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Geraldine Liot

Robert-Alain Le Pr

Toilon D'

Marco Emanuele

Nathalie Wolkoff

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Pasquale Sileo

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Laure

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J'adresse mes remerciements au Dr Marie-Christine Chartier-Harlin qui m'a accueillie dans son équipe dès le Master 2. Je la remercie d'avoir été si patiente et d'avoir donné beaucoup de son temps pour m'aider dans toutes les étapes de mon doctorat. Merci de m'avoir transmis votre esprit critique et vos bons conseils. Les mots me manquent pour parvenir à décrire avec justesse l'enthousiasme, la gentillesse et la soif d'enseigner de mon directeur de thèse. Le Dr Jean-Marc Taymans a contribué à ce que cette thèse se déroule dans la meilleure ambiance possible malgré les échecs présents à différents moments depuis mon Master 2 jusqu'à la fin de mon Doctorat. Merci Jean-Marc. Ces années de Doctorat ont été un énorme plaisir pour moi. J'ai réalisé mon rêve d'enfant : participer à la recherche scientifique (Merci l'émission C'est pas sorcier).

Abstract

Parkinson's disease (PD) is the most common neurodegenerative motor disease. Mutations in the Leucine Rich Repeat Kinase 2 (LRRK2) gene are linked to autosomal dominant parkinsonism, and genomic variation at the LRRK2 locus is associated with increased risk for sporadic PD. LRRK2 is a multi-phosphorylated protein and reduced phosphorylation is reported in PD patient brains as well as for some disease mutant forms of LRRK2.

Dephosphorylation leads to alterations in LRRK2 interactions and subcellular localization. PD is characterized by impaired intracellular trafficking. However, the link between LRRK2 phosphorylation and membrane trafficking is not fully understood.

The idea behind this project is to understand the consequences of phosphorylation or dephosphorylation of LRRK2 on its cellular functions.

To this purpose, we generated LRRK2 phosphorylation site mutants and studied how these impacted LRRK2 catalytic activity, neurite growth, localization, protein binding and lysosomal physiology in cell models.

We show that phosphorylation of RAB8a and RAB10 substrates are reduced with phosphomimicking forms of LRRK2, while RAB29 induced activation of LRRK2 kinase activity is enhanced for phosphodead forms of LRRK2 (LRRK2 S860/910/935/955/973/976A).

Considering the hypotheses that PD pathology is associated to increased LRRK2 kinase activity, our results suggest that for its heterologous phosphorylation sites, LRRK2 phosphorylation correlates to protective phenotypes and LRRK2 dephosphorylation correlates to deleterious phenotypes.

Abstract

La maladie de Parkinson (MP) est la maladie motrice neurodégénérative la plus fréquente.

Une des causes de la MP est la présence de mutations pathogènes ou de polymorphismes associés à un risque accru de MP au sein du locus du gène LRRK2 (Leucine Rich Repeat Kinase 2). Sa protéine LRRK2 est multiphosphorylée. Or, des défauts de sa phosphorylation sont observées dans le cerveau de tels patients. Cette déphosphorylation de LRRK2 à certains sites entraîne des modifications de ses interactions et de sa localisation cellulaire. Sachant qu'une altération du trafic intracellulaire est observée dans la MP, il mieux comprendre le lien entre la phosphorylation de LRRK2 et le trafic membranaire est nécessaire.

Cette étude a pour objectif de comprendre les conséquences de la phosphorylation ou de la déphosphorylation de LRRK2 sur ses fonctions cellulaires.

Pour cela nous avons généré des mutants des sites de phosphorylation de LRRK2 et étudié l'impact de ceux-ci sur l'activité catalytique de LRRK2, la croissance des neurites, la localisation, l'interaction protéine-protéine et la physiologie lysosomale dans des modèles cellulaires.

Nous montrons que la phosphorylation des substrats RAB8a et RAB10 est réduite avec les formes phosphomimétiques de LRRK2 (S860/910/935/955/973/976D), tandis que l'activation de l'activité kinase de LRRK2 induite par RAB29 est renforcée pour les formes déphosphomimétiques de LRRK2 (LRRK2 S860/910/935/955/973/976A). Compte tenu des hypothèses selon lesquelles la pathologie de la MP est associée à une activité kinase accrue de LRRK2, nos résultats suggèrent que pour ses sites de phosphorylation hétérologues, la phosphorylation de LRRK2 est corrélée à des phénotypes protecteurs et la déphosphorylation de LRRK2 à des phénotypes délétères. Figure 37: Assessment of interaction between LRRK2 and 14-3-3. Table 1: Non-motor symptoms of Parkinson's disease. Parkinson's disease (PD) is the second most common neurodegenerative disease across the world affecting 0.1 to 0.2% of people, with up to 1% of people affected over the age of 60 [START_REF] Tysnes | Epidemiology of Parkinson's disease[END_REF]. With the aging of the population and the lengthening of the life expectancy, the amount of people with PD will considerably increase from 2010 to 2050 and there will be close to 10 million people by 2030 [START_REF] Dorsey | Projected number of people with Parkinson disease in the most populous nations, 2005 through 2030[END_REF][START_REF] Bach | Projected numbers of people with movement disorders in the years 2030 and 2050[END_REF].

List of figures

List of Tables

Description of the pathology

The first descriptions of a state reminiscent of PD date back to 1000 BC and come from traditional Indian texts and ancient Chinese sources [START_REF] Manyam | Paralysis agitans and levodopa in "Ayurveda": ancient Indian medical treatise[END_REF]. In 1817, Shaking Palsy was described by James Parkinson in an essay on the shaking palsy [START_REF] Parkinson | An Essay on the Shaking Palsy[END_REF]. James

Parkinson described by visual inspection 6 patients with resting tremor, postural instability, paralysis and reduced muscle strength and an evolution of the disease over time The term PD was popularized by William Sanders and Jean-Martin Charcot who distinguished PD from multiple sclerosis and other tremor-like diseases. One century later, pathological structures, later named Lewy bodies (LB), were identified in post-mortem analysis of PD patient brains by Frederic Lewy [START_REF] Lewy | Paralysis agitans. I. Pathologische Anatomie[END_REF]. Some years later, Constantin Tretiakoff identified a depigmentation of the Substantia nigra as another pathological hallmark of the disease [START_REF] Tretiakoff | Contribution a l'etude de l'anatomie pathologique du locus niger de Soemmering avec quelques deductions relatives a la pathogenie des troubles du tonus musculaire et de la maladie de Parkinson[END_REF].

Clinical features

The diagnosis of Parkinson's disease is most often established after the appearance of cardinal motor symptoms, a phase of the disease where up to 50% of dopaminergic cells in the nigro-striatal system are lost [START_REF] Dauer | Parkinson's disease: mechanisms and models[END_REF]. Symptoms include the progressive appearance of bradykinesia, associated with rigidity and resting tremor with characteristic parkinsonian asymmetry. Most patients also develop cognitive and behavioural dysfunctions in parallel with motor symptoms. Non-motor symptoms that can appear before the diagnosis of PD which is usually pronounced with the appearance of motor dysfunctions. The non-motor symptoms will be more pronounced as the disease progress. The disability emerges from the combination and progression of non-motor and motor symptoms of PD. Adapted from [START_REF] Poewe | Parkinson disease[END_REF]

Symptoms

Resting tremor: It is the symptom of Parkinson's disease that is most directly associated with the pathology by the broad public. Interestingly, 20% of parkinsonian patients do not present tremors. The other 80% have limb tremor with a majority of resting pill-rolling tremor of the hand. The tremor oscillates at 4 to 6Hz at rest and disappears during initiation of movement and worsens with stress or intense concentration [START_REF] Jankovic | Parkinson's disease: clinical features and diagnosis[END_REF].

Bradykinesia: It is characterized by the decline of voluntary movement, and in some cases, leads to the suppression of movement (akinesia). It is identified by an inexpressive face, shuffle while walking, difficulty with repetitive tasks. It is almost always accompanied by rigidity.

Rigidity: It is a muscular hypertonia in which the muscle cannot stretch and relax normally, the patient also feels a weakness in the muscles. More than 90% of people with PD experience rigidity.

Postural instability:

The postural instability appears in the early stage or later in the disease evolution. It is one of the most disabling symptoms, which can lead to falls (in 62% of patients) and serious injuries [START_REF] Stolze | Falls in frequent neurological diseases[END_REF].

Freezing: After several years from the appearance of PD, up to 60% of patients experience freezing of movements. Freezing varies from the inability to move for several seconds/minutes, to speak or the interruption of a movement.

Non-motor symptoms: For many years, PD was considered primarily as a motor disease.

Today, important NMS are known to develop prior to motor dysfunctions (Figure 1). More than 50% of PD patients experience dysfunctions of their autonomic function daily. They are summarized in table 1.

Abnormalities of sensation

-Impairment of olfaction (90% of PD patients) [START_REF] Doty | Olfactory dysfunction in parkinsonism: A general deficit unrelated to neurologic signs, disease stage, or disease duration[END_REF] -Visual acuity, contrast sensitivity, colour discrimination [START_REF] Armstrong | Visual Symptoms in Parkinson's Disease[END_REF] -Pain (76%) [START_REF] Valkovic | Pain in Parkinson's disease: A cross-sectional study of its prevalence, types, and relationship to depression and quality of life[END_REF] -Abnormal tactile pressure perception [START_REF] Kesayan | Abnormal tactile pressure perception in Parkinson's disease[END_REF] Cognitive changes -Depression (17%) [START_REF] Reijnders | A systematic review of prevalence studies of depression in Parkinson's disease[END_REF] -Anxiety (67%) [START_REF] Menza | Parkinson's disease and anxiety: Comorbidity with depression[END_REF] -Apathy (40%) [START_REF] Den Brok | Apathy in Parkinson's disease: A systematic review and meta-analysis[END_REF] -Mild cognitive impairment (18%) [START_REF] Nicoletti | Incidence of mild cognitive impairment and dementia in Parkinson's disease: The Parkinson's disease cognitive impairment study[END_REF] -Hallucinations

Autonomic dysfunction

-Orthostatic hypotension (60%) [START_REF] Hellman | Continuous non-invasive monitoring to detect covert autonomic dysfunction in Parkinson's disease[END_REF] -Gastrointestinal dysfonction (80-90%) [START_REF] Fasano | Gastrointestinal dysfunction in Parkinson's disease[END_REF] -Urinary symptoms (25-50%) [START_REF] Winge | Lower urinary tract dysfunction in patients with parkinsonism and other neurodegenerative disorders[END_REF] -Sexual dysfunction [START_REF] Bronner | Correlates of quality of sexual life in male and female patients with Parkinson disease and their partners[END_REF] -Thermoregulation [START_REF] Swinn | Sweating dysfunction in Parkinson's disease[END_REF] Sleep disturbances review in [START_REF] Schrempf | Sleep Disorders in Parkinson's Disease[END_REF] -Insomnia (50%) [START_REF] Lee | Measuring symptom load in Idiopathic Parkinson's disease[END_REF] -Excessive daytime sleepiness -Restless legs syndrome (0-50%) [START_REF] Krishnan | Restless legs syndrome in Parkinson's disease: A case-controlled study[END_REF] -REM sleep behaviour disorder (30-50%) [START_REF] Gagnon | REM sleep behavior disorder and REM sleep without atonia in Parkinson's disease[END_REF] Other symptoms -Excessive drooling (70%) [START_REF] Barbe | Self-assessment of oral health, dental health care and oral health-related quality of life among Parkinson's disease patients[END_REF] -Fatigue (50%) [START_REF] Siciliano | Fatigue in Parkinson's disease: A systematic review and meta-analysis[END_REF] -Swallowing difficulties (41%) [START_REF] Hartelius | Speech and Swallowing Symptoms Associated with Parkinson's Disease and Multiple Sclerosis: A Survey[END_REF] Table 1: Non-motor symptoms of Parkinson's disease. The prevalence of symptoms for PD patient is indicated in brackets.

Anatomical pathology of Parkinson's disease

Organization of the basal ganglia

First mentioned by Claude Galien (129-201 AD) the basal ganglia (BG) were associated to the control of movement in the 19 th century. Edouard Brissaud (1852Brissaud ( -1909) ) was the first to associate the tremor in PD to the substantia nigra. The BG refers to a group of interconnected structures in the brain that control voluntary motor movement and some cognitive functions such as procedural learning, non-associative learning, behaviours and emotions. The BG can be divided into 4 structures:

-Striatum: It is the largest structure of the basal ganglia, composed of the caudate nucleus and putamen. It constitutes the principal entry of the basal ganglia and receives inputs from the cortex, substantia nigra and other brain regions. The striatum is mainly composed of GABAergic neurons called Medium Spiny Neurons (MSNs) and cholinergic interneurons.

-Pallidum: or Globus pallidus, it can be divided into two structures, globus pallidus externus (GPe) and globus pallidus internus (GPi). Mainly composed of GABAergic neurons, the GPe is the major output of the striatum and the GPi is the main output for direct and indirect pathways of the BG.

-Substantia nigra: It is composed of the Substantia Nigra Pars Compacta (SNpc) and Substantia Nigra Pars Reticulata (SNpr). They differ by the neuron composition and projection, the SNpc is composed of dopaminergic neurons projecting to the striatum.

The SNpr is composed of GABAernic neurons that project to the thalamus.

-Subthalamic nucleus: the neurons of the subthalamic nucleus are glutamatergic neurons and target the GPe/GPi, SNpc/SNpr and the striatum.

These structures form a transmission loop that start in the cortex, are regulated in the basal ganglia and projected to the motor cortex. This control loop is called Cortico-basal gangliathalamo-cortical loop (CBGTC loop).

The cortex sends glutamatergic excitatory projections to the striatum. The neurons from the cortex are connected to the striatal GABAergic neurons (MSN). From the striatum, two pathways differ; the direct or excitatory pathway that promotes movement or the indirect pathways that inhibits movement.

When no voluntary movement is intended, the globus pallidus internus and the substantia nigra pars reticulate (GPi/SNpr) are activated, thereby inhibiting the thalamus resulting in the absence of movement. When a voluntary movement is initiated, the striatum is activated resulting in the inhibition of the GPi/SNpr, thereby disinhibiting the thalamus and allowing excitation of the motor cortex and the movement. The SNpc acts in this loop by fine tuning this circuit through its projections to the Striatum (Figure 2).

Dopaminergic neuron loss

Since the first description of a depigmentation of the substantia nigra (SN) observed in postmortem PD patient brains, comprehension of the mechanisms resulting in massive cell death of the dopaminergic neurons of the SN are a priority. When motor dysfunction appears in a patient, it has been estimated than more than 50% of the dopaminergic neurons are depleted.

As these neurons are rich in the dark pigment neuromelanin, this explains the depigmentation of the SN (Figure 3, A) that can be observed in PD patient brains. In the surviving neurons, Dr. The death of the dopaminergic neurons in the SN will reduce the fine control of the direct and indirect pathways. The inhibition induced in healthy conditions by the SNpc on the indirect pathways is weakened, resulting in a higher inhibition of the GPe, reduced inhibition of STN and enhanced activation of the GPi/SNpr that further inhibits the thalamus and inhibits movement. In parallel, the direct pathway will no longer be activated by the SNpc lifting the inhibition of the GPi/SNpr, causing higher inhibition of the thalamus decreasing the voluntary movement (Figure 4, A, B).

Lewy bodies

In 1998, Spillantini, Goedert and their team were able to discover that alpha-synuclein was one of the major components of the LB in patients with alpha-synuclein mutation but also in sporadic patients without any alpha-synuclein mutations [START_REF] Spillantini | α-Synuclein in Lewy bodies[END_REF]. Alphasynuclein was around this period linked to hereditary PD with two missense mutations, A53T

identified in an Italian family [START_REF] Polymeropoulos | Mutation in the α-synuclein gene identified in families with Parkinson's disease[END_REF] and A30P identified in a German family [START_REF] Krüger | Ala30Pro mutation in the gene encoding α-synuclein in Parkinson's disease[END_REF]. LBs are not specific to PD patients, it is possible to find LBs in different pathologies, such as Lewy Body Dementia, Pick's disease and others. The exact role of the LBs are still unknown; it also has been suggested that LBs could be a marker and not the cause of neurodegeneration [START_REF] Corti | Parkinson's disease: What do the genes involved in familial forms tell us?[END_REF]. Recently, analysis of LBs from PD patient brains show accumulation of organelles with lipid membranes, ubiquitin and many other components [START_REF] Shahmoradian | Lewy pathology in Parkinson's disease consists of crowded organelles and lipid membranes[END_REF]. They are the histopathological markers of PD [START_REF] Forno | Neuropathology of Parkinson's Disease[END_REF].

Pathology outside the substantia nigra

In PD, accumulation of Lewy bodies extends beyond the CNS, affecting the enteric nervous system (ENS) [START_REF] Wakabayashi | Lewy bodies in the enteric nervous system in Parkinson's disease[END_REF][START_REF] Braak | Gastric α-synuclein immunoreactive inclusions in Meissner's and Auerbach's plexuses in cases staged for Parkinson's disease-related brain pathology[END_REF]. The ENS is often considered as the second brain. It contains up to 600 million neurons (200 times less than a human brain) [START_REF] Furness | The enteric nervous system and neurogastroenterology[END_REF], with similar neurotransmitters. Half of the dopamine in the body is produced by the ENS [START_REF] Eisenhofer | Substantial production of dopamine in the human gastrointestinal tract[END_REF]. The control of gastro-intestinal mobility and secretion is controlled by parasympathetic and sympathetic stimulations [START_REF] Cersosimo | Neural control of the gastrointestinal tract: Implications for Parkinson disease[END_REF]. The parasympathetic stimulation is mainly ensured by the vagus nerve. Interestingly, Lewy pathology was also detected in the dorsal nucleus of the vagus nerve [START_REF] Benarroch | Involvement of vagal autonomic nuclei in multiple system atrophy and Lewy body disease[END_REF][START_REF] Braak | Gastric α-synuclein immunoreactive inclusions in Meissner's and Auerbach's plexuses in cases staged for Parkinson's disease-related brain pathology[END_REF]. Braak observed LB in the dorsal nucleus of the vagus nerve in all patients and he postulated that this structure was at the crossroads of the pathological process (Figure 5, A-C). An additional hypothesis suggests that PD disease starts in the gut due to disruption of intestinal permeability that allows microbial products to increase inflammation and alpha-synuclein accumulation in enteric neurons [START_REF] Braak | Staging of brain pathology related to sporadic Parkinson's disease[END_REF][START_REF] Shen | The Association Between the Gut Microbiota and Parkinson's Disease, a Meta-Analysis[END_REF] (Figure 5, C).

Braak suggested that the distribution of alpha-synuclein inclusions can spread according to a determined anatomical path that can be divided in 6 steps depending on the progress of the pathology [START_REF] Braak | Staging of brain pathology related to sporadic Parkinson's disease[END_REF].

The process would start within the olfactory bulb and the ENS with the appearance of Lewy neurites and alpha-synuclein aggregates. It is considered to be the pre-symptomatic phase (Stage 1-2). From the brainstem, alpha-synuclein inclusions would be present in the substantia nigra and the hypothalamus and LB lesion start in the SNpc, starting to affect motor function with the appearance of motor dysfunctions (Stage 3-4). In the stages 5-6 alpha-synuclein inclusions are present in the total neocortex and cell death occurs in the SNpc (Figure 5, B) [START_REF] Halliday | Milestones in Parkinson's disease--clinical and pathologic features[END_REF]. Not only the neurons are affected in PD. Supporting cells such as glial cells are important components of the brain, crucial for the regulation of inflammation and for the health of neurons, and are also affected (see next section).

Alterations in glial cells

Glial cells are non-neuronal cells present in the brain, spinal cords and the peripheral nervous system. They are associated to the neurons and represent 50% of total cells in the brain. There are 3 main types of glial cells in the brain that can all regulates cell homeostasis regulate the adverse event that happen in PD.

-Oligodendrocytes: Oligodendrocytes represent 5 to 8% of total cells in the adult Central

Nervous System (CNS) [START_REF] Levine | The oligodendrocyte precursor cell in health and disease[END_REF]. They generate myelin around axons, accelerating the electric transmission of signals. The oligodendrocytes support the integrity of axons covered by myelin by releasing glial trophic support and extracellular vesicles (EVs) [START_REF] Nave | Myelination and support of axonal integrity by glia[END_REF][START_REF] Frühbeis | Oligodendrocytes support axonal transport and maintenance via exosome secretion[END_REF]. Demyelination can lead to axonal disruption, death of oligodendrocytes and can also induce neuronal death.

-Microglia: Microglia are the brain macrophages and form the principal immune defence in the brain. They also contribute to the brain development by regulating the elimination of synapses. The phenomenon, called synapse pruning is controlled by environmental factors, mainly by learning in childhood [START_REF] Paolicelli | Synaptic pruning by microglia is necessary for normal brain development[END_REF]. The outgrowth of microglial processes isolates injured cells and prevent the expansion of the lesion in the brain. It helps to maintain brain homeostasis in the microenvironment around the injury [START_REF] Hines | Microglia processes block the spread of damage in the brain and require functional chloride channels[END_REF]. After injury, microglia produce an inflammatory response to prevent infections. Microglia release proinflammatory cytokines (TNFα,etc). Some of the released cytokines have multiple functions: regulating neurite outgrowth [START_REF] Parish | The Role of Interleukin-1, Interleukin-6, and Glia in Inducing Growth of Neuronal Terminal Arbors in Mice[END_REF], metabolism [START_REF] Gavillet | Modulation of astrocytic metabolic phenotype by proinflammatory cytokines[END_REF], ion channel activity [START_REF] Viviani | Cytokines and neuronal channels: A molecular basis for age-related decline of neuronal function?[END_REF]. High expression levels or mutant forms of alpha-synuclein can induce activation of microgial cells (Lee et al., 2010b).

iPSC-derived macrophages from PD patients with A53T mutations or alpha-synuclein triplication reduced phagocytosis capability, supporting the hypothesis of defective microglial function in PD patients [START_REF] Haenseler | Excess αsynuclein compromises phagocytosis in iPSC-derived macrophages[END_REF].

-Astrocytes: Astrocytes represent the majority of the glial cells. Several roles have been attributed to astrocytes. They regulate the homeostasis of the brain microenvironment by controlling the extracellular levels of glutamate and GABA. They also regulate the brain water homeostasis though their aquaporins. Neurons depend on astrocytes for oxygen/nutrient supply. Astrocytes control the development and the survival of dopaminergic neurons by secretion of Glial-Derived Neurotrophic Factor (GDNF) [START_REF] Lin | GDNF: a glial cell line-derived neurotrophic factor for midbrain dopaminergic neurons[END_REF]. They also contribute to the permeability of the Blood Brain Barrier (BBB) that protects the brain from pathogenic agents, toxins, hormones. Blood vessels with oligodendrocytes, microglia and astrocytes form the neurovascular units. The end feet of the basal process completely surround the blood vessel capillaries to allow high exchanges thought the gap-junctions. In neurodegenerative diseases, such as PD or AD, the neurovascular unit is disrupted [START_REF] Gray | Striatal blood–brain barrier permeability in Parkinson's disease[END_REF]. The permeability of the BBB is decreased, reducing the filtering of harmful substances to the brain (Kaneko et al., 2020).

With age, many aspects of the glial homeostasis are altered [START_REF] Streit | Microglial senescence: does the brain's immune system have an expiration date?[END_REF]. Age is one of the most important factors for PD. Many PD related genes are known to affect the functions of glial cells as presented above. Neuroprotection given by the glial cells are slowly decreased with the progress of the pathology. Development of neuroprotective compounds for the glial cells could help to prevent or slow the disease progression.

Treatments for PD

At present, PD still has no cure. The treatments currently available help to improve the patient's quality of life, with most of the treatments aimed at countering the loss of dopamine in the striatum (Figure 6). When L-DOPA is combined with non-brain permeable AADC inhibitors to promote L-DOPA conversion in the CNS, this results in higher brain dopamine levels. From [START_REF] Poewe | Parkinson disease[END_REF].

L-DOPA

Levodopa was first synthetized in 1911 by Polish biochemist Casimir Funk. 50 years later, two neurologists performed a clinical trial of this compound on 20 patients presenting a parkinsonian syndrome. They were able to detect an impressive improvement of all motor symptoms [START_REF] Birkmayer | Der L-Dioxyphenylalanin (=L-DOPA)-Effekt beim Parkinson-Syndrom des Menschen: Zur Pathogenese und Behandlung der Parkinson-Akinese[END_REF]. If the bradykinesia, and rigidity responds to L-DOPA, the efficacy concerning the tremor is weaker. Concerning the non-motor symptoms, the response is poor or absent. L-DOPA side effects vary, from dyskinesia (30-50% of patients) [START_REF] Schrag | Dyskinesias and motor fluctuations in Parkinson's disease A community-based study[END_REF], to diarrhea and constipation. The first year of treatment with L-DOPA is a real relief for PD patients, usually the response to treatment is good to excellent without motor fluctuations. This period is referred as the honeymoon. With disease progression, L-DOPA response will be less and less steady with appearance of symptoms that cannot be reversed with L-DOPA treatment such as the non-motor symptoms [START_REF] Poewe | Parkinson disease[END_REF].

Other treatments

After the discovery of L-DOPA treatment in 1961, other compounds or surgery has been developed to increase the quality of life of PD patients (Figure 7).

Dopamine agonism

Dopamine agonists offer an alternative treatment to L-DOPA with a higher half-life that allows a longer distribution in the organism. They are less well tolerated but they delay the appearance of dyskinesias.

Non-dopaminergic treatment

-Anticholinergic drugs can be used for the treatment of tremor. In older patient, this treatment is avoided due to the side effects such as hallucination, confusions and dementia [START_REF] Ffytche | Psychosis in Parkinson's Disease[END_REF].

-Antiglutamatergic drugs are used to treat dyskinesia induced by L-DOPA treatment. It increases the release of dopamine and blocks its reabsorption. By themselves, antiglutamatergic drugs do not display significant efficacy in the treatment of PD [START_REF] Crosby | Amantadine in Parkinson's disease[END_REF].

-Adenosine receptor antagonists are believed to be a new potential treatment for PD.

In the basal ganglia, neurons express a huge proportion of A2A receptors outside of the nerve terminals. Studies in animals show an improvement of motor functions but in humans the effects on PD motor dysfunctions was less promising [START_REF] Hickey | Adenosine A2A Antagonists in Parkinson's Disease: What's Next?[END_REF]. This treatment is still in development and effects on neuroprotection and nonmotor symptoms still need to be investigated.

-Surgical treatment Deep Brain Stimulation is used to re-establish an equilibrium in the basal ganglia thalamocortical circuit by placing an active electrode in the globus pallidus or subthalamic nucleus to inhibit their activity. The electrodes are connected to a stimulating impulse generator battery located in the abdomen. The surgery was approved by the US Food and Drug Administration in 2002 for the treatment of PD tremor and advanced PD [START_REF] Dostrovsky | Mechanisms of deep brain stimulation[END_REF]. The surgery implicates risks, such as infection, stroke, bleeding or seizures. It is considered to be the best therapeutic advancement since the levodopa. This treatment shows a remarkable improvement in the quality of life of PD patient.

-Neuronal grafting is currently an emerging method in development. This could be used to replace neuronal loss in PD patients. It uses stem cells as a source of dopaminergic progenitor cells. Several trials are ongoing [START_REF] Stoker | Recent developments in the treatment of Parkinson's Disease[END_REF].

