Keywords: ALS-FTLD, C9ORF72, Dipeptide Repeats, Hexanucleotide Repeat Expansions, Zebrafish

Amyotrophic Lateral Sclerosis (ALS) is a devastative neurodegenerative disorder that affects nerve cells in the brain and the spinal cord and that is ultimately fatal as there is curative treatment.

The most common known cause of ALS is a hexanucleotide repeat expansion (HRE) in the first intron of the C9ORF72 gene. Consequently, unraveling C9ORF72-mediated pathogenicity in ALS is fundamental to explore new avenues for the treatment of ALS. Proposed mechanisms concerning C9ORF72 mutation are loss and gain of function, including aggregation of dipeptide repeats (DPRs) translated through a non-ATG-initiated translation of the HRE. One of the hypotheses concerning the function of C9ORF72 would be a role in autophagy, which is also linked with other ALS monogenic causes, such as VCP, OPTN, TBK1, UBQLN2 and SQSTM1 genes. Particularly, SQSTM1/p62 protein is an essential actor and regulator of the initiation of the autophagy pathway. SQSTM1/p62-positive inclusions have been detected in patients carrying the C9ORF72 HRE, leading to the hypothesis of a functional common purpose between these genes.

To investigate the pathogenic mechanisms induced by C9ORF72 and SQSTM1 mutations in ALS, we developed a zebrafish model to study sqstm1 haploinsufficiency, a model of c9orf72 knockdown being already available. We observed that loss of function of the sqstm1 zebrafish orthologue leads to a specific motor phenotype, associated with shorter motor neuron axons and reduced swimming capacity, similarly to what was previously demonstrated for c9orf72 knockdown in zebrafish. To elucidate the common cellular mechanisms underlying motor neuron degeneration in ALS, c9orf72 and sqstm1 zebrafish models were utilized to analyze their epistatic interactions. We found that c9orf72 and sqstm1 partial inhibitions have an additive effect and that human C9ORF72 can rescue the phenotype induced by sqstm1 knockdown. These results indicate that both proteins belong to the same pathway and that c9orf72 acts downstream of sqstm1. Also, coherently with a role of C9ORF72 in autophagy degradation pathway, we found that C9orf72 depletion in mouse motor neurons primary cultures leads to the early death of motor neurons associated with their inability to product autophagosomes.

Finally, to develop a vertebrate model that recapitulates the different mechanisms associated with the C9ORF72 HRE pathogenicity, we combined the inhibition of c9orf72 with the expression of plasmids containing the five DPR repeats (poly(PA), poly(PR), poly(GA), poly(GP) et poly(GR)). Combination of c9orf72 knockdown with DPR expression induces a robust motor phenotype characterized by locomotor defects and paralysis. Focusing on poly(GP) (glycine-proline) repeats, we observed that the loss of function of c9orf72 is essential to inhibit poly(GP) clearance, which is associated with SQSTM1/p62 accumulation, severe motor neurons abnormalities with shortened axons and motor neuron death. These phenotypes can be rescued by the inhibition of caspase 9, an essential regulator of apoptosis. Also, inducing autophagy in zebrafish embryos by rapamycin treatment leads to an improvement of the clearance of poly(GP) and of SQSTM1/p62, which is associated with restored swim and axonal length, thus confirming the role of C9ORF72 in autophagy.

These results indicate that DPR toxicity is related to lowered expression of C9ORF72, suggesting that both gain and loss of function synergize in the C9ORF72 HRE pathogenicity, and thus opening novel avenues for potential treatment of ALS.

Résumé

La sclérose latérale amyotrophique (SLA) est une maladies neurodégénérative dévastatrice qui touche les cellules nerveuses du cerveau et de la moelle épinière. Il n'existe pas de traitement curatif pour la SLA et son issue est fatale.

