uncertainty of about ±0.5°. Detection of the annihilation event happens through simultaneous detection of these photons by opposite detectors, termed coincidence detection (Figure 6) [START_REF] Cherry | Use of positron emission tomography in animal research[END_REF][START_REF] Miller | Synthesis of 11C, 18F, 15O, and 13N radiolabels for positron emission tomography[END_REF][START_REF] Van Camp | Quantification of Brain Function and Neurotransmission System In Vivo by Positron Emission Tomography: A Review of Technical Aspects and Practical Considerations in Preclinical Research[END_REF]. The time-window for coincidence detection, called the coincidence window, is traditionally 5-10 ns and based on the time it takes to cross the field of view of the scanner (3 ns) [START_REF] Van Camp | Quantification of Brain Function and Neurotransmission System In Vivo by Positron Emission Tomography: A Review of Technical Aspects and Practical Considerations in Preclinical Research[END_REF].

The raw data collected by a PET scanners is thus a list of these coincidence events, representing a line in space, and flagged with a time stamp [START_REF] Van Camp | Quantification of Brain Function and Neurotransmission System In Vivo by Positron Emission Tomography: A Review of Technical Aspects and Practical Considerations in Preclinical Research[END_REF]. Coincidence detection can also detect photons that do not stem from the same annihilation event. These accidental coincidences arise when two separate annihilation events cause two unrelated photons to hit opposing detectors within the coincidence window. These accidental, and false, coincidences need to be corrected for in order to prevent overestimation or mislocalisation of the isotope concentration [START_REF] Van Camp | Quantification of Brain Function and Neurotransmission System In Vivo by Positron Emission Tomography: A Review of Technical Aspects and Practical Considerations in Preclinical Research[END_REF]. Another important correction is for photon attenuation, which reduces the number of coincidences and can thus underestimate the signal [START_REF] Chow | Attenuation correction for small animal PET tomographs[END_REF].

C h a p t e r 1 -I n t r o d u c t i o n | 37

Advantages and disadvantages of PET imaging

PET imaging has several advantages over other in vivo imaging techniques. First of all PET imaging is very versatile; radiotracers against many different substances exist (See paragraph 4.4, page 39). Additionally, PET imaging is a very translational tool that can be used in many different animal species [START_REF] Cherry | Use of positron emission tomography in animal research[END_REF][START_REF] Van Camp | Quantification of Brain Function and Neurotransmission System In Vivo by Positron Emission Tomography: A Review of Technical Aspects and Practical Considerations in Preclinical Research[END_REF][START_REF] Herfert | Quantitative Rodent Brain Receptor Imaging[END_REF]. PETs in vivo nature allows for longitudinal study design, which allows one to follow disease or therapy progression, as well as a minimisation of animal numbers [START_REF] Cherry | Use of positron emission tomography in animal research[END_REF][START_REF] Herfert | Quantitative Rodent Brain Receptor Imaging[END_REF].

PET imaging is very sensitive and can detect nanomolar concentrations of radiotracer [START_REF] Van Camp | Quantification of Brain Function and Neurotransmission System In Vivo by Positron Emission Tomography: A Review of Technical Aspects and Practical Considerations in Preclinical Research[END_REF], with high specificity and high resolution [START_REF] Abbasi Gharibkandi | Radiotracers for imaging of Parkinson's disease[END_REF]. Since PET imaging is a truly quantitative technique [START_REF] Cherry | Use of positron emission tomography in animal research[END_REF][START_REF] Miller | Synthesis of 11C, 18F, 15O, and 13N radiolabels for positron emission tomography[END_REF][START_REF] Abbasi Gharibkandi | Radiotracers for imaging of Parkinson's disease[END_REF], it can give information about physiological and biochemical events by monitoring the distribution and concentration of the radiotracer in the body over time [START_REF] Cherry | Use of positron emission tomography in animal research[END_REF][START_REF] Miller | Synthesis of 11C, 18F, 15O, and 13N radiolabels for positron emission tomography[END_REF]. It thus provides metabolic information rather than structural information [START_REF] Miller | Synthesis of 11C, 18F, 15O, and 13N radiolabels for positron emission tomography[END_REF]. And lastly, it can do so without interfering in the biological activity of the molecule of interest [START_REF] Miller | Synthesis of 11C, 18F, 15O, and 13N radiolabels for positron emission tomography[END_REF].

One of the main challenges of PET imaging is the short half-lives of the radioisotopes [START_REF] Miller | Synthesis of 11C, 18F, 15O, and 13N radiolabels for positron emission tomography[END_REF][START_REF] Abbasi Gharibkandi | Radiotracers for imaging of Parkinson's disease[END_REF], which necessitate rapid synthesis methods. The labelled probe has to be synthesised, purified, analysed, and formulated within timescale of roughly three isotope half-lives [START_REF] Miller | Synthesis of 11C, 18F, 15O, and 13N radiolabels for positron emission tomography[END_REF][START_REF] Van Camp | Quantification of Brain Function and Neurotransmission System In Vivo by Positron Emission Tomography: A Review of Technical Aspects and Practical Considerations in Preclinical Research[END_REF], which corresponds to 60 minutes for [ 11 C] and only 6 minutes for [ 15 O]. This is less of an issue for [ 18 F], which has a half-life of 110 minutes [START_REF] Miller | Synthesis of 11C, 18F, 15O, and 13N radiolabels for positron emission tomography[END_REF]. Within the synthesis time, also the radionuclide purity needs to be ensured, which can require extra purification steps [START_REF] Abbasi Gharibkandi | Radiotracers for imaging of Parkinson's disease[END_REF]. These technical challenges also hinder the development and optimisation of new probes [START_REF] Cherry | Use of positron emission tomography in animal research[END_REF].

C h a p t e r 1 -I n t r o d u c t i o n | 38 A major disadvantage of PET imaging is the radioactive nature of the technique and the high costs that are paired with it [START_REF] Van Camp | Quantification of Brain Function and Neurotransmission System In Vivo by Positron Emission Tomography: A Review of Technical Aspects and Practical Considerations in Preclinical Research[END_REF][START_REF] Abbasi Gharibkandi | Radiotracers for imaging of Parkinson's disease[END_REF]. These arise from the heavy infrastructure [START_REF] Miller | Synthesis of 11C, 18F, 15O, and 13N radiolabels for positron emission tomography[END_REF][START_REF] Van Camp | Quantification of Brain Function and Neurotransmission System In Vivo by Positron Emission Tomography: A Review of Technical Aspects and Practical Considerations in Preclinical Research[END_REF], and the many different human competences that are required; including chemists, physicists, biologists, physicians, and imaging processing specialists [START_REF] Van Camp | Quantification of Brain Function and Neurotransmission System In Vivo by Positron Emission Tomography: A Review of Technical Aspects and Practical Considerations in Preclinical Research[END_REF]. PET imaging calls for cyclotron facilities for radioisotope production, radiochemical laboratories with shielded hot cells and automated chemical units for radioligand preparation, and PET scanners, preferably all in close proximity to limit synthesis time and time to imaging [START_REF] Miller | Synthesis of 11C, 18F, 15O, and 13N radiolabels for positron emission tomography[END_REF][START_REF] Van Camp | Quantification of Brain Function and Neurotransmission System In Vivo by Positron Emission Tomography: A Review of Technical Aspects and Practical Considerations in Preclinical Research[END_REF]. A part of this heavy infrastructure is needed to reduce exposure to radioactivity for all involved parties [START_REF] Van Camp | Quantification of Brain Function and Neurotransmission System In Vivo by Positron Emission Tomography: A Review of Technical Aspects and Practical Considerations in Preclinical Research[END_REF].

Lastly, PET imaging in small animals has inherent physical limitations that affect the accuracy of quantification [START_REF] Van Camp | Quantification of Brain Function and Neurotransmission System In Vivo by Positron Emission Tomography: A Review of Technical Aspects and Practical Considerations in Preclinical Research[END_REF]; which are described above (4.1 Principles of PET imaging). Additionally, PET imaging has a limited time resolution which challenges the detection of fast molecular and functional changes [START_REF] Herfert | Quantitative Rodent Brain Receptor Imaging[END_REF]. These complex biological processes, fast or slow, are described by very simplified pharmacokinetical models, which must be taken into account for data interpretation [START_REF] Herfert | Quantitative Rodent Brain Receptor Imaging[END_REF].

Radiotracer properties

When producing a new radiotracer, there are several things to take into account, e.g. the radioligand production time, ability to pass the blood-brain barrier (BBB) while its radiometabolites should not, and interference with biological processes [START_REF] Van Camp | Quantification of Brain Function and Neurotransmission System In Vivo by Positron Emission Tomography: A Review of Technical Aspects and Practical Considerations in Preclinical Research[END_REF].

The ability of a radiotracer to cross the BBB is determined by it lipophilicity, molecular weight, cross-sectional area, hydrogen-bonding capacity, formal charge [START_REF] Pike | PET radiotracers: crossing the blood-brain barrier and surviving metabolism[END_REF][START_REF] Van Camp | Quantification of Brain Function and Neurotransmission System In Vivo by Positron Emission Tomography: A Review of Technical Aspects and Practical Considerations in Preclinical Research[END_REF]. Moderate lipophilicity is sufficient for good cerebral uptake, while still ensuring low non-specific binding. High lipophilicity has the disadvantage of favouring binding to blood proteins, which reduced the tracer free fraction in blood plasma [START_REF] Pike | PET radiotracers: crossing the blood-brain barrier and surviving metabolism[END_REF]. Passive entry into the brain is promoted by a low molecular weight [START_REF] Pike | PET radiotracers: crossing the blood-brain barrier and surviving metabolism[END_REF], a small cross-sectional area [START_REF] Fischer | Blood-brain barrier permeation: Molecular parameters governing passive diffusion[END_REF][START_REF] Seelig | The role of size and charge for blood-brain barrier permeation of drugs and fatty acids[END_REF], a low hydrogen-bonding capacity [START_REF] Pike | PET radiotracers: crossing the blood-brain barrier and surviving metabolism[END_REF], and lack of a formal charge [START_REF] Seelig | The role of size and charge for blood-brain barrier permeation of drugs and fatty acids[END_REF]. A radiotracer's ability to stay in the brain is a combination of its capacity for passive entry versus its susceptibility for expulsion by drug efflux pumps [START_REF] Raub | P-glycoprotein recognition of substrates and circumvention through rational drug design[END_REF][START_REF] Pike | PET radiotracers: crossing the blood-brain barrier and surviving metabolism[END_REF]. The radiotracer should thus be poor substrate for drug efflux pumps, like P-glycoprotein [START_REF] Van Camp | Quantification of Brain Function and Neurotransmission System In Vivo by Positron Emission Tomography: A Review of Technical Aspects and Practical Considerations in Preclinical Research[END_REF].

Radiotracers are vulnerable to metabolisation. Ideally, this metabolism happens outside the brain, and produces radiometabolites that cannot cross the BBB due to lower lipophilicity with low or no interaction with the target protein [START_REF] Pike | PET radiotracers: crossing the blood-brain barrier and surviving metabolism[END_REF], these radiometabolites should not accumulate in the liver or any other organs, where they can cause irradiation issues; ideally they are excreted via the urinary tract [START_REF] Kristensen | Safety and efficacy of radiopharmaceuticals[END_REF]. The metabolisation of a radiotracer can be determined through ex vivo analyses [START_REF] Pike | PET radiotracers: crossing the blood-brain barrier and surviving metabolism[END_REF]for example Peyronneau et al., 2012).

PET imaging aims to study the normal functioning of the body, meaning that when using a PET tracer it should not affect the biology of the brain. Therefore, radiotracers are used at tracer doses, ~5% of receptor occupancy, and not at pharmacological doses. The specific activity (the ratio between activity and mass) has to be taken into account when deciding on the injected dose to assure tracer dose. However, in the generation of Focus 220 scanners, radiotracer dose needs to C h a p t e r 1 -I n t r o d u c t i o n | 39 compensate for a loss of sensitivity in rodent studies, due the increase resolution needed for their small brains [START_REF] Cherry | Use of positron emission tomography in animal research[END_REF], resulting in a 25-fold higher dose as compare to humans.

Lastly, animals are generally anaesthetised for PET imaging, which can cause interference between the anaesthetic and the radiotracer, e.g. FDG-PET and xylazine, which can introduce variability if the anaesthesia is not constant between animals.

The two most common PET isotopes are [ 11 C] and [ 18 F], with a half-life of 20 minutes and 110 minutes, respectively [START_REF] Abbasi Gharibkandi | Radiotracers for imaging of Parkinson's disease[END_REF]. Due to its short half-life, [ 11 C] radiotracers can only be used synthesised on-site. The longer half-life of 18 F makes is suitable for use at imaging centres that do not have on-site cyclotron (Abbasi Gharibkandi and Hosseinimehr, 2019). However, a major disadvantage of [ 18 F] labelled radiotracer is the occurrence of defluorination, which produced an [ 18 F] ion that binds extremely well to bone tissue, including skull [START_REF] Pike | PET radiotracers: crossing the blood-brain barrier and surviving metabolism[END_REF]. The resulting accumulation of radioactivity in the skull is problematic for subsequent quantification; together with the spatial resolution of PET imaging it results in a partial volume effect, i.e. a 'spillover' in the adjacent brain tissue. This can be avoided by choosing proper labelling sites [START_REF] Pike | PET radiotracers: crossing the blood-brain barrier and surviving metabolism[END_REF].

PET imaging targets in PD

As mentioned above, PET tracers can be used to monitor receptors, transporters, neurotransmitter synthesis and enzymes, intracellular proteins, and aggregated proteins in vivo (Figure 5) [START_REF] Van Camp | Quantification of Brain Function and Neurotransmission System In Vivo by Positron Emission Tomography: A Review of Technical Aspects and Practical Considerations in Preclinical Research[END_REF][START_REF] Abbasi Gharibkandi | Radiotracers for imaging of Parkinson's disease[END_REF][START_REF] Herfert | Quantitative Rodent Brain Receptor Imaging[END_REF]. Here, the most relevant targets in the context of PD will be discussed, with a focus on tracers that were used in the present study.

Glucose metabolism

[ 18 F]2-fluoro-2-deoxy-D-glucose ([ 18 F]FDG) is the most frequently used tracer in clinical PET imaging, but has also widely been used in animal studies [START_REF] Cherry | Use of positron emission tomography in animal research[END_REF]. [ 18 F]FDG visualises regional glucose metabolism, which has shown to be altered in many diseases and disorders [START_REF] Phelps | Positron emission tomography provides molecular imaging of biological processes[END_REF], and can be used in humans and many different animal species [START_REF] Cherry | Use of positron emission tomography in animal research[END_REF].

In humans, a PD related pattern of glucose metabolism measured with [ 18 F]FDG has been reported. This corresponds to relative increased metabolism of the thalamus, globus pallidus/putamen, cerebellum and pons, and relative hypometabolism of the occipital, temporal, parietal, and frontal cortices [START_REF] Eidelberg | The metabolic topography of parkinsonism[END_REF][START_REF] Ma | Abnormal metabolic network activity in Parkinson's disease: test-retest reproducibility[END_REF]reviewed in Meles et al., 2017reviewed in Meles et al., , 2020;; independent study by [START_REF] Matthews | FDG PET Parkinson's disease-related pattern as a biomarker for clinical trials in early stage disease[END_REF]. A correlation was observed between this pattern and PD patient cognitive skills [START_REF] Trošt | Correlations of Neuropsychological and Metabolic Brain Changes in Parkinson's Disease and Other α-Synucleinopathies[END_REF].

Similarly, in MPTP monkeys (paragraph 2.2, page 24) a correlation between altered [ 18 F]FDG pattern and motor deficits was observed [START_REF] Ma | Abnormal metabolic brain networks in a nonhuman primate model of parkinsonism[END_REF]. In nigral 6-OHDA lesioned rats (see paragraph on 6-hydroxydopamine, page 24) significant glucose hypo-metabolism was observed in the ipsilateral sensory motor cortex using [ 18 F]FDG-PET [START_REF] Casteels | Metabolic-dopaminergic mapping of the 6-hydroxydopamine rat model for Parkinson's disease[END_REF]. The multivariate pattern approach, as used by Eidelberg and colleagues (1994), has been further developed and applied in rat models of PD. Devrome and colleagues (2019) observed a PD specific pattern of glucose metabolism in an adeno-associated viral vector based α-synuclein rat model. This pattern overlapped to a large extent with the human PD related pattern [START_REF] Devrome | Identifying a glucose metabolic brain pattern in an adeno-associated viral vector based rat model for Parkinson's disease using 18F-FDG PET imaging[END_REF]. In a similar viral vector-mediated overexpression model of PD, bilateral hypermetabolism was observed in the C h a p t e r 1 -I n t r o d u c t i o n | 40 striatum at 2wpi, ipsilateral nigral hypermetabolism at 4wpi, ipsilateral striatal hypometabolism at 6wpi, and contralateral striatal hypermetabolism at 9wpi [START_REF] Crabbé | Temporal changes in neuroinflammation and brain glucose metabolism in a rat model of viral vector-induced α-synucleinopathy[END_REF]. Preclinical [ 18 F]FDG-PET imaging can support the validation of disease specific brain patterns of glucose metabolism.

L-aromatic amino acid decarboxylase (AADC) enzymatic activity

AADC is the second enzyme in the biosynthetic pathway of several monoamine neurotransmitters; dopamine, norepinephrine, and serotonin. AADC rapidly converts L-DOPA to dopamine, which can be subsequently converted to norepinephrine or serotonin [START_REF] Dejesus | Aromatic L-amino acid decarboxylase turnover in vivo in rhesus macaque striatum: a microPET study[END_REF].

L-3,4-dihydroxy-6-[ 18 F]fluoro-phenylalanine ([ 18 F]FDOPA) is a commonly used PET tracer in PD patient imaging [START_REF] Ruottinen | Striatal [18F]fluorodopa utilization after COMT inhibition with entacapone studied with PET in advanced Parkinson's disease[END_REF] and visualises dopamine synthesis, storage and turnover [START_REF] Garnett | Dopamine visualized in the basal ganglia of living man[END_REF]. The chemical structure of [ 18 F]FDOPA is closely related to L-DOPA, and undergoes all the same metabolic steps; decarboxylation by L-aromatic amino acid decarboxylase (AADC), storage by vesicular monoamine transporters (VMAT), and catabolism by monoamine oxidase and monoamine oxidase and catechol-O-methyl-transferase (COMT) [START_REF] Melega | L-6-[18F]fluoro-dopa metabolism in monkeys and humans: biochemical R e f e r e n c e s | 201 parameters for the formulation of tracer kinetic models with positron emission tomography[END_REF][START_REF] Endres | Affinities of dopamine analogs for monoamine granular and plasma membrane transporters: implications for PET dopamine studies[END_REF]. However, AADC and COMT are abundantly present in the peripheral organs, such as liver and kidney [START_REF] Rahman | Aromatic L-amino acid decarboxylase activity in central and peripheral tissues and serum of rats with L-DOPA and L-5-hydroxytryptophan as substrates[END_REF][START_REF] Männistö | Catechol O-methyltransferase: characterization of the protein, its gene, and the preclinical pharmacology of COMT inhibitors[END_REF]. Peripheral metabolisation of [ 18 F]FDOPA by COMT results in 3-O-methyl-[ 18 F]FDOPA, which can cross the BBB and thus reduce signal-to-background ratio and lower image contrast. Either plasma metabolite analysis or peripheral COMT inhibitors (e.g. entecapone, tolcapone, or opicapone) are therefore required for [ 18 F]FDOPA quantification [START_REF] Ruottinen | Striatal [18F]fluorodopa utilization after COMT inhibition with entacapone studied with PET in advanced Parkinson's disease[END_REF][START_REF] Becker | Comparative assessment of 6-[18F]fluoro-L-m-tyrosine and 6-[18F]fluoro-Ldopa to evaluate dopaminergic presynaptic integrity in a Parkinson's disease rat model[END_REF]. Blocking of peripheral AADC (via e.g. carbidopa or benserazide) prior to [ 18 F]FDOPA administration is required to inhibit peripheral metabolism of [ 18 F]FDOPA in PD patients [START_REF] Melega | L-6-[18F]fluoro-dopa metabolism in monkeys and humans: biochemical R e f e r e n c e s | 201 parameters for the formulation of tracer kinetic models with positron emission tomography[END_REF][START_REF] Ruottinen | Striatal [18F]fluorodopa utilization after COMT inhibition with entacapone studied with PET in advanced Parkinson's disease[END_REF][START_REF] Li | The role of the LRRK2 gene in Parkinsonism[END_REF], and non-human primates [START_REF] Melega | L-6-[18F]fluoro-dopa metabolism in monkeys and humans: biochemical R e f e r e n c e s | 201 parameters for the formulation of tracer kinetic models with positron emission tomography[END_REF][START_REF] Pate | Correlation of striatal fluorodopa uptake in the MPTP Monkey with dopaminergic indices[END_REF][START_REF] Dejesus | Evaluation of fluorinated m-tyrosine analogs as PET imaging agents of dopamine nerve terminals: comparison with 6-fluoroDOPA[END_REF].

In PD patients, [ 18 F]FDOPA showed reduced uptake in the caudate and putamen [START_REF] Holtbernd | Dopaminergic correlates of metabolic network activity in Parkinson's disease[END_REF][START_REF] Akamatsu | A revisit to quantitative PET with 18F-FDOPA of high specific activity using a high-resolution condition in view of application to regenerative therapy[END_REF]Daryl J. Wile et al., 2017), and the substantia nigra was clearly visualised [START_REF] Akamatsu | A revisit to quantitative PET with 18F-FDOPA of high specific activity using a high-resolution condition in view of application to regenerative therapy[END_REF]. Furthermore, efforts are being undertaken to increase quantification methods, for example by integrating blood metabolomics [START_REF] Glaab | Integrative analysis of blood metabolomics and PET brain neuroimaging data for Parkinson's disease[END_REF] or applying other quantification methods [START_REF] Alves | Dual time point method for the quantification of irreversible tracer kinetics: A reference tissue approach applied to [18F]-FDOPA brain PET[END_REF].

In rats [ 18 F]FDOPA was shown to successfully visualise unilateral 6-ODHA lesions; peripheral COMT inhibitors were combined with peripheral AADC inhibitors for these studies. [START_REF] Kyono | Use of [18F]FDOPA-PET for in vivo evaluation of dopaminergic dysfunction in unilaterally 6-OHDA-lesioned rats[END_REF][START_REF] Walker | Measuring dopaminergic function in the 6-OHDA-lesioned rat: a comparison of PET and microdialysis[END_REF][START_REF] Walker | In-vivo measurement of LDOPA uptake, dopamine reserve and turnover in the rat brain using [18F]FDOPA PET[END_REF][START_REF] Becker | Comparative assessment of 6-[18F]fluoro-L-m-tyrosine and 6-[18F]fluoro-Ldopa to evaluate dopaminergic presynaptic integrity in a Parkinson's disease rat model[END_REF]. As in non-human primate, a significant correlation between [ 18 F]FDOPA and striatal dopamine levels was observed in rats [START_REF] Kyono | Use of [18F]FDOPA-PET for in vivo evaluation of dopaminergic dysfunction in unilaterally 6-OHDA-lesioned rats[END_REF][START_REF] Walker | Measuring dopaminergic function in the 6-OHDA-lesioned rat: a comparison of PET and microdialysis[END_REF]. However, [ 18 F]FDOPA was unable to discriminate between a partial and complete 6-OHDA lesion [START_REF] Becker | Comparative assessment of 6-[18F]fluoro-L-m-tyrosine and 6-[18F]fluoro-Ldopa to evaluate dopaminergic presynaptic integrity in a Parkinson's disease rat model[END_REF].

Alternatively, 6-[ 18 F]fluoro-m-tyrosine ([ 18 F]FMT) has a 10-fold greater affinity for AADC than [ 18 F]FDOPA and is not a substrate for COMT [START_REF] Dejesus | Evaluation of fluorinated m-tyrosine analogs as PET imaging agents of dopamine nerve terminals: comparison with 6-fluoroDOPA[END_REF]. [ 18 F]FMT is transformed by AADC in 6-[ 18 F]-m-tyramine ([ 18 F]FMTA) in the blood plasma, and is subsequently oxidized to 6-[ 18 F]fluorom-hydroxyphenylacetic acid ([ 18 F]FHPAA) by mono-amine oxidase. Even though these two metabolites cannot cross the BBB, peripheral inhibition of AADC is still necessary (via e.g. carbidopa or benserazide) [START_REF] Endres | Affinities of dopamine analogs for monoamine granular and plasma membrane transporters: implications for PET dopamine studies[END_REF][START_REF] Becker | Comparative assessment of 6-[18F]fluoro-L-m-tyrosine and 6-[18F]fluoro-Ldopa to evaluate dopaminergic presynaptic integrity in a Parkinson's disease rat model[END_REF].

C h a p t e r 1 -I n t r o d u c t i o n | 41
In a clinical setting, it was shown that [ 18 F]FMT-PET imaging inversely correlated with motor scores of PD patients [START_REF] Gallagher | A within-subject comparison of 6-[18F]fluoro-m-tyrosine and 6-[fluoro-L-dopa in Parkinson's disease[END_REF]. Compared to [ 18 F]FDOPA, [ 18 F]FMT was also found to have higher extrastriatal uptake in regions with specific uptake (such as amygdala, pallidum, thalamus, and hippocampus), which might be useful for the examination of non-motor symptoms [START_REF] Li | The role of the LRRK2 gene in Parkinsonism[END_REF]. In mice, like [ 18 F]FDOPA, [ 18 F]FMT failed to clearly visualise the mouse striatum [START_REF] Honer | Comparison of [18F]FDOPA, [18F]FMT and [18F]FECNT for imaging dopaminergic neurotransmission in mice[END_REF].

To the best of our knowledge, only a single study so far has been conducted using [ 18 F]FMT in rats. Becker and colleagues (2017) have shown that [ 18 F]FMT can distinguish between partial and complete 6-OHDA lesioned rats, which was not discernible using [ 18 F]FDOPA. However, in contrast to the human and macaque studies, inhibition of peripheral AADC activity was necessary for both [ 18 F]FMT and [ 18 F]FDOPA. The study suggests thus that [ 18 F]FMT could be more sensitive to detect dopaminergic lesions, and can be used to investigate presynaptic dopamine integrity and AADC activity. This lesion most likely has to exceed 25% loss of dopaminergic terminals to be detectable with [ 18 F]FMT [START_REF] Becker | Comparative assessment of 6-[18F]fluoro-L-m-tyrosine and 6-[18F]fluoro-Ldopa to evaluate dopaminergic presynaptic integrity in a Parkinson's disease rat model[END_REF].

Dopamine transporter

Dopamine transporter (DAT) is expressed on the presynaptic membrane in the synaptic terminal of dopaminergic neurons. Several cocaine analogues exist as radioligand targeting DAT [START_REF] Abbasi Gharibkandi | Radiotracers for imaging of Parkinson's disease[END_REF][START_REF] Ikeda | Dopamine Transporter Imaging in Parkinson Disease: Progressive Changes and Therapeutic Modification after Anti-parkinsonian Medications[END_REF]. In clinics, DAT-SPECT (using [ 123 I]FP-CIT) is used widely for the differential diagnosis of PD and other neurodegenerative parkinsonism from essential tremor, drug-induced parkinsonism, or other neurological diseases that do not involve the nigrostriatal dopaminergic system [START_REF] Brooks | Molecular imaging of dopamine transporters[END_REF][START_REF] Varrone | SPECT Molecular Imaging in Familial Parkinson's Disease[END_REF][START_REF] Ikeda | Dopamine Transporter Imaging in Parkinson Disease: Progressive Changes and Therapeutic Modification after Anti-parkinsonian Medications[END_REF]. Examples of DAT-PAT radioligands are [ 18 F]LBT999 [START_REF] Dollé | Synthesis, radiosynthesis and in vivo preliminary evaluation of [11C]LBT-999, a selective radioligand for the visualisation of the dopamine transporter with PET[END_REF][START_REF] Chalon | The story of the dopamine transporter PET tracer [18F]LBT-999: From conception to clinical use[END_REF] and [ 18 F]FE-PE2I [START_REF] Schou | Synthesis, radiolabeling and preliminary in vivo evaluation of [18F]FE-PE2I, a new probe for the dopamine transporter[END_REF][START_REF] Varrone | In vitro autoradiography and in vivo evaluation in cynomolgus monkey of [18F]FE-PE2I, a new dopamine transporter PET radioligand[END_REF]. DAT-PET is becoming a valuable clinical tool to replace DAT-SPECT; in a clinical study [ 18 F]FE-PE2I was found to yield excellent basic diagnostic differentiation in early-stage PS, at least as good as [ 123 I]FP-CIT (Jakobson [START_REF] Mo | Dopamine transporter imaging with [18F]FE-PE2I PET and [123I]FP-CIT SPECT-a clinical comparison[END_REF], while a clinical trial to compare [ 18 F]LBT999 to [ 123 I]FP-CIT is planned to start in 2020 (NCT04265209). I will focus here on [ 18 F]LBT999 since this radioligand was used in my PhD research.

(E)-N-(4-fluorobut-2-enyl)-2β-carbomethoxy-3β-(4'-tolyl)nortropane, was first utilised as an 11 C radiotracer ([ 11 C]LBT999). In baboons it showed a high and rapid uptake mainly localised in striatal regions [START_REF] Chalon | Pharmacological characterization of (E)-N-(4-fluorobut-2-enyl)-2betacarbomethoxy-3beta-(4'-tolyl)nortropane (LBT-999) as a highly promising fluorinated ligand for the dopamine transporter[END_REF]. The fluorinated version ([ 18 F]LBT999) can be synthesized using either two-step [START_REF] Dollé | Synthesis, radiosynthesis and in vivo preliminary evaluation of [11C]LBT-999, a selective radioligand for the visualisation of the dopamine transporter with PET[END_REF] or one-step radiosynthesis [START_REF] Dollé | One-step radiosynthesis of [18F]LBT-999: A selective radioligand for the R e f e r e n c e s | 186 visualization of the dopamine transporter with PET[END_REF].

In healthy volunteers [ 18 F]LBT999 was shown to have high symmetric uptake in the caudate and putamen, and low uptake in the cerebellum [START_REF] Arlicot | PET imaging of Dopamine Transporter with 18F-LBT999: first human exploration[END_REF]. In non-human primates, [ 18 F]LBT999 showed a similar pattern; high uptake in the caudate and putamen, moderate uptake in the midbrain, and low uptake in the cortex and cerebellum [START_REF] Saba | 18F]LBT-999, a new radioligand to study the dopamine transporter with PET: Characterization in baboons[END_REF]. Furthermore, [ 18 F]LBT999 showed reduced uptake in both the caudate and putamen in early PD patients as compared to controls. Using striatal ratios it was even possible to differentiate patients from controls [START_REF] Santiago-Ribeiro | A simplified method for the diagnosis of striatal dopaminergic dysfunction using PET with a new fluorine DAT tracer, the 18F-LBT-999[END_REF].

In rats, [ 18 F]LBT999 showed specific binding to DAT and allowed quantification with high reproducibility, sensitivity, and specificity in the striatum. In the nigral area the variability was higher and the reliability lower [START_REF] Sérrière | In vivo PET quantification of the dopamine transporter in rat brain with [18F]LBT-999[END_REF]. Thereafter, the same group has shown that DAT density, as seen by [ 18 F]LBT999, was reduced in 6-OHDA lesioned rats [START_REF] Sérrière | Assessment of the Protection of Dopaminergic Neurons by an α7 Nicotinic Receptor Agonist, PHA 543613 Using[END_REF][START_REF] Vetel | Extensive exploration of a novel rat model of Parkinson's disease using partial 6-hydroxydopamine lesion of dopaminergic neurons suggests new therapeutic approaches[END_REF] C h a p t e r 1 -I n t r o d u c t i o n | 42 al., 2019). Grealish and colleagues (2014) have taken it one step further and have shown that [ 18 F]LBT999 is sensitive enough to observe functional improvement after cell grafting in 6-OHDA lesioned rats [START_REF] Grealish | Human ESC-derived dopamine neurons show similar preclinical efficacy and potency to fetal neurons when grafted in a rat model of Parkinson's disease[END_REF].

Vesicular monoamine transporter 2

VMAT2 has a critical role in maintaining catecholamine and serotonin levels in central nervous system. VMAT2 is expressed in all monoaminergic neurons of the brain, including those expressing dopamine, serotonin (5-HT), norepinephrine, epinephrine, and histamine. In 2006, VMAT2-PET imaging was proposed as a potential biomarker for PD [START_REF] Bohnen | Positron emission tomography of monoaminergic vesicular binding in aging and Parkinson disease[END_REF].

[ 11 C]fluoropropyl-(+)-dihydrotetrabenazine is a well characterised radiotracer for vesicular monoamine transporter II (VMAT2) (reviewed by [START_REF] Kilbourn | Classics in Neuroimaging: Radioligands for the Vesicular Monoamine Transporter 2[END_REF]. [ 18 F]fluoropropyl-(+)dihydrotetrabenazine ([ 18 F]DTBZ, also termed [ 18 F]AV-133) is the 18 F-version of this radiotracer [START_REF] Kung | Characterization of optically resolved 9-fluoropropyl-dihydrotetrabenazine as a potential PET imaging agent targeting vesicular monoamine transporters[END_REF]. VMAT2 transports dopamine, and norepinephrine and serotonin, from the cytoplasm to the secretory vesicles [START_REF] Schuldiner | A molecular glimpse of vesicular monoamine transporters[END_REF]. [ 18 F]DTBZ was shown to have a high specific binding ratio and metabolic stability [START_REF] Kilbourn | Pharmacokinetics of [18F]fluoroalkyl derivatives of dihydrotetrabenazine in rat and monkey brain[END_REF].

In healthy volunteers, [ 18 F]DTBZ showed the highest brain uptake levels in the striatum [START_REF] Lin | Brain Imaging of Vesicular Monoamine Transporter Type 2 in Healthy Aging Subjects by 18F-FP-(+)-DTBZ PET[END_REF]. [ 18 F]DTBZ demonstrated decreased binding potential in the striatal regions of PD patients [START_REF] Okamura | In vivo measurement of vesicular monoamine transporter type 2 density in Parkinson disease with 18F-AV-133[END_REF][START_REF] Lin | In vivo detection of monoaminergic degeneration in early parkinson disease by 18F-9-fluoropropyl-(1)-dihydrotetrabenzazine PET[END_REF]. Subsequently, Hsiao and colleagues (2014) showed that this decrease in PD patients, and thus the decrease in VMAT2, was inversely correlated with disease severity [START_REF] Hsiao | Correlation of Parkinson disease severity and 18F-DTBZ positron emission tomography[END_REF].

In 6-OHDA lesioned baboons [ 18 F]DTBZ showed a reduction of VMAT2 in the lesioned striatum, which was confirmed by autoradiography [START_REF] Zhu | Imaging of VMAT2 binding sites in the brain by 18F-AV-133: The effect of a pseudo-carrier[END_REF]. In the 6-OHDA lesioned rat, [ 18 F]DTBZ showed a clear and definitive loss of VMAT2 binding in the ipsilateral striatum [START_REF] Wang | In vivo studies of the SERT-selective [18F]FPBM and VMAT2-selective []AV-133 radiotracers in a rat model of Parkinson's disease[END_REF].

[ 18 F]DTBZ has been used to validate a novel PD rat model [START_REF] Weng | 18F]FP-(+)-DTBZ PET study in a lactacystin-treated rat model of Parkinson disease[END_REF], while in the MPTP mouse model the radiotracer has been used to investigate therapeutic outcomes [START_REF] Weng | Quantitative analysis of the therapeutic effect of magnolol on MPTP-induced mouse model of Parkinson's disease using in vivo 18F-9-fluoropropyl-(+)dihydrotetrabenazine PET imaging[END_REF].

In an AAV2/6-Synapsin/WPRE-WT-α-synuclein rat model, [ 11 C]DTBZ showed asymmetrical striatal binding in the absence of dopaminergic neurodegeneration in the substantia nigra, while S129-phosphorylated α-synuclein was observed in the dopaminergic terminals [START_REF] Phan | Early synaptic dysfunction induced by α-synuclein in a rat model of Parkinson's disease[END_REF]. This indicates that DTBZ is sensitive enough to detect early synaptic dysfunction caused by αsynuclein overexpression.

Dopamine receptors

While previous targets are located at the presynaptic side of the dopaminergic synapse, D 2 -and D 3 -receptors (D 2 R/D 3 R) are located at the postsynaptic side. Dopaminergic neuronal loss induces a dopamine depletion in the synapse and subsequent increase of D 2 R binding sites and affinity [START_REF] Mach | Challenges in the development of dopamine D2-and D3selective radiotracers for PET imaging studies[END_REF]. Therefore, D 2 R PET ligands are of interest to evaluate restoration of dopamine levels at the level of the synapse after dopamine replacement therapies (for example [START_REF] Palfi | Long-term safety and tolerability of ProSavin, a lentiviral vector-based gene therapy for Parkinson's disease: a dose escalation, open-label, phase 1/2 trial[END_REF]for example Fukai et al., 2019).

The preferential ligand to image D 2 R is [ 11 C]raclopride, which is in direct competition with endogenous dopamine. In PD patients, [ 11 C]raclopride binding is reduced due to a combination of upregulation of D 2 R/D 3 R and reduced occupancy by endogenous dopamine [START_REF] Arena | Optimizing diagnosis in Parkinson's disease: Radionuclide imaging[END_REF]. 18 F]fallypride) is another dopamine D 2 -and D 3 -receptor [START_REF] Mukherjee | Preliminary assessment of extrastriatal dopamine D-2 receptor binding in the rodent and nonhuman primate brains using the high affinity radioligand, 18F-fallypride[END_REF] antagonist first described by Mukherjee and colleagues (1995). They showed high striatal uptake of [ 18 F]fallypride in rats and specific displacement by haloperidol in macaque brain (see page 140) [START_REF] Mukherjee | Fluorinated benzamide neuroleptics--III. Development of (S)-N-[(1-allyl-2pyrrolidinyl)methyl]-5-(3-[18F]fluoropropyl)-2, 3-dimethoxybenzamide as an improved dopamine D-2 receptor tracer[END_REF]. Subsequently the same group demonstrated that specific binding of [ 18 F]fallypride is reduced after amphetamine-induced dopaminergic release [START_REF] Mukherjee | Evaluation of d-amphetamine effects on the binding of dopamine D-2 receptor radioligand, 18F-fallypride in nonhuman primates using positron emission tomography[END_REF], through direct competition with dopamine for binding to the D 2 R and D 3 R (Abbasi Gharibkandi and Hosseinimehr, 2019).

C h a p t e r 1 -I n t r o d u c t i o n | 43 Alternatively, (S)-N-[(1-allyl-2-pyrrolidinyl)methyl]-5-(3-[ 18 F]fluoropropyl)-2,3-dimethoxy- benzamide ([
[ 18 F]fallypride has been successfully used to study the D 2 R and D 3 R changes in the striatum in PD patients [START_REF] Fisher | Treadmill exercise elevates striatal dopamine D2 receptor binding potential in patients with early Parkinson's disease[END_REF][START_REF] Stark | Nigrostriatal and Mesolimbic D2/3 Receptor Expression in Parkinson's Disease Patients with Compulsive Reward-Driven Behaviors[END_REF][START_REF] Stark | 18F]fallypride characterization of striatal and extrastriatal D2/3 receptors in Parkinson's disease[END_REF]. Unlike [ 11 C]raclopride, [ 18 F]fallypride is also capable of identifying extrastriatal dopamine receptors [START_REF] De Paulis | The discovery of epidepride and its analogs as high-affinity radioligands for imaging extrastriatal dopamine D(2) receptors in human brain[END_REF][START_REF] Elsinga | PET tracers for imaging of the dopaminergic system[END_REF][START_REF] Stark | 18F]fallypride characterization of striatal and extrastriatal D2/3 receptors in Parkinson's disease[END_REF]. However, recently Karalija and colleagues (2019) have demonstrated reliable extrastriatal [ 11 C]raclopride binding in older adults [START_REF] Karalija | High long-term test-retest reliability for extrastriatal 11C-raclopride binding in healthy older adults[END_REF].

In rats, [ 18 F]fallypride showed increased brain uptake in the ipsilteral to a 6-ODHA lesion, indicating D 2 R and D 3 R upregulation [START_REF] Choi | Evaluation of dopamine transporters and D2 receptors in hemiparkinsonian rat brains in vivo using consecutive PET scans of [18F]FPCIT and []fallypride[END_REF]. Mann and colleagues (2018) confirmed these results and continued by showing with [ 18 F]fallypride that botulinum neurotoxin A can reverse the effect of 6-OHDA [START_REF] Mann | 18F]fallypride-PET/CT Analysis of the Dopamine D₂/D₃ Receptor in the Hemiparkinsonian Rat Brain Following Intrastriatal Botulinum Neurotoxin A Injection[END_REF].

Serotonin

Alternatively, biomarkers could be used to predict the progression of PD and the occurrence of non-motor symptoms. The serotonin system is a very interesting target for this since it is involved in several non-motor symptoms of PD, like depression, anxiety, and apathy [START_REF] Dujardin | Neuropsychiatric Disorders in Parkinson's Disease: What Do We Know About the Role of Dopaminergic and Non-dopaminergic Systems?[END_REF]. Moreover, Millot and colleagues (2020) have shown that serotonergic lesioning in non-human primates aggravates MPTP-induced parkinsonian motor-symptoms. It must be noted, that, to a lesser extent, also dopaminergic neurons where lesioned using MDMA [START_REF] Millot | Prior MDMA administration aggravates MPTP-induced Parkinsonism in macaque monkeys[END_REF].

Much like DAT, the serotonin transpoter regulates the concentration of free serotonin in the synaptic cleft (Leung, 2004). One example of a radioligand to visualise serotonin transporter binding is [ 11 [START_REF] Wilson | Novel Radiotracers for Imaging the Serotonin Transporter by Positron Emission Tomography: Synthesis, Radiosynthesis, and in Vitro and ex Vivo Evaluation of 11 C-Labeled 2-(Phenylthio)araalkylamines[END_REF]. Using [ 11 C]DASB, it was found that apathetic PD patients show greater serotonergic alterations compared to non-apathetic patients, indicating that serotonergic degeneration has a prominent role in neuropsychiatric symptoms of early PD [START_REF] Maillet | The prominent role of serotonergic degeneration in apathy, anxiety and depression in de novo Parkinson's disease[END_REF].

C]N,N-Dimethyl-2-(2-amino-4-cyanophenylthio)benzylamine ([ 11 C]DASB)

TSPO imaging in the context of PD

18kDa translocator protein (TSPO) is expressed on the outer mitochondrial membrane of activated microglial and astrocytes [START_REF] Papadopoulos | Translocator protein (18kDa): new nomenclature for the peripheraltype benzodiazepine receptor based on its structure and molecular function[END_REF][START_REF] Venneti | The peripheral benzodiazepine receptor (Translocator protein 18 kDa) in microglia: From pathology to imaging[END_REF], where it is part of the mitochondrial transition pore. TSPO is expressed in astrocytes, microglia, macrophages but also by endothelial cell, and in peripheral organs [START_REF] Banati | Visualising microglial activation in vivo[END_REF][START_REF] Bonsack | TSPO: An evolutionarily conserved protein with elusive functions[END_REF]. Increased densities of activated microglia and astrocytes during neuroinflammatory events result in higher regional densities of TSPO [START_REF] Furube | Brain Region-dependent Heterogeneity and Dose-dependent Difference in Transient Microglia Population Increase during Lipopolysaccharide-induced Inflammation[END_REF], making this protein the PET imaging biomarker of neuroinflammation [START_REF] Chauveau | Comparative evaluation of the translocator protein radioligands 11C-DPA-713, 18F-DPA-714, and 11C-PK11195 in a rat model of acute neuroinflammation[END_REF][START_REF] Owen | Imaging Brain Microglial Activation Using Positron Emission Tomography and Translocator Protein-Specific Radioligands[END_REF][START_REF] Abbasi Gharibkandi | Radiotracers for imaging of Parkinson's disease[END_REF].

C h a p t e r 1 -I n t r o d u c t i o n | 44 [ 11 C](R)-PK11195
is the first generation radioligand that targets TSPO, which has been used since the 80's, however, the clinical usefulness is limited due to low brain bioavailability, non-specific binding, and the relatively short half-life of 11 C (Best et al., 2019). In PD patients, overall increased [ 11 C](R)-PK11195 binding was observed in the pons, basal ganglia (including substantia nigra) and frontal and temporal cortical regions, which remained stable over 2 years [START_REF] Gerhard | In vivo imaging of microglial activation with [11C](R)-PK11195 PET in idiopathic Parkinson's disease[END_REF] reviewed by [START_REF] Bartels | Neuroinflammation in the pathophysiology of Parkinson's disease: Evidence from animal models to human in vivo studies with [11C]-PK11195 PET[END_REF][START_REF] Kang | Noninvasive PK11195-PET Image Analysis Techniques Can Detect Abnormal Cerebral Microglial Activation in Parkinson's Disease[END_REF].

Low specific binding and quantification issues of [ 11 C](R)-PK11195 have led to the development of a whole generation of new ligands, so called 2 nd generation ligands. However, in humans, a polymorphism in TSPO has been demonstrated to underlie different binding affinities of second generation ligands to 18kDa TSPO [START_REF] Bonsack | TSPO: An evolutionarily conserved protein with elusive functions[END_REF][START_REF] Best | New and Old TSPO PET Radioligands for Imaging Brain Microglial Activation in Neurodegenerative Disease[END_REF][START_REF] Werry | Recent developments in TSPO PET imaging as a biomarker of neuroinflammation in neurodegenerative disorders[END_REF], which has not been observed in rodents.

N,N-diethyl-2-(2-(4-(2[18F]-fluoroethoxy)phenyl)5,7dimethylpyrazolo[1,5a]pyrimidin-3- yl)acetamide ([ 18 F]DPA714
) is a second generation radioligand that targets TSPO, developed by James and colleagues (2008). Over the last ten years, different research groups within the CEA have validated [ 18 F]DPA714; from rodents [START_REF] Chauveau | Comparative evaluation of the translocator protein radioligands 11C-DPA-713, 18F-DPA-714, and 11C-PK11195 in a rat model of acute neuroinflammation[END_REF] to non-human primates [START_REF] Lavisse | 18F]DPA-714 PET imaging of translocator protein TSPO (18 kDa) in the normal and excitotoxically-lesioned nonhuman primate brain[END_REF][START_REF] Van Camp | Assessment of simplified methods for quantification of [ 18 F]-DPA-714 using 3D whole-brain TSPO immunohistochemistry in a non-human primate[END_REF], and into humans [START_REF] Lavisse | Optimized Quantification of Translocator Protein Radioligand 18 F-DPA-714 Uptake in the Brain of Genotyped Healthy Volunteers[END_REF][START_REF] García-Lorenzo | Validation of an automatic reference region extraction for the quantification of [ F]DPA-714 in dynamic brain PET studies[END_REF][START_REF] Wimberley | Impact of endothelial 18-kDa translocator protein on the quantification of 18 F-DPA-714[END_REF]. Even though the TSPO PET signal is mostly attributed to activated microglia, more recently it was demonstrated that also activated astrocytes contribute significantly to the [ 18 F]DPA714 PET signal [START_REF] Lavisse | Reactive Astrocytes Overexpress TSPO and Are Detected by TSPO Positron Emission Tomography Imaging[END_REF][START_REF] Van Camp | Assessment of simplified methods for quantification of [ 18 F]-DPA-714 using 3D whole-brain TSPO immunohistochemistry in a non-human primate[END_REF]. Other studies underlined this finding by showing that both activated microglia and astrocytes are positive for TSPO [START_REF] Ory | PET imaging of TSPO in a rat model of local neuroinflammation induced by intracerebral injection of lipopolysaccharide[END_REF][START_REF] Pannell | Imaging of translocator protein upregulation is selective for proinflammatory polarized astrocytes and microglia[END_REF].

In rats, multiple groups have used [ 18 F]DPA714 to image LPS-based neuroinflammatory models of different strengths, and suggesting different quantification methods [START_REF] Ory | PET imaging of TSPO in a rat model of local neuroinflammation induced by intracerebral injection of lipopolysaccharide[END_REF][START_REF] Ory | Quantification of TSPO overexpression in a rat model of local neuroinflammation induced by intracerebral injection of LPS by the use of [18F]DPA-714 PET[END_REF][START_REF] Sridharan | Comparative Evaluation of Three TSPO PET Radiotracers in a LPS-Induced Model of Mild Neuroinflammation in Rats[END_REF][START_REF] Vieira | Volume-of-interest-based supervised cluster analysis for pseudo-reference region selection in [18F]DPA-714 PET imaging of the rat brain[END_REF]. In a non-human primate model of neuroinflammation, a clear correlation between TSPO expression, as measured by optical density, and binding of [ 18 F]DPA was observed. Furthermore, [ 18 F]DPA714 showed a strong colocalisation with CD86 + microglia, and in a lesser extend with GFAP + astrocytes [START_REF] Lavisse | 18F]DPA-714 PET imaging of translocator protein TSPO (18 kDa) in the normal and excitotoxically-lesioned nonhuman primate brain[END_REF]. PET imaging in healthy volunteers suggested that [ 18 F]DPA714 is a promising radioligand with good stability and expected biodistribution [START_REF] Arlicot | Initial evaluation in healthy humans of [ 18F]DPA-714, a potential PET biomarker for neuroinflammation[END_REF][START_REF] Lavisse | Optimized Quantification of Translocator Protein Radioligand 18 F-DPA-714 Uptake in the Brain of Genotyped Healthy Volunteers[END_REF]; high binding in the thalamus and low binding in the cerebellum [START_REF] Lavisse | Optimized Quantification of Translocator Protein Radioligand 18 F-DPA-714 Uptake in the Brain of Genotyped Healthy Volunteers[END_REF], similar to the distribution in non-human primates [START_REF] Lavisse | 18F]DPA-714 PET imaging of translocator protein TSPO (18 kDa) in the normal and excitotoxically-lesioned nonhuman primate brain[END_REF].