-Neuroprotective compounds are in development. Currently, no drug is known to stop disease progression, however some compounds are believed to be able to slow the disease progression such as immunotherapies (to decrease the amount of alphasynuclein aggregations in cells) [START_REF] Zella | Emerging Immunotherapies for Parkinson Disease[END_REF], iron chelation (to act on ferroptosis, an iron dependent cell death) [START_REF] Ward | Is Chelation Therapy a Potential Treatment for Parkinson's Disease?[END_REF], LRRK2 kinase inhibition and more [START_REF] Salamon | Neuroprotection in Parkinson's disease: facts and hopes[END_REF].

Cause of pathology

The exact aetiology of PD is poorly known, although the cause of the pathology is multifactorial (Figure 8). Except few direct links between toxic molecule such as MPTP, PD etiology is always an association of different parameters; environmental factors (pollution, habits, etc) and genetic factors summarized in Table 2.

Genetics

Before 1997, PD was not commonly considered as a non-genetic disease, heritability was ambiguous due to the lack of conclusive results in studies on twins [START_REF] Tanner | Parkinson disease in twins: An etiologic study[END_REF]. In the last quarter century, genetic studies of disease transmission in families have identified mutations in the coding sequence or other changes such as gene multiplications of many genes as genetic causes of parkinsonism. Still, 10% of PD patient report the presence of PD in their family [START_REF] Thomas | Parkinson's disease[END_REF]. Many genes are identified as genetically linked for PD (Table 2). [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]. The identification of genetic determinants in PD is a great help that allows the identification of molecular pathways that can be implicated in the pathophysiology of PD. Follow up analyses are fundamental to associate loci to a biological function. Research on biological functions of genetic determinants were able to uncover that SNCA, RAB29, MAPT, BST1, GAK, LRRK2 and HLA-DRB5 and others are implicated in cellular pathways such as vesicular trafficking, cytoskeleton and synaptic functions [START_REF] Escott-Price | Polygenic risk of Parkinson disease is correlated with disease age at onset[END_REF][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].

Gene Clinical phenotypes Autosomal dominant inheritance

Non-genetic factors

The total contribution of the environment in the pathogenesis of PD is not fully understood and difficult to quantify. Correlation studies are often used to identify relationships between environmental factors such as exposure to a compound such as pesticides or pollutant and the development of PD. Some identified mechanisms mediated by environmental risk factors are regulation of the inflammation or oxidative stress in cells or direct action on dopamine pathways. Factors that protect against PD have also been identified such as specific diets or lifestyle, with mechanisms involving action on oxidative stress.

Risks factors

Neurotoxins: Some synthetic molecules can cause PD-like phenotypes. One such compound is MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), accidentally discovered after the production and injection of a synthetic opioid intended as a recreational drug. It specifically destroys neurons in the substantia nigra and causes permanent PD symptoms [START_REF] Langston | 1-Methyl-4phenylpyridinium ion (MPP+): Identification of a metabolite of MPTP, a toxin selective to the substantia nigra[END_REF][START_REF] Nonnekes | MPTP-induced parkinsonism: an historical case series[END_REF].

Pesticides: Several studies pinpoint the link between PD and exposure to pesticides such as rotenone or paraquat [START_REF] Tanner | Rotenone, Paraquat, and Parkinson's Disease[END_REF].

Methamphetamine: Methamphetamine binds to dopamine transporter resulting in a higher extracellular dopamine. It has been shown to damage dopaminergic neurons in animal models and a retrospective study in human demonstrate a 3 fold increased risk of PD in methamphetamine user [START_REF] Curtin | Methamphetamine/amphetamine abuse and risk of Parkinson's disease in Utah: A population-based assessment[END_REF].

Cancer: A 44% increased risk to develop PD has been discovered in patients that had developed a melanoma [START_REF] Olsen | Malignant melanoma and other types of cancer preceding parkinson disease[END_REF]. Interestingly, an inverse correlation between smoker-related cancer and PD was found [START_REF] Bajaj | Parkinson's disease and cancer risk: a systematic review and meta-analysis[END_REF].

Traumatic brain injury: It results in a disruption of the blood brain barrier and accumulation of alpha-synuclein in the brain [START_REF] Delic | Biological links between traumatic brain injury and Parkinson's disease[END_REF].

Positive risk factors

Caffein: In several studies, regular coffee consumption were associated with a lower PD risk [START_REF] Hu | Coffee and tea consumption and the risk of Parkinson's disease[END_REF][START_REF] Hong | The Effect of Caffeine on the Risk and Progression of Parkinson's Disease: A Meta-Analysis[END_REF]. The lowering of the risks due to coffee is robust and seems to be linked with sex hormones, the effect is higher in men compared to women [START_REF] Palacios | Caffeine and risk of Parkinson disease in a large cohort of men and women[END_REF]. The effect is also greater in women not under postmenopausal hormones compared to women taking hormones [START_REF] Ascherio | Caffeine, postmenopausal estrogen, and risk of Parkinson's disease[END_REF].

Tobacco: In a prospective study, a lowering by 70% of risk to develop PD was found [START_REF] Morozova | Variations in gender ratios support the connection between smoking and Parkinson's disease[END_REF][START_REF] Li | Association between cigarette smoking and Parkinson's disease: A meta-analysis[END_REF]. In addition to these studies, results from an American cohort identify 2 polymorphisms implicated in interaction between genes and tobacco in PD risk. The SNPs are located in two genes, RXRA and SLC17A6. Interestingly, RXRA encodes the retinoic acid transport (Implicated in dopamine system) and SLC17A6 the vesicular glutamate transporter [START_REF] Lee | Smoking and Parkinson disease: Evidence for gene-by-smoking interactions[END_REF].

Urate: Uric acid (Urate) elevation in serum is linked to protection against dopaminergic neuron death [START_REF] Guerreiro | Protection of midbrain dopaminergic neurons by the end-product of purine metabolism uric acid: potentiation by low-level depolarization[END_REF]. Urate is known to be a strong antioxidant, so a higher concentration of urate could help preventing the increased oxidative stress in cells of PD patients. Meta-analysis suggest a decreased risk to develop PD when having high urate plasma concentration [START_REF] Weisskopf | Plasma urate and risk of Parkinson's disease[END_REF].

Physical activity: Physical exercise is often associated with increased quality of life and lifespan. In this way, a 12.6 years follow-up of 43.000 volunteers indicates that a medium level of physical exercise lowers PD risk [START_REF] Yang | Physical activity and risk of Parkinson's disease in the Swedish National March Cohort[END_REF]. Also, physical exercise is known to affect positively mitochondrial health [START_REF] Memme | Exercise and mitochondrial health[END_REF]) and the immune system [START_REF] Nieman | The compelling link between physical activity and the body's defense system[END_REF].

Physiological dysfunction involved in Parkinson's disease

Mitochondrial dysfunction and oxidative stress

The mitochondrion is the powerhouse of the cell, producing adenoside triphosphate (ATP) a molecule that provides the energy necessary for the chemical reactions of the cell metabolism. Mitochondria are also regulators of cell cycle, cell death, and signalling [START_REF] Horbay | Mitochondrial dynamics during cell cycling[END_REF]. Due to the central role of mitochondria, modification of its activity could result in modification of ATP production, increase of ROS and decrease of cell viability [START_REF] Murphy | How mitochondria produce reactive oxygen species[END_REF]. In an analysis of neurons from postmortem tissue of idiopathic PD, Complexes 1 and 2 were affected [START_REF] Mizuno | Deficiencies in Complex I subunits of the respiratory chain in Parkinson's disease[END_REF][START_REF] Grünewald | Mitochondrial DNA Depletion in Respiratory Chain-Deficient Parkinson Disease Neurons[END_REF]. Alphasynuclein accumulation observed in PD results in the inhibition of mitochondrial complex I leading to an increase of ROS production [START_REF] Mullin | α-Synuclein and Mitochondrial Dysfunction in Parkinson's Disease[END_REF]. Another pathophysiological hallmark of PD, is the accumulation of iron in the SN of PD patients [START_REF] Hirsch | Iron and Aluminum Increase in the Substantia Nigra of Patients with Parkinson's Disease: An X-Ray Microanalysis[END_REF]. Iron (Fe 2+ ) produces ROS through the Fenton reaction and increased ROS production also contributes to mitochondrial dysfunction [START_REF] Wu | Mitochondrial dysfunction in neurodegenerative diseases and drug targets via apoptotic signaling[END_REF]. Many PD associated genes have been linked with mitochondrial dysfunctions, such as impairment of mitochondrial fission, biogenesis, production of ROS, mitophagy, production of ATP and calcium homeostasis (Figure 9). Alpha-synuclein is reported to bind to outer-mitochondrial membrane (OMM) proteins VDAC1, TOM40, TOM20. Interestingly, levels of VDAC1 are reduced in substantia nigra of PD patients [START_REF] Chu | Abnormal alpha-synuclein reduces nigral voltage-dependent anion channel 1 in sporadic and experimental Parkinson's disease[END_REF]. Overexpression of SNCA causes mitochondrial toxicity through increased size of mitochondria containing laminated bodies and decreased (3-(4,5dimethythiazolyl)-2.5-diphenyl-2H-tetrazolium-bromide) MTT levels leading to oxidative stress [START_REF] Hsu | Α-Synuclein Promotes Mitochondrial Deficit and Oxidative Stress[END_REF]. Alpha-synuclein A53T can be found in the membrane of mitochondria and result in the inhibition of complex-1 and increase the mitophagy [START_REF] Chinta | Mitochondrial alphasynuclein accumulation impairs complex I function in dopaminergic neurons and results in increased mitophagy in vivo[END_REF][START_REF] Risiglione | Alpha-Synuclein and Mitochondrial Dysfunction in Parkinson's Disease: The Emerging Role of VDAC[END_REF].

Protein aggregation

Alpha-synuclein aggregation is a major pathological feature of PD, as these aggregates are an abundant component of Lewy bodies [START_REF] Spillantini | α-Synuclein in Lewy bodies[END_REF]. As mentioned above, alpha synuclein was found to have a direct involvement in PD pathogenesis given its implication in autosomal dominant forms of PD [START_REF] Polymeropoulos | Mutation in the α-synuclein gene identified in families with Parkinson's disease[END_REF][START_REF] Singleton | alpha-Synuclein locus triplication causes Parkinson's disease[END_REF][START_REF] Chartier-Harlin | α-synuclein locus duplication as a cause of familial Parkinson's disease[END_REF][START_REF] Martinez | Genome-wide scan linkage analysis for Parkinson's disease: the European genetic study of Parkinson's disease[END_REF][START_REF] Zarranz | The new mutation, E46K, of alpha-synuclein causes Parkinson and Lewy body dementia[END_REF]. Overexpression of alphasynuclein in cell culture induces cell toxicity and neuronal death [START_REF] Zhou | Overexpression of human α-synuclein causes dopamine neuron death in rat primary culture and immortalized mesencephalon-derived cells[END_REF] affecting cellular pathways and organelle homeostasis such as mitochondria, dopamine signalling and lysosomal degradation [START_REF] Minakaki | The Convergence of Alpha-Synuclein, Mitochondrial, and Lysosomal Pathways in Vulnerability of Midbrain Dopaminergic Neurons in Parkinson's Disease[END_REF]. Toxic species of alpha-synuclein can cause alteration of autophagy at different stages of this process [START_REF] Xilouri | Autophagy and Alpha-Synuclein: Relevance to Parkinson's Disease and Related Synucleopathies[END_REF]. Upon aggregation, alpha-synuclein can acquire different conformations resulting in different aggregated forms (oligomers, different fibrillary strains, amorphous aggregates) (Figure 10,A). [START_REF] Espay | Revisiting protein aggregation as pathogenic in sporadic Parkinson and Alzheimer diseases[END_REF][START_REF] Mehra | α-Synuclein misfolding and aggregation: Implications in Parkinson's disease pathogenesis[END_REF] When alpha-synuclein aggregates are formed, they have a negative impact on many cellular pathways such as the SNAREs complex assembly, lipid binding, dopamine synthesis and transport, vesicle synthesis and neurotransmitter release (reviewed in [START_REF] Burré | Cell biology and pathophysiology of αsynuclein[END_REF][START_REF] Sarchione | Alpha-Synuclein and Lipids: The Elephant in the Room?[END_REF]) (Figure 10,B). Alpha-synuclein fibrils can spread from cell to cell, spreading alpha-synuclein aggregation through the brain. It was therefore described as a prion-like disease progression. Preformed fibrils can be internalize and contribute to formation of inclusions in primary cultures [START_REF] Volpicelli-Daley | Exogenous α-Synuclein Fibrils Induce Lewy Body Pathology Leading to Synaptic Dysfunction and Neuron Death[END_REF].

Inflammation

Evidences are emerging implicating neuroinflammation in the appearance and progression of PD. Neuroimaging and analysis of post mortem brain samples show microglial activation, with increased expression of Major histocompatibility complex II (MHCII) following alpha-synuclein pathology [START_REF] Tansey | Immune system responses in Parkinson's disease: Early and dynamic[END_REF]. Astrocytes are also affected by alpha-synuclein aggregation. Indeed, transmission from neurons to astrocytes has been discovered through analysis of post mortem brain samples [START_REF] Braak | Development of α-synuclein immunoreactive astrocytes in the forebrain parallels stages of intraneuronal pathology in sporadic Parkinson's disease[END_REF]Lee et al., 2010c) and alphasynuclein has been found in lysosomal structures, possibly affecting autophagy and protein degradation in astrocytes [START_REF] Loria | α-Synuclein transfer between neurons and astrocytes indicates that astrocytes play a role in degradation rather than in spreading[END_REF]. Activations of these astrocytes in PD leads to the increase of pro-inflammatory cytokines (IL1α, C1q and TNFα), adhesion molecules and increased production of ROS. Production of these molecules creates a vicious circle in the inflammatory system that will increase inflammation in the brain (Figure 11). In light of the role of neuroinflammation in PD, therapies acting on the modulation of neuroinflammation are hypothesized to have a neuroprotective role in PD [START_REF] Hirsch | Neuroinflammation in Parkinson's disease: a target for neuroprotection?[END_REF].

Cell death and stress regulation

For the moment, no therapy has been found to stop the death of neurons in PD. Research to discover compounds that target cell death is a priority therapeutic approach in the context of PD. Different cell death mechanisms have been reported in PD pathogenesis:

Apoptotic cell death: Apoptosis is a programmed cell death which is vital for numerous processes such as cell turnover, development and regulation of the immune system, embryonic development and others [START_REF] Kerr | Apoptosis: A Basic Biological Phenomenon with Wideranging Implications in Tissue Kinetics[END_REF]. It is the main mechanism of cell death in PD and it is characterized by the accumulation of fragmented DNA and apoptotic chromatin that was identified in dopaminergic neurons of PD patients in post-mortem studies [START_REF] Tompkins | Apoptotic-Like Changes in Lewy-Body-Associated Disorders and Normal Aging in Substantia Nigral Neurons[END_REF]. Also in post-mortem studies, elevated caspase-3 activity and protein expression was shown in the SNpc [START_REF] Mogi | Caspase activities and tumor necrosis factor receptor R1 (p55) level are elevated in the substantia nigra from Parkinsonian brain[END_REF]. Dopaminergic cell death can be prevented in in vitro PD models with apoptotic and caspase inhibitors [START_REF] Mb | An anti-Parkinson's disease drug, Npropargyl-1(R)-aminoindan (rasagiline), enhances expression of anti-apoptotic bcl-2 in human dopaminergic SH-SY5Y cells[END_REF][START_REF] Iaccarino | Apoptotic mechanisms in mutant LRRK2-mediated cell death[END_REF].

Diverse alterations of mitochondria lead to mitochondria-mediated apoptosis involving increased ROS production, cytochrome c release, ATP depletion, caspase-9/caspase-3 activation [START_REF] Fiskum | Mitochondrial mechanisms of neural cell death and neuroprotective interventions in Parkinson's disease[END_REF]. Genetic determinants of PD have been linked with mitochondrial deficits including Parkin, LRRK2, PINK1, and DJ-1 [START_REF] Klein | Genetics of Parkinson's Disease[END_REF].

Autophagy: Autophagy is a degradative process of the cell, through which abnormally folded proteins or defective cellular components of the cells will be sequestered in a double membranous vesicle, called autophagosome, and then degraded by the fusion with lysosomes [START_REF] Klionsky | Autophagy as a Regulated Pathway of Cellular Degradation[END_REF]. There are 3 different classes of autophagy: macroautophagy, microautophagy and chaperone-mediated autophagy [START_REF] Mizushima | Autophagy: Renovation of Cells and Tissues[END_REF].

Interestingly, numerous genes linked to PD play a role in the regulation of autophagy (Figure 12). Analysis of brains from Parkinson's patients have shown reduced HSC70 (Role in protein folding and acts as a cargo protein between different organelles) et LAMP-2A (Role in chaperone-mediated autophagy and phagosome maturation) [START_REF] Alvarez-Erviti | Chaperone-Mediated Autophagy Markers in Parkinson Disease Brains[END_REF] as well as an increased proportion of autophagosomes [START_REF] Dehay | Pathogenic lysosomal depletion in Parkinson's disease[END_REF] resulting in defective autophagy. Ferroptotic cell death: Ferroptosis is an iron-dependent cell death involving toxic accumulation of lipid peroxides. Iron-chelation prevents cell death induced by ferroptosis (Do [START_REF] Van | Ferroptosis, a newly characterized form of cell death in Parkinson's disease that is regulated by PKC[END_REF]. Interestingly, iron accumulation occurs in SNpc of PD patients and can be detected using magnetic resonance imaging [START_REF] Lhermitte | Original Papers: ON THE OCCURRENCE OF ABNORMAL DEPOSITS OF IRON IN THE BRAIN IN PARKINSONISM WITH SPECIAL REFERENCE TO ITS LOCALISATION[END_REF][START_REF] Graham | Brain iron deposition in Parkinson's disease imaged using the PRIME magnetic resonance sequence[END_REF]. Iron can accelerate the oxidation of dopamine and forms quinones and free radicals which are known to be involved in neurodegeneration [START_REF] Chen | Oxidative stress in neurodegenerative diseases[END_REF]. The increased production of lipid peroxides cause accumulation of alpha-synuclein [START_REF] Angelova | Lipid peroxidation is essential for α-synuclein-induced cell death[END_REF]. Alpha-synuclein aggregation and Lewy body formation is promoted by iron accumulation, dopamine oxidation and H2O2 [START_REF] Castellani | Sequestration of iron by Lewy bodies in Parkinson's disease[END_REF].

Calcium homeostasis: Calcium is finely regulated and it is a common component of signalling pathways in neurons. It can be taken up from the extracellular space and stored mainly in the endoplasmic reticulum and in the mitochondria. Excessive calcium regulation triggers necrosis and apoptosis [START_REF] Hansford | Physiological role of mitochondrial Ca2+ transport[END_REF][START_REF] Orrenius | Regulation of cell death: the calciumapoptosis link[END_REF]. Dopaminergic neuron health can be compromised by upregulation of calcium levels [START_REF] Blandini | Neurodegeneration and energy metabolism: from chemistry to clinics[END_REF]Chan et al., 2007) or downregulation of cellular calcium content [START_REF] Salthun-Lassalle | Rescue of mesencephalic dopaminergic neurons in culture by low-level stimulation of voltagegated sodium channels[END_REF]. The increased calcium concentration can trigger the activation of tyrosine hydroxylase, producing increased amount of dopamine that leads to cellular damage due to higher dopamine autooxidation [START_REF] Michel | Toxicity of 6-hydroxydopamine and dopamine for dopaminergic neurons in culture[END_REF][START_REF] Rittenhouse | Role of N-and L-type calcium channels in depolarization-induced activation of tyrosine hydroxylase and release of norepinephrine by sympathetic cell bodies and nerve terminals[END_REF] and increased posttranslational modification of alpha-synuclein [START_REF] Martinez-Vicente | Dopamine-modified α-synuclein blocks chaperone-mediated autophagy[END_REF]. The calcium overload can activate calpains (calcium-dependent cysteine proteases). Thus, inhibition of calpains in A30P alpha-synuclein mice results in decreased alpha-synuclein aggregates [START_REF] Dufty | Calpain-Cleavage of-Synuclein Connecting Proteolytic Processing to Disease-Linked Aggregation[END_REF][START_REF] Diepenbroek | Overexpression of the calpain-specific inhibitor calpastatin reduces human alpha-Synuclein processing, aggregation and synaptic impairment in [A30P]αSyn transgenic mice[END_REF].

Unfolded protein response (UPR): To manage increases in misfolded or abnormal proteins, cells can use the Unfolded protein response or ER stress pathway. It is a complex signal transduction pathway that begins with three ER stress activation sensors, inositolrequiring protein 1 (IRE1), protein kinase RNA-like ER kinase (PERK) and activating transcription factor 6 (ATF6). Activation of UPR is needed to control ER homeostasis [START_REF] Hetz | The unfolded protein response: controlling cell fate decisions under ER stress and beyond[END_REF]. Under constant ER stress, UPR will trigger pro-apoptotic pathways resulting in death of the cells. In PD, it is hypothesized that accumulation of misfolded alpha-synuclein may trigger this UPR mechanism leading to increased dopaminergic cell death. UPR makers have been described in synucleinopathy models [START_REF] Matus | Protein folding stress in neurodegenerative diseases: a glimpse into the ER[END_REF] and alpha-synuclein toxic oligomers accumulate in the ER during disease progression [START_REF] Colla | Accumulation of toxic α-synuclein oligomer within endoplasmic reticulum occurs in αsynucleinopathy in vivo[END_REF]. ER-stress and activation of UPR response take place in dopaminergic neurons of PD patients, discovered in brain samples from patients [START_REF] Hoozemans | Activation of the unfolded protein response in Parkinson's disease[END_REF].

Lysosomal dysfunction

Lysosomes are membrane bound organelles whose primary role are the degradations of cellular component and endocytosed particles. They are present in all cells except red blood cells. The degradation capacity of lysosomes is enabled by 60 different enzymes acting in a luminal solution at pH 4.5-5 [START_REF] Xu | Lysosomal Physiology[END_REF].

The accumulation of protein aggregates in the brain of PD patients support the idea of a defect in the degradative pathway. Indeed, reports show accumulation of autophagic vesicles with lysosomal reduction in PD patient brains [START_REF] Chu | Alterations in lysosomal and proteasomal markers in Parkinson's disease: Relationship to alphasynuclein inclusions[END_REF][START_REF] Crews | Selective molecular alterations in the autophagy pathway in patients with lewy body disease and in models of α-synucleinopathy[END_REF][START_REF] Dehay | Pathogenic lysosomal depletion in Parkinson's disease[END_REF]. The increase of ROS from mitochondria causes abnormal membrane permeabilization and disruption of its integrity [START_REF] Vila | Lysosomal membrane permeabilization in Parkinson disease[END_REF]. Autophagy use lysosomes for its degradative properties. Genetic studies also provide numerous evidences of lysosomes/autophagy disruption in PD as seen in table 3, presenting the genes linked with lysosomal/autophagy degradative pathways. The defective function of lysosomes contributes to PD pathogenesis. Lysosomes can degrade alpha-synuclein [START_REF] Cuervo | Impaired Degradation of Mutant -Synuclein by Chaperone-Mediated Autophagy[END_REF] and perturbations in degradative capacity of lysosomes can result in increased alpha-synuclein level in cells. Different components of the trafficking machinery are PD risks factors, leading to alterations of their pathways. PD risks factors such as GBA, VPS35, GAK, RAB29 and LRRK2 impaired the vesicular trafficking in PD leading to increased ROS production, enhancing the aggregation of alpha-synuclein, discussed in section 2.4. (Figure 13) [START_REF] Ebanks | Vesicular Dysfunction and the Pathogenesis of Parkinson's Disease: Clues From Genetic Studies[END_REF]. Several PD genes have been associated with dysregulation of trafficking machinery, which affect lysosomal homeostasis, decreasing its degradative properties. The impairment of mitophagy increases the release of ROS (Reactive Oxygen Species) which enhances alpha-synuclein aggregation. The alpha-synuclein aggregation promotes the disruption of vesicular trafficking as well as lysosomal degradation properties. Adapted from [START_REF] Klein | Is Parkinson's disease a lysosomal disorder?[END_REF] Part 2: LRRK2

Discovery of LRRK2 as a genetic determinant of PD

The identification of LRRK2 started in 2002. Several families presenting a dominant PD inheritance pattern had been identified however the causative gene locus and gene was not known at that time. A genome-wide linkage analysis of a Japanese family (the so-called Sagamihara kindred) identified the PARK8 locus on chromosome 12q12 [START_REF] Funayama | A new locus for Parkinson's Disease (PARK8) maps to chromosome 12p11.2-q13.1[END_REF]. Sequencing of the locus led in 2004 to the identification by two independent groups of mutations in the gene LRRK2 as the cause of PARK8 PD [START_REF] Paisán-Ruíz | Cloning of the gene containing mutations that cause PARK8-linked Parkinson's disease[END_REF]Zimprich et al., 2004).

LRRK2 functional domains

The LRRK2 gene is composed of 51 exons and encodes a 2527 amino acid protein with a molecular mass of 286 kDa. LRRK2 belongs to the ROCO protein family, consisting in humans of 4 homologs (DAPK1, MASL1, LRRK1 and LRRK2). This family of proteins is defined by the presence of a ROC (Ras Of Complex proteins)/ GTPase domain followed by a COR (C-terminal of ROC) domain [START_REF] Bosgraaf | Roc, a Ras/GTPase domain in complex proteins[END_REF]. Another catalytic domain, KIN, is present in LRRK2 and acts as a kinase. These catalytic domains are surrounded by protein-binding domains (ARM-ANK-LRR-WD40) (Figure 14,A). [START_REF] Marchand | LRRK2 Phosphorylation, More Than an Epiphenomenon[END_REF][START_REF] Myasnikov | Structural analysis of the full-length human LRRK2[END_REF].

Protein-Protein interaction domains

LRRK2 is known to interact with various proteins (See section 2.5) through its protein-binding domains (ARM-ANK-LRR-WD40) localized in its N and C terminal regions.

ARM Domain: This domain is found in various proteins from plants to mammals, it consists of approximately 42 repetitive amino acid modular elements. A minimum of 6 repeats is enough to form a functional structure to allow protein interaction [START_REF] Hatzfeld | The Armadillo Family of Structural Proteins[END_REF]. The motif was first discovered in the armadillo locus (homologue of mammalian β-catenin) in Drosophila melanogaster [START_REF] Riggleman | Molecular analysis of the armadillo locus: uniformly distributed transcripts and a protein with novel internal repeats are associated with a Drosophila segment polarity gene[END_REF]. This motif is conserved through evolution and it has been found in diverse species from yeast to human. The role of the ARM domain is to act as binding modules for numerous interacting proteins. Specifically, the ARM domain of LRRK2 is composed of 14 putative armadillo repeats [START_REF] Sejwal | Cryo-EM analysis of homodimeric full-length LRRK2 and LRRK1 protein complexes[END_REF] and it is reported to interact with vesicular proteins such as the RABs at residues 386-392 (Figure 14,C) [START_REF] Mcgrath | LRRK2 binds to the Rab32 subfamily in a GTP-dependent manner via its armadillo domain[END_REF][START_REF] Myasnikov | Structural analysis of the full-length human LRRK2[END_REF].