La cause connue la plus fréquente de SLA est une expansion d'une répétition hexanucléotidique (« hexanucleotide repeat expansion », HRE) dans le premier intron du gène C9ORF72. Par conséquent, identifier les mécanismes pathogéniques associés à la mutation du gène C9ORF72 dans la SLA est fondamental pour identifier de nouvelles pistes thérapeutiques. Les mécanismes proposés concernant cette mutation sont la perte et le gain de fonction, notamment via l'agrégation de peptides (« dipeptide repeats », DPR) qui sont issus de la traduction de l'HRE via un mécanisme non-ATG dépendant. Une des hypothèses concernant la fonction de C9ORF72 est un rôle dans l'autophagie, un mécanisme qui est également associé à d'autres causes monogéniques de SLA, comme les gènes VCP, OPTN, TBK1, UBQLN2 et SQSTM1. En particulier, la protéine SQSTM1/p62 est un acteur et régulateur essentiel de l'initiation de l'autophagie. Des inclusions SQSTM1/p62-positives ont été observées dans les tissus des patients porteurs de l'HRE dans le gène C9ORF72, suggérant que C9ORF72 et SQSTM1 auraient une finalité commune.

Afin d'étudier les mécanismes pathologiques induits par les mutations de C9ORF72 et SQSTM1 dans la SLA, nous avons développé un modèle de poisson zèbre pour étudier l'haploinsuffisance de sqstm1 (un modèle d'inhibition de c9orf72 chez le poisson zèbre étant déjà disponible). Nous avons observé que la perte de fonction du gènes orthologue sqstm1 induit un phénotype moteur caractéristique, associé à un raccourcissement des projections axonales des neurones moteurs, similairement à ce qui a été préalablement démontré pour le knockdown de c9orf72 chez le poisson zèbre. Afin d'élucider les mécanismes cellulaires communs responsables de la dégénérescence des neurones moteurs dans la SLA, nous avons analysé les interactions épistatiques des gènes c9orf72 et sqstm1. Nous avons observé que la surexpression de la version humaine de C9ORF72 permet la restauration du phénotype obtenu par l'inhibition de sqstm1. Ces résultats montrent que c9orf72 et sqstm1 appartiennent à une voie cellulaire commune, et que c9orf72 agit en aval de sqstm1. De plus, confirmant le rôle de C9ORF72 dans l'autophagie, nous avons observé que l'inhibition de C9orf72 dans des cultures primaires de motoneurones de souris entraine une mort prématurée de ces cellules, associée à un défaut de formation des autophagosomes.

Enfin, pour développer un modèle vertébré qui récapitule les différents mécanismes associés à la pathogénicité de la mutation du gène C9ORF72, nous avons combiné l'inhibition partielle de c9orf72 avec l'expression de plasmides contenant les DPR (poly(PA), poly(PR), poly(GA), poly(GP) et poly(GR)). La combinaison du knockdown de c9orf72 et de l'expression des DPR induit un phénotype moteur robuste, caractérisé par des défauts locomoteurs et une paralysie. En se focalisant sur les répétitions de GP (glycine-proline), nous avons observé que la perte de fonction de c9orf72 entraine l'inhibition de la clairance de ces peptides, qui est associée à une accumulation de SQSTM1/p62 ainsi qu'à des anormalités sévères des motoneurones qui présentent des axones raccourcis et meurent. Ces dernières caractéristiques ont pu être restaurées par l'inhibition de la caspase 9, un régulateur essentiel de l'apoptose. De plus, nous avons observé que la rapamycine, un activateur de l'autophagie, améliore la clairance des répétitions de GP et de SQSTM1/p62, ainsi que le phénotype moteur, confirmant ainsi le rôle de C9ORF72 dans cette voie cellulaire.

Ces résultats montrent que la toxicité des DPR est due à la diminution de l'expression de C9ORF72, suggérant que la perte et le gain de fonction de C9ORF72 agissent en synergie dans la pathogénicité de la mutation de C9ORF72, ouvrant ainsi de nouvelles perspectives de traitement de la SLA.

CHAPTER 1 -INTRODUCTION 1.