To the best of our knowledge, the use of [ 18 F]DPA714 in PD patients has not been published yet. However, Terada and colleagues have reported significant increases of BP nd in the occipital, temporal and parietal cortex of PD patients using [ 11 C]DPA713, which was increased even further 1 year later [START_REF] Terada | Extrastriatal spreading of microglial activation in Parkinson's disease: a positron emission tomography study[END_REF]. Similarly, other studies using [ 11 C](R)-PK11195 have shown increased binding levels in the pons, basal ganglia (including substantia nigra and striatum), and frontal and temporal cortical regions [START_REF] Ouchi | Microglial activation and dopamine terminal loss in early Parkinson's disease[END_REF][START_REF] Gerhard | In vivo imaging of microglial activation with [11C](R)-PK11195 PET in idiopathic Parkinson's disease[END_REF][START_REF] Edison | Microglia, amyloid, and glucose metabolism in Parkinson's disease with and without dementia[END_REF][START_REF] Iannaccone | In vivo microglia activation in very early dementia with Lewy bodies, comparison with Parkinson's disease[END_REF], which remained stable over 2 years [START_REF] Gerhard | In vivo imaging of microglial activation with [11C](R)-PK11195 PET in idiopathic Parkinson's disease[END_REF].

Context and aims of the PhD Thesis

EVALUATION OF THE DOP AMINERGIC SYSTEM USI NG IN VIVO PET IMAGING AND POST-MORTEM

ANALYSES IN THE CONT EXT OF MILD NEURODEGENERATIVE AND NEUROI NFLAMMATORY PD MODELS

Scientific context: BrainMatTrain program

Even though cell replacement therapy has great potential, there is still suboptimal cell survival, differentiation, and reinnervation of host tissue. Studies report a <20% survival rate for grafted fetal tissue and <10% for grafted human iPSC-derived dopaminergic neuron progenitor cells [START_REF] Castilho | Improving the survival of grafted embryonic dopamine neurons in rodent models of Parkinson's disease[END_REF][START_REF] Bruggeman | Harnessing stem cells and biomaterials to promote neural repair[END_REF]. Biomaterials may have great potential to overcome these problems with cell-based therapies and might propel the field forward. Biomaterials can be utilised to provide physical support during implantation and differentiation, as well as to deliver relevant tropic factors to both grafted and host tissue [START_REF] Bruggeman | Harnessing stem cells and biomaterials to promote neural repair[END_REF]. Biomaterials of interest are hydrogels; 3D networks of hydrated polymers [START_REF] Bruggeman | Harnessing stem cells and biomaterials to promote neural repair[END_REF]. Hydrogels are one of two main components of the extracellular matrix [START_REF] Frantz | The extracellular matrix at a glance[END_REF], making them well-suited to mimic the extracellular environment [START_REF] Bruggeman | Harnessing stem cells and biomaterials to promote neural repair[END_REF]. Indeed, GDNFcontaining hydrogels were found to increase survival, striatal re-innervation, and functional efficacy of grafted fVM cells in 6-OHDA-lesioned rats [START_REF] Moriarty | Encapsulation of primary dopaminergic neurons in a GDNF-loaded collagen hydrogel increases their survival, re-innervation and function after intrastriatal transplantation[END_REF][START_REF] Moriarty | Encapsulation of young donor age dopaminergic grafts in a GDNF-loaded collagen hydrogel further increases their survival, reinnervation, and functional efficacy after R e f e r e n c e s | 202 intrastriatal transplantation in hemi-Parkinsonian rats[END_REF].

"Development of Biomaterial-based Delivery Systems for Parkinson's Disease-An Integrated Pan-European Approach", BrainMatTrain, was an Intensive Training Network program financed under the Marie-Sklowdoska-Curie Actions, which financed the current PhD work. The goal of the BrainMatTrain project 3,4 was to develop a multi-modal collagen-based hydrogel that incorporates both neuroprotective and anti-inflammatory moieties to help improve graft survival in a PD environment. BrainMatTrain, which has officially finished December 2019, was an interdisciplinary project to study the use of biomaterials from fundamental to translational preclinical research, and up to commercial development.

Within this network I have focused on the improvement and characterisation of rodent models and their in vivo biomarkers, with the ultimate aim to follow the therapeutic efficacy of the multimodal hydrogels in vivo. In order to achieve this, I have worked closely together with Francesco Gubinelli on the characterisation of the models and their biomarkers. Additionally, I have visited our partners at Lund University to gain more insight into the technical difficulties that come with cell grafting in rodent models.

Aim of the PhD Thesis

As described above, the brain pathology of Parkinson's disease is characterized by a progressive dopaminergic neurodegeneration, accumulation of α-synuclein aggregates, and the presence of neuroinflammation. To this day there is no treatment that can slow or stop PD progression; treatment only exists to alleviate the symptoms. Animal models are often used to learn more about disease progression and in search for novel treatments. As discussed above, there are many different animal models that can be employed to model specific traits of PD, ranging from neurotoxic and neuroinflammatory-causing substances to PD-related gene overexpression.

PD is a progressive disease with a long prodromal phase that emerges later in life. Especially the prodromal to early, mild stages of PD are ideal targets for treatments to slow or stop PD progression. Even though this represents an important phase within the physiopathology of PD, only few imaging studies exists on mild models of PD revealing the course of dopamine pathology.

The "Neurodegenerative Diseases Laboratory" of MIRCen evaluates neurodegeneration, protein aggregation, neuroinflammation, and genetic predisposition and their interaction using different models ranging from cell cultures to viral-vector induced rodent and primate models. At MIRCen we have access to several techniques to study our models, including in vivo MR and PET imaging, behavioural assessment, and post-mortem immunohistochemistry and RT-qPCR.

Behavioural studies are widely accessible in preclinical research and therefore, first choice to evaluate neuronal loss in vivo, even though, moderate neuronal loss does not induce a pronounced behavioural phenotype and requires sensitive behavioural tests that are consistently performed. PET imaging offers the advantage to image dopaminergic neuronal loss in vivo and has been put forward as the translational imaging technique in both preclinical and clinical studies. However, preclinical PET imaging in these mild rodent models is highly challenging; neuronal loss is only moderate, and thus the choice of image post-processing will be crucial to obtain optimal sensitivity of detection. Preclinical rodent studies offer the access to post-mortem analysis, which allows validation of in vivo results. The strength, and the major challenge within this project, was to combine in vivo behavioural analyses and PET imaging with post-mortem analysis, which were all performed on the same animals. With the combibation of these tools, we aimed for an extensive comprehension of in vivo imaging markers.

In this PhD thesis, I aimed to evaluate in vivo the effects of 1-α-synuclein overexpression, 2α-synuclein and LRRK2 co-expression, and 3-mild neuroinflammation, on the dopaminergic system and dopaminergic neuronal cell loss.

To this end, PET imaging and behavioural studies have been selected as the main in vivo tools.

In vivo data were validated by post-mortem techniques evaluating gene-(by RT-qPCR) and protein-expression (by immunohistochemistry).

Data are presented as mean ± standard error of the mean (SEM), unless stated otherwise. Data is presented as %-change or as relative difference (Δ%), further referred to as asymmetry or asymmetry ratio. While %-change infers a directionality of the difference as compared to the contralateral hemisphere, relative difference does not give a directionality, and can thus be used with varying contralateral data.

The number of rats is indicated by n. Statistical analysis was performed using Statistica 13 software (Statsoft; Tulsa, USA). Prior to analysis, the data were assessed for normality and homogeneity of variance. Difference between the injected and non-injected side were analysed using the student t-test. If multiple groups were compared an ANOVA test was used, followed by Scheffé post-hoc analysis. Spearman correlation analyses were performed using R software. Annotations used to indicate level of significance are as follows: *P < 0.05, **P < 0.01, ***P < 0.001.

Animals

All animal experimental procedures were performed in accordance with the French regulation (French Act Rural Code R 214/87-131; authorization n°B 92-032-02) and complied with Standards for Humane Care and Use of Laboratory Animals of the Office of Laboratory Animal Welfare (OLAW -n°A5826-01) and the European Union (EU Directive 2010-63/EEC). All experimental procedures performed in this PhD work received approval from the local ethical committee (CETEA n°44), and the French Ministry of Education and Research (approvals SYNCHNEURO: …, LRRKSYN: APAFIS#1372-2015080415269690v02, NEUROMODEL: APAFIS#389-20150327162135690v02). All efforts were made to minimize animal suffering and animal care was supervised by veterinarians and animal technicians skilled in rodent healthcare and housing.

All animals used for the presented studies are male Sprague Dawley rats (JANVIER; St Berthevin, France), which arrived with a mean weight of ~280 grams, and acclimatised one week before start of any study. Rats were randomly allocated to experimental groups.

Housing

Rats were housed in a temperature-controlled (22±1°C, humidity 50%) room maintained on a 12/12 hour light/dark cycle. Food and water were provided ad libitum. Animals were housed, if possible, in pairs in standard individual ventilated rat cages with either corn cob or wood chip bedding. Enrichment was provided in the form of tissue paper, and a wooden chewing block.

Anaesthesia & monitoring

All experimental procedures, except behavioural testing, have been performed under general anaesthesia. Overall, anaesthesia was induced with 4% isoflurane (Iso-Vet; Coumon d'Auvergne, France) in 100% O 2 . To prevent corneal desiccation, ophthalmic ointment was regularly applied on the eyes during all procedures.

For stereotactic injections, anaesthesia was maintained with an intrapereotoneal (i.p.) injection of a mixture of ketamine (75 mg/kg; Imalgène 1000; Merial, Lyon, France) and xylazine (5 mg/kg; Rompun™; Bayer Animal Health). As analgesia, lidocaine (Xylovet; Ceva Santé Animale, Libourne, France; 7 mg/kg) was administered subcutaneously under the scalp prior to the stereotactic surgery. After surgical intervention, anaesthesia was reversed by subcutaneous injection of atipamezole (0,5 mg/kg; Antisedan; Vetoquinol, France).

For in vivo PET imaging and MR imaging studies, maintenance of anaesthesia was assured with 1.5-2.5% isoflurane in O 2 .

During stereotactic injection and PET imaging, body temperature was monitored with a rectal probe and a feedback coupled heating blanket (Homeothermic Blanket System; Harvard Apparatus Ltd.), while using regulated water flow in the MRI bed during magnetic resonance (MR) imaging. Respiratory rate was monitored during MR imaging using PC SAM software (Small Animal Instruments, Stony Brook, USA), or using a home-made respiration monitoring system and an oxygen saturation/heartbeat monitoring system (PureSAT® 2500A VET; Nonin, USA or CapnoTrue® AMP; Bluepoint Medical, Germany) during PET imaging.

Stereotactic surgery

Anaesthetised rats were placed on the stereotactic frame and injected using 34-gauge blunttip canula linked to a 10 μl Hamilton syringe (Hamilton; Reno, USA) by a polyethylene catheter and a micro pump (Micro4 Controller; WPI Inc.). After injection the needle was left in place for an additional 5 minutes and then slowly withdrawn.

At the end of the surgical procedure, the site of incision was cleaned and the scalp was closed using Michel wound clips. After anaesthesia reversal, rats were rehydrated with a subcutaneous injection of warm saline (10 ml/kg) and left in a ventilated heating box (28°C) until they fully recovered from anaesthesia. Post-surgery analgesia was insured by the administration of paracetamol (1.6 mg/ml; Doliprane) in the drinking water for 48 hours.

Complete lesion model: 6-OHDA

The gold-standard rat model for Parkinson's disease (PD) is the 6-hydroxydopmine (6-OHDA) model. As stated above (Chapter 1: 2.1, page 24), 6-OHDA has been used since the sixties [START_REF] Ungerstedt | 6-hydroxy-dopamine induced degeneration of central monoamine neurons[END_REF], and it can cause rapid and complete neurodegeneration if injected into the medial forebrain bundle (MFB) [START_REF] Cadet | Free radicals and the pathobiology of brain dopamine systems[END_REF].

Here, we used this widely studied complete lesion model to train the experimenter and evaluate, validate, and where needed, improve our methods, for the use on milder PD models. To induce a severe unilateral dopaminergic lesion, 6-ODHA (Sigma-Aldrich, France) was injected in the MFB (Figure 7). 6-OHDA was injected into two separate locations at a rate of 0.5 μl/min with a final mass of 17.5 μg of 6-OHDA per rat; 2*2.5 μl 6-OHDA solution at a concentration of 3.5 μg/μl, diluted in phosphate buffered saline (PBS). 6-OHDA was unilateral injected into the right MFB at the following stereotactic coordinates [START_REF] Paxinos | The Rat Brain in Stereotaxic Coordinates[END_REF]: anteroposterior -3.8/-4.4 mm inter-aural, mediolateral -1.6/-1.4 mm from bregma, and ventral -7.8/-8.0 mm from the dura, with the tooth bar set at -3.9 mm.

The complete lesion model for validation and optimisation

For validation and optimisation of novel and existing techniques, six 6-OHDA rats have been used for behavioural testing, PET imaging, immunohistochemistry, and real-time quantitative PCR (RT-qPCR) (Figure 8). The apomorphine-induced rotation test was performed at 5 weeks postinjection (wpi), the cylinder test at 6wpi, and the PhenoTyper test at 7wpi. AADC-PET imaging was performed at 6 months post-injection. All animals were sacrificed at 17 months post-injection. Immunohistochemistry was performed using primary antibodies directed against dopamine transporter (DAT), ionized calcium-binding adapter molecule 1 (IBA1), tyrosine hydroxylase (TH), vesicular monoamine transporter 2 (VMAT2), and S129-phosphorylated α-synuclein (pS129-α-syn), for further details see section 6.2, page 71. For RT-qPCR primers were used against aromatic L-amino acid decarboxylase (AADC), DAT, IBA1, TH, and VMAT2, for further details see section 6.3, page 75.

Data is presented as %-change and as relative difference (Δ), further referred to as asymmetry or asymmetry ratio.

Mild dopaminergic lesion model: α-synuclein overexpression

Previously, our lab developed a viral vector-induced α-synuclein overexpressing rodent PD model; overexpressing either A53T mutant human α-synuclein protein (see Chapter 5,page 108,Cresto et al.,submitted) or WT human α-synuclein (unpublished data) in the substantia nigra (2.5x10 10 viral particles). In order to aggravate and/or accelerate this model, in the context of my PhD, we doubled the viral vector dose, as well as the number of injection sites per substantia nigra; thus increasing the viral genome count four-fold.

For AAV production, plasmids constructs were packaged into AAV2/6 capsids as previously described [START_REF] Berger | Repair of Rhodopsin mRNA by Spliceosome-Mediated RNA Trans-Splicing: A New Approach for Autosomal Dominant Retinitis Pigmentosa[END_REF]. AAVs coded for WT or A53T α-synuclein, under the control of the PGK promotor. Briefly, viral particles were produced by transient co-transfection of HEK-293T cells with an adenovirus helper plasmid (pXX6-80), an AAV packaging plasmid carrying the rep2 and cap8 genes, and the AAV2 transfer vector containing the above-mentioned expression (WT or A53T-αsynuclein) cassettes. Seventy-two hours after transfection, recombinant vectors were purified and concentrated from the cell lysate and supernatant by ultracentrifugation on an iodixaniol density gradient, followed by dialysis against PBSMK (0.5 mM MgCl2 and 1.25 mM KCl in PBS). Concentration of the vector stocks was estimated by quantitative PCR following the method described by Aurnhammer and colleagues (2012) and expressed as viral genomes per ml of concentrated stocks (vg/ml).

Recombinant AAVs coding for either wild-type (WT) or A53T mutated human αsynuclein were injected into the left substantia nigra of rats, at a rate of 0.25 μl/min, using a double needle injection approach (Figure 9). A total of 6 μl of viral vector solution was injected per rat, evenly divided over the two injection sites. Viruses were injected at a concentration of 5.0x10 10 viral particles per injection site, giving a final concentration of 1.0x10 11 viral particles per rat.

The following stereotactic coordinates [START_REF] Paxinos | The Rat Brain in Stereotaxic Coordinates[END_REF] were used: anteroposterior +2.4/+3.4 mm inter-aural, mediolateral +2.0 mm from bregma, and ventral -7.8 mm from the skull, with the tooth bar set at -3.3 mm. The coordinates were verified on age-matched animals prior to stereotactic injection with AAV2/6-PGK-α-synuclein, using blue dye injection and subsequent slicing of frozen brains.

In Chapter 5 (page 108), the synergistic effect of A53T-α-synuclein and leucine-rich repeat kinase 2 (LRRK2) overexpression on the survival of dopaminergic neurons in the substantia nigra is evaluated. For this, AAVs coding for A53T-α-synuclein was co-injected with AAVs coding for GFP, G2019S-ΔLRRK2 or DK-ΔLRRK2 (dead kinase) at the same stereotactic coordinates. Further details on the LRRK2 vectors can be found in the paper included in Chapter 5.

Neuroinflammation model: LPS

Lipopolysaccharide (LPS) from Escherichia coli can cause a systemic or local neuroimflammatory response, as previously described in Chapter 1: 2.3, page 25. Although LPS is an established model for neuroinflammation, it has only recently been developed and validated in our lab, in collaboration with my colleague Dr. Gubinelli (PhD 2016-2019), in the context of the BrainMatTrain project. LPS (L2880, Sigma-Aldrich, France) was diluted with PBS to reach a concentration of 5 μg/μl. Each animal was unilaterally injected in the left striatum with 2 μl of solution, hence 10μg of LPS per rat, at a rate of 0.5 μl/min (Figure 10). The following stereotactic coordinates were applied [START_REF] Paxinos | The Rat Brain in Stereotaxic Coordinates[END_REF]: anteroposterior +0.5 mm from bregma, mediolateral +3 mm from bregma, and ventral -4.3 mm from the dura, with the tooth bar set at -3.3 mm.

Behavioural analyses

Prior to all behavioural testing, the animals were habituated to experimenter's handling and interaction during a 5-day habituation period, during 2 minutes every day. All behavioural experimentation was recorded using a small camera and EthoVision XT software (Noldus Information Technology, Wageningen, the Netherlands).

Apomorphine-induced rotation test

Apomorphine is a non-selective dopamine agonist, which binds to both D 1 and D 2 -receptors [START_REF] Schwarting | THE UNILATERAL 6-HYDROXYDOPAMINE LESION MODEL IN BEHAVIORAL BRAIN RESEARCH[END_REF][START_REF] Millan | Differential Actions of Antiparkinson Agents at Multiple Classes of Monoaminergic Receptor. I. A Multivariate Analysis of the Binding Profiles of 14 Drugs at 21 Native and Cloned Human Receptor Subtypes[END_REF], resulting in rotational behaviour in unilateral dopaminergic lesion models. Literature suggests that a severe unilateral lesion (>80-90% dopaminergic nerve terminal loss [START_REF] Hudson | Correlation of apomorphine-and amphetamine-induced turning with nigrostriatal dopamine content in unilateral 6-hydroxydopamine lesioned rats[END_REF][START_REF] Schwarting | THE UNILATERAL 6-HYDROXYDOPAMINE LESION MODEL IN BEHAVIORAL BRAIN RESEARCH[END_REF]) or a large dopamine depletion (>95%; [START_REF] Chang | Biochemical and anatomical characterization of forepaw adjusting steps in rat models of Parkinson's disease: studies on medial forebrain bundle and striatal lesions[END_REF]) are required to induce rotational behaviour after apomorphine administration. Below this threshold rotational behaviour will not occur.

For this test we used the Rotameter set-up (Panlab-Harvard Apparatus, Spain), which can hold up to 4 rats at the same time, and was placed in a operator-separate room with lighting at 90 lux. The individual bowls were separated from each other by opaque plastic walls (±30 cm), while an infrared camera was mounted above the bowls to observe the rats from the operator-separate control room. Apomorphine (10 mg/ml; Sigma-Aldrich, France) was diluted 1:10 in saline to create a stock solution of 1 mg/ml. Each rat received an i.p. apomorphine injection while awake, with a dose of 0.75 mg/kg. Rats were immediately placed in the Rotameter harness, and counting started as soon as all rats were placed in the Rotameter apparatus. Clockwise and counter-clockwise rotations were recorded for 60 minutes, after which rats were placed back in their home cages and surveyed for another 30 minutes. Clockwise rotations were subtracted from counter-clockwise rotations to arrive at the net counter-clockwise rotations.

Preliminary evaluation in a complete lesion model

Whilst the amphetamine-induced rotation test has previously been employed in the lab with rat PD models, apomorphine has not been used before. The general setup of the apparatus and analysis method is the same; however, the time-window of assessment of the rotations has a crucial impact on the evaluation of the results, as demonstrated below.

Comparison of clockwise (CW) and counter-clockwise (CCW) rotations over a 60 minute period demonstrated significant more counter-clockwise rotations (+2489±515%; Δ179±7%; n=6; p=0.0007). We observed that the average counter-clockwise rotations per 3-minute interval dropped drastically after 39 minutes (Figure 11A). The elimination half-life of apomorphine is 28 minutes [START_REF] Symes | Time-course of apomorphine in the brain of the immature rat after apomorphine injection[END_REF], which corresponds with this observation. Therefore, it has been decided to assess only the first 30 minutes of the apomorphine-induced rotation test.

Comparison of clockwise and counter-clockwise rotations over the first 30 minute period resulted in an even larger difference of +8746±2874%, or a relative difference of Δ194±2% (n=6; p=0.0003). Even though the total and net number of rotations decreased (Figure 11B), the RPM values increased (Figure 11C). The test indicates that our 6-OHDA animals have a severe lesion, which was later on confirmed by histological assessment (section 6.2.6, page 73). The cylinder test evaluates forelimb usage by counting the number of touches by the forepaws -either left, right, or simultaneous -with the wall of a clear glass cylinder (5L, Ø20 cm). The animals were placed in the glass cylinder in an operator-separate room (lights at 90 lux), with a video camera placed below the cylinder. The animals were recorded during 5 minutes. Video recordings were analysed by counting the number of contacts with the wall of the clear glass cylinder, for each forepaw, alone or simultaneous. The number of impaired forelimb contacts -contralateral to the injection -was expressed as a percentage of total forelimb contacts, as described in the paper by Gombash and colleagues [START_REF] Gombash | Morphological and behavioral impact of AAV2/5-mediated overexpression of human wildtype alpha-synuclein in the rat nigrostriatal system[END_REF]; Control rats should score around 50% in this test. For statistical analyses a student t-test with unequal variance was performed against this hypothetical 50% score. Literature has shown that forelimb use is significantly correlated with striatal TH levels, as seen by optical density measurements [START_REF] Gombash | Morphological and behavioral impact of AAV2/5-mediated overexpression of human wildtype alpha-synuclein in the rat nigrostriatal system[END_REF].

Preliminary evaluation in a complete lesion model

The cylinder is frequently used in our lab to study unilateral PD models (unpublished data).

We confirmed that in healthy control groups, rats have an average Gombash score that is not significantly different from the hypothetic 50% score (Figure 12, 49%; WT: n=15; p=0.50). In the complete 6-ODHA model, the cylinder test showed significantly reduced use of -54±9% of the contralateral forepaw at 6wpi (Figure 12; n=6; p=0.0019), or a left-right asymmetry ratio of (Δ116±18%). These data correspond to what has previously been published on the cylinder test in a complete 6-ODHA lesion model [START_REF] Decressac | Comparison of the behavioural and histological characteristics of the 6-OHDA and α-synuclein rat models of Parkinson's disease[END_REF].

Spontaneous locomotor activity: PhenoTyper

Spontaneous locomotor activity was recorded in PhenoTyper cages (Noldus Information Technology, Wageningen, the Netherlands). The PhenoTyper is an instrumented home cage in which rodent behaviour is automatically monitored through a video-based observation system. The cages are 45 cm x 45 cm x 45 cm, and are made of transparent Perspex walls with an opaque black Prespex Cages are equipped with a water bottle and feeding station, with ad libitum food and water provided. Each cage also contains a top unit with built-in hardware for video tracking (digital infrared-sensitive video camera and infrared lights). Six PhenoTyper cages were connected via two sequential Quad units to a single computer. Rats were introduced into the PhenoTyper cages at 10h00, during the light phase, and monitored for 23 consecutive hours. Ethovision XT (Noldus Information Technology, Wageningen, the Netherlands) was used for data acquisition and for analysis. The following parameters were assessed: total distance moved, mean velocity, and time spend moving.

Preliminary evaluation in a complete lesion model

In MIRCen, the PhenoTyper cages have been used before in Alzheimer disease models (unpublished data), but they have never been used in a PD model.

Here, we compared spontaneous movement over 23 hours in 6-OHDA rats (n=6) with healthy age-matched controls (n=3), using PhenoTyper cages (Noldus Information Technology, Wageningen, the Netherlands). We did not observe any significant differences in total distance moved (Figure 13A; n=6/2; p=0.342), mean velocity (Figure 13B; n=6/2; p=0.343), or time spend moving (Figure 13C; n=6/2; p=0.788).

Sgroi and colleagues ( 2014) claim that locomotor activity in 6-OHDA animals (10μg in 4 μl ascorbic acid 0.1%) stabilised 2 weeks post-injection. But, they do not report if locomotor activity differed before and after surgery. To the best of our knowledge, significant differences in locomotor behaviour using PhenoTyper cages have only been reported after levodopa treatment [START_REF] Nikolaus | Relationship between L-DOPA-induced reduction in motor and exploratory activity and degree of DAT binding in the rat[END_REF][START_REF] Sgroi | Spontaneous locomotor activity and L-DOPAinduced dyskinesia are not linked in 6-OHDA parkinsonian rats[END_REF]. This makes spontaneous locomotor activity tracking using the PhenoTyper not an ideal analysis technique for our models. We have therefor not used it in subsequent studies.

Forelimb akinesia: Adjusting step test

The adjusting step test allows assessing asymmetry in motor abilities. The adjusting step test was performed after the example of Olsson and colleagues (1995). Briefly, rats are held by the torso fixing one forelimb, while the other forepaw touches the table. Rats are then moved across the table C h by an experienced experimenter at a speed of around 5 sec/m, sequentially in the forehand and backhand direction. This is repeated until at least two good runs are recorded for both forelimbs, with a maximum of 5 minutes per rat. Video recordings are analysed at a lower speed to count the number of steps for each trial. An average of four trials per forepaw was used for statistical analysis:

The adjusting step test has previously succesfully been used in our lab on PD models (unpublished data). Olsson and colleagues (1995) showed that, in a 6-OHDA rat model, the contralateral forepaw is severely affected in the adjusting step test, in both the backhand and forehand direction, which resulted in a dragging paw when the rats was moved sideways by the experimenter [START_REF] Olsson | Forelimb akinesia in the rat Parkinson model: differential effects of dopamine agonists and nigral transplants as assessed by a new stepping test[END_REF]. Even though one study suggested that large depletion of striatal dopamine is necessary before rats can no longer make the adjusting steps [START_REF] Chang | Biochemical and anatomical characterization of forepaw adjusting steps in rat models of Parkinson's disease: studies on medial forebrain bundle and striatal lesions[END_REF], more recent literature suggests that the adjusting step test is capable to detect partial lesion, and might be more sensitive than the cylinder test. Rats injected with preformed fibrils, inducing 48% loss of THpositive neurons in the ipsilateral substantia nigra, showed reduced use of the contralateral forepaw in the adjusting step test but no in the cylinder test [START_REF] Patterson | Time course and magnitude of alpha-synuclein inclusion formation and nigrostriatal degeneration in the rat model of synucleinopathy triggered by intrastriatal α-synuclein preformed fibrils[END_REF].

We have used the adjusting step test in the WT and A53T-α-synuclein model, results of which are presented and discussed in Chapter 3 (page 86).

Sensorimotor integration: Vibrissae-evoked forelimb-placing test

The vibrissae-evoked forelimb-placing test allows assessing sensorimotor integration. The test was performed after the example of Schallert and colleagues (2000). Rats were held in the similar grip as for the adjusting steps test, while vibrissae were stimulated by brushing the edge of a table by a forward movement. In a control animal, this quickly elicits an ipsilateral forepaw touch on the table; animals with a unilateral lesion will show impaired limb placing ability, but no deficit on the unimpaired side. One session contains three successful trials on every side, or a maximum test time of three minutes. The outcome score is calculated as a percentage of touches or misses on total attempts:

The vibrissae-evoked forelimb-placing test has, to the best of my knowledge, never been performed in our lab before. Schallert and colleagues (2000) have demonstrated that 6-OHDA lesioned rats, with 88% reduction of dopamine concentration in the striatum, show a severe significant deficit using the vibrissae-evoked forelimb-placing test [START_REF] Schallert | CNS plasticity and assessment of forelimb sensorimotor outcome in unilateral rat models of stroke, cortical ablation, parkinsonism and spinal cord injury[END_REF]. In a Huntington disease model, Lelos and colleagues (2016) observed a significant motor deficit using the vibrissae-evoked forelimbplacing test, but not using the cylinder test, suggesting higher sensitivity of the vibrissae-evoked forelimb-placing test compared to the cylinder test [START_REF] Lelos | Direct Comparison of Rat-and Human-Derived Ganglionic Eminence Tissue Grafts on Motor Function[END_REF].

We have used the adjusting step test in the WT and A53T-α-synuclein model, results of which are presented and discussed in Chapter 3 (page 86). To the best of our knowledge, only a few studies have reported on the use of the PhenoTyper in PD models, showing only relevant results after levodopa treatment. In our hands, the PhenoTyper did not demonstrate any significant deficits in spontaneous locomotor activity in the complete 6-OHDA lesion model, and thus we do not expect to detect any spontaneous deficits in milder lesion models. Therefore, we have not further evaluated this test in any of the other models.

Conclusion and choice of behavioural tests

Even though the apomorphine-induced rotation test showed very promising results in the 6-OHDA model, it is known from literature that a large lesion or severe dopamine depletion are required for this test to show significant turning behaviour. Since we do not expect such an extensive lesion in our α-synuclein overexpression models, we have opted not to use it for the models evaluated in this PhD study.

The cylinder test is easy to put in place, robust and little time-consuming. For these reasons, this was the behavioural test of choice. According to literature, the adjusting step test and vibrissaeevoked forelimb-placing test have good potential to identify mild lesions and motor deficits, with a higher sensitivity as compared to the cylinder test. Therefore, we have included these tests in the evaluation of α-synuclein overexpression models (Chapters 3, 4, and 5).

Magnetic resonance Imaging

MR acquisition

For each study, anatomical 3-dimensional (3D) T 2 -weighted MR images were acquired on a separate group of age-matched animals using a horizontal 11.7 T scanner (Bruker, Ettlingen, Germany) and ParaVision 6 software (Bruker, Germany). Animals were fixed in prone position in a MRI-compatible stereotactic rat frame to prevent head motion. 3D T2-weighted images were acquired using a turbo-spin echo sequence with the following parameters: TE, 45 ms; 17 echo averages; TR, 10000 ms; in-plane resolution 200x200 μm; 100 slices with 300 μm thickness; resulting in an acquisition time of 20 min.

Image processing

Image processing was performed using PMOD® software (PMOD 3.804; PMOD Technologies LLC, Switzerland). All processes were performed by the same operator for all animals.

Creation of MRI template

MRI templates of age-matched animals (Figure 14) were created to the example of Vállez Garcia and colleagues (2015). This was performed for the studies of Chapters 3 and 4(Early timepoint: n=4; bw=580±11g, Late: n=8; body weight (bw)=484±8g), Chapter 5(n=4; bw≈567g), and Chapter 6 (n=3; bw=450±20g).

Briefly, a brain mask was applied to individual T2-weighted MR images. These masked MR images were then automatically co-registered to a representative image using rigid transformations using the FuseIt module of PMOD® software. Next, the co-registered images were averaged into a single image. This averaged image was subsequently mirrored in axial orientation. This mirrored image was then automatically co-registered to the initial averaged image using the FuseIt module of Subsequently masked images were co-registered to a representative image using rigid transformations and averaged. This average MRI was mirrored, and co-registered to the original average MR image. Lastly, the mirrored and original averaged MR image were averaged again, created our final MRI template.

Co-registration of Atlas to MRI template

To define anatomical areas of interest, an existing MRI Atlas was adapted to the home-made MRI templates that were created for each individual study (Figure 15). To this end, the home-made MRI template was automatically co-registered to the T2-weighted MRI template of the Schiffer/PMOD Rat Brain Atlas, using affine transformations in the FuseIt module of PMOD® software [START_REF] Schiffer | Serial microPET measures of the metabolic reaction to a microdialysis probe implant[END_REF]. The inverse transformation was subsequently used to place the Schiffer/PMOD Rat Brain Atlas [START_REF] Schiffer | Serial microPET measures of the metabolic reaction to a microdialysis probe implant[END_REF] on our MRI template.

Creation of BrainMask

To mask outer-brain signal on PET images, a BrainMask was created based on the Schiffer/PMOD Rat Brain Atlas [START_REF] Schiffer | Serial microPET measures of the metabolic reaction to a microdialysis probe implant[END_REF]. For this, the individual regions of interest (ROIs) were fused into one large ROI covering the whole brain, which was subsequently dilated by 2 pixels. This BrainMask has corresponding localisation to the home-made MRI templates. ]FMT) is a PET tracer that is metabolised by aromatic L-amino acid decarboxylase (AADC), which is involved in the production of dopamine (Chapter 1: 4.4.2, page 40). [ 18 F]FMT was synthesized as described by Lemaire and colleagues (2015). Briefly, the radiosynthesis of [ 18 F]FMT was realised using a multi-step approach after 18 F substitution of a small deactivated aromatic ring. Its conversion into an 18 F-fluorobenzyl halide compound was followed by the enantioselective formation of a new chiral carbon-carbon bond by phase transfer catalysis. Subsequent hydrolysis and HPLC purification afforded [ 18 F]FMT with a radiochemical yield of 30 ± 5% (decay corrected; mean±95% confidence interval). At the end of synthesis the enantiomeric purity was ≥ 96%, the specific activity was more than 555 GBq/µmol and the radioactivity concentration more than 1.1 GBq/mL.

[ 18 F]FMT injected volume was diluted in physiological water, and the average injected volume was 1 ml (range 0.5-1.7 ml). The injected doses (ID), body weight, and benserazide dose per study are shown in Table 3. ]LBT999) binds reversibly to dopamine transporter (DAT), responsible for presynaptical dopamine reuptake (Chapter 1: 4.4.3, page 41). [ 18 F]LBT999 was synthesized as described by Dollé and colleagues (2007). Briefly, LBT999 was labelled with 18F at its 4-fluo-2-butenyl element via direct substitution of the chloro-analog. The mixture was purified by HPLC using ammonium acetate and the desired fraction was collected and diluted in water. [ 18 F]LBT999 was trapped using a light cartridge, eluted with ethanol, and subsequently diluted with 0.9% NaCl. The radiochemical yields of 18 F were about 30-40%, and a radiochemical purity of >90%. Before usage, the tracer was diluted in 0.9% NaCl to have a maximum ethanol content of 15%.

[ 18 F]LBT999 was diluted in physiological water and the average injected volume was 0.8 ml (range 0.5-1.1 ml). Since [ 18 F]LBT999 is eluted with ethanol, dilutions needed to be made to not 4. 18 F]DPA714) binds to 18kD translocator protein (TSPO), located on the outer membrane of mitochondria, and upregulated during inflammatory events (Chapter 1: 4.4.7, page 43). [ 18 F]DPA714 was synthesised according to procedures previously reported [START_REF] Damont | Radiosynthesis of [18F]DPA-714, a selective radioligand for imaging the translocator protein (18 kDa) with PET[END_REF][START_REF] James | DPA-714, a new translocator protein-specific ligand: Synthesis, radiofluorination, and pharmacologic characterization[END_REF]. Briefly, DPA714 was labelled with 18 F at its 2-fluoroethyl moiety using atosyloxy-for-fluorine nucleophilic aliphatic substitution, using a commercially available GE TRACERLab FX-FN synthesizer (GE Medicam Systems; Buc, France) [START_REF] Kuhnast | PBR111 and [ 18F]FEDAA1106-Selective radioligands for imaging TSPO 18kDa with PET: Automated radiosynthesis on a TRACERLAb FX-FN synthesizer and quality controls[END_REF]. [ 18 F]DPA714 was formulated in physiological saline containing less than 10% of ethanol at a concentration of 61±13 MBq/mL (2.46±1.65 nmol/mL; mean±SD). Radiochemical purity of [ 18 F]DPA714 was greater than 99% and specific radioactivity ranging from 100 to 165 GBq/μmol (n=3; mean±SD).

[ 18 F]DPA714[N,N-diethyl-2-(2-(4-(2-[18F]fluoroethoxy)phenyl)-5,7-dimethylpyrazolo[1, 5- a]pyrimidin-3-yl)acetamide] ([
[ 18 F]DPA714 injected volume was varied to compensate for changes in activity and diluted in physiological water; average injected volumes was 1.2 ml (range 1.0-1.5 ml). Since [ 18 F]DPA714 is eluted with ethanol, dilutions needed to be made to not exceed 15% of ethanol. The injected doses (ID), body weight, RAS, and ethanol dose per chapter are shown in Table 5.

Image acquisition and reconstruction

PET image aqcuisition was performed on a MicroPET® Focus 220 system (resolution: 1.5 × 1.5 × 1 mm; Siemens, France). Animals were placed within the scanner using a home-made stereotacticlike frame compatible with PET acquisition in prone position. Radioligands were injected into the tail vein, using an injection pump (Pump11 Elite; Harvard Apparatus Ltd.), at a rate of 1 mL/min via a 26gauge catheter.

A dynamic emission scan was started simultaneously with the intravenous bolus injection of [ 18 F]FMT, [ 18 F]LBT999, or [ 18 F]DPA714, and lasted 60 min, 90 min, or 90 min, respectively. The data were acquired in list-mode. The time coincidence window was set to 6 ns, and the levels of energy discrimination were set to 350-650 keV.

For image reconstruction, PET data was normalised for detector inhomogeneities, corrected for scatter, attenuation, and radioactivity decay, and reconstructed using Fourier rebinning and ordered subset expectation maximization 2D (16 subsets and 4 iterations). The following 24 timeframes were used for 90 min scans: 4 x 15 sec; 4 x 30 sec; 2 x 60 sec; 5 x 2 min; 3 x 5 min; 6 x 10 min, while the last 3 timeframes were eliminated for 60 min scans.

Arterial input function and metabolite analysis

Metabolite corrected arterial input functions were measured in a separate group of animals (n=3), only for the [ 18 F]DPA714 study. Blood samples (≈50 μL) were collected at selected timepoints (see Table 6) from the femoral artery to establish [ 18 F]DPA714 arterial kinetics of the whole blood, and plasma. First, whole blood samples were counted using a gamma counter (Wizard 2480; PerkinElmer, France), and next radioactivity was measured in cell-free plasma (≈20 μL) after centrifugation (5 min, 2054 g, 4°C), Larger samples (500 μL) were collected at 5, 10, 20, 40, 60 and 90 min after radiotracer injection for further metabolite analysis [START_REF] Peyronneau | Metabolism and Quantification of [ 18 F]DPA-714, a New TSPO Positron Emission Tomography Radioligand[END_REF]. Briefly, the cartridge (60-mg HLB Oasis SPE cartridge; Waters) was conditioned with 1 mL of acetonitrile and equilibrated with 5 mL of purified water. Plasma samples (200 μL) were diluted to 400 μL with an aqueous solution containing 4% hydrochloric acid. The radioactivity was measured in the resulting solution, which was directly applied to the HLB cartridge. The cartridge was then sequentially washed with 1 mL of water and acetonitrile /water (35/65, v/v) solution. [ 18 F]DPA714 was finally eluted with 1 mL of acetonitrile. The radioactivity was measured in all the collected fractions as well as in the cartridge. The radioactivity due to unchanged [ 18 F]DPA714 was expressed as the percentage of the sum of radioactivity found in the eluted fractions or as the fraction of the initial radioactivity measured in each plasma sample [START_REF] Peyronneau | Metabolism and Quantification of [ 18 F]DPA-714, a New TSPO Positron Emission Tomography Radioligand[END_REF].

For each individual animal, the parental fraction was fitted using a 1-exponential decay function, and then applied to plasma input function. Time activity curves (TACs) of the parental [ 18 F]DPA714 plasma fraction were expressed in standard uptake values (SUV). Parent [ 18 F]DPA714 plasma exposure was estimated in all tested conditions by calculating the area under the curve (AUC) from 0 to 90 min. AUC expressed in SUV x min is inversely correlated to [ 18 F]DPA714 plasma clearance (Cl = dose/AUC).

Image processing

Image processing was performed using PMOD® software (PMOD 3.804; PMOD Technologies LLC, Switzerland), by the same operator for all animals.

Image pre-processing and segmentation

Dynamic PET images were summed ([ 18 F]FMT: 60 min, [ 18 F]LBT999: 5 min, and [ 18 F]DPA714: 90 min), and subsequently smoothed with a Gaussian filter (1x1x1mm) and cropped (20*15*30mm). The resulting summed-images were manually co-registered onto the MRI template (paragraph 4.2.1, page 57) using only rigid transformations (PET2MRI) (Figure 16).

Next, the inverse affine transformations (revPET2MRI) where used to place the MRI Atlas (4.2.2, page 58) and MRI BrainMask (4.2.3, page 58) on the raw dynamic PET image, hence without altering the PET images. The voxels outside of the BrainMask were masked to reduce outer-brain signal. Segmentation based on the MRI Atlas was used for all subsequent analyses.

These image pre-processing steps were executed using alternatively PVIEW and PFUSIT modules of the PMOD® software.

Relative quantification

Relative quantification was performed using the VIEW module of PMOD® software. Time activity curves (TACs), representing the radioligand uptake in VOIs over time, were extracted from dynamic PET images and expressed in standard uptake values (SUV, Figure 17):

In a subsequent step, average SUV was calculated within a specified timeframe corresponding to pseudo-equilibrium uptake of the radioligand; and relative SUV values can be calculated as the ratio of SUV in a VOI relative to the SUV uptake within the (pseudo-) reference region. The reference region being defined as the region that does not express the target of interest, and which displays similar radioligand uptake and distribution as the VOI.

This relative quantification method has been successfully employed previously in our lab in rodent studies for both [ 18 F]LBT999 [START_REF] Grealish | Human ESC-derived dopamine neurons show similar preclinical efficacy and potency to fetal neurons when grafted in a rat model of Parkinson's disease[END_REF] and [ 18 F]DPA714 [START_REF] Lavisse | Reactive Astrocytes Overexpress TSPO and Are Detected by TSPO Positron Emission Tomography Imaging[END_REF]. However, during this PhD, we have used absolute quantification methods with the aim to be more sensitive and precise in our quantification, which was not previously done for any of these tracers in rodents in our lab.

Absolute quantification

TACs were used for subsequent absolute quantification through kinetic modelling. We chose to calculate quantitative parametric images using the PXMOD module of PMOD® software, in which the pixel intensity corresponds to the quantitative measure. Though less sensitive than quantification based on individual regional TACs, this allows to; evaluate eventual regional changes and offers the possibility of voxel-based analysis (Chapter 5).

Overall, we have chosen simplified quantification methods based on (pseudo-) reference regions; for [ 18 F]FMT and [ 18 F]LBT999 simplified quantification methods have been extensively evaluated [START_REF] Eberling | PET 6-[F]fluoro-L-m-tyrosine Studies of Dopaminergic Function in Human and Nonhuman Primates[END_REF][START_REF] Sérrière | In vivo PET quantification of the dopamine transporter in rat brain with [18F]LBT-999[END_REF]. As reported in literature, the cerebellum was used as the reference region for [ 18 F]FMT and [ 18 F]LBT999 quantification.

Quantification of TSPO is more complex; firstly, since there is no reference region available that does not express TSPO, and secondly, TSPO-PET ligands have complex blood pharmacokinetics. Previous studies by Ory and colleagues [START_REF] Ory | Quantification of TSPO overexpression in a rat model of local neuroinflammation induced by intracerebral injection of LPS by the use of [18F]DPA-714 PET[END_REF] demonstrated the contralateral striatum is a better pseudo-reference region than the cerebellum. However, there is no clear consensus whether simplified quantification methods are equally or more sensitive than full quantification using metabolite-corrected plasma input curves. Therefore, we conducted a pilot study for [ 18 We aimed to evaluated the feasibility of simultaneous PET imaging and whole blood measurements. The findings for this pilot study will be described in section 5.5.2 (page 68).

Patlak plot -[ 18 F]FMT

The Patlak plot is a graphical analysis method suitable for systems with irreversible trapping of the radiotracer [START_REF] Patlak | Graphical evaluation of blood-to-brain transfer constants from multiple-time uptake data[END_REF][START_REF] Peters | Graphical analysis of dynamic data: the Patlak-Rutland plot[END_REF]. The Patlak plot can be used if the plasma activity is known, resulting in a plot of the transformed TAC against a "normalised time" (PMOD, 2009d): However, we did not have information on the plasma activity and therefore had to use the reference region method, which was the cerebellum. A prerequisite for the reference region is that the tracer is not irreversibly being trapped [START_REF] Patlak | Graphical Evaluation of Blood-to-Brain Transfer Constants from Multiple-Time Uptake Data. Generalizations[END_REF]. In the Patlak reference plot, the plasma data is then replaced by the TAC of the reference tissue. The slope of the linear regression now has the following relation with the equilibrium constant K eq (PMOD, 2009d):

The Patlak graphical reference plot has also been successfully applied on rat [ 18 F]FMT data by Becker and colleagues [START_REF] Becker | Comparative assessment of 6-[18F]fluoro-L-m-tyrosine and 6-[18F]fluoro-Ldopa to evaluate dopaminergic presynaptic integrity in a Parkinson's disease rat model[END_REF].

Logan plot -[ 18 F]LBT999

The Logan plot is a second graphical analysis method, but unlike the Patlak plot, it is specific for reversible receptor systems [START_REF] Logan | Graphical Analysis of Reversible Radioligand Binding from Time-Activity Measurements Applied to [ N -11 C-Methyl]-(-)-Cocaine PET Studies in Human Subjects[END_REF]. The Logan plot can be used if the plasma activity is known (PMOD, 2009c):

However, for the final study, we did not collect plasma activity information, and had to use the Logan reference tissue method [START_REF] Logan | Distribution volume ratios without blood sampling from graphical analysis of PET data[END_REF]. This is possible since the distribution volume ratio (DVR) can directly be calculated from the graphical method using data from a reference region (C'(t)) and an average tissue-to-plasma clearance (k' 2 ):

In this case DVR is the regression slope and the intercept int' which is constant after equilibration time t* (PMOD, 2009b).

PXMOD was first allowed to find the best fit, with liberty in the equilibration time t*. Next, the average equilibration time t* per group was calculated and forced for absolute quantification of each single pixel.

Previously, Sérrière and colleagues (2014) have compared several methods of analysis for [ 18 F]LBT999 PET imaging, and successfully used the Logan reference tissue model, amongst others, in healthy rats [START_REF] Sérrière | In vivo PET quantification of the dopamine transporter in rat brain with [18F]LBT-999[END_REF].

MRTM0 -[ 18 F]DPA714

Previously, Ory and colleagues (2015) have used the simplified reference tissue model (SRTM) [START_REF] Lammertsma | Simplified reference tissue model for PET receptor studies[END_REF] to quantify [ 18 F]DPA714 PET data [START_REF] Ory | PET imaging of TSPO in a rat model of local neuroinflammation induced by intracerebral injection of lipopolysaccharide[END_REF]. In a subsequent paper, Ory and colleagues (2016) suggest that full kinetic modelling is possible using SRTM or multi-linear reference tissue model (MRTM) with the contralateral striatum as the reference region, and a scan duration of at least 90 minutes [START_REF] Ory | Quantification of TSPO overexpression in a rat model of local neuroinflammation induced by intracerebral injection of LPS by the use of [18F]DPA-714 PET[END_REF].

However, in our experiments we did not meet this last constraint; due to time limitations our scan lasted 60 minutes. In order to select the most suitable model, I have therefore reanalysed three existing 90 minutes [ 18 F]DPA714 PET data sets [START_REF] Lavisse | Reactive Astrocytes Overexpress TSPO and Are Detected by TSPO Positron Emission Tomography Imaging[END_REF] using the SRTM and MRTM model as suggested by Ory and colleagues (2016), and compared the results to quantification of the same datasets, but only using the first 60 minutes of data acquisition. This comparison revealed that Ichise's non-invasive plot (MRTM 0 [START_REF] Ichise | Noninvasive quantification of dopamine D2 receptors with iodine-123-IBF SPECT[END_REF]) at 60 minutes showed BPnd values closest to SRTM and MRTM at 90 minutes; data correlated well, even though there was a slight underestimation.