ANK Domain: This domain consists of a 33-residue sequence motif. It was initially discovered in Drosophila and in yeast [START_REF] Breeden | Similarity between cell-cycle genes of budding yeast and fission yeast and the Notch gene of Drosophila[END_REF]. The ankyrin repeats locate in many proteins with different functions. The repeat is present in Bacteria, Archaea and Eukaryotes and in the viral genome. It serves as a scaffold for interactions and the folding of this domain play important roles such as binding to RABs and VARP protein [START_REF] Mosavi | The ankyrin repeat as molecular architecture for protein recognition[END_REF][START_REF] Barrick | Biological regulation via ankyrin repeat folding[END_REF][START_REF] Araki | Physiological and pathological functions of LRRK2: implications from substrate proteins[END_REF]. Six putative ankyrin repeats have been discovered in LRRK2 [START_REF] Sejwal | Cryo-EM analysis of homodimeric full-length LRRK2 and LRRK1 protein complexes[END_REF]. This domain is also an interacting domain with RAB proteins [START_REF] Purlyte | Rab29 activation of the Parkinson's disease-associated LRRK2 kinase[END_REF].

LRR Domain: Leucine-Rich Repeats are characterized by 20-30 amino acid residues with a rich proportion of Leucine, constituting β-sheet and α-helix folds that when repeated adopt the form of a horseshoe. 375 human proteins have been identified with LRR domains, and most of the LRR proteins remain uncharacterized. LRR motifs are implicated in protein-protein interactions [START_REF] Ng | Leucine-rich repeat (LRR) proteins: Integrators of pattern recognition and signaling in immunity[END_REF]. 14 putative Leucine Rich Repeats were detected in LRRK2 [START_REF] Vancraenenbroeck | Expression, purification and preliminary biochemical and structural characterization of the leucine rich repeat namesake domain of leucine rich repeat kinase 2[END_REF][START_REF] Sejwal | Cryo-EM analysis of homodimeric full-length LRRK2 and LRRK1 protein complexes[END_REF]. Structural analysis of this domain reveals a modulation that stabilizes the inactive state of the KIN domain by protecting ATP entry by maintaining the ATP binding cleft open [START_REF] Myasnikov | Structural analysis of the full-length human LRRK2[END_REF]. WD40 Domain: WD repeats were first identified in the beta-subunit of heterotrimeric G proteins as a repetitive sequence of 43 amino acids. The length is approximately 40 amino acids and often ended by a WD dipeptide [START_REF] Fong | Repetitive segmental structure of the transducin beta subunit: homology with the CDC4 gene and identification of related mRNAs[END_REF]. The repeats form a β-sheet that fold to β-propellers. WD40 domains can be found in a broad range of species from bacteria to humans [START_REF] Xu | Structure and function of WD40 domain proteins[END_REF]. Seven putative WD40 repeats in the C-terminal domain of LRRK2 were detected. WD40, with ROC-COR domain, contributes to dimerization of LRRK2 [START_REF] Jorgensen | The WD40 domain is required for LRRK2 neurotoxicity[END_REF]. The LRRK2 WD40 domain is also reported to mediate protein interaction to bind synaptic vesicles [START_REF] Piccoli | Leucine-Rich Repeat Kinase 2 Binds to Neuronal Vesicles through Protein Interactions Mediated by Its C-Terminal WD40 Domain[END_REF].

GTPase domain

The GTPase domain of LRRK2 (the ROC domain) represents a small fraction of the full-length protein (7% of total), from amino acids 1320 to 1516. Five regions can be identified in the GTPase domain of LRRK2, a P-loop domain, a switch I and Switch II region, G4 and G5 motifs.

The ROC-GTPase domain of LRRK2 binds both GTP and GDP [START_REF] Civiero | Biochemical characterization of highly purified leucine-rich repeat kinases 1 and 2 demonstrates formation of homodimers[END_REF][START_REF] Taymans | The GTPase function of LRRK2[END_REF] and it is capable of hydrolysing GTP [START_REF] Ito | GTP Binding Is Essential to the Protein Kinase Activity of LRRK2, a Causative Gene Product for Familial Parkinson's Disease †[END_REF][START_REF] Lewis | The R1441C mutation of LRRK2 disrupts GTP hydrolysis[END_REF][START_REF] Daniëls | Insight into the mode of action of the LRRK2 Y1699C pathogenic mutant[END_REF] (Figure 15). It can be hypothesized that ROC, in analogy to other Ras family proteins, can shuttle between inactive GDP-bound state and active GTP-bound state and that ROC may be the activator of LRRK2 kinase. Interestingly, the ROC domain of LRRK2 has been found to be necessary for kinase activity of LRRK2 [START_REF] Ito | GTP Binding Is Essential to the Protein Kinase Activity of LRRK2, a Causative Gene Product for Familial Parkinson's Disease †[END_REF], however detailed analysis found that this is independent of its GDP or GTP bound state suggesting that the ROC GTPase cycle does not regulate LRRK2 kinase activity [START_REF] Liu | Kinetic mechanistic studies of wild-type leucine-rich repeat kinase 2: characterization of the kinase and GTPase activities[END_REF][START_REF] Taymans | LRRK2 kinase activity is dependent on LRRK2 GTP binding capacity but independent of LRRK2 GTP binding[END_REF]. The catalytic core domains, including ROC and COR domains, are known to be a hotspot for pathogenic mutations of LRRK2 such as N1437H, R1441C/G/H, Y1699C, and R1628P reported to increase risk for PD and cause alteration of GTPase activity. The R1441C/G/H and Y1699C induce reduced GTPase activity [START_REF] West | Parkinson's disease-associated mutations in LRRK2 link enhanced GTP-binding and kinase activities to neuronal toxicity[END_REF][START_REF] Daniëls | Insight into the mode of action of the LRRK2 Y1699C pathogenic mutant[END_REF][START_REF] Liao | Parkinson diseaseassociated mutation R1441H in LRRK2 prolongs the "active state" of its GTPase domain[END_REF].

Functional mutations in the ROC domain, K1347A and T1348N, that abolish GTP binding capacity also abolish the kinase activity of LRRK2 [START_REF] Ito | GTP Binding Is Essential to the Protein Kinase Activity of LRRK2, a Causative Gene Product for Familial Parkinson's Disease †[END_REF][START_REF] Taymans | LRRK2 kinase activity is dependent on LRRK2 GTP binding capacity but independent of LRRK2 GTP binding[END_REF]. The mutant R1398L increases the GTPase activity but this does not result in increased kinase activity. Structural analysis of the ROC-COR domain demonstrates that mutations are found in or near the interface between ROC and COR domain [START_REF] Daniëls | Insight into the mode of action of the LRRK2 Y1699C pathogenic mutant[END_REF][START_REF] Myasnikov | Structural analysis of the full-length human LRRK2[END_REF]. Mutations in the ROC/COR domain tandem can alter the affinity of GTP and disturb the GTPase activity [START_REF] Ito | GTP Binding Is Essential to the Protein Kinase Activity of LRRK2, a Causative Gene Product for Familial Parkinson's Disease †[END_REF][START_REF] Biosa | GTPase activity regulates kinase activity and cellular phenotypes of parkinson's diseaseassociated LRRK2[END_REF]. Interestingly, the mutant R1398H is associated with a decreased risk for developing PD, and unlike the R1398L or Q, its GTPase activity is enhanced [START_REF] Nixon-Abell | Protective LRRK2 R1398H Variant Enhances GTPase and Wnt Signaling Activity[END_REF][START_REF] Gopalai | LRRK2 N551K and R1398H variants are protective in Malays and Chinese in Malaysia: A case-control association study for Parkinson's disease[END_REF]. The R1441H mutation is reported to result in a twofold reduced GTPase activity in vitro [START_REF] Liao | Parkinson diseaseassociated mutation R1441H in LRRK2 prolongs the "active state" of its GTPase domain[END_REF].

Kinase domain

The kinase activity of LRRK2 has received comparatively more attention than LRRK2 GTPase activity. This is due to the gain of function mutations discovered in familial and sporadic PD patients, leading to increased kinase activity. LRRK2 is a serine/threonine kinase which catalyses the transfer of a phosphate from ATP to the serine or threonine in proteins. The ANK and LRR domain of LRRK2 interact with kinase domain through three sites [START_REF] Myasnikov | Structural analysis of the full-length human LRRK2[END_REF]. Interestingly, one of these sites includes a mutation, N2081D, that increases the risk for Crohn's 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].

LRRK2 kinase activity is a confirmed regulator in LRRK2 in signalling pathways. Indeed, many LRRK2 substrates are found dysregulated in disease (See section 2.4). Through it kinase domain, LRRK2 also phosphorylates itself, including at a cluster of autophosphorylation sites in the ROC domain [START_REF] Greggio | The Parkinson's disease kinase LRRK2 autophosphorylates its GTPase domain at multiple sites[END_REF][START_REF] Marchand | LRRK2 Phosphorylation, More Than an Epiphenomenon[END_REF]. Pathogenic variants have been found in the kinase domain of LRRK2, including mutations, G2019S and I2020T, N2081D [START_REF] Hui | Functional variants in the LRRK2 gene confer shared effects on risk for Crohn's disease and Parkinson's disease[END_REF]. These variants are known to increase the kinase activity of LRRK2. It has been proposed that hyperactive kinase could cause elevated phosphorylation of LRRK2's targets and lead to cell homeostasis disruption [START_REF] Greggio | Kinase activity is required for the toxic effects of mutant LRRK2/dardarin[END_REF]. In vitro studies and mice studies have linked elevated kinase activity of LRRK2 with various toxic effects such as autophagy alteration, neurite shortening, degeneration of DA neurons and organellar dysfunctions [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]Lee et al., 2010a;[START_REF] Madureira | LRRK2: Autophagy and Lysosomal Activity[END_REF].

In this way, selective kinase inhibitors were developed to reduce the toxic effect of LRRK2 [START_REF] Anand | Investigation of leucine-rich repeat kinase 2 : enzymological properties and novel assays[END_REF][START_REF] Deng | Characterization of a selective inhibitor of the Parkinson's disease kinase LRRK2[END_REF][START_REF] Ramsden | Chemoproteomics-Based Design of Potent LRRK2-Selective Lead Compounds That Attenuate Parkinson's Disease-Related Toxicity in Human Neurons[END_REF]. LRRK2 kinase inhibition results in a decreased kinase activity and are followed by a decreased heterologous phosphorylation [START_REF] Dzamko | Inhibition of LRRK2 kinase activity leads to dephosphorylation of Ser 910 /Ser 935 , disruption of 14-3-3 binding and altered cytoplasmic localization[END_REF]. LRRK2 dephosphorylation of S910-S935-S973 has been detected in human PD brain lysates [START_REF] Dzamko | LRRK2 levels and phosphorylation in Parkinson's disease brain and cases with restricted Lewy bodies[END_REF]. It is also interesting to note that recently, increased kinase activity was observed in human Idiopathic PD post-mortem brain of substantia nigra, independently of LRRK2 mutations [START_REF] Di Maio | LRRK2 activation in idiopathic Parkinson's disease[END_REF]. This result reinforces the potential use of LRRK kinase inhibitors in sporadic PD. See more about Kinase inhibition in section 2.8. LRRK2 kinase activity is regulated by LRRK2 regulators. Two PD risk factors, RAB29 and VPS35, implicated in vesicular trafficking physically interact with LRRK2 [START_REF] Macleod | RAB7L1 interacts with LRRK2 to modify intraneuronal protein sorting and Parkinson's disease risk[END_REF][START_REF] Steger | Phosphoproteomics reveals that Parkinson's disease kinase LRRK2 regulates a subset of Rab GTPases[END_REF]. The overexpression of RAB29 induces an elevated S1292 autophosphorylation with a LRRK2 recruitment to the TGN [START_REF] Purlyte | Rab29 activation of the Parkinson's disease-associated LRRK2 kinase[END_REF]. In a study in human PBMCs carrying the VPS35 D620N pathogenic mutation, an elevated LRRK2-mediated T73-RAB10 phosphorylation is reported as well as LRRK2 mediated-phosphorylation of RAB8a, RAB10 and RAB12 [START_REF] Mir | The Parkinson's disease VPS35[D620N] mutation enhances LRRK2-mediated Rab protein phosphorylation in mouse and human[END_REF]. Interestingly, this emerging evidence suggests a relationship between LRRK2, RAB29 and VPS35 in regulating the retromer complex and neurite growth. Expression of LRRK2 G2019S or RAB29 knock down results in neurite shortening in rat primary neurons, and this can be prevented by overexpression of VPS35. Moreover, expression of VPS35 D620N mimics the neurite shortening induced by LRRK2 G2019S [START_REF] Macleod | RAB7L1 interacts with LRRK2 to modify intraneuronal protein sorting and Parkinson's disease risk[END_REF]. Genetic variations at the LRRK2 locus have been reported to be implicated in PD development with missense mutations being the most common cause of familial PD. The first reported mutation was discovered in a German-Canadian and Western Nebraska family [START_REF] Zimprich | Mutations in LRRK2 cause autosomal-dominant parkinsonism with pleomorphic pathology[END_REF]. After this discovery, other mutations have been reported in different families, from all around the globe. In 2005, a mutation in the activation loop of LRRK2 at the position 2019 was reported [START_REF] Kachergus | Identification of a novel LRRK2 mutation linked to autosomal dominant parkinsonism: evidence of a common founder across European populations[END_REF]. This missense mutation changes a glycine residue to a serine, noted G2019S. This mutation is the most common LRRK2 mutation linked to PD. LRRK2 G2019S segregates with PD in 4% of familial cases and 1% of sporadic PD worldwide [START_REF] Healy | Phenotype, genotype, and worldwide genetic penetrance of LRRK2-associated Parkinson's disease: a case-control study[END_REF]. Most LRRK2 PD mutations are located in the catalytic core of the protein and potentiate the kinase activity of LRRK2. The pathogenic mutants R1441C/G/H, Y1699C cause decreased GTPase activity with decreased phosphorylation at S910, S935 and increase kinase activity leading to the hypothesis of a potential interplay between the kinase activity and GTPase activity of LRRK2 [START_REF] Taymans | The GTPase function of LRRK2[END_REF].

Involvement of LRRK2 in Parkinson's disease

Apart from pathogenic mutations, protective risk factors have also been discovered in the GTPase domain (R1398H) but also in the ARM domain (N551K) of LRRK2 [START_REF] Gopalai | LRRK2 N551K and R1398H variants are protective in Malays and Chinese in Malaysia: A case-control association study for Parkinson's disease[END_REF] (Figure 16). Interestingly, these two protective mutants lead to a reduced RAB10 phosphorylation in Peripheral Blood Mononuclear Cells (PBMC) of healthy carriers without change of S935 phosphorylation level [START_REF] Wang | Understanding LRRK2 kinase activity in preclinical models and human subjects through quantitative analysis of LRRK2 and pT73 Rab10[END_REF]. Many additional PD risk factor mutations in LRRK2 have been reported, although many remain to be confirmed and few of them are subjected to deeper investigations (Table 4).

LRRK2 functions

Improving our comprehension of the precise role of LRRK2 is crucial for the development of new therapies. As seen before, LRRK2 is subjected to modifications, such as its phosphorylation rate, in sporadic PD. Moreover, LRRK2 mutations represent a nonnegligeable proportion of PD patients. By studying the protein-protein interaction network of LRRK2 as well as exploring the phenotypes of different LRRK2 mutations, it is possible to deduce the involvement of LRRK2 in diverse pathways resumed in Figure 17. It is possible to identify a pathway involving LRRK2 by looking at the pathways of its cellular partners. In Table 5, a non-comprehensive list of LRRK2's cellular partners are given. Partners are grouped based on their implication in specific pathways.

Protein

Relation to LRRK2 Potential role reference

ArfGAP1

GAP-like protein for LRRK2. LRRK2 genetic modifier LRRK2.

Kinase substrate.

Golgi to ER retrograde vesicular sorting. GTPase activating protein (GAP) for LRRK2. [START_REF] Xiong | GTPase Activity Plays a Key Role in the Pathobiology of LRRK2[END_REF][START_REF] Xiong | ArfGAP1 Is a GTPase Activating Protein for LRRK2: Reciprocal Regulation of ArfGAP1 by LRRK2[END_REF]Stafa et

RPS27

LRRK2 kinase substrate 40S ribosomal subunit [START_REF] Martin | Ribosomal protein s15 phosphorylation mediates LRRK2 neurodegeneration in Parkinson's disease[END_REF] Table 5: Identified cellular partners of LRRK2. From [START_REF] Erb | LRRK2 and the Endolysosomal System in Parkinson's Disease[END_REF][START_REF] Jeong | Pathological Functions of LRRK2 in Parkinson's Disease[END_REF]. In RED are indicated the proteins involved in vesicular trafficking. In ORANGE the proteins linked with protein translation. In BLUE proteins related to microtubules. In GREEN proteins linked with mitochondria. In GRAY proteins linked with LRRK2 regulation.

From this list, it is possible to identify pathways in which LRRK2 regulates a large number of factors. By performing an Interaction Network Analysis, Tomkins and colleagues were able to link LRRK2 to functions in intracellular organization, transport and protein metabolism [START_REF] Tomkins | Comparative Protein Interaction Network Analysis Identifies Shared and Distinct Functions for the Human ROCO Proteins[END_REF]. Other reports show implications in the regulations of autophagy or Golgi-related functions [START_REF] Beilina | Unbiased screen for interactors of leucine-rich repeat kinase 2 supports a common pathway for sporadic and familial Parkinson disease[END_REF]). We will discuss in this section the different pathways where LRRK2 has been linked.

Vesicular trafficking:

From synthesis of proteins to their release, the intracellular membrane system communicates via vesicles, which is a structure enclosed by a lipid bilayer, similar to the plasma membrane.

A vesicle can fuse to the plasma membrane to release its content in the extracellular space (exocytosis) or fuse with intracellular organelles. A vesicle can also be released out of the cell, for examples via specialized endosomes (Multi-Vesicular Bodies), containing intraluminal vesicles. The MVBs can be degraded via fusion with the lysosomes or released by fusing with the plasma membrane, this fusion will lead to the release of extracellular vesicles [START_REF] Hessvik | Current knowledge on exosome biogenesis and release[END_REF]. A vesicle can be formed from the invagination of the plasma membrane and

RGS2

Interacting partner of LRRK2 GTPase Activating Protein for LRRK2 [START_REF] Dusonchet | A Parkinson's disease gene regulatory network identifies the signaling protein RGS2 as a modulator of LRRK2 activity and neuronal toxicity[END_REF] 14-3-3

PP1 PP2A

Phosphatase of LRRK2 Regulate LRRK2 phosphorylation state [START_REF] Lobbestael | Identification of protein phosphatase 1 as a regulator of the LRRK2 phosphorylation cycle[END_REF][START_REF] Drouyer | Protein phosphatase 2A holoenzymes regulate leucine-rich repeat kinase 2 phosphorylation and accumulation[END_REF] form intracellular vesicles (Endocytosis). These mechanisms are regulated by many different protein classes such as adaptor proteins, tethering proteins and coat proteins, Soluble Nethylmaleimide-Sensitive factor Attachment protein Receptors (SNAREs) and RAB-GTPases, to name a few. 18). The curvature of membrane will increase the tension on both membranes leading to hemifusion and yielding to a fusion pore. The fusion pore expands until the complete release of tension and complete fusion [START_REF] Chen | SNARE-mediated membrane fusion[END_REF].

The RAB-GTPase proteins localize to diverse compartments in cells in a close relationship to membranes. They regulate membrane trafficking events such as vesicle formation, vesicle transport and membrane fusion [START_REF] Søreng | Membrane Trafficking in Autophagy[END_REF]. RAB proteins localize at specific organelles, regulating their transport or processes such as autophagy [START_REF] Ao | Regulation of autophagy by the Rab GTPase network[END_REF]. LRRK2 is found at SNARE complexes and interacts with different RAB proteins that regulates membrane trafficking in the cells (Figure 19).

LRRK2 is found in a homogenous distribution throughout the cytoplasm and associated with organellar membranes and the microtubule network [START_REF] Biskup | Localization of LRRK2 to membranous and vesicular structures in mammalian brain[END_REF]. LRRK2 can localize to the Trans-Golgi network (TGN) where one of its interactors, RAB29, regulates TGN structure. The overexpression of RAB29 recruits LRRK2 to the TGN and induces it fragmentation and causes centrosomal cohesion deficits by accumulation of phosphorylated RAB8a [START_REF] Fujimoto | Parkinson's disease-associated mutant LRRK2 phosphorylates Rab7L1 and modifies trans-Golgi morphology[END_REF][START_REF] Madero-Pérez | RAB7L1-Mediated Relocalization of LRRK2 to the Golgi Complex Causes Centrosomal Deficits via RAB8A[END_REF][START_REF] Purlyte | Rab29 activation of the Parkinson's disease-associated LRRK2 kinase[END_REF].

In addition to the many vesicular traffic interactors with which LRRK2 interacts, the role of LRRK2 in the regulation of vesicular trafficking has been confirmed in animal models. LRRK2

KO mice display increased lysosomal markers such as cathepsin D, lipofuscin and p62 in the kidney but not in the brain [START_REF] Herzig | LRRK2 protein levels are determined by kinase function and are crucial for kidney and lung homeostasis in mice[END_REF][START_REF] Tong | Loss of leucinerich repeat kinase 2 causes age-dependent bi-phasic alterations of the autophagy pathway[END_REF][START_REF] Baptista | Loss of Leucine-Rich Repeat Kinase 2 (LRRK2) in Rats Leads to Progressive Abnormal Phenotypes in Peripheral Organs[END_REF][START_REF] Wallings | LRRK2 interacts with the vacuolar-type H+-ATPase pump a1 subunit to regulate lysosomal function[END_REF]. Overexpression of LRRK2 G2019S leads to a reduction in RAB7 activity and delays early endosomal trafficking [START_REF] Gómez-Suaga | LRRK2 delays degradative receptor trafficking by impeding late endosomal budding through decreasing Rab7 activity[END_REF]. G2019S also results in the accumulation of multi vesicular bodies (MVB) and autophagic vacuoles (Plowey et al., 2008a;[START_REF] Alegre-Abarrategui | LRRK2 regulates autophagic activity and localizes to specific membrane microdomains in a novel human genomic reporter cellular model[END_REF][START_REF] Gómez-Suaga | Leucine-rich repeat kinase 2 regulates autophagy through a calcium-dependent pathway involving NAADP[END_REF]. Similar results are obtained in the cerebral cortex of G2019S mice and in iPSC derived dopaminergic neurons from G2019S carriers [START_REF] Ramonet | Dopaminergic Neuronal Loss, Reduced Neurite Complexity and Autophagic Abnormalities in Transgenic Mice Expressing G2019S Mutant LRRK2[END_REF][START_REF] Sánchez-Danés | Disease-specific phenotypes in dopamine neurons from human iPS-based models of genetic and sporadic Parkinson's disease[END_REF][START_REF] Reinhardt | Genetic Correction of a LRRK2 Mutation in Human iPSCs Links Parkinsonian Neurodegeneration to ERK-Dependent Changes in Gene Expression[END_REF]. The observations made with pathogenic mutations of LRRK2 emphasize its role in regulation of vesicle trafficking.

Autophagy:

Dysregulation of autophagic flux in neurons, in a context of proteinopathies such as PD, leads to the alteration of the degradation system and accumulation of toxic species such as alpha synuclein. LRRK2 have been linked with different autophagy step. It is possible to divide autophagy in different steps, phagophore formation, maturation with completion of the autophagosome, fusion with lysosome, and at the final step with the degradation of autolysosomes content (Figure 20). LRRK2 has been found to act on all of these steps.

To initiate autophagy, phosphorylated p62 binds to LC3 and ubiquitinated proteins and therefore are associated to the nascent phagophore membrane. LRRK2 phosphorylates p62 and expression of pathogenic mutants (N1437H, R1441C/G/H, Y1699C, and G2019S) result in an increased p62 phosphorylation. Overexpression of p62 increased LRRK2 degradation through autophagic pathways [START_REF] Park | Interplay between Leucine-Rich Repeat Kinase 2 (LRRK2) and p62/SQSTM-1 in Selective Autophagy[END_REF]. LRRK2 is able to phosphorylate Leucyl-tRNA synthetase 1 (LRS), which is responsible of mTOR activation [START_REF] Bonfils | Leucyl-tRNA Synthetase Controls TORC1 via the EGO Complex[END_REF]Ho et al., 2018b). Expression of LRS phosphomutant T293D (a phosphomimicking variant at the LRRK2 phosphosite) in cells increased the expression of LC3B and p62 and result in accumulation of alpha-synuclein (Ho et al., 2018b). The same observation was established in brain lysate from G2019S mice (Ho et al., 2018b). Expression of WT-LRRK2 and G2019S-LRRK2 in rat primary cortical neuron culture induced decreases of LC3 puncta compared to non-transgenic cultures after induction of lysosomal biogenesis, indicating that LRRK2 expression as well as G2019S inhibits the production of autophagosomes. Authors where able to block this effect with LRRK2 kinase inhibition [START_REF] Su | Inhibition of excessive mitochondrial fission reduced aberrant autophagy and neuronal damage caused by LRRK2 G2019S mutation[END_REF][START_REF] Manzoni | mTOR independent regulation of macroautophagy by Leucine Rich Repeat Kinase 2 via Beclin-1[END_REF][START_REF] Wallings | LRRK2 interacts with the vacuolar-type H+-ATPase pump a1 subunit to regulate lysosomal function[END_REF].

To form autolysosomes, the fusion between a lysosome and autophagosomes is needed. In iPSC-derived LRRK2-G2019S astrocytes, colocalization between markers of autophagosomes (LC3) and lysosomes (LAMP1) is decreased, suggesting a reduced fusion event [START_REF] Di Domenico | Patient-Specific iPSC-Derived Astrocytes Contribute to Non-Cell-Autonomous Neurodegeneration in Parkinson's Disease[END_REF]. The same results were described in SH-SY5Y expressing G2019S mutant [START_REF] Obergasteiger | Kinase inhibition of G2019S-LRRK2 enhances autolysosome formation and function to reduce endogenous alpha-synuclein intracellular inclusions[END_REF]. After LRRK2 kinase inhibition, the number of autolysosomes in these cells was lowered, due to alteration of autophagosomes/lysosome fusion [START_REF] Saez-Atienzar | The LRRK2 inhibitor GSK2578215A induces protective autophagy in SH-SY5Y cells: involvement of Drp-1-mediated mitochondrial fission and mitochondrial-derived ROS signaling[END_REF].