Amyotrophic Lateral Sclerosis

Clinical features

of patients with ALS, having been shown in clinical trials to increase median survival of three months [START_REF] Jaiswal | Riluzole and edaravone: A tale of two amyotrophic lateral sclerosis drugs[END_REF]. Another drug, edaravone have recently come to market (Hardiman & van den Berg, 2017) ALS is characterized by the progressive degeneration of both upper motor neurons (UMN, projecting from the cortex to the brainstem and the spinal cord) and lower motor neurons (LMN, projecting from the brainstem or the spinal cord to muscles), with denervation and muscle wasting (see Fig. 1). Considerable heterogeneity exists in ALS clinical presentation. The initial presentation of ALS varies between patients, with different ages of onset, progression rates, and survival. Also, some display a spinal-onset disease with muscle weakness of the limbs, while other patients present bulbar-onset disease characterized by dysarthria and dysphagia (difficulties with speech and swallowing) (van Es et al, 2017;[START_REF] Hardiman | Edaravone: a new treatment for ALS on the horizon?[END_REF].

Primary symptoms of ALS are motor dysfunctions, but up to 50% of patients develop also cognitive and/or behavioural impairment, including frontotemporal lobar degeneration (FTLD) that appears in 13% of ALS cases [START_REF] Phukan | The syndrome of cognitive impairment in amyotrophic lateral sclerosis: A population-based study[END_REF][START_REF] El-Brolosy | Cognitive changes predict functional decline in ALS: A population-based longitudinal study[END_REF].

Jean-Martin Charcot 1825-1893

National Library of Medicine Amyotrophic Lateral Sclerosis (ALS), colloquially known as Charcot's disease, who first described the disease (or Lou Gherig's disease in United States and motor neuron disease in United Kingdom), is a rare, devastating and fatal neurodegenerative disease of the motor system.

The only widely available drug is riluzole that prolongs survival

Diagnosis

According to the revised El Escorial and Airlie House criteria, the diagnosis of ALS requires the presence of signs of UMN and LMN degeneration by clinical, electrophysiological or neuropathologic examination, as well as progressive spread of signs within a region or to other regions.

These features need to occur together with the absence of electrophysiological, nor neuroimaging evidences, of other disease processes (such as stroke, multiple sclerosis, tumor or myelopathy) that might explain the signs of LMN and/or UMN degenerations and the observed clinical signs [START_REF] Brooks | El Escorial revisited: Revised criteria for the diagnosis of amyotrophic lateral sclerosis[END_REF][START_REF] Traynor | Clinical features of amyotrophic lateral sclerosis according to the El Escorial and Airlie House diagnostic criteria: A population-based study[END_REF]. Clinical examinations should be repeated at least every six months to assess progression. Clinical features that support the diagnosis of ALS include: abnormal pulmonary and/or larynx functions, speech and/or swallowing studies that are not explained by other causes, abnormal strength test in clinically uninvolved muscles, abnormal muscle biopsy with evidence of denervation [START_REF] Brooks | El Escorial revisited: Revised criteria for the diagnosis of amyotrophic lateral sclerosis[END_REF][START_REF] Traynor | Clinical features of amyotrophic lateral sclerosis according to the El Escorial and Airlie House diagnostic criteria: A population-based study[END_REF].

Conclusive diagnosis of ALS usually takes place approximately one year after the beginning of symptoms [START_REF] Zoccolella | Predictors of delay in the diagnosis and clinical trial entry of amyotrophic lateral sclerosis patients: A population-based study[END_REF]. To date, the diagnosis relies mostly on electromyography and on the exclusion of differential diseases: there is no definitive diagnostic test for ALS.

Therefore, the identification of a molecular biomarker would be useful for the earlier diagnosis of the disease. Although not integrated into standard clinical practice, cerebrospinal fluid (CSF)

and serum Nefl (neurofilament light chain) levels are relevant in supporting the diagnosis [START_REF] Xu | Expanded GGGGCC repeat RNA associated with amyotrophic lateral sclerosis and frontotemporal dementia causes neurodegeneration[END_REF][START_REF] Verde | Neurofilament light chain in serum for the diagnosis of amyotrophic lateral sclerosis[END_REF]. Also, with new sequencing technologies, clinical genetic testing becomes more feasible and could offer the possibility of a definitive diagnosis for some mutations. However, in absence of medical treatment, predictive genetic test is a sensitive issue, similar to most other neurodegenerative disorders.