MRTM 0 is based on the Logan plot and has been developed for receptor studies. The model requires a region without receptors and a receptor-rich region and assumes that the nondisplaceable distribution volumes are equal in those regions [START_REF] Ichise | Noninvasive quantification of dopamine D2 receptors with iodine-123-IBF SPECT[END_REF]PMOD, 2009a), which we have found in the contralateral and ipsilateral striatum, respectively.

If the Logan plot is applied for both TACs of the receptor-lacking and receptor-rich region, the input curve can be eliminated and the following expression is found:

V and V' are the total distribution volumes of C tissue (t) and C' tissue (t), k' 2 is the clearance constant, and int' is the intercept which is constant after equilibration time t* [START_REF] Ichise | Noninvasive quantification of dopamine D2 receptors with iodine-123-IBF SPECT[END_REF]PMOD, 2009a).

PXMOD was first allowed to find the best fit, with liberty in the equilibration time t*. Next, the average equilibration time t* per group was calculated and forced for absolute quantification of each single pixel.

Absolute quantification based on automated arterial input measurements simultaneous to [ 18 F]DPA714 PET imaging are described in detail on page 68, where a pilot study is described.

Image post-processing and segmentation

The resulting [ 18 F]FMT parametric images were smoothed (2x2x2mm) before further processing, but not the [ 18 F]LBT999 or [ 18 F]DPA714 images. The "revPET2MRI" transformation was applied to put the atlas on parametric images in order to extract the average Ki or BPnd values per VOI from the MRI atlas.

To illustrate the results per study, averaged parametric images have been created per group. To that end, the individual parametric images were moved to MRI template space, using the PET2MRI transformation described above. Next, the parametric PET images were averaged and subsequently fused to the MRI template. In doing so, the orignial PET data have not been modified or transformed before absolute quantification, but only for the rendering of an average quantified PET image.

Methodological improvements

[ 18 F]FMT

As explained previously, (Chapter 1: 1.1.2, page 15) L-DOPA is converted into dopamine by the AADC enzyme [START_REF] Musacchio | Enzymes Involved in the Biosynthesis and Degradation of Catecholamines[END_REF], and ultimately degraded by COMT into a biological inactive molecule [START_REF] Eisenhofer | Catecholamine metabolism: a contemporary view with implications for physiology and medicine[END_REF]). [ 18 F]FDOPA (Chapter 1: 4.4.2, page 40) is closely related to L-DOPA and undergoes the same metabolic steps [START_REF] Melega | L-6-[18F]fluoro-dopa metabolism in monkeys and humans: biochemical R e f e r e n c e s | 201 parameters for the formulation of tracer kinetic models with positron emission tomography[END_REF][START_REF] Endres | Affinities of dopamine analogs for monoamine granular and plasma membrane transporters: implications for PET dopamine studies[END_REF]; [ 18 F]FDOPA is completely metabolized by peripheral AADC and COMT, resulting in radioactive metabolites that can cross the BBB. Peripheral metabolism has to be blocked in order to achieve brain uptake of [ 18 F]FDOPA [START_REF] Ruottinen | Striatal [18F]fluorodopa utilization after COMT inhibition with entacapone studied with PET in advanced Parkinson's disease[END_REF][START_REF] Kyono | Use of [18F]FDOPA-PET for in vivo evaluation of dopaminergic dysfunction in unilaterally 6-OHDA-lesioned rats[END_REF][START_REF] Walker | Measuring dopaminergic function in the 6-OHDA-lesioned rat: a comparison of PET and microdialysis[END_REF][START_REF] Walker | In-vivo measurement of LDOPA uptake, dopamine reserve and turnover in the rat brain using [18F]FDOPA PET[END_REF][START_REF] Becker | Comparative assessment of 6-[18F]fluoro-L-m-tyrosine and 6-[18F]fluoro-Ldopa to evaluate dopaminergic presynaptic integrity in a Parkinson's disease rat model[END_REF]. However, this does not eliminate the formation of [ 18 F]FDOPA metabolites in the brain.

In contrast to [ 18 F]FDOPA, [ 18 F]FMT (Chapter 1: 4.4.2, page 40) has the advantage of being a substrate for AADC, but with only minimal affinity for COMT [START_REF] Jordan | 6-[18F]fluoro-L-m-tyrosine: metabolism, positron emission tomography kinetics, and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine lesions in primates[END_REF]. Therefore, it is considered that [ 18 F]FMT does not require the use of COMT inhibitors prior to PET imaging [START_REF] Barrio | Radiofluorinated L-m-tyrosines: New in-vivo probes for central dopamine biochemistry[END_REF][START_REF] Dejesus | Evaluation of fluorinated m-tyrosine analogs as PET imaging agents of dopamine nerve terminals: comparison with 6-fluoroDOPA[END_REF]. However, we found that blocking with benserazide, a peripheral AADC inhibitor, was not successful in almost 30% of the animals (6 out of 20) in [ 18 F]FMT PET scans, leaving us unable to perform absolute quantification in these animals. This led us to investigate the several parameters that could influence the efficacy of benserazide; anaesthesia and injection route.

Pilot study on intravenous injection of benserazide in a complete lesion model

Our first aim was to evaluate if blocking of peripheral AADC could be more efficient after intravenous administration. However, to the best of our knowledge, little is known about the toxicity of benserazide after intravenous injection; therefore, in a pilot study (n=4) we injected only 5mg/kg of benserazide intravenously 30 minutes before radiotracer injection. In the bulk of our experiments, we injected animals ~30 minutes before radiotracer injection intraperitoneally with 10mg/kg of benserazide, after the example of Becker and colleagues [START_REF] Becker | Comparative assessment of 6-[18F]fluoro-L-m-tyrosine and 6-[18F]fluoro-Ldopa to evaluate dopaminergic presynaptic integrity in a Parkinson's disease rat model[END_REF].

Quantification of [ 18 F]FMT PET imaging using the cerebellum as the reference region (section 5.4.3, page 63) showed that Ki values were not different from studies using intraperitoneal injection of benserazide (Chapter 3, n=4/5; p=0.10). Average Ki value of the striatum contralateral to 6-OHDA injection after intraperitoneal benserazide injection was 0.012±0.001, while intravenously it was 0.010±0.002 (contralateral to AAV2/6-PGK-WT/A53T-α-synuclein injection).

A significant decrease of -58±3% of AADC enzymatic rate in the ipsilateral caudate putamen was observed as compared to the contralateral caudate putamen (Figure 18A; Δ81±6%; n=4; p=0.0017) in 6-OHDA rats.

It was therefore concluded that chancing the injection route of benserazide had no beneficial effect on peripheral AADC inhibition by benserazide, and thus the intraperitoneal injection of benserazide would be used in future experiments.

AADC quantification in extrastriatal brain regions

In addition to the striatum, we also observed significant left-right differences in several extrastriatal regions: hypothalamus (Figure 18A, -33±6%; Δ40±8%; n=4; p=0.021), thalamus (-18±3%; Δ20±4%; p=0.029), and cortex (-20±4%; Δ23±5%; p=0.002).

Previously, Honer and colleagues (2006) observed highest radioactivity concentrations in the striatum, followed by the thalamic nuclei and the substantia nigra in MPTP mice [START_REF] Honer | Comparison of [18F]FDOPA, [18F]FMT and [18F]FECNT for imaging dopaminergic neurotransmission in mice[END_REF]. Taken together with our data, this indicates that [ 18 F]FMT could possibly quantify extrastriatal brain regions, however, this was outside the scope of the PhD work [START_REF] Li | The role of the LRRK2 gene in Parkinsonism[END_REF].

Pilot study on different anaesthesia regimes in a complete lesion model

Secondly, we aimed to evaluate if the AADC inhibition by benserazide would be impacted by isoflurane. To that end, we evaluated if another anaesthesia regime, under continuous medetomidine infusion, could improve success rate of [ 18 F]FMT PET imaging. Medetomidine (Domitor) was first subcutaneous injected as a bolus (0.05 mg/kg) after anaesthetic induction by isoflurane, and subsequently perfused (0.1 mg/kg/h). Benserazide injection protocol was equal to described above: 5mg/kg of benserazide intravenously injected 30 minutes before radiotracer injection.

Quantification of [ 18 F]FMT PET imaging using the cerebellum as the reference region (section 5. 4.3, page 63) showed no significant differences between the use of isoflurane (ki=0.013±0.002) and medetomidine (Ki=0.011±0.002) (Figure 18B,n=4;p=0.42).

Conclusion

Taken together, intravenous administration of benserazide or medetomidine anaesthesia do not improve the blocking rate of peripheral AADC using benserazide. Thus, we have continued to use the same parameters for our studies as suggested by Becker and colleagues [START_REF] Becker | Comparative assessment of 6-[18F]fluoro-L-m-tyrosine and 6-[18F]fluoro-Ldopa to evaluate dopaminergic presynaptic integrity in a Parkinson's disease rat model[END_REF].

In future experiments, it should be tested if peripheral inhibition of COMT increases the success rate of quantifiable [ 18 F]FMT PET scans.

Aims

Previously, it has been suggested by Napieczynska and colleagues (2018) that online recording of the blood radioactivity through an arteriovenous shunt allows precise measurement of the arterial input function (AIF) without losing the blood volume. Furthermore, combining the continuous data acquisition with a single manually collected blood sample was suggested to be the most accurate approach for subsequent quantitative data analysis [START_REF] Napieczynska | Impact of the arterial input function recording method on kinetic parameters in small-animal PET[END_REF]. Our aim here was to evaluate the feasibility of simultaneous acquisition of PET images and the arterial input function after [ 18 F]DPA714 injection using the commercially available Twilite system (Swisstrace; Zurich, Switzerland), to the example of [ 18 F]FDG PET experiments [START_REF] Weber | A femoral arteriovenous shunt facilitates arterial whole blood sampling in animals[END_REF]. Specifically, in this pilot study, we aimed to 1) measure the plasma to blood conversion function, and 2) compare the "gold-standard" manual AIF with the automated input function, and 3) identify dispersion or other parameters.

Experimental setup

A commercially available Twilite system (Swisstrace; Zurich, Switzerland) was used to measure the whole blood concentration of [ 18 F]DPA714 (Figure 19). We used the same experimental setup as previously described [START_REF] Weber | A femoral arteriovenous shunt facilitates arterial whole blood sampling in animals[END_REF]. Briefly, polyethylene catheters (PE20; prefilled with 50 UI/mL heparinized saline) were implanted, under isoflurane anaesthesia in 100% oxygen, in the femoral artery and vein of five animals to create an arteriovenous shunt. A continuous blood flow through the shunt was assured using a peristaltic pump (Ismatec®, Germany) at a standard flow rate of 0.332ml/min. At the arterial side, the PE20 catheter was placed in a cassette that entered the coincidence detector; at the venous side, butterfly catheters allowed for radiotracer injection in the arteriovenous shunt using an automated injection pump, and withdrawal of blood samples. Whole blood concentration of the radioligand was continuously measured online using PMOD® software (PMOD 3.804; PMOD Technologies LLC, Switzerland). Additional blood samples (500μL) were manually withdrawn from the shunt at [START_REF] Singleton | alpha-Synuclein locus triplication causes Parkinson's disease[END_REF][START_REF] Pais N-Ru Z | Cloning of the Gene Containing Mutations that Cause PARK8-Linked Parkinson's Disease[END_REF][START_REF] Kalia | Clinical Correlations With Lewy Body Pathology in LRRK2-Related Parkinson Disease[END_REF]40,60 and 90 min after injection to count in parallel, and on the same animal, the whole blood and plasma concentration, and to determine the parental fraction of [ 18 After the start of the PET acquisition, the radiotracer is injected via an infusion pump as a fast bolus, and the activity in the whole blood is counted using an online blood counting system. For this, a catheter is placed in the femoral artery of the animal and subsequently passes through the blood counter, in which the radioactivity from the blood is counted. The peristaltic pump ensures a constant and continuous blood flow through the arterial-venous shunt. Manual blood sampling can be conducted using an arterial catheter to correct the blood curve for the plasma free fraction and the contribution from metabolites. Image from Herfert and colleagues (2019).

Results

Plasma to whole blood conversion

The parental fraction was highly consistent for the three animals. [ 18 F]DPA714 metabolises rapidly with only 50% of the parental fraction present 30 minutes post-tracer injection, and 20% 90 minutes after injection (Figure 20A).

Blood input curves were highly consistent amongst the three animals (Figure 20B), with the whole-blood input function displaying larger area under the curve (AUC) compared to the plasma input curve (Figure 20). The Plasma over Blood (PoB) curves showed up to almost tenfold higher uptake in the whole blood compared to the plasma, which equilibrated during the 90 minutes period to almost equally whole-blood plasma ratios. This PoB ratio is much lower as compared to the NHP, but similar to what we know for mice (unpublished data). Note that the time points for metabolite sampling created an important noise in the whole-blood curves. This is most probably due to an error in the decay correction, as sampling of the larger blood volumes required more time. Here, we used the onset of the blood sampling for decay correction, which results in overestimation of the wholeblood signal if sampling takes longer than the other samples. This creates an important noise in the PoB curve, and a more correct decay correction should be taken into account for future experiments. The dashed grey line (Figure 20D) is a rough estimate of what could be expected for a plasma to whole-blood conversion function. Despite high consistent input functions between the three animals, we observed a high variability in the PoB ratios. This variability, together with the noise in the whole blood curves, did not allow estimating a proper fit for the PoB curve.

Comparison of the manual and the automated input function

Of the four automated input acquisitions tested, the first three displayed serious coagulation in the arteriovenous shunt already at 30 minutes after tracer injection, despite heparinization of the catheters. Shortening of the arteriovenous shunt to almost half its length and reducing the death volume wherever possible, combined with a rigorous heparinization protocol (flushing every 15 minutes), allowed for a full 90 minutes automated acquisition of the whole blood input function.

The peak of the automated input curve was higher than the peak obtained by manual blood sampling (Figure 21). However, the peak width was broader, indicating that dispersion effects occur, which need to be corrected for. More problematically, the tail of the whole-blood input curve, acquired over the entire 90 minute period, showed increasing noise over time, strongly suggesting that [ 18 F]DPA714 was sticking to the PE20 tubing.

Discussion

We conducted a first pilot experiment to evaluate the feasibility of simultaneous blood sampling and PET imaging using the [ 18 F]DPA714 radioligand. With this pilot experiment, we obtained preliminary data, which are the basis for future experiments required to further develop this tool.

First, we observed that whole blood to plasma conversion data shows a high variability amongst animals. These preliminary data suggest that manual blood samples for each animal are required to scale each whole blood to plasma conversion correctly. These results are in concordance with the suggestion of Napieczynska and colleagues (2018) combining the continuous data with a single manually collected blood sample is the most accurate approach for the data analysis [START_REF] Napieczynska | Impact of the arterial input function recording method on kinetic parameters in small-animal PET[END_REF].

Secondly, these preliminary data showed that the implication of the Twilite system for the collection of automated [ 18 F]DPA714 input functions requires large methodological improvements; such as the evaluation of other catheter materials (e.g. Teflon), denoising algorithms (suggested by [START_REF] Santangelo | Modelling arterial input functions using data acquired with an MR-compatible sampler: a validation study using [18F[END_REF], and correction for dispersion. The development of these technical aspects were out of the scope of my PhD.

Taken together, it was decided to perform absolute quantification based on the reference tissue model as previously described by Ory andcolleagues (2015, 2016), and detailed previously on page 65.

Post-mortem studies 6.1 Brain tissue collection

All animals were ultimately sacrificed for subsequent immunohistological and biochemical analyses. Animals were deeply anaesthetized with 4% isoflurane before receiving a lethal dose of pentobarbital (180 mg/kg; Hexagon). Animals were then transcardially perfused with 280 ml 4% paraformaldehyde (PFA) in 0.01 M PBS at a rate of 28 ml/min. The brains were collected and postfixated for 24h in 4% PFA at +4°C and transferred to a 30% sucrose solution in PBS for cryoprotection. All brains were cut into 40 μm coronal sections using a freezing microtome (CM1900, Leica, Germany). Serial sections of the striatum and substantia nigra were stored at -20°C in antifreeze solution (30% sucrose, 30% ethylene glycol in PBS) until use.

Immunohistological analyses and quantification

Immunohistochemistry

For free-floating immunohistochemistry, sections were incubated for 20 min with 0.3% H 2 O 2 in PBS. Where necessary, antigen retrieval protocol was executed before H 2 O 2 incubation; 20min at 85° in citrate (pH 6.0). After rinsing sections were incubated 1 h in blocking solution containing 4.5% normal goat serum (NGS) in PBS with 0.2% Triton™ X-100 (PBST). Sections were subsequently transferred into primary antibody solution (in 3% NGS in PBST) for incubation overnight at 4°C (unless otherwise stated). After rinsing, sections were incubated with the appropriate secondary antibody (Vector Laboratories) diluted to 1:1000 into blocking solution for 1 h at room temperature followed by 1 h incubation in Vectastatin ABC Kit (Vector Laboratories) and revelation using the DAB Peroxidase Substrate Kit with nickel (Vector Laboratories). The following day, sections were dehydrated in consecutive ethanol baths (50%, 70%, 96%, absolute) and xylene before sealing the cover slip with Eukitt mounting medium.

We used primary antibodies directed against S129 phosphorylated α-synuclein (pS129-α-syn; 1:5.000; ab51253; Abcam), DAT (antigen retrieval; 1:200; AB1766; Chemicon), GFAP (1:10.000; Z0334; DAKO), IBA1 (1:3.000; 019_19741; WAKO), TH (2 night incubation; 1:2.000; MAB318; Millipore), Vimentin (1:2.000; IF01; Calbiochem), and VMAT2 (3 night incubation; 1:5.000; 496-515; Calbiochem).

Stereology

Stereological cell counting was performed using the optical fractionator principle on a Leica microscope (DM6000B & CTR6500, Leica, Germany). The amount of TH + neurons per substantia nigra was calculated using Mercator Software (Explora Nova, France). Squares (100 x 100 μm) were randomly systematically placed 80 μm apart in the manually outlined substantia nigra. Quantification was performed on serial sections separated by 200 μm covering the whole substantia nigra (anterior to posterior).

Optical density

The stained sections were digitalised using an ImageScanner III (GE Healthcare, USA) with Epson scan software (Epson, USA) at 2400dpi. Subsequent analysis was performed using Fiji-ImageJ software (open source, GitHub). In Fiji-Image the images were first transformed to 8-bit format and the scale was set. The area of interests were manually outlined for every section, including a blank region per microscope slide. The build-in function of Fiji-ImageJ was subsequently used to provide the light intensity per area of interest. Lastly, staining intensity was measured as optical density after background subtraction using the formula displayed below:

Immunofluorescence

The protocol for immunofluorescence is similar to the one for immunohistochemistry. Briefly, after rinsing sections were incubated 30 minutes in blocking solution containing 4.5% normal goat serum (NGS) in PBS with 0.2% Triton™ X-100. Sections were subsequently transferred into primary antibody solution (in 3% NGS in PBST) for incubation overnight at 4°C. After rinsing, sections were incubated with the appropriate secondary antibody (Invitrogen) diluted to 1:1000 into blocking solution for 1 hour at room temperature. Subsequently, sections were stained for DAPI for 5 minutes at room temperature and mounted in fluorescent mounting medium. We used primary antibody against IBA1 (1:1000; 019_19741; WAKO).

Fluorescent intensity

The fluorescently stained sections were digitalised using laser confocal microscopy (SP8, Leica, Germany. Subsequent analysis was performed using Fiji-ImageJ software (open source, GitHub). The images were first converted to 8-bit format and the scale was set, subsequently the area of interest was delineated. The fluorescent intensity was obtained using the standard intensity measurement in Fiji-ImageJ.

Preliminary evaluation in a complete lesion model

Tyrosine hydroxylase TH immunohistochemistry was performed after the example of [START_REF] Cresto | The C-terminal domain of LRRK2 with the G2019S mutation is sufficient to produce neurodegeneration of dopaminergic neurons in vivo[END_REF]. Even though TH immunohistochemistry had been employed successfully before, I have attempted to increase penetrance of the antibody by using pre-treatments of the tissue (EDTA or citrate at 85°C) and by increasing incubation time. However, none of this yielded a better result than achieved with the protocol described by Cresto and colleagues (2020).

The density of TH-positive cells was first assessed using optical density analyses following immunohistochemistry in the substantia nigra (Figure 23A), the density of TH-positive fibres was assessed using optical density analysis in the striatum (Figure 23B). This showed a significant decrease of 50%, equal to an asymmetry of Δ67% of TH density in the substantia nigra (-50±3%; Δ67±6%; n=6; p<0.001), and a significant decrease of 77%, equal to an asymmetry of Δ129% of THpositive fibre density in the striatum (-77±6%; Δ129±14%; n=6; p<<0.001).

These data suggest that the loss of dopaminergic fibres in the striatum is larger than the reduction of dopaminergic cells in the substantia nigra. Although surprising, this could be in line with research by Phan and colleagues (2017), who observed synaptic dysfunction before neurodegeneration in a α-synuclein overexpression model [START_REF] Phan | Early synaptic dysfunction induced by α-synuclein in a rat model of Parkinson's disease[END_REF]. Nevertheless, others suggest more equal dopaminergic neurodegeneration in the substantia nigra and striatal TH fibre loss [START_REF] Decressac | Comparison of the behavioural and histological characteristics of the 6-OHDA and α-synuclein rat models of Parkinson's disease[END_REF].

More likely, this phenomenon is induced by the less sensitive method of assessing dopaminergic neuronal loss through TH + optical density in the substantia nigra instead of cell counting via unbiased stereology. This is supported by our behavioural results (paragraph 3.1, page 52); the presence of rotational behaviour in the apomorphine-induced rotation test suggests a severe unilateral lesion (>80-90% dopaminergic nerve terminal loss; [START_REF] Hudson | Correlation of apomorphine-and amphetamine-induced turning with nigrostriatal dopamine content in unilateral 6-hydroxydopamine lesioned rats[END_REF][START_REF] Schwarting | THE UNILATERAL 6-HYDROXYDOPAMINE LESION MODEL IN BEHAVIORAL BRAIN RESEARCH[END_REF]). Indeed, using a different data set (from Chapter 4), comparison between results obtained through stereology and those obtained through TH optical density in the substantia nigra showed only a weak correlation between the two measures (Figure 22), even though both showed a significant asymmetry between ipsilateral and contralateral side. It also appeared that optical density underestimates the loss of TH-positive neurons. Thus, although optical density analysis is much less time consuming, we recommend using unbiased stereological techniques for assessment of THpositive neurons in the substantia nigra.

S129phosporylated-α-synuclein

The antibody against S129-phosphorylated α-synuclein has previously been employed in our lab, thus no further optimisation of the protocol was required.

Here, we have shown no significant signal corresponding to S129phosporylated-α-synuclein in 6-OHDA injected animals, in either the substantia nigra nor in the striatum.

Inflammation markers

All immunohistochemistry against markers for inflammation has been performed after the example of [START_REF] Gubinelli | Evaluation of an in situ polymerizing hydrogel scaffold as a brain delivery system for Parkinson's disease therapeutics[END_REF]. We have also attempted to performed immunohistochemistry against TSPO, however we have not succeeded in finding an antibody that worked in our hands.

Here, analysis in the striatum of IBA1 as an inflammatory marker revealed no significant increase in 6-OHDA animals (Figure 23F; n=6; p=0.09). Thus, we can say that there is no major inflammatory events going in the 6-OHDA model 1 year after injection, even though there is broad neurodegeneration.

Dopaminergic system

While all antibodies to visualise the dopaminergic system have been employed before in our lab, none had been used in rat PD models. Therefore, pilot stainings were performed to determine the optimum concentration, incubation time, and pre-treatment. While this was straightforward for VMAT2 (3 night incubation; 1:5.000; 496-515; Calbiochem), it proved more difficult for DAT, a high antibody concentration and rigorous pre-treatment was required with citrate (pH 6. AB1766; Chemicon). We have also attempted to perform immunohistochemistry against AADC, however we have not succeeded in finding an antibody that worked in our hands.

In the 6-OHDA model, the large scale dopaminergic neurodegeneration and fibre loss is accompanied by a strong reduction in DAT protein density (Figure 24A; Δ77±7%; n=6; p=0.003). Additionally, we observed a considerable reduction of VMAT density in the striatum (Figure 24B; Δ95±5%; n=6; p<0.001).

Real-time quantitative PCR

For transcriptomic analysis, striata and substantia nigra was first dissected by hand from the coronal 40 μm sections, using a surgical scalpel and a binocular microscope. These were then used to extract mRNA using E.Z.N.A.® FFPE RNA Kit (Omega Biotek, Georgia, USA) following the supplier's heat extraction guidelines. mRNA levels were measured using Nanodrop (ThermoFischer Scientific), and quality was validated using Bioanalyzer (Agilent). mRNA was then reverse-transcribed into cDNA using SuperScript™VILO™ cDNA Synthesis Kit (Vilo Life Technologies).

RT-qPCR was performed using iTaq ™ Universal SYBR® Green Supermix (Bio-Rad) and primers (Eurofin Genomics) specific to different targets on 0.35-1 ng of cDNA, using 10 nM of primers (see Table 7). Reactions were run in triplicates in 384-well PCR plates (Bio-Rad or 4Titute), using a mix of cDNA and a household gene (Ppia or Rplp0) primer as an inter-plate control.

Data were analysed using Bio-Rad CFX Maestro software (Bio-Rad). Cycle threshold (Ct) values were generated in regression mode. Results are shown as relative normalised expression.

Preliminary evaluation in a complete lesion model

Dopaminergic system

Although RT-qPCR against TH and VMAT has been used before in our lab, AADC and DAT had not been investigated before. Multiple primers for each target were designed by an expert in our lab, and subsequently I identified the most suitable one.

Here, significant differences were shown in mRNA levels for all tested dopaminergic markers between the ipsilateral and contralateral substantia nigra after 6-OHDA injection into the MFB (Figure 25). Both TH (Δ197%; n=4/6; p<<0.001) and AADC (Δ163%; n=2/6; p<<0.001) are involved in dopamine synthesis (Chapter 1: 1.1.2, page 15), while DAT is for presynaptic dopamine reuptake (Δ197%; n=3/6; p<<0.001), and VMAT is for intercellular transport (197%; n=4/6; p<<0.001).

Inflammation markers

Although most inflammation markers had already been validated and used by Dr. [START_REF] Gubinelli | Evaluation of an in situ polymerizing hydrogel scaffold as a brain delivery system for Parkinson's disease therapeutics[END_REF] in his PhD thesis, I have identified the most suitable option for use against TSPO. These inflammation markers are used in Chapter 6.

Here, there is no significant difference in IBA1 mRNA levels between the ipsilateral and contralateral substantia nigra 1 year after 6-OHDA injection into the MFB (Figure 25

Correlation between different markers

Pairwise Spearman correlation analysis, between different measures including left-right side, demonstrated different associations between post-mortem dopaminergic markers, FMT-PET imaging and behaviour. The results of this analysis are graphically represented in a correlation plot, in which the color corresponds to the strength and sign of the correlation (Figure 26). Expression of all dopaminergic markers are correlated; especially the association between TH and VMAT is strong (r=0.98). Surprisingly, whilst immunohistochemistry did not show any significant neuroinflammation in the SN, IBA1 expression showed relatively mild inverse association with the dopaminergic markers In vivo measures, FMT-PET and gombash score correlate positively with dopaminergic expression markers, and inversely with the expression of Iba1. Even though these data must be considered with caution -the unilateral complete 6OHDA model induces an "off/on" situation in the affected/healthy nigrostriatal system-this approach will be applied in future studies.

Background and aims

As shown previously in the introduction (Table 2), several groups have published on the overexpression of wild-type (WT) or A53T-α-synuclein in the substantia nigra using viral vectors. These have shown varying degrees of dopaminergic cell death in the substantia nigra and fibre loss in the striatum, while all showed α-synuclein pathology. However, to the best of our knowledge, not many studies have compared overexpression of WT-α-synuclein to overexpression of A53T-αsynuclein.

In our lab, two AAV2/6 viral vectors have been developed under the PGK promoter encoding WT and mutant A53T human α-synuclein protein (AAV2/6-PGK-WT-α-synuclein and AAV2/6-PGK-A53T-α-synuclein). Cresto and colleagues (in progress) have injected AAV2/6-PGK-A53T-α-synuclein unilaterally in the substantia nigra and showed significant dopaminergic neurodegeneration in the substantia nigra only occurs 15 weeks post-injection. Dopaminergic neurons often displayed accumulation of pS129-α-synuclein and stained positive for ThioS, suggesting the formation of aggregates. In addition, a 40% decrease of dopaminergic neurons was observed in the substantia nigra at 15 weeks post-injection (wpi), and consequenlty 20% less use of the contralateral forepaw in the cylinder test.

Nevertheless, an incubation time of 15 weeks post-injection makes this model less optimal to evaluate therapeutic strategies. In order to accelerate this timeline, we increased the viral vector dose, as well as the number of injection sites per hemisphere, using a double injection strategy. As a result the viral genome count was four-fold.

Here, we aimed to evaluate the impact of viral vector-induced expression of wild-type or mutant-A53T α-synuclein on the survival of dopaminergic neurons in the nigrostriatal pathway. For this, we used in vivo behavioural analyses and dopaminergic PET markers, which were compared with post-mortem analyses measuring neuronal death and α-synuclein aggregation.

Study design

Fifteen rats were unilaterally injected in the substantia nigra with a viral vector overexpressing wild-type (WT-α-syn; n=7) or mutated (A53T-α-syn; n=8) human α-synuclein, using a double injection approach (AAV2/6-PGK-WT-α-synuclein or AAV2/6-PGK-A53T-α-synuclein; 2x5.00E+10 vgc).

Animals were studied 10 to 12 weeks post injection (wpi; Figure 27) with PET imaging, using a ligand substrate for aromatic L-amino acid decarboxylase (AADC) (6[ 18 F]fluoro-L-m-tyrosine ([ 18 F]FMT)), or a ligand for dopamine transporter (DAT), [ 18 F]LBT999. Anatomical MRI scans were performed for PET coregistration and quantification. Behavioural evaluation was performed using the cylinder test, adjusting steps test, and vibrissae test. The in vivo studies were followed by postmortem stereology for tyrosine hydroxylase (TH)-positive cells in the substantia nigra.

Statistical analyses were performed using paired student t-tests to compare the ipsilateral and contralateral side in imaging studies, RT-qPCR, and immunohistochemical analyses, while for behavioural analysis a student t-test with unequal variance was used to compare against the hypothetic 50% value (chapter). All data were calculated as asymmetry ratios, according to the following formula:

Results

Behavioural analysis

Using the cylinder test, we observed a modest but significant motor deficit in the WT-αsynuclein group at 4 weeks post injection (wpi) (Figure 28 left; Δ39±12%; n=7; p=0.041), which could not be demonstrated at 10wpi (n=7; p=0.20). On the contrary, the A53T-α-synuclein group presented a statistically significant asymmetry in forepaw use at 4wpi (Figure 28 right; Δ45±7%; n=8; p<0.001), which was even more pronounced at 10wpi (Δ79±22%; n=8; p=0.034).

PET imaging of the dopaminergic system

Averaged parametric Ki images representing the AADC enzymatic rate in the striatum are shown in Figure 29 for both the WT-α-synuclein group (Figure 29A) and the A53T-α-synuclein group at 10-12wpi (Figure 29B). We did not observe a significant asymmetry in AADC metabolism between the striata ipsilateral and contralateral to AAV-injection for the WT-α-synuclein group (Figure 29C; n=3; p=0.32). A trend was observed for the A53T-α-synuclein group, although it did not reach statistical significance (n=2). 5 Averaged parametric images representing non-displaceable binding potential (BP nd ) of [ 18 F]LBT999 to DAT in the striatum 10-12wpi in WT-α-synuclein and A53T-α-synuclein groups are shown in figures Figure 29D and Figure 29E, respectively. The A53T-α-synuclein group showed significant asymmetry in DAT binding between the ipsilateral and contralateral striatum (Figure 29F; Δ54±8%; n=4; p=0.003). In the WT-α-synuclein groups the left-right asymmetry in DAT binding was smaller, with a larger heterogeneity (Δ32±13%), not reaching the significance level (n=3; p=0.051). 5 Due to the difficulties in blocking with benserazide, a pepipheral AADC inhibitor, we could not perform absolute quantification in all scanned animals (n=4). More details can be found in chapter 2, page 66.

Post-mortem analysis of neurodegeneration 6

The number of TH-positive cells in the SN was assessed by unbiased stereology. This revealed no significant difference for animals injected with WT-α-synuclein (Figure 30 left; n=7; p=0.31). For animals injected with A53T-α-synuclein we observed a significant asymmetry of Δ33% in the number of TH-positive cells in the injected SN as compared to the contralateral SN (Figure 30 right; Δ33±10%; n=8; p=0.009).

Discussion

Behaviour At 4wpi, we observed a significant asymmetry in forepaw use in the cylinder test for both the WT-α-synuclein and A53T-α-synuclein group. This transient reduction in contralateral forepaw use might be due to side-effects of the stereotactic surgery, rather than being an effect of the WT-αsynuclein overexpression. This is underlined by the fact that the motor deficits in both the WT-αsynuclein and A53T-α-synuclein group are very similar at 4wpi (WT: Δ39±12%, A53T: Δ45±7%), while this motor deficit is no longer seen at 12wpi in the WT-α-synuclein group, and increases for the A53T-α-synuclein group. This disappearance of a motor deficit at 12wpi in the WT-α-synuclein group is probably due to recovery after surgery. For the A53T-α-synuclein group the deficit remains at 12wpi and tends to aggravate when compared to the 4 week timepoint.

At the early timepoint after surgery, it might be difficult to discern between the effect of the surgery and/or injection, and that of the overexpression of WT or A53T-α-synuclein overexpression. Indeed in the present experiments we did not test the impact of the surgery on the performance of the rat during the cylinder test. However, Dr Cresto reported in her PhD dissertation that intranigral injection of PBS does not results in behavioural asymmetry in the cylinder test at 15wpi. In line with this, transduction of the substantia nigra with AAV-PGK-WT/G2019-LRRK2 does not induce significant C h a p t e r 3 -R e s u l t s o n α -s y n u c l e i n o v e r e x p r e s s i o n | 84 asymmetry either. Similarly, in the first series of experiments that were published [START_REF] Cresto | The C-terminal domain of LRRK2 with the G2019S mutation is sufficient to produce neurodegeneration of dopaminergic neurons in vivo[END_REF], the motor performance of rats intranigrally injected with AAVs coding for LRRK2 indicated no asymmetry when assessed with the CatWalk system (Noldus), suggesting that AAV-injections at the titers used here do not produce major aspecific asymmetric use of the forepaws. However, it cannot completely be ruled out that at 4wpi the mild asymmetry seen in the WT-α-synuclein group is at least in part due to the injection procedure. To test the effects of the stereotaxic surgery our results should have been compared to non-injected and PBS-injected animals.

At 12wpi, the lack of a motor deficit for the WT-α-synuclein group cannot likely be explained by habituation to, or lack of interest for, the behavioural tests. Since the cylinder test is based on the ratio between left and right forepaw use, this cannot affect the outcome measure. However, a significant reduction of overall touches was observed for both groups (for more detailed information see the Supplementary data, Figure 31 (page 86)). The absence of asymmetry in this group at 12 wpi suggests that if an initial "dysfunction" of nigral neurons had occurred at 4 weeks, compensatory mechanisms prevailed to restore "normal" neuronal functions.

In conclusion, the motor asymmetry seen after AAV injection is with high probability induced by the transgene and its neurotoxic effects on neuronal function and /or neurogeneration.

AADC-PET imaging

We were not able to demonstrate a significant asymmetry in the A53T-α-synuclein group, even though our results are suggestive for an asymmetric AADC enzymatic activity in the A53T-α-synuclein group. Different reasons can be put forward. First, we could only quantify AADC enzymatic activity in a small number of animals, due to insufficient blocking of peripheral AADC activity with benserazide, resulting in a low statistical power of this study. Next, there is a low signal to background-ratio in our [ 18 F]FMT PET images (Figure 29A,B). Smoothing of the PET images was required to enable quantification of K i ; this might have caused a loss of sensitivity in our quantification. The interanimal variability of the effect of benzerazide in rats is not explained so far, but represent another difficulty to use FMT in rat models.

To the best of our knowledge, only a few studies exist using [ 18 F]FMT PET imaging in rodent PD models [START_REF] Honer | Comparison of [18F]FDOPA, [18F]FMT and [18F]FECNT for imaging dopaminergic neurotransmission in mice[END_REF][START_REF] Becker | Comparative assessment of 6-[18F]fluoro-L-m-tyrosine and 6-[18F]fluoro-Ldopa to evaluate dopaminergic presynaptic integrity in a Parkinson's disease rat model[END_REF]. Honer and colleagues (2006) were unable to demonstrate dopaminergic neuronal loss, in a MPTP mouse model, using [ 18 F]FMT PET imaging. This might be attributed to the spill-in effect from surrounding brain regions displaying high radiotracer uptake, such as the basal brain areas, which possibly represent thalamic nuclei and/or basal ganglia (e.g. substantia nigra) [START_REF] Honer | Comparison of [18F]FDOPA, [18F]FMT and [18F]FECNT for imaging dopaminergic neurotransmission in mice[END_REF]. Becker and colleagues (2017) have used [ 18 F]FMT to study both the complete and partial 6-OHDA lesion models. They observed a significant decrease in [ 18 F]FMT uptake in the ipsilateral striatum as compared to the contralateral striatum for both the complete and partial 6-ODHA model [START_REF] Becker | Comparative assessment of 6-[18F]fluoro-L-m-tyrosine and 6-[18F]fluoro-Ldopa to evaluate dopaminergic presynaptic integrity in a Parkinson's disease rat model[END_REF]. It must be noted that the partial lesion has a 72% loss of dopamine content in the striatum, as compared to 99% reduction in the full lesion group, making it still a very severe model. Becker and colleagues could not demonstrate a significant difference between their partial and complete lesion [START_REF] Becker | Comparative assessment of 6-[18F]fluoro-L-m-tyrosine and 6-[18F]fluoro-Ldopa to evaluate dopaminergic presynaptic integrity in a Parkinson's disease rat model[END_REF].

[ 18 F]FDOPA, another PET tracer widely used to measure AADC enzymatic activity, has so far never been used in α-synuclein overexpression models. Nonetheless, in the complete 6-OHDA lesion model, Walker and colleagues (2013) showed that [ 18 F]FDOPA PET quantification was correlated with dopamine metabolite concentration [START_REF] Walker | Measuring dopaminergic function in the 6-OHDA-lesioned rat: a comparison of PET and microdialysis[END_REF] 2011) used varying degrees of the partial 6-OHDA lesion model and showed a significant correlation between [ 18 F]FDOPA uptake and dopamine concentration [START_REF] Kyono | Use of [18F]FDOPA-PET for in vivo evaluation of dopaminergic dysfunction in unilaterally 6-OHDA-lesioned rats[END_REF].

DAT-PET imaging

We demonstrate a significantly decreased binding of [ 18 F]LBT999 to DAT in the A53T-αsynuclein group, but not in the WT-α-synuclein group. Comparison of in vivo with post-mortem data showed an asymmetry ratio of Δ54% compared to Δ34% neuronal loss. Van der Perren and colleagues (2015) have demonstrated a gradual decrease of DAT binding using [ 18 F]-FECT PET imaging in their α-synuclein overexpression model. They show a ~50% decrease of DAT binding after ~60% reduction of TH-positive cells in the substantia nigra (Van der Perren et al., 2015 AAV2/7-CMV/Synapsin-A53T-α-synuclein).

[ 18 F]LBT999 has never been used in α-synuclein overexpression models before, though, it has succesfully been used in the partial 6-OHDA model. Sérrière and colleagues (2015) have demonstrated a significant decrease in DAT binding in the ipsilateral striatum using [ 18 F]LBT999 [START_REF] Sérrière | Assessment of the Protection of Dopaminergic Neurons by an α7 Nicotinic Receptor Agonist, PHA 543613 Using[END_REF]. Vetel and colleagues (2019) used [ 18 F]LBT999 to characterize a novel 6-OHDA lesion model; they showed a reproducible ~64% decrease in DAT binding in the striatum associated with a ~64% reduction of TH-positive cells in the substantia nigra [START_REF] Vetel | Extensive exploration of a novel rat model of Parkinson's disease using partial 6-hydroxydopamine lesion of dopaminergic neurons suggests new therapeutic approaches[END_REF]. Grealish and colleagues ( 2014) have taken it one step further and have used [ 18 F]LBT999 to observe functional improvement after cell grafting in 6-OHDA lesioned rats [START_REF] Grealish | Human ESC-derived dopamine neurons show similar preclinical efficacy and potency to fetal neurons when grafted in a rat model of Parkinson's disease[END_REF].

Taken together, our results suggest that the DAT tracer [ 18 F]LBT999 is more sensitive to detect mild dopaminergic neuronal loss as compared to the AADC substrate [ 18 F]FMT. The increased signal to noise-ratio and easier usage make [ 18 F]LBT999 the preferred radiotracer of the two studied here. Additionally, [ 18 F]LBT999 has been extensively studied in rodent and non-human primate models, showing an ability to precisely quantify dopaminergic neuron loss and even assess the beneficial effects of therapeutic approaches [START_REF] Grealish | Human ESC-derived dopamine neurons show similar preclinical efficacy and potency to fetal neurons when grafted in a rat model of Parkinson's disease[END_REF]reviewed by Chalon et al., 2019).

Neuronal loss

In the present study, we did not observe any dopaminergic neuron loss in the substantia nigra after the expression of AAV2/6-PGK-WT-α-synuclein at 12wpi. In contrast, we demonstrated that overexpression of AAV2/6-PGK-A53T-α-synuclein lead to -27±6% of neuronal loss at 12wpi (asymmetry: Δ33±10%). Several other research groups have also reported on AAV-based overexpression of WT and A53T-α-synuclein in the substantia nigra, leading to various degrees of dopaminergic neuron loss in the substantia nigra and fibre loss in the striatum (Table 2).

The lack of a motor deficit for the WT-α-synuclein was consistent with results from the postmortem analyses, which showed no significant loss of dopaminergic neurons in the substantia nigra. Literature has shown that WT-α-synuclein overexpression not always induces significant dopaminergic cell death [START_REF] Sanchez-Guajardo | Microglia acquire distinct activation profiles depending on the degree of α-synuclein neuropathology in a rAAV based model of Parkinson's disease[END_REF][START_REF] Phan | Early synaptic dysfunction induced by α-synuclein in a rat model of Parkinson's disease[END_REF]. Even so, multiple groups have shown neurodegeneration after overexpression of WT-α-synuclein [START_REF] Decressac | Comparison of the behavioural and histological characteristics of the 6-OHDA and α-synuclein rat models of Parkinson's disease[END_REF][START_REF] Gombash | Morphological and behavioral impact of AAV2/5-mediated overexpression of human wildtype alpha-synuclein in the rat nigrostriatal system[END_REF]. Since the resulting phenotype is mild as compared to A53Tα-synuclein overexpression, it might be possible that the selected timepoint of this study was too early. For future experiments, it would thus be beneficial to study multiple, more distant, timepoints (e.g. 16, 20, and 24 weeks post-injection).

Interestingly, although in most cases deficits induced with WT-α-synuclein are smaller than with A53T-α-synuclein, at least one group showed the reverse; Lo Bianco and colleagues (2002) C h a p t e r 3 -R e s u l t s o n α -s y n u c l e i n o v e r e x p r e s s i o n | 86 observed more dopaminergic cell and fibre loss after WT-α-synuclein overexpression using lenti-viral vectors with a PGK promoter as compared to A53T-α-synuclein overexpression.

Van der Perren and colleagues (2015), using AAV2/7-CMV/Synapsin-A53T-α-synuclein expression in the sunstantia nigra, demonstrated significant dopaminergic cell loss in the substantia nigra (up to 80%) at 10wpi, leading to a significant motor deficit in the cylinder test (-50%) ( [START_REF] Van Der Perren | Longitudinal follow-up and characterization of a robust rat model for Parkinson's disease based on overexpression of alpha-synuclein with adeno-associated viral vectors[END_REF]. The dopaminergic neurodegeneration we have observed in our experiments is much smaller than that found by other groups. However, the severity of the model also highly depends on the viral vector (e.g. AAV2, AAV6, AAV2/6), its promoter (e.g. PGK, CBA), and its enhancer elements (e.g. WPRE), the viral vector load, injection volume, and injection site and coordinates.

Conclusion

In conclusion, we have shown that A53T-α-synuclein overexpression, but not WT-α-synuclein, is able to generate neuronal loss and dopamine deficiency 12 weeks post-injection, which is detectable in vivo by DAT-PET imaging and the cylinder test, and post-mortem by TH + stereology. The double injection AAV2/6-PGK-A53T-α-synuclein model results in a shorter time-window to develop a behavioural deficit and dopaminergic neuron loss in the substantia nigra, which makes this model more interesting for the evaluation of potential new therapeutic strategies. Interestingly, we observed larger asymmetry with DAT PET (Δ54±8%) than in neuronal loss (-27±6%; Δ33±10%), suggesting additional downregulation of DAT protein. We further aim to characterize this model longitudinally, and to identify dopaminergic synaptic compensation mechanisms.

Supplementary behavioural data

Longitudinal evaluation of cylinder test

Longitudinal evaluation of the cylinder test revealed that the WT-αsynuclein and A53T-α-synuclein animals had a significant lower amount of total touches at 10wpi as compared to 4wpi (WT: Δ69±11%; n=7; p<0.001, A53T: Δ53±15%; n=8; p=0.002). Interestingly, on an individual level, we observe that while for some rats of the WT-α-synuclein group the motor symptoms seem to aggravate, for others they diminish or even disappear over time (Figure 31).

Adjusting steps test and vibrissae test

Neither the WT-α-synuclein nor the A53T-α-synuclein group showed a behavioural deficit using the adjusting step test at 4wpi (Figure 32B, WT: n=7; p=0.19, A53T: n=8; p=0.07) or the vibrissae test at 10wpi (Figure 32C, WT: n=7; p=0.52, A53T: n=8; p=0.07).

We did not observe a behavioural deficit using the adjusting steps test. However, Gombash and colleagues (2013) and Decressac and colleagues (2012) showed a significant deficit in their respective WT-α-synuclein models before positive outcome of the cylinder test [START_REF] Decressac | Comparison of the behavioural and histological characteristics of the 6-OHDA and α-synuclein rat models of Parkinson's disease[END_REF][START_REF] Gombash | Morphological and behavioral impact of AAV2/5-mediated overexpression of human wildtype alpha-synuclein in the rat nigrostriatal system[END_REF], suggesting that the adjusting steps test is more sensitive as compared to the cylinder test to detect motor symptoms in PD models. The discrepancy between our results and literature might be explained by the low replicability for the adjusting steps test in our hands. Indeed, the angle of the forepaw-restriction hold was decidedly variable between animals and runs. This inconsistency caused more false negatives since rats were not sufficiently stimulated to make the adjusting steps. The improve this, the rats could be trained longer beforehand to get accustomed to the forepaw-restriction hold, making it easier for the experimenter. Furthermore, a treadmill could be employed (as described by [START_REF] Chang | Biochemical and anatomical characterization of forepaw adjusting steps in rat models of Parkinson's disease: studies on medial forebrain bundle and striatal lesions[END_REF], which would equalise the speed and make the experimenter stationary, which could also make this test easier to perform consistently.

Similarly, like the adjusting steps test, the vibrissae test is experimenter-dependent, and therefore very variable in its execution, mainly in terms of hold, angle, and distance to the table. The lack of reproducibility induces a lack of sensitivity; hence, these tests were not used for further studies.

Part i. Evaluation of α-synuclein-induced neurodegeneration 1. Background and aims

Previously, we have compared two pathologically relevant rodent PD models based on overexpression of wildtype (WT) or A53T-mutant human α-synuclein protein in the substantia nigra through viral vectors (Chapter 3). This work has shown that A53T-α-synuclein, but not WT-αsynuclein, is able to generate neuronal loss and dopamine deficiency at 12 weeks post-injection (wpi). We demonstrated changes in the dopaminergic system in vivo through DAT-PET imaging corresponding to dopaminergic neuronal loss shown with post-mortem by TH + stereology.

In the present study, we continued the evaluation of the double injection AAV2/6-PGK-A53Tα-synuclein model, with a focus on the effect of α-synuclein accumulation on the dopaminergic projection neuron. To that end, we performed extensive comparison of in vivo and post-mortem data.

In addition to the previously discussed 12 week timepoint (Chapter 3), we evaluated neuronal loss in the substantia nigra and the projection neurons at an earlier timepoint (8wpi), and added PBS control animals. In addition to the previous chapter, post-mortem analyses were completed with more extensive immunohistochemical analyses and RT-qPCR.

Study design

Four cohorts of rats (n total =32) were unilaterally injected in the SN with AAV2/6 viral vector coding for mutated (A53T) human α-synuclein (AAV2/6-PGK-A53T-α-synuclein; 2x5.00E+10 vgc) or with PBS (Figure 33A). Animals were evaluated at an early (n=2x8; 6-8 weeks post-injection (wpi)) or the late timepoint (n=2x8; 10-13wpi) (Figure 33B). The late A53T-α-synuclein cohort consist of the same animals as described in Chapter 3 (page 79). At each timepoint, the same animals underwent in vivo behaviour and PET imaging, and post-mortem immunohistological and RT-qPCR analyses.