Maintenance of lysosomal pH (around 4.5-5) is a crucial step for the activity of enzymes and degradation of lysosomal content [START_REF] Hu | The endosomal-lysosomal system: from acidification and cargo sorting to neurodegeneration[END_REF]. Interestingly, LRRK2 interacts with the ATP6V0A1 proton pump, a risk factor for PD [START_REF] Chang | A meta-analysis of genome-wide association studies identifies 17 new Parkinson's disease risk loci[END_REF]. ATP6V0A1 is involved in the acidification of various organelles [START_REF] Chang | A meta-analysis of genome-wide association studies identifies 17 new Parkinson's disease risk loci[END_REF][START_REF] Wallings | LRRK2 interacts with the vacuolar-type H+-ATPase pump a1 subunit to regulate lysosomal function[END_REF]. Interestingly, in cultured primary cortical neurons, the interaction between LRRK2-R1441C and ATPV60A1 is reduced, with reduced acidification of lysosomes followed by a reduction in lysosomal degradation [START_REF] Wallings | LRRK2 interacts with the vacuolar-type H+-ATPase pump a1 subunit to regulate lysosomal function[END_REF]. In LRRK2-G2019S derived fibroblasts as well as postmortem tissue, enlarged lysosomes were observed [START_REF] Henry | Pathogenic LRRK2 mutations, through increased kinase activity, produce enlarged lysosomes with reduced degradative capacity and increase ATP13A2 expression[END_REF]. In HEK293T cells, lysosomal enzymes Cathepsin B and L activities was lowered with G2019S expression [START_REF] Henry | Pathogenic LRRK2 mutations, through increased kinase activity, produce enlarged lysosomes with reduced degradative capacity and increase ATP13A2 expression[END_REF]. These two enzymes, when inhibited, abolish the degradation of alpha-synuclein and promote its aggregation [START_REF] Mcglinchey | Cysteine cathepsins are essential in lysosomal degradation of α-synuclein[END_REF].

Synaptic function:

BAC transgenic mice expressing LRRK2 G2019S are reported to present age dependent decreases in release and uptake of dopamine without loss of dopaminergic neurons in SNpc at 12 months of age [START_REF] Li | Neurobiology of Disease Enhanced Striatal Dopamine Transmission and Motor Performance with LRRK2 Overexpression in Mice Is Eliminated by Familial Parkinson's Disease Mutation G[END_REF]. LRRK2 G2019S was responsible of degeneration of dopaminergic terminals as well as downregulation of dopaminergic homeostasis [START_REF] Thomas | Parkinson's disease[END_REF]. LRRK2 participates in different protein pathways regulating synaptic vesicle trafficking at the presynaptic and postsynaptic terminals.

LRRK2, by its interaction can modulate exocytosis events:

RAB3a, 3b, 3c and 3d regulate exocytosis events and lysosome exocytosis [START_REF] Lledo | Rab3 proteins: key players in the control of exocytosis[END_REF][START_REF] Vieira | Rab3a and Rab10 are regulators of lysosome exocytosis and plasma membrane repair[END_REF]. RAB10 phosphorylation by LRRK2 induces lysosomal release [START_REF] Vieira | Rab3a and Rab10 are regulators of lysosome exocytosis and plasma membrane repair[END_REF][START_REF] Kuwahara | Roles of lysosomotropic agents on LRRK2 activation and Rab10 phosphorylation[END_REF]. LRRK2 can also interact with AP3B1 which is a component of the AP3 complex to regulate synaptic vesicle generation from endosomes [START_REF] Cheung | Adaptor Protein Complexes 1 and 3 Are Essential for Generation of Synaptic Vesicles from Activity-Dependent Bulk Endosomes[END_REF].

LRRK2 modulates the SNARE complex during synaptic transmission by a negative regulation of snapin and SNAP-25 [START_REF] Yun | LRRK2 phosphorylates Snapin and inhibits interaction of Snapin with SNAP-25[END_REF].

Endocytosis events can also be regulated by LRRK2 interactors:

RAB5 is an important player in the regulation of clathrin-mediated endocytosis [START_REF] He | Dynamics of phosphoinositide conversion in clathrin-mediated endocytic traffic[END_REF]. RAB5 is also a regulator of the readily releasable pool, a pool of vesicles at the presynaptic bouton that can be easily deployed compared to other vesicles [START_REF] Hoopmann | Endosomal sorting of readily releasable synaptic vesicles[END_REF]. Finally, RAB5 controls AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid)

receptor internalization in a clathrin-dependent manner [START_REF] Brown | NMDA Receptor-Dependent Activation of the Small GTPase Rab5 Drives the Removal of Synaptic AMPA Receptors during Hippocampal LTD[END_REF]. RAB8a modules EGFR trafficking and EGFR (Epidermal Growth Factor Receptor) internalization is disrupted by LRRK2 G2019S, and the deficits of EGFR trafficking induced by LRRK2 G2019S can be rescued by RAB7 overexpression [START_REF] Gómez-Suaga | LRRK2 delays degradative receptor trafficking by impeding late endosomal budding through decreasing Rab7 activity[END_REF][START_REF] Rivero-Ríos | The G2019S variant of leucine-rich repeat kinase 2 (LRRK2) alters endolysosomal trafficking by impairing the function of the GTPase RAB8A[END_REF].

Due to the hyperactivity of LRRK2, increased phosphorylation of auxilin (DNAJ6) was reported in patient-derived iPSC dopaminergic neurons carrying G2019S, R1441C/G [START_REF] Nguyen | LRRK2 phosphorylation of auxilin mediates synaptic defects in dopaminergic neurons from patients with Parkinson's disease[END_REF]. This led to an impairment of clathrin binding leading to disrupted synaptic vesicle endocytosis. LRRK2 is able to interact with and phosphorylate NSF (N-ethylmaleimide

Sensitive Fusion), this interaction increases LRRK2 kinase activity and SNARE complex dissociation, leading to alteration of synaptic vesicle recycling [START_REF] Belluzzi | LRRK2 phosphorylates pre-synaptic N-ethylmaleimide sensitive fusion (NSF) protein enhancing its ATPase activity and SNARE complex disassembling rate[END_REF]. LRRK2

directly phosphorylates synaptojanin-1 that leads to the disruption of the endophilin-synaptojanin1 interaction leading to a decreased endocytosis [START_REF] Pan | Parkinson's diseaseassociated LRRK2 hyperactive kinase mutant disrupts synaptic vesicle trafficking in ventral midbrain neurons[END_REF]. Synaptojanin-1 is a crucial protein for coating of endocytosed vesicles [START_REF] Cremona | Essential Role of Phosphoinositide Metabolism in Synaptic Vesicle Recycling[END_REF]. LRRK2 is reported to phosphorylate a partner of synaptojanin-1, endophilin A, which is a protein hub for clathrin-independent endocytosis [START_REF] Matta | LRRK2 Controls an EndoA Phosphorylation Cycle in Synaptic Endocytosis[END_REF][START_REF] Arranz | LRRK2 functions in synaptic vesicle endocytosis through a kinasedependent mechanism[END_REF][START_REF] Ap | Endophilin marks and controls a clathrinindependent endocytic pathway[END_REF][START_REF] Renard | Endophilin-A2 functions in membrane scission in clathrin-independent endocytosis[END_REF].

Translation:

Dysregulation of eIF2 signaling and eIF2 phosphorylation has been demonstrated in sporadic and familial PD patients [START_REF] Mutez | Involvement of the immune system, endocytosis and EIF2 signaling in both genetically determined and sporadic forms of Parkinson's disease[END_REF]. It has been suggested that key PD protein could interact with the translation machinery, resulting in its deregulation [START_REF] Taymans | Deregulation of protein translation control, a potential game-changing hypothesis for Parkinson's disease pathogenesis[END_REF].

LRRK2 is reported to interact with a subset of translation initiation factors (eIF2C1, eIF2C2, eIF4B, and eIF3J) and ribosomal proteins (S15, S20 and SA) and elongation factor eEF1D [START_REF] Dorval | LRRK2 in transcription and translation regulation: Relevance for Parkinson's disease[END_REF][START_REF] Martin | Ribosomal protein s15 phosphorylation mediates LRRK2 neurodegeneration in Parkinson's disease[END_REF][START_REF] Reyniers | Differential protein protein interactions of LRRK1 and LRRK2 indicate roles in distinct cellular signaling pathways[END_REF] (Figure 21). Drosophila models harboring LRRK2 pathogenic mutants presented alteration in cap-dependent mRNA translation. LRRK2 is able to interact and phosphorylate 4E-BP1 leading to the release of eiF4E from the complex. eiF4E binds to the cap-structure of mRNA and allows the translation, while excess phosphorylation of 4E-BP will increase cap-dependent protein translation and dysregulation of gene expression such as Furin-1, required for synaptic function [START_REF] Dorval | LRRK2 in transcription and translation regulation: Relevance for Parkinson's disease[END_REF][START_REF] Penney | LRRK2 regulates retrograde synaptic compensation at the Drosophila neuromuscular junction[END_REF]. G2019S is responsible, in drosophila, of increased phosphorylation of Ribosomal Protein S15, resulting in upregulation of protein synthesis and dopaminergic neurodegeneration in aged drosophila [START_REF] Martin | Ribosomal protein s15 phosphorylation mediates LRRK2 neurodegeneration in Parkinson's disease[END_REF]. LRRK2 also interacts with argonaute proteins that are essential for the regulation of translation/degradation of mRNA [START_REF] Gehrke | Pathogenic LRRK2 negatively regulates microRNA-mediated translational repression[END_REF]. In fibroblasts from PD patients (G2019S and sporadic cases), protein synthesis is reduced by 40%, a phenomenon that can be reverted with LRRK2 kinase inhibition [START_REF] Katsuda | Protein synthesis is suppressed in sporadic and familial Parkinson[END_REF].

Ribosome profiling of G2019S Human Induced Pluripotent Stem Cell (hiPSC)-derived dopaminergic neurons presented alteration of genes involved in calcium homeostasis, which could lead to disruption of mitochondrial respiration, increased ROS production [START_REF] Nicholls | Oxidative stress and energy crises in neuronal dysfunction[END_REF][START_REF] Surmeier | Calcium and Parkinson's disease[END_REF][START_REF] Kim | Defects in mRNA Translation in LRRK2-Mutant hiPSC-Derived Dopaminergic Neurons Lead to Dysregulated Calcium Homeostasis[END_REF].

Figure 21: LRRK2 impact on protein translation and calcium homeostasis. LRRK2 pathogenic mutations have been shown to regulate proteins from the translation mechanism. LRRK2 is reported to impact the global translation, increasing the metabolic demand of the cell leading to an increased cellular stress. LRRK2 has been shown to specifically increase the 5'UTR translation, increasing the calcium influx in the cell. Increased calcium content led to impairment of mitochondrial functions and is associated to neuronal stress. Adapted from [START_REF] Kim | Defects in mRNA Translation in LRRK2-Mutant hiPSC-Derived Dopaminergic Neurons Lead to Dysregulated Calcium Homeostasis[END_REF].

Mitochondria:

Mitochondrial dysfunction have been observed in PD patients including PD patients carrying LRRK2 mutations [START_REF] Bose | Mitochondrial dysfunction in Parkinson's disease[END_REF][START_REF] Hsieh | Functional Impairment in Miro Degradation and Mitophagy Is a Shared Feature in Familial and Sporadic Parkinson's Disease[END_REF]. LRRK2 is present on the outer membrane of mitochondria [START_REF] Biskup | Localization of LRRK2 to membranous and vesicular structures in mammalian brain[END_REF]. iPSC-derived neural cells from LRRK2 G2019S carriers presented damaged mitochondrial DNA [START_REF] Sanders | LRRK2 mutations cause mitochondrial DNA damage in iPSC-derived neural cells from Parkinson's disease patients: Reversal by gene correction[END_REF][START_REF] Howlett | LRRK2 G2019S-induced mitochondrial DNA damage is LRRK2 kinase dependent and inhibition restores mtDNA integrity in Parkinson's disease[END_REF].

This damaged DNA can result from higher ROS production. Some reports suggest a small ROS production with LRRK2 pathogenic mutations [START_REF] Heo | LRRK2 enhances oxidative stress-induced neurotoxicity via its kinase activity[END_REF][START_REF] Angeles | Mutations in LRRK2 increase phosphorylation of peroxiredoxin 3 exacerbating oxidative stressinduced neuronal death[END_REF], but mutants increased the susceptibility to oxidative stress [START_REF] Cooper | Pharmacological Rescue of Mitochondrial Deficits in iPSC-Derived Neural Cells from Patients with Familial Parkinson's Disease[END_REF].

Mitochondria fuse and divide constantly and the balance between fusion and fission constitutes one part of mitochondrial health regulation. Maintenance of the mitochondrial dynamics is crucial for ATP production, Ca 2+ homeostasis, ROS production and apoptosis [START_REF] Detmer | Functions and dysfunctions of mitochondrial dynamics[END_REF]. LRRK2 has been shown to regulate fusion/fission event. For instance, LRRK2 G2019S promotes mitochondrial fission and elongated mitochondria [START_REF] Mortiboys | Mitochondrial impairment in patients with Parkinson disease with the G2019S mutation in LRRK2[END_REF]Ho et al., 2018a[START_REF] Ho | G2019s LRRK2 promotes mitochondrial fission and increases TNFα-mediated neuroinflammation responses[END_REF]. In primary cortical neurons, LRRK2 G2019S induces mitochondrial fragmentation through interaction with dynamin like protein 1 (DLP1), a mitochondrial fission protein [START_REF] Niu | Leucine-rich repeat kinase 2 disturbs mitochondrial dynamics via Dynamin-like protein[END_REF][START_REF] Su | Inhibition of excessive mitochondrial fission reduced aberrant autophagy and neuronal damage caused by LRRK2 G2019S mutation[END_REF], which is an important factor for normal morphology of mitochondria [START_REF] Pitts | The Dynamin-like Protein DLP1 Is Essential for Normal Distribution and Morphology of the Endoplasmic Reticulum and Mitochondria in Mammalian Cells[END_REF].

LRRK2 has been shown to regulate mitophagy in iPSC-derived cells. LRRK2 G2019S slows down the transport of the damaged mitochondria on the cytoskeleton by impairing the kinesin motor complex, reducing the autophagosome engulfment and its degradation [START_REF] Hsieh | Functional Impairment in Miro Degradation and Mitophagy Is a Shared Feature in Familial and Sporadic Parkinson's Disease[END_REF]. LRRK2 mutants G2019S and R1441C have been shown to regulate the PINK1/parkindependent mitophagy [START_REF] Wauters | LRRK2 mutations impair depolarization-induced mitophagy through inhibition of mitochondrial accumulation of RAB10[END_REF]. LRRK2 G2019S and R1441C increased RAB10 phosphorylation, impairing the binding with optineurin that binds to depolarized mitochondria and promotes their degradation [START_REF] Lazarou | The ubiquitin kinase PINK1 recruits autophagy receptors to induce mitophagy[END_REF]. These effects can be reversed with LRRK2 kinase inhibition [START_REF] Wauters | LRRK2 mutations impair depolarization-induced mitophagy through inhibition of mitochondrial accumulation of RAB10[END_REF].

Microtubule dynamics and neurite outgrowth:

LRRK2 G2019S and I2020T overexpression in cultured cells or in vivo cause neurite shortening and reduced process branching [START_REF] Macleod | The familial Parkinsonism gene LRRK2 regulates neurite process morphology[END_REF]. LRRK2 pathogenic mutants are able to co-localize with microtubules in an organized and repetitive fashion [START_REF] Kett | LRRK2 Parkinson disease mutations enhance its microtubule association[END_REF]. LRRK2

can interact with and phosphorylate beta-tubulin and activate tubulin polymerization in presence of Microtubule Associated Proteins (MAPs) [START_REF] Gillardon | Leucine-rich repeat kinase 2 phosphorylates brain tubulin-beta isoforms and modulates microtubule stability -a point of convergence in Parkinsonian neurodegeneration?[END_REF]. In Drosophila, expression of LRRK2 G2019S enhances Tau phosphorylation and mislocated Tau, resulting in dendrite degeneration [START_REF] Lin | LRRK2 G2019S Mutation Induces Dendrite Degeneration through Mislocalization and Phosphorylation of Tau by Recruiting Autoactivated GSK3[END_REF]. Expression of LRRK2 G2019S in differentiated SH-SY5Y induced neurite shortening and generated an increase accumulation of autophagic vacuoles in the soma as well as in the neurites (Plowey et al., 2008b). Cultured neurons from G2019S mice reveal alterations of neurite complexity and autophagic vacuole accumulation [START_REF] Ramonet | Dopaminergic Neuronal Loss, Reduced Neurite Complexity and Autophagic Abnormalities in Transgenic Mice Expressing G2019S Mutant LRRK2[END_REF]. When a crucial gene for the autophagosome formation (ATG7) is repressed in mice, accumulation of alpha-synuclein and LRRK2 is found in the presynaptic space [START_REF] Friedman | Disrupted Autophagy Leads to Dopaminergic Axon and Dendrite Degeneration and Promotes Presynaptic Accumulation of -Synuclein and LRRK2 in the Brain[END_REF]. This supports the idea of a disrupted transport and autophagy regulation that will lead to alpha-synuclein accumulation and disruption of the neurite's complexity.

Alpha-synuclein aggregation:

LRRK2 is reported to accelerate the propagation of alpha-synuclein aggregates in multiple models. In the alpha-synuclein A53T mouse model, the overexpression of LRRK2 increases the rate of alpha-synuclein accumulation [START_REF] Lin | Leucine-Rich Repeat Kinase 2 Regulates the Progression of Neuropathology Induced by Parkinson's-Disease-Related Mutant α-synuclein[END_REF] and KO of LRRK2 in mice overexpressing alpha-synuclein reduces the synuclein-induced dopaminergic neurodegeneration [START_REF] Daher | Abrogation of -synuclein-mediated dopaminergic neurodegeneration in LRRK2-deficient rats[END_REF]. In LRRK2 KO mice, at 20 months of age, accumulation and aggregation of alphasynuclein is increased in the kidney due to autophagy impairment [START_REF] Tong | Loss of leucinerich repeat kinase 2 causes age-dependent bi-phasic alterations of the autophagy pathway[END_REF]. Ablation of the LRRK2 kinase activity with pharmacological treatment enhance the proportion of alphasynuclein directed to the lysosomes of alpha-synuclein transgenic mice [START_REF] Daher | Leucine-rich Repeat Kinase 2 (LRRK2) Pharmacological Inhibition Abates α-Synuclein Gene-induced Neurodegeneration[END_REF][START_REF] Bae | LRRK2 kinase regulates α-synuclein propagation via RAB35 phosphorylation[END_REF]. A mechanism due to another RAB substrate of LRRK2, RAB35 is known to regulates exosome secretion but its precise role in the protein degradation needs to be defined [START_REF] Hsu | Regulation of exosome secretion by Rab35 and its GTPase-activating proteins TBC1D10A-C[END_REF][START_REF] Bae | The LRRK2-RAB axis in regulation of vesicle trafficking and αsynuclein propagation[END_REF]. From (Bae and Lee, 2020).

LRRK2 phosphorylation

LRRK2 is a highly phosphorylatable protein (Figure 23). First, LRRK2 exists as a phosphorylated protein in mammalian cells under basal conditions as observed after metabolic labelling of LRRK2-expressing cells with radioactive phosphate or by detection of LRRK2 in phosphoprotein isolates from cell culture [START_REF] Greggio | Mutations in LRRK2/dardarin associated with Parkinson disease are more toxic than equivalent mutations in the homologous kinase LRRK1[END_REF][START_REF] Lobbestael | Identification of protein phosphatase 1 as a regulator of the LRRK2 phosphorylation cycle[END_REF][START_REF] Reyniers | Differential protein protein interactions of LRRK1 and LRRK2 indicate roles in distinct cellular signaling pathways[END_REF]. Second, additional phosphorylation potential above the cellular phosphorylation of LRRK2 can be observed when purified LRRK2 is submitted to in vitro autophosphorylation. Third, in a similar fashion, in vitro incubation of LRRK2 with a separate kinase, such as protein kinase A (PKA), can also lead to additional phosphorylation of LRRK2 [START_REF] Muda | Parkinson-related LRRK2 mutation R1441C/G/H impairs PKA phosphorylation of LRRK2 and disrupts its interaction with 14-3-3[END_REF]. The notion that LRRK2 is a highly phosphorylated protein is confirmed by phosphosite mapping studies via mass spectrometry, showing at least 74 phosphorylation sites on isolated LRRK2 protein, corresponding to almost 3% of all amino acid residues of the protein [START_REF] Greggio | The Parkinson's disease kinase LRRK2 autophosphorylates its GTPase domain at multiple sites[END_REF][START_REF] Kamikawaji | Identification of the autophosphorylation sites of LRRK2[END_REF][START_REF] Gloeckner | Phosphopeptide Analysis Reveals Two Discrete Clusters of Phosphorylation in the N-Terminus and the Roc Domain of the Parkinson-Disease Associated Protein Kinase LRRK2[END_REF][START_REF] Pungaliya | Identification and characterization of a leucine-rich repeat kinase 2 (LRRK2) consensus phosphorylation motif[END_REF]. Phosphorylation sites include a majority of serines (59%), followed by 37% threonines and some tyrosines (4%). Further compilation of the reported LRRK2 phosphosites indicates that 37 are reported by two or more separate studies, meaning that half of the reported LRRK2 phosphorylation sites still await independent confirmation. As is the case for many kinases, LRRK2 can autophosphorylate, and as for any phosphorylated kinase, it is therefore possible to divide the phosphorylated sites into two groups, the heterologous phosphorylation sites and the autophosphorylation sites. In addition, one can distinguish phosphosites that are observed from LRRK2 directly isolated from cells or tissues without any further manipulations (cellular phosphorylation sites) from sites that are submitted to additional in vitro phosphorylation. Sites that are qualified as autophosphorylation sites are confirmed when their phosphorylation rates increase after in vitro phosphorylation, while this is not the case for heterologous phosphorylation sites. Using these criteria, 60% of the identified LRRK2 phosphorylation sites are autophosphorylation sites and 36% are heterologous, while the remaining 4% of sites have been identified as both autophosphorylation and PKA phosphorylation sites (threonine 833, serine 1443, and serine 1444) [START_REF] Gloeckner | Phosphopeptide Analysis Reveals Two Discrete Clusters of Phosphorylation in the N-Terminus and the Roc Domain of the Parkinson-Disease Associated Protein Kinase LRRK2[END_REF][START_REF] Pungaliya | Identification and characterization of a leucine-rich repeat kinase 2 (LRRK2) consensus phosphorylation motif[END_REF][START_REF] Muda | Parkinson-related LRRK2 mutation R1441C/G/H impairs PKA phosphorylation of LRRK2 and disrupts its interaction with 14-3-3[END_REF].

Looking at the distribution of the phosphorylated residues across the LRRK2 protein, one prominent phosphorylation cluster is located between the ANK and the LRR domain at serines S860, S910, S935, S955, S973, and S976 for the most studied sites. The importance of the heterologous phosphorylation sites for LRRK2 function has been supported by the findings that 14-3-3 binding to LRRK2 is dependent on S910 and S935 phosphorylation and that LRRK2 phosphorylation levels at heterologous phosphorylation sites affect subcellular distribution of LRRK2.

The LRRK2 autophosphorylation occurs on at least 20 different serine or threonine residues located in and around the ROC domain and some in the kinase domain. While in vitro phosphorylation has revealed a large number of autophosphorylation sites, it remains unclear which proportion of these exist under physiological conditions. One example of an autophosphorylation site identified in cells and in vivo is the S1292 site that is positively modulated in LRRK2 mutants (N1437H, R1441C/G/H, Y1699C, G2019S, and I2020T) [START_REF] Reynolds | LRRK2 kinase activity and biology are not uniformly predicted by its autophosphorylation and cellular phosphorylation site status[END_REF][START_REF] Steger | Phosphoproteomics reveals that Parkinson's disease kinase LRRK2 regulates a subset of Rab GTPases[END_REF].

Regulators of LRRK2 phosphorylation: Kinases

The first kinase reported as a candidate to regulate LRRK2 phosphorylation was PKA in 2007 [START_REF] Ito | GTP Binding Is Essential to the Protein Kinase Activity of LRRK2, a Causative Gene Product for Familial Parkinson's Disease †[END_REF]. The authors of the study were able to identify PKA as an upstream kinase responsible for the phosphorylation of LRRK2 in HEK293 cells using two different potent inhibitors of PKA. They also showed that PKA efficiently phosphorylates LRRK2 K1906M kinaseinactive mutant. Several years later, two independent groups confirmed PKA as a kinase acting on the S910 and S935 sites [START_REF] Li | Phosphorylation-Dependent 14-3-3 Binding to LRRK2 Is Impaired by Common Mutations of Familial Parkinson's Disease[END_REF][START_REF] Muda | Parkinson-related LRRK2 mutation R1441C/G/H impairs PKA phosphorylation of LRRK2 and disrupts its interaction with 14-3-3[END_REF] and also on the S1444 site both in vitro and in cellulo [START_REF] Muda | Parkinson-related LRRK2 mutation R1441C/G/H impairs PKA phosphorylation of LRRK2 and disrupts its interaction with 14-3-3[END_REF]. Treatment with PKA activator forskolin increased phosphorylation at S910 as well as at S1444. The S1444 phosphorylation site was proposed as a new alternate 14-3-3 binding phosphosite. However, another study showed an opposite effect of PKA activation on LRRK2, with a decrease of phosphorylation at S910, S935, S955, and S973 and reduced 14-3-3 binding on LRRK2 overexpressed in HEK293 T-Rex cells and endogenous LRRK2 in A549 lung derived cell lines [START_REF] Reynolds | LRRK2 kinase activity and biology are not uniformly predicted by its autophosphorylation and cellular phosphorylation site status[END_REF]. Finally, PKA activation or inhibition had no effect on the level of phosphorylation at pS935 [START_REF] Hermanson | Screening for Novel LRRK2 Inhibitors Using a High-Throughput TR-FRET Cellular Assay for LRRK2 Ser935 Phosphorylation[END_REF]. Further work will be needed to better decipher the role of PKA in the regulation of LRRK2 phosphorylation. Interestingly, the recent literature supports the notion of a functional cross-regulation between LRRK2 and PKA that might be cell type specific [START_REF] Parisiadou | LRRK2 regulates synaptogenesis and dopamine receptor activation through modulation of PKA activity[END_REF][START_REF] Russo | Leucine-rich repeat kinase 2 controls protein kinase A activation state through phosphodiesterase 4[END_REF].

Dzamko et al. showed that the inhibitor of Ikappa B kinases (IKKα and β) phosphorylates the S910 and S935 sites in macrophages derived from bone marrow during stimulation of Toll-like receptor signalling [START_REF] Dzamko | The IkappaB Kinase Family Phosphorylates the Parkinson's Disease Kinase LRRK2 at Ser935 and Ser910 during Toll-Like Receptor Signaling[END_REF]. Further data indicate that IKKβ is also a potential kinase regulating LRRK2 phosphorylation in SH-SY5Y and HEK293 cells [START_REF] Hermanson | Screening for Novel LRRK2 Inhibitors Using a High-Throughput TR-FRET Cellular Assay for LRRK2 Ser935 Phosphorylation[END_REF]. Intriguingly, LRRK2 dephosphorylation induced by kinase inhibition with LRRK2-IN1 and CZC25146 was completely prevented by LPS stimulation [START_REF] Dzamko | The IkappaB Kinase Family Phosphorylates the Parkinson's Disease Kinase LRRK2 at Ser935 and Ser910 during Toll-Like Receptor Signaling[END_REF]. Finally, Chia et al. provided the evidence that Casein Kinase 1-alpha (CK1α) is a physiologically upstream kinase regulator of LRRK2 at the constitutive phosphorylation sites using an unbiased siRNA kinome screen in HEK-293T cells as well as in the mouse brain with ex vivo experiment [START_REF] Chia | Phosphorylation of LRRK2 by casein kinase 1α regulates trans-Golgi clustering via differential interaction with ARHGEF7[END_REF]. In addition, the repression of the expression or inhibition of CK1α led to a decrease in phosphorylation at S910 and S935 as well as an increase in the association of ARHGEF7 with LRRK2, which decreased GTP binding. Treatment with siRNAs targeting CK1α also reduced RAB29-dependent Golgi fragmentation caused by LRRK2, indicating that phosphorylation of heterologous LRRK2 sites modulates recruitment of LRRK2 within the TGN [START_REF] Chia | Phosphorylation of LRRK2 by casein kinase 1α regulates trans-Golgi clustering via differential interaction with ARHGEF7[END_REF]. A number of additional upstream kinases have been proposed [START_REF] Lobbestael | Phosphorylation of LRRK2: from kinase to substrate[END_REF].