Epidemiology

Epidemiological studies are complex in ALS due to several factors, including determination of the precise onset, low incidence and high variability in causative mechanisms and hence in symptoms of the disease. These factors make it essential to design rigorous inclusion criteria to get unbiased patient cohorts [START_REF] Rooney | Benefits, pitfalls, and future design of population-based registers in neurodegenerative disease[END_REF].

ALS is the most frequently occurring of the neuromuscular degenerative disorder. Most population-based studies have come from high-quality European patient registers combined to form the European ALS Epidemiology Consortium (EURALS) [START_REF] Liu | Ubiquitylation of Autophagy Receptor Optineurin by HACE1 Activates Selective Autophagy for Tumor Suppression[END_REF].

Studies have established that the incidence of ALS in Europe is quite uniform at 2.16 per 100 000 person-years. [START_REF] Liu | Ubiquitylation of Autophagy Receptor Optineurin by HACE1 Activates Selective Autophagy for Tumor Suppression[END_REF]. Men have higher incidence of disease (3.0 per 100 000 person-years; 95% than do women (2.4 per 100 000 person-years; 95% CI 2. 2-2.6), although the incidence between men and women is about the same in familial disease.

The overall population-based lifetime risk of ALS is 1:400 for women and 1:350 for men. Peak age at onset is 58-63 years for sporadic disease and 47-52 years for familial diseases. [START_REF] Liu | Ubiquitylation of Autophagy Receptor Optineurin by HACE1 Activates Selective Autophagy for Tumor Suppression[END_REF]Al-Chalabi & Hardiman, 2013). Despite the clinical heterogeneity, median survival time of ALS patients is 3-5 years [START_REF] Traxinger | Prognosis and epidemiology of amyotrophic lateral sclerosis[END_REF]. In some regions (such as Guam and the Kii peninsula of Japan) the reported incidence was very high but has reduced substantially over the past 30 years for reasons that remain unclear [START_REF] Wiederholt | Neuroepidemiologic research initiatives on Guam: Past and present[END_REF].

Histopathology

The neuropathological hallmark of ALS disease is the presence of inclusions composed of abnormal protein aggregates in the cytoplasm of remaining and degenerating motor neurons and other cell types in the affected CNS regions: in the spinal cord, the hippocampus, the cerebellum and frontal and temporal cortices (Kabashi & Durham, 2006;[START_REF] Blokhuis | Protein aggregation in amyotrophic lateral sclerosis[END_REF][START_REF] Götzl | Impaired protein degradation in FTLD and related disorders[END_REF]Ruegsegger & Saxena, 2016;[START_REF] Medinas | Proteostasis disturbance in amyotrophic lateral sclerosis[END_REF]. In most types of ALS, TAR DNA-binding protein is part of these inclusions, although the gene encoding for TDP-43 (TARDBP) is mutated in only 4% of ALS [START_REF] Kabashi | TARDBP mutations in individuals with sporadic and familial amyotrophic lateral sclerosis[END_REF][START_REF] Sreedharan | TDP-43 mutations in familial and sporadic amyotrophic lateral sclerosis[END_REF][START_REF] Van Deerlin | TARDBP mutations in amyotrophic lateral sclerosis with TDP-43 neuropathology: a genetic and histopathological analysis[END_REF]. Indeed, approximately 97% of ALS patients have TDP-43 proteinopathy, with depletion of TDP-43 from the nucleus and formation of phosphorylated and ubiquitinated cytoplasmic proteinaceous aggregates, in neurons and glial cells, in the primary motor cortex, motor nuclei of the brainstem, spinal cord, and the associated whitematter tracts [START_REF] Arai | TDP-43 is a component of ubiquitin-positive tau-negative inclusions in frontotemporal lobar degeneration and amyotrophic lateral sclerosis[END_REF][START_REF] Neumann | Ubiquitinated TDP-43 in Frontotemporal Lobar Degeneration and Amyotrophic Lateral Sclerosis[END_REF][START_REF] Hasegawa | Phosphorylated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis[END_REF]) (Fig. 2).

.