PET imaging was performed using 6-[ 18 F]fluoro-L-m-tyrosine ([ 18 F]FMT) -a substrate for AADC, and [ 18 F]-LBT999 ([ 18 F]LBT) -a radioligand for dopamine transporter (DAT). AADC enzymatic rate (Ki) and DAT binding (BP ND ) were calculated using Patlak and Logan graphical methods respectively, employing the cerebellum as a reference region. For behavioural analysis, rats underwent the cylinder test during 5 minutes, in which contralateral and ipsilateral forepaw use was compared. Immediately after the in vivo studies, rats were sacrificed for histological studies and biochemical analyses. RT-qPCR studied gene expression of hydroxylase (TH), ionised calcium-binding adaptor molecule 1 (IBA1), aromatic L-amino acid decarboxylase (AADC), dopamine transporter (DAT), vesicular monoamine transporter 2 (VMAT2) in the substantia nigra. Immunohistochemical studies evaluated protein expression of hydroxylase (TH), phosphorylated serine 129 α-synuclein (pS129-αsyn), IBA1, DAT, VMAT in the substantia nigra and/or striatum. More detailed information about the methods can be found in Chapter 2.

Statistical analyses were performed using paired student t-tests to compare the ipsilateral and contralateral side in imaging studies, RT-qPCR, and immunohistochemical analyses, while for behavioural analysis a student t-test with unequal variance was used to compare against the hypothetic 50% value. All data is presented as asymmetry data:

Results

Behavioural analysis

The cylinder test revealed a clear motor deficit at both the early (Figure 34, Δ76±23%; n=7; p=0.025) and the late timepoint (Δ79±22%; n=8; p=0.034) after AAV2/6-PGK-A53T-α-synuclein injection. Neither the early (n=8; p=0.32), nor the late PBS injected animals (n=8; p=0.14) show a significant motor deficit.

PET imaging of the dopaminergic system

At the early timepoint, FMT-PET imaging did not show any significant left-right asymmetry in AADC enzymatic activity in the striatum after AAV2/6-PGK-A53T-α-synuclein injection, as we have previously shown for the late timepoint (Figure 35A,B, Early: n=4; p=0.25, Late: n=2).

In contrast, the significant asymmetry in DAT binding potential in the ipsilateral versus contralateral striatum was also observed at the early timepoint in A53T-α-synuclein rats (Figure 35C,D, Δ33±7%; n=6; p=0.027), as we have previously shown for the late timepoint (Δ54±8%; n=4; p=0.003).

Post-mortem analyses: α-synuclein, inflammation, and the dopaminergic system pS129-α-synuclein accumulation

We observed the presence of pS129-α-synuclein protein in the substantia nigra at the early timepoint after AAV2/6-PGK-A53T-α-synuclein injection as compared to the contralateral side (Figure 36A,B, Δ71±12%; n=7; p=0.004), and a smaller, but still significant, accumulation at the late timepoint (Δ34±9%; n=8; p=0.012).

The late PBS groups did not show a significant accumulation of pS129-α-synuclein in the substantia nigra (n=8; p=0.28).

Tyrosine hydroxylase

At the early timepoint, we observed a significant difference of TH-positive positive neurons between the ipsilateral and contralateral substantia nigra after AAV2/6-PGK-A53T-α-synuclein injection, as measured by unbiased stereological counting (Figure 36C,D, Δ17±5%; n=6; p=0.013). At the late timepoint after AAV2/6-PGK-A53T-α-synuclein injection the difference in TH-positive neurons between the ipsi-and contralateral substantia nigra was stronger, as measured by unbiased TH stereology (Δ33±10%; n=8; p=0.009). This coincided with a borderline significant asymmetry of TH protein in the striatum at the late timepoint (Figure 36E,F, Δ40±12%; n=8; p=0.056). RT-qPCR showed no significant difference in TH mRNA levels in the substantia nigra at either timepoint after AAV2/6-PGK-A53T-α-synuclein injection (Figure 36G, Early: n=7/6; p= 0.13, Late: n=4/6; p=0.16).

The early PBS group did not show a significant asymmetry in TH-positive positive neurons in the substantia nigra, as measured by unbiased stereological counting (n=4; p=0.98). Neither did we observe a significant asymmetry in TH protein in the striatum at the early timepoint, as measured by optical density (n=8; p=0.51), nor at the late timepoint for PBS rats (n=8; p=0.44). RT-qPCR showed no significant difference in TH mRNA levels in the substantia nigra at either timepoint after PBS injection (Early: n=8/8; p=0.20, Late: n=8/8; p=0.99).

Neuroinflammation 7

At the early timepoint we demonstrated a minor but significant increase of IBA1 protein in the substantia nigra after AAV2/6-PGK-A53T-α-synuclein injection (Figure 36H,I, Δ6±2%; n=7, p=0.010). This result was not confirmed by RT-qPCR, which did not show significantly increased IBA1-mRNA expression in the substantia nigra at the early timepoint (Figure 36J, n=7/7, p=0.27). At the late timepoint, however, we did observe a significant increase of IBA1 mRNA levels (Δ38%; n=3/6; p=0.025).

With RT-qPCR we did not observe a significant difference at either the early timepoint (n=8/8; p=0.72), nor at the late timepoint for the PBS injected animals (n=8/8; p=0.88).

Correlation between post-mortem data and PET imaging

At the early timepoint after AAV2/6-PGK-A53T-α-synuclein injection we observed positive associations between the different post-mortem dopaminergic makers (Figure 38C), while they showed a moderate negative association with IBA1 expression. In vivo DAT-PET showed weak associations with the post-mortem dopaminergic markers. Interestingly, however, DAT-PET showed a strong negative association with IBA1 expression at the early timepoint. At the late timepoint we observe the same overall pattern (Figure 38D). Notably, the associations between post-mortem dopaminergic markers and IBA1 expression seem stronger, while the association between in vivo DAT-PET and IBA1 expression seems weaker.

In the PBS group at the early timepoint we observed a less distinct pattern of associations (Figure 38A). While at the late timepoint we again observe positive associations between postmortem dopaminergic markers and a mild negative association with IBA1 expression (Figure 38B).

Discussion

Behavioural analysis

With this work, we aimed to evaluate the progression of neuronal loss in the substantia nigra and the dopaminergic system in our double injection AAV2/6-PGK-A53T-α-synuclein model. Using behavioural testing a motor deficit was shown at the early timepoint that remained stable at the late timepoint, with equal strength and variability. The severity of the motor deficit was mild comparable to what we observed in a complete lesion model of 6-ODHA (Δ116±18%, Chapter 2: 3.2.1, page 54). Our double AAV2/6-PGK-A53T-α-synuclein injection displayed a comparable motor deficit at 8wpi as measured in a rAAV2/7-CMV-A53T-α-synuclein model at 3wpi, reported by Van der Perren and colleagues (2015). However, unlike our model, these authors performed a longitudinal study in the same animals and showed a progressive increase of the motor deficit (-25% at 3wpi, -50% at 4wpi) ( [START_REF] Van Der Perren | Longitudinal follow-up and characterization of a robust rat model for Parkinson's disease based on overexpression of alpha-synuclein with adeno-associated viral vectors[END_REF]. The lack of evolution of the motor deficit between the 8wpi and 12wpi group cannot be attributed to a plateau-effect, since our 6-OHDA data, confirmed by others [START_REF] Schallert | CNS plasticity and assessment of forelimb sensorimotor outcome in unilateral rat models of stroke, cortical ablation, parkinsonism and spinal cord injury[END_REF], shows that the use of the contralateral forepaw in the cylinder test can reduce much below what we have observed in the double injection AAV2/6-PGK-A53T-α-synuclein model. Lastly, it could be that the cylinder test lacks sensitivity to demonstrate the differences between our two groups. Van der Perren and colleagues (2015) used a similar induction method, but their model develops much faster and stronger, making the differences between timepoints larger. To circumvent the question of sensitivity, the corridor test could be included in future studies. Although it measures sensorimotor integration, it is said to be highly sensitive to deficits in the dopaminergic system in PD rat models [START_REF] Dowd | The Corridor Task: a simple test of lateralised response selection sensitive to unilateral dopamine deafferentation and graft-derived dopamine replacement in the striatum[END_REF][START_REF] Fitzsimmons | Further validation of the corridor task for assessing deficit and recovery in the hemi-Parkinsonian rat: restoration of bilateral food retrieval by R e f e r e n c e s | 188 dopamine receptor agonism[END_REF].

PET imaging of the dopaminergic system

With AADC-PET we did not observe a significant loss of dopaminergic fibre, despite a clear trend as discussed in Chapter 3. In literature it has been suggested that AADC could be affected by compensation mechanisms in order to increase the amount of dopamine available in the synapse [START_REF] Lee | In vivo positron emission tomographic evidence for compensatory changes in presynaptic dopaminergic nerve terminals in Parkinson's disease[END_REF][START_REF] Blesa | Compensatory mechanisms in Parkinson's disease: Circuits adaptations and role in disease modification[END_REF][START_REF] Kaasinen | Striatal dopamine in Parkinson disease: A meta-analysis of imaging studies[END_REF]; and thus raise synaptic dopamine levels. However, our data showed no change in AADC-mRNA expression levels at the early timepoint. At the late timepoint we observed a significant asymmetry for left/right AADC-mRNA expression, which is most probably due to a decrease of the AADC mRNA level in the ipsilateral striatum rather than an increase in the contralateral striatum. Even so, since there is translational and posttranslational modification, there are still multiple ways in which final protein concentration can be modified (e.g. [START_REF] Chang | Alternative splicing in the coding region of human aromatic L-amino acid decarboxylase mRNA[END_REF][START_REF] Duchemin | Phosphorylation and activation of brain aromatic L-amino acid decarboxylase by cyclic AMP-dependent protein kinase[END_REF]. Thus, a lack of compensation mechanism at the mRNA expression level does not conclusively show that there is no AADC compensation mechanisms at all. Since PET imaging shows the accumulation of all previous processes, it could still be that the lack of asymmetry we observe with AADC-PET imaging is due to a combination of a reduced number of nerve terminals and a functional increase of AADC protein.

We have shown a significant left-right asymmetry in DAT binding using PET imaging. The difference in left-right binding (Δ54±8%) was markedly larger at the late timepoint as compared to the early timepoint (Δ33±7%), which coincides with increasing size of the dopaminergic lesion. However, DAT-PET shows a much larger asymmetry at the late timepoint (Δ54±8%) than suggested by the loss of dopaminergic neurons alone (Δ33±10%). DAT regulates synaptic dopamine levels; and thus, reduced DAT expression increases the amount of dopamine available in the synapse. DAT is thought to compensate for dopaminergic neuronal loss in the substantia nigra and the accompanying loss of dopaminergic fibres in the striatum [START_REF] Lee | In vivo positron emission tomographic evidence for compensatory changes in presynaptic dopaminergic nerve terminals in Parkinson's disease[END_REF]. At the late timepoint, we indeed observe an asymmetry in DAT mRNA and protein levels in the substantia nigra and striatum, respectively. Thus, our results at the late timepoint are in concordance with the idea of DAT compensation.

At the early timepoint, we do not observe such an compensatory effect of either DAT mRNA or protein levels. However, DAT-PET still shows a much larger asymmetry (Δ33±7%) than the loss of dopaminergic neurons (Δ17±5%) suggests. Recently it has been shown that extracellular monomeric and fibrillar α-synuclein can potentiate DAT binding and pre-endocytic clustering of DAT on the cell surface, thereby facilitating DAT endocytosis and down-regulating its transporter activity [START_REF] Kobayashi | Extracellular α-synuclein enters dopaminergic cells by modulating flotillin-1-assisted dopamine transporter endocytosis[END_REF]. Down-regulation of striatal DAT has also been suggested after intra-MFB 6-OHDA injection in rats [START_REF] Sossi | Dopamine transporter relation to levodopa-derived synaptic dopamine in a rat model of Parkinson's: an in vivo imaging study[END_REF]. Conversely, striatal DAT binding was increased during the premotor phase in MPTP-treated non-human primates [START_REF] Vezoli | Increased DAT binding in the early stage of the dopaminergic lesion: A longitudinal [11C]PE2I binding study in the MPTP-monkey[END_REF]. Other studies observed no compensation mechanisms for striatal DAT in MPTP-treaded monkeys [START_REF] Bezard | Relationship between the appearance of symptoms and the level of nigrostriatal degeneration in a progressive 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned macaque model of Parkinson's disease[END_REF], mice [START_REF] Bezard | Adaptive changes in the nigrostriatal pathway in response to increased 1methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced neurodegeneration in the mouse[END_REF], or the partial 6-OHDA lesion rat model [START_REF] Dentresangle | Increased extracellular DA and normal evoked DA release in the rat striatum after a partial lesion of the substantia nigra[END_REF].

Using PET imaging in PD patients a greater reduction in DAT binding was observed as compared to AADC activity ([ 18 F]DOPA) or VMAT2 binding; suggestive of a down-regulation of DAT in the striatum [START_REF] Lee | In vivo positron emission tomographic evidence for compensatory changes in presynaptic dopaminergic nerve terminals in Parkinson's disease[END_REF][START_REF] Adams | PET in LRRK2 mutations: comparison to sporadic Parkinson's disease and evidence for presymptomatic compensation[END_REF]Daryl J Wile et al., 2017). Additionally, a downregulation of DAT mRNA in the striatum has been reported [START_REF] Uhl | Dopamine transporter messenger RNA in Parkinson's disease and control substantia nigra neurons[END_REF][START_REF] Joyce | Differential modification of dopamine transporter and tyrosine hydroxylase mRNAs in midbrain of subjects with Parkinson's, Alzheimer's with parkinsonism, and Alzheimer's disease[END_REF]. Interestingly, reduced DAT binding can already be observed during the prodromal phase, using PET imaging, possibly reflecting prodromal compensatory mechanisms [START_REF] Adams | PET in LRRK2 mutations: comparison to sporadic Parkinson's disease and evidence for presymptomatic compensation[END_REF][START_REF] Stoessl | Positron emission tomography in premotor Parkinson's disease[END_REF]Daryl J Wile et al., 2017).

Neuronal loss & α-synuclein aggregation

Here we have injected a viral vector coding for A53T-α-synuclein into the substantia nigra, which led to significant dopaminergic neuronal loss in the substantia nigra at 8wpi (-15±4%, Δ17±5%), as measured by unbiased TH + -stereology. This loss of dopaminergic neurons was mild compared to a complete lesion 6-ODHA model (-93.6±1.2%) [START_REF] Decressac | Comparison of the behavioural and histological characteristics of the 6-OHDA and α-synuclein rat models of Parkinson's disease[END_REF]. Our model is also milder compared to other viral vector overexpression models of α-synuclein. Decressac and colleagues (2012) showed around 70% loss of the dopaminergic neurons in a WT-αsynuclein model [START_REF] Decressac | Comparison of the behavioural and histological characteristics of the 6-OHDA and α-synuclein rat models of Parkinson's disease[END_REF]; AAV2/6-Synapsin/WPRE-WT-αsynuclein), while Van der Perren and colleagues (2015) showed around 70% loss in their A53T-αsynuclein overexpression model already at 2.5wpi. However, this last group also described a low vector dose model, which is much more in line with the size of the reduction of TH + cells we observe, which is around 30% at 12wpi (Van der Perren et al., 2015; AAV2/7-Synapsin/CMV-A53T-αsynuclein). This demonstrates that besides the construction of the viral vector, also the titer plays a major role.

In the striatum, we have observed an almost significant reduction of TH-fibres at the late timepoint, while we observed significant neuronal loss in the substantia nigra (-27±6%, Δ33±10%). Decressac and colleagues (2012) demonstrated an almost equal reduction in striatal fibres as in dopaminergic cells in the substantia nigra. Noteworthy, our model seems to affect mostly the distal side of the striatum (visual observation), while Decressac's model seems to affect the medial striatum [START_REF] Decressac | Comparison of the behavioural and histological characteristics of the 6-OHDA and α-synuclein rat models of Parkinson's disease[END_REF]. This difference is most probably due to different stereotactic injection coordinates in the substantia nigra, engaging different areas in the striatum [START_REF] Haber | The place of dopamine in the cortico-basal ganglia circuit[END_REF].

Overall, we have shown here that our model progresses over time, with both an increase in the loss of TH + neurons in the substantia nigra and TH + fibres in the striatum.

In our studies, the loss of dopaminergic neurons in the substantia nigra was accompanied by an increase of α-synuclein phosphorylated at serine 129. Previously, studies have shown that approximately 90% of α-synuclein in Lewy Bodies is phosphorylated at serine 129 (Van der [START_REF] Van Der Perren | Longitudinal follow-up and characterization of a robust rat model for Parkinson's disease based on overexpression of alpha-synuclein with adeno-associated viral vectors[END_REF]. At the early timepoint we also observed increased levels of pS129-α-synuclein in the striatum, which might suggest relocation or even spreading of α-synuclein aggregates [START_REF] Gerfen | The Neuroanatomical Organization of the Basal Ganglia[END_REF]. Similarly, Van der Perren and colleagues (2015) observed dopaminergic neuronal loss and striatal fibre loss after nigral overexpression of α-synuclein (Van der [START_REF] Van Der Perren | Longitudinal follow-up and characterization of a robust rat model for Parkinson's disease based on overexpression of alpha-synuclein with adeno-associated viral vectors[END_REF].

Interestingly, we observed a larger increase of pS129-α-synuclein in both the substantia nigra and the striatum at the early timepoint as compared to the late timepoint, which might be attributed to neuronal loss. Nevertheless, Van der Perren and colleagues (2015) observed an initial increase in the percentage of aggregate positive cells which remained stable at later timepoints despite the occurance of severe neuronal loss [START_REF] Van Der Perren | Longitudinal follow-up and characterization of a robust rat model for Parkinson's disease based on overexpression of alpha-synuclein with adeno-associated viral vectors[END_REF].

We observed a small but significant inflammation at the early timepoint in the substantia nigra, which was not measurable by RT-qPCR. At the late timepoint we observed an upregulation of IBA1 mRNA in the substantia nigra using RT-qPCR. Previously, Chung and colleagues (2009) observed early microglial activation in the striatum, but not in the substantia nigra, after α-synuclein overexpression in the substantia nigra [START_REF] Chung | Dynamic Changes in Presynaptic and Axonal Transport Proteins Combined with Striatal Neuroinflammation Precede Dopaminergic Neuronal Loss in a Rat Model of AAV -Synucleinopathy[END_REF].

Taken together the decrease of pS129-α-synuclein and the increase of inflammation over time, we hypothesize that neurodegeneration is mainly driven by α-synuclein overexpression at our early timepoint. Neuroinflammation seems to occur secondary to neuronal death. Based on their results, Chung and colleagues (2009) suggested that neuroinflammation is involved already before neuronal death occurs in their AAV2-Synapsin-A53T-α-synuclein model, and forms a positive feedback-loop to aggravate neurodegeneration [START_REF] Chung | Dynamic Changes in Presynaptic and Axonal Transport Proteins Combined with Striatal Neuroinflammation Precede Dopaminergic Neuronal Loss in a Rat Model of AAV -Synucleinopathy[END_REF]. However, they did not report on α-synuclein aggregation in their model.

Conclusion

Here, we evaluated double injection of AAV2/6-PGK-A53T-α-synuclein as a rat PD model at two different timepoints. We have shown progressive loss of dopaminergic neurons in the substantia nigra, in combination with increasing neuroinflammation. The in vivo PET imaging and post-mortem analyses support the presence of potential DAT compensation mechanisms. Future research will have to unveil whether these compensation effects occur at the ipsilateral and/or contralateral synapse.

Part ii. Exploration of compensation mechanisms in mild neurodegenerative models

Background and aims

We have previously suggested that compensation effects might impact PET imaging markers. Here, we aimed to identify whether these compensation mechanisms occur at the ipsilateral and/or contralateral hemisphere.

Since the analyses of the data are still ongoing, we have decided not to integrate the data obtained in this additional A53T-α-synuclein overexpressing group with the previous study. Unbiased stereological assessment is still ongoing 8 and should give confirmation of the lesion size and compatibility of these complementary data.

Study design

In parallel to the PBS 10-12wpi (reported above), one cohort of animals (n=10) was injected in the substantia nigra with AAV2/6 viral vector coding for mutated (A53T) human alpha-synuclein (n=10) or with PBS (n=8) (Figure 39A). Animals were evaluated at the late timepoint (Figure 39B; n=18; 10-12wpi). Animals underwent in vivo behaviour and PET imaging, and post mortem immunohistological and RT-qPCR analysis. The late PBS is the same as shown above, while the A53Tα-synuclein is a new cohort; the post-mortem analyses were performed at the same time to be able to compare. More detailed information about the methods can be found above (page 100) and in Chapter 2.

Statistical analyses were performed using paired student t-tests to compare the ipsilateral and contralateral side in imaging studies, RT-qPCR, and immunohistochemical analyses, while for behavioural analysis a student t-test with unequal variance was used to compare against the hypothetic 50% value. All data are presented as asymmetry ratios, calculated using the following formula:

Results

Behavioural analysis

Using the cylinder test, we observed significant deficits in spontaneous forepaw usage after AAV2/6-PGK-A53T-α-synuclein injection (Figure 40, Δ36±10%; n=8; p=0.013), while the PBS injected group showed no motor deficit (n=8; p=0.14).

PET imaging of the dopaminergic system

Using [ 18 F]FMT-PET imaging, we did not observe significant left-right asymmetry in AADC enzymatic activity in the striatum after AAV2/6-PGK-A53T-α-synuclein injection (Figure 41A,B,n=5;p=0.10).

Interestingly, if we combine these data with the previously obtained data for the late timepoint, we observe a small but significant asymmetry (Δ13±4%; n=7; p=0.030).

Post-mortem analyses: α-synuclein, inflammation, and the dopaminergic system pS129-α-synuclein accumulation

We demonstrated a significant increase of pS129-α-synuclein protein in the substantia nigra of A53T-α-synuclein injected animals (Figure 42A, Δ57±16%; n=9; p=0.007). As expected, in the PBS group at the late timepoint we did not observe a significant difference (n=8; p=0.28). Similarly, in the striatum there was a significant increase of pS129-α-synuclein in A53T-α-synuclein injected animals (Figure 42B, Δ13±3%; n=9; p=0.006). In the PBS group at the late timepoint we did not observe a significant asymmetry (n=8; p=0.08); interestingly, there was a significant difference between the ipsilateral PBS-injected animals and contralateral A53T-α-synuclein injected animals (Δ23%; n=8/9; p=0.013).

Tyrosine hydroxylase

Using optical density to rapidly estimate the amount of TH-positive cells in the substantia nigra, we did not observe a significant asymmetry after either A53T-α-synuclein injection (Figure 42C,n=9;p=0.50; or PBS injection (n=8; p=0.08; non-parametric). In the striatum, we did not observe any TH asymmetry at the late timepoint using optical density analysis for A53T-αsynuclein (Figure 42E,n=9;p=0.28) or PBS injected animals (n=8; p=0.44). RT-qPCR showed a significant asymmetry in TH mRNA expression in the substantia nigra in the A53T-α-synuclein injected animals (Figure 42D, Δ29%; n=8/8; p=0.008), while there was no asymmetry in PBS animals (n=8/8; p=0.99).

Neuroinflammation

In the striatum, we did not observe any asymmetry for IBA1 at the late timepoint after A53T-αsynuclein injection (Figure 42G,n=9;p=0.20) or PBS injection using optical density analysis (n=8; p=0.44). Using RT-qPCR we observed no significant asymmetry in IBA1 levels in the substantia nigra in the A53T-α-synuclein or PBS group (Figure 42F

Aromatic L-amino acid decarboxylase

Using RT-qPCR we did not observe a significant asymmetry in AADC mRNA expression in the substantia nigra of A53T-α-synuclein animals (Figure 43A

Dopamine transporter

We did not observe a significant asymmetry in DAT protein levels in the striatum for A53T-αsynuclein injected animals (Figure 43C,n=9;p=0.30), nor for the PBS injected animals (n=8; p=0.71). RT-qPCR showed significant asymmetry of DAT mRNA expression levels in the substantia nigra in A53T-α-synuclein injected animals (Figure 43B, Δ20%; n=8/8; p=0.042), while PBS animals showed no asymmetry (n=8/8; p=0.45).

Vesicular monoamine transporter 2

We did not show any significant asymmetry in VMAT2 protein levels in the striatum after A53T-α-synuclein injection (Figure 43E,n=9;p=0.20), nor after PBS injection (n=8; p=0.47). Similarly, using RT-qPCR, we did not observe significant asymmetry in VMAT2 mRNA expression in the substantia nigra for A53T-α-synuclein injected animals (Figure 43D,n=8/8;p=0.33), nor for PBS injected animals (n=8/8; p=0.95). With this work we aimed to study the above observed compensation effect in more detail. We used our double injection AAV2/6-PGK-A53T-α-synuclein rat model at the late timepoint (10-12wpi), and PBS injected animals as a control. Using behavioural testing we observed a motor deficit for A53T-α-synuclein rats, while the PBS injected rats did not show a motor deficit. Conversely, using in vivo AADC-PET imaging we did not observe a significant asymmetry. Using post-mortem, we demonstrated significant presence of phosphorylated α-synuclein in both the substantia niga and striatum, and a reduction of TH mRNA in the substantia nigra. We did not observe neuroinflammation, as assessed by IBA1 mRNA and protein levels, nor did we observe significant changes in AADC and VMAT2 protein density in the striatum, or VMAT2 mRNA expression in the substantia nigra.

Previsously, we have also shown a motor deficit at 10-13wpi after nigral AAV2/6-PGK-A53T-αsynuclein injection. Morever, previously we had already observed an increase in pS129-α-synuclein protein density in the substantia nigra, and an asymmetry in DAT mRNA expression in the substantia nigra. Conversely, again we did not demonstrate asymmetry using AADC-PET, nor did we observe asymmetry in IBA1 mRNA expression in the substantia nigra or TH and VMAT2 protein density in the striatum.

However, we have previously demonstrated asymmetry in AADC and VMAT2 mRNA expression in the substantia nigra, as well as DAT protein density in the striatum, which we were unable to replicate here. Thus, we were unable to conclusively show the presense of DAT compensation mechanisms here. The lack of compensation mechanisms in our current experiment might be explain by the lack of dopaminergic neuron loss in the substantia nigra. However, a less sensitive method of analysis was used due to a lack of time. As shown in Chapter 2: 6.2.6 (page 73), optical density underestimates neuronal loss as compared to unbiased stereological counting. It is therefore not conclusive that there is indeed no neurodegeneration in the nigrostriatal pathway. Further research by way of unbiased stereology will be done to answer this question.

Conclusion

Here, we aimed to investigate whether the previously observed compensation mechanisms occur at the ipsilateral and/or contralateral hemisphere. However, we were not able to demonstrate any compensation mechanism. Further research has to show if the neuronal loss in the new cohort is comparable to the first cohort.

Nevertheless, a very mild PD model, like described here, might be reflective of a prodromal phase, thus making it very interesting for research on neuroprotective agents; the dopaminergic neurons are suffering, but have not perished yet. Additionally, this mild model might be used the research the interplay between A53T-α-synuclein overexpression and other PD-related gene mutations, e.g. the putative enhancing effect of the A53T-α-synuclein mutation and LRRK2 [START_REF] Cresto | The unlikely partnership between LRRK2 and α-synuclein in Parkinson's disease[END_REF].

Chapter 5: Results on the interplay between LRRK2 and α-synuclein

RESUTLS ON THE INTERPLAY BETWEEN LRRK2 AND α-SYNUCLEIN

Background and aims

Leucine-rich repeat kinase 2 (LRRK2) is a kinase of which rare mutations have shown to cause autosomal dominant PD (Chapter 1: 2.5, page 27). The overlap between clinical phenotypes associated with mutations in SNCA and LRRK2 suggests that these proteins play an important role in both sporadic and genetic PD [START_REF] Cresto | The Unlikely Partnership Between LRRK2 and α-Synuclein in Parkinson's Disease[END_REF]. Our lab is interested in the potential synergistic interaction between α-synuclein and LRRK2 and their role in the susceptibility to develop PD. Conversely, inhibition of LRRK2 kinase activity is envisioned as one of the main therapeutic targets in PD.

The main mode of action of LRRK2 is through its kinase domain; rendered constitutively active through the G2019S. Previously, it was shown by the team of Emmanuel Brouillet from our lab that overexpression of the C-terminal domain of LRRK2 alone did not affect dopaminergic neuronal survival at 6 months post-injection, while the constituently active G2019S form of LRRK2 resulted in a significant reduction of 30% of TH-positive neurons in the substantia nigra of rats (AAV2/9-PGK/WPRE-ΔLRRK2 WT/G2019S ) [START_REF] Cresto | The C-terminal domain of LRRK2 with the G2019S mutation is sufficient to produce neurodegeneration of dopaminergic neurons in vivo[END_REF]. In Chapter 4, we have shown that A53T-α-synuclein overexpression results in significant dopaminergic neurodegeneration in the rat substantia nigra. The hypothesis is that mutated G2019S-LRRK2 increases the toxicity of A53T-α-synuclein overexpression. Previous studies performed in our lab at 15wpi have demonstrated that G2019S-ΔLRRK2 alone did not produce significant dopaminergic neurodegeneration. Co-overexpression of A53T-α-synuclein and G2019S-ΔLRRK2 induced a greater dopaminergic neurodegeneration than overexpression of A53T-α-synuclein alone. These results show that the effect of exacerbated A53T-α-synuclein toxicity by G2019S-ΔLRRK2 overexpression might be due to cell-autonomous mechanisms involving the the LRKK2 kinase domain. Alternatively, the interplay between A53T-α-synuclein and LRRK2 might be due to non-cell-autonomous mechanisms, such as neuroinflammation.

This study was subsequently repeated using AAV2/6-PGK-A53T-α-synuclein model as characterized in Chapter 4. The post-mortem data at 6wpi complementing the existing 15wpi data have been summarised in a paper entitled "The C-terminal fragment of LRRK2 with the G2019S substitution increases the neurotoxicity of mutant A53T α-synuclein in dopaminergic neurons in vivo", which was submitted to Translational Neurodegeneration (page 148). For this work, I was involved in the experiment conducted at 6 weeks post-injection; I have assisted with the stereotactic injections, and with the transcranial perfusions, cut all the brains, and contributed to the immunohistochemical analyses.

In the present chapter, we aimed to evaluate if DAT-PET imaging allows to discriminate A53Tα-synuclein-induced dopaminergic neuron loss from that induced by co-overexpression of A53T-αsynuclein and G2019S-ΔLRRK2.

Study design

Briefly, four cohorts of rats (n total =48) were unilaterally injected in the SN with AAV2/6 viral vector coding for mutated (A53T) human α-synuclein in combination with a viral vector coding for GFP, G2019S-LKKR2 or a dead-kinase form of LRRK2 (DK-LRRK2); control animals were injected with PBS (Figure 44A,B). Animals were evaluated at the early timepoint (n=4x12; 6-8 weeks post-injection (wpi, Figure 44C). Animals underwent in vivo behaviour and PET imaging, and post-mortem immunohistological evaluation.

For behavioural analysis, rats were subjected to the cylinder test for 5 minutes, in which contralateral and ipsilateral forepaw use was compared. PET imaging was performed using [ 18 F]LBT999 -a radioligand for dopamine transporter (DAT). DAT binding (BP ND ) was calculated using Logan's graphical method, employing the cerebellum as a reference region. Immediately after the in vivo studies, rats were sacrificed for histological studies. Immunohistochemical studies evaluated protein expression of tyrosine hydroxylase (TH). Unbiased stereology was performed by a single experimenter, due to time constraints, not all contralateral substantia nigras were counted. More detailed information about the methods can be found in Chapter 2, and in the submitted paper (page 148).

Inter-group statistical analyses were performed using repeated measures ANOVA, to compare the ipsilateral and contralateral side across all groups in imaging studies and immunohistochemical analyses. For post-hoc analyses between groups, Fisher's least significant difference test (LSD) was used. For behavioural analysis ANOVA was used to compare against the Gombash score of the PBS group. Within-group statistical analyses were performed using paired student-tests to compare the ipsilateral and contralateral side within groups in imaging studies and immunohistochemical analyses. Data are presented as asymmetry ratios:

Results

Behavioural analysis

The cylinder test (Figure 45A) revealed a significant difference from the PBS group for the A53T/GFP (Δ62±17%; n=12; p=0.005) and A53T/GS group (Δ58±10%; n=12; p=0.021), but not the A53T/DK group (n=12; p=0.37). Noteworthy, the A53T/GFP group was also significantly different from the A53T/DK group (Δ41±10%; n=12; p=0.044).

PET imaging of the dopaminergic system

Ipsi-and contralateral BPnd measures between groups

We did not demonstrate a group effect or a left-right effect in the DAT non-displaceable binding potential in the striata after expression of the different viral vectors in the substantia nigra (Figure 46; n=7/7/7/5; p=0.21). Surprisingly, we observed a high variability in BP nd between different vector groups.

However, if we compare the ipsilateral and contralateral hemisphere of each group individually, we a statistically significant asymmetry in the A53T/GFP (Δ21±2%; n=7; p=0.001), A53T/GS (Δ15±3%; n=7; p=0.003), and A53T/DK groups (Δ17±4%; n=7; p=0.003), but not the PBS group (n=5; p=0.25).

BPnd-ratio between groups

To account for the variability in BP nd between vector groups, we calculated the BP nd -ratio, using the contralateral striatum as a control side (Figure 47). Despite the absence of a significant difference between BP nd -ratios, we observed a trend similar to the behavioural data, showing the lowest BP nd -ratio for A53T/GFP (0.81±0.02), followed by A53T/GS (0.86±0.03), A53T/DK (0.87±0.03), and PBS (0.94±0.04). As the BP nd -ratios were derived from BP nd -measures from the entire striatum, these data were suggestive of local and modest BP nd changes.

However, if we compare the ipsilateral over contralateral ratio of each group individually to the hypothetical 1 value, we observe statistically significant differences in the A53T/GFP (n=7; p<<0.001), A53T/GS (n=7; p=0.002), and A53T/DK groups (n=7; p=0.004), but not the PBS group (n=5; p=0.16).

Post-mortem analysis: neurodegeneration

Dopaminergic neuronal loss was assessed by unbiased stereology in the injected and noninjected substantia nigra, performed by a single operator. We did not observe stastically significant dopaminergic neuronal loss in the injected substantia nigra of the experimental groups as compared to the PBS group, using one-way ANOVA (Figure 48, A53T/GFP: n=12; p=0.84, A53T/GS: n=12; p=0.61, A53T/DK: n=12; p=0.52). However, if we compare the ipsilateral and contralateral hemisphere of each group individually, we observe stastically significant asymmetry in both the A53T/GS (Δ21%; n=12/5; p=0.035) and A53T/DK groups (Δ30%; n=12/4; p=0.008), but not the A53T/GFP group (n=12/6; p=0.21) or PBS (n=12/7; p=0.49).

Discussion

In addition to the post-mortem study submitted, we have evaluated the synergistic effect of G2019S-ΔLRRK2 or DK-ΔLRRK2 and A53T-α-synuclein overexpression in vivo in the substantia nigra. G2019S-ΔLRRK2 co-overexpression with A53T-α-synuclein did not induce a larger behavioural deficit than observed after co-overexpression of A53T-α-synuclein and GFP. Similarly, the DAT-BP nd -ratio was not more different in the G2019S-ΔLRRK2 co-overexpression group. Combined overexpression of A53T/DK induced a behavioural phenotype close to PBS animals, even though DAT-BP nd -ratio was comparable to the G2019S-ΔLRRK2 group. Our results show that DAT-BP nd -ratios globally followed the same pattern as the cylinder test showed. Surprisingly, the post-mortem data obtained by unbiased TH + stereology showed an opposite pattern; a tendency for increased dopaminergic neurodegeneration after co-overexpression of A53T-α-synuclein and G2019S-ΔLRRK2 or DK-ΔLRRK2. Since viral vector load was equal in all groups, these results suggest an interplay between ΔLRRK2 and A53T-α-synuclein toxicity. These findings support the hypothesis from the paper (appendix 2, page 148); the C-terminal domain of G2019S-LRRK2 (G2019S-ΔLRRK2) is sufficient to augment the toxic effects of A53T-α-synuclein through a cell-autonomous mechanism involving the catalytic activity of its kinase domain. Furthermore, the effect of G2019S-ΔLRRK2 upon A53T-α-synuclein toxicity is likely specific or a particular interplay between these two proteins, and the crosstalk occurs within the same neurons. However, the results also indicdate that even in an experimental condition where the two proteins are expressed in high levels (approximately 5-50 fold the level of endogenous α-synuclein and LRRK2, respectively), the effect of G2019S-ΔLRRK2 on A53T-α-synuclein is moderate. Thus, it is reasonable to speculate that in a condition with physiological levels of expression of LRRK2 and α-synuclein, the cell-autonomous crosstalk between the two proteins might be of moderate importance in dopaminergic neurons. Non-cell autonomous mechanisms involving interaction of dopaminergic neurons with neighbouring glial cells and immune cells may have more important roles in patients and transgenic animal models (Cresto et al., in progress).

Discrepancies between in vivo and post-mortem data might be due to several compensation mechanisms. TH + stereology seems to suggest an increase of TH-positive neurons in the contralateral substantia nigra, which could be explained by an increase in TH expression per cell, resulting in greater visibility and thus a bias in TH stereological counting. Little is known about TH compensation effects in the substantia nigra. RT-qPCR analyses measuring at the mRNA levels of TH in the substantia nigra could elucidate this question. A few older reports have described TH compensation mechanisms in the substantia nigra in MPTP mice and humans. Greenwood and colleagues (1991) showed that in MPTP-treated mice cytoplasmic TH protein was increased, resulting in stable levels of dopamine and DOPAC even with the presence neuronal loss [START_REF] Greenwood | Increased dopamine synthesis in aging substantia nigra neurons[END_REF]. Joyce and colleagues (1997) demonstrated loss of TH protein in the substantia nigra of PD patients, accompanied by a severe loss of neurons expression TH and DAT mRNA. Remaining neurons showed a shift to higher concentrations of TH mRNA, but a shift to lower concentrations of DAT mRNA per cell [START_REF] Joyce | Differential modification of dopamine transporter and tyrosine hydroxylase mRNAs in midbrain of subjects with Parkinson's, Alzheimer's with parkinsonism, and Alzheimer's disease[END_REF].

The discrepancy between in vivo and post-mortem data could also suggest that ΔLRRK2 itself impacts different cell mechanisms at a functional level, influencing DAT-BP nd -ratios and behavioural phenotype, but not influence neuronal cell death. In support of this view, Zhou and colleagues (2011) have shown that temporal overexpression of G2019S-LRRK2 impairs DAT-mediated dopamine reuptake. However, they did not observe a direct interaction between DAT and G2019S-LRRK2 [START_REF] Zhou | Temporal expression of mutant LRRK2 in adult rats impairs dopamine reuptake[END_REF]. Morevover, two studies described reduced DAT-binding using PET imaging in asymptomatic LRRK2 mutation carriers [START_REF] Adams | PET in LRRK2 mutations: comparison to sporadic Parkinson's disease and evidence for presymptomatic compensation[END_REF][START_REF] Nandhagopal | Progression of dopaminergic dysfunction in a LRRK2 kindred: A multitracer PET study[END_REF], thus indicating that LRRK2 might influence DAT binding, but does not influence neuronal death. Interestingly, Penney and colleagues (2016) demonstrated compensation mechanisms in neuromuscular junctions of the fruitfly after post-synaptic LRRK2 overexpression; enhancement of presynaptic neurotransmitter release by increasing the size o the release ready pool of vesicles [START_REF] Penney | LRRK2 regulates retrograde synaptic compensation at the Drosophila neuromuscular junction[END_REF].

We cannot exclude compensation mechanisms of the contralateral hemisphere contributing to inter-animal or inter-group differences. Assessment of DAT protein levels in the striatum and mRNA expression in the substantia nigra, using immunohistochemical and biochemical techniques, respectively, could give more conclusive information about potential DAT compensation mechanisms in co-overexpression models of LRRK2 and α-synuclein.

We have shown that overexpression of A53T-α-synuclein, both alone and in combination with G2019S-ΔLRRK2, led to a significant motor deficit as measured with the cylinder test. Conversely, combined overexpression of A53T-α-synuclein and DK-ΔLRRK2 does not lead to a significant motor deficit. This could suggest that the dead kinase form of ΔLRRK2 might be neuroprotective. Others, indeed, observed that a dead kinase form of LRRK2 alone reduced neuronal toxicity [START_REF] Greggio | Kinase activity is required for the toxic effects of mutant LRRK2/dardarin[END_REF][START_REF] Smith | Kinase activity of mutant LRRK2 mediates neuronal toxicity[END_REF]. Additionally, LRRK2 knock-out rats show reduced neurodegeneration after α-synuclein overexpression compared to wild-type rats [START_REF] Daher | Abrogation of α-synuclein-mediated dopaminergic neurodegeneration in LRRK2-deficient rats[END_REF]. In a subsequent paper Daher and colleagues (2015) suggested that a LRRK2 kinase inhibitor has neuroprotective effects in wild-type rats overexpressing α-synuclein [START_REF] Daher | Leucine-rich Repeat Kinase 2 (LRRK2) Pharmacological Inhibition Abates α-Synuclein Gene-induced Neurodegeneration[END_REF]. These findings highlight the potential of LRRK2 as a putative therapeutic neuroprotective target.

Finally, we have explored different methods to analyse the acquired DAT-PET data; "standard" BP nd quantification, BP nd -ratios. Using BP nd quantification we were not able to show between-group differences, but we did observe a tendency for within-group asymmetry. To account for the variability in absolute BP nd values we have calculated BP nd -ratios. A next step could be to use statistical parametric mapping.

Previously, Casteels and colleagues (2006) used statistical parametric mapping in 6-OHDA rats to demonstrate severe hypometabolism in the ipsilateral sensorimotor cortex, using [ 18 F]FDG, and a striatal decrease in DAT availability, using [ 18 F]FECT [START_REF] Casteels | Construction and evaluation of multitracer small-animal PET probabilistic atlases for voxel-based functional mapping of the rat brain[END_REF]. Moreover, statistical parametric mapping has been used to quantify and map abnormalities in DAT activity in early PD patients [START_REF] Ma | Parametric mapping of [18F]FPCIT binding in early stage Parkinson's disease: a PET study[END_REF]. More recently, Wang and colleagues (2017) have demonstrated the use of statistical parametric mapping using [ 11 C]β-CFT (DAT), [ 11 C]Raclopride (D 2 R), and [ 18 F]FDG (glucose metabolism). They conclude that statistical parametric mapping of static PET/CT scan data is potentially very usefull for clinical use [START_REF] Wang | Suite PET/CT neuroimaging for the diagnosis of Parkinson's disease: statistical parametric mapping analysis[END_REF].

Conclusion

Taken together, these data are indicative of an interplay between ΔLRRK2 and A53T-αsynuclein toxicity. Moreover, they point towards an important role for the kinase domain of LRRK2 in this synergistic toxicity. This makes LRRK2, especially its kinase domain, an interesting target for therapeutic strategies. Future studies could shed more light on the role of compensation mechanism in this co-overexpression model.

Supplementary data: Voxel-wise BPnd-ratio changes between groups

In an attempt to pick up local, intra-striatal BP nd -ratio changes, we calculated parametric BP ndratio images. To that end, we flipped each individual image, co-registered both flipped and nonflipped images, and finally divided the original by the co-registered flipped image. The striatum contralateral to the injection side was masked (Figure 49A). SPM analysis using ANOVA was performed on the masked BP nd -ratio images of the ipsilateral striatum. Nevertheless the local differences lacked statistical power to reach a significance level of p<0.001 (Figure 49B, no correction for multiple comparisons, no cluster size). Nevertheless, T-score images reflect same tendency as observed with overall BPnd ratios. Additionnally, T-score images show that BPnd is not homogeneously decreased over the entire striatum, underlying the interest of voxel-wise analysis. The results are highly suggestive of regional differences between groups, and warrent further investigation.

Background and aims

Besides dopaminergic cell loss in the substantia nigra, a second hallmark of Parkinson's Disease (PD) is the presence of neuro-inflammation, more specifically activated microglial cells [START_REF] Mcgeer | Reactive microglia are positive for HLA-DR in the substantia nigra of Parkinson's and Alzheimer's disease brains[END_REF]. Nevertheless, it remains a major question in PD whether neuro-inflammation is a consequence or a cause of dopaminergic cell loss [START_REF] Tansey | Neuroinflammation in Parkinson's disease: Its role in neuronal death and implications for therapeutic intervention[END_REF]. In a healthy situation, glial cells support neuronal viability, while in an activated state they are responsible for pro-and antiinflammatory responses [START_REF] Kim | Regional difference in susceptibility to lipopolysaccharide-induced neurotoxicity in the rat brain: role of microglia[END_REF]. Literature suggests that glial activation and peripheral pro-inflammatory mediators contribute to the pathogenesis of PD [START_REF] Kempuraj | Neuroinflammation Induces Neurodegeneration[END_REF].

As outlined in the introduction, LPS can be considered as the model of choice to evaluate neuroinflammation induced neurodegeneration on the dopaminergic neurons. Susceptibility of dopaminergic neuronal loss is dependent on the injected dose, administration route and location, and the time-window during which the model is evaluated. This model is generally used to validate new PET radioligands for neuroinflammation [START_REF] Ory | PET imaging of TSPO in a rat model of local neuroinflammation induced by intracerebral injection of lipopolysaccharide[END_REF][START_REF] Pottier | PET imaging of cannabinoid type 2 receptors with[END_REF][START_REF] Sridharan | Comparative Evaluation of Three TSPO PET Radiotracers in a LPS-Induced Model of Mild Neuroinflammation in Rats[END_REF][START_REF] Berdyyeva | PET Imaging of the P2X7 Ion Channel with a Novel Tracer [18F]JNJ-64413739 in a Rat Model of Neuroinflammation[END_REF], or to evaluate neuroprotective or anti-inflammatory strategies.

Here, we aimed to characterize the neuro-inflammatory response and its impact on the dopaminergic neurons in a moderate intrastriatal LPS model, before induction of dopaminergic neurodegeneration. To that end, we combined in vivo PET imaging and post-mortem analyses.

Study design

Twelve adult male Sprague Dawley rats were injected unilaterally in the striatum with 10 µg lipopolysaccharides (LPS; Figure 50A), and were studied using in vivo PET imaging and post-mortem techniques. Animals underwent PET imaging at 5 or 7 days post injection (dpi), and were sacrificed immediately after completion of the PET scan (Figure 50B).

In vivo PET imaging was performed using a ligand for translocator protein (TSPO); [ 18 F]DPA714 [N,N-diethyl-2-(2-(4-(2-[18F]fluoroethoxy)phenyl)-5,7-dimethylpyrazolo [1,5-a]pyrimidin-3-yl)acetamide] ([ 18 F]DPA714) during 60 minute long continuous scans (n=9).

Immunohistochemistry was performed using primary antibodies directed against ionized calcium-binding adapter molecule 1 (IBA1), glial fibrillary acidic protein (GFAP), vimentin, dopamine transporter (DAT), and tyrosine hydroxylase (TH). RT-qPCR was performed using primers for aromatic L-amino acid decarboxylase (AADC), DAT, GFAP, IBA1, TH, tumour necrosis factor alpha (TNFα), 18 kDa translocator protein (TSPO), vimentin, and vesicular monoamine transporter 2 (VMAT2).

More detailed information on the methodology can be found in Chapter 2. Statistical analyses were performed using paired student t-tests to compare the ipsilateral and contralateral side in imaging studies, RT-qPCR, and immunohistochemical analyses. Inflammation markers are presented as %-change, while dopaminergic marker data are presented as asymmetry ratios:

C h a p t e r 6 -R e s u l t s o n n e u r o i n f l a m m a t i o n | 119

Results

In vivo and post-mortem analyses of neuroinflammation

PET imaging using [ 18 F]DPA714, a ligand for 18kD translocator protein (TSPO; Figure 51A), demonstrated a significant increase of the binding potential (BPnd) (Figure 51B; +262±74%; n=9; p=0.0011) in the LPS-injected striatum as compared to the contralateral non-injected side at 5 (n=3) or 7dpi (n=6). These results were coherent with those obtained by RT-qPCR, showing increased TSPO mRNA expression in the ipsilateral striatum (Figure 52B; +236%; n=12/11; p<<0.001). Post-mortem analyses of other inflammatory markers revealed a drastic increase in the density of activated microglia in the injected striatum using anti-IBA1 immunohistochemistry (Figure 52C; +322±25%; n=12; p<<0.001). These results were underlined by RT-qPCR, showing increased IBA1 mRNA expression in the ipsilateral striatum (Figure 52D; +371%; n=12/11; p<<0.001). Additionally, we observed an increased density of activated reactive astrocytes in the LPS-injected striatum C h a p t e r 6 -R e s u l t s o n n e u r o i n f l a m m a t i o n | 120 (Vimentin: Figure 52E; n=12; p<0.001, GFAP: Figure 52G; +107±11%; n=12; p<<0.001). These findings were consistent with those obtained by RT-qPCR, showing increased expression of vimentin and GFAP mRNA in the ipsilateral striatum (Vimentin: Figure 52F; n=12/10; p<<0.001, GFAP: Figure 52H; +181%; n=12/10; p<<0.001). In parallel, mRNA expression of the pro-inflammatory cytokine TNFα was strongly increased in the ipsilateral striatum (Figure 52I;+277%;n=11/11;p<<0.001).