Regulators of LRRK2 phosphorylation: Phosphatases

The phosphoregulation of LRRK2 is a complex mechanism (Figure 24). The rapid induction of LRRK2 dephosphorylation after LRRK2 kinase inhibition suggests the involvement of protein phosphatases. Moreover, cAMP stimulation downregulated LRRK2 phosphorylation that suggests that a phosphatase may be activated in HEK293 but also in A549 cells [START_REF] Hermanson | Screening for Novel LRRK2 Inhibitors Using a High-Throughput TR-FRET Cellular Assay for LRRK2 Ser935 Phosphorylation[END_REF].

The search of phosphatases related to LRRK2 pathophysiology has seen some advances in recent years. Regarding phosphatases regulating heterologous phosphorylation sites, only the alpha catalytic subunit of Protein Phosphatase 1 (PPP1CA) has been demonstrated to regulate phosphorylation of LRRK2 at S910, S935, S955, and S973 [START_REF] Lobbestael | Identification of protein phosphatase 1 as a regulator of the LRRK2 phosphorylation cycle[END_REF]. Indeed, pharmacological inhibition of Protein Phosphatase 1 (PP1) with Calyculin A (CalA) prevented the dephosphorylation of LRRK2 induced by LRRK2 kinase inhibitors. Interestingly, the effects of PPP1CA on LRRK2 phosphorylation were confirmed in several cell types HEK-293T, SH-SY5Y, NIH 3T3, A549, and U-2 OS but also in mouse primary cortical neurons. This shows that PPP1CA is active as an LRRK2 phosphatase independent of the cell type tested. Moreover, under LRRK2 dephosphorylation conditions, the association between PP1 and LRRK2 is increased, for example: during treatment with LRRK2 kinase inhibitors or in the presence of LRRK2 mutants with low level of phosphorylation [START_REF] Lobbestael | Identification of protein phosphatase 1 as a regulator of the LRRK2 phosphorylation cycle[END_REF]. Furthermore, a study on LRRK2 and oxidative stress [START_REF] Mamais | Arsenite stress down-regulates phosphorylation and 14-3-3 binding of leucine-rich repeat kinase 2 (LRRK2), promoting self-association and cellular redistribution[END_REF]) also highlighted the importance of the physiological role of PP1 in the dephosphorylation of LRRK2. Arsenite-mediated stress leads to a reduction in the phosphorylation of LRRK2 at S910 and S935 in cell culture, and this reduction is reversed by CalA treatment. In addition, CalA counteracted arsenite and H2O2-induced S935

dephosphorylation, but only arsenite induced an increase association of PPP1CA with LRRK2 [START_REF] Mamais | Arsenite stress down-regulates phosphorylation and 14-3-3 binding of leucine-rich repeat kinase 2 (LRRK2), promoting self-association and cellular redistribution[END_REF]. PP1 target specificity is driven by the association of regulatory subunits [START_REF] Bollen | The extended PP1 toolkit: Designed to create specificity[END_REF]. We do not yet know which regulatory subunits form the active PP1 holoenzyme responsible for catalyzing LRRK2 dephosphorylation. Therefore, a key issue to understand how LRRK2 dephosphorylation is regulated is to identify the composition of the PP1 holoenzyme by identifying the LRRK2-specific subunits that form the active PP1 holoenzyme that acts on LRRK2. LRRK2 is also able to regulate the activity of PKA by a direct interaction with its ROC domain or by an indirect manner, by acting on the phosphodiesterase 4 (PDE4). PPP1CA has been confirmed to act on LRRK2. The holoenzyme PP2A could regulate the phosphorylation at S1292. The phosphorylation of the N-ter sites allows the interaction with 14-3-3. If phosphorylated by PAK6, the binding to LRRK2 is abolished. RAB29 interacts with LRRK2 in the Trans-Golgi network; this interaction leads to an increased phosphorylation of the N-ter and the kinase activity of LRRK2. LRRK2 can phosphorylate RAB29 and avoid LRRK2 activation, creating an inactivation loop. From [START_REF] Marchand | LRRK2 Phosphorylation, More Than an Epiphenomenon[END_REF] It is unclear which phosphatases are regulating LRRK2 phosphosites outside the ANK-LRR interdomain region. However, PP2A has been identified as a partner interacting with LRRK2 [START_REF] Athanasopoulos | Identification of protein phosphatase 2A as an interacting protein of leucine-rich repeat kinase 2[END_REF]. This study reports that LRRK2 interacts with all three subunits of PP2A and that this is mediated by the ROC domain in cultured cells. This is consistent with the recent report by Sim and colleagues who identified in a Drosophila model the three components of PP2A that are required to form a functional holoenzyme, i.e., scaffolding, regulatory, and catalytic subunits, as a modulator of LRRK2 function. Although PP2A has been found to be responsible LRRK2 dephosphorylation [START_REF] Athanasopoulos | Identification of protein phosphatase 2A as an interacting protein of leucine-rich repeat kinase 2[END_REF][START_REF] Drouyer | Protein phosphatase 2A holoenzymes regulate leucine-rich repeat kinase 2 phosphorylation and accumulation[END_REF]. In addition, silencing of the catalytic subunit of PP2A by shRNA aggravated cell degeneration in SH-SY5Y cells expressing the LRRK2 R1441C variant as well as in cultured cortical neurons derived from G2019S overexpressing transgenic mice. Interestingly, relevance of PP2A as an LRRK2 phosphatase for the regulation of S1292 phosphorylation site could be demonstrated by pharmacological and genetic approaches in mutant LRRK2 flies [START_REF] Sim | Identification of PP2A and S6 Kinase as Modifiers of Leucine-Rich Repeat Kinase-Induced Neurotoxicity[END_REF]. Pharmacological activation with either ceramide or fingolimod (FTY720)

ameliorates their disease-associated phenotypes. In addition, under conditions of PP2A subunit overexpression, LRRK2 phosphorylation at S1292 was found reduced. This is consistent with a report demonstrating that S1292 dephosphorylation is mediated by phosphatases that are sensitive to CalA and okadaic acid [START_REF] Reynolds | LRRK2 kinase activity and biology are not uniformly predicted by its autophosphorylation and cellular phosphorylation site status[END_REF].

Phosphorylation phenotypes of LRRK2

While the global picture of how LRRK2 phosphorylation levels at its various phosphorylation sites influence LRRK2 function is still incomplete, several studies have shown that changes in LRRK2 phosphorylation influences LRRK2 biochemical or cellular properties. Links between LRRK2 phosphorylation and disease or pathological mechanisms are being established in different ways: by monitoring LRRK2 phosphorylation in patient-derived samples, disease models, and study of phosphomutant forms of LRRK2 and how these affect cellular phenotypes. To investigate the links between LRRK2 phosphorylation and its kinase activity, phosphomutants are used. When testing for autophosphorylation activity of the S910A/S935A mutant, no change in S1292 autophosphorylation was observed in cells compared to WT [START_REF] Reynolds | LRRK2 kinase activity and biology are not uniformly predicted by its autophosphorylation and cellular phosphorylation site status[END_REF]. Other phosphorylation site mutants or combinations of phosphorylation site mutants from the S935 cluster on LRRK2 kinase activity remain to be tested. Mutant S2032A, T2035A, and S2032A/T2035A showed a reduced autophosphorylation activity, assessed by in vitro autophosphorylation with 32P-labeled ATP (Li et al., 2010a). The overall conclusion here is that specific LRRK2 phosphorylation sites may affect LRRK2 kinase activity. Conversely, there is not a uniform correlation between LRRK2 phosphorylation and its kinase activity. Besides kinase activity, GTP-binding and GTPase activity may also be influenced by LRRK2 phosphorylation levels. Of particular interest are the autophosphorylation sites that are clustered in and around the ROC GTPase domain and several sites map to G-box motifs that mediate GTP binding, which point to the possibility that autophosphorylation may affect GTPase functions [START_REF] Webber | Autophosphorylation in the leucine-rich repeat kinase 2 (LRRK2) GTPase domain modifies kinase and GTP-binding activities[END_REF][START_REF] Taymans | The GTPase function of LRRK2[END_REF]. In particular, some phosphomimetic mutants such as T1491D and T1503D showed impaired GTP binding, although GTP binding is unchanged for another phosphomimetic LRRK2 mutant, T1410D [START_REF] Kamikawaji | Identification of the autophosphorylation sites of LRRK2[END_REF][START_REF] Webber | Autophosphorylation in the leucine-rich repeat kinase 2 (LRRK2) GTPase domain modifies kinase and GTP-binding activities[END_REF]. By extension, a potential role of heterologous phosphorylation sites of LRRK2 on its GTP-related functions cannot be excluded and has been studied in the present work.

LRRK2's interactors

The phosphorylation at S910 and S935 sites, as well as the S1444 site, have been shown to be responsible for the interaction of LRRK2 with 14-3-3 proteins [START_REF] Dzamko | Inhibition of LRRK2 kinase activity leads to dephosphorylation of Ser 910 /Ser 935 , disruption of 14-3-3 binding and altered cytoplasmic localization[END_REF][START_REF] Nichols | 14-3-3 binding to LRRK2 is disrupted by multiple Parkinson's disease-associated mutations and regulates cytoplasmic localization[END_REF][START_REF] Li | Phosphorylation-Dependent 14-3-3 Binding to LRRK2 Is Impaired by Common Mutations of Familial Parkinson's Disease[END_REF][START_REF] Muda | Parkinson-related LRRK2 mutation R1441C/G/H impairs PKA phosphorylation of LRRK2 and disrupts its interaction with 14-3-3[END_REF]. Indeed, phosphodead mutations (substitution of the serine residue for alanine) at S910 and S935, but not at S955 and S973, lead to a strong reduction of 14-3-3 binding [START_REF] Doggett | Phosphorylation of LRRK2 serines 955 and 973 is disrupted by Parkinson's disease mutations and LRRK2 pharmacological inhibition[END_REF]. Moreover, if 14-3-3 binding is blocked using difopein (dimeric fourteen-three-three peptide inhibitor), LRRK2 appears to be dephosphorylated at S910 and S935 [START_REF] Fraser | LRRK2 secretion in exosomes is regulated by 14-3-3[END_REF][START_REF] Zhao | LRRK2 dephosphorylation increases its ubiquitination[END_REF]. Therefore, it has been suggested that 14-3-3 interaction could protect against dephosphorylation at these two phosphorylation sites and influence the subcellular localization of LRRK2 in the cell [START_REF] Nichols | 14-3-3 binding to LRRK2 is disrupted by multiple Parkinson's disease-associated mutations and regulates cytoplasmic localization[END_REF][START_REF] Li | Phosphorylation-Dependent 14-3-3 Binding to LRRK2 Is Impaired by Common Mutations of Familial Parkinson's Disease[END_REF]. The absence of 14-3-3 binding to LRRK2 when S910 and S935 sites are dephosphorylated induces accumulations of LRRK2 in the cytoplasm of cells. Accumulation types include filamentous "skein-like" structures [START_REF] Dzamko | Inhibition of LRRK2 kinase activity leads to dephosphorylation of Ser 910 /Ser 935 , disruption of 14-3-3 binding and altered cytoplasmic localization[END_REF][START_REF] Nichols | 14-3-3 binding to LRRK2 is disrupted by multiple Parkinson's disease-associated mutations and regulates cytoplasmic localization[END_REF][START_REF] Reyniers | Differential protein protein interactions of LRRK1 and LRRK2 indicate roles in distinct cellular signaling pathways[END_REF] and/or punctate accumulations [START_REF] Chia | Phosphorylation of LRRK2 by casein kinase 1α regulates trans-Golgi clustering via differential interaction with ARHGEF7[END_REF]. Likewise, pathogenic mutants that exhibit a reduction in phosphorylation at S910 and S935 sites (N1437H, R1441C/G/H, Y1699C, I2020T, and the risk factor G2385R, but not the G2019S variant) display a similar loss of 14-3-3 binding and relocalization of LRRK2 to cytoplasmic accumulations pools and filamentous skein-like structures [START_REF] Dzamko | Inhibition of LRRK2 kinase activity leads to dephosphorylation of Ser 910 /Ser 935 , disruption of 14-3-3 binding and altered cytoplasmic localization[END_REF][START_REF] Nichols | 14-3-3 binding to LRRK2 is disrupted by multiple Parkinson's disease-associated mutations and regulates cytoplasmic localization[END_REF][START_REF] Deng | Characterization of a selective inhibitor of the Parkinson's disease kinase LRRK2[END_REF][START_REF] Doggett | Phosphorylation of LRRK2 serines 955 and 973 is disrupted by Parkinson's disease mutations and LRRK2 pharmacological inhibition[END_REF]. The brain is the tissue with the highest 14-3-3 concentration [START_REF] Boston | Human 14-3-3 Protein: Radioimmunoassay, Tissue Distribution, and Cerebrospinal Fluid Levels in Patients with Neurological Disorders[END_REF]. The role of 14-3-3 proteins in neurodegeneration has been reviewed in [START_REF] Shimada | Neuroprotective function of 14-3-3 proteins in neurodegeneration[END_REF] and is known to affect protein localization and activity through its binding to targeted substrates. Interestingly, there is an additional layer of regulation of 14-3-3 proteins that affects LRRK2 phosphorylation. Indeed, 14-3-3γ is phosphorylated by PAK6

(kinase 6 activated by p21), a serine/threonine kinase [START_REF] Civiero | PAK6 phosphorylates 14-3-3γ to regulate steady state phosphorylation of LRRK2[END_REF]. Phosphorylated 14-3-3γ is no longer able to bind S935 site, thus causing its dephosphorylation.

Several teams have demonstrated an interaction of LRRK2 with RAB29 [START_REF] Macleod | RAB7L1 interacts with LRRK2 to modify intraneuronal protein sorting and Parkinson's disease risk[END_REF][START_REF] Beilina | Unbiased screen for interactors of leucine-rich repeat kinase 2 supports a common pathway for sporadic and familial Parkinson disease[END_REF][START_REF] Liu | LRRK2 phosphorylates membrane-bound Rabs and is activated by GTP-bound Rab7L1 to promote recruitment to the trans-Golgi network[END_REF][START_REF] Purlyte | Rab29 activation of the Parkinson's disease-associated LRRK2 kinase[END_REF]. This interaction takes place in the ANK domain of LRRK2 and regulates the heterologous phosphorylation sites of the S935 cluster [START_REF] Purlyte | Rab29 activation of the Parkinson's disease-associated LRRK2 kinase[END_REF]. Purlyte et al. discovered that all RAB29 binding-deficient ankyrin domain LRRK2 variants are also dephosphorylated on these heterologous phosphorylation sites. In addition, the loss of endogenous RAB29 in A549 cells moderately reduces the phosphorylation of these sites. However, these data do not exclude the possibility that another Golgi resident, a protein kinase or phosphatase, regulates the phosphorylation of these sites. The LRRK2 kinase activity seems also to be regulated by RAB29 through the phosphorylation of the S935 cluster. In fact, the kinase activity of LRRK2 is reduced when a phosphomimetic mutant of RAB29 is expressed but no change is found with the dephosphomimetic form of RAB29. In particular, RAB29 is itself phosphorylated by LRRK2, suggesting that RAB29 binding to LRRK2 may mediate a potential positive feedback loop between LRRK2 phosphorylation at the S935 cluster and LRRK2 kinase activity, although further work would be required to confirm this [START_REF] Purlyte | Rab29 activation of the Parkinson's disease-associated LRRK2 kinase[END_REF]. LRRK2 has other RABs as substrate but none of these have yet been reported to increase LRRK2's kinase activity.

Involvement of LRRK2 in other pathologies

Cancer

As seen in section2.3-translation, LRRK2 is able to regulate translation through regulation of eIF4E-binding protein is known to be a specific target of the Drosophila homologue of mTOR [START_REF] Imai | Phosphorylation of 4E-BP by LRRK2 affects the maintenance of dopaminergic neurons in Drosophila[END_REF]. The deregulation of mTOR by LRRK2 might occur in some cancers [START_REF] Looyenga | Chromosomal amplification of leucine-rich repeat kinase-2 (LRRK2) is required for oncogenic MET signaling in papillary renal and thyroid carcinomas[END_REF]. In type 1 papillary renal cell carcinoma, LRRK2 was amplified and overexpressed (mRNA, DNA and protein level) [START_REF] Gera | AKT Activity Determines Sensitivity to Mammalian Target of Rapamycin (mTOR) Inhibitors by Regulating Cyclin D1 and c-myc Expression[END_REF]. Another evidence of a possible implication of LRRK2 in cancer is the number of mutations identified in the catalogue of Somatic mutations

In cancer database (human cancer mutation database) founded in a large number of tumour types.

Leprosy

Leprosy is a chronic disease caused by bacteria, Mycobacterium leprae. The disease progression is known to be dependent of the host genetic background [START_REF] Fava | Pleiotropic effects for Parkin and LRRK2 in leprosy type-1 reactions and Parkinson's disease[END_REF] and patients are subjected to Type 1 reaction leading peripheral nerve damage. LRRK2 has been identified by GWAS to be associated to leprosy [START_REF] Zhang | Genomewide Association Study of Leprosy[END_REF]. The LRRK2 variant, R1628P, was recurrent in patients without Type 1 and seems to confer a protection against Type 1 reaction in leprosy.

Crohn's disease

LRRK2 N551K and R1398H are protective risk factors for Crohn's 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] as well as PD. Hui and Collaborators also identified the risk factor N2081D, that increases the kinase activity of LRRK2 and reduced acetylation of alpha-tubulin and decrease lysosomal pH whereas the pH was increased for N551K and R1398H. These results present similarities between PD and Crohn's disease with LRRK2 mutants [START_REF] Hui | Functional variants in the LRRK2 gene confer shared effects on risk for Crohn's disease and Parkinson's disease[END_REF].

Kinase inhibition

Many pharmaceutical groups have since developed inhibitors that target the LRRK2 kinase function converging on ATP active site and describe at type I inhibitor or more recently type II inhibitor (non-ATP-competitive inhibitor). Starting in 2006, different scaffolds against the ATP site were publish [START_REF] Deng | Leucine-rich repeat kinase 2 inhibitors: a patent review (2006 -2011)[END_REF][START_REF] Kethiri | Leucine-rich repeat kinase 2 inhibitors: a review of recent patents (2011 -2013)[END_REF][START_REF] Galatsis | Leucine-rich repeat kinase 2 inhibitors: a patent review (2014-2016)[END_REF]. In 2018, the first clinical trial (NCT03710707) was announced by Denali Therapeutics Inc., followed by a second clinical trial in 2019 (NCT04056689).The structures of DNL201 and DNL151 are not published yet [START_REF] Konstantinidou | The tale of proteolysis targeting chimeras (PROTACs) for Leucine-Rich Repeat Kinase 2 (LRRK2)[END_REF].

As mentioned in the section on LRRK2 Phosphatases, pharmacological inhibition of LRRK2 kinase activity induces LRRK2 dephosphorylation. Moreover, the induction of LRRK2 ubiquitination has been observed after LRRK2 pharmacological kinase inhibition followed by decreased protein levels, due to proteasomal degradation [START_REF] Zhao | LRRK2 dephosphorylation increases its ubiquitination[END_REF][START_REF] Lobbestael | Pharmacological LRRK2 kinase inhibition induces LRRK2 protein destabilization and proteasomal degradation[END_REF]. This suggests that one of the consequences of prolonged LRRK2 dephosphorylation at the S935 cluster may be LRRK2 degradation, although this effect may be tissue and condition specific. In rats, administration of LRRK2 kinase inhibitor PFE360 in food leads to a decrease of LRRK2 total protein level in the brain but not in lung [START_REF] Kelly | The G2019S mutation in LRRK2 imparts resiliency to kinase inhibition[END_REF]. In contrast, loss of LRRK2 protein level was not detected in mouse brain, consistent with results reported in other studies using MLi-2-treated mice [START_REF] Fell | MLi-2, a potent, selective, and centrally active compound for exploring the therapeutic potential and safety of LRRK2 kinase inhibition[END_REF]. At the phenotypic level, LRRK2 kinase inhibitors induced abnormal cytoplasmic accumulation of secretory lysosome in the lungs but no change in the kidney in non-human primates [START_REF] Fuji | Effect of selective LRRK2 kinase inhibition on nonhuman primate lung[END_REF]. Six hours of treatment with inhibitors of CK1α, an upstream kinase of the S935 cluster, induced dephosphorylation of S935 and protein destabilization. In fact, CK1α inhibition is able to destabilize LRRK2 mutant R1441G/I2020T and also mutant without ARM domain [START_REF] De Wit | Inhibition of LRRK2 or Casein Kinase 1 Results in LRRK2 Protein Destabilization[END_REF]. While these data suggest the notion that LRRK2 dephosphorylation at S935 cluster may be a priming event for LRRK2 degradation, the reality of the mechanism is likely more complex. Loss of phosphorylation does not seem to be enough to destabilize the protein; LRRK2 dephosphomutant at six heterologous sites for S908A/910A/935A/955A/973A/976A does not show reduced basal expression levels, but this mutant is still degraded after 24 h of pharmacological inhibition in cell culture [START_REF] De Wit | Inhibition of LRRK2 or Casein Kinase 1 Results in LRRK2 Protein Destabilization[END_REF]. Other examples of discrepancies in LRRK2 expression in different conditions include KI mice for the kinase dead variant of LRRK2, D1994S, that display decreased protein levels. However, those observations are not replicated in cells [START_REF] Herzig | LRRK2 protein levels are determined by kinase function and are crucial for kidney and lung homeostasis in mice[END_REF]. Also, R1441G and Y1699C mutants with low GTPase activity and reduced steady-state phosphorylation at the S935 cluster have an increased basal level of ubiquitination compared to the I2020T mutant that shows normal

GTPase activity and increased kinase activity [START_REF] De Wit | Inhibition of LRRK2 or Casein Kinase 1 Results in LRRK2 Protein Destabilization[END_REF]. Pharmacological inhibition does not affect the ubiquitination level of those mutants and no destabilization is found after 48 h of kinase inhibitor treatment by MLi-2 or PFE-475. Ubiquitination level of those mutants can be restored with the PP1 and PP2A inhibitor CalA. Intriguingly, the N-terminal LRRK2 sequence as well as the LRRK2 S935 phosphosite is involved in inhibitor-induced LRRK2 destabilization. Indeed, a truncated form of LRRK2 (170-kDa) that lacks the ARM domain is dephosphorylated on S1292 after kinase inhibition but not destabilized. Nevertheless, this version of LRRK2 does not present a phosphorylation at S935 [START_REF] De Wit | Inhibition of LRRK2 or Casein Kinase 1 Results in LRRK2 Protein Destabilization[END_REF]. Due to the LRRK2 protein destabilization observed in certain conditions after kinase inhibition, it should be noted that some of these phenotypes may correspond to phenotypes observed in LRRK2 KO animals. For instance, increased number and size of lysosomes in kidney proximal tubule cells and lamellar bodies in lung type II cells is found in LRRK2 KO mice [START_REF] Herzig | LRRK2 protein levels are determined by kinase function and are crucial for kidney and lung homeostasis in mice[END_REF], while similar findings are made in LRRK2 KO rats [START_REF] Baptista | Loss of Leucine-Rich Repeat Kinase 2 (LRRK2) in Rats Leads to Progressive Abnormal Phenotypes in Peripheral Organs[END_REF]. Further research should be performed to further determine the hypothesized parallel between LRRK2 kinase inhibition and LRRK2 KO.

Part 3: Objectives

Understanding the pathophysiological mechanisms involved in Parkinson's disease is a crucial issue in the search for a cure. The LRRK2 protein is intimately linked to PD pathophysiology and constitutes an interesting therapeutic target for novel PD therapies. Indeed, expression of LRRK2 pathogenic mutations such as G2019S lead to deleterious phenotypes in cells and animal models. The phosphorylation of LRRK2 that is the focus of this PhD thesis is a key property of LRRK2 that may be central to LRRK2 pathogenic mechanisms, although some We hypothesize that dysregulation of LRRK2 phosphorylation could contribute to PD pathogenesis by modification of it control of proteins in charge of maintaining cellular homeostasis, in particular processes related to vesicular physiology or lysosomal dysfunction.

The overall objective of this PhD thesis is to determine whether a specific phosphorylation profile of LRRK2 can drive LRRK2 into a toxic state.

To achieve this evaluation, we generated expression constructs of phosphomutant forms of LRRK2, i.e. LRRK2 that is mutated at its phosphorylation sites in order to mimic phosphorylation or dephosphorylation and studied how these impacted (1) LRRK2 biochemical properties (Catalytic activities, protein binding, protein phosphorylation), (2) its subcellular localization, (3) known cellular phenotypes of LRRK2 such as lysosomal protein degradation, neurite outgrowth, or RAB29 induced kinase activation (Figure 25).

Together, these results will help us to increase the knowledge on the precise role of LRRK2 phosphorylation and how it may be associated to deleterious phenotypes. Taken together, these results will provide new insight between LRRK2 phosphorylation and PD pathophysiology. Differentiation was induced with RPMI 1640 medium with 1% fetal horse serum and 0.5% fetal horse serum containing 50µg/mL of NGF, medium was changed every 48H. Differentiation was completed when neurites network is fully developed, after 10 days of differentiation.

1.2.2) Site directed mutagenesis

Mutations (S910A, S910D, S935A, S935D, S955A, S955D, S973A, S973D, S1292A, S1292D, S860/910/935/955/973/976A, S860/910/935/955/973/976D) were introduced using site directed mutagenesis or synthesized on demand at a cloning service company (eZyvec, Lille,

1.3) Cell transfections

Cells were plated and at transfected with FuGENE® 6 Transfection Reagent (Promega Corporation, Madison, USA) at 30% confluency with a ratio 3:1 or with polyethylenimine (PEI) (Sigma Aldrich, Darmstadt, Germany), at 50% confluency with 1µg of DNA for 3µL of PEI (1 µg/µL). Culture medium was changed after 24H of incubation with transfection agents.

1.4) Cell transduction

To obtain stable expression in our cell models we use transduction with lentiviral particles.

This expression method allows us to maintain our cell lines overtime with identical protein expression through time.

1.4.1) Lentiviral particle production

Lentiviral particles encoding 3xFLAG-LRRK2; mCherry-LRRK2 construct were prepared as described [START_REF] Lobbestael | Immunohistochemical detection of transgene expression in the brain using small epitope tags[END_REF]. A mixture of 3 different plasmids is needed, packaging plasmid, envelop plasmid and the transfer plasmid encoding the sequence of interest.

HEK293T are triple transfected with these plasmids at 60% confluency. Medium is refreshed at 24H post-transfection with 10mL of Optimen 0% FCS. 48H later, cell supernatant containing LV particles is filtered through 0.45µm filter and concentrated with Amicon filter unit (Merck Millipore, USA). Lentiviral particles concentration is measured by ELISA quantification of the capsid protein P24 (Anti-HIV p24, Eurobio).

1.4.2) Cell transduction and selection

Cells are plated into P24 dish, with same amount on lentiviral particles for each LRRK2 construction. Alpha-synuclein LVs co-express a resistance to blasticidin, allowing us to select the cells transduced with the lentivectors. Transduced cells were selected with 15µg/ml of blasticidin for one week. For LRRK2 constructs, we use Fluorescent Activated Cell Sorter cytometer to sort the cells positive for mCherry-LRRK2.

2) Molecular biology

2.1) Western Blot

2.1.1) Protocol

Quantity of lyzed samples is determined with Pierce BCA protein assay kit (Thermo scientific, The quantification of the evolution of phosphorylation is done with the use of ImageQuant software (GE Healthcare Life Sciences, USA), for each phosphorylation site the levels observed for the phosphomutants are compared to the level of the wild form of the protein to allow us to evaluate the variations in the level of phosphorylation of the other phosphorylation sites.

2.1.2) List of antibodies

2.1.3) LRRK2 purification

HEK293T expressing 3xFLAG-LRRK2 were lysed in IP lysis buffer (Tris 20mM pH7.4, NaCL 150mM, EDTA 1mM pH 8.8, Glycerol 10%, Triton x-100 1%, protease/phosphatase inhibitor 1X) and incubated with anti-FLAG-M2-magnetic beads (Sigma-Aldrich, USA) overnight at 4°C. Beads were washed in different buffer (4X IP lysis buffer) followed by (2X elution buffer, Tris pH 7.4 25mM, NaCl 200mM, MgCl2 5mM, DTT 1mM, Triton X-100 0.02%). LRRK2 proteins are eluted from the beads by competition with 0.1µg/µL of 3xFLAG peptide in elution buffer for 30mn at 4°C on a vertical agitator. Quantities of purified protein are quantified by silver staining using standard dilution of BSA.

2.2) LRRK2 enzymatc activity tests

2.2.1) in vitro kinase assays

-32 P incorporation:

The kinase activities of purified LRRK2 were measured at 30°C in Kinase assay buffer consisting of 25mM Tris (pH7.5), 15mM MgCl2, 20mM β-glycerol phosphate, 1mM sodium fluoride, 1mM EGTA, 1mM sodium orthovanadate, 2mM DTT and 0.1mg/ml BSA. 0.1µM purified LRRK2 and 75µM LRRKtide (LRRK generic substrate) were used in the assay. The reaction was initiated by addition of 25µM 32P-γ-ATP (4Ci/mmol). At different time points until 30min of reaction, the reaction mixtures were quenched in 100mM ice-cold EDTA. The quenched samples were then spotted on P81 phosphocellulose paper discs pre-rinsed with 75mM ice-cold phosphoric acid and further washed with phosphoric acid to remove free ATP. The paper discs were then air dried before scintillation counting.

-Phosphorylation of LRRK2 substrate: 40ng of purified LRRK2 proteins are mixed with 200ng of recombinant RAB8a (Abcam) and 100µM of ATP in kinase buffer (Tris pH 7.4 25mM, MgCl2 10mM, DTT 2mM, Triton X-100 0.02%) for 1H at 37°C. Phosphorylation of RAB8a is assessed by SDS-PAGE, followed by western blotting for total and phosphorylated RAB8a.

2.2.2) GTPase assay

LRRK2 WT and mutants are produced in HEK293T cells and purified on beads as described in 2.2.1. Hydrolysis of GTP is measured at 25°C in a solution (20mM HEPES pH 7.5, 150mM NaCl, 5mM MgCl2, 5% glycerol and 1mM DTT). The resulting solution containing GTP and newly produced GDP is separated on a column (C18-reversed phase, Phenomenex, Jupiter 5μm C18 300 Å) coupled with an HPLC system (Waters). An absorbance measurement is made at 254nm, the surface of the GDP is converted into concentration with a comparison to a standard curve.

2.3) Co-localisation LRRK2/RAB8a

2.4) Immunocytochemistry

SH-SY5Y stably expressing 3xFLAG-LRRK2 WT and its mutants are splitted and transfer to P24 plate containing glass coverslips coated with Cell tak solution (292.5µL of bi-carbonate buffer, 2.5µL of NaOH 1M, 5µL of Cell Tak). Once desired confluence obtained, cells are washed one time with cold PBS and fixed for 20mn with paraformaldehyde 4%. Coverslips are washed with PBS and permeabilized for 5mn with triton 0.1% solution and cells are blocked with PBS + 0.5% BSA for 20mn. Primary antibody is incubated overnight at 4°C, cells are washed 3 times with PBS and coverslips are incubated with secondary antibody for 2H at room temperature with fluorescent antibodies (See table 6 for antibodies concentration). Coverslips are placed on glass slides with a drop of mounting medium (ProLong Diamond black Antifade Mountant with DAPI, Invitrogen). Coverslips are visualized through confocal microscope Zeiss LSM 710 and analysed with IMARIS software.

3) Lysosomes

3.1) Isolation of lysosomes from cells

HEK293T cells express 3xFLAG-LRRK2 cultured for 24H in a medium containing an iron-coupled dextran. This compound accumulates in lysosomes. After a washing time of 24H in a medium without dextran the cells are centrifuged 60g for 5mn then washed with PBS. A mechanical lysis is performed with a Dounce Tight followed by 8 passes in a 23 G needle. After a 400G centrifugation for 10 min the lysate is placed on a magnetic column (MS Coumns, Miltenyi Biotec) (Figure 28). The lysosome-enriched fraction is then collected by detaching the column from the magnetic base in PBS/0.1mM sucrose buffer. The samples are then analysed by western blot.

Figure 28: Lysosomes isolation with dexomag40 treatment. Briefly, cells uptake a ferromagnetic compound, dexomag40, that will be integrated into the endosomal system. After 24H of wash, lysosomes are enriched with the ferromagnetic compounds. After the lysis, magnetic lysosomes are collected on a magnetic stand for further analysis.

3.2) Image Stream analysis

Imaging of lysosomal density is measured by flow cytometry-microscopy. Image Stream MK2 instrument integrates flow cytometer coupled with fluorescence microscopy it allows the measurement at single cell cytometry data and microscopy. This allows us to measure at high throughput the quantification of lysosomes density for a representative large number of cultured cells.

Lysosomal density and lysosomal enzyme activity

5 million PC12 cells are centrifuged 500G, 5mn and resuspended in 1mL of medium containing 10µg of Hoechst 33258 (Invitrogen). After 30mn of incubation at 37°C cells are centrifuged 500G for 5mn and incubated in medium containing 75nM of Lysotracker Deep Red (ThermoFisher, USA) and 75µg of FDGlu (Tebu-Bio, France) for 1H at room temperature. Cells are centrifuged and resuspended in 20µL of PBS and EDTA 0.5M. Cells are analysed by ImageStream MKII at 60X magnification and spectral compensation was carried out of single stained controls. Data were analysed with IDEAS Software (EMD Millipore).

4) Incucyte analysis

To analyse the neurite complexity, we used the IncuCyte live-cell imaging system (Sartorius, UK). 4000 PC12 cells stably expressing mCherry were seeded on collagen coated Costar® 24well Clear TC-treated Multiple Well Plates (Corning, USA). Differentiation was induced with RPMI 1640 medium containing 1% fetal horse serum and 0.5% fetal bovine serum + 50 µg/mL NGF. This medium was refreshed every 48H until full differentiation at day 10. Plates were scanned by the Incucyte system using a 20x objective and 36 images were taken per well.

Assessment of neurite length and branch points was performed using the Incucyte®Neurotrack analysis software module. Average Neurite length and branch points per cell were calculated by dividing total neurite length or branch points per well by the cell confluence expressed in mm².

5) Statistical analysis

All data presented is this document represents mean ± SEM (Standard Error of Mean) with a minimum of three independent replicate for each experiment. Exact number of replicates are indicated in the legend of the figures. Statistical tests were performed with GraphPad Prism 7. Significance is assessed by different tests, as presented in the results section.

Part 5: Results

Characterization of LRRK2 phosphomutants

To investigate the links between LRRK2 phosphorylation and its functions, phosphomutants were used. In cells, LRRK2 is constantly phosphorylated at ANK-LRR domain [START_REF] West | Parkinson's disease-associated mutations in LRRK2 link enhanced GTP-binding and kinase activities to neuronal toxicity[END_REF].

Single change was able to modified the localization of LRRK2 within the cell by inducing accumulation of LRRK2 in the cytoplasm (S910A; S935A; S910A/S935A) [START_REF] Doggett | Phosphorylation of LRRK2 serines 955 and 973 is disrupted by Parkinson's disease mutations and LRRK2 pharmacological inhibition[END_REF].

Phosphorylation changes can also lead to decreased of its kinase activity or GTP binding and destabilization. Phosphorylation changes are known to modify adjacent phosphorylation sites, S910A or 935A dephosphorylation induce diminution of S973 phosphorylation [START_REF] Doggett | Phosphorylation of LRRK2 serines 955 and 973 is disrupted by Parkinson's disease mutations and LRRK2 pharmacological inhibition[END_REF]. Despite the number of described LRRK2 pathogenic mutant's phosphorylation profile, there is little evidence establishing a direct link between the loss of a specific phosphorylation site and pathway dysregulation. Expression in HEK293T was assayed and on each expressed protein, LRRK2 phosphoantibodies was applied (S910, S935, S955, S973, S1292). LRRK2 phosphorylation mutant are expressed in the same proportion, independently from their phosphorylation profile. Specific antibodies against phosphorylated serine seems unable to bind to mutated sites except for the S910D and a strong reduction in binding can be observed with mutant S935A and S935D (Figure 29). Phosphodead mutant S910A, S935A and S955A change the phosphorylation of adjacent serine (Figure 30,A,B,D), matching previous observations [START_REF] Nichols | 14-3-3 binding to LRRK2 is disrupted by multiple Parkinson's disease-associated mutations and regulates cytoplasmic localization[END_REF][START_REF] Doggett | Phosphorylation of LRRK2 serines 955 and 973 is disrupted by Parkinson's disease mutations and LRRK2 pharmacological inhibition[END_REF].

By comparison, phosphomimicking mutants of these same sites presents the same effect on neighbouring Serines as the dephosphorylation mutants, except S910D which does not display altered S935 phosphorylation. We tested our phosphorylation mutants for S1292 autophosphorylation site, representative of the kinase activity of LRRK2. Individually, S910 S935, S955, and S973 had no significant effect on the phosphorylation rate of the S1292 autophosphorylation site (Figure 31). Unexpectedly, our mutants LRRK2 6xA and 6xD presented a 30% reduced phosphorylation a S1292 (Figure 31). Other dephosphorylation mutant (S910/S935/S955/S973A) induce a kinase activation (pSer1292 p = 0.066; pThr1491 p = 0.097; pThr2483 p = 0.055) [START_REF] Reynolds | LRRK2 kinase activity and biology are not uniformly predicted by its autophosphorylation and cellular phosphorylation site status[END_REF].

Interestingly, there were no detection of increased phosphorylation after induction of phosphorylation mutants (Figure 32). Phosphorylation mutants seems to only induce a reduction of phosphorylation level over the other phosphorylation sites. We then tested whether or not LRRK2 6xD could trigger dephosphorylation of RAB29, a PD risk factor that has previously been reported as an interactor and substrate of LRRK2 [START_REF] Beilina | Unbiased screen for interactors of leucine-rich repeat kinase 2 supports a common pathway for sporadic and familial Parkinson disease[END_REF][START_REF] Reyniers | Differential protein protein interactions of LRRK1 and LRRK2 indicate roles in distinct cellular signaling pathways[END_REF]. Measurement of endogenous RAB29 phosphorylation was no completed du to performance of phosphor-RAB29 antibody (Figure 34). Expression of the LRRK2 phosphorylation mutants 6xA and 6xD did not induce changes in RAB29-T72 phosphorylation compared to LRRK2 WT (Figure 34). RAB29 phosphorylation interact with LRRK2 on a different domain than RAB8a and RAB10. It has been reported that RAB29 can bind to LRRK2 in the ankyrin domain [START_REF] Purlyte | Rab29 activation of the Parkinson's disease-associated LRRK2 kinase[END_REF] while another RAB, RAB35, has been reported to bind to the ARM domain [START_REF] Myasnikov | Structural analysis of the full-length human LRRK2[END_REF].

In this experiment, we decided to also test for effects of RAB29 on LRRK2, given previous reports that RAB29 overexpression activates autophosphorylation of LRRK2 at S1292 [START_REF] Purlyte | Rab29 activation of the Parkinson's disease-associated LRRK2 kinase[END_REF]. We found that expression of RAB29 led to an activation of LRRK2-S1292 phosphorylation of LRRK2 6xA that was 3-fold higher compared RAB29 induced activation of LRRK2-WT or LRRK2-6xD (Figure 35).

In vitro catalytic assay

We show that our phosphorylation mutant can induce phosphorylation changes on RABs substrates, as well as a decrease autophosphorylation at S1292. Phosphorylation mutant of autophosphorylation sites are known to decrease GTP binding (T1343A, T1348A/D, T1349D, T1357A/D, S1403A, T1404A, T1410A T1503A/D) [START_REF] Greggio | The Parkinson's disease kinase LRRK2 autophosphorylates its GTPase domain at multiple sites[END_REF][START_REF] Webber | Autophosphorylation in the leucine-rich repeat kinase 2 (LRRK2) GTPase domain modifies kinase and GTP-binding activities[END_REF][START_REF] Kamikawaji | Differential effects of familial parkinson mutations in LRRK2 revealed by a systematic analysis of autophosphorylation[END_REF]. To test whether if our phosphorylation mutants induce change of catalytic activity of LRRK2, we assay our mutants for in vitro kinase activity (LRRKtide peptide phosphorylation in the presence of 32P-γ-ATP) and GTPase activity (hydrolysis of 32P-γ-GTP). Mutation of phosphorylation sites S1292A, S1292D had no effect on the incorporation of 32Pγ-ATP and 32P-γ-GTP. However, in the multi-mutant LRRK2 6xD kinase activity trend to decrease (P = 0.0549) (Figure 36,A,B). As we identified LRRK2 6xD to present, in cellulo, a decreased phosphorylation at RAB8a and RAB10, we tested LRRK2 multi-mutant for in vitro kinase assay on recombinant RAB8a. The loss of T72 RAB8 and S1292 phosphorylation decreased obtain in cellulo were not replicated by only using the two purified partners (Figure 36,C,D,E). We hypothesized that the decreased phosphorylation of RAB8a and S1292 could only happened when LRRK2 6xD a part of a protein complex or in a close relationship with membranes.

Interaction with 14-3-3

LRRK2 phosphorylation is an important factor allowing the interaction with 14-3-3. LRRK2 S910A/S935A abolish the binding to 14-3-3 through overlay assay [START_REF] Nichols | 14-3-3 binding to LRRK2 is disrupted by multiple Parkinson's disease-associated mutations and regulates cytoplasmic localization[END_REF][START_REF] Doggett | Phosphorylation of LRRK2 serines 955 and 973 is disrupted by Parkinson's disease mutations and LRRK2 pharmacological inhibition[END_REF]. LRRK2 kinase inhibition which result in a decreased phosphorylation of the ANK-LRR domain disrupt the interaction with 14-3-3 protein. Interaction with 14-3-3 is known to induce localization change in the cells [START_REF] Dzamko | Inhibition of LRRK2 kinase activity leads to dephosphorylation of Ser 910 /Ser 935 , disruption of 14-3-3 binding and altered cytoplasmic localization[END_REF].

We first use Micro Scale Thermophoresis (MST) that allow us a quick identification of binding between overexpress fluorescent protein and recombinant partners, here 14-3-3zeta. Binding with LRRK2-WT was confirmed (Figure 37,A,B). Surprisingly, binding with dephosphomimic mutant 6xA was confirmed, as well as 6xD mutant. . (C) HEK293T were transfected with 14-3-3 or FLAG-LRRK2 in combination with 14-3-3, as indicated. After coimmunoprecipitation with anti-FLAG beads, the immunoprecipitate (IP) and input samples were analysed by SDS-PAGE and western using the indicated antibodies.

To confirm the binding with LRRK2 6xA and 14-3-3 with performed Co-immunoprecipitation of 3xFLAG-LRRK2 and myc-14-3-3zeta in HEK293T.

We were able to co-immunoprecipitate LRRK2 with 14-3-3, more importantly, no change in interaction was confirmed with LRRK2 dephosphomimic 6xA and phosphomimic 6xD. Our findings suggested that phosphorylation of LRRK2 need a specific pattern to guide it interaction with 14-3-3. As previously reported, dephosphomimic mutant S910/935A mutant result in a lost binding with 14-3-3 while our multi mutant that contain the mutated site 910/935A do not replicate the previous finding.

Localization of LRRK2

Localisation of LRRK2 phosphomutants in cells

LRRK2 localisation in cells has repeatedly been shown to correlate with 14-3-3 binding. It is possible to change the diffuse cytoplasmic distribution of LRRK2 by kinase inhibition of LRRK2, this treatment induces disruption of both 14-3-3 binding and the diffuse cytoplasmic distribution of LRRK2 and creates filamentous LRRK2 accumulations in cells described as skein like, puncta or amorphous. The new subcellular distribution of LRRK2 in cells may be responsible for the interaction of LRRK2 to specific interactors in specific compartment.

To test if the phosphorylation of LRRK2 can trigger a specific localization of LRRK2 we expressed our LRRK2 phosphorylation mutants in HEK293T and subjects them to immunocytochemistry and immunofluorescence analysis.

Localization of S973A phosphorylation mutant is in agreement with the localization already reported [START_REF] Doggett | Phosphorylation of LRRK2 serines 955 and 973 is disrupted by Parkinson's disease mutations and LRRK2 pharmacological inhibition[END_REF] and same as the S973N, PD associated mutation [START_REF] Nichols | 14-3-3 binding to LRRK2 is disrupted by multiple Parkinson's disease-associated mutations and regulates cytoplasmic localization[END_REF]. We reported no significant effect on the cellular localization of LRRK2 mutants (Figure 38). Interestingly, the dephosphorylated mutant LRRK2 6xA does not present skein like LRRK2 positive accumulations without MLi-2 treatment.

Binding to 14-3-3 is not altered by S860A, S955A, S973A, S976A, S973A/S976A phosphorylation mutant [START_REF] Nichols | 14-3-3 binding to LRRK2 is disrupted by multiple Parkinson's disease-associated mutations and regulates cytoplasmic localization[END_REF]). 14-3-3 binding is dissociated after a kinase inhibition resulting of an ANK-LRR dephosphorylation. For instance, no consensus prone the direct link between phosphorylation and loss of 14-3-3 binding. Mimicking the dephosphorylation of 6 sites in the ANK-LRRK domain does not result in a loss of binding with 14-3-3 (Figure 37,C). In agreements with our findings, no change of interaction was detected between LRRK2 6xA and 14-3-3, consistent with the presence of a homogenous cytoplasmic distribution with LRRK2 6xA and 6xD.

Analysis of LRRK2 phosphomutants in lysosome enriched fractions in cell culture

LRRK2 is found to stabilize RAB8a and RAB10 on lysosomes depending on their phosphorylation. LRRK2 can be mobilized to enlarged lysosomes to interact and phosphorylates its substrates RAB8a and RAB10 [START_REF] Eguchi | LRRK2 and its substrate Rab GTPases are sequentially targeted onto stressed lysosomes and maintain their homeostasis[END_REF]. Repair of damaged endolysosomes is dependent of LRRK2's recruitment of RAB8a, this recruitment prevent the lysophagy of damaged endolysosome [START_REF] Herbst | LRRK2 activation controls the repair of damaged endomembranes in macrophages[END_REF]. To test if the dephosphorylation of RAB8a and RAB10 was due to a miss localization of LRRK2 to a positive RAB8a/RAB10 compartment, we induce this know colocalization with chloroquine in SH-SY5Y stably expressing 3xFLAG-LRRK2. We found no changes in the subcellular distribution of LRRK2 6xA or 6xD compared to WT in any of the conditions tested (Figure 39). To further investigate LRRK2 relocalization to the lysosomes, we measure abundance of LRRK2 in purified lysosomes (Figure 40). In our experiment, we were able to identify 3xFLAG-LRRK2

and its phosphorylation mutant in the isolated lysosomes (Figure 40 Under chloroquine treatment, LRRK2 6xD conserved a dephosphorylated RAB8a (Figure 41).

The difference with the previous finding by Eguchi and colleagues could be explained by a higher expression level of LRRK2 resulting in a bigger proportion of LRRK2 in the lysosomes at the basal level. Lysosomal Glucocerebrosidase (GBA) activity in live PC12 cells were measured using the quenchable substrate FDGlu that allow us to measure in real time the activity of this enzyme [START_REF] Migita | Selection of transduced CD34+ progenitors and enzymatic correction of cells from Gaucher patients, with bicistronic vectors Communicated by Roscoe 0[END_REF]. LRRK2 G2019S and R1441C are known to decrease the activity of this enzymes in iPSC derived cells due to regulation of RAB10 [START_REF] Ysselstein | LRRK2 kinase activity regulates lysosomal glucocerebrosidase in neurons derived from Parkinson's disease patients[END_REF].

Overexpression of RAB10 is known to increase the activity of GBA, but overexpression of phosphomimick RAB10 T72E had no effect on GBA activity [START_REF] Ysselstein | LRRK2 kinase activity regulates lysosomal glucocerebrosidase in neurons derived from Parkinson's disease patients[END_REF]. In our experiment, LRRK2 overexpression or its phosphorylation had no effect on lysosomal GBA activity (Figure 42,C).

LRRK2 phosphomutants and neurite outgrowth

LRRK2 kinase activity is known to affect neurite length, mutants G2019S and R1441G cause neurites lengths reductions in primary cultured hippocampal neurons and increased RAB10 phosphorylation [START_REF] Lavalley | 14-3-3 Proteins regulate mutant LRRK2 kinase activity and neurite shortening[END_REF][START_REF] Fan | R1441G but not G2019S mutation enhances LRRK2 mediated Rab10 phosphorylation in human peripheral blood neutrophils[END_REF]. We generated PC12 cell line stably expressing mCherry-LRRK2 to measure the neurite length of each construct, after 10 days of differentiation no change in neurites length or neurites branch points was shown between our phosphorylation mutants (Figure 43,A,B).

Introduction

In section 5.2.1, we presented a phosphorylation mutant, LRRK2 6xA, hyperactivated by overexpression of RAB29. LRRK2 binding to RAB29 allows recruitment of the complex to the trans Golgi network and allows LRRK2 to interact with others factors that can result in a change of trans Golgi network morphology [START_REF] Macleod | RAB7L1 interacts with LRRK2 to modify intraneuronal protein sorting and Parkinson's disease risk[END_REF][START_REF] Beilina | Unbiased screen for interactors of leucine-rich repeat kinase 2 supports a common pathway for sporadic and familial Parkinson disease[END_REF]. LRRK2 was recently reported to be part of the Golgi-associated retrograde protein (GARP) complex, promoting retrograde transport through interaction with SNARE proteins, Syntaxin-6 (t-SNARE) and VAMP4 (v-SNARE) [START_REF] Beilina | The Parkinson's Disease Protein LRRK2 Interacts with the GARP Complex to Promote Retrograde Transport to the trans-Golgi Network[END_REF]. Interestingly, LRRK1, the closest homolog of LRRK2, can interact with a protein from the SNARE family VAMP7. This interaction is

proposed to be at the ANK-LRR interface of LRRK1 (Figure 44) [START_REF] Toyofuku | Leucine-Rich Repeat Kinase 1 Regulates Autophagy through Turning On TBC1D2-Dependent Rab7 Inactivation[END_REF].

VAMP7 is expressed in all tissues and is enriched in neuronal cells including mesencephalic neurons and particularly in the somatodendritic compartment where most of the neurons of the dopaminergic pars compacta are affected in PD [START_REF] Coco | Subcellular localization of tetanus neurotoxin-insensitive vesicle-associated membrane protein (VAMP)/VAMP7 in neuronal cells: evidence for a novel membrane compartment[END_REF]. VAMP7 has also been implicated in lysosome secretion via the LRRK1/VARP complex that are in competition [START_REF] Wang | Biomechanical control of lysosomal secretion via the VAMP7 hub: a tug-of-war mechanism between VARP and LRRK1[END_REF]. At present, the precise relationship between LRRK2 and VAMP7 is not known.

Since the region of interaction between LRRK1 and VAMP7 is, the ANK and LRR domain is conserved in LRRK2. Thus it is in this region where the mutations of LRRK2 6xA are localized.

Given this information's, we choose to evaluate LRRK2 as an interactor of VAMP7.

Hypothesis

As described in the first part of the thesis, LRRK2 is at the interface of different membrane fusion events in the cells. LRRK2 pathogenic mutations have been shown to decrease fusion events such as autophagosome/lysosome fusion. Due to the implication of LRRK2 in the SNARE complex machinery and the homology with LRRK1, we hypothesize that LRRK2 could interact with VAMP7, inducing changes on the neurite's complexity. Thus, the phosphorylation of LRRK2 in the ANK-LRR domain could be a trigger for the interaction/disruption of the interaction with VAMP7 leading to modification of trafficking event in the cells.

Aims

To evaluate the interaction between LRRK2 and VAMP7 we (1) performed a Coimmunoprecipitation between our LRRK2 and VAMP7 (2) assessed whether this interaction is could be controlled by the phosphorylation status of LRRK2 phosphorylation profile and (3) whether loss of VAMP expression can lead to changes in neurite complexity in differentiated PC12 cells.