A more comprehensive study of neuro-inflammation in this same LPS model was performed by my colleague Dr Gubinelli in the context of his PhD dissertation [START_REF] Gubinelli | Evaluation of an in situ polymerizing hydrogel scaffold as a brain delivery system for Parkinson's disease therapeutics[END_REF], which is added in the supplementary data (page 127).

C h a p t e r 6 -R e s u l t s o n n e u r o i n f l a m m a t i o n | 121

Post-mortem analyses: dopaminergic system

Although we were unable to quantify TH fibre loss in the striatum due to technical difficulties using immunohistochemistry, we observed a small, but significant decrease of TH mRNA of Δ28% in the injected striatum using RT-qPCR (Figure 53A; n=10/11; p=0.045).

Nevertheless, preliminary results obtained by Dr. Gubinelli at 30dpi indicated that there was neither a loss of TH fibre loss in the striatum (Figure 53B; n=9; p=0.060), nor a reduction in the number of TH-positive neurons in the substantia nigra pars compacta as measured by unbiased stereology (Figure 53C; n=6/8; p=0.26; unpaired student t-test). Immunohistological evaluation of the dopaminergic system did not reveal any significant asymmetry in the density of DAT protein in the LPS-injected striatum (Figure 54A; n=12; p=0.75). RT-qPCR analysis of DAT mRNA expression in the striatum confirmed these results (Figure 54B; n=3/3; p=0.50). It must be noted that the experimental number for the DAT RT-qPCR analysis was slightly lower due to some technical difficulties. Neither did we observe a significant asymmetry in AADC mRNA expression (Figure 54C

Correlation between in vivo and post-mortem data

Spearman correlation analysis of between post-mortem measures and in vivo individual TSPO-BP nd data showed postive associations between all different inflammation related markers (Figure 55A). Interestingly, expression of TNFα was highly correlated with expression of GFAP (r=0.96, Figure 55A,B), IBA1 (r=0.80, Figure 55A,B), and TSPO (r=0.81, Figure 55A,C). Consitent with these observations, TSPO-BP nd showed positive associations with the expression of inflammatory markers (r BP-TSPO = 0.9, r BP-TNFα = 0.85, r BP-GFAP = 0.84, r BP-Iba1 = 0.85, Figure 55A,C).

Amongst the dopaminergic expression markers only AADC and TH showed a positive association (Figure 55A,E). Additionally, expression of VMAT2, AADC, and TH showed comparable negative associations with the expression of inflammation-related markers, including BP nd (Figure 55A,D

Discussion

Inflammation in a striatal LPS model

We observed significantly increased TSPO binding in vivo, using [ 18 F]DPA714 PET imaging in the LPS-injected striatum at 5-7dpi. A likely interpretation of LPS-induced TSPO changes is an increase in the number of TSPO-expressing microglia as suggested in the peripheral induced LPS mouse model [START_REF] Furube | Brain Region-dependent Heterogeneity and Dose-dependent Difference in Transient Microglia Population Increase during Lipopolysaccharide-induced Inflammation[END_REF]. In line with this, it was shown in the LPS model using fluorescence-activated cell sorting on radioligand-treated tissue that not the radioactivity per microglial cell was increased, but the number of microglia per gram of tissue increased [START_REF] Tournier | Fluorescence-activated cell sorting to reveal the cell origin of radioligand binding[END_REF]. However, we cannot attribute the observed [ 18 F]DPA714 PET signal to microglia alone, since Lavisse and colleagues (2012) have shown that also reactive astrocytes overexpress TSPO and have a role in the [ 18 signal. Additionaly, our post-mortem data showed that LPS-induced neuroinflammation does not only lead to an increased density of activated microglia, but to increased densities of reactive astrocytes as well, which is in line with previous reports, showing that LPS-induced proinflammatory microglia can activate astrocytes [START_REF] Liddelow | Neurotoxic reactive astrocytes are induced by activated microglia[END_REF]. Ory and co-workers (2015) and Herera and colleagues (2000) reported on the presence of GFAP positive cells with low TSPO immunoreactivity in the periphery of the LPS-induced neuroinflammatory lesion [START_REF] Herrera | The single intranigral injection of LPS as a new model for studying the selective effects of inflammatory reactions on dopaminergic system[END_REF][START_REF] Ory | PET imaging of TSPO in a rat model of local neuroinflammation induced by intracerebral injection of lipopolysaccharide[END_REF]. Here we show, using a mild model of neuroinflammation, that astrocytes are present within the LPSinduced neuroinflammatory lesion, even though to a lesser extend than the presence of microglia. In addition, we showed a strong association between the expression of TSPO-and astrocytic markers.

These reactive astrocytes and microglia produce neurotoxic molecules which, in turn, can damage the blood-brain barrier (BBB) integrity (reviewed by [START_REF] Sofroniew | Astrocyte barriers to neurotoxic inflammation[END_REF]. Indeed, Ory and colleagues (2015), who studied a high-dose (50µg) LPS-induced inflammation model, demonstrated a loss of BBB integretry at the LPS-injected hemisphere at 7 days post-injection using dynamic contrast enhanced MRI. Even though we have not performed in vivo or ex vivo analyses of BBB integrity, Ory and colleagues (2015) have shown that despite BBB disruption in their model, it does not affect tracer uptake during PET imaging. They have demonstrated that TSPO binding and BBB breakdown followed different patterns, whilst in vivo PET data and post-mortem autoradiography data were positively correlated [START_REF] Ory | PET imaging of TSPO in a rat model of local neuroinflammation induced by intracerebral injection of lipopolysaccharide[END_REF].

It is known that activated microglia can have both beneficial and deleterious effects on neuronal survival [START_REF] Liu | Lipopolysaccharide Animal Models for Parkinson's Disease[END_REF]Kalia and Lang, 2015). The latter is attributed to the production of a variety of neurotoxic factors including interleukin-1 (IL-1), IL-6, tumour necrosis factor alpha (TNFα), nitric oxide, prostaglandin E2, and superoxide [START_REF] Kim | Microglia, major player in the brain inflammation: their roles in the pathogenesis of Parkinson's disease[END_REF]. These factors might cause neuronal damage, which, in turn, induces microglial activation, leading to a positive reinforcement loop [START_REF] Streit | Reactive microgliosis[END_REF][START_REF] Liu | Lipopolysaccharide Animal Models for Parkinson's Disease[END_REF].

We observed a strong increase of TNFα mRNA expression in the ipsilateral striatum. This proinflammatory cytokine is expressed by reactive microglia, which in turn activate astrocytes, but also by infiltrating macrophages. CD68 protein is highly expressed by circulating macrophages and tissue macrophages (e.g. microglia) [START_REF] Holness | Molecular cloning of CD68, a human macrophage marker related to lysosomal glycoproteins[END_REF]. Ory and colleagues (2015) observed CD68-positive cells in the ipsilateral striatum [START_REF] Ory | PET imaging of TSPO in a rat model of local neuroinflammation induced by intracerebral injection of lipopolysaccharide[END_REF], while Herrera and colleagues (2000) observed many integrin alpha M (ITGAM)-positive cells with macrophage morphology in the core of the lesion [START_REF] Herrera | The single intranigral injection of LPS as a new model for studying the selective effects of inflammatory reactions on dopaminergic system[END_REF]. This corresponds to the results presented here on TNFα mRNA, in combination with increased mRNA levels for TNFα and ITGAM shown by Mr. [START_REF] Gubinelli | Evaluation of an in situ polymerizing hydrogel scaffold as a brain delivery system for Parkinson's disease therapeutics[END_REF], this confirms that, at least part of, the macrophages contribute to the observed PET signal.

Dopaminergic markers

TH mRNA can normally be found in the striatum, in the axons of dopamine synthesising neurons [START_REF] Melia | Detection and regulation of tyrosine hydroxylase mRNA in catecholaminergic terminal fields: possible axonal compartmentalization[END_REF][START_REF] Aschrafi | Disruption of the axonal trafficking of tyrosine hydroxylase mRNA impairs catecholamine biosynthesis in the axons of sympathetic neurons[END_REF]. Our transcriptomic data revealed a small decrease of TH expression in the synaptic terminals of the striatum after striatal LPS injection, while preliminary data obtained at 30 days post-injection did not show any dopaminergic neurodegeneration, consistent with other studies using 10 μg striatal LPS-injection [START_REF] Hoban | Further characterisation of the LPS model of Parkinson's disease: A comparison of intra-nigral and intra-striatal lipopolysaccharide administration on motor function, microgliosis and nigrostriatal neurodegeneration in the rat[END_REF]. Additionally, we observed negative associations with the inflammatory expression markers. These data are suggestive of neuroinflammation-induced suffering of the dopaminergic afferents. We did not observe a reduction in striatal DAT protein levels, nor for striatal DAT, AADC, or VMAT mRNA levels. Nevertheless, we observed negative associations between the expression of TH, AADC, and VMAT2 and the inflammatory markers (e.g. IBA1, GFAP, and TNFα).

Previously, it has been shown that intranigral LPS injection induces reduced DAT mRNA expression in the substantia nigra [START_REF] De Pablos | Dopamine-dependent neurotoxicity of lipopolysaccharide in substantia nigra[END_REF], while again others suggest increased DAT activity in the nucleus accumbens and medial prefrontal cortex after peripheral LPS injection [START_REF] Van Heesch | Lipopolysaccharide increases degradation of central monoamines: an in vivo microdialysis study in the nucleus accumbens and medial prefrontal cortex of mice[END_REF].

Interestingly, Herrera and colleagues (2000) found increased levels of dopamine and DOPAC in the LPS-injected striatum [START_REF] Herrera | The single intranigral injection of LPS as a new model for studying the selective effects of inflammatory reactions on dopaminergic system[END_REF], while many others found increased levels of dopamine and/or DOPAC in several other brain regions after peripheral LPS administration [START_REF] Dunn | Endotoxin-induced activation of cerebral catecholamine and serotonin metabolism: comparison with interleukin-1[END_REF][START_REF] Lavicky | Endotoxin administration stimulates cerebral catecholamine release in freely moving rats as assessed by microdialysis[END_REF][START_REF] Borowski | Lipopolysaccharide, central in vivo biogenic amine variations, and anhedonia[END_REF][START_REF] Mohankumar | Lipopolysaccharide-induced changes in monoamines in specific areas of the brain: blockade by interleukin-1 receptor antagonist[END_REF][START_REF] Van Heesch | Lipopolysaccharide increases degradation of central monoamines: an in vivo microdialysis study in the nucleus accumbens and medial prefrontal cortex of mice[END_REF]. This could be explained by increased dopamine turnover, which can be be evaluated by measuring the dopamine concentration over DOPAC and HVA concentrations. An increased dopamine turnover could indicate increased TH activity and/or levels. Indeed, Nolan and colleagues (2000) demonstrated increased activity of TH after systemic LPS injection [START_REF] Nolan | Lipopolysaccharide administration produces time-dependent and regionspecific alterations in tryptophan and tyrosine hydroxylase activities in rat brain[END_REF]. However, this is in discrepancy with our results and of others [START_REF] De Pablos | Dopamine-dependent neurotoxicity of lipopolysaccharide in substantia nigra[END_REF], since we observed a small decrease of TH mRNA expression. Nevertheless, TH activity can be regulated independently by mRNA phosphorylation.

Others have reported on the associations between inflammation markers and markers for the dopaminergic system. A PET study in asymptomatic MPTP non-human primates demonstrated a decrease in VMAT BP nd which was associated with an increase in TSPO levels as shown with [ 11 C]PK11195. It was suggested that decreased VMAT2 is an early pathogenic event which might precede nigrostriatal degeneration [START_REF] Chen | VMAT2 and dopamine neuron loss in a primate model of Parkinson's disease[END_REF]. Furthermore, Glaab and colleagues (2019) observed both metabolic changes associated with inflammation and voxel-wise changes using [ 18 F]FDOPA-PET in PD patients [START_REF] Glaab | Integrative analysis of blood metabolomics and PET brain neuroimaging data for Parkinson's disease[END_REF].

Conclusion

In conclusion, we have used [ 18 F]DPA714 PET imaging to show increased TSPO binding in a LPSinduced neuroinflammatory rat model. We have shown that unilateral striatal injection of 10μg LPS does not lead to dopaminergic neuron loss in the substantia nigra nor fibre loss in the striatum. Even though, we have shown no changes in the dopaminergic markers, we demonstrated an inverse correlation between AADC, TH, and VMAT2 and IBA1 gene expression. In addition, these dopaminergic markers were inversely associated wiht TSPO-BP nd and microglial density. These data strongly support the hypothesis that neuroinflammatory events underlie suffering of dopaminergic neurons.

Figure 56). Additionally, Dr. [START_REF] Gubinelli | Evaluation of an in situ polymerizing hydrogel scaffold as a brain delivery system for Parkinson's disease therapeutics[END_REF] showed a significant increase of GFAP (Figure 57) and vimentin positive cells after LPS injection (Figure 58).

Moreover, several inflammation markers were assessed using RT-qPCR, all of which displayed a significant increase in the LPS-injected animals (Figure 59); C1qa and C1 are components of the complement system, CCL2 is recruites cells on the insult-site, IBA1 is a marker for microglial activation, IL1b is a mediator of inflammatory response, ITGAM is a marker for activated macrophages and phagocytosis, and TNFα is a proinflammatory cytokine. Conversely, NeuN, specific neuronal marker expressed in all striatal neurons, showed a significant decrease.

EVALUATION OF THE DOP AMINERGIC SYSTEM USI NG IN VIVO PET IMAGING AND POST-MORTEM ANALYSES IN THE CONTEXT OF MILD NEURODEG ENERATIVE AND NEUROI NFLAMMATORY PD MODELS

In this PhD thesis I aimed to evaluate the effects of α-synuclein overexpression and neuroinflammation on dopaminergic neuron loss and the dopaminergic system. To achieve this I have used both in vivo techniques, i.e. behavioural assessment and PET imaging, complimented with post-mortem techniques, i.e. immunohistochemistry and RT-qPCR, obtained from the same animals. By combinging these tools in the same animals, we also searched for a better comprehension of the underlying mechanisms of the in vivo biomarkers.

Interplay inflammation, α-synuclein, LRRK2, and neuronal loss

Using viral vector strategies, we have first overexpressed different forms of α-synuclein in the substantia nigra of rats, and later expressed α-synuclein in combination with different mutant forms of LRRK2. We have shown that overexpression of human WT-α-synclein in the substantia nigra through viral vectors (AAV2/6-PGK-WT-α-synuclein) does not generate detectable neuronal loss in the substantia nigra, nor does it generate in vivo motor deficits or changes in the dopaminergic system as seen by in vivo PET imaging. In contrast, overexpression of A53T-α-synuclein in the substantia nigra, using AAV2/6-PGK-A53T-α-synuclein viral vector approach, resulted in significant αsynuclein aggregation in the substantia nigra as soon as 8wpi, but not in the striatum. In our model, α-synuclein aggregation induced a mild but progressive degeneration of dopaminergic neurons in the substantia nigra but limited fibre loss in the striatum. This coincided with initially moderate, followed by a more pronounced, microglial response. These data suggest that, in this model, neuronal loss is induced by A53T-α-synuclein overexpression and that neuroinflammation might occur secondary to neuronal loss. Additionally, overexpression of AAV2/6-PGK-A53T-α-synuclein in combination with AAV2/6-PGK-G2019S-LRRK2 generated an added toxicity as demonstrated by unbiased stereology. LRRK2 interacts with α-synuclein via many direct and indirect pathways, which have recently been reviewed by Cresto and colleagues (2018). Our data show the potential of LRRK2 as a therapeutic target, but also underline the complexity of the use of in vivo biomarkers to accurately measure LRRK2-induced toxicity.

There are multiple putative mechanisms by which α-synuclein causes toxicity in the neurons, which ultimately leads to neurodegeneration. Amongst those, literature reports on genomic damage, mitochondrial dysfunction, lipid membrane interactions and disruption, lipid synthesis and metabolism, impaired vesicular trafficking, proteostasis disturbances, and nuclear dysfunction [START_REF] Zhang | New Perspectives on Roles of Alpha-Synuclein in Parkinson's Disease[END_REF][START_REF] Bernal-Conde | Alpha-Synuclein Physiology and Pathology: A Perspective on Cellular Structures and Organelles[END_REF][START_REF] Fanning | Parkinson's disease: proteinopathy or lipidopathy?[END_REF][START_REF] Han | Proteostasis of α-Synuclein and Its Role in the Pathogenesis of Parkinson's Disease[END_REF][START_REF] Vasquez | A multi-faceted genotoxic network of alpha-synuclein in the nucleus and mitochondria of dopaminergic neurons in Parkinson's disease: Emerging concepts and challenges[END_REF]. Interestingly, α-synuclein can also cause neuroinflammatory events by activating microglia and inducing migration [START_REF] Bliederhaeuser | Age-dependent defects of alpha-synuclein oligomer uptake in microglia and monocytes[END_REF][START_REF] Zhang | New Perspectives on Roles of Alpha-Synuclein in Parkinson's Disease[END_REF].

Moreover, it has been demonstrated that aggravated neuroinflammatory responses eventually lead to specific loss of the dopaminergic neurons, and therefore may have potential as therapeutic targets [START_REF] Wang | Neuroinflammation in Parkinson's disease and its potential as therapeutic target[END_REF]. To that end, LPS is the tool of choice to generate systemic and/or neuroinflammation and evaluate its impact on the survival of the dopaminergic neurons. Here we performed a single-site, unilateral injection of a low dose of LPS (10µg) in the striatum, to evaluate the impact of neuroinflammation on the expression of defined dopaminergic markers. We induced an acute neuroinflammatory reaction in the striatum, reaching maximum C h a p t e r 7 -G e n e r a l D i s c u s s i o n | 132 intensity at 7dpi without any dopaminergic neuronal loss in the substantia nigra or synaptic loss in the striatum at 7dpi nor at 30dpi. Nevertheless, we observed a significant inverse correlation between inflammation markers and markers for dopamine production (TH, AADC) and storage (VMAT2) in this inflammatory-based model. These data support the hypothesis that neuroinflammation alters dopaminergic markers and eventually can induce dopaminergic neuron loss. Epidemiological studies that have associated the frequent use of non-steroidal antiinflammatory drugs (e.g. aspirin, ibuprofen) with a lower risk for PD [START_REF] Chen | Nonsteroidal anti-inflammatory drugs and the risk of Parkinson disease[END_REF][START_REF] Chen | Nonsteroidal antiinflammatory drug use and the risk for Parkinson's disease[END_REF][START_REF] Noyce | Meta-analysis of early nonmotor features and risk factors for Parkinson disease[END_REF], are in support of these results, supporting the hypothesis that neuroinflammation is a potential target in PD therapeutical strategies.

PET imaging to measure PD progression

In all the above-mentioned models PET imaging was applied. We imaged the most commonly used presynaptic dopaminergic targets, measuring AADC enzymatic activity and DAT binding. Ont he other hand, we imaged neuroinflammation using the gold standard neuroinflammation marker, 18kD TSPO. DAT-PET imaging, using [ 18 F]LBT999, and AADC-PET, using [ 18 F]FMT, was performed in viral vector models transducing AAV2/6-PGK-WT-α-synuclein or AAV2/6-PGK-A53T-α-synuclein. DAT-PET imaging, but not AADC-PET imaging, was able to measure mild neuronal loss in the A53T-α-synuclein model as soon as 8 weeks post-injection (wpi). We observed a larger asymmetry at 12wpi, which corresponded to an increased dopaminergic neuronal loss. Here, we showed that DAT-PET was more sensitive than robust behavioural testing. Surprisingly, DAT-PET imaging on models overexpressing A53T-α-synuclein in combination with G2019S-or dead kinase-ΔLRRK2 overexpression, showed asymmetrical DAT-PET binding per group, however, we were unable do demonstrate between-group differences. Statistical parametric mapping analyses on DAT BP nd -ratios underlined regional heterogeneity of DAT BP nd -ratios within the striatum. In addition, we showed that DAT BP nd -ratios followed the same tendency as behavioural measurements. Surprisingly, in this study, in vivo data followed the opposite pattern of post-mortem data.

The combination of in vivo and post-mortem data in our studies suggests that DAT-PET does not merely reflect neuronal loss, but might be influenced by α-synuclein accumulation [START_REF] Kobayashi | Extracellular α-synuclein enters dopaminergic cells by modulating flotillin-1-assisted dopamine transporter endocytosis[END_REF] and regulatory gene expression mechanisms [START_REF] Shumay | Genomic features of the human dopamine transporter gene and its potential epigenetic states: Implications for phenotypic diversity[END_REF]. Compensatory mechanisms are being reported in the literature to occur at molecular and synaptic levels, both pre-and postsynaptically of the dopaminergic neuron to counterbalance the loss of the dopaminergic neuron [START_REF] Adams | PET in LRRK2 mutations: comparison to sporadic Parkinson's disease and evidence for presymptomatic compensation[END_REF][START_REF] Blesa | Compensatory mechanisms in Parkinson's disease: Circuits adaptations and role in disease modification[END_REF][START_REF] Fu | Joint pattern analysis applied to PET DAT and VMAT2 imaging reveals new insights into Parkinson's disease induced presynaptic alterations[END_REF].

Postsynaptic compensation mechanisms are mainly reported at the level of the dopamine receptor; demonstrating increased binding to post-synaptic D 2 /D 3 R as observed in asymptomatic 6-OHDA-lesioned rats in both the ipsilateral and contralateral hemisphere [START_REF] Nikolaus | Bilateral increase in striatal dopamine D2 receptor density in the 6hydroxydopamine-lesioned rat: a serial in vivo investigation with small animal PET[END_REF][START_REF] Sahin | Differential dopamine receptor occupancy underlies L-DOPA-induced dyskinesia in a rat model of Parkinson's disease[END_REF]. Bezard and colleagues (2001) showed biphasic compensation mechanisms of D 2 R-like biding using autoradiography in MPTP-treated non-human primates; an initial decrease in the presymptomatic period was followed by an upregulation of postsynaptic receptors commencing when striatal dopaminergic homeostasis is broken [START_REF] Bezard | Relationship between the appearance of symptoms and the level of nigrostriatal degeneration in a progressive 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned macaque model of Parkinson's disease[END_REF]. PET imaging studies visualising D 2 /D 3 R can benefit from these post-synaptic compensation mechanisms to measure the functional outcome of endogenous dopamine replenishment after gene therapy [START_REF] Palfi | Long-term safety and tolerability of ProSavin, a lentiviral vector-based gene therapy for Parkinson's disease: a dose escalation, open-label, phase 1/2 trial[END_REF] or cell replacement therapies [START_REF] Sahin | Differential dopamine receptor occupancy underlies L-DOPA-induced dyskinesia in a rat model of Parkinson's disease[END_REF].

C h a p t e r 7 -G e n e r a l D i s c u s s i o n | 133 Presynaptic compensation mechanisms have been reported at the level of dopamine synthesis, but also at the level of DAT expression and availability. These compensatory mechanisms occur to counterbalance the dopaminergic loss [START_REF] Blesa | Compensatory mechanisms in Parkinson's disease: Circuits adaptations and role in disease modification[END_REF][START_REF] Herfert | Quantitative Rodent Brain Receptor Imaging[END_REF].

Even though we were not able to demonstrate significantly increased expression of TH in the contralateral substantia nigra, the left-right asymmetry measured in the A53T-α-synuclein model at 12wpi might possibly be due to an upregulation in the contralateral side rather than an increased neuronal loss ipsilateral to AAV-induced A53T-α-synuclein overexpression.

Comparison of in vivo and post-mortem data at 8wpi revealed that the asymmetry observed with both behaviour (Δ76±23%) and DAT-PET (Δ33±7%) was larger than observed with post-mortem stereological counting (Δ17±5%). At this timepoint, no change was observed in DAT gene-, or protein expression. At the late timepoint (13wpi), the behavioural deficit remained stable (Δ79±22%) but the asymmetry observed in DAT-PET was more pronounced (Δ54±8%). Again, both in vivo measures revealed higher differences than post-mortem DAT gene-and protein expression data, which were both around similar levels (Δ30%). Interestingly, DAT post-mortem data were coherent with stereological counting in the SN (Δ33±10%) and TH optical density data in the striatum (Δ40±12%).

This phenomenon of exacerbated DAT decrease was also observed MPTP-treated non-human primates [START_REF] Bezard | Relationship between the appearance of symptoms and the level of nigrostriatal degeneration in a progressive 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned macaque model of Parkinson's disease[END_REF]. Reduced DAT availability has been demonstrated to reduce presynaptic dopamine uptake, in order to increase dopamine availability in the synapse [START_REF] Vezoli | Increased DAT binding in the early stage of the dopaminergic lesion: A longitudinal [11C]PE2I binding study in the MPTP-monkey[END_REF][START_REF] Blesa | Compensatory mechanisms in Parkinson's disease: Circuits adaptations and role in disease modification[END_REF]. Recently, a DAT-SPECT study on 105 de novo PD patient demonstrated that lower dopamine transporter binding in early-onset PD predicts the later development of motor complications. In this study, DAT-binding was not related to severity of motor symptoms, suggesting age-related differences in striatal compensatory mechanisms in PD [START_REF] Palermo | Dopamine Transporter, Age, and Motor Complications in Parkinson's Disease: A Clinical and Single-Photon Emission Computed Tomography Study[END_REF]. Nevertheless, other studies report on adverse compensation effects. Del-Bel and colleagues (2014) observed a decrease of dopamine concentration in the ipsilateral striatum after a 6-OHDA lesioning in rats, accompanied by an increase of dopamine concentration in the contralateral striatum [START_REF] Del-Bel | Counteraction by nitric oxide synthase inhibitor of neurochemical alterations of dopaminergic system in 6-OHDA-lesioned rats under L-DOPA treatment[END_REF]. Similarly, Vezoli and colleagues (2014) show an unexpected increase of DAT-BP nd in the striatum using [ 11 C]PE2I before the onset of motor symptoms in MPTP treated non-human primates [START_REF] Vezoli | Increased DAT binding in the early stage of the dopaminergic lesion: A longitudinal [11C]PE2I binding study in the MPTP-monkey[END_REF]. In our co-overexpression model of A53T-α-synuclein and DK-or G2019S-ΔLRRK2, DAT BP nd -ratios also demonstrated an opposite tendency as observed by post-mortem data. Previously, Adams and colleagues (2005) demonstrated in a multitracer (AADC and DAT) PET study that the in vivo phenotype of LRRK2-patients is indistinguishable from that of sporadic PD patients, despite the pathological heterogeneity of the condition [START_REF] Adams | PET in LRRK2 mutations: comparison to sporadic Parkinson's disease and evidence for presymptomatic compensation[END_REF].

Alltogether these data suggest that compensatory mechanisms of DAT availability occur very early in the pathology, and probably at the level of the synapse before the appearance of compensatory mechanism regulated by gene expression. It has been suggested that these compensatory changes, including downregulation of the DAT and upregulation of AADC activity, might delay the onset of parkinsonian symptoms [START_REF] Adams | PET in LRRK2 mutations: comparison to sporadic Parkinson's disease and evidence for presymptomatic compensation[END_REF], and probably have a major impact on therepeutical approaches for PD [START_REF] Navntoft | How compensation breaks down in Parkinson's disease: Insights from modeling of denervated striatum[END_REF].

In contrast to DAT, VMAT2 density is highly related to terminal degeneration and VMAT2-PET imaging might be less prone to presynaptic compensation events [START_REF] Decressac | Comparison of the behavioural and histological characteristics of the 6-OHDA and α-synuclein rat models of Parkinson's disease[END_REF] In our studies, post-mortem data on VMAT2 protein-density did not demonstrate any left-right difference at either 8 or 13wpi. Similar to DAT gene-expression, VMAT2 geneexpression showed a significant left/right difference (Δ42%) at the late timepoint. Unfortunately, we did not have acces to VMAT2-PET radioligands. However, [ 18 to decrease in striatal regions of PD patients [START_REF] Okamura | In vivo measurement of vesicular monoamine transporter type 2 density in Parkinson disease with 18F-AV-133[END_REF][START_REF] Lin | In vivo detection of monoaminergic degeneration in early parkinson disease by 18F-9-fluoropropyl-(1)-dihydrotetrabenzazine PET[END_REF], which was correlated with disease severity [START_REF] Hsiao | Correlation of Parkinson disease severity and 18F-DTBZ positron emission tomography[END_REF].

All together, this underlies the use of multitracer PET studies (e.g. [START_REF] Fu | Joint pattern analysis applied to PET DAT and VMAT2 imaging reveals new insights into Parkinson's disease induced presynaptic alterations[END_REF] to contribute in imaging the patterns of different mechanisms underlying disease initiation or progression.

Future studies

In future studies I would recommend to investigate the nature of the small inflammatory response that was observed in the A53T-α-synuclein overexpression model, as was done for the LPS model. Vice versa, I would recommend to study the dopaminergic system more in depth in the LPSinduced inflammation model, especially using in vivo DAT versus VMAT-PET imaging. Additionally, I think studying the development of the LPS model, using a longitudinal design, would be beneficial, and could possibly provide answers about the cause and consequence of neuroinflammation and neurodegeneration.

In the context of the BrainMatTrain program I have focussed on the characterisation of rodent PD models and the understanding of in vivo biomarkers, with the overall aim to use these models for therapeutical assessement of multi-modal hydrogels containing cell grafts. The models described here all have their own strengths and weaknesses with regards to the evaluation of multi-model hydrogels.

The intanigral 6-OHDA model that was used here for methodological improvements generates near complete loss of the dopaminergic neurons in the substantian nigra and the synaptic terminals. This makes the 6-ODHA model very suitable to study graft survival, striatal re-innervation, and functional efficacy [START_REF] Moriarty | Encapsulation of primary dopaminergic neurons in a GDNF-loaded collagen hydrogel increases their survival, re-innervation and function after intrastriatal transplantation[END_REF][START_REF] Moriarty | Encapsulation of young donor age dopaminergic grafts in a GDNF-loaded collagen hydrogel further increases their survival, reinnervation, and functional efficacy after R e f e r e n c e s | 202 intrastriatal transplantation in hemi-Parkinsonian rats[END_REF]. However, even though it is the gold-standard model for PD, it does not generate its extensive neuronal loss in a pathologically relevant way. Here, we have implementen several models based on hallmarks of PD; neuroinflammation and PD-related gene overexpression. As discussed above, the LPS-based neuroinflammation model allows the study of effects of neuroinflammation on dopaminergic cell and fibre loss. This model would be well suited to study the anti-inflammatory effects of a multi-modal hydrogel.

Lastly, we have used overexpression of PD-related genes through viral vectors. AAV2/6-PGK-WT-α-synuclein did not generate neuronal loss in our hands, making it less ideal to study multi-model hydrogels. AAV2/6-PGK-A53T-α-synuclein transduction, on the other hand, did generate significant progressive dopaminergic neuronal loss, changes in the dopaminergic system as seen by in vivo PET imaging, and a slight neuroinflammation. These qualities give the A53T-α-synuclein great potential to evaluate the efficacy neuroprotective and disease altering therapeutic strategies. The AAV2/6-PGK-A53T-α-synuclein model can also be used to study neurotoxic or neuroprotectice interactive effects, as we have aimed to for A53T-α-synuclein and G2019S-LRRK2 [START_REF] Cresto | The C-terminal domain of LRRK2 with the G2019S mutation is sufficient to produce neurodegeneration of dopaminergic neurons in vivo[END_REF]. Moreover, the relatively slow disease progression also means that it is possible to study the model in a prodromallike phase, where less than 30% of the dopaminergic neurons have died. This could be especially useful in the study of early biomarkers for PD. neurotransmitters and receptors [START_REF] Pienaar | Parkinson's disease: Insights from non-traditional model organisms[END_REF][START_REF] Duty | Animal models of Parkinson's disease: A source of novel treatments and clues to the cause of the disease[END_REF]. The connectome of C. elegans has been fully mapped; however it has a limited size of 302 neurons, of which only 8 are dopaminergic [START_REF] White | The Structure of the Nervous System of the Nematode Caenorhabditis elegans[END_REF][START_REF] Pienaar | Parkinson's disease: Insights from non-traditional model organisms[END_REF][START_REF] Duty | Animal models of Parkinson's disease: A source of novel treatments and clues to the cause of the disease[END_REF][START_REF] Konnova | Animal Models of Parkinson's Disease, Parkinson's Disease: Pathogenesis and Clinical Aspects[END_REF]. C. elegans has homologues for parkin, LRRK2, PINK1, and DJ-1, thus accounting for most of the major PD genes [START_REF] Duty | Animal models of Parkinson's disease: A source of novel treatments and clues to the cause of the disease[END_REF][START_REF] Jagmag | Evaluation of Models of Parkinson's Disease[END_REF][START_REF] Cooper | Modeling Parkinson's disease in C. elegans[END_REF]. Dopaminergic neurons of C. elegans exposed the neurotoxins 6-hydroxydopamine (6-OHDA) and rotenone underwent apoptosis [START_REF] Nass | Neurotoxin-induced degeneration of dopamine neurons in Caenorhabditis elegans[END_REF], while MPTP leads to decrease motility and degeneration of dopaminergic neurons [START_REF] Braungart | Caenorhabditis elegans MPP+ Model of Parkinson's Disease for High-Throughput Drug Screenings[END_REF]. The discrepancy between motor symptoms and dopaminergic cells death shows that there is no clear-cut correlation between the two in C. elegans. C. elegans expressing wild-type or mutated human SNCA show behavioural deficits and reduced neuronal dopamine levels [START_REF] Kuwahara | Familial Parkinson mutant alpha-synuclein causes dopamine neuron dysfunction in transgenic Caenorhabditis elegans[END_REF][START_REF] Cooper | Modeling Parkinson's disease in C. elegans[END_REF]. The later could be rescued by administration of dopamine [START_REF] Kuwahara | Familial Parkinson mutant alpha-synuclein causes dopamine neuron dysfunction in transgenic Caenorhabditis elegans[END_REF].

RNA interference can easily be applied, since the nematodes can be fed on agar plates containing bacteria that express small interfering RNA (siRNA), to create knock-downs of targeted genes [START_REF] Pienaar | Parkinson's disease: Insights from non-traditional model organisms[END_REF]. RNA interference can be used on a large scale to screens for genes involved in specific pathways. For example, a RNA interference screen has identified a handful of genes that caused severe growth-and motor abnormalities SNCA transgenic C. elegans after knock-down [START_REF] Kuwahara | A systematic RNAi screen reveals involvement of endocytic pathway in neuronal dysfunction in α-synuclein transgenic C . elegans[END_REF].

Although C. elegans shows promising PD models, it must be noted that the anatomical and functional connectivity is much simpler than in mammals, and the relation of behavioural deficits might be limited [START_REF] Pienaar | Parkinson's disease: Insights from non-traditional model organisms[END_REF][START_REF] Duty | Animal models of Parkinson's disease: A source of novel treatments and clues to the cause of the disease[END_REF]. However, C. elegans could be used to study the interaction between environment and genetic factors [START_REF] Duty | Animal models of Parkinson's disease: A source of novel treatments and clues to the cause of the disease[END_REF]. For example; DJ-1 and parkin mutations have been shown to increase the susceptibility to rotenone [START_REF] Ved | Similar Patterns of Mitochondrial Vulnerability and Rescue Induced by Genetic Modification of α-Synuclein, Parkin, and DJ-1 in Caenorhabditis elegans[END_REF]. A more elaborate review of the use of C. elegans in PD research has been written by [START_REF] Cooper | Modeling Parkinson's disease in C. elegans[END_REF].

Fruit fly (Drosophila melanogaster)

The fruit fly (Drosophila melanogaster) has a significantly bigger neuron pool than C. elegans, containing around 135.000 neurons, which is currently being mapped [START_REF] Konnova | Animal Models of Parkinson's Disease, Parkinson's Disease: Pathogenesis and Clinical Aspects[END_REF], and includes dopaminergic neurons. As in vertebrates, dopamine modulates motor behaviour and circadian rhythms, amongst others [START_REF] Riemensperger | Behavioral consequences of dopamine deficiency in the Drosophila central nervous system[END_REF]. This motor ability can be affected by oral administration of paraquat, an herbicide and neurotoxin (see below); Drosophila exposed to paraquat developed parkinsonian symptoms including resting tremor, bradykenesia, and postural instability. This phenotype could be rescued by treatment with dopamine or levodopa. Interestingly, male Drosophila where earlier and more severely affected than their female counterparts [START_REF] Chaudhuri | Interaction of genetic and environmental factors in a Drosophila parkinsonism model[END_REF]. Similarly, rotenone (another neurotoxin, see below) induced dopaminergic neurodegeneration and motor impairment, the latter could be rescued by levodopa [START_REF] Coulom | Chronic exposure to rotenone models sporadic Parkinson's disease in Drosophila melanogaster[END_REF].

Drosophila has homologues for most PD-related genes, i.e. PINK1, Parkin, DJ-1, and LRRK2 [START_REF] Whitworth | Drosophila models pioneer a new approach to drug discovery for Parkinson's disease[END_REF], and models of PD are most commonly genetic models. Although no homologue for the SNCA gene is present in Drosophila, the human wild-type or mutant form can be over-expressed in transgenic flies. [START_REF] Feany | A Drosophila model of Parkinson's disease[END_REF] have shown that this leads to adult onset dopaminergic neuron loss, motor dysfunction (reversible by levodopa ( [START_REF] Feany | A Drosophila model of Parkinson's disease[END_REF]. The Drosophila genetic toolbox allows for tissue or neuron specific dominant mutations on one hand, and targeted point mutations or deletions for recessive mutations on the other hand [START_REF] West | Neurophysiology of Drosophila models of Parkinson's disease[END_REF]. See [START_REF] Pienaar | Parkinson's disease: Insights from non-traditional model organisms[END_REF] and [START_REF] West | Neurophysiology of Drosophila models of Parkinson's disease[END_REF] for more detailed reviews on genetic PD research in Drosophila. In short, parkin, LRRK2, and PINK1 mutations all lead to a reduced amount dopaminergic neurons and behavioural phenotypes, which can be reversed by levodopa, while DJ-1 mutations have no effect on this [START_REF] Duty | Animal models of Parkinson's disease: A source of novel treatments and clues to the cause of the disease[END_REF].

The short lifespan and easy husbandry of Drosophila make them great models for rapid screening of pharmacological therapeutics [START_REF] Duty | Animal models of Parkinson's disease: A source of novel treatments and clues to the cause of the disease[END_REF]. Although several pathways are conserved, the specific modifiers for PD still need to be verified in human PD. Another major downside of Drosophila is the lack of a α-synuclein homologue [START_REF] Dawson | Genetic Animal Models of Parkinson's Disease[END_REF].

Zebrafish (Danio rerio)

The zebrafish (Danio rerio) is a vertebrate species with a high reproduction rate and small adult size (3-4cm), which allows for simple and economic husbandry. Additionally, the embryos are transparent and develop outside the mother [START_REF] Pienaar | Parkinson's disease: Insights from non-traditional model organisms[END_REF]. The zebrafish brain shows important similarity to the human brain; i.e. homologues for midbrain and ventral tegmental neurons, and a striatum-like region in the procencaphalon (Rink andWullimann, 2001, 2002;[START_REF] Pienaar | Parkinson's disease: Insights from non-traditional model organisms[END_REF]. Furthermore, zebrafish have homologues for several PD related genes, most notably parkin, PINK1, DJ-1, and LRRK2 [START_REF] Jagmag | Evaluation of Models of Parkinson's Disease[END_REF]. Antisense morpholino oligonucleotides can be used, via microinjection in to single-cell embryos, to knockdown PD-related genes. This has been successfully shown for DJ-1 in combination with H 2 O 2 and proteasome inhibitor MG132; leading to widespread neurodegeneration [START_REF] Bretaud | p53-dependent neuronal cell death in a DJ-1-deficient zebrafish model of Parkinson's disease[END_REF], parkin; leading to dopaminergic neurodegeneration, which can be exacerbated by MPTP [START_REF] Flinn | Complex I deficiency and dopaminergic neuronal cell loss in parkin-deficient zebrafish (Danio rerio)[END_REF], and PINK1; leading to disrupted patterning of dopaminergic neurons and motor dysfunction [START_REF] Xi | Impaired dopaminergic neuron development and locomotor function in zebrafish with loss of pink1 function[END_REF]. Interestingly, LRRK2 seems the most promising gene to model PD in zebrafish [START_REF] Duty | Animal models of Parkinson's disease: A source of novel treatments and clues to the cause of the disease[END_REF]; deletion of the WD40 domain resulted in dopaminergic neuron loss and motor dysfunction [START_REF] Xi | Modeling Neurodegeneration in Zebrafish[END_REF].

Due to the aquatic nature of zebrafish, hydrophilic substances can easily be added to the water and subsequently be absorbed through the skin and gills. This can be utilised to create models or perform drug screens and tests [START_REF] Pienaar | Parkinson's disease: Insights from non-traditional model organisms[END_REF]. For example, the neurotoxin MPTP reduces catecholaminergic innervations in larvae [START_REF] Thirumalai | Endogenous Dopamine Suppresses Initiation of Swimming in Prefeeding Zebrafish Larvae[END_REF], and induces dopaminergic neurodegeneration in zebrafish embryos, shown as a reduction of TH+ cells [START_REF] Lam | Zebrafish embryos are susceptible to the dopaminergic neurotoxin MPTP[END_REF]. Interestingly, exposure to exogenous dopamine reduced spontaneous swimming behaviour in zebrafish larvae, while pharmacologically blocking D 2 -Receptors increased this swimming behaviour [START_REF] Thirumalai | Endogenous Dopamine Suppresses Initiation of Swimming in Prefeeding Zebrafish Larvae[END_REF]. Alternatively, zebrafish can be injected with toxic substances; both 6-OHDA and MPTP significantly reduced the levels of dopamine and noradrenaline in adult brains, while also negatively affecting motor behaviour [START_REF] Anichtchik | Neurochemical and behavioural changes in zebrafish Danio rerio after systemic administration of 6-hydroxydopamine and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine[END_REF], while Rotenone and paraquat seemed to have no significant effects [START_REF] Bretaud | Sensitivity of zebrafish to environmental toxins implicated in Parkinson's disease[END_REF].

A major downside of the zebrafish as a model organism for PD is that many genetic mutation result in affected swimming behaviour, however, the neural mechanism for this behaviour is currently unknown, thus limiting the translatability to humans. Although less used than the above described species, anurans (frogs and toads) have some valuable characteristics; they are easily raised, produce enormous single-celled eggs, have external development, have relatively large embryos which are easy to manipulate, and have transparent tadpoles [START_REF] Pienaar | Parkinson's disease: Insights from non-traditional model organisms[END_REF]. Additionally, the entire genome of Xenopus laevis has been sequenced [START_REF] Session | Genome evolution in the allotetraploid frog Xenopus laevis[END_REF]. This includes a parkin homolog that might be subject to posttranslational modifications like phosphorylation [START_REF] Horowitz | Immunodetection of Parkin protein in vertebrate and invertebrate brains: a comparative study using specific antibodies[END_REF]. Anurans have a brain region homologues to the striatum [START_REF] Endepols | Dorsal striatopallidal system in anurans[END_REF] and the substantia nigra pars compacta, containing dopaminergic neurons that innervate the striatum [START_REF] Marin | Basal Ganglia Organization in Amphibians: Development of Striatal and Nucleus Accumbens Connections With Emphasis on the Catecholaminergic Inputs[END_REF]. The substantia nigra is named for its darkly pigmented colour in humans, however most traditional model animals have very little to no neuromelanin. Since anurans do express neuromelanin they might be very suited to study the role of neuromelanin in PD [START_REF] Pienaar | Parkinson's disease: Insights from non-traditional model organisms[END_REF].

Bilateral 6-OHDA injection, discussed in more detail below, causes a behavioural deficit as well as cognitive symptoms, and a decrease in TH-positive cells in Hyla versicolor [START_REF] Endepols | 6-hydroxydopamine lesions in anuran amphibians: A new model system for Parkinson's disease?[END_REF]. Alternatively, consecutive intraperitoneal injections of MPTP, see below, caused numerous motor deficits, including rigidity, slowness, and freezing in Rana Clamitans Clamitans and Rana Pipiens. The dopamine concentration and melanin index were also affected by the MPTP injections (A Barbeau et al., 1985;A. Barbeau et al., 1985). Interestingly, paraquat causes similar symptoms in Rana Pipiens, but dopamine concentrations first rise before dropping (A Barbeau et al., 1985).

Pharmacological models

Mammalian models have the benefit of resembling the human body and brain more closely. Many well-established experimental models exist for rodents. And while mice have the advantage of easier genomic manipulation, rats have a larger brain, which allows for better stereotactic accuracy [START_REF] Cherry | Use of positron emission tomography in animal research[END_REF].

The first models to be used were pharmacological models. They generally cause dopamine depletion in the striatum, leading to PD-like motor symptoms. The downsides of pharmacological models are reversibility of the effects and relative short periods effect of time. Additionally, they do not cause neurodegeneration in the nigrostriatal pathway. Despite the limited applicability of the these model, they have led to some ground-breaking results.

Reserpine

The reserpine models was already used as early as 1957 (Carlsson, Lindqvist and Magnusson), and is thus the earliest model used in PD research [START_REF] Duty | Animal models of Parkinson's disease: A source of novel treatments and clues to the cause of the disease[END_REF][START_REF] Tieu | A guide to neurotoxic animal models of Parkinson's disease[END_REF]. Reserpine's mode of action is through temporary blocking vesicular monoamine transporter (VMAT2) [START_REF] Duty | Animal models of Parkinson's disease: A source of novel treatments and clues to the cause of the disease[END_REF] through magnesium and ATP-dependent mechanisms [START_REF] Bezard | Experimental models of Parkinson's disease: From the static to the dynamic[END_REF], which leads to global depletion of monoamines, e.g. noradrenaline, serotonin, and dopamine [START_REF] Duty | Animal models of Parkinson's disease: A source of novel treatments and clues to the cause of the disease[END_REF]. This temporary depletion of catecholamines has been shown to lead to PDlike motor deficits in many mammals, including mouse, rat, and monkey [START_REF] Bezard | Experimental models of Parkinson's disease: From the static to the dynamic[END_REF]. In their ground-breaking research Carlsson and colleagues showed that these motor symptoms can be successfully reversed by levodopa [START_REF] Carlsson | 3,4-Dihydroxyphenylalanine and 5-Hydroxytryptophan as Reserpine Antagonists[END_REF]. The reserpine model has also greatly contributed to our knowledge of the link between monoamines and PD [START_REF] Duty | Animal models of Parkinson's disease: A source of novel treatments and clues to the cause of the disease[END_REF]. The effects of reserpine are relatively transient, excluding the possibility to test repeated drug administration [START_REF] Duty | Animal models of Parkinson's disease: A source of novel treatments and clues to the cause of the disease[END_REF][START_REF] Tieu | A guide to neurotoxic animal models of Parkinson's disease[END_REF]. However, the biggest drawback of the reserpine model is the lack of dopaminergic cell loss in the substantia nigra, which limits the therapeutic strategies to symptomatic treatments. Indeed, the reserpine model has led to many of the dopaminergic drugs currently used in clinics (e.g. pramipexole, ropinirole, and levodopa), thus showing the strong predictive value of this model [START_REF] Duty | Animal models of Parkinson's disease: A source of novel treatments and clues to the cause of the disease[END_REF].

Haloperidol

Like reserpine model, the haloperidol model has already been used since a long time. Haloperidol's mode of action is through antagonizing dopamine D 2 (and D 1 ) receptor in medium spiny neurons, which results in a block of striatal dopamine transmission, leading to muscle rigidity and catalepsy (posture fixation) in rodents [START_REF] Kobayashi | EFFECTS OF L-DOPA AND BROMOCRIPTINE ON HALOPERIDOL-INDUCED MOTOR DEFICITS IN MICE[END_REF][START_REF] Bezard | Experimental models of Parkinson's disease: From the static to the dynamic[END_REF][START_REF] Duty | Animal models of Parkinson's disease: A source of novel treatments and clues to the cause of the disease[END_REF]. Additionally, acute administration of haloperidol leads to depletion of striatal dopamine, noradrenalin, and serotonin in rats [START_REF] Kulkarni | In vivo microdialysis studies of striatal level of neurotransmitters after haloperidol and chlorpromazine administration[END_REF].

Again, like reserpine, the effects of haloperidol are transient, which limits their usefulness in long-term studies. Additionally, they only display limited symptomatic overlap with PD, limiting the use in neuroprotective and neuroreparative studies [START_REF] Bezard | Experimental models of Parkinson's disease: From the static to the dynamic[END_REF][START_REF] Duty | Animal models of Parkinson's disease: A source of novel treatments and clues to the cause of the disease[END_REF]. Despite the limited applicability, several drugs currently in use have shown efficacy in the haloperidol model (e.g. levodopa and carbidopa [START_REF] Kobayashi | EFFECTS OF L-DOPA AND BROMOCRIPTINE ON HALOPERIDOL-INDUCED MOTOR DEFICITS IN MICE[END_REF], and pramipexole [START_REF] Maj | The behavioural effects of pramipexole, a novel dopamine receptor agonist[END_REF]).