Results

In order to assess whether LRRK2 can physically interact with VAMP7 we performed coimmunoprecipitation of mCherry-LRRK2 with GFP-VAMP7. We show a decreased interaction with VAMP7 with dephosphorylated LRRK2 6xA while the phosphorylation mutant 6xD conserves the interaction with VAMP7 (Figure 45). LRRK2 interacts with VAMP7, in a phosphorylation dependent manner. Indeed, LRRK2

phosphorylation mutant 6xA presented a decreased binding with VAMP7, an important factor for fusion event in cell [START_REF] Wojnacki | Role of VAMP7-Dependent Secretion of Reticulon 3 in Neurite Growth[END_REF]. No change was detected in size of neurites or branch points in PC12 lacking of VAMP2, VAMP4 or VAMP7. We were able to detect a trend in a deficit of neurite complexity (neurite length and neurite branch points) in PC12 VAMP7 KO models, as previously described (Figure 46) [START_REF] Wojnacki | Role of VAMP7-Dependent Secretion of Reticulon 3 in Neurite Growth[END_REF]. In comparison to LRRK2 WT, the phosphorylation mutant of LRRK2 6xA and 6xD doesn't seems to regulates neurites shortening or number of branch points (Figure 46,47). We demonstrate that VAMP7 KO alone can reduce the size of neurites and the number of branch points in differentiated PC12 (Figure 47, D) (Figure 48,D). In the VAMP7 KO model, the overexpression of LRRK2, independently of its phosphorylation status, increase the neurite length and number of branch points. Previous data indicates that several grey zones persist as to whether LRRK2 dephosphorylation linked to its toxicity. LRRK2 is dephosphorylated in PD patient brains [START_REF] Dzamko | LRRK2 levels and phosphorylation in Parkinson's disease brain and cases with restricted Lewy bodies[END_REF], and in cells with LRRK2 pathogenic mutations [START_REF] Marchand | LRRK2 Phosphorylation, More Than an Epiphenomenon[END_REF]. A particular case is for LRRK2-G2019S, the most common LRRK2 mutation, where heterologous phosphorylation is unchanged in cell culture but reduced in brains, lungs and kidney of G2019S knock-in mice [START_REF] Ito | Lack of correlation between the kinase activity of LRRK2 harboring kinase-modifying mutations and its phosphorylation at Ser910, 935, and Ser955[END_REF]. Dephosphorylation also occurs in cells/tissues treated with LRRK2 type I inhibitors, that have also shown in several studies the ability to abolish adverse phenotype induced by LRRK2 pathogenic mutations such as LRRK2 mutant induced neurite shortening [START_REF] Lavalley | 14-3-3 Proteins regulate mutant LRRK2 kinase activity and neurite shortening[END_REF]. Dephosphorylation of the N-TER region does not occur with Type II LRRK2 inhibitors, stabilizing LRRK2 in an open conformation [START_REF] Tasegian | Impact of Type II LRRK2 inhibitors on signalling and mitophagy[END_REF]. Induction of filamentous accumulations of LRRK2, where LRRK2 binds to microtubules, is possible with LRRK2 stabilized in a closed conformation [START_REF] Deniston | Structure of LRRK2 in Parkinson's disease and model for microtubule interaction[END_REF][START_REF] Tasegian | Impact of Type II LRRK2 inhibitors on signalling and mitophagy[END_REF]. In our experiment, LRRK2 6xA and 6xD can also present filamentous cellular accumulations after treatment with MLi-2, a type I LRRK2 kinase inhibitor, suggesting that immobilization of LRRK2 in a closed conformation by MLi-2 is independent of the phosphorylation state of LRRK2 ANK-LRR domain. Interestingly, our previous work showed that cellular treatment with type I inhibitors of LRRK2 kinase leads to rapid recruitment of phosphatases to the LRRK2 complex (subunits of both protein phosphatase 1 and 2A holoenzymes), suggestive of a conformational change in LRRK2. We postulated that such a conformational change enables LRRK2 dephosphorylation, and given our observation that LRRK2 phosphomutants remain sensitive to type I inhibitor induced filamentous accumulations, we postulate that this phenomenon is related to a conformational change in LRRK2 rather than to dephosphorylation of LRRK2 per se. A recent paper from Myasnikov presents the highest resolution of LRRK2 structure, unfortunately, the region between the ANK and LRR remains to be solved. Our mutations are localised to this unresolved region. The absence of resolution could be due the high dynamic conformational change due to the phosphorylation change.

Looking at cellular kinase activity, when we tested effects of the individual LRRK2 phosphosite mutants on phosphorylation of the LRRK2 substrates RAB8a and RAB10 in cells, we had no or low changes, while a significant decrease was observed for the 6xD mutant, consistent with a decreased kinase activity of LRRK2 in cells when it is phosphorylated. Intriguingly, when tested in vitro this result was not as pronounced as the in cellulo experiment. It is possible that, in cellulo¸ yet unidentified partners act as a sensor for the phosphorylation motif at the ANK-LRR. We hypothesized that the decreased dephosphorylation of RAB8a and S1292

preferentially occurs when LRRK2 6xD is found in a cellular context, perhaps via the aid of additional proteins as a part of a LRRK2 complex.

While we showed in cellulo that LRRK2's phosphorylation profile specifically regulates the phosphorylation of RAB8a and RAB10, we did not detect any effect on RAB29 phosphorylation. It has been reported that RAB29 binds to LRRK2 via the ankyrin domain and increased RAB29 expression is a trigger to activate LRRK2 kinase activity [START_REF] Purlyte | Rab29 activation of the Parkinson's disease-associated LRRK2 kinase[END_REF].

While the exact mechanisms of RAB29 induced activation of LRRK2 remains to be elucidated, our observation that the phosphodead variant of LRRK2 is significantly more activated than the phosphomimicking variant of LRRK2 suggests that LRRK2 phosphorylation is a modulator of LRRK2 activation. Further work would be warranted to explore the link between RAB29 mediated activation of LRRK2 and LRRK2 phosphorylation. For instance, a recent study has modelled binding between RAB38 and ARM domain, using a high resolution structure of LRRK2 [START_REF] Myasnikov | Structural analysis of the full-length human LRRK2[END_REF], a similar analysis could be performed for LRRK2 and RAB29. Also, RAB29 overexpression recruits LRRK2 to the Trans Golgi network and this is assumed to modulate kinases/phosphatases and stimulate LRRK2's activity [START_REF] Purlyte | Rab29 activation of the Parkinson's disease-associated LRRK2 kinase[END_REF].

Another phenotypic effect of LRRK2 mutations is its ability to affect neurite outgrowth, however the role of LRRK2 phosphorylation in this phenotype was never explored with phosphorylation mutants. LRRK2 G2019S is reported to reduce stable contact between growth-cones [START_REF] Onishi | LRRK2 mediates axon development by regulating Frizzled3 phosphorylation and growth cone-growth cone communication[END_REF] and G2019S impair neurite outgrowth in mice [START_REF] Winner | Adult neurogenesis and neurite outgrowth are impaired in LRRK2 G2019S mice[END_REF]. LRRK2 G2019S also increases the phosphorylation of RAB8a/RAB10 that are crucial factors required in rat hippocampal neurons to control neurite outgrowth [START_REF] Huber | A deficiency of the small GTPase rab8 inhibits membrane traffic in developing neurons[END_REF][START_REF] Wang | Lgl1 Activation of Rab10 Promotes Axonal Membrane Trafficking Underlying Neuronal Polarization[END_REF]. In brief, a protein from the trans Golgi network, TRIO, regulates the axonal guidance by interacting with RABIN8, a guanine exchange factor (GEF) that activates RAB8/RAB10 [START_REF] Villarroel-Campos | Rab GTPase signaling in neurite outgrowth and axon specification[END_REF]. RAB8a interaction with RABIN8 is dependent of the phosphorylation of RAB8a's S111 phosphorylation site [START_REF] Pourjafar-Dehkordi | Phosphorylation of Ser111 in Rab8a Modulates Rabin8-Dependent Activation by Perturbation of Side Chain Interaction Networks[END_REF] the Rab8-RABIN8 interaction is also blocked with RAB8a T72 phosphorylation [START_REF] Mamais | Pathogenic mutations in LRRK2 sequester Rab8a to damaged lysosomes and regulate transferrin-mediated iron uptake in microglia Running Title LRRK2 modulates iron uptake in glial cells[END_REF] and this interaction promotes neurite outgrowth [START_REF] Homma | Rabin8 regulates neurite outgrowth in both GEF activitydependent and -independent manners[END_REF]. Despite the reduced RAB8a and RAB10 phosphorylation in the presence of LRRK2-6xD, no significant effect on neurite complexity and length was detected in the tested conditions of NGF induced neurite outgrowth in PC12 cells. Our result suggests that other mechanisms may be at play besides RAB phosphorylation in LRRK2-mediated regulation of neurite complexity and further work is warranted such as testing different experimental conditions (such as testing other neurite forming cell types) and/or analyzing finer phenotypes such as growth-cone formation deficits.

LRRK2 kinase activity has been associated with the regulation of lysosomal properties.

Enhanced kinase activity results in enlarged lysosomes and reduced GBA activity and reduced lysosomal degradative properties [START_REF] Henry | Pathogenic LRRK2 mutations, through increased kinase activity, produce enlarged lysosomes with reduced degradative capacity and increase ATP13A2 expression[END_REF][START_REF] Hockey | Dysregulation of lysosomal morphology by pathogenic LRRK2 is corrected by TPC2 inhibition[END_REF][START_REF] Ysselstein | LRRK2 kinase activity regulates lysosomal glucocerebrosidase in neurons derived from Parkinson's disease patients[END_REF][START_REF] Obergasteiger | Kinase inhibition of G2019S-LRRK2 enhances autolysosome formation and function to reduce endogenous alpha-synuclein intracellular inclusions[END_REF]. Our results show that LRRK2 phosphorylation at S860/910/935/955/973/976 is not associated with modification of glucocerebrosidase activity. Interestingly, our results suggest that this cluster of Serine residues is involved in determining cellular lysosome numbers, as both the phosphodead and the phosphomimicking variants showed reduced numbers on lysosomes in PC12 cells (Figure 9, B). Further work is needed to investigate the link between phosphorylation change and lysosomal dysfunction.

Our results also suggest that the localization of LRRK2 to damaged lysosomes is not dependent on its ANK-LRR phosphorylation status. In [START_REF] Eguchi | LRRK2 and its substrate Rab GTPases are sequentially targeted onto stressed lysosomes and maintain their homeostasis[END_REF] LRRK2, in absence of chloroquine treatment, is not detected in the lysosomal compartment. We were able to detect LRRK2 with and without chloroquine treatment in lysosomes. LRRK2 6xD shows a nonstatistically significant tendency to be less abundant in the lysosomes, with or without chloroquine treatment. The difference with the previous finding by Eguchi and colleagues could be explained by a higher expression level of LRRK2 resulting in a bigger proportion of LRRK2 in the lysosomes at the basal level. It is important to correlate these results with microscopy imaging, without chloroquine, LRRK2 and RAB8a are not present in the same vesicular structures. Therefore, we conclude that LRRK2 can be recruited to lysosomes independently of its phosphorylation, on the conditions tested.

As its homologue LRRK1, we demonstrate that LRRK2 can interact with VAMP7. We show that this interaction is disrupted by LRRK2 6xA dephosphorylation mutant but not with the phosphorylation mutant 6xD. Tyrosine phosphorylation of VAMP7 increase its activity and lead to exocytosis of resting pool and neurites growths [START_REF] Burgo | Increased activity of the vesicular soluble N-ethylmaleimide-sensitive factor attachment protein receptor TI-VAMP/VAMP7 by tyrosine phosphorylation in the Longin domain[END_REF]. Our results show decreased neurites complexity in PC12 KO for VAMP7 as previously described [START_REF] Wojnacki | Role of VAMP7-Dependent Secretion of Reticulon 3 in Neurite Growth[END_REF]. It was possible to restore the neurites deficits induced by the KO of VAMP7 in PC12 by overexpressing LRRK2, independently of its phosphorylation profile. Further investigation is required to verify if VAMP7 is phosphorylated by LRRK2 and what the precise mechanisms are for the LRRK2:VAMP7 interaction and how this affects neurite complexity.

Reduced phosphorylation of RAB8a and RAB10 observed in the presence of LRRK2 6xD phosphomimicking could be accompanied by reduced RAB35 phosphorylation, however this would need to be verified. Recent resolution of LRRK2 structure in its inactive state indicate that RAB proteins may bind to the ARM domain of LRRK2 [START_REF] Myasnikov | Structural analysis of the full-length human LRRK2[END_REF]. In its inactive conformation, the ARM is remotely positioned relative to the kinase domain that is also covered by the LRR domain that wraps around the ATP binding cleft and block its use. The coordination of the RAB with the ARM domain suggests that LRR and ARM would shift positions in the active state to allow access of the RAB to the kinase.

Perspectives

The new insight provided in this thesis demonstrate the importance of the phosphorylation profile of LRRK2 in the physiology of LRRK2 in PD and opens several perspectives for future research. For instance, this thesis has studied specifically the phosphorylation sites of the ANK-LRR interdomain region, that are only a fraction of the many LRRK2 phosphorylation sites (see figure 23). As we focused out work on the ANK-LRR interdomain region, it would be worth using the phosphorylation mutant approach outside of this hotspot. Assays should be performed in such a way that all LRRK2 phosphorylation sites are measured simultaneously in order to identify a subset of sites that act in parallel (for instance, a subset of sites always phosphorylated or not). Phosphorylation changes in specific functional subsets of phosphosites could be important for the regulation of specific pathways.

Autophosphorylation sites tend to be increased in the disease while heterologous sites are

decreased. An interesting experiment would be to construct a phosphorylation mutant with constant dephosphorylation of autophosphorylation sites and constant phosphorylation of heterologous sites (as well as the other way around) and then study the behaviour of the protein. The results of such modification could be, as suggested by [START_REF] Myasnikov | Structural analysis of the full-length human LRRK2[END_REF], a modification of its structure. They described that the addition of negative changes by phosphorylation of S1292 could lead to the modification of the interface of LRR with KIN domain [START_REF] Myasnikov | Structural analysis of the full-length human LRRK2[END_REF]. Therefore, it would be interesting to test single phosphorylation for its effect on structural properties and with modification of binding to partners (Protein, ATP, GTP/GDP), which is dependent on its 3D structure. These experiments would allow us to understand the relationship between LRRK2 structure and its phosphorylation status and point to mechanisms explaining how a toxic effect could be derived from an altered phosphorylation profile.

As such, monitoring the phosphorylation status of LRRK2 or its targets could have the potential to define a biomarker for Parkinson's disease [START_REF] Taymans | LRRK2 detection in human biofluids: potential use as a Parkinson's disease biomarker?[END_REF]. Several studies have begun to survey the phosphorylation state of LRRK2 in different fluids of parkinsonian patients [START_REF] Rideout | The Current State-of-the Art of LRRK2-Based Biomarker Assay Development in Parkinson's Disease[END_REF]. A more complete description of the relationship between LRRK2 phosphorylation and its association with a deleterious or beneficial state could open the way to new therapeutic strategies.

Today, the strategies developed focus on the kinase activity of LRRK2. Acting directly on the kinase function modifies the phosphorylation state of all LRRK2 substrates. We were able to demonstrate in our work that the phosphorylation state of LRRK2 can act on the phosphorylation of certain substrates while others substrates are unaffected. In the future, the development of compounds that can block the action of LRRK2 phosphatases or its kinases could lock LRRK2 into a certain phosphorylation profile and thus enable it to remain in a less pernicious state. To this end, a better description of LRRK2 phosphorylation regulation dynamics is also needed to identify the precise phosphatases and kinases.

Considering our results showing different phosphorylation profile between RAB8a/RAB10 and RAB29 in the presence of LRRK2 phosphomutants, it would be worth to investigate the phosphorylation of other RABs and substrates of LRRK2 with our phosphorylation mutants.

Several RABs are involved in pathogenic mechanisms such as RAB35 for alpha-synuclein release, RAB11/RAB43 regulating TRAPPII complex and other substrates [START_REF] Bae | LRRK2 kinase regulates α-synuclein propagation via RAB35 phosphorylation[END_REF][START_REF] Jenkins | The substrate specificity of the human TRAPPII complex's Rab-guanine nucleotide exchange factor activity[END_REF]. It is known that LRRK2 can regulate alpha-synuclein secretion by RAB35 phosphorylation. A possible experiment to carry out is to test if RAB35 phosphorylation is dependent on LRRK2 phosphorylation profile and if this modification led to alteration of alphasynuclein secretion. In our laboratory, we are currently working on this hypothesis by performing measurements of the alpha-synuclein release in cell culture in response to different experimental conditions (PhD thesis of Alessia Sarchione under the supervision of Dr Chartier-Harlin). The preliminary data she obtained in collaboration with myself and Dr Taymans suggest that LRRK2 phosphorylation is able to modulate alpha-synuclein release.

This thesis also reveals that binding of the v-SNARE VAMP7 to LRRK2 is dependent on the LRRK2 phosphorylation profile. VAMP7 is an important factor regulating fusion events in cells.

This study should be extended to the whole family of VAMP proteins such as VAMP4, considered as a potential PD risk factor. Our collaborators are currently working on this hypothesis (PhD of Francesca Filippini under the supervision of Dr Thierry Galli). In our study, we were not able to detect differences in the neurite's complexity of our PC12 between 6xA and 6xD mutants. As described by [START_REF] Wojnacki | Role of VAMP7-Dependent Secretion of Reticulon 3 in Neurite Growth[END_REF], VAMP7 is factor regulating axonal growth and neuronal polarity. Further work is needed to identify a potential role of LRRK2 phosphorylation the membrane fusion mechanism.

This thesis work also observed a reduced number of lysosomes per cell in the cells overexpressing LRRK2 6xA and 6xD relative to WT, with no differences between phosphorylated or dephosphorylated LRRK2. We also observed that these two mutants do not reduce the activity of GBA in lysosomes. A more precise description of the structure of the lysosomes and its content (RAB phosphorylation, LRRK2 kinase or phosphates, expression of ATPases…) after expression of LRRK2 phosphomutants could pinpoint importance of LRRK2 phosphorylation on lysosomal physiology. In this study, we did not test the degradative properties of lysosomes in the presence of LRRK2 phosphomutants, this could contribute to further documenting deleterious or beneficial states of LRRK2 relative to PD pathology.

We believe that the complex regulation of LRRK2 phosphorylation is a crucial factor regulating diverse processes in cells. Developing new tools to study LRRK2 phosphorylation states and correlate these to disease related phenotypes will open new opportunities to develop new therapeutic and diagnostic approaches for PD.

Conclusions

Considering the postulates that enhanced kinase activity and increase of RABs phosphorylation are detrimental processes, our study results are consistent with the hypothesis that phosphorylation of LRRK2 at the ANK-LRR phosphosites corresponds to a protective state for LRRK2 while dephosphorylation at these sites correspond to a deleterious state. Future research should pursue the investigations on this hypothesis, notably to understand the precise role of LRRK2 phosphorylation on disease related processes such as LRRK2 effects on membrane fusion, endosomal trafficking, neurite complexity and release of alpha-synuclein.

Figure 1 :

 1 Figure 1: Evolution of PD symptoms.

Figure 2 :

 2 Figure 2: Basal ganglia structure and connectivity.

Figure 3 :

 3 Figure 3: Evidence of PD in the human brain.

Figure 4 :

 4 Figure 4: Neuronal circuitry causing motor dysfunctions in PD.

Figure 5 :

 5 Figure 5: Representation of the PD Braak staging system.

Figure 6 :

 6 Figure 6: Drugs used in clinical practice to treat PD symptoms.

Figure 7 :

 7 Figure 7: milestone of PD treatment.

Figure 8 :

 8 Figure 8: Component of PD factors.

Figure 9 :

 9 Figure 9: Mitochondrial dysfunction in PD.

Figure 10 :

 10 Figure 10: Representation of alpha synuclein aggregation leading to toxic species and neuronal death.

Figure 11 :

 11 Figure 11: Neuroinflammation in PD.

Figure 12 :Figure 13 :

 1213 Figure 12: Implication of PD related genes on autophagy Figure 13: PD genetic risk factors regulate the trafficking machinery and increase cell toxicity.

Figure 14 :

 14 Figure 14: LRRK2 domains and structure.

Figure 15 :

 15 Figure 15: LRRK2 GTP binding and activation model.

Figure 16 :

 16 Figure 16: LRRK2's pathogenic mutations.

Figure 17 :

 17 Figure 17: Cellular Functions of LRRK2.

Figure 18 :

 18 Figure 18: Basic principles of membrane trafficking.

Figure 19 :

 19 Figure 19: LRRK2 RAB substrates are implicated in the regulation of vesicular trafficking.

Figure 20 :

 20 Figure 20: LRRK2 and the autophagy.

Figure 21 :

 21 Figure 21: LRRK2 impact on protein translation and calcium homeostasis.

Figure 22 :

 22 Figure 22: LRRK2-RAB mediated alpha-synuclein propagation.

Figure 23 :

 23 Figure 23: Schematic representation of LRRK2 domains and phosphorylation sites.

Figure 24 :

 24 Figure 24: Phosphoregulation of LRRK2.

Figure 25

 25 Figure 25: Objectives.

Figure 26 :

 26 Figure 26: Plasmid map of vectors used in this study.

Figure 27 :

 27 Figure 27: Construction of phosphomutant of LRRK2.

Figure 28 :

 28 Figure 28: Lysosomes isolation with dexomag40 treatment.

Figure 29 :

 29 Figure 29: Phosphorylation state of LRRK2 phosphorylation mutant.

Figure 30 :

 30 Figure 30: In cellulo phosphorylation profile of LRRK2 phosphorylation mutants.

Figure 31 :

 31 Figure 31: In cellulo auto-phosphorylation profile of LRRK2 phosphorylation mutants.

Figure 32 :

 32 Figure 32: Representation of phosphorylation effect in HEK293T.

Figure 33 :

 33 Figure 33: In cellulo phosphorylation profile of LRRK2 phosphorylation mutants.

Figure 34 :

 34 Figure 34: In cellulo phosphorylation of RAB29 by LRRK2.

Figure 35 :

 35 Figure 35: In cellulo LRRK2 activation by RAB29.

Figure 36 :

 36 Figure 36: Catalytic activities of LRRK2 phosphorylation mutants.

Figure 38 :

 38 Figure 38: Localization of LRRK2 phosphorylation mutants in cells.

Figure 39 :

 39 Figure 39: LRRK2 and RAB8a are localized to vesicular structures after chloroquine treatment.

Figure 40 :

 40 Figure 40: Isolation of lysosomes from HEK293T.

Figure 41 :

 41 Figure 41: RAB phosphorylation under chloroquine treatment.

Figure 42 :

 42 Figure 42: Lysosotracker analysis PC12 cells expressing WT and phosphomutant LRRK2 treated with and without chloroquine via imaging flow cytometry (ImageStream MKII, Amnis®).

Figure 43 :

 43 Figure 43: LRRK2 phosphorylation do not affect neurites complexity in PC12 cells.

Figure 44 :

 44 Figure 44: Interaction simulation between LRRK2 and VAMP7.

Figure 45 :

 45 Figure 45: Co-immunoprecipitation with mCherry-LRRK2 and GFP-VAMP7.

Figure 46 :

 46 Figure 46: Neurite complexity in PC12 VAMPs KO cells.

Figure 47 :

 47 Figure 47: Neurite's lengths and VAMP KO.

Figure 48 :

 48 Figure 48: Neurites branch points and VAMP KO

Figure 1 :

 1 Figure 1: Evolution of PD symptoms.Non-motor symptoms that can appear before the diagnosis of PD which is usually pronounced with the appearance of motor dysfunctions. The non-motor symptoms will be more pronounced as the disease progress. The disability emerges from the combination and progression of non-motor and motor symptoms of PD. Adapted from[START_REF] Poewe | Parkinson disease[END_REF]

Figure 2 :

 2 Figure2: Basal ganglia structure and connectivity. Dopamine projections from the SNpc are represented in blue. Dopamine projections from the SNc activate the direct pathway (green) and inactivate the indirect pathway (Red). This results in higher inhibition of GPi and reduces its inhibition on the thalamus, promoting the movement. Adapted from[START_REF] Nelson | Reassessing Models of Basal Ganglia Function and Dysfunction[END_REF][START_REF] Mcgregor | Circuit Mechanisms of Parkinson's Disease[END_REF].

F

  .H Lewy, identified in 1912 inclusions called Lewy bodies. In the clinic it is possible to indirectly monitor the death of the dopaminergic neurons with imaging techniques such as DATscan (Cerebral Scintigraphy of striatal dopamine transporter) or [18F]-Dopa PET that measures the density of dopaminergic neurons in the brain (Figure 3, B, C).

Figure 3 :

 3 Figure 3: Evidence of PD in the human brain. (A) Brain section showing depigmentation of the substantia nigra and Lewy body inclusion from PD patient (Red arrow). (B) MRI imaging of SNpc showing reduced signal intensity. (C) DAT-scan showing reduced dopaminergic innervation in striata of a PD patient (right panel) compared to control (left panel). Adapted from[START_REF] Politis | Neuroimaging in Parkinson disease: From research setting to clinical practice[END_REF].

Figure 4 :

 4 Figure 4: Neuronal circuitry causing motor dysfunctions in PD. (A) With disruption of dopaminergic neurons from the SNc, the direct pathway activity is reduced and indirect pathway is increased. The STN increases its stimulation of the GPi/SNr while inhibition from the dMSN is reduced, leading to higher inhibition of the thalamus causing inhibition of the movement. (B) Stimulation spikes differences between controls and PD patients in different structures of the basal ganglia. Adapted from (Mcgregor and Nelson, 2019).

Figure 5 :

 5 Figure 5: Representation of the PD Braak staging system. (A) Vagus nerve or pneumogastric nerve is in charge of parasympathetic stimulation controlling the digestive tract. (B) Disease progression has been observed in the brain with the progression of Lewy body pathology through the brain and can be divided in 6 stages of progression. (C) Lewy body pathology is hypothesized to start in the gut after increased alpha-synuclein aggregation resulting from disrupted function of intestinal barrier.

Figure 6 :

 6 Figure 6: Drugs used in clinical practice to treat PD symptoms. Enhancing dopamine at the synapse can be achieved with catechol-O-methyltransferase (COMT) inhibitors that will reduce catecholamine degradation or Monoamine oxidase type B (MAOB) inhibitors that reduce monoamine oxidation. Treatment with L-DOPA leads to its transformation into dopamine by amino acid decarboxylase (AADC).When L-DOPA is combined with non-brain permeable AADC inhibitors to promote L-DOPA conversion in the CNS, this results in higher brain dopamine levels. From[START_REF] Poewe | Parkinson disease[END_REF].

Figure 7 :

 7 Figure 7: milestone of PD treatment. Since 19 th century, diverse advances have been achieved in the research field of PD therapies. Development of compounds, neurosurgery and innovative treatment has greatly improved the quality of life of PD patients. L-DOPA, COMT (catechol-Omethyltransferase), MAOB (monoamine oxidase type B), mGlu (metabotropic glutamate receptor), NAM (negative allosteric modulator). Adapted from[START_REF] Charvin | Therapeutic strategies for Parkinson disease: beyond dopaminergic drugs[END_REF].

Figure 8 :

 8 Figure 8: Component of PD factors. PD pathogenesis results from interactions between ages of patients, environmental factors (Protectives and risks factors) and genetics components. Examples of environmental factors are indicated as protective or risk factors for PD. Loci implicated in PD are indicated as genetics component.

Figure 9 :

 9 Figure 9: Mitochondrial dysfunction in PD. Different system of the mitochondria can be affected in PD. Different proteins implicated in dysregulation of mitochondrial functions are indicated in boxes. Adapted from (Park et al., 2018).

Figure 10 :

 10 Figure 10: Representation of alpha synuclein aggregation leading to toxic species and neuronal death. (A) Alpha-synuclein monomers evolve into partially folded secondary structures, contributing to the formation of oligomers, protofibrils and finally fibrils. (B) Alternatively, or complementarily, secondary molecular changes created after protein aggregation combine with oligomers and fibrils to hasten cell death (lower panel). T-0 = time zero; α-syn = α-synuclein. Adapted from[START_REF] Espay | Revisiting protein aggregation as pathogenic in sporadic Parkinson and Alzheimer diseases[END_REF][START_REF] Mehra | α-Synuclein misfolding and aggregation: Implications in Parkinson's disease pathogenesis[END_REF]

Figure 11 :

 11 Figure 11: Neuroinflammation in PD. Illustration of the production of different neuroinflammatory compounds by astrocytes, microglia and dopaminergic neurons and indication of a crosstalk between each cell type. Adapted from (Pajares et al., 2020).

Figure 12 :

 12 Figure12: Implication of PD related genes on autophagy. Autophagy has been shown to be regulated by different PD genes. SNCA, LRRK2, DJ-1 and VPS35 affect the macroautophagy as well as the chaperone mediated autophagy. The fusion and homeostasis of autophagolysosome is modulated by GBA, ATP13A12 and VPS35. Adapted from[START_REF] Hou | Autophagy in Parkinson's Disease[END_REF].