Racemetirosine

Racemetirosine, also called α-methyl-ρ-tyrosine, inhibits tyrosine hydroxylase (TH), and thus dopamine and noradrenaline synthesis [START_REF] Spector | BLOCKADE OF ENDOGENOUS NOREPINEPHRINE SYNTHESIS BY ALPHA-METHYL-TYROSINE, AN INHIBITOR OF TYROSINE HYDROXYLASE[END_REF][START_REF] Bezard | Experimental models of Parkinson's disease: From the static to the dynamic[END_REF][START_REF] Duty | Animal models of Parkinson's disease: A source of novel treatments and clues to the cause of the disease[END_REF][START_REF] Tieu | A guide to neurotoxic animal models of Parkinson's disease[END_REF]. Similar as described above, this leads to a transient dopamine depletion without any neuronal loss [START_REF] Tieu | A guide to neurotoxic animal models of Parkinson's disease[END_REF]. Racemetirosine can be used in combination with reserpine to potentiate its effects [START_REF] Carlsson | Dramatic synergism between MK-801 and clonidine with respect to locomotor stimulatory effect in monoamine-depleted mice[END_REF][START_REF] Duty | Animal models of Parkinson's disease: A source of novel treatments and clues to the cause of the disease[END_REF].

Toxin-based mammalian models

Before the first genes involved in PD were identified, neurotoxin models were used to study the disease. Unlike pharmacological models, toxin-based models generally do cause neurodegeneration of the nigrostriatal pathway. While most of these models have the drawback to only portray PD in a limited, singular way, they have still taught us valuable lessons about PD. Of the neurotoxins, 6-hydroxydopamine (6-OHDA) was the first to be discovered and is the classic rodent model [START_REF] Jackson-Lewis | Animal models of Parkinson's disease[END_REF], followed by [START_REF] Chartier-Harlin | alpha-Synuclein locus duplication as a cause of familial Parkinson's disease[END_REF]3,6tetrahydropyridine (MPTP), which alone is clearly linked to human parkinsonism [START_REF] Dauer | Parkinson's Disease: Mechanisms and Models[END_REF]. Most of the other toxins used to model PD are pesticide based [START_REF] Jackson-Lewis | Animal models of Parkinson's disease[END_REF] and are believed to act through reactive oxygen species (ROS) formation [START_REF] Dauer | Parkinson's Disease: Mechanisms and Models[END_REF].

MPTP

See Chapter 1: 2.2, page 24.

Rotenone

Rotenone is a naturally occurring broad spectrum insecticide and pesticide [START_REF] Dauer | Parkinson's Disease: Mechanisms and Models[END_REF][START_REF] Tieu | A guide to neurotoxic animal models of Parkinson's disease[END_REF][START_REF] Jackson-Lewis | Animal models of Parkinson's disease[END_REF][START_REF] Jagmag | Evaluation of Models of Parkinson's Disease[END_REF][START_REF] Konnova | Animal Models of Parkinson's Disease, Parkinson's Disease: Pathogenesis and Clinical Aspects[END_REF]. Rotenone acts through inhibition of mitochondrial complex I, at the same site as MPP+, and thereby blocking the mitochondrial electron transport chain [START_REF] Dauer | Parkinson's Disease: Mechanisms and Models[END_REF][START_REF] Duty | Animal models of Parkinson's disease: A source of novel treatments and clues to the cause of the disease[END_REF][START_REF] Jagmag | Evaluation of Models of Parkinson's Disease[END_REF]. Rotenone is highly lipophilic and can thus easily cross the blood-brain barrier and enter cells, allowing for easy systemic injections [START_REF] Betarbet | Chronic systemic pesticide exposure reproduces features of Parkinson's disease[END_REF]. Its short half-life (3-5 days) necessitates repeated injections [START_REF] Jackson-Lewis | Animal models of Parkinson's disease[END_REF]. Chronic systemic rotenone exposure has been shown to cause dopaminergic neuron degeneration as well as motor symptoms and formation of intracellular Lewybody-like inclusions [START_REF] Betarbet | Chronic systemic pesticide exposure reproduces features of Parkinson's disease[END_REF].

However, there are several problems with the rotenone rat model which shake the validity of the model [START_REF] Cicchetti | Environmental toxins and Parkinson's disease: what have we learned from pesticide-induced animal models?[END_REF]. Firstly, it is difficult to replicate studies due to the high mortality rate [START_REF] Fleming | Behavioral and immunohistochemical effects of chronic intravenous and subcutaneous infusions of varying doses of rotenone[END_REF], due to systemic toxicity [START_REF] Duty | Animal models of Parkinson's disease: A source of novel treatments and clues to the cause of the disease[END_REF]. Secondly, it has been shown that chronic administration at low doses causes motor deficit, while failing to show dopaminergic cell dead [START_REF] Fleming | Behavioral and immunohistochemical effects of chronic intravenous and subcutaneous infusions of varying doses of rotenone[END_REF]. This could either point towards early stage PD modelling, or the involvement of other pathways and cell types. Thirdly, even though chronic rotenone treatment has been associated with sleep disturbances, an equally large effect has been shown after vehicle treatment (DMSO/PEG), meaning that these sleep disturbances cannot be attributed to dopaminergic cell loss [START_REF] García-García | Sleep disturbances in the rotenone animal model of Parkinson disease[END_REF]. Lastly, besides dopaminergic cell death, many other cell types and regions have been shown to be affected by chronic rotenone treatment, suggesting more general mitochondrial failure is involved than previously assumed [START_REF] Höglinger | Chronic systemic complex I inhibition induces a hypokinetic multisystem degeneration in rats[END_REF]. A redeeming quality is the extensive microglial activation in both the substantia nigra and striatum [START_REF] Sherer | Selective microglial activation in the rat rotenone model of Parkinson's disease[END_REF]. Despite the limitations, pesticide models like rotenone can be used to study the effect of environmental factors on PD, for example the interaction with other risk factors like age or genetic mutations [START_REF] Konnova | Animal Models of Parkinson's Disease, Parkinson's Disease: Pathogenesis and Clinical Aspects[END_REF], or the relationship between aggregate formation and neuronal death [START_REF] Dauer | Parkinson's Disease: Mechanisms and Models[END_REF]. N,N'-dimethyl-4-4-4'-bipiridinium (paraquat) is a widely used herbicide that shows structural resemblance to MPP+ [START_REF] Snyder | Neurology: Predicting Parkinson's disease[END_REF]. But whereas MPP+ acts through blockage of mitochondrial complex I [START_REF] Nicklas | Inhibition of NADH-linked oxidation in brain mitochondria by 1-methyl-4-phenyl-pyridine, a metabolite of the neurotoxin, 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine[END_REF], paraquat acts through redox mediated oxidative stress [START_REF] Bonneh-Barkay | Redox cycling of the herbicide paraquat in microglial cultures[END_REF], generating reactive oxygen species, leading to damage of lipids, proteins, DNA, and RNA [START_REF] Jackson-Lewis | Animal models of Parkinson's disease[END_REF]. It is still unclear how paraquat crosses the blood-brain barrier [START_REF] Konnova | Animal Models of Parkinson's Disease, Parkinson's Disease: Pathogenesis and Clinical Aspects[END_REF], but it appears to be mediated by the neutral amino acid transport system [START_REF] Shimizu | Carrier-mediated processes in blood-brain barrier penetration and neural uptake of paraquat[END_REF]. Unlike MPP+, paraquat is A p p e n d i x 1 -A n i m a l m o d e l s o f P D | 142 not a substrate for Dopamine Transporter, raising the question how paraquat enters dopaminergic neurons [START_REF] Tieu | A guide to neurotoxic animal models of Parkinson's disease[END_REF].

Paraquat

Chronic systemic injections of paraquat in mice [START_REF] Mccormack | Environmental risk factors and Parkinson's disease: selective degeneration of nigral dopaminergic neurons caused by the herbicide paraquat[END_REF] and rats [START_REF] Cicchetti | Systemic exposure to paraquat and maneb models early Parkinson's disease in young adult rats[END_REF] have been shown to lead to dopaminergic cell loss in the substantia nigra. Interestingly, in mice, this is not accompanied by dopamine depletion in the striatum or motor deficits, which the authors speculate might be due to compensatory mechanisms [START_REF] Mccormack | Environmental risk factors and Parkinson's disease: selective degeneration of nigral dopaminergic neurons caused by the herbicide paraquat[END_REF]. However, higher α-synuclein levels have been observed, as well as aggregate formation [START_REF] Manning-Bog | The Herbicide Paraquat Causes Up-regulation and Aggregation of α-Synuclein in Mice[END_REF]. In rats, again, there was no dopamine depletion observed, but some motor deficits were present. Additionally, microglial activation was observed, indicating that dopaminergic cell loss might be due to inflammation [START_REF] Cicchetti | Systemic exposure to paraquat and maneb models early Parkinson's disease in young adult rats[END_REF].

It has to be kept in mind that high doses of paraquat cause pulmonary fibrosis [START_REF] Bismuth | Paraquat Poisoning[END_REF], which could show PD-unrelated motor deficits [START_REF] Konnova | Animal Models of Parkinson's Disease, Parkinson's Disease: Pathogenesis and Clinical Aspects[END_REF]. Additionally, there is some controversy about the cell death and specificity or paraquat [START_REF] Jackson-Lewis | Animal models of Parkinson's disease[END_REF][START_REF] Jagmag | Evaluation of Models of Parkinson's Disease[END_REF]. Paraquat's strengths lie in the possibility to study Lewy body formation and the role of α-synuclein [START_REF] Jackson-Lewis | Animal models of Parkinson's disease[END_REF].

Other toxin-based models

Maneb 2-(dithiocarboxy)aminoethylcarbamodithioato(2-)-kS, kS' manganese is a fungicide more commonly known as Maneb [START_REF] Jagmag | Evaluation of Models of Parkinson's Disease[END_REF]. It preferentially inhibits complex III of the mitochondrial respiration chain [START_REF] Zhang | Manganese ethylene-bis-dithiocarbamate and selective dopaminergic neurodegeneration in rat: a link through mitochondrial dysfunction[END_REF]. Maneb has been shown to evoke a motor deficit in mice, while no decrease in tyrosine hydroxylase (TH) or DAT immunoreactivity were observed [START_REF] Thiruchelvam | The nigrostriatal dopaminergic system as a preferential target of repeated exposures to combined paraquat and maneb: Implications for Parkinson's disease[END_REF]. The same group has also shown that Maneb can exacerbate the effect of paraquat; only when both toxins were combined they observed a reduction in TH protein levels [START_REF] Thiruchelvam | The nigrostriatal dopaminergic system as a preferential target of repeated exposures to combined paraquat and maneb: Implications for Parkinson's disease[END_REF]. In Sprague-Dawley rats the results were more variable, however, a reduction in striatal dopamine levels was observed [START_REF] Tinakoua | The impact of combined administration of paraquat and maneb on motor and non-motor functions in the rat[END_REF]. DOPAL 3,4-dihydroxyphenylacetaldehyde (DOPAL) is an intermediate of the dopamine metabolism. DOPAL is created by oxidative deamination of dopamine by monoamine oxidase. DOPAL is normally cleaved by aldehyde dehydrogenase (ALDH1A1) into 3,4-dihydroxyphenylacetic acid (DOPAC) [START_REF] Panneton | The neurotoxicity of DOPAL: Behavioral and stereological evidence for its role in Parkinson disease pathogenesis[END_REF]. Increased levels of DOPAL [START_REF] Mattammal | Confirmation of a dopamine metabolite in parkinsonian brain tissue by gas chromatography-mass spectrometry[END_REF] and decreased levels of ALDH1A1 [START_REF] Galter | ALDH1 mRNA: Presence in human dopamine neurons and decreases in substantia nigra in Parkinson's disease and in the ventral tegmental area in schizophrenia[END_REF] have been reported in PD patients. Panneton and colleagues (2010) have shown a decrease of dopaminergic neurons in the substantia nigra of rats following DOPAL injections into the same region. Additionally, they show a motor deficit and reduced striatal dopamine levels [START_REF] Panneton | The neurotoxicity of DOPAL: Behavioral and stereological evidence for its role in Parkinson disease pathogenesis[END_REF], as well as α-synuclein accumulation [START_REF] Burke | Aggregation of α-synuclein by DOPAL, the monoamine oxidase metabolite of dopamine[END_REF].

Amphetamines; Methamphetamine and MDMA

Methamphetamine can enter dopaminergic neurons via uptake by dopamine transporter, or via diffusion [START_REF] Tieu | A guide to neurotoxic animal models of Parkinson's disease[END_REF]. Inside the cells, methamphetamine interacts with VMAT2, which incapacitates the vesicular proton gradient, leading to dopamine oxidation in the cytosol, generating reactive oxygen species and leading to oxidative stress [START_REF] Larsen | Methamphetamine-induced degeneration of dopaminergic neurons involves autophagy and upregulation of dopamine synthesis[END_REF]. Additionally, this dopamine release from the vesicles into the cytosol leads to an influx of dopamine into the synaptic cleft, via reverse transport through DAT [START_REF] Sulzer | Amphetamine and Other Weak Bases Act to Promote Reverse Transport of Dopamine in Ventral Midbrain Neurons[END_REF] increase locomotor activity [START_REF] Tieu | A guide to neurotoxic animal models of Parkinson's disease[END_REF]. Interestingly, it is unclear if methamphetamine toxicity originates from oxidative stress or from hyperthermia [START_REF] Tieu | A guide to neurotoxic animal models of Parkinson's disease[END_REF].

Repeated administration of methamphetamine has been shown to cause a continuing reduction of striatal dopamine levels in rats (and guinea pig) [START_REF] Wagner | Methamphetamine-induced changes in brain catecholamines in rats and guinea pigs[END_REF], as well as TH levels in the substantia nigra [START_REF] Trulson | Effects of chronic methamphetamine on the nigral-striatal dopamine system in rat brain: Tyrosine hydroxylase immunochemistry and quantitative light microscopic studies[END_REF]. Methamphetamine acts more upon the dopaminergic nerve terminals than the cell bodies, unlike paraquat (cell bodies), and MPTP and 6-OHDA (both) [START_REF] Tieu | A guide to neurotoxic animal models of Parkinson's disease[END_REF]. Although, this model is not believed to be very reliable [START_REF] Jackson-Lewis | Animal models of Parkinson's disease[END_REF], some believe it might hold value in combination with other neurotoxins likes MPTP or paraquat [START_REF] Jagmag | Evaluation of Models of Parkinson's Disease[END_REF] 3,4-methylenedioxymethamphetamine (MDMA or Ecstasy) is an amphetamine analogue that causes reduced levels of TH and DAT in the striatum [START_REF] Granado | Early loss of dopaminergic terminals in striosomes after MDMA administration to mice[END_REF] and substantia nigra of mice [START_REF] Granado | Persistent MDMA-induced dopaminergic neurotoxicity in the striatum and substantia nigra of mice[END_REF]. Notably, while rats, non-human primates, and humans show severe effects of MDMA, the damage is localized to the serotonin system, with the dopaminergic system being spared [START_REF] Green | The Pharmacology and Clinical Pharmacology of 3,4-Methylenedioxymethamphetamine (MDMA, "Ecstasy")[END_REF].

Isoquinoline derivatives

Isoquinoline and its derivatives are naturally occurring in plants and animals, including in mammalian brains. Some derivatives can cross the blood-brain barrier, but they can also be synthesized from dopamine in the brain [START_REF] Tieu | A guide to neurotoxic animal models of Parkinson's disease[END_REF]. These compounds are structurally similar to MPTP, and their mechanism of toxicity has been shown to be very similar to MPP+, i.e. affinity for dopamine transporter and complex I inhibition [START_REF] Tieu | A guide to neurotoxic animal models of Parkinson's disease[END_REF]. However, derivatives can have different effects in different animal species, and, more importantly, they can easily be converted into other derivatives, some of which might even have neuroprotective properties [START_REF] Tieu | A guide to neurotoxic animal models of Parkinson's disease[END_REF]. It was also described that isoquinoline derivatives down-regulate tyrosine hydroxylase, thus confounding neuronal loss with TH down-regulation [START_REF] Lorenc-Koci | Effect of 1,2,3,4,-tetrahydroisoquinoline administration under conditions of CYP2D inhibition on dopamine metabolism, level of tyrosine hydroxylase protein and the binding of [3H]GBR 12,935 to dopamine transporter in the rat nigrostriatal, dopaminergic sy[END_REF].

PD hallmark-based models

While neurotoxin-based PD models are currently the most widely used models, they do not offer much information about disease progression and reversal of neuronal damage. While these models have taught us about the pathogenic mechanisms underlying neurodegeneration in PD, they have not shown us how to stop or slow its progression. Another way to constitute PD models is focussed on its hallmarks; formation of Lewy bodies and presence of activated microglia. These models have a high construct validity [START_REF] Duty | Animal models of Parkinson's disease: A source of novel treatments and clues to the cause of the disease[END_REF].

Proteasomal inhibitors

The work on proteasomal inhibitors to model PD is fuelled by two key findings from last decade; a mutation in α-synuclein (A53T) responsible for a familial from of PD [START_REF] Polymeropoulos | Mutation in the α-Synuclein Gene Identified in Families with Parkinson's Disease[END_REF], and the presence of α-synuclein in Lewy bodies [START_REF] Spillantini | a-Synuclein in Lewy bodies[END_REF]. The discovery of two additional mutations of the ubiquitin-proteasome system (UCH-L1 [START_REF] Leroy | The ubiquitin pathway in Parkinson's disease[END_REF], and parkin [START_REF] Kitada | Mutations in the parkin gene cause autosomal recessive juvenile parkinsonism[END_REF]) in familial forms is PD further propelled interest [START_REF] Duty | Animal models of Parkinson's disease: A source of novel treatments and clues to the cause of the disease[END_REF].

In vitro, proteasomal inhibitors (lactacystin and ubiquitin aldehyde) caused concentrationdependent dopaminergic neurodegeneration. Lactacystin administration, affecting 26/20S proteasomes, was shown to lead to accumulation of α-synuclein and ubiquitin, as well as Lewy body-A p p e n d i x 1 -A n i m a l m o d e l s o f P D | 144 like inclusions (Kevin St P. McNaught et al., 2002). Subsequently, the same group was able to show that lactacystin injection into the substantia nigra of rats leads to PD-associated motor symptoms, reversible by apomorphine. Similarly as in vitro, in vivo they observed dose-dependent dopaminergic neurodegeneration and the aggregation of α-synuclein into Lewy body-like inclusions (Kevin St. P. McNaught et al., 2002). Later work of the group showed similar effects using systemic injection of epoxomicin (naturally occurring proteasome inhibitor) or PSI (synthetic) in adult rats [START_REF] Mcnaught | Systemic exposure to proteasome inhibitors causes a progressive model of Parkinson's disease[END_REF]. Animals developed progressive motor-symptoms reversible by apomorphine, striatal dopamine depletion, dopaminergic cells loss, and Lewy body-like inclusion containing α-synuclein. Interestingly, this model also showed neuroinflammation in the substantia nigra [START_REF] Mcnaught | Systemic exposure to proteasome inhibitors causes a progressive model of Parkinson's disease[END_REF].

Although proteasomal inhibition to model PD seemed very promising, other groups were unable to replicate McNaughts' findings using PSI [START_REF] Kordower | Failure of proteasome inhibitor administration to provide a model of Parkinson's disease in rats and monkeys[END_REF][START_REF] Manning-Bo | Lack of nigrostriatal pathology in a rat model of proteasome inhibition[END_REF]. If the variability issue with proteasomal inhibitors can be addressed it could be a valuable model to test neuroprotective strategies due to its high construct validity [START_REF] Duty | Animal models of Parkinson's disease: A source of novel treatments and clues to the cause of the disease[END_REF].

Lipopolysaccharide

See Chapter 1: 2.3, page 25.

Genetic-based models

The discovery of a mutation (A53T) in the α-synuclein gene (SNCA) that is responsible for a familial from of PD [START_REF] Polymeropoulos | Mutation in the α-Synuclein Gene Identified in Families with Parkinson's Disease[END_REF] led to the use of genetic based models. Even though genetic cases of PD are relatively rare and represent only a small group of patients [START_REF] Dauer | Parkinson's Disease: Mechanisms and Models[END_REF][START_REF] Jackson-Lewis | Animal models of Parkinson's disease[END_REF], several autosomal dominant (SNCA and LRRK2) and autosomal recessive (PINK1, Parkin, DJ-1) mutations have been identified [START_REF] Jackson-Lewis | Animal models of Parkinson's disease[END_REF]. Studying genetic alterations of PD-related genes can reveal information about molecular pathways involved in PD [START_REF] Cooper | Modeling Parkinson's disease in C. elegans[END_REF].

α-synuclein

See Chapter 1: 2.4, page 26.

LRRK2

See Chapter 1: 2.5, page 27.

Autosomal recessive genes

Apart from the aforementioned autosomal dominant genes, there are several autosomal recessive PD-related genes. Of these, Parkin is the most common, accounting for almost half of the early-onset cases of PD [START_REF] Jackson-Lewis | Animal models of Parkinson's disease[END_REF]; others are PINK1 and DJ-1.

Parkin

Mutations in the Parkin gene (PARK2) [START_REF] Kitada | Mutations in the parkin gene cause autosomal recessive juvenile parkinsonism[END_REF] account for almost half of the earlyonset familial cases of PD [START_REF] Lücking | Association between Early-Onset Parkinson's Disease and Mutations in the Parkin Gene[END_REF][START_REF] Jackson-Lewis | Animal models of Parkinson's disease[END_REF], and there are over 100 mutations known for the Parkin gene [START_REF] Lücking | Association between Early-Onset Parkinson's Disease and Mutations in the Parkin Gene[END_REF], most of which are A p p e n d i x 1 -A n i m a l m o d e l s o f P D | 145 loss-of-function [START_REF] Konnova | Animal Models of Parkinson's Disease, Parkinson's Disease: Pathogenesis and Clinical Aspects[END_REF]. Parkin is involved in the ubiquitin proteasome system as an E3 ubiquitin ligase [START_REF] Shimura | Familial Parkinson disease gene product, parkin, is a ubiquitin-protein ligase[END_REF][START_REF] Zhang | Parkin functions as an E2-dependent ubiquitin-protein ligase and promotes the degradation of the synaptic vesicle-associated protein, CDCrel-1[END_REF]. In Drosophila, Parkin mutations lead to a reduced amount of dopaminergic neurons, as well behavioural phenotypes [START_REF] Duty | Animal models of Parkinson's disease: A source of novel treatments and clues to the cause of the disease[END_REF], while Parkin knockout flies were shown to have decreased TH levels and dopaminergic neurodegeneration [START_REF] Dawson | Genetic Animal Models of Parkinson's Disease[END_REF]. However, most knockout models of Parkin in mice did not result in any nigrostriatal, dopaminergic, or behavioural pathology [START_REF] Perez | Parkin-deficient mice are not a robust model of parkinsonism[END_REF]. Interestingly, overexpression of mutated (Q311X) Parkin in mice leads to dopaminergic neuronal loss in the substantia nigra, synaptic loss in the striatum, a reduction of striatal dopamine levels, Lewy body-like inclusions, and behavioural deficits [START_REF] Lu | Bacterial Artificial Chromosome Transgenic Mice Expressing a Truncated Mutant Parkin Exhibit Age-Dependent Hypokinetic Motor Deficits, Dopaminergic Neuron Degeneration, and Accumulation of Proteinase K-Resistant -Synuclein[END_REF]. Conversely, more recently, wild-type and mutant Parkin overexpression were shown to induce moderate neurodegeneration in rats [START_REF] Van Rompuy | Long-Term Overexpression of Human Wild-Type and T240R Mutant Parkin in Rat Substantia Nigra Induces Progressive Dopaminergic Neurodegeneration[END_REF].

PINK1

The gene for Phosphatase and tensin (PTEN) homolog-induced novel kinase 1 (PINK1) is the second most commonly mutated in early-onset PD [START_REF] Konnova | Animal Models of Parkinson's Disease, Parkinson's Disease: Pathogenesis and Clinical Aspects[END_REF]. Similar to Parkin, PINK1 mutations are recessive, and lead to loss-of-function [START_REF] Dawson | Genetic Animal Models of Parkinson's Disease[END_REF]. PINK1 is normally localised in the mitochondria [START_REF] Silvestri | Mitochondrial import and enzymatic activity of PINK1 mutants associated to recessive parkinsonism[END_REF], where it recruits Parkin and increases its ubiquination activity and induces Parkin-mediated mitophagy [START_REF] Lazarou | PINK1 drives Parkin self-association and HECT-like E3 activity upstream of mitochondrial binding[END_REF]. Again like Parkin, Pink1 mutations in Drosophila lead to a reduced amount of dopaminergic neurons, as well behavioural phenotypes [START_REF] Duty | Animal models of Parkinson's disease: A source of novel treatments and clues to the cause of the disease[END_REF], with a small reduction of dopaminergic neurons [START_REF] Dawson | Genetic Animal Models of Parkinson's Disease[END_REF]. And again Pink1 knockout mice do not exhibit a major pathology [START_REF] Kitada | Impaired dopamine release and synaptic plasticity in the striatum of PINK1deficient mice[END_REF]. However, more recently it was shown Pink1 knockout in rats leads to progressive dopaminergic neurodegeneration, as well motor deficits [START_REF] Dave | Phenotypic characterization of recessive gene knockout rat models of Parkinson's disease[END_REF]. Additionally, Pink1 knockout and knockdown mice were shown to be more sensitive to systemic MPTP treatment, resulting in greater neurodegeneration in the substantia nigra and reduced fibre density in the striatum [START_REF] Haque | Inactivation of Pink1 Gene in Vivo Sensitizes Dopamine-producing Neurons to 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and Can Be Rescued by Autosomal Recessive Parkinson Disease Genes, Parkin or DJ-1[END_REF]. This effect could be reversed by viral mediated overexpression of Parkin or DJ-1 (see below). This suggests a role for Pink1 in neuronal survival, and similar roles for Parkin and DJ-1, either acting parallel or downstream of Pink1 [START_REF] Haque | Inactivation of Pink1 Gene in Vivo Sensitizes Dopamine-producing Neurons to 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and Can Be Rescued by Autosomal Recessive Parkinson Disease Genes, Parkin or DJ-1[END_REF].

DJ-1

DJ-1 is a redox sensitive molecular chaperone [START_REF] Moore | Lessons from Drosophila models of DJ-1 deficiency[END_REF], whose mutations play a role in early-onset PD [START_REF] Bonifati | Mutations in the DJ-1 gene associated with autosomal recessive early-onset parkinsonism[END_REF]; causing loss of function [START_REF] Macedo | The DJ-1L166P mutant protein associated with early onset Parkinson's disease is unstable and forms higher-order protein complexes[END_REF][START_REF] Moore | A missense mutation (L166P) in DJ-1, linked to familial Parkinson's disease, confers reduced protein stability and impairs homo-oligomerization[END_REF]. DJ-1 plays a role in the inhibition of α-synuclein aggregation [START_REF] Shendelman | DJ-1 is a redox-dependent molecular chaperone that inhibits alphasynuclein aggregate formation[END_REF]. It is expressed widely throughout the whole body, and localised in the cytosol, and mitochondria [START_REF] Zhang | Mitochondrial localization of the Parkinson's disease related protein DJ-1: implications for pathogenesis[END_REF]. In Drosophila, DJ-1 knockdown in dopaminergic neurons (through RNAi) led to age-dependent dopaminergic loss [START_REF] Yang | Inactivation of Drosophila DJ-1 leads to impairments of oxidative stress response and phosphatidylinositol 3-kinase/Akt signaling[END_REF]. As seen in Parkin and Pink1, DJ-1 knockout mice did not display any major abnormalities in dopaminergic neuronal survival [START_REF] Goldberg | Nigrostriatal dopaminergic deficits and hypokinesia caused by inactivation of the familial Parkinsonism-linked gene DJ-1[END_REF]. More recently it has been shown that DJ-1 knockout rats show progressive dopaminergic neurodegeneration and motor abnormalities [START_REF] Dave | Phenotypic characterization of recessive gene knockout rat models of Parkinson's disease[END_REF]. Interestingly, knockdown of DJ-1 in mice increased sensitivity to MPTP [START_REF] Kim | Hypersensitivity of DJ-1-deficient mice to 1-methyl-4-phenyl-1,2,3,6tetrahydropyrindine (MPTP) and oxidative stress[END_REF].

Even though the autosomal recessive models or Parkin, PINK1, and DJ-1 do not seem very promising or useful at first sight, they could shed light on the early stages of nigrostriatal dysfunction [START_REF] Dawson | Genetic Animal Models of Parkinson's Disease[END_REF]. The lack of neurodegeneration in rodent knockout models could point towards compensatory mechanisms which prevents manifestation of PD symptoms (Jackson-A p p e n d i x 1 -A n i m a l m o d e l s o f P D | 146 [START_REF] Jackson-Lewis | Animal models of Parkinson's disease[END_REF]. In the future, studying mutations observed in PD patients could be of interest to better understand the mechanisms of Parkin, PINK1, and DJ-1 [START_REF] Dawson | Genetic Animal Models of Parkinson's Disease[END_REF]. Moreover, these genes are of particular interest to evaluate the interaction between genetic mutations and environmental factors. Early in the course of the disease, the most obvious symptoms are movementrelated, including shaking (resting tremor), rigidity, and slowness of movement. The neuropathological hallmarks of PD are characterized by the progressive loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNc) and the presence of neuronal aggregates (Lewy bodies) and dystrophic Lewy neurites containing the protein α--syn) [1]. There is no treatment to delay -syn aggregation and the preferential death of DA neurons is unknown. PD is mainly a sporadic neurodegenerative disorder but approximately 10% of the cases are of genetic origin. Several genes have been identified as causative factors.

THE C-TERMINAL FRAGMENT OF LRRK2 WITH THE G2019S

Duplication, triplication, and rare mutations (A53T, A30P, E46K, H50Q, G51D, A53E) in the SNCA -syn protein have been found in families with dominantly-inherited PD and are associated with early-onset forms of PD, -syn aggregation [START_REF] Chartier-Harlin | alpha-Synuclein locus duplication as a cause of familial Parkinson's disease[END_REF][3][START_REF] Lesage | Parkinson's disease: from monogenic forms to genetic susceptibility factors[END_REF][START_REF] Singleton | alpha-Synuclein locus triplication causes Parkinson's disease[END_REF]. The A53T [START_REF] Polymeropoulos | Mutation in the α-Synuclein Gene Identified in Families with Parkinson's Disease[END_REF], A30P [START_REF] Krüger | AlaSOPro mutation in the gene encoding α-synuclein in Parkinson's disease[END_REF] and E46K [START_REF] Zarranz | The new mutation, E46K, of α-synuclein causes parkinson and Lewy body dementia[END_REF] substitutions have been the most studied so far. Compelling evidence -syn takes center stage in PD and plays a key role via various aggregated forms, including abnormally phosphorylated aggregates that produce multiple cellular alterations, eventually leading to the death of DA neurons [START_REF] Saito | Accumulation of Phosphorylated α-Synuclein in Aging Human Brain[END_REF].

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of both familial and sporadic PD [START_REF] Pais N-Ru Z | Cloning of the Gene Containing Mutations that Cause PARK8-Linked Parkinson's Disease[END_REF]11]. There are also variants in the LRRK2 locus that are considered to be risk factors for developing PD. The most prevalent mutation in LRRK2 is the G2019S substitution, accounting for 5 to 6% of AD familial PD and 1 to 2% of de novo generic PD cases [START_REF] Gilks | A common LRRK2 mutation in idiopathic Parkinson's disease[END_REF]13]. The cases of patients harboring the G2019S and other mutations are clinically indistinguishable from idiopathic PD cases, including the presence of Lewy bodies (LBs) in most cases [START_REF] Biskup | Zeroing in on LRRK2-linked pathogenic mechanisms in Parkinson's disease[END_REF][START_REF] Yahalom | Motor progression of Parkinson's disease with the leucine-rich repeat kinase 2 G2019S mutation: Disease progression in PD with G2019S mutation[END_REF]. Although G2019S patients show similar clinical manifestations as those of sporadic patients [START_REF] Cresto | The Unlikely Partnership Between LRRK2 and α-Synuclein in Parkinson's Disease[END_REF], several studies have shown subtle differences [START_REF] Ben Romdhan | LRRK2 G2019S Parkinson's disease with more benign phenotype than idiopathic[END_REF]18]. Some have reported the presence of LBs in symptomatic LRRK2 mutation carriers and LRRK2 can be found in LBs [START_REF] Zimprich | Mutations in LRRK2 cause autosomal-dominant parkinsonism with pleomorphic pathology[END_REF], although this is still a subject of debate, as other neuropathological studies have instead reported the absence of detectable LBs in a subpopulation of PD patients with LRRK2 mutations [START_REF] Kalia | Clinical Correlations With Lewy Body Pathology in LRRK2-Related Parkinson Disease[END_REF]. In general, although LRRK2 variants or mutations are considered to be risk factors for developing PD, the onset of symptoms in LRRK2 carriers has been found to be similar to that of idiopathic PD cases [START_REF] Brockmann | Clinical and brain imaging characteristics in leucine-rich repeat kinase 2-associated PD and asymptomatic mutation carriers[END_REF][START_REF] Oosterveld | Greater motor progression in patients with Parkinson disease who carry LRRK2 risk variants[END_REF]. The mechanisms underlying LRRK2 neurotoxicity are still unknown. It is now generally accepted that the G2019S mutation increases LRRK2 kinase activity (both autophosphorylation and phosphorylation of exogenous kinase substrates) and that neurotoxicity originates from such increased activity [START_REF] Lee | Inhibitors of Leucine Rich Repeat Kinase 2 (LRRK2) Protect Against LRRK2-Models of Parkinson's Disease[END_REF][START_REF] West | Parkinson's disease-associated mutations in LRRK2 link enhanced GTP-binding and kinase activities to neuronal toxicity[END_REF].

The central role of -syn in PD pathogenesis has led to the hypothesis of a functional (and possibly physical) interaction between LRRK2 and -syn [for a review [START_REF] Cresto | The unlikely partnership between LRRK2 and αsynuclein in Parkinson's disease[END_REF]. Indeed, LRRK2 toxicity may require the presence of -syn and, conversely, the presence of variant/mutant LRRK2 could increase the risk and/or impact ofsynucleopathy in PD. The level of kinase activity of LRRK2 could thus be a modifier of -syn toxicity. If this is true, the therapeutic implication would be extremely important: the regulation of LRRK2 kinase activity could be theoretically beneficial in slowing disease progression not only in individuals harboring LRRK2 mutations, but also in idiopathic PD. Recent experiments in transgenic mouse models of LRRK2 and -syn support these hypotheses. The results of experiments in genetic models of mutant or wildtype LRRK2, in particular the effect of the pharmacological blockade of the kinase activity of LRRK2G2019S in these models, suggest that LRRK2 may increase -syn toxicity [START_REF] Daher | Abrogation of α-synuclein-mediated dopaminergic neurodegeneration in LRRK2-deficient rats[END_REF][START_REF] Daher | Leucine-rich Repeat Kinase 2 (LRRK2) Pharmacological Inhibition Abates α-Synuclein Gene-induced Neurodegeneration[END_REF][START_REF] Lin | Leucine-rich repeat kinase 2 regulates the progression of neuropathology induced by Parkinson's-disease-related mutant alpha-synuclein[END_REF].

However, the mechanisms underlying the "protoxic" effect of LRRK2 (especially the G2019S mutation) on -syn toxicity are not known and the exact role of the kinase domain has not been completely demonstrated. Pharmacological intervention suggests that the kinase activity of LRRK2 likely contributes to the synergy (for a review [START_REF] Cresto | The Unlikely Partnership Between LRRK2 and α-Synuclein in Parkinson's Disease[END_REF]). In addition, it is not known whether the potentiation of -syn toxicity by the presence of LRRK2G2019S is related solely to a cellautonomous mechanism. Alternatively, LRRK2expressing cells that surround DA neurons, especially microglial cells, astrocytes, and cells of the immune system likely play a role. However, pharmacological intervention with LRRK2 inhibitors, pan-cellular knock out, or transgenic animals expressing wildtype LRRK2 or mutant LRRK2 cannot easily distinguish between cellautonomous and non-cell-autonomous mechanisms in vivo.

Here, we attempted to precisely address these questions in a relevant cellular context by studying the effect of the C-terminal domain of human LRRK2 harboring the G2019S mutation ( LRRK2G2091S) or its inactive form, (mutations G2019S plus D1994A, called DK) LRRK2DK, on the neurotoxicity of human -syn with the A53T mutation (α-synA53T). We performed experiments using AAVs that lead to a moderate overexpression of the various forms of LRRK2 and human -synA53T alone or in combination in DA neurons of the SNc in adult rats. Quantitative histological evaluation showed that although -synA53Tinduced neurotoxicity likely through a mechanism involving the catalytic activity of the kinase domain.

Materials and methods

Viral construction and production

Adeno-associated viral vectors (AAVs). Plasmid constructs were packaged into AAV2/6 capsids as described previously [58]. Briefly, viral particles were produced by co-transfection of HEK-293T cells with (1) an adenovirus helper plasmid (pXX6-80), [START_REF] Chartier-Harlin | alpha-Synuclein locus duplication as a cause of familial Parkinson's disease[END_REF] an AAV packaging plasmid carrying the rep2 and cap8 genes, and (3) the AAV2 expression vector containing the transgene. Seventy-two hours following transfection, recombinant vectors were purified and concentrated from cell lysates and supernatants by ultracentrifugation on an iodixaniol density gradient followed by dialysis against PBSMK (0.5 mM MgCl2 and 1.25 mM KCl in PBS). The concentration of vector stocks was estimated by real-time-PCR following the method described by Aurnhammer et al. [START_REF] Aurnhammer | Universal real-time PCR for the detection and quantification of adeno-associated virus serotype 2-derived inverted terminal repeat sequences[END_REF] and expressed as viral genomes per ml of concentrated stocks (Vg/ml). AAVs coding for human ∆LRRK2 (WT, G2019S, and "kinase dead), α-syn A53T, and GFP were produced. For stereotactic injections, the animals were deeply anaesthetized with 4% isoflurane, followed by a mixture of ketamine (75 mg/kg) and xylazine (5 mg/kg), and placed in a stereotactic frame. Recombinant adeno-associated viral vectors were injected unilaterally into the SNc, at the following stereotactic coordinates: +3.4 mm anterior to the interaural zero and ±2.0 mm lateral to bregma, at a depth of -7.8 mm relative to the skull, with the tooth bar set at -3.3 mm. We injected 4 µl of virus at a concentration of 2.5x10 10 viral particles per site for single injections and 2.5x10 10 viral particles of each vector for co-injections for a total of 5x10 10 viral particles per site, with a 34-gauge blunt-tipped needle linked to a 10-μl Hamilton syringe by a polyethylene catheter at a rate of 0.25 μl/min using an automatic pump (CMA-4004). The needle was left in place for five minutes and then slowly withdrawn.

Stereotactic injection

Tissue processing

For all procedures, rats were first deeply anesthetized by isoflurane inhalation, followed by the intraperitoneal injection of a lethal dose of sodium pentobarbital.

For immunohistochemistry, rats were transcardially perfused with 300 ml 4% paraformaldehyde in phosphate buffer at a rate of 30 ml/min. After perfusion, the brain of each rat was quickly removed and immersed in ice-cold 4% paraformaldehyde/0.1X PBS for at least 24 h, before transfer to 15% sucrose for 24 h and then 30% sucrose the next day, for cryoprotection. The brains were then cut into 40-μm sections on a freezing microtome (CM1900, Leica, Germany). Serial sections of the striatum and midbrain were stored in antifreeze solution (30% sucrose/30% ethylene glycol in PBS) until use.

Immunohistological analysis and quantification

Immunohistochemistry Sections were removed from the antifreeze solution and washed in PBS. Endogenous peroxidase activity was quenched by transferring them to 1% H 2 O 2 and incubation for 30 min at room temperature (RT) and washing them three times with PBS for 10 min each. The sections were then blocked by incubation with 4.5% normal goat serum for 30 min in PBS-T (0.2% Triton X-100 in PBS) and then incubated overnight with primary antibody in 3% normal goat serum in PBS-T at 4°C with gentle shaking. The following primary antibodies were used for the present study: antityrosine hydroxylase (TH) antibody: MAB318 clone LNC1, Merk-Millipore, 1:3000; anti-hemagglutinin tag (HA), Covance clone 11, 1:1000; anti-human α-synuclein, syn 211, 1:1000; anti-phospho-α-synS129, ab51253, Abcam, 1:5000]. The next day, the sections were removed from the primary antibody solution, washed three times, and incubated for 1 h at RT with the appropriate biotinylated secondary antibody in PBS-T (Vector Laboratories, Burlingame, CA, USA, 1:1000). The sections were then washed and incubated with ABC complex solution in PBS-T (1:250, reagents A and B combined in a 1:1 ratio, Vector Laboratories) for 1 h. The sections were then incubated with DAB for 30 s to 1 min and mounted on slides in Eukitt mounting medium.

Cell counting

Optical fractionator sampling was carried out on a Zeiss microscope AxioPlan. Midbrain dopaminergic neurons were outlined on the basis of TH immunolabelling with reference to a coronal atlas of the rat brain (Paxinos and Watson, 6 th edition). TH-positive cells were counted by unbiased stereology in the entire SNc and the number of positive neurons per section was calculated using Mercator Software (Explora Nova, France). We placed 100 × 100 μm grids in a systematically random manner, 80 × 80 μm apart, with a 3-µm offset from the surface of the section. Quantification was performed on 12 serial sections spaced by 200 µm, corresponding to the entire SNc.

The phosphorylation of α-syn on S129 (P-αsyn S129 ) was evaluated by counting the number of P-α-syn S129 -positive neurons in the SNc using stereology methods. The SNc was delimited by Nissl staining and the grids (250 x 250 µm) placed, with a space of 100 x 100 µm. Quantification was performed on six serial sections spaced by 400 µm, corresponding to the entire SNc. In the striatum, a threshold was applied to select only the P-α-syn S129 -positive neurons by immunostaining and quantification performed on three slices, corresponding to the beginning, middle, and end of the striatum.

Immunofluorescence

The procedure used was similar to that for immunohistochemistry, but without the incubation in 1% H 2 O 2 . The primary antibodies used for the immunofluorescence procedure were the same as previously described (IBA1, Dako, 1:1000). Sections were first incubated with the primary antibody overnight at 4°C. The next day, they were incubated with a fluorescent secondary antibody (Alexa Fluor 594-labeled goat anti-rabbit IgG or Alexa Fluor 488-labeled goat anti-rabbit IgG (1:1000, Life Technologies)) for 1h at RT. Sections were then washed and incubated overnight at 4°C with another primary antibody. Finally, they were incubated with a second fluorescent secondary antibody (Alexa Fluor 488-labeled goat anti-mouse IgG or 594-labeled goat anti-mouse IgG (1:1000, Life Technologies)) for 1h at RT. The sections were stained with DAPI, washed, and mounted in a fluorescence mounting medium. Images were acquired with a laser confocal microscope (SP8, Leica, Germany) or an epifluorescence microscope (DM8000, Leica, Germany).

Colocalization

The percentage of co-localization between ΔLRRK2 and α-syn was determined by counting the number of cells co-expressing both ΔLRRK2 and α-syn proteins divided by the number of cells expressing α-syn alone. Images were acquired with a laser confocal microscope (SP8, Leica, Germany). On the same acquisitions, the levels of ΔLRRK2 and α-syn proteins were evaluated on three coronal sections in the SNc. Twenty cells coexpressing both ΔLRRK2 and α-syn proteins were delineated per animal using image J software and the mean fluorescence intensity in the red and in green channels (corresponding to ΔLRRK2 and αsyn proteins, respectively) was measured in each cell.

Fluorescence intensity measurement

Striatal dopaminergic innervation at 15 weeks was quantified by measuring the fluorescence intensity of TH-immunoreactive terminals on three coronal striatal sections. The sections were observed by epifluorescence microscopy at a magnification of 63X and the fluorescence intensity determined using MorphoStrider software (Explora Nova, France).

Microglia area measurement

The area occupied by microglia was evaluated by confocal microscopy at a magnification of 20X in the dorso-medial part of the striatum and in the SN pars reticulata. A threshold was applied and the area of 20 microglia cells measured per acquisition. Three acquisitions per animal were used.

Statistical analysis

Normal distribution of data was checked by Shapiro test. Data were analyzed by two-tailed, one-way analysis of variance (ANOVA) performed with Statistica software (Statsoft Inc., Tulsa, Oklahoma, USA) and, when appropriate, LSD post-hoc correction for multiple comparisons was applied. Unpaired Student's t-tests were performed for pairwise comparisons. The annotations used to indicate the level of significance are as follows: *p < 0.05, **p < 0.01, ***p < 0.001.

Results

Determination of the experimental conditions to detect potential synergy between AAV-a-syn A53T and AAV-LRRK2 G2019S toxicity

We investigated whether human LRRK2 can increase the toxicity of human -syn in DA neurons through cell-autonomous mechanisms. We used AAVs, which allow selective expression in neuronal cells and efficiently transduce a large volume of brain tissue due to their good diffusion properties. In a previous study [START_REF] Cresto | The C-terminal domain of LRRK2 with the G2019S mutation is sufficient to produce neurodegeneration of dopaminergic neurons in vivo[END_REF], we showed that the C-terminal portion of human LRRK2 G2019S (LRRK2 G2019S , 1330-2527 aa) retains, at least in part, the biochemical properties of full-length LRRK2 G2019S , including higher kinase activity than the wildtype fragment. In addition, we found that overexpression of the C-terminal portion of human LRRK2 G2019S in the adult rat SNc, using AAVs, produced partial (~30%) but significant loss of DA neurons at 25 weeks post-transduction, whereas overexpression of the wildtype form of LRRK2 (LRRK2 WT ) was not toxic. Here, we used a similar approach using a slightly larger fragment (aa 1283-2527) (Fig. 1A).

We studied the effects of AAV-ΔLRRK2 G2019S , AAV-ΔLRRK2 WT , and AAV-ΔLRRK2 G2019S/D1994A alone at 15 weeks PI. We injected 4 µl of viral vector solution in all cases. A final amount of 2.5x10 10 viral particles per site and per vector was used. Each AAV was injected unilaterally into the SNc (2.5 10 10 vg). In addition to the three experimental groups, a control group received injections of vehicle (PBS/pluronic acid). The integrity of the nigrostriatal pathway was assessed using unbiased stereology to count the number of DA neurons displaying tyrosine hydroxylase (TH) staining in the injected part of the SNc (Fig. 1). Observation at low-magnification revealed no major loss of THpositive cells in any of the groups injected with AAVs encoding the LRRK2 fragments (Fig. 1B). The total number of TH-positive cells in the SNc did not differ significantly between the control group (PBS) and AAV-LRRK2 WT , AAV-LRRK2 G2019S , or AAV-LRRK2 G2019S/D1994A groups (Fig. 1C). Thus, these results suggest that the ΔLRRK2 fragments alone did not trigger significant neurodegeneration of DA neurons at 15 weeks PI.

We sought an injection protocol that leads to mild degeneration, in such a way that potential "pro-toxic" effect of LRRK2 constructs could be easily detected, as we wanted to investigate whether AAVs encoding the different ΔLRRK2 constructs could increase the toxicity of AAV-syn A53T . We conducted pilot experiments to determine the appropriate dose (titers) of AAV-syn alone that would lead to progressive and partial loss of DA neurons. Quantification of THpositive cells in the SNc showed a moderate loss of DA neurons (~30%) at 12 and 15 weeks after transduction with AAV--syn A53T (2.5x10 10 particles post-injection (PI)) (Supplementary Fig. S1A-B). After transduction, DA neurons often displayed accumulation of -syn phosphorylated at its serine 129 (p-synS129). The cells positive for p-synS19 were also positive for ThioS suggesting that these accumulations were aggregates (Supplementary Fig. S1C).

Thus, a co-injection protocol with AAV-αsyn A53T and AAV-∆LRRK2 G2019S and the evaluation of DA cell loss at 15 weeks PI appeared to be suitable for the detection of the potential synergy of toxicity between the two pathological transgenes.

Effects of co-transduction with AAV-αsyn A53T and AAV-∆LRRK2 G2019S

We next investigated whether the presence of the various LRRK2 fragments (wildtype, G2019S, or G2019S/D1994A) in DA neurons could modify the toxicity of human α-syn A53T using this co-transduction paradigm (with 2.5x10 10 particles for each vector).

We first studied the neurotoxic effects produced by AAV-α-syn A53T in the presence or absence of AAV-∆LRRK2 G2019S at 15 weeks PI. We assessed the co-localization of human -syn A53T and LRRK2 fragments in the SNc after cotransduction, as we wanted to investigate the combined effects of -syn A53T and the various LRRK2 fragments in DA neurons. Analysis by confocal microscopy showed that human α-syn expression in the SNc was high in TH-positive neurons (Fig. 2A). On average, 70% of neurons coexpressed both human -syn A53T and the LRRK2 fragments (Fig. 2B). We next evaluated the toxic effects of human -syn A53T in the presence or absence of AAV-ΔLRRK2 G2019S . AAVα-syn A53T alone produced a significant 38% loss of TH-positive cells, as measured by unbiased stereology in the SNc at 15 weeks PI (mean count ± SEM: Control, 12,344 ± 734;

AAV-α-syn A53T , 7,555 ± 527). The coinjection of AAV-syn A53T with AAV-GFP (as a control of viral load) induced a 46% loss of DA neurons, which was not statistically different from that obtained with AAV-α-syn A53T alone (6,601 ± 360). The coinjection of AAV-asyn A53T and AAV-LRRK2 G2019S induced a loss (-55%) of DA neurons (mean count ± SEM: 5,585 ± 355) that was significantly greater than that measured in the two other groups injected with AAV--syn A53T (Fig. 3A). We evaluated the impact of SNc cell loss on the level of dopaminergic terminals in the dorso-medial striatum using THimmunofluorescence in both the α-syn A53T /GFP and α-syn A53T /∆LRRK2 G2019S groups (Fig. 4A). These measurements were performed in the dorsal striatum, which receives projections from the injected SNc region. TH immunoreactivity in the striatum in both the α-syn A53T /GFP and αsyn A53T /∆LRRK2 G2019S groups was 15% lower than in the control group (PBS). This small -syn A53Tinduced loss of TH-positive fibers was similar in the GFP and ∆LRRK2 G2019S groups (Fig. 4C).

We also counted the number of SNc cells showing P-α-synS129 immunoreactivity, a marker of α-syn aggregation, in the different groups (Fig. 3C). The number of P-α-synS129-positive cells was significantly lower in the group co-infected with AAV-α-syn A53T and AAV-LRRK2 G2019S than that of the groups infected with AAV-αsyn A53T alone or in combination with AAV-GFP (Fig. 3D). We also evaluated P-α-synS129 immunoreactivity in the striatum. Small P-α-synS129 immuno-positive objects with an elongated form or with a pearl necklace-like shape, reminiscent of neuritelike aggregates were seen in the striatum (Fig. 3E). Consistent with the results obtained in the SNc, we found lower levels of aggregated α-syn in the striatum of rats co-infected with AAV-α-syn A53T and AAV-LRRK2 G2019S than in those infected with AAV-α-syn A53T / GFP (Fig. 3E-F).

Differential effects of AAV-∆LRRK2 G2019S and AAV-∆LRRK2 G2019S/D1994A on AAV-α-syn A53T toxicity

We next investigated whether the effect of AAV-ΔLRRK2 GS on AAV-α-syn A53Tinduced toxicity was dependent on the kinase activity of the LRRK2 construct. We thus compared the effect of ΔLRRK2 G2019S with that of the dead kinase form ∆LRRK2 DK . We examined an earlier time point PI (6 weeks) for these experiments. We reasoned that, although the kinase activity of ∆LRRK2 DK is "dead", it may lead to cellular disturbances in long-term experiments because of its potential dominant-negative effect on endogenous LRRK2. We first compared the levels of transgene expression after transduction with αsyn A53T and AAV-ΔLRRK2 G2019S or AAV-∆LRRK2 DK . Quantitative immunofluorescence analysis showed that almost the entire SNc was infected by AAV-α-syn A53T when co-infected with either AAV-ΔLRRK2 GS or AAV-∆LRRK2 DK (Fig. 5). We also re-evaluated the colocalization of the LRRK2related transgenes and αsyn A53T (Fig. 6). In total, 76% of neurons expressed both α-syn A53T and ΔLRRK2 (Fig. 6C), consistent with our observations in the experiments described above (see Fig. 1). We evaluated the level of fluorescence intensity corresponding to ∆LRRK2-HA staining at higher magnification. ΔLRRK2 GS and ∆LRRK2 DK were expressed at the same level in SNc neurons (Fig. 6C). In addition, human α-syn A53T protein was expressed at the same levels in neurons co-expressing ΔLRRK2 GS and ∆LRRK2 DK (Fig. 6C).

We then assessed the loss of DA neurons produced by AAV--syn A53T when co-injected with either AAV-ΔLRRK2 G2019S or AAV-∆LRRK2 DK . The loss of DA neurons induced by human α-syn A53T was significantly lower in the presence of ∆LRRK2 DK than that in the presence of ΔLRRK2 GS (Fig. 7A-B). The number of cells with P-α-synS129 immunoreactivity was similar in the ∆LRRK2 DK and ΔLRRK2 G2019S groups. Finally, we carried out a preliminary characterization of the status of microglial cells at this early time point (6 weeks PI), by immunohistochemistry using a validated marker (Iba1), because of the role of neuroinflammation in neurodegeneration observed in human αsyn A53T models. As expected, microglial cells in rats overexpressing human -syn A53T appeared more reactive as compared to rats injected with vehicle. The quantification of immunofluorescence levels in the SN and striatum showed that human αsyn A53T significantly activated microglia. However, overexpression of ΔLRRK2 G2019S and ΔLRRK2 DK did not have any impact on the microglial activation induced by the mutant human α-syn (Supplementary Fig. S2).

Finally, we investigated whether the "protoxic" effect of ΔLRRK2 G2019S on -syn A53T could also be detected for other aggregating proteins. Using a different approach with lentiviral vectors in mice, we tested whether the different forms of ΔLRRK2 could modify the neurotoxicity produced by the N-terminal domain of human huntingtin (Htt) with a pathological expansion of its polyglutamine (Q) region (Htt-N171-82Q) [START_REF] Ruiz | Viral-mediated overexpression of mutant huntingtin to model HD in various species[END_REF]. Lentiviral vectors were injected into the striatum of wildtype mice to produce local cell loss within the six weeks following transduction, as previously described [START_REF] Francelle | Loss of the thyroid hormone-binding protein Crym renders striatal neurons more vulnerable to mutant huntingtin in Huntington's disease[END_REF][START_REF] Galvan | The striatal kinase DCLK3 produces neuroprotection against mutant huntingtin[END_REF]. The striatal lesions were characterized by the loss of neuronal markers DARPP32 and COX (not shown) as well as NeuN (Fig. 8A-B). Localization of the lesions in the striatum coincided with that of the expression of LRRK2 fragments detected using the HA-tag (Fig. 8C). Quantitative analysis of these histological markers showed that none of the ΔLRRK2 forms significantly modified the volume of the striatal lesions produced by mutant Htt (Fig. 8B). In addition, overexpression of the mutant Httfragment led to the accumulation of inclusions containing ubiquitin (mostly nuclear) (data not shown). Quantification of the presence of ubiquitin positive inclusions did not indicated differences within groups (Fig. 8 D). We also selectively detected mutant Htt aggregates using the EM48 antibody which specifically recognizes the aggregated form of the N-terminal domain of mutant Htt [START_REF] Damiano | A role of mitochondrial complex II defects in genetic models of Huntington's disease expressing N-terminal fragments of mutant huntingtin[END_REF][START_REF] Diguet | Normal aging modulates the neurotoxicity of mutant huntingtin[END_REF][START_REF] Gutekunst | Nuclear and neuropil aggregates in Huntington's disease: relationship to neuropathology[END_REF]. ΔLRRK2 G2019S significantly increased by 44% the number of EM48-positive aggregates when compared to the control group (LV-LacZ), an effect not seen with the WT or DK forms (Fig. 8D-E).

Our results show that the synergistic effect of ΔLRRK2 G2019S on the toxicity of human -syn A53T towards DA neurons depends on its kinase activity. Importantly, ΔLRRK2 G2019S overexpression did not significantly increase the neurotoxic effects produced by a different aggregating protein, mutant Htt, suggesting that the effect of ΔLRRK2 G2019S on -syn A53T -induced neurotoxicity is not due to a general increase in the vulnerability of the neurons.

Discussion

The mechanisms leading to the degeneration of DA neurons in LRRK2 mutation gene carriers with PD are unknown. It is generally accepted that the LRRK2 G2019S mutation leads to increased kinase activity and that such abnormally increased kinase activity could lead to cell death [START_REF] Lee | Inhibitors of Leucine Rich Repeat Kinase 2 (LRRK2) Protect Against LRRK2-Models of Parkinson's Disease[END_REF][START_REF] Greggio | Kinase activity is required for the toxic effects of mutant LRRK2/dardarin[END_REF][START_REF] Smith | Kinase activity of mutant LRRK2 mediates neuronal toxicity[END_REF][START_REF] West | Parkinson's disease-associated mutations in LRRK2 link enhanced GTP-binding and kinase activities to neuronal toxicity[END_REF].

In addition, a role for LRRK2 in -syn toxicity has been suggested [START_REF] Daher | Abrogation of α-synuclein-mediated dopaminergic neurodegeneration in LRRK2-deficient rats[END_REF][START_REF] Daher | Leucine-rich Repeat Kinase 2 (LRRK2) Pharmacological Inhibition Abates α-Synuclein Gene-induced Neurodegeneration[END_REF][START_REF] Lin | Leucine-rich repeat kinase 2 regulates the progression of neuropathology induced by Parkinson's-disease-related mutant alpha-synuclein[END_REF]. Indeed, neuropathological evaluation of the brain of PD patients with LRRK2 mutations shows in many cases the presence of bona fide LBs and Lewy neurites [START_REF] Giasson | Biochemical and pathological characterization of Lrrk2[END_REF]. However, the role of the kinase in the crosstalk between LRRK2 and -syn, especially how the kinase activity of LRRK2 G2019S modulates -syn neurotoxicity in the SNc is unknown. In addition, the respective roles of cellautonomous and non-cell-autonomous mechanisms are largely unknown in this crosstalk.

Here, we used an AAV-based approach to selectively target SNc DA neurons and investigated how the C-terminal domain of LRRK2, harboring the G2019S mutation, with increased kinase activity, could modify the loss of DA neurons induced by the overexpression of α-syn A53T in the rat SNc. Under our experimental conditions, AAV-LRRK2 G2019S alone did not induce the loss of DA neurons and AAV-α-syn A53T alone induced a partial loss. The loss of DA cells produced by co-expression of LRRK2 G2019S and α-syn A53T was significantly higher than that measured in rats injected with AAV-α-syn A53T alone or coinjected with a control vector (AAV-GFP).

Conversely, overexpression of the inactive "kinase dead" form LRRK2 DK , at levels similar to those of LRRK2 G2019S , did not alter the toxicity of -synA53T. Although preliminary, quantitative characterization of microglial reactivity induced by human -syn A53T in SNc and striatum did not show obvious changes attributable to LRRK2 fragments. These novel findings further support the hypothesis that the C-terminal domain of LRRK2 G2019S is sufficient to augment the toxic effects of -syn A53T through a cellautonomous mechanism involving the catalytic activity of its kinase domain.

The histological evaluation we performed after transduction of the SNc with AAV and AAV-LRRK2 G2019S show that both transgenes were overexpressed in DA neurons. In both cases, approximately 70% of the SNc was infected. After coinjection, co-localization of both transgenes was found in most neurons in the SNc. Neuropathological evaluation at 15 weeks PI after transduction with AAV--syn A53T showed the partial loss of DA neurons, based on the detection of TH-positive cells. This likely reflects neuronal loss, as suggested in our previous work [START_REF] Cresto | The C-terminal domain of LRRK2 with the G2019S mutation is sufficient to produce neurodegeneration of dopaminergic neurons in vivo[END_REF].

The relevance of overexpressing the C-terminal domain of LRRK2 versus the full-length protein is potentially debatable. Indeed, LRRK2 G2019S lacks N-terminal domains that are known to play crucial roles in LRRK2 function.

we previously showed that overexpression of the LRRK2 G2019S fragment using AAVs triggers neurodegeneration of DA neurons six months posttransduction, whereas the LRRK2 WT fragment, expressed at similar high levels, was devoid of obvious neurotoxicity [START_REF] Cresto | The C-terminal domain of LRRK2 with the G2019S mutation is sufficient to produce neurodegeneration of dopaminergic neurons in vivo[END_REF]. Cell death produced by this fragment which has a detectable kinase activity is likely independent of the interaction with RAB10, since the LRRK2 fragment was found unable to interact with RAB10, in contrary to full-length LRRK2 fragment [START_REF] Cresto | The C-terminal domain of LRRK2 with the G2019S mutation is sufficient to produce neurodegeneration of dopaminergic neurons in vivo[END_REF]. Thus, it is conceivable that the overexpression of LRRK2 G2019S leads to abnormal/higher phosphorylation of substrates, relative to that by the overexpression of LRRK2 WT , that is at least partially in common with that of full-length LRRK2 [40][START_REF] Gloeckner | The Parkinson disease causing LRRK2 mutation I2020T is associated with increased kinase activity[END_REF][START_REF] Greggio | The Parkinson Disease-associated Leucine-rich Repeat Kinase 2 (LRRK2) Is a Dimer That Undergoes Intramolecular Autophosphorylation[END_REF][START_REF] Ito | Re-examination of the dimerization state of leucine-rich repeat kinase 2: predominance of the monomeric form[END_REF].

We investigated whether LRRK2 G2019S effect on -syn A53T was specific of these two pathological proteins, or only resulted from a nonspecific vulnerability of neurons expression the LRRK2 fragment. We tested in a different cellular context the effect of LRRK2 G2019S against mutant Htt in the striatum using a simple approach with lentiviral vectors. In this case, the degeneration of striatal neurons induced by mutant Htt within the 6 weeks PI was not significantly changed by the overexpression of LRRK2 G2019S . Thus the effect of LRRK2 G2019S upon -syn A53T toxicity is likely specific of a particular interplay between these two proteins.

The present experimental paradigm using AAVs allowed addressing the question of the potential cell-autonomous exacerbation of syn A53T toxicity by the kinase activity of LRRK2 directly in the SNc and only in neurons. In contrast, other viral vector models (HSV or adeno) also transduce different cell types in the striatum [START_REF] Melrose | Impaired dopaminergic neurotransmission and microtubule-associated protein tau alterations in human LRRK2 transgenic mice[END_REF][START_REF] Ramonet | Dopaminergic Neuronal Loss, Reduced Neurite Complexity and Autophagic Abnormalities in Transgenic Mice Expressing G2019S Mutant LRRK2[END_REF]. Here, we directly investigated whether there is a functional interaction between AAV-αsyn A53T and-LRRK2 G2019S in DA neurons. Our results show the existence of such an interaction, as overexpression of LRRK2 G2019S significantly enhanced the neurotoxic effects of -syn A53T in rat SNc. Lin et al. showed that the overexpression of LRRK2 (wildtype or with the G2019S mutation) in forebrain neurons (striatum and cerebral cortex) increased the toxicity of -syn A53T in transgenic animals [START_REF] Lin | Leucine-rich repeat kinase 2 regulates the progression of neuropathology induced by Parkinson's-disease-related mutant alpha-synuclein[END_REF]. In these doubletransgenic mice, the authors found significant degeneration of the striatum and cortex and enhanced accumulation of -syn aggregates. This proved the existence of a functional cross talk between -syn and LRRK2 in neurons in vivo when the proteins are expressed at relatively high levels. Pathological transgenes were not expressed in the SNc and no DA degeneration was detected in these models. The CamKII promoter used to drive the expression of the tetracycline transactivator (tTA), which activates the TetO promoter of the LRRK2 and -syn A53T transgenes in these mice, is likely not active in SNc DA neurons, as endogenous expression of CamKII in neurons of the SNc is lower than that observed in forebrain neurons. In LRRK2 knockout rats, the toxicity induced by AAV coding for -syn is lower than that in wildtype rats [START_REF] Daher | Leucine-rich Repeat Kinase 2 (LRRK2) Pharmacological Inhibition Abates α-Synuclein Gene-induced Neurodegeneration[END_REF]. Daher et al. found no synergy between the transgenes following the crossbreeding of other transgenic models in which the promoters driving LRRK2 G2019S and α-syn A53T expression were different (Prion and CMV respectively) [START_REF] Daher | Neurodegenerative phenotypes in an A53T αsynuclein transgenic mouse model are independent of LRRK2[END_REF]. These observations and our results suggest that the crosstalk between LRRK2 and -syn can occur in the same neurons. Thus, our results show that synergy between LRRK2 and -syn depends, at least in part, on cellautonomous mechanisms. However, our results also indicate that even in an experimental condition where the two proteins are expressed in high levels (approximately 5-50 fold the level of endogenous -syn and LRRK2 respectively), the effect of LRRK2 G2019S on -synA53T is moderate. Thus, it is reasonable to speculate that in a condition where physiological levels of expression of LRRK2 and -syn, the cell-autonomous crosstalk between the two proteins might be of moderate importance in DA neurons. Non-cell autonomous mechanisms involving interaction of DA neurons with neighboring glial cells and immune cells may have more important roles in patients and transgenic animal models. For example, it has been recently shown that the seeding of -syn aggregates by the exposure of neurons to -syn fibrils is higher in neurons expressing mutant LRRK2 [START_REF] Volpicelli-Daley | S-LRRK2 Expression Augments α-Synuclein Sequestration into Inclusions in Neurons[END_REF]. The level of LRRK2 activity in microglial cells may also regulate protoxic phenomena associated with α-syn-induced neuroinflammation [START_REF] Daher | Abrogation of α-synuclein-mediated dopaminergic neurodegeneration in LRRK2-deficient rats[END_REF][START_REF] Maekawa | Leucine-rich repeat kinase 2 (LRRK2) regulates α-synuclein clearance in microglia[END_REF][START_REF] Russo | LRRK2 and neuroinflammation: partners in crime in Parkinson's disease?[END_REF]. LRRK2 plays a key role in the immune system [START_REF] Dzamko | LRRK2 and the Immune System[END_REF]. A single nucleotide polymorphism (N2081D) in the region coding for kinase domain of LRRK2 is a major risk factors for Crohn disease, a form of inflammatory bowel disease [START_REF] Hui | Functional variants in the LRRK2 gene confer shared effects on risk for Crohn's disease and Parkinson's disease[END_REF].

We investigated whether LRRK2 G2019S effect on -syn A53T was specific of these two pathological proteins, or only resulted from a nonspecific vulnerability of neurons expression the LRRK2 fragment. We tested in a different cellular context the effect of LRRK2 G2019S against mutant Htt in the striatum using a simple approach with lentiviral vectors. In this case, the degeneration of striatal neurons induced by mutant Htt within the 6 weeks PI was not significantly changed by the overexpression of LRRK2 G2019S . Thus the effect of LRRK2 G2019S upon -syn A53T toxicity is likely specific of functional interplay between these two proteins.

The neuronal mechanisms underlying the synergy between LRRK2 and -syn are ill defined. It is possible that LRRK2 --syn A53T interplay involves a direct physical interaction between the two proteins [START_REF] Guerreiro | LRRK2 interactions with α-synuclein in Parkinson's disease brains and in cell models[END_REF], although, indirect effects leading to a functional interplay is more often discussed. In addition, the role of the kinase domain of LRRK2 is unclear. It is generally accepted that the higher kinase activity of LRRK2 G2019S , relative to that of wild-type LRRK2, leads to neurodegeneration through increased phosphorylation of substrates, possibly through multifactorial cellular changes, including the disruption of microtubule assembly, mitochondrial defects, and alterations in protein translation [START_REF] Rosenbusch | Activation Mechanism of LRRK2 and Its Cellular Functions in Parkinson's Disease[END_REF]. However, whether the increased kinase activity of LRRK2 mutations plays a key role is still a subject of debate. As already mentioned, various transgenic rodent models expressing LRRK2 G2019S have been developed and extensively characterized. These models display no (or very limited) degeneration of DA cells in the SNc. Various experimental approaches clearly demonstrate that the severity of the resulting toxicity is dependent on the level of expression of LRRK2 G2019S [START_REF] Skibinski | Mutant LRRK2 Toxicity in Neurons Depends on LRRK2 Levels and Synuclein But Not Kinase Activity or Inclusion Bodies[END_REF]. There is limited evidence obtained in vivo that shows a relationship between the higher kinase activity of LRRK2 G2019S and neurotoxicity to dopaminergic cells of the SNc. The overexpression of LRRK2 G2019S (or LRRK2 G2019S ) in the SNc was found to induce the loss of DA neurons using HSV and adenovirus models injected into the striatum [START_REF] Lee | Inhibitors of Leucine Rich Repeat Kinase 2 (LRRK2) Protect Against LRRK2-Models of Parkinson's Disease[END_REF][START_REF] Dusonchet | A Rat Model of Progressive Nigral Neurodegeneration Induced by the Parkinson's Disease-Associated G2019S Mutation in LRRK2[END_REF], as well as in our previous study with AAV-LRRK2 G2019S injected into the SNc [START_REF] Cresto | The C-terminal domain of LRRK2 with the G2019S mutation is sufficient to produce neurodegeneration of dopaminergic neurons in vivo[END_REF].

Only a few studies have directly addressed the role of the kinase in the interaction between -syn and LRRK2 toxicity. It was shown that neuroinflammation and neurodegeneration produced by the transduction of the SNc with AAV--syn is significantly attenuated in LRRK2 KO rats relative to that in wildtype littermates. In these experiments, the role of the kinase activity was not assessed [START_REF] Daher | Abrogation of α-synuclein-mediated dopaminergic neurodegeneration in LRRK2-deficient rats[END_REF]. More recently, Daher et al. showed that the toxicity of AAV-α-synuclein in the SNc was higher in transgenic LRRK2 G2019S than wildtype rats. Interestingly, treatment of both genotypes with the LRRK2 inhibitor PF-06447475 (PF) reduced the toxicity of -syn [START_REF] Daher | Leucine-rich Repeat Kinase 2 (LRRK2) Pharmacological Inhibition Abates α-Synuclein Gene-induced Neurodegeneration[END_REF]. This suggests that the exacerbation of -syn toxicity by LRRK2 G2019S could result from elevated catalytic activity of the kinase. However, LRRK2 inhibitors, including PF, destabilize LRRK2, leading to reduced cellular levels of the protein [START_REF] Lobbestael | Pharmacological LRRK2 kinase inhibition induces LRRK2 protein destabilization and proteasomal degradation[END_REF]. Thus, protection by PF against combined -syn/LRRK2 G2019S toxicity may result from the reduction of LRRK2 levels rather than actual inhibition of the catalytic activity of the kinase. Here, we found that overexpression of the inactive protein LRRK2 G2019S/D1994S did not alter the toxicity AAV-αsyn A53T , whereas expression of LRRK2 G2019S increased the toxicity of AAV-α-syn A53T towards DA neurons. In our experimental system where both proteins have high expression levels, we were able to verify that the difference between the toxicity of LRRK2 G2019S and LRRK2 DK was not related to a difference in protein levels. Quantitative confocal analysis showed that the levels of the two proteins were similar in DA neurons in vivo. Thus, it is likely that the difference between the effects of LRRK2 WT and LRRK2 G2019S is mainly due to their different kinase activities.

Conclusion

Our results suggest that the C-terminal domain of LRRK2 G2019S containing the ROC-COR, Kinase and WD40 domains is sufficient to potentiate the toxicity of human -syn A53T in DA neurons in vivo and that this effect is kinasedependent. This cell-autonomous mechanism may additively or synergistically acts with other noncell-autonomous mechanisms, especially those involving neuro-inflammation to trigger the death of DA neurons in PD. Our findings further support the view that inhibition of the kinase activity of LRRK2 might be beneficial to slow α-syndependent mechanisms in PD.

Appendix 3 -Lists of figures and tables

ÉVALUATION DU SYSTEME DOPAMINERGIQUE EN UT ILISANT L 'IMAGERIE TEP IN VIVO ET DES ANALYSES POST MORTEM DANS LE CONTEXTE D'UN MOD PD NEURODEGENERATIF ET NEUROINFLAMMATOIRE L EGER

La maladie de Parkinson (MP) est le deuxième trouble neurodégénératif le plus commenté, affectant plus de 10 millions de patients dans le monde. La pathologie cérébrale de la maladie de Parkinson est caractérisée par une perte progressive des neurones dopaminergiques de la substantia nigra et une accumulation d'agrégats d'a-synucléine. Une autre caractéristique de la MP est la présence de neuroinflammation. Le plus grand facteur de risque de MP est l'âge, mais les facteurs génétiques et environnementaux jouent également un rôle. Dans cette thèse de doctorat, je visais à évaluer, in vivo et post mortem, les effets de trois facteurs sur le système dopaminergique: 1) la surexpression α-synucléine, 2) la co-expression α-synucléine et LRRK2, et 3) la neuroinflammation légère , sur le système dopaminergique et la perte de cellules neuronales dopaminergiques.

À cette fin, l'imagerie par tomographie par émission de positons (TEP) et les études comportementales ont été sélectionnées comme principaux outils in vivo. J'ai utilisé l'imagerie TEP [18F] LBT999 et [18F] FMT pour évaluer les niveaux striataux de transporteur de dopamine (DAT) et l'enzyme AADC synthétisant la dopamine, respectivement. Pour évaluer la neuroinflammation, j'ai utilisé le [18F] DPA714 pour évaluer la liaison du TSPO à 18 kDa. Les données in vivo ont été validées par des techniques post mortem évaluant l'expression des gènes (RT-qPCR) et des protéines (immunohistochimie). Tout d'abord, j'ai mis en place, évalué et amélioré plusieurs techniques existantes dans notre laboratoire en utilisant le modèle 6-OHDA standard. Ensuite, les résultats de mon travail montrent que la surexpression de la WT-α-synucléine humaine dans la substantia nigra par le biais de vecteurs viraux (AAV2 / 6-PGK-WT-α-synucléine) ne génère pas de perte neuronale détectable dans la substantia nigra, pas plus que la il génère des déficits moteurs in vivo ou des modifications du système dopaminergique comme le montre l'imagerie TEP in vivo 10 à 12 semaines après l'injection (wpi). D'autre part, j'ai démontré ici que la surexpression de A53T-α-synucléine dans la substantia nigra, en utilisant une approche de vecteur viral AAV2 / 6-PGK-A53T-α-synucléine, a entraîné une agrégation significative de la α-synucléine dans la substantia nigra dès 8wpi, mais pas dans le striatum. Des analyses microscopiques quantitatives montrent que l'agrégation A53T-α-synucléine induit une dégénérescence légère mais progressive des neurones dopaminergiques dans la substantia nigra. La perte de fibres dopaminergiques dans le striatum détectée par l'immunohistochimie de la tyrosine hydroxylase (TH) reste cependant modérée. L'imagerie DAT-PET, mais pas l'imagerie AADC-PET, a pu mesurer la perte neuronale progressive. À 12 ppp, nous avons observé des réductions significatives de l'ARNm de DAT dans la substantia nigra et de la protéine DAT dans le striatum. Ensemble, nos données in vivo et post mortem suggèrent que les mécanismes compensatoires de la disponibilité du DAT se produisent très tôt dans la pathologie, et probablement au niveau de la synapse avant l'apparition d'un mécanisme compensatoire régulé par l'expression des gènes. Une réduction des niveaux de DAT, combinée à des niveaux normaux de TH et d'AADC, pourrait normaliser les concentrations de dopamine synaptique dans le striatum. En outre, cette perte neuronale dopaminergique a coïncidé avec une réponse initialement modérée, suivie d'une réponse microgliale plus prononcée.

Dans un modèle de vecteur viral de co-surexpression de AAV2 / 6-PGK-A53T-α-synucléine et AAV2 / 6-PGK-G2019S-LRRK2, nous n'avons pas observé de neurotoxicité supplémentaire de LRRK2 muté G2019S en toxicité A53T-α-synucléine en utilisant évaluation comportementale in vivo et imagerie TEP. Cependant, la stéréologie TH a démontré que la co-surexpression de G2019S-ΔLRRK2 et A53T-α-synucléine a légèrement augmenté la neurodégénérescence par rapport à la cosurexpression de A53T-α-synucléine et GFP, ce qui pourrait suggérer que même si G2019S-ΔLRRK2 augmente la mort cellulaire , il déclenche également un mécanisme compensatoire qui est visible avec DAT-PET et l'analyse comportementale.

Dans un modèle aigu suivant l'injection de LPS dans le striatum, l'imagerie TSPO-PET in vivo et les analyses post mortem, nous avons observé une réponse neuroinflammatoire dans le striatum, indiquant l'implication de microglies, d'astrocytes et, éventuellement, d'infiltration de macrophages. De plus, l'analyse post mortem a révélé l'absence de perte neuronale dopaminergique dans la substantia nigra et de perte synaptique dans le striatum. Néanmoins, j'ai observé une corrélation inverse significative entre les marqueurs de l'inflammation (expression TSPO-PET et IBA1) et les marqueurs pour la production de dopamine (TH) et le stockage (VMAT2). Ces données soutiennent l'hypothèse selon laquelle la neuroinflammation peut altérer la fonctionnalité du système dopaminergique, quelle que soit la présence de perte de neurones dopaminergiques. De plus, dans la littérature, il a été démontré que les réponses neuro-inflammatoires aggravées conduisent finalement à une perte spécifique des neurones dopaminergiques et peuvent donc avoir un potentiel en tant que cibles thérapeutiques.

En conclusion, les résultats de ma thèse confirment l'intérêt du PET pour démontrer les dommages fonctionnels in vivo, qui ne peuvent pas être démontrés post mortem dans des modèles animaux de PD. Dans l'ensemble, cela sous-tend l'utilisation d'études de TEP multi-racers pour contribuer à l'imagerie des modèles de différents mécanismes sous-jacents à l'initiation ou à la progression de la maladie.

Dans le cadre du programme BrainMatTrain, je me suis concentré sur l'amélioration et la caractérisation des modèles PD de rongeurs dans le but général d'utiliser ces modèles pour l'évaluation thérapeutique d'hydrogels multimodaux contenant des greffes cellulaires. Les modèles décrits ici ont tous leurs propres forces et faiblesses en ce qui concerne l'évaluation des hydrogels multimodèles. Le modèle 6-OHDA intanigral qui a été utilisé ici génère une perte presque complète des neurones dopaminergiques dans le substantif nigra et les terminaisons synaptiques, mais pas de manière pathologiquement pertinente. Ici, nous avons également utilisé deux modèles basés sur les caractéristiques de PD; neuroinflammation et surexpression des gènes liés à la PD. Comme discuté cidessus, le modèle de neuroinflammation basé sur le LPS permet l'étude des effets de la neuroinflammation sur la perte de cellules et de fibres dopaminergiques. Enfin, le modèle A53T-αsynucléine a un grand potentiel pour évaluer l'efficacité des stratégies thérapeutiques neuroprotectrices et modifiant la maladie. De plus, la progression relativement lente de la maladie signifie également qu'il est possible d'étudier le modèle dans une phase de type prodromique, où moins de 30% des neurones dopaminergiques sont morts. Cela pourrait être particulièrement utile dans l'étude des premiers biomarqueurs de la MP.

EVALUATION OF THE DOP AMINERGIC SYSTEM USI NG IN VIVO PET IMAGING AND POST-MORTEM ANALYSES IN THE CONTEXT OF MILD NEURODEGENERATI VE AND NEUROINFLAMMATORY PD MODELS

Thesis summary in English

The cerebral pathology of Parkinson's disease is characterized by a progressive loss of dopaminergic neurons of the substantia nigra, and an accumulation of α-synuclein aggregates. neuroinflammation and genetic predisposition contribute to PD as the main confounding factors. In this PhD thesis, I aimed to evaluate, in vivo and post-mortem, the effects of three factors on the dopaminergic system: 1) α-synuclein overexpression, 2) α-synuclein and LRRK2 co-expression, and 3) mild neuroinflammation, on the dopaminergic system and dopaminergic neuronal cell loss.

To this end, positron emission tomography (PET) imaging and behavioural studies have been selected as the main in vivo tools. I have used [ 18 F]LBT999 and [ 18 F]FMT PET imaging to evaluate striatal levels of dopamine transporter (DAT) and the dopamine-synthesising AADC enzyme, respectively. To assess neuroinflammation, I have used [ 18 F]DPA714 to evaluate 18kDa TSPO binding. The in vivo data were validated by post-mortem techniques evaluating the expression of genes (RT-qPCR) and proteins (immunohistochemistry).

The results of my work show that overexpression of human WT-α-synuclein in the substantia nigra through viral vectors (AAV2/6-PGK-WT-α-synuclein) does not generate detectable neuronal loss in the substantia nigra, nor does it generate in vivo motor deficits or changes in the dopaminergic system as seen by in vivo PET imaging. On the other hand, I have demonstrated here that overexpression of A53T-α-synuclein in the substantia nigra, using an AAV2/6-PGK-A53T-α-synuclein viral vector approach, resulted in significant α-synuclein aggregation in the substantia nigra as soon as 8wpi, but not in the striatum. Quantitative microscopic analyses show that A53T-α-synuclein aggregation induced a mild but progressive degeneration of dopaminergic neurons in the substantia nigra. The loss of dopaminergic fibres in the striatum as detected by immunohistochemistry of tyrosine hydroxylase (TH) remains, however, moderate. DAT-PET imaging, but not AADC-PET imaging, was able to measure the progressive neuronal loss. Taken together, our in vivo and post-mortem data suggest that DAT-PET does not only reflect neuronal loss induced by α-synuclein accumulation, but also functional compensation mechanisms of the dopaminergic synapse. A reduction in DAT levels, combined with normal TH and AADC levels, could normalise the synaptic dopamine concentrations in the striatum. In addition, this dopaminergic neuronal loss coincided with in initially moderate, followed by a more pronounced microglial response. Finally, in a viral vector model of cooverexpression of AAV2/6-PGK-A53T-α-synuclein and AAV2/6-PGK-G2019S-LRRK2, we did not observe added neurotoxicity of G2019S mutated LRRK2 to A53T-α-synuclein toxicity.

In an acute neuroinflammatory model following LPS injection in the striatum, post-mortem analysis revealed the absence of dopaminergic neuronal loss in the substantia nigra and synaptic loss in the striatum. Nevertheless, I observed a significant inverse correlation between inflammation markers (TSPO-PET and IBA1 expression) and markers for dopamine production (TH) and storage (VMAT2). These data support the hypothesis that neuroinflammation may impair the functionality of the dopaminergic system, regardless of the presence dopaminergic neuron loss.

In summary, my thesis results confirm the interest of PET in demonstrating functional damage in vivo, which cannot be demonstrated post-mortem in animal models of PD.

Thesis summary in French

La pathologie cérébrale de la maladie de Parkinson (MP) est caractérisée par une perte neuronale progressive des neurones dopaminergiques de la substance noire pars compacta (SNc) et une accumulation d'α-synucléine agrégée. L'inflammation et la prédisposition génétique contribuent à la MP en tant que facteurs principaux de comorbidité. Dans cette thèse, j'ai étudié, in vivo et post mortem, les effets de trois facteurs sur le système dopaminergique: 1) l'agrégation d'α-synucléine, 2) la toxicité additionnelle des mutations LRRK2 et 3) une neuroinflammation modérée. Pour ce faire, l'imagerie par Tomographie par Emission de Positons (TEP) et des tests comportementaux ont été utilisés. J'ai utilisé les radiotraceurs [18F]LBT-999 et [18F]FMT pour mesurer respectivement les taux cérébraux du Transporteur de la dopamine (DAT) et de l'AADC, l'enzyme de synthèse de la dopamine. Pour évaluer l'inflammation j'ai utilisé le [18F]DPA714, un radiotraceur de référence pour la neuroinflammation radioligand du TSPO 18kDa,. Les résultats obtenus ont été validés par des techniques post mortem évaluant l'expression des gènes (RT-qPCR) et des protéines (immunohistochimie).

Les résultats de mes travaux montrent que la surexpression de l'α-synucleine humaine « sauvage » obtenue par transduction de la substance noire par des vecteurs viraux (AAV2/6-PGK-WT-α-synuclein) ne génère ni perte neuronale détectable dans la substance noire, ni déficits moteur, ni changements majeurs du système dopaminergique observé par imagerie TEP in vivo. D'autre part, utilisant cette même stratégie d'expression par vecteur viral (AAV2/6-PGK-A53T-α-synucléine), j'ai démontré une agrégation significative de l'α-synucléine mutée (A53T) dans la substance noire dès 8 semaines post-injection, celle-ci restant très faible dans le striatum. Les analyses quantitatives en microscopie montrent que la surexpression de l'α-synucléine induit une dégénérescence modérée mais significative des neurones dopaminergiques, s'aggravant au cour du temps. La perte des fibres dopaminergiques détectées par l'immunohistochimie de la Tyrosine Hydroxylase (TH) reste cependant très modérée dans le striatum. Les résultats obtenus par imagerie TEP montrent une réduction du DAT dans le striatum, au contraire de l'AADC dont les niveaux restent stables. Ainsi, les données in vivo combinées aux observations post-mortem suggèrent que l'imagerie TEP du DAT ne reflète pas seulement la perte neuronale induite par l'α-synucléine, mais aussi une dérégulation fonctionnelle de la synapse dopaminergique. Celle-ci pourrait permettre, via la baisse du transporteur, et en présence de niveau normaux de TH et AADC le maintien des concentrations synaptiques de dopamine dans le striatum. J'ai également montré que cette perte neuronale coïncidait avec une réponse inflammatoire initialement modérée, suivie d'une réponse microgliale plus prononcée. Enfin, dans le modèle viral de co-expression des vecteurs AAV2/6-PGK-A53T-αsynucléine et AAV2/6-PGK-G2019S-LRRK2, nous n'avons pas observé que la présence de LRRK2 mutée augmentait la toxicité de l'α-synucléine.

Dans le modèle de neuroinflammation induite par l'injection de LPS, l'imagerie TEP et les analyses post-mortem montrent l'absence de perte des neurones dopaminergiques. Néanmoins, j'ai mis en évidence une corrélation inverse significative entre les marqueurs de sévérité de l'inflammation (l'imagerie TEP des TSPO, expression IBA1) et les marqueurs de production (TH) et de stockage (VMAT2) de la dopamine. Ces données renforcent l'hypothèse que la neuroinflammation altère la fonctionnalité du système dopaminergique, indépendamment de la perte neuronale dopaminergique.

Thesis summary in Dutch

De cerebrale pathologie van de ziekte van Parkinson (PD) wordt gekenmerkt door een progressief verlies van de dopaminerge neuronen in de substantia nigra en de accumulatie van αsynucleïne-aggregaten. Neuroinflammatie en genetische aanleg zijn de belangrijkste comorbiditeitsfactoren voor PD. In dit proefschrift heb ik getracht om zowel in vivo als post-mortem de effecten van drie factoren op het dopaminerge systeem en dopaminerge neuronale overleving te evalueren: 1) α-synucleïne overexpressie, 2) co-overexpressie van α-synucleïne en LRRK2, en 3) milde neuroinflammatie. Daartoe zijn Positron Emissie Tomografie (PET) beeldvorming en gedragsstudies geselecteerd als de belangrijkste in vivo technieken. Ik heb [ 18 F]LBT-999 en [ 18 F]FMT PET beeldvorming gebruikt om dopamine transporter (DAT) en het dopamine-synthetiserend enzym AADC, respectievelijk, te evalueren in het striatum. Om de neuroinflammatie te evalueren, heb ik [ 18 F]DPA714 gebruikt om 18kDa TSPO binding in beeld te brengen. De in vivo resultaten werden vervolgens gevalideerd door post-mortem technieken die gen-(door RT-qPCR) en eiwitexpressie (door immunohistochemie) evalueren op dezelfde modellen.

In het resulterende werk heb ik aangetoond dat overexpressie van humane WT-α-syncleïne in de substantia nigra door middel van virale vectoren (AAV2/6-PGK-WT-α-synuclein) hier geen detecteerbaar verlies van dopaminerge neuronen genereert, noch in vivo motorische symptomen veroorzaakt of andere veranderingen in het dopaminerge systeem zoals in beeld gebracht met in vivo PET-beeldvorming. Anderzijds heb ik hier aangetoond dat overexpressie van A53T-α-synucleïne in de substantia nigra, met behulp van een AAV2/6-PGK-A53T-α-synuclein virale vector benadering, resulteerde in significante α-synucleïne-aggregatie in de substantia nigra reeds na 8 weken, maar niet in het striatum. Kwantitatieve microscopische analyse laat zien dat α-synucleïne-aggregatie een milde maar progressieve degeneratie van dopaminerge neuronen in de substantia nigra veroorzaakte. Het verlies van dopaminerge vezels in het striatum, zoals gezien met immunohistochemie van tyrosine hydroxylase (TH) bleef echter beperkt. PET-beeldvorming van DAT, maar niet van AADC, gaf een duidelijke aanwijzing voor progressief verlies van de dopaminerge neuron. De combinatie van de in vivo en post-mortem data suggereert dat DAT-PET-beeldvorming niet alleen het neuronaal verlies reflecteert, veroorzaakt door α-synucleïne accumulatie, maar ook beïnvloed wordt door functionele aanpassingsmechanismen van de dopaminerge synaps. Een vermindering van DAT expressie, in combinatie met normale TH en AADC expressie, zou de hoeveelheid dopamine in de synapse kunnen normaliseren. In parallel werd aanvankelijk een matige response waargenomen gevolgd door een meer uitgesproken microgliale respons. Daarnaast heb ik geen verergerd toxisch effect gemeten van co-overexpressie van G2019S gemuteerd LRRK2 en A53Tα-synucleïne op A53T-α-synucleïne toxiciteit.

In een acuut neuro-inflammatoir model geïnduceerd door intrastriatale LPS-injectie heb ik in post-mortem een afwezigheid van dopaminerge neuronaal verlies waargenomen in de substantia nigra alsmede een afwezigheid van synaptisch verlies in het striatum. Desalniettemin heb ik een significante inverse correlatie waargenomen tussen inflammatiemarkers (TSPO-PET en IBA1 expressie) en markers voor dopamine productie (TH) en opslag (VMAT2). Deze data ondersteunen de hypothese dat neuroinflammatie de functionaliteit van dopaminerge markers kan veranderen, ongeacht de aanwezigheid van neuronaal verlies. Samengevat laat mijn thesis dus zien dat er een belang is voor het gebruik van PET beeldvorming om functionele schade aan het dopaminerge systeem weer te geven in vivo, die niet aangetoond kan worden door middel van post-mortem technieken in PD diermodellen. Pendant ma thèse, j'ai évalué les effets de l'α-synucléine et de l'inflammation modérée sur les neurones DA dans des modèles animaux de la MP. J'ai utilisé une méthode d'imagerie nucléaire, la tomographie d'émission de positons (TEP) et des analyses post mortem évaluant l'expression de différents gènes et protéines dans ces modèles.

J'ai démontré que l'α-synucléine mutée induit une mort des neurones DA, et une faible réponse inflammatoire.

À l'inverse, l'inflammation modérée n'entraîne pas la mort de ces neurones. Dans les deux cas, la TEP montre de plus des atteintes fonctionnelles des neurones DA, qui ne sont pas détectées par les analyse post mortem, soulignant l'intérêt crucial de cette méthode non invasive pour mieux caractériser la dégénérescence dans les modèles animaux de la MP.

Title : Evaluation of the dopaminergic system using in vivo PET imaging and post-mortem analyses in the context of mild neurodegenerative and neuroinflammatory PD models Keywords : Parkinson's disease, dopamine, neuroimaging, compensation, nigrostriatal system Abstract : Parkinson's disease (PD) is characterised by the aggregation of α-synuclein, a progressive loss of the dopamine-producing neurons, and the presence of neuroinflammation During my PhD studies, I evaluated the impact of α-synuclein aggregation and moderate neuroinflammation on dopaminergic neurons in animal models of PD. To this end, I have used a nuclear imaging method, positron emission tomography (PET), and post-mortem analyses to assess the expression of different genes and proteins in these models.

I have demonstrated that mutated αsynuclein induces mild progressive death of dopamine neurons, coupled with a weak inflammatory response.

Conversely, mild neuroinflammation did not lead to loss of dopamine neurons. PET imaging showed functional damage to the dopamine neurons, which was not detectable by post-mortem analyses. This highlights the crucial interest PET imaging to better characterise neurodegeneration in animals models of PD.

Figure 1 :

 1 Figure 1: Clinical symptoms and time course of PD progression. Diagnosis of PD occurs with the onset of motor symptoms, but can be preceded by a prodromal phase of over 20 years. EDS=excessive daytime sleepiness, MCI=mild cognitive impairment, RBD=REM sleep behaviour disorder. Figure from (Kalia and Lang, 2015).

Figure 2 :

 2 Figure 2: Schematic representation of the basal ganglia. Exhitatory glutamatergic pathways are shown in green, and inhibitory GABA-ergic pathways in red. Dopaminergic pathways are shown in blue. GPe = globus pallidus external, GPi = globus pallidus internal, STN = subthalamic nucleus, SNr = substantia nigra reticulata, SNpc = substantia nigra pars compacta. (adaptation of Basal ganglia diagram, 2013)

Figure 3 :

 3 Figure 3: Braak staging in PD patients. First the peripheral nervous system, olfactory system, and medulla are effected, followed by the pons and spinal cord grey matter. In stage 3/4 the midbrain -including the SN pars compacta-, basal forebrain, and amygdala display Lewy pathology, which spreads further to the rest of the limbic system, thalamus, and temporal cortex. In stage 5 and 6 multiple cortical regions show Lewy pathology. Figure from (Doty, 2012).

Figure 4 :

 4 Figure 4: Schematic representation of prion-like mechenisms for α-synuclein. α-synuclein can adopt mulitpe conformations, some of which are capable of interaction with molecules of the same conformation. These can form stable inter-molecular interactions and create assemblies that are structurally well-denined with highly specific bonds. (Figure from Melki, 2018)

Table 1 :

 1 Overview of the main toxin-based animal models for PD. This table is a simplified overview, showing the most commen uses of the models: also time window studied, dosage, and other factors can influence the pathology in the animal models. SN = substantia nigra, MFB = medial forebrain bundle, STR = striatum. (1)

Table 2 :

 2 Overview of the use of α-synuclein overexpressing models using viral vector approaches in literature. The first line (bold) indicates the AAV2/6-PGK-A53T-α-synuclein model that was studied at 15 weeks post-injection in the submitted paper (page 108). AAV = adeno virus-associated vector, LV = lentivirus. CBA = chicken β-actin, CMV = cytomegalovirus, PGK = phosphoglycerate kinase, WPRE = woodchuck hepatitis virus posttranscriptional regulatory element. WT = wildtype. pi = post-injection. nm = not mentioned. C h a p t e r 1 -I n t r o d u c t i o n | 29

Figure 5 :

 5 Figure 5: Schematic overview of PET imaging targets for PD. A PET radiotracer is injected intravenously and diffuse freely through the body. Radiotracers can selectively bind to elements in the dopamine synthesis (AADC), neurotransmitter vesicles (VMAT2), protein aggregates, presynaptical transporters (DAT), post-synaptical receptors (D 2 R), or glial mitochondrial proteins.

Figure 6 :

 6 Figure 6: Schematic representation of positron emission tomography imaging. Positrons are emitted from the radionuclide and annihilate upon interaction with electrons, giving rise to gamma ray photon emission in opposite directions. Detection of the annihilation event happens through simultaneous detection of these photons by opposite detectors, termed coincidence detection. Figure from Cherry and Gambhir (2001).

  C h a p t e r 2 -M e t h o d o l o g y & I m p r o v e m e n t s | 49

Figure 7 :

 7 Figure 7: Schematic overview of our complete lesion model. 6-OHDA is unilaterally injected in two separate locations near the MFB, connecting the substantia nigra and striatum.

Figure 8 :

 8 Figure 8: Schematic overview of timeline for our complete lesion model.

Figure 9 :

 9 Figure 9: Schematic overview of our mild dopaminergic lesion model. AAVs coding for WT or A53T α-synuclein are unilaterally injected into two sites of the substantia nigra.

Figure 10 :

 10 Figure 10: Schematic overview of our neuroimflammatory model. LPS is unilaterally injected into the striatum.

Figure 11 :

 11 Figure 11: Behavioural assessment with apomorphine-induced rotation test in 6-OHDA rat model at 5wpi. A) Counterclockwise rotations per animal (grey) and group average (black) over time. B) Group average total clockwise (grey) and counter-clockwise (black) rotations over a 60 minute period (left) or the first 30 minute period (right). C) Group average rotations per minute for total clockwise (grey) and counter-clockwise (black) rotations over a 60 minute period (left) or the first 30 minute period (right). Results are expressed as means ± the standard error of the mean (SEM). Paired student t-test **p<0.01, ***p<0.001.

3. 2

 2 Spontaneous forelimb use: Cylinder test C h a p t e r 2 -M e t h o d o l o g y & I m p r o v e m e n t s | 54

Figure 12 :

 12 Figure 12: Behavioural assessment with cylinder test in 6-OHDA rat model at 6wpi and healthy controls. Average percentage of contralateral (red/grey) and ipsilateral (blue) forepaw contacts with the cylinder during 5 minutes. Student t-test with unequal variance was performed against the hypothetical 50% score. Results are expressed as means ± the standard error of the mean (SEM). Student t-test with unequal variance **p<0.01.

Figure 13 :

 13 Figure 13: Behavioural assessment with PhenoTyper activity cages in 6-ODHA rats and healthy control rats at 7wpi. A) Group averages for 6-OHDA (red) and healthy controls (grey) of total distance moved in meters during 23 hours. B) Average velocity in m/h during 23 hours. C) Average time spend moving in hours during 23 hours. Results are expressed as means ± the standard error of the mean (SEM). Student t-test, non-significant.

  a p t e r 2 -M e t h o d o l o g y & I m p r o v e m e n t s | 56

  C h a p t e r 2 -M e t h o d o l o g y & I m p r o v e m e n t s | 57

  C h a p t e r 2 -M e t h o d o l o g y & I m p r o v e m e n t s | 58 PMOD® software. Finally, the average-MR-image and the co-registered-mirrored-average-MR-image were averaged into a single image, resulting in the final MRI template.

Figure 14 :

 14 Figure 14: Schematic representation of the creation of the MRI template. First, all individual MRI are masked.Subsequently masked images were co-registered to a representative image using rigid transformations and averaged. This average MRI was mirrored, and co-registered to the original average MR image. Lastly, the mirrored and original averaged MR image were averaged again, created our final MRI template.