Figure 13 :

 13 Figure 13: PD genetic risk factors regulate the trafficking machinery and increase cell toxicity.Several PD genes have been associated with dysregulation of trafficking machinery, which affect lysosomal homeostasis, decreasing its degradative properties. The impairment of mitophagy increases the release of ROS (Reactive Oxygen Species) which enhances alpha-synuclein aggregation. The alpha-synuclein aggregation promotes the disruption of vesicular trafficking as well as lysosomal degradation properties. Adapted from[START_REF] Klein | Is Parkinson's disease a lysosomal disorder?[END_REF]

Figure 14 :

 14 Figure 14: LRRK2 domains and structure. (A) LRRK2 is composed of 7 domains. Domains flanking the catalytic core (ROC-COR-KIN) are implicated in protein-protein interaction, Leucine-Rich repeat "LRR", Ankyrin repeat "ANK", Amardillo repeat "ARM" in the N-terminal segment of LRRK2 and the WD40 domain at the C-terminus. (B) High resolution structure of full-length LRRK2 depicting the 3D organisation of LRRK2 domains. (C) High resolution structure of homodimeric form of LRRK2 interacting with RAB partners connecting LRRK2 to membrane bound state. Adapted from[START_REF] Marchand | LRRK2 Phosphorylation, More Than an Epiphenomenon[END_REF][START_REF] Myasnikov | Structural analysis of the full-length human LRRK2[END_REF].

Figure 15 :

 15 Figure 15: LRRK2 GTP binding and activation model. LRRK2 ROC domain contains a GDP/GDP binding motif. LRRK2 catalyses the hydrolysis of GTP to GDP. The GTPase function of LRRK2 is proposed to act as a molecular switch. GTP binding is associated to LRRK2 conformational changes leading to connection with effectors.

Figure 16 :

 16 Figure 16: LRRK2's pathogenic mutations. Pathogenic variants of LRRK2 are indicated in red, rare variants pathogenic in blue and protective variants are indicated in green. Adapted from[START_REF] Goveas | Mind the Gap: LRRK2 Phenotypes in the Clinic vs[END_REF]

Figure 17 :

 17 Figure17: Cellular Functions of LRRK2. This scheme compiles different cellular events that have been reported to be influenced by LRRK2 in physiology or in disease. This representation associate finding from different research groups and from different cellular/animal models. It is not yet determined if all of these events are all disrupted by LRRK2 at the same time in the same cell model.

Figure 18 :

 18 Figure18: Basic principles of membrane trafficking. Intracellular trafficking involved different coat proteins and interaction with SNARE protein and RABs. Relationship between these proteins allow docking and fusion event on plasma membrane. From[START_REF] Søreng | Membrane Trafficking in Autophagy[END_REF].

Figure 19 :

 19 Figure 19: LRRK2 RAB substrates are implicated in the regulation of vesicular trafficking. At the Golgi, RAB1, 29 and 43 regulate the trafficking between the Golgi, TGN and ER. The secretion is regulated by RAB3a, 8, 10 35. The endosomal system transport and maturation is regulated by RAB8a, 10, 12, 35. Adapted From (Bae and Lee, 2020).

Figure 20 :

 20 Figure 20: LRRK2 and the autophagy. At different steps indicated in brackets, LRRK2 has been linked with autophagy. (1) Once the cargo is fully encapsulated by a bi-layered membrane the autophagosome (2) fuses with the lysosome (3) to produce the autolysosome (4). Proteins/organelles are degraded via lysosomal enzymes. The diagram shows how LRRK2 wildtype and the two most common LRRK2 mutations are likely to impact autophagic flow and lysosomal protein degradation. LRRK2 is represented by its respective domains (ANK, LRR, ROC, COR, Kinase, and WD-40). LRRK2 mutations are represented by asterisks in the respective domains where they are located. G2019S, yellow asterisk in kinase domain; R1441C, purple asterisk in ROC domain; interactions represented by arrows; flow of autophagic phases represented by dashed arrows. From (Madureira et al., 2020).

Figure 22 :

 22 Figure 22: LRRK2-RAB mediated alpha-synuclein propagation. Secretion of alpha-synuclein is possible through secretory organelles derived from late endosomes or directly from the lysosomes. From (Bae and Lee, 2020).

Figure 23 :

 23 Figure 23: Schematic representation of LRRK2 domains and phosphorylation sites. Parts of known phosphorylation site are indicated. Heterologous phosphorylation sites are indicated in blue, autophosphorylation site are indicated in red. The most studied sites are indicated in bold. Adapted from (Marchand et al., 2020).

Figure 24 :

 24 Figure24: Phosphoregulation of LRRK2. Phosphoregulation of LRRK2 protein put together a lot of different partners, and some of those partners can also be regulated by LRRK2 itself. On the upstream regulation, the inhibitory phosphatases are localized on the left and the activating kinases are localized on the right. Kinases and phosphatases are implicated in the regulation of the N-ter phosphorylation sites (S910/935/955/973). N-ter sites and S1444 are phosphorylated by PKA while LRRK2 is also able to regulate the activity of PKA by a direct interaction with its ROC domain or by an indirect manner, by acting on the phosphodiesterase 4 (PDE4). PPP1CA has been confirmed to act on LRRK2. The holoenzyme PP2A could regulate the phosphorylation at S1292. The phosphorylation of the N-ter sites allows the interaction with 14-3-3. If phosphorylated by PAK6, the binding to LRRK2 is abolished. RAB29 interacts with LRRK2 in the Trans-Golgi network; this interaction leads to an increased phosphorylation of the N-ter and the kinase activity of LRRK2. LRRK2 can phosphorylate RAB29 and avoid LRRK2 activation, creating an inactivation loop. From[START_REF] Marchand | LRRK2 Phosphorylation, More Than an Epiphenomenon[END_REF]

  questions remain. Post-mortem analysis of PD patient brains revealed dephosphorylation of LRRK2, and similar observations are made after treatments with experimental therapies under clinical trials. It is not clear if phosphorylation or dephosphorylation is associated to healthy of deleterious conditions. The elucidation of the cellular phenotypes of LRRK2 phosphorylation has the potential to lead us to further understand the link between phosphorylation of LRRK2 and PD and open future venues to develop novel treatments.

Figure 25 :

 25 Figure 25: Objectives. Depicted is a schematic representation of the sub-aims of the project. (1) Could LRRK2 phosphorylation profile be in charge its biochemical properties? (2) Is LRRK2 phosphorylation in charge of its localization? (3) Is LRRK2 phosphorylation profile a regulator of neurite outgrowth, a regulating factor of the lysosomal homeostasis?

  France). Alanine (A) substitution result in complete dephosphorylation while Aspartate (D) mimic the phosphorylation of a phospho-serine (Figure27, A). pLV.CSJ-mCherry-LRRK2 plasmids were generated by excising the 3flag sequence (restriction sites to include) and ligating the mCherry sequence (obtained by PCR using the pLV-mCherry and primers with sequences). All coding sequences were sequenced (Eurofins genomics) and digested with EcoRI and EcroRV enzymes (New England Biolabs).

Figure 27 :

 27 Figure 27: Construction of phosphomutant of LRRK2. (A) structures of the amino acid serine, a phosphorylated serine, mimicking aspart since the phosphorylation of serine and alanine that mimick a dephosphorylated serine. (B) All phosphomutants generated by site-directed mutagenesis from the plasmid pLV.CSJ-3xFLAG-LRRK2. (C) Localization of the different positions of the generated phosphomutants, heterologous phosphorylation sites are in blue and autophosphorylation in Red.

  USA). 10µg of proteins are mixed with NuPAGE LDS sample buffer 4X (Life technologies, USA) and reduced 10mn at 95°C. Proteins samples are loaded into a Novex 4-20% Tris-Glycine mini gel (Fisher Scientific, USA) into a Tris-Glycine running buffer (Life technologies, USA) for 45mn at 225V. The proteins are transferred on a PVDF membrane low fluorescence (Bio Rad, USA) for 16H at 5V. Membranes are blocked in milk 5% in TNT 1X (15mM Tris base, 0.14M NaCl, 0.5M tween 20, pH 8) for 1H at room temperature. The membrane and then incubated with selected antibodies in Signal Boost solution (Merck Millipore, USA) for 16H at 4°C. After 4 washes in TNT 1X membranes are incubated with secondary antibodies for 2H at RT, washed in TNT and imaged with Typhoon FLA 9500.

First

  , to determine if the phosphorylation motif of LRRK2 could induce new phosphorylation profile we mutated the Serine (S) residues of position 910, 935, 955, 973, and 1292 to Alanine (A) to mimic non-phosphorylated Serines and to Aspartate (D) to mimic phosphorylated Serine. A compound mutant was also generated to mimic phosphorylated/nonphosphorylated LRRK2 presenting 6 mutations of Serines to Alanines or Aspartates in the heterologous phosphorylation cluster noted 6xA or 6xD (Serines 860/910/935/955/973/976).

Figure 29 :

 29 Figure 29: Phosphorylation state of LRRK2 phosphorylation mutant. HEK293T cells were trans transfected with the Wild Type human LRRK2 as well as the phosphorylation mutant of LRRK2. The phosphorylation state of LRRK2 was analysed by Western Blot. The quantification of the signals was obtained by Typhoon FLA 9500 imaging.

Figure 30 :

 30 Figure 30: In cellulo phosphorylation profile of LRRK2 phosphorylation mutants. Western blot analysis of HEK293T cell lysates after transfection of LRRK2 phosphomutants. The ratio of phosphorylated site over total LRRK2 signal is shown in (A) for S910, (B) for S935, in (C) for S955 and in (D) for S973. The data represents the mean ± SEM from four independent experiments. The quantification of the signals was done by using Typhoon FLA 9500 and ImageQuant software. The data was analysed by One-way-ANOVA with Dunnet's multiple comparison. (*** P <0.001), (**** P <0.0001).

Figure 32 :

 32 Figure 32: Representation of phosphorylation effect in HEK293T. Data from quantified western blot are merged and modification of phosphorylation are shown in red for decreased phosphorylation, X when the phosphorylation is not possible with phospho-antibodies, = when no change is observed.

Figure 31 :

 31 Figure 31: In cellulo auto-phosphorylation profile of LRRK2 phosphorylation mutants. Western blot analysis of HEK293T cell lysates after transfection of LRRK2 phosphomutants. The ratio of 1292 phosphorylated site over total LRRK2 signal is shown. The data represents the mean ± SEM from four independent experiments. The quantification of the signals was done by using Typhoon FLA 9500 and ImageQuant software. The data was analysed by One-way-ANOVA with Dunnet's multiple comparison. (*** P <0.001), (**** P <0.0001).

Figure 34 :

 34 Figure 34: In cellulo phosphorylation of RAB29 by LRRK2. Western blot analysis of HEK293T cell lysates after transfection of LRRK2 phosphomutants and 2myc-Rab29. The ratio of phosphorylated site over total protein signal is shown. Phosphorylation level of RAB29 T72. The data represents the mean ± SEM from four independent experiments. The quantification of the signals was done by using Imager 600 and ImageQuant software.

Figure 35 :

 35 Figure 35: In cellulo LRRK2 activation by RAB29. Western blot analysis of HEK293T cell lysates after transfection of LRRK2 phosphomutants and 2myc-RAB29. The ratio of phosphorylated site over total protein signal is shown. LRRK2 autophosphorylation site S1292 was quantified over total LRRK2 after transfection of 2myc-RAB29. The data represents the mean ± SEM from 3 independent experiments. The quantification of the signals was done by using Imager 600 and ImageQuant software. The data was analysed by One-way-ANOVA with Dunnet's multiple comparison. (* P <0.05).

Figure 36 :

 36 Figure 36: Catalytic activities of LRRK2 phosphorylation mutants. The kinase activities of purified LRRK2 were measured by addition of 32P-γ-ATP for 30mn (A) and 32P-γ-GTP for 180mn (B) and then quenched. Samples were assay for scintillation counting. (C) Silver staining of purified LRRK2 proteins. (D) Purified proteins are incubated with recombinant RAB8 (Life technologies) and ATP for 60mn. Measurement of LRRK2 autophosphorylation level pS1292 (D) and level of phosphorylation of RAB8a (E). The data represents the mean ± SEM from three independent experiments and analysed by ANOVA Kruskal Wallis test; comparison to WT. (# p=0.0549).

Figure 37 :

 37 Figure 37: Assessment of interaction between LRRK2 and 14-3-3. (A) MST raw data showing shift between after incubation of mCherry-LRRK2 + 14-3-3. (B) Binding check comparison between LRRK2 WT/6xA and 6xD with and without ligand. Significant shift signal noise-to-noise ratio is calculated by Noise = 3x S/N, with S/N = Response amplitude √ ∑ (𝑟 𝑖 -𝑟 ̅)² 𝑖 𝑛-1

Figure 38 :

 38 Figure 38: Localization of LRRK2 phosphorylation mutants in cells. HEK293T cells were transfected with 3xFLAG-LRRK2 construct. After 48H of transfection, cells were treated with DMSO or MLi-2 10nM for 2H followed by paraformaldehyde fixation. Representative fluorescent imaging of the indicated phosphosite mutants is shown with and without inhibitor treatment.

Figure 39 :

 39 Figure 39: LRRK2 and RAB8a are localized to vesicular structures after chloroquine treatment. (A) SH-SY5Y stably expressing LRRK2 were treated with 50µM of chloroquine for 24H prior fixation. (B) Quantification of the percentage of cells presenting RAB8a positive LRRK2 vesicles. Data are mean ± SEM (n = 3 independent experiments, 100 -130 cells were analysed per mutant in each experiment).

  , B) without chloroquine treatment. Chloroquine treatment do not significatively increased the proportion of 3xFLAG-LRRK2 in the isolated lysosomes (Figure 40, B).

Figure 40 :

 40 Figure 40: Isolation of lysosomes from HEK293T. Abundance of LRRK2 in purified lysosomes. Lysosomes were magnetically isolated from HEK293T cells expressing 3× FLAG-LRRK2 and phosphorylation mutants. Cells were treated with Chloroquine 50µM for 24H to induce LRRK2 enrichment to lysosomes. Levels of LRRK2 in the Flow through and in the lysosomes isolated are quantified by western blotting (A). Relative abundance of LRRK2 in Flow Through (B) and in isolated lysosomes (C) is normalized with LAMP2 levels. The data represents the mean ± SEM from three independent experiments and analyzed with one sample t test (* p < 0.05).

Figure 41 :

 41 Figure 41: RAB phosphorylation under chloroquine treatment. HEK293T were transfected with 3xFLAG-LRRK2 constructs and treated with chloroquine (50µM for 24H) 48H post transfection. Lysates were analysed by western blot with the indicated antibodies.

Figure 42 :

 42 Figure 42: Lysosotracker analysis PC12 cells expressing WT and phosphomutant LRRK2 treated with and without chloroquine via imaging flow cytometry (ImageStream MKII, Amnis®). PC12 cells were incubated with lysotracker for 1H and subjected to ImageStream analysis. (A) Representative images of PC12 cells labelled with lysotracker, showing red-coloured positive spots corresponding to lysosomes. (B) Quantification of the number of spots imaged per cell. Data represent the mean spots detected from each genotype. (n= 4 independent experiments, 3000 -5000 cells were analysed per mutant in each experiment).

Figure 43 :

 43 Figure 43: LRRK2 phosphorylation does not affect neurite complexity in PC12 cells. PC12 transduced wit mCherry-LRRK2 are coated on P24 plate and differentiations are followed by Incucyte and analysis of neurites complexity was performed when cells were fully differentiated, 10 days of differentiation are needed to obtain differentiated cells. (Incucyte, sartorius). Phase-contrast imaging of differentiated PC12 is show (A) and measurement of neurites length and neurites branch points was assay (B). The data represents the mean ± SEM from three independent experiments.

Figure 44 :

 44 Figure 44: Interaction simulation between LRRK2 and VAMP7.

Figure 45 :

 45 Figure 45: Co-immunoprecipitation with mCherry-LRRK2 and GFP-VAMP7. HEK293T were transfected with GFP-VAMP7 or FLAG-LRRK2 in combination with GFPVAMP7, as indicated. After coimmunoprecipitation with GFP-trap beads, the immunoprecipitate (IP) and input samples were analysed by SDS-PAGE and western blotnusing the indicated antibodies.

Figure 46 :

 46 Figure 46: Neurite complexity in PC12 VAMPs KO cells. PC12 cells of different genotypes (WT, VAMP2 KO, VAMP4 KO and VAMP7 KO) expressing mCherry-empty are analyzed with Incucyte and analysis of neurites complexity was performed when cells were fully differentiated, 10 days of differentiation are needed to obtain differentiated cells (Incucyte, sartorius). Representative phasecontrast images are shown of the differentiated PC12 cells at day 10 taken by live cell imaging as described in materials and methods. Scale bar, 100 µm (B) Neurite length and neurite branch points were measured by automated quantification as described in materials and methods. The data represents the mean ± SEM from three independent experiments. The data was analysed by Oneway-ANOVA with Dunnet's multiple comparison with indicated P value.

Figure 47 :

 47 Figure 47: Neurite's lengths and VAMP KO. PC12 transduced wit mCherry-LRRK2 are coated on P24 plate and differentiations are followed by Incucyte and analysis of neurites complexity was performed when cells were fully differentiated, 10 days of differentiation are needed to obtain differentiated cells. (Incucyte, sartorius). Phase-contrast imaging of differentiated PC12 transduced with LRRK2 phosphorylation mutants are shown and measurement of neurites length is shown for PC12 navies (A), PC12 VAMP2 KO (B), PC12 VAMP4 KO (C), PC12 VAMP7KO (D). The data was analysed by One-way-ANOVA with Dunnet's multiple comparison. (** P <0.01).

Figure 48 :

 48 Figure 48: Neurites branch points and VAMPKO. PC12 transduced wit mCherry-LRRK2 are coated on P24 plate and differentiations are followed by Incucyte and analysis of neurites complexity was performed when cells were fully differentiated, 10 days of differentiation are needed to obtain differentiated cells. (Incucyte, sartorius). Phase-contrast imaging of differentiated PC12 transduced with LRRK2 phosphorylation mutants are shown and measurement of neurites branch points is shown for PC12 navies (A), PC12 VAMP2 KO (B), PC12 VAMP4 KO (C), PC12 VAMP7KO (D). The data was analysed by One-way-ANOVA with Dunnet's multiple comparison. (** P <0.01).

Table 2 :

 2 Classification of hereditary parkinsonism.

Table 3 :

 3 Overview of PD genes connected to autophagy-lysosomal pathways.

Table 4 :

 4 LRRK2 mutations found in patients.

Table 5 :

 5 Identified cellular partners of LRRK2.

Table 6 :

 6 List of antibodies used in the present study.

	List of abbreviations
	A2AR	Adenosine receptor 2A
	AD	Alzheimer Disease
	ALP	Autophagy-Lysosomal Pathway
	AR	Autosomal Recessive
	ATP	Adenosine Triphosphate
	BBB	Blood Brain Barrier
	BG	Basal Ganglia
	CalA	Calyculin A
	CBGTC	Cortico-Basal Ganglia-Thalamo-Cortical
	CK1α	Casein Kinase 1-alpha
	CMA	Chaperone-Mediated Autophagy
	CNS	Central Nervous System
	DBS	Deep Brain Stimulation
	ENS	Enteric Nervous System
	EV	Extracellular Vesicle
	GBA	Glucocerebrosidase
	GDNF	Glial-Derived Neurotrophic Factor
	GPe	Globus Pallidus externa
	GPi	Globus Pallidus interna
	GWAS	Genome Wide Association Study
	hiPSC	Human Induced Pluripotent Stem Cell
	IL	Interleukin
	LB	Lewy Bodies
	LRS	Leucyl-tRNA Synthetase 1

Table 2 : Classification of principal hereditary parkinsonism. PD

 2

	genes are associated to their

Table 3 : Overview of PD genes connected to autophagy-lysosomal pathways

 3

	Locus	Inheritance Protein		Involvement in ALP
	SNCA	AD	Alpha-		Inhibition of CMA and TFEB sequestration
			synuclein
	LRRK2	AD	LRRK2		Inhibition of CMA and macroautophagy
	VPS35	AD	VPS35		Retromer formation
	DNAJC13	AD	DNAJC13		Endosomal pathway
	PARK2	AR	parkin		Mitophagy
	PARK6	AR	PINK1		Mitophagy
	PARK7	AR	Dj-1		Mitophagy/oxidative stress
	PARK9	AR	ATP13A2		Lysosomal cation homeostasis and lipid flippase
	SYNJ1	AR	SYNJ1		Endosomal pathway
	ATP6AP2	X-linked	ATP6AP2		Lysosomal acidification
	GBA	/	GBA		Lysosomal enzyme (ceramide metabolism)
	SMPD1	/	SMPD1		Lysosomal enzyme (ceramide metabolism)
	PARK16	/	RAB29		Golgi apparatus trafficking
	SCARB2	/	LIMP2		Mannose-6-phosphate independent trafficking receptor for GBA
	MCCC1/LAMP3	/	MCCC1	and	LAMP3: lysosomal membrane protein MCCC1: mitochondrial protein
			LAMP3

. CMA: chaperonemediated autophagy. ALP: autophagy-lysosomal pathways. TFEB: Transcription Factor EB. Adapted from

[START_REF] Bourdenx | What lysosomes actually tell us about Parkinson's disease?[END_REF]

.

Table 4 : LRRK2 mutations found in patients.

 4 LRRK2 mutations reported in PD patients are classified according to their domain location. Changes in catalytic activities are indicated and the hallmarks of the mutations are described if observed in patients' cells.

	Mutation	Catalytic activities	Hallmarks	References
	ARM-ANK-LRR domain
	A397T	Unknown		(Kishore et al., 2019)
	G472R	Unknown		(Kishore et al., 2019)
	L550W	Unknown		(Kishore et al., 2019)
	N551K	Unknown	Reduced pRAB10	(Ross et al., 2011; Wang et al.,
				2021)
	R793M	Unknown		(Covy et al., 2009)
	L1165P	Unknown		(Covy et al., 2009)
	ROC domain
	R1398H	Decreased kinase	Reduced pRAB10	(Chen et al., 2011; Nixon-Abell
		activity	Decrease in active GTP-bound	et al., 2016; Wang et al., 2021)
			LRRK2
	N1437H	Increased kinase		(Puschmann et al., 2012)
		activity
	R1441C	Increased kinase	Reduction in lysosomal GBA	(Haugarvoll et al.,
		activity	activity.	2008)(Ysselstein et al., 2019)
			Increased pRAB10.
	R1441G	Increased kinase	Reduction in lysosomal GBA	(Simón-Sánchez et al.,
		activity	activity.	2006)(Ysselstein et al., 2019)
	R1441H	Increased kinase		(Ferreira et al., 2007)
		activity
	R1441S			(Mata et al., 2016)
	COR domain
	R1628P	Unknown		(Oosterveld et al., 2015)
	S1647T	Unknown		(Oosterveld et al., 2015)
	M1646T	Unknown		(Sosero et al., 2021)
	Y1699C	Increased kinase		(Khan et al., 2005)
		activity
	S1761R			(Lorenzo-Betancor et al., 2012)
	Kinase domain
	D1887G	Unknown		(Kishore et al., 2019)
	G2019S	Increased kinase	mtDNA damage in iPSC	(Kachergus et al., 2005;
		activity	derived NPC and neural cells.	Sanders et al., 2014; Hockey et
			Reduction in lysosomal GBA	al., 2015; Schwab et al., 2017;
			activity.	Ysselstein et al., 2019)
			Impaired mitochondrial
			respiration.
			Enlarged lysosomes.
	I2020T	Increased kinase		(Funayama et al., 2005)
		activity
	WD40 domain
	G2294R	Unknown	Decreased RAB8a and RAB10	(Ogata et al., 2021)
			levels in macrophages
	G2385R	Decreased kinase	Alteration of synaptic vesicle	(Di Fonzo et al., 2006; Carrion
		activity	trafficking.	et al., 2017; Rudenko et al.,
				2017)

Table 6 : List of antibodies used in the present study.

 6

	Name	Target	Type	Clone	Concentration	Concentration
					for WB	for ICC
	N241A/34	Total	Monoclonal N241A/34	1µg/mL	1µg/mL
		LRRK2
	MJFR11	pS955-	Monoclonal MJF-R11 (75-1)	1/1000
		LRRK2
	MJFR12	pS973-	Monoclonal MJF-R12 (37-1)	1/1000
		LRRK2
	MJFR19	pS1292-	Monoclonal MJFR-19-7-8	1/1000
		LRRK2
	UDD1	pS910-	Monoclonal UDD1 15(3)	1/1000
		LRRK2
	UDD2	pS935-	Monoclonal UDD2 10(12)	1/1000
		LRRK2
	FLAG M2	FLAG tag	Monoclonal M2	1/1000	1/1000
	Myc	Myc tag	Monoclonal 4A6	1/2000
	GFP	GFP tag	Monoclonal		1/1000
	RAB8a	Total	Monoclonal D22D8	1/1000	1/200
		RAB8A
	RAB8a p	RAB8a-	Monoclonal MJF-R20	1/1000
	T72	pT72
	RAB10	Total	Monoclonal D36C4	1/1000
		RAB10
	pRAB10 p	pT73-	Monoclonal MJF-R21	1/1000
	T73	RAB10

  The question of the phenotypic consequences of LRRK2 phosphorylation or dephosphorylation has remained mostly unanswered. Here, we present results that show that changes at the phosphosites in the ANK-LRR interdomain region affect LRRK2 activity in cells,

	including cellular kinase activity, RAB29 induced activation of LRRK2 differently following it
	phosphorylation profile.
	Using a phosphomutant approach, we first made a survey of LRRK2 phosphomutant variants,
	including modifications of individual sites compared to modifications of multiple sites. While
	some of the phosphodead mutants [Serine (S) to Alanine (A)] have been reported before, few
	reports have tested phosphomimicking mutants [(Serine (S) to Aspartate (D)] of LRRK2 and
	our report is also the first to include side by side comparisons of phosphodead with
	phosphomimicking mutants. For those constructs that have been previously reported, such as
	the S910A or S935A mutants, our observations were similar to published observations, namely
	that these mutants affect the phosphorylation rate at other sites in the S910/S935 cluster,
	underscoring the robustness of our results. The phosphomutant approach was thus
	implemented to verify which known phenotypes of LRRK2 are modified by LRRK2
	phosphorylation.

References

Remerciements

Acknowledgements

Passages have been modified from my own contributions to published papers (Marchand et al., 2020;Sarchione et al., 2021) in sections "2.4 LRRK2 phosphorylation" -"2.5 LRRK2's interactors" -"2.8 Kinase inhibition" and in figures 24.

Part 4: Materials and methods

Kinase activity of LRRK2 phosphomutant

In whole cell lysate

To assess a potential deficit in kinase activity of these two particular mutants (6xA/6xD), we measured in total cell lysate the phosphorylation of two known substrates of LRRK2, RAB8a and RAB10. LRRK2 is able to phosphorylation of RAB8a at T72 and RAB10 at T73.

In HEK293T, we were able to detect a decreased phosphorylation for both RAB8a and RAB10 in cellulo for LRRK2 6xD but not for LRRK2 6xA (Figure 33,A,B).

Parts 6: LRRK2 interaction with VAMPs Discussion