Figure 15 : 59 5

 1559 Figure 15: Schematic representation of coregistration of the Schiffer rat brain atlas to the MRI template.

  C h a p t e r 2 -M e t h o d o l o g y & I m p r o v e m e n t s | 60 exceed 15% of ethanol. The injected doses (ID), body weight, RAS, and ethanol dose per chapter are shown in Table

Figure 16 :

 16 Figure 16: Schematic representation of PET image pre-processing and segmentation. Individual dynamic PET images were first summed and subsequently smoothened. Next, the summed-smoothened individual PET images were co-registered to

  F]DPA714.

Figure 17 :

 17 Figure 17: Representative example of DAT PET Time Activity Curve (TAC). The vertical axis represents SUV values, and the horizontal axis time (s).

  C h a p t e r 2 -M e t h o d o l o g y & I m p r o v e m e n t s | 65

  C h a p t e r 2 -M e t h o d o l o g y & I m p r o v e m e n t s | 66

  C h a p t e r 2 -M e t h o d o l o g y & I m p r o v e m e n t s | 67

Figure 18 :

 18 Figure 18: AADC-PET analysis on 6-OHDA complete lesion model using isoflurane and medetomidine anaesthesia at 28wpi. A) Average AADC enzymatic rate per indicated brain area for the ipsilateral (red) and contralateral (blue) striatum. B) Average AADC enzymatic rate per type of anaesthesia. Results are expressed as means ± the standard error of the mean (SEM). Paired student t-test. *p<0.05, **p<0.01.

  F]DPA714 as described above (paragraph 5.3, page 61). C h a p t e r 2 -M e t h o d o l o g y & I m p r o v e m e n t s | 69

Figure 19 :

 19 Figure19: Schematic illustrations of an online blood counter setup and manual blood sampling to obtain an arterial input function from a rat. After the start of the PET acquisition, the radiotracer is injected via an infusion pump as a fast bolus, and the activity in the whole blood is counted using an online blood counting system. For this, a catheter is placed in the femoral artery of the animal and subsequently passes through the blood counter, in which the radioactivity from the blood is counted. The peristaltic pump ensures a constant and continuous blood flow through the arterial-venous shunt. Manual blood sampling can be conducted using an arterial catheter to correct the blood curve for the plasma free fraction and the contribution from metabolites. Image fromHerfert and colleagues (2019).

Figure 20 :

 20 Figure 20: Plasma to whole blood conversion. A) [ 18 F]DPA714 metabolisms shown as the parental fraction over time. B) Blood input curve in SUV for whole blood (blue) and plasma (grey) over time. C) Calculation of the total area under the curve for graph B. D) Plasma over blood curve. Results are expressed as means.

Figure 21 :

 21 Figure 21: Comparison of the manual (blue) and the automated input function (grey). The radioactivity (kBq/cc) plotted over time (s). Results are expressed as means.

  C h a p t e r 2 -M e t h o d o l o g y & I m p r o v e m e n t s | 72

  C h a p t e r 2 -M e t h o d o l o g y & I m p r o v e m e n t s | 73

  C h a p t e r 2 -M e t h o d o l o g y & I m p r o v e m e n t s | 74

Figure 22 :

 22 Figure 22: Graphs showing correlation between TH stereology and optical density in the substantia nigra. A) Correlation per animal per substantia nigra between the amount of TH-positive cells as measured by stereology, and the TH optical density. B) Correlation between the left/right differences between measurements taken by stereology and optical density. Each point is an individual animal. Correlation calculated with Spearman correlation, non-significant.

Figure 23 :

 23 Figure 23: Histological evaluation of the effect of a complete 6-OHDA lesion on TH and IBA1 in both the substantia nigra and striatum. A) Average TH optical density in ipsilateral (red) and contralateral (blue) substantia nigra and representative histological image. B) Average TH optical density in the striatum. C) Average IBA1 optical density in the striatum. Representative images are taken from a single animal. Results are expressed as means ± the standard error of the mean (SEM). Paired student t-test ***p<0.001.

  0 at 85°C; 1:200; C h a p t e r 2 -M e t h o d o l o g y & I m p r o v e m e n t s | 75

Figure 24 :

 24 Figure 24: Histological evaluation of the effect of a complete 6-OHDA lesion on the dopaminergic system in the striatum. A) Average DAT optical density in the ipsilateral (red) and contralateral (blue) striatum and a representative histological image. B) Average VMAT2 optical density. Representative images are taken from a single animal. Results are expressed as means ± the standard error of the mean (SEM). Paired student t-test **p<0.01, ***p<0.001.

  ; n=3/6; p=0.30). C h a p t e r 2 -M e t h o d o l o g y & I m p r o v e m e n t s | 77

Figure 25 :

 25 Figure25: mRNA levels of dopaminergic and inflammatory markers in the substantia nigra in 6-OHDA animals. mRNA levels in the ipsilateral (red) and contralateral substantia nigra (blue) in intra-MFB 6-OHDA lesioned rats as measured by RT-qPCR. Results are expressed as means ± the standard error of the mean (SEM). Student t-test ***p<0.001.

Figure 26 :Chapter 3 :

 263 Figure 26: Spearman correlation between in vivo data and different post-mortem data. Spearman correlation coefficients are marked in the corresponding case; blue signifies a positive association, while red signifies a negative association. * p<0.01, # p<0.05.

  C h a p t e r 3 -R e s u l t s o n α -s y n u c l e i n o v e r e x p r e s s i o n | 80 More detailed information about the methods can be found in Chapter 2.

Figure 27 :

 27 Figure 27: Schematic overview of the study design. A) Animals were injected with either AAV2/6-PGK-WT-α-synuclein or AAV2/6-PGK-A53T-α-synuclein in the substantia nigra using a double injection approach. B) Timeline of the different experimental analyses.

Figure 28 :

 28 Figure 28: Behavioural analysis using the cylinder test at 4 and 12wpi for both the WT-and A53T-α-synuclein groups. Relative use of the contralateral (yellow/orange) and ipsilateral forepaw (green). Results are expressed as means ± the standard error of the mean (SEM). Student t-test with unequal variance *p<0.05, ***p<0.001.

  C h a p t e r 3 -R e s u l t s o n α -s y n u c l e i n o v e r e x p r e s s i o n | 82

Figure 29 : 18 F

 2918 Figure 29: in vivo PET analyses of both the WT-and A53T-α-synuclein groups using [ 18 F]FMT (AADC) and [ 18 F]LBT999 (DAT). A) Average of quantified AADC-PET images for the WT-α-synuclein and B) A53T-α-synuclein animals. C) AADC enzymatic rate for ipsilateral (yellow/orange) and contralateral striata (green) for both WT-and A53T-α-synuclein injected animals at 11wpi. D) Average of quantified DAT-PET images for the WT-α-synuclein and E) A53T-α-synuclein animals. F) DAT non-displaceable binding potential for ipsilateral and contralateral striata for both WT-and A53T-αsynuclein injected animals at 12wpi. Results are expressed as means ± the standard error of the mean (SEM). Paired student t-test **p<0.01

Figure 30 :

 30 Figure 30: Stereological analysis of the amount TH-positive neurons in the substantia nigra. Average number of cells counted by stereology for ipsilateral (yellow/orange) and contralateral striata (green) for both WT-and A53T-α-synuclein injected animals. Results are expressed as means ± the standard error of the mean (SEM). Paired student t-test **p<0.01.

  Kyono and C h a p t e r 3 -R e s u l t s o n α -s y n u c l e i n o v e r e x p r e s s i o n | 85 colleagues (

Figure 31 :

 31 Figure 31: Use of the contralateral forepaw per animal at both 4wpi and 12wpi. Every line show the evolution of a single animal.

Figure 32 :

 32 Figure 32: Percentage of successful forepaw use in A) the Adjusting steps test and B) the Vibrissae test. Relative use of the contralateral (yellow/orange) and ipsilateral forepaw (green). Results are expressed as means ± the standard error of the mean (SEM). Paired student t-test, non-significant.

Figure 33 :

 33 Figure 33: Schematic overview of study design. A) AAV2/6-PGK-A53T-α-synuclein or PBS is injected in the substantia nigra in two locations. Data are collected from the substantia nigra and the striatum. B) Schematic overview of time schedule for the experiment. Rats were studied at 6-8wpi (early) or 10-13wpi (late).

Figure 34 :

 34 Figure 34: Behavioural analysis using the cylinder test at the early (8wpi) and late (10wpi) timepoints for both the A53Tα-synuclein groups and PBS groups. Relative use of the contralateral (greey/orange) and ipsilateral forepaw (green). Results are expressed as means ± the standard error of the mean (SEM). Student t-test with unequal variance *p<0.05.

Figure 35 :

 35 Figure 35: in vivo PET analyses of A53T-α-synuclein animals, at both the early and late timepoint, using [ 18 F]FMT (AADC) and [ 18 F]LBT999 (DAT). A) Average of quantified AADC-PET images for the early (left) and late timepoint (right) of A53T-αsynuclein injected animals. B) AADC enzymatic rate for ipsilateral (orange) and contralateral striata (green) for both early and late timepoints after A53T-α-synuclein injection. C) Average of quantified DAT-PET images for the A53T-α-synuclein animals, at both the early (left) and late timepoint (right). D) DAT non-displaceable binding potential for ipsilateral and contralateral striata for A53T-α-synuclein injected animals, at both the early and late timepoint. Results are expressed as means ± the standard error of the mean (SEM). Paired student t-test *p<0.05, **p<0.01.

Figure 36 :

 36 Figure 36: Post-mortem analyses of the pS129-α-synuclein, neuroinflammation, and neurodegeneration in the substantia nigra and striatum. A) Representative immunohistochemical images of pS129-α-synuclein staining. B) Quantification of optical density of pS129-α-synuclein staining in the ipsilateral (grey/orange) and contralateral (green) substantia nigra. C) Representative immunohistochemical image of TH staining in the striatum. D) Representative immunohistochemical images of TH staining, and E) quantification through unbiased stereological counting. F) Relative TH mRNA expression levels in the substantia nigra. G) Quantification using optical density measurements. H) Representative immunofluoresence image of IBA1 staining, and I) quantification through fluorescent intensity in the substantia nigra. J) Relative IBA1 mRNA expression levels in the substantia nigra. Results are expressed as means ± the standard error of the mean (SEM). Paired student t-test *p<0.05, **p<0.01.

Figure 37 :

 37 Figure 37: Post-mortem analyses of the dopaminergic system in the substantia nigra and striatum. A) Representative immunohistochemical images of DAT staining in the striatum, and C) quantification using optical density measurements of the ipsilateral (green/orange) and contralateral (green) striatum. B) Relative AADC mRNA expression levels in the substantia nigra. D) Relative DAT mRNA expression levels in the substantia nigra. E) Representative immunohistochemical images of VMAT2 staining in the striatum, and F) quantification using optical density measurements. G) Relative DAT mRNA expression levels in the substantia nigra. Results are expressed as means ± the standard error of the mean (SEM). Paired student t-test *p<0.05, **p<0.01.

Figure 38 :

 38 Figure 38: Spearman correlation between in vivo data and different post-mortem data. A) Shows the early PBS group, while B) shows the late PBS group. C) Shows the early AAV2/6-PGK-A53T-α-synuclein injection group, while D) shows this group at the late timepoint. Spearman correlation coefficients are marked in the corresponding case; blue signifies a positive associations, while red signifies a negative associations. * p<0.01, # p<0.05.

Figure 39 :

 39 Figure 39: Schematic overview of study design. A) AAV2/6-PGK-A53T-α-synuclein or PBS is injected in the substantia nigra in two locations. Data are collected from the substantia nigra and the striatum. B) Schematic overview of time schedule for the experiment.

Figure 40 :

 40 Figure 40: Behavioural analysis using the cylinder test at late timepoint (10wpi) for both the A53T-α-synuclein and PBS groups. Relative use of the ipsilateral (grey/brown) and contralateral forepaw (green). Results are expressed as means ± the standard error of the mean (SEM). Student t-test with unequal variance *p<0.05.

Figure 41 :

 41 Figure 41: in vivo PET analyses of A53T-α-synuclein animals, at 11wpi, using [ 18 F] (AADC). A) Average of quantified AADC-PET images of A53T-α-synuclein injected animals. B) AADC enzymatic rate for ipsilateral (brown) and contralateral striata (green) 11 weeks after A53T-α-synuclein injection. Results are expressed as means ± the standard error of the mean (SEM). The colour scale represent Ki (1/min). Paired student t-test, non-significant.

  , A53T: n=8/8; p=0.25, PBS: n=8/8; p=0.30). C h a p t e r 4 -R e s u l t s o n a m i l d n e u r o d e g e n e r a t i o n m o d e l | 103

Figure 42 :

 42 Figure 42: Post-mortem analyses of the pS129-α-synuclein, neuroinflammation, and neurodegeneration in the substantia nigra and striatum. A) Quantification of immunohistochemical pS129-α-synuclein staining in the ipsilateral (grey/brown) and contralateral (green) substantia nigra, and B) in the striatum, both assessed with optical density measurements. C) Stereological quantification of immunohistochemical TH staining in the substantia nigra. D) Relative TH mRNA expression levels in the substantia nigra. E) Optical denstity quantification of immunohistochemical TH staining in the striatum. F) Relative IBA1 mRNA expression levels in the substantia nigra. G) Optical denstity quantification of immunohistochemical TH staining in the striatum. Results are expressed as means ± the standard error of the mean (SEM). Paired student t-test **p<0.01.

  , n=8/8; p=0.062) nor in PBS injected animals (n=8/8; p=0.42). C h a p t e r 4 -R e s u l t s o n a m i l d n e u r o d e g e n e r a t i o n m o d e l | 104

Figure 43 :

 43 Figure 43: Post-mortem analyses of the dopaminergic system in the substantia nigra and striatum. A) Relative AADC mRNA expression levels in the ipsilateral (grey/brown) and contralateral (green) substantia nigra. B) Relative DAT mRNA expression levels in the substantia nigra. C) Quantification of immunohistochemical DAT staining in the striatum, as assessed with optical density measurements. D) Relative VMAT2 mRNA expression levels in the substantia nigra. E) Quantification of immunohistochemical VMAT2 staining in the striatum, as assessed with optical density measurements. Results are expressed as means ± the standard error of the mean (SEM). Paired student t-test *p<0.05.

Figure 44 :

 44 Figure 44: Schematic overview of study design. A) Viral vectors or PBS is injected in the substantia nigra in two locations. Data are collected from the substantia nigra and the striatum. B) Representation of viral vectors used for the different experimental groups. C) Schematic overview of time schedule for the experiment. Rats were studied at 6-8wpi (early).

Figure 45 :

 45 Figure 45: Behavioural analysis using the cylinder test at 8wpi for all experimental groups and the PBS group.Relative use of the contralateral (orange/grey) and ipsilateral forepaw (green). Results are expressed as means ± the standard error of the mean (SEM). ANOVA with Scheffé post-hoc, *p<0.05, **p<0.01.

Figure 46 :

 46 Figure 46: in vivo PET analysis of all experimental groups and PBS group using [ 18 F]LBT999 (DAT). A) Average of quantified DAT-PET images for experimental groups and PBS animals. B) DAT non-displaceable binding potential for ipsilateral (orange/grey) and contralateral striata (green). Results are expressed as means ± the standard error of the mean (SEM). ANOVA did not show significant differences. Within group paired student t-test ##p<0.01 in intragroup comparision.

Figure 47 :

 47 Figure 47: DAT-PET BP nd -ratio per group. Ratio was calculated by division of the average BP nd of the ipsilateral striatum by the average BP nd of the contralateral striatum. Results are expressed as means ± the standard error of the mean (SEM). ANOVA did not show significant differences. Within group paired student t-test with unequal variance, ##p<0.01, ###p<0.001 in intragroup comparisons.

Figure 48 :

 48 Figure 48: Post-mortem analysis of neurodegeneration in the substantia nigra. Quantification of the number of THpositive cells in the ipsilateral (orange/gre) and contralateral substantia nigra (green) using unbiased stereological counting. Results are expressed as means ± the standard error of the mean (SEM). ANOVA did not show significant differences. Within group paired student t-test #p<0.05, ##p<0.01, in intragroup comparisons.

Figure 49 :Chapter 6 :

 496 Figure 49: Voxel-wise DAT-PET BP nd -ratio changes. A) Creation of the voxel-wise DAT-PET BP nd -ratio images per rat. B) Tmaps created by SPM showing voxel-wise analysis of the DAT-PET BP nd -ratio images versus the PBS group. Nonsignificant.

Figure 50 :

 50 Figure 50: Schematic overview of study design. A) LPS is injected in the striatum, and data are collected from both striata. B) Schematic overview of time schedule for the experiment. Rats were studied at 5-7dpi.

Figure 51 :

 51 Figure 51: In vivo analysis of TSPO as neuroinflammation marker in the striatum. A) Average quantified TSPO-PET images in non-displacable binding potential in different orientations. B) Quantification of TSPO-PET imaging in the striatum. The ipsilateral striatum (orange) and the contralateral striatum (green) at 7 days post-LPS injection. Results are expressed as means ± the standard error of the mean (SEM). Paired student t-test **p<0.01.

Figure 52 :

 52 Figure 52: Post-mortem analyses of neuroinflammation markers in the striatum. A) Relative TSPO mRNA expression levels in the striatum. B) Quantification of optical density of IBA1 staining in the striatum. C) Relative IBA1 mRNA expression levels in the striatum. D) Quantification of optical density of vimentin staining in the striatum. E) Relative vimentin mRNA expression levels in the striatum. F) Quantification of optical density of GFAP staining in the striatum. G) Relative GFAP mRNA expression levels in the striatum. H) Relative TNFα mRNA expression levels in the striatum. The ipsilateral striatum (orange) and the contralateral striatum (green) at 7 days post-LPS injection. Results are expressed as means ± the standard error of the mean (SEM). Paired student t-test ***p<0.001.

Figure 53 :

 53 Figure 53: Post-mortem analyses of dopaminergic neurodegeneration markers. A) Relative TH mRNA expression levels in LPS injected striata (orange) or the contralateral side (green) at 7dpi. B) Quantification of TH through optical density in the striatum following LPS (orange) or PBS injection (grey) at 30dpi. C) Quantification of TH-positive cells in the substantia nigra through unbiased stereology following LPS (orange) of PBS injection (grey) at 30dpi. Results are expressed as means ± the standard error of the mean (SEM). A) Paired student t-test *p<0.05, B,C) unpaired student ttest, non-significant.

  ; n=9/9; p=0.29), nor for VMAT2 mRNA expression in the striatum at 7dpi (Figure 54D; n=11/11; p=0.14). C h a p t e r 6 -R e s u l t s o n n e u r o i n f l a m m a t i o n | 123

Figure 54 :

 54 Figure 54: Post-mortem analyses of dopaminergic markers in the striatum. A) Quantification of DAT through optical density in LPS injected striata (orange) or the contralateral side (green) at 7dpi. Relative B) DAT, C) AADC, and D) VMAT2 mRNA expression levels in the striatum following LPS injection at 7dpi. Results are expressed as means ± the standard error of the mean (SEM). Unpaired student t-test, non-significant.

  ). C h a p t e r 6 -R e s u l t s o n n e u r o i n f l a m m a t i o n | 124

Figure 55 :

 55 Figure 55: Spearman correlation between in vivo individual PET BP nd data and post-mortem expression data. A) Spearman correlation coefficients are marked in the corresponding case; blue signifies a positive correlation, while red signifies a negative correlation. * p<0.01, # p<0.05. B) Correlation graph between expression of GFAP (light blue/red) and IBA1 (dark blue), and TNFα. C) Correlation graph between TSPO-BP nd (light blue/red) and TSPO expression (dark blue), and TNFα. D) Correlation graph between TH and IBA1. E) Correlation graph between TH and AADC.

  F]DPA714 PET C h a p t e r 6 -R e s u l t s o n n e u r o i n f l a m m a t i o n | 125

  C h a p t e r 6 -R e s u l t s o n n e u r o i n f l a m m a t i o n | 126

  C h a p t e r 6 -R e s u l t s o n n e u r o i n f l a m m a t i o n | 128

Figure 56 :

 56 Figure 56: Histological evaluation of IBA1 in LPS-injected, PBS-injected, or non-injected animals at 7dpi. A) Representative photomicrographs showing striatal IBA1 staining in the striatum of the animals of the three experimental groups. B) Striatal area occupied by IBA1+ cells were evaluated in relation to the striatal area and expressed as percentage. One-way ANOVA was used for between-group comparison and Bonferroni post hoc correction for multiple comparisons was applied. n=6/group. Scale bar: 50 μm. ***p<0.001. Figure by Dr. Gubinelli (2019).

Figure 57 :

 57 Figure 57: Representative striatal GFAP expression in LPS, PBS, or non-injected rats at 7dpi. Confocal Z-stack pictures were taken in different points of the striatum close to the injection point. Scale bar: 50 μm. Figure by Dr. Gubinelli (2019).

Figure 58 :

 58 Figure 58: Vimentin histological evaluation after striatal injection of LPS, PBS or non-injection at 7dpi. A) Representative striatal vimentin staining for LPS and PBS-injected groups. B) Striatal optical density for the evaluated groups. Kruskal-Wallis test was used for between-group comparison and Dunn's test of multiple comparisons was applied. n=6/group. Scale bar: 50 μm. *p<0.05, ***p<0.001. Figure by Dr. Gubinelli (2019).

Figure 59 :

 59 Figure 59: Striatal mRNA expression of different genes related to the inflammatory response. Mann-Whitney U test was used for between-group comparison. n=6/group. *p<0.05, **p<0.01. Figure by Dr. Gubinelli (2019).

  F]DTBZ binding potential has been shown C h a p t e r 7 -G e n e r a l D i s c u s s i o n | 134

  Pendleton et A p p e n d i x 1 -A n i m a l m o d e l s o f P D | 138 al., 2002)), and Lewy Body-like inclusions

  A p p e n d i x 1 -A n i m a l m o d e l s o f P D | 140

  , ultimately leading to A p p e n d i x 1 -A n i m a l m o d e l s o f P D | 143

Fig. 1

 1 Fig. 1 Immunohistochemistry for tyrosine hydroxylase (TH). A The C-terminal fragment of LRRK2, called ΔLRRK2, was generated in different forms, WT: G2019S, or dead kinase (DK). B-C, The three fragments were cloned into an AAV and unilaterally injected into the rat SNc. Fifteen weeks PI, TH immunohistochemistry was performed (B). The number of TH-positive neurons was evaluated by stereology (C), with no obvious toxicity due to the LRRK2 fragments at 15 weeks PI. Results are expressed as means ± the standard error of the mean (SEM). N = 10 animals/group. ANOVA and PLSD post hoc test. n.s.: not significant. Scale bars: 750 μm and 400 μm.

Fig. 2

 2 Fig. 2 Histological evaluation of transgene expression in the SNpc at 15 weeks post-injection. (A) Evaluation of α-syn (in green) transduction in the SNpc. TH staining, in red. Scale bar: 500 μm. (B) Measurement of the number of neurons expressing both α-syn and ΔLRRK2GS. The higher magnification shows cytoplasm localization of ΔLRRK2GS. Results are expressed as means ± the standard error of the mean (SEM). Scale bars: 40X = 60 μm, 63X zoom 4 = 10 μm.

Fig. 3

 3 Fig. 3 Immunohistochemistry for tyrosine hydroxylase (TH) and phospho-α-synS129 aggregates at 15 weeks postinjection. (A-D) Evaluation of the number of TH-positive and P-α-synS129-positive neurons by stereology in the SNpc. Scale bars: 750 μm, 400 μm. (E-F) Determination of the percentage of the area occupied by P-α-synS129positive staining in the striatum at 15 weeks PI. Results are expressed as means ± the standard error of the mean (SEM). N = 10 animals/group. ANOVA and PLSD post hoc test. *P < 0.05, ***P < 0.001. Scale bars: 200 μm,50 μm.

Fig. 4

 4 Fig. 4 Immunohistochemistry for tyrosine hydroxylase (TH). (A) Evaluation of striatal fiber loss in the dorso-medial striatum by TH-immunofluorescence. Scale bar: 1000 μm. (B-C) Quantification of the mean grey value at a magnification of 5X. Results are expressed as means ± the standard error of the mean (SEM). N = 10 animals/group. ANOVA and PLSD post hoc test. **P < 0.01 (A-B) scale bar: 1000 μm. (B-C) scale bar: 100 μm.

Fig. 5

 5 Fig. 5 Measurement of transduction volume. A, confocal images to delineate the SNpc based on TH staining (in red), reported in the green channel, corresponding to the human α-syn protein when coexpressed with LRRK2G2019S (GS) and the dead kinase form of LRRK2. Scale bar: 1000 μm. B, Quantification of the fraction (%) of the SNpc expressing human α-syn protein after cotransduction with LRRK2G2019S or LRRK2DK. Note that the level of overexpression of human α-syn protein is similar between the two conditions. Results are expressed as means ± the standard error of the mean (SEM). N = 8 animals/group. No statistical difference, Student t test.

Fig. 6

 6 Fig. 6 Co-localization and expression of ΔLRRK2 and α-syn. A-B, Double immuno-fluorescence of ΔLRRK2 and α-syn at low and high-magnification, showing co-expression of both transgenes at similar levels by most cells. Scale bars: 200 μm, magnifications = 50 μm and 10 μm. C, Quantification of the percentage of co-localization and protein levels (mean fluorescence) for two different groups. Results are expressed as means ± the standard error of the mean (SEM). N = 8 animals/group. No statistical difference, Student t test.

Fig. 7

 7 Fig. 7 Immunohistochemistry for tyrosine hydroxylase (TH) and phospho-α-synS129 aggregates at six weeks post-injection. (A-D) Evaluation of the number of THpositive and P-α-synS129-positive neurons in the SNc by stereology. Results are expressed as means ± the standard error of the mean (SEM). N = 10 animals/group. ANOVA and Fisher PLSD post hoc test. *, P < 0.05. Scale bar: 750 μm, 400 μm.

Fig. 8

 8 Fig. 8 Effect of the various forms of ΔLRRK2 on the toxicity of mutant Htt Mice received a bilateral intrastriatal injection of a mixture of two lentiviral vectors (LV): LV-Htt171-82Q with LV-LacZ (CT, control), LV-ΔLRRK2WT (WT), LV-LRRK2G2019S (GS) or LV-ΔLRRK2G2019S/A1994E (DK). Six weeks after injection, brains were processed for histological evaluation by immunohistochemistry (ICH) for the HA-tag, NeuN, ubiquitin and mutant Htt aggregates using the EM48 antibody to evaluate Htt171-82Q-dependent neurotoxicity. (A) Camera Lucida representation of the rostro-caudal extension of striatal lesions produced by the mutant Htt fragment as seen using NeuN ICH. Dark gray spots in the striatum represent area with loss of NeuN staining (lesions). (B) Histograms of the volumes of the striatal lesions for the four groups. (C) Photomicrographs of the mouse striatum showing that lesions superimposed with HA tag-positive areas in the 3 LLRK2 groups. (D, E) Histograms showing quantification of mutant Htt inclusions (ubiquitin) and aggregates (EM48). Note the absence of major effects of LLRK2 constructs on mutant Htt toxicity, except for a mild increase of the number of EM48 aggregates in the LLRK2G2019S group. Results are expressed as the mean ± the standard error of the mean (SEM). N = 10-12/group. One-way ANOVA and post hoc Fisher's PLSD test. *, p<0.05, Scale bars: A, 1 mm ; C, 500 μm.

Supp Fig 1

 1 Results of the pilot experiments carried out to set up the AAV--synA53T model, characterized by partial degeneration of DA neurons in the SNc of adult rats. (A, B) AAV--synA53T produced partial loss of TH-positive neurons in the SNc at 12 and 15 weeks post injection. Stereological cell count in B shows significant loss compared to control rats of the same age (injected with PBS). Neurons in the SNc after infection were found to be immune-positive for the phosphorylated form of -syn at serine 129 (P-synS129) and thioflavin S (ThioS) fluorescence suggesting aggregation of -synS129. Results are expressed as the mean ± the standard error of the mean (SEM). One-way ANOVA and post hoc Fisher's PLSD test. *, p<0.01. Scale bar in C, 10 μm.

Supp Fig 2

 2 Evaluation of microglial activation (microglia fluorescent area) based on IBA1 immunofluorescence in the SNc (A-B) and striatum (C-D). Results are expressed as the percentage of staining of the control group (PBS). Results are expressed as the mean ± the standard error of the mean (SEM). N = 8 animals/group. ANOVA and PLSD post hoc test (SNc) and Kruskal-Wallis and Mann-Whitney test (striatum). *, p < 0.05; **, p<0.01. Scale bar: low magnification, 200 μm; high magnifications, 50 μm.

Figure 1 :

 1 Figure 1: Clinical symptoms and time course of PD progression.. ........................................................

Figure 2 :

 2 Figure 2: Schematic representation of the basal ganglia. .....................................................................

Figure 3 :

 3 Figure 3: Braak staging in PD patients.. .................................................................................................

Figure 4 :

 4 Figure 4: Schematic representation of prion-like mechenisms for α-synuclein. .................................

Figure 5 :

 5 Figure 5: Schematic overview of PET imaging targets for PD. ..............................................................

Figure 6 :

 6 Figure 6: Schematic representation of positron emission tomography imaging. .................................

Figure 7 :

 7 Figure 7: Schematic overview of our complete lesion model. ..............................................................

Figure 8 :

 8 Figure 8: Schematic overview of timeline for our complete lesion model. ..........................................

Figure 9 :

 9 Figure 9: Schematic overview of our mild dopaminergic lesion model. ...............................................

Figure 10 :

 10 Figure 10: Schematic overview of our neuroimflammatory model. .....................................................

Figure 11 :

 11 Figure 11: Behavioural assessment with apomorphine-induced rotation test in 6-OHDA rat model at 5wpi. ......................................................................................................................................................

Figure 12 :

 12 Figure 12: Behavioural assessment with cylinder test in 6-OHDA rat model at 6wpi and healthy controls. .................................................................................................................................................

Figure 13 :

 13 Figure 13: Behavioural assessment with PhenoTyper activity cages in 6-ODHA rats and healthy control rats at 7wpi. ..............................................................................................................................

Figure 14 :

 14 Figure 14: Schematic representation of the creation of the MRI template. .........................................

Figure 15 :

 15 Figure 15: Schematic representation of co-registration of the Schiffer rat brain atlas to the MRI template. ...............................................................................................................................................

Figure 16 :

 16 Figure 16: Schematic representation of PET image pre-processing and segmentation. ......................

Figure 17 :

 17 Figure 17: Representative example of DAT PET Time Activity Curve (TAC). .........................................

Figure 18 :

 18 Figure 18: AADC-PET analysis on 6-OHDA complete lesion model using isoflurane and medetomidine anaesthesia at 28wpi. ............................................................................................................................

Figure 19 :

 19 Figure 19: Schematic illustrations of an online blood counter setup and manual blood sampling to obtain an arterial input function from a rat. .........................................................................................

Figure 20 :

 20 Figure 20: Plasma to whole blood conversion. .....................................................................................

Figure 21 :

 21 Figure 21: Comparison of the manual (blue) and the automated input function (grey). .....................

Figure 22 :

 22 Figure 22: Graphs showing correlation between TH stereology and optical density in the substantia nigra. ......................................................................................................................................................

Figure 23 :

 23 Figure 23: Histological evaluation of the effect of a complete 6-OHDA lesion on TH and IBA1 in both the substantia nigra and striatum. ........................................................................................................

Figure 24 :

 24 Figure 24: Histological evaluation of the effect of a complete 6-OHDA lesion on the dopaminergic system in the striatum. ..........................................................................................................................

Figure 25 :

 25 Figure 25: mRNA levels of dopaminergic and inflammatory markers in the substantia nigra in 6-OHDA animals. .................................................................................................................................................

Figure 26 :

 26 Figure 26: Spearman correlation between in vivo data and different post-mortem data. ...................

Figure 27 :

 27 Figure 27: Schematic overview of the study design. .............................................................................

Figure 28 :

 28 Figure 28: Behavioural analysis using the cylinder test at 4 and 12wpi for both the WT-and A53T-αsynuclein groups. ...................................................................................................................................

Figure 29 :

 29 Figure 29: in vivo PET analyses of both the WT-and A53T-α-synuclein groups using [ 18 F]FMT (AADC) and [ 18 F]LBT999 (DAT). ..........................................................................................................................

Figure 30 :

 30 Figure 30: Stereological analysis of the amount TH-positive neurons in the substantia nigra. ............

Figure 31 :

 31 Figure 31: Use of the contralateral forepaw per animal at both 4wpi and 12wpi................................

Figure 32 :

 32 Figure 32: Percentage of successful forepaw use in A) the Adjusting steps test and B) the Vibrissae test. ........................................................................................................................................................

Figure 33 :

 33 Figure 33: Schematic overview of study design. ...................................................................................

Figure 34 :

 34 Figure 34: Behavioural analysis using the cylinder test at the early (8wpi) and late (10wpi) timepoints for both the A53T-α-synuclein groups and PBS groups. .......................................................................

Figure 35 :

 35 Figure 35: in vivo PET analyses of A53T-α-synuclein animals, at both the early and late timepoint, using [ 18 F]FMT (AADC) and [ 18 F]LBT999 (DAT). .....................................................................................

Figure 36 :

 36 Figure 36: Post-mortem analyses of the pS129-α-synuclein, neuroinflammation, and neurodegeneration in the substantia nigra and striatum. ....................................................................

Figure 37 :

 37 Figure 37: Post-mortem analyses of the dopaminergic system in the substantia nigra and striatum.

Figure 38 :

 38 Figure 38: Spearman correlation between in vivo data and different post-mortem data. ...................

Figure 39 :

 39 Figure 39: Schematic overview of study design. .................................................................................

Figure 40 :

 40 Figure 40: Behavioural analysis using the cylinder test at late timepoint (10wpi) for both the A53T-αsynuclein and PBS groups. ...................................................................................................................

Figure 41 :

 41 Figure 41: in vivo PET analyses of A53T-α-synuclein animals, at 11wpi, using [ 18 F] (AADC). ..............

Figure 42 :

 42 Figure 42: Post-mortem analyses of the pS129-α-synuclein, neuroinflammation, and neurodegeneration in the substantia nigra and striatum.. .................................................................

Figure 43 :Figure 44 :

 4344 Figure 43: Post-mortem analyses of the dopaminergic system in the substantia nigra and striatum

Figure 45 :

 45 Figure 45: Behavioural analysis using the cylinder test at 8wpi for all experimental groups and the PBS group. ...........................................................................................................................................

Figure 46 :

 46 Figure 46: in vivo PET analysis of all experimental groups and PBS group using [18 F]LBT999 (DAT). .

Figure 47 :

 47 Figure 47: DAT-PET BP nd -ratio per group. Ratio was calculated by division of the average BP nd of the ipsilateral striatum by the average BP nd of the contralateral striatum. .............................................

Figure 48 :

 48 Figure 48: Post-mortem analysis of neurodegeneration in the substantia nigra................................

Figure 49 :

 49 Figure 49: Voxel-wise DAT-PET BP nd -ratio changes. ............................................................................

Figure 50 :

 50 Figure 50: Schematic overview of study design. .................................................................................

Figure 51 :

 51 Figure 51: In vivo analysis of TSPO as neuroinflammation marker in the striatum. ...........................

Figure 52 :

 52 Figure 52: Post-mortem analyses of neuroinflammation markers in the striatum. ...........................

Figure 53 :

 53 Figure 53: Post-mortem analyses of dopaminergic neurodegeneration markers. .............................

Figure 54 :

 54 Figure 54: Post-mortem analyses of dopaminergic markers in the striatum. .....................................

Figure 55 :

 55 Figure 55: Spearman correlation between in vivo individual PET BP nd data and post-mortem expression data. ..................................................................................................................................

Figure 56 :

 56 Figure 56: Histological evaluation of IBA1 in LPS-injected, PBS-injected, or non-injected animals at 7dpi. .....................................................................................................................................................

Figure 57 :

 57 Figure 57: Representative striatal GFAP expression in LPS, PBS, or non-injected rats at 7dpi. .........

Figure 58 :

 58 Figure 58: Vimentin histological evaluation after striatal injection of LPS, PBS or non-injection at 7dpi. .............................................................................................................................................................

Figure 59 :

 59 Figure 59: Striatal mRNA expression of different genes related to the inflammatory response. ......

Figure 60 :

 60 Figure 60: Homologues for familial PD genes in model species.........................................................

Table 1 :Appendix 4 -

 14 Overview of the main toxin-based animal models for PD. ...................................................... Table 2: Overview of the use of α-synuclein overexpressing models using viral vector approaches in literature. ............................................................................................................................................... Table 3: Overview of experimental parameters for [ 18 F]FMT PET imaging. ......................................... Table 4: Overview of experimental parameters for [ 18 F]LBT999 PET imaging. ..................................... Table 5: Overview of experimetal parameters for [ 18 F]DPA714 PET imaging. ...................................... Table 6: List of timepoints for manual blood sampling ......................................................................... Table 7: List of primers used for RT-qPCR ............................................................................................. A p p e n d i x 4 -L o n g F r e n c h s u m m a r y | 174 French summary -long

  R e f e r e n c e s | 220

  R e f e r e n c e s | 221

  Évaluation du système dopaminergique en utilisant l'imagerie TEP in vivo et des analyses post mortem dans le contexte d'un mod PD neurodégénératif et neuroinflammatoire léger Mots clés : La maladie de Parkinson, dopamine, neuroimagerie, compensation, système nigrostriatal Résumé : La maladie de Parkinson (MP) est caractérisée par l'agrégation de l'α-synucléine, la perte des neurones qui synthétisent la dopamine (neurone DA), et une inflammation cérébrale.

Table 3 : Overview of experimental parameters for [ 18 F]FMT PET imaging.

 3 The column for "animals scanned" shows the total number of animals successfully scanned per group, and in brackets the number quantifiable PET scans.

	Chapter	Group	Animals	Average	ID range	Body	Benserazide
			scanned	ID (MBq)	(MBq)	weight	dose
						(g)	(mg/kg)
	Ch 2	6-ODHA	4 (4)	48±3	41-54	612±12	5.14±0.05 i.v.
	Ch 3	WT	4 (3)	40±1	36-43	579±23	9.99±0.20
	Ch 4	Early A53T 7 (4)	45±4	31-61	542±12	9.95±0.04
	Ch 3 and 4 Late A53T 4 (2)	43±2	40-46	589±21	11.24±1.2
	Ch 4 add. Late A53T 6 (5)	49±1	43-53	569±15	9.98±0.10
	5.1.2 [ 18 F]LBT999
	8-((E)-4-fluoro-but-2-enyl)-3β-p-tolyl-8-aza-bicyclo[3.2.1]octane-2β-carboxylic acid methyl
	ester ([ 18

Table 4 : Overview of experimental parameters for [ 18 F]LBT999 PET imaging.

 4

	Chapter	Group	Animals	Average	ID range	Body	Injected
			scanned	ID (MBq)	(MBq)	weight	mass
						(g)	(nmoles)
	Ch 3	WT	3	60±1	58-62	566±21	7.86±3.26
	Ch 4	Early A53T 6	46±2	40-52	546±15	9.90±4.0
	Ch 3 and 4 Late A53T 4	57±2	54-63	590±21	7.55±2.54
	Ch 5	A53T/GFP	7	52±2	45-62	565±22	4.46±0.82
		A53T/GS	7	52±3	45-66	587±14	3.44±0.81
		A53T/DK	7	53±3	39-61	580±13	6.16±1.71
		PBS	5	49±3	39-56	538±27	6.67±1.99
	5.1.3 [ 18 F]DPA714

Table 5 : Overview of experimetal parameters for [ 18 F]DPA714 PET imaging.

 5

	Chapter	Group	Animals	scanned	Average	ID (MBq)	ID range	(MBq)	Body	weight (g)	Injected	mass	(nmoles)	Ethanol	concentra	tion (%)
	Ch 6	LPS	9		61±4	39-82	375±5	2.46±0.46 12±1

C h a p t e r 2 -M e t h o d o l o g y & I m p r o v e m e n t s | 61

Table 6 : List of timepoints for manual blood sampling

 6

	Timepoint	Amount
	(min)	(μL)
	00:05
	00:10
	00:15
	00:30
	00:45
	01:00
	01:15
	01:30
	01:45
	02:00
	03:00
	05:00
	07:30
	10:00
	15:00
	20:00
	30:00
	40:00
	50:00
	60:00
	70:00
	80:00
	90:00

Table 7 : List of primers used for RT-qPCR

 7

	Gene/Primer	Forward sequence	Reverse sequence
	Ppia / Cyclo (housekeeping)	ATGGCAAATGCTGGACCAAA	GCCTTCTTTCACCTTCCCAAA
	Rplp0 (housekeeping)	CAGGCGTCCTCATTAGAG	ATCTGCTGCATCTGCTTGGAG
	Hprt1 (housekeeping)	GGACCTCTCGAAGTGTTGGATAC	CCCTGAAGTGCTCATTATAGTCAA
	AADC / DDC	GTGACCTCTGGTGGCAATGA	GCTTTCTGCTGTGTCCCGAT
	DAT / Slc6a3	GCCCATTTATGCGACCTACAA	CCAGTGACGCAGCGTGA
	GFAP	AATGACTATCGCCGCCAAC	CTCCTGGTAACTCGCCGACT
	IBA1	CCAGCCTAAGACAACCAGCGTC	GCTGTATTTGGGATCATCGAGGAA
	TH	GCCCTACCAAGATCAAACCTAC	GGCGCTGGATACGAGAG
	TNFα	AAATGGGCTCCCTCTCATCAGTTC	TCTGCTTGGTGGTTTGCTACGAC
	TSPO / PBR	CAGTGTCCTTCACGGAGCAG	CGGGTACCCAGGATTGAGAC
	Vimentin	GCAAAGCAGGAGTCAAACGA	AATTCTCTTCCATTTCACGCATCT
	VMAT2	CAGTCACAGGCGAGCCAGAGT	AGCAGCGCAAGGAACACGAT

http://www.curamdevices.ie/curam/research/eu-projects/currentprojects/h2020---mariecurie/brainmattrain/

[START_REF] Chartier-Harlin | alpha-Synuclein locus duplication as a cause of familial Parkinson's disease[END_REF] The BrainMatTrain project is funded by the European Union Horizon 2020 Programme (H2020-MSCA-ITN-2015) under the Marie Skłodowska-Curie Initial Training Network and Grant Agreement No. 676408.

http://www.curamdevices.ie/curam/research/eu-projects/currentprojects/h2020---mariecurie/brainmattrain/

[START_REF] Lesage | Parkinson's disease: from monogenic forms to genetic susceptibility factors[END_REF] The BrainMatTrain project is funded by the European Union Horizon 2020 Programme (H2020-MSCA-ITN-2015) under the Marie Skłodowska-Curie Initial Training Network and Grant Agreement No. 676408.

Due to the global situation concerning COVID-19, analysis of pS129-α-synuclein for the WT-α-synuclein animals has been temporarily put on hold.

Unbiased stereology is ongoing, but halted due to the current COVID-19 pandemic and consequent confinemt.

Acknowledgements / Dankwoord

Acknowledgements / Dankwoord ........................................................................................................... Niet-wetenschappelijke samenvatting .................................................................................................... Non-scientific summary...........................................................................................................................

Acknowledgements/Funding

This work was funded by rolling grants from the CEA and the CNRS. The research leading to these results received funding from la Fondation de France (Parkinson Committee, AAP 2010, Engt 00016819 and AAP 2014, Engt 2014-0052580). Noemie Cresto and Pauline Roost are recipients of PhD fellowships from the Association France Parkinson (2016 and 2019 respectively). This work benefited from support from the national "Translational Research Infrastructure for Biotherapies in Neurosciences" (NeurATRIS, "Investissement d'Avenir", ANR-11-INBS-0011).

Chapter 4: Results on a mild progressive neurodegeneration model

RESULTS ON A MILD PROGRESSIVE NEURODEGENERATION MODEL

Aromatic L-amino acid decarboxylase

We did not measure an asymmetry in AADC mRNA expression in the substantia nigra after AAV2/6-PGK-A53T-α-synuclein injection using RT-qPCR at the early timepoint (Fout! Verwijzingsbron niet gevonden.A, n=7/7; p=0.36). At the late timepoint, however, we do observe a significant difference in AADC mRNA levels between the ipsilateral and contralateral substantia nigra (Δ25%, n=3/6, p=0.043).

For PBS injected animals we did not observe a significant asymmetry in AADC mRNA expression in the substantia nigra using RT-qPCR at either the early (n=8/7; p=0.67), nor at the late timepoint (n=8; p=0.42).

Dopamine transporter

At the early timepoint after AAV2/6-PGK-A53T-α-synuclein injection, we did not observe a significant asymmetry in DAT mRNA expression in the substantia nigra (Fout! Verwijzingsbron niet gevonden.D, n=7/7; p=0.17), nor in DAT protein levels in the striatum (Fout! Verwijzingsbron niet gevonden.B,C, n=7; p=0.13). However, at the late timepoint, we did demonstrate a significant left/right difference in DAT mRNA levels in the substantia nigra (Δ30%; n=4/5; p=0.027) and DAT protein levels in the striatum (Δ29±6%; n=8; p=0.005).

For the PBS groups we did not observe significant asymmetry in DAT mRNA expression in the substantia nigra at either timepoint (Early: n=8/7; p=0.93, Late: n=8/8; p=0.45). Similarly, we did not observe significant asymmetry in DAT protein levels in the striatum after PBS injection (Early: n=8; p=0.42, Late: n=8; p=0.71).

Vesicular monoamine transporter 2

We did not observe a significant asymmetry in VMAT2 mRNA levels in the substantia nigra at the early timepoint after AAV2/6-PGK-A53T-α-synuclein injection (Fout! Verwijzingsbron niet gevonden.G, n=7/6; p=0.47), while there was a significant asymmetry at the late timepoint (Δ42%; n=4/5, p=0.024). However, we did not demonstrate a significant left/right difference in VMAT2 protein levels in the striatum at the early timepoint (Fout! Verwijzingsbron niet gevonden.E,F, n=7; p=0.16) nor the late timepoint in the A53T-α-synuclein animals (n=8; p=0.34).

In the early PBS group we observed a small, but significant asymmetry in VMAT2 mRNA expression in the substantia nigra (Δ18%; n=8/8, p=0.045), while at the late timepoint we did not observe asymmetry (n=8/8; p=0.95). Similarly, we did not observe significant asymmetry in VMAT2 protein levels in the striatum after PBS injection (Early: n=8; p=0.65, Late: n=8; p=0.47).

Supplementary data by Dr. Gubinelli

Similar to what is shown here, Dr. Gubinelli (2019) showed in his doctoral thesis a significant increase of IBA1 cells 7 days increase of IBA1 cells 7 days post-LPS injection as compared to PBS-injected or non-injected animals, and measured by %and measured by %-occupied area of the total striatum after immunohistochemical staining (

Appendix 1: Animal Models of PD

ANIMAL MODELS OF PD

A p p e n d i x 1 -A n i m a l m o d e l s o f P D | 136 Even though, as described above, some important discoveries have been done on human samples, PD autopsy material is rare and precious. Animal models allow for study of different aspects of the disease, and explore both disease progression and evaluate possible treatment strategies. At the moment, there is still no perfect model available that mimics all pathological aspects of PD. The current available models can be broadly divided in four types; 1) pharmacological models, 2) toxinbased models, 3) PD hallmark-based models, and 4) genetic-based models.

Non-mammalian models of PD

Several non-mammalian model species have respective homologues for familial PD genes like LRRK2, Parkin, PINK1, and DJ-1 (Figure 60) [START_REF] Pienaar | Parkinson's disease: Insights from non-traditional model organisms[END_REF][START_REF] Jagmag | Evaluation of Models of Parkinson's Disease[END_REF]. These models could be considered for rapid testing of possible treatment targets and relative effects of genetic and environmental factors on PD pathology [START_REF] Pienaar | Parkinson's disease: Insights from non-traditional model organisms[END_REF][START_REF] Duty | Animal models of Parkinson's disease: A source of novel treatments and clues to the cause of the disease[END_REF][START_REF] Konnova | Animal Models of Parkinson's Disease, Parkinson's Disease: Pathogenesis and Clinical Aspects[END_REF]. Even though they are limited in the way they represent PD symptoms [START_REF] Duty | Animal models of Parkinson's disease: A source of novel treatments and clues to the cause of the disease[END_REF], non-mammalian models have time and economic advantages over traditional rodent or non-human primate models [START_REF] Pienaar | Parkinson's disease: Insights from non-traditional model organisms[END_REF][START_REF] Konnova | Animal Models of Parkinson's Disease, Parkinson's Disease: Pathogenesis and Clinical Aspects[END_REF]. The non-mammalian models are not thought to replace mammalian models, but precede or work alongside each other [START_REF] Pienaar | Parkinson's disease: Insights from non-traditional model organisms[END_REF]. Nematodes (Caenorhabditis elegans) are small transparent roundworms that have a relatively short lifespan (2-3 weeks), fast reproduction, and high progeny number (~350), leading to easy and economic colony maintenance [START_REF] Pienaar | Parkinson's disease: Insights from non-traditional model organisms[END_REF][START_REF] Cooper | Modeling Parkinson's disease in C. elegans[END_REF]. C. elegans can be self-fertilizing, thus creating genetically identical offspring [START_REF] Cooper | Modeling Parkinson's disease in C. elegans[END_REF]. Their basic cell biology and biochemistry is similar to mammals, including Appendix 2 -The C-terminal fragment of LRRK2 with the G2019S substitution increases the neurotoxicity of mutant A53T α-synuclein in dopaminergic neurons in vivo