In some ALS cases, other types of aggregates are observed, such as misfolded superoxide dismutase 1 (SOD1) protein and hyaline proteins in patients carrying a mutation in SOD1 gene [START_REF] Kato | Recent advances in research on neuropathological aspects of familial amyotrophic lateral sclerosis with superoxide dismutase 1 gene mutations: Neuronal Lewy body-like hyaline inclusions and astrocytic hyaline inclusions[END_REF] Post-mortem examinations have also revealed that ALS patients exhibit a loss of motor neurons in the motor cortex and in the ventral horn of the spinal cord, as well as a neuroinflammatory reaction consisting of diffuse astrocytic activation visible by histological labelling, microglial proliferation and infiltration and muscle denervation [START_REF] Philips | Neuroinflammation in amyotrophic lateral sclerosis: Role of glial activation in motor neuron disease[END_REF]) (Fig. 2)

Genetics

Traditionally, ALS is divided into sporadic (sALS, 90-95%) and familial (fALS, 5-10%) forms.

About 10% of sALS patients have mutations in genes that are also known to be associated with fALS [START_REF] Hamasaki | The risk to relatives of patients with sporadic amyotrophic lateral sclerosis[END_REF].

Identification of ALS genes has been accomplished through identification of the disease loci on the chromosomes, by linkage analysis of large pedigrees and subsequent positional sequencing.

In the 2000's, the advent of next generation sequencing techniques have highly accelerated the discovery of ALS genetic basis. To date, approximately 40 genes have been identified as being mutated in ALS patients. An overview of all currently known monogenic causes is provided in Table 1. However, in about 60-80% of patients with familial ALS, a mutation in the major established causal ALS genes can be identified, of which C9ORF72 (DeJesus-Hernandez et al, 2011a; Renton et al, 2011a), SOD1 [START_REF] Rosen | Nuclear accumulation of mRNAs underlies G4C2-repeatinduced translational repression in a cellular model of C9orf72 ALS[END_REF], FUS (encoding fused in sarcoma) [START_REF] Kwiatkowski | Mutations in the FUS/TLS gene on chromosome 16 cause familial amyotrophic lateral sclerosis[END_REF][START_REF] Vance | Mutations in FUS, an RNA processing protein, cause familial amyotrophic lateral sclerosis type 6[END_REF], and TARDBP [START_REF] Kabashi | TARDBP mutations in individuals with sporadic and familial amyotrophic lateral sclerosis[END_REF][START_REF] Sreedharan | TDP-43 mutations in familial and sporadic amyotrophic lateral sclerosis[END_REF][START_REF] Van Deerlin | TARDBP mutations in amyotrophic lateral sclerosis with TDP-43 neuropathology: a genetic and histopathological analysis[END_REF] are the most common (Table 1). Amongst these, the GGGGCC hexanucleotide repeat expansion (HRE) mutation in the 5' non-coding region of C9ORF72 is by far the biggest contributor as it is mutated in 23-47 % of fALS and 4-21 % of sALS (DeJesus-Hernandez et al, 2011a;Renton et al, 2011a) (please refer to section 4. for more details about C9ORF72 mutation). Interestingly, many genes associated with ALS are pleiotropic (in which a single genetic mutation has multiple phenotypic manifestations) (Table 1). The most glaring example is the C9ORF72 HRE, which is clearly linked to ALS and FTLD, but also to parkinsonism [START_REF] Lesage | C9orf72 repeat expansions are a rare genetic cause of parkinsonism[END_REF][START_REF] Luigetti | Frontotemporal dementia, Parkinsonism and lower motor neuron involvement in a patient with C9ORF72 expansion[END_REF][START_REF] O'dowd | C9ORF72 expansion in amyotrophic lateral sclerosis/frontotemporal dementia also causes parkinsonism[END_REF]), Huntington's disease [START_REF] Hensman | C9orf72 expansions are the most common genetic cause of Huntington disease phenocopies[END_REF][START_REF] Kostić | C9orf72 expansion as a possible genetic cause of Huntington disease phenocopy syndrome[END_REF][START_REF] Koutsis | C9ORF72 hexanucleotide repeat expansions are a frequent cause of Huntington disease phenocopies in the Greek population[END_REF], Alzheimer's disease [START_REF] Kohli | Repeat expansions in the C9ORF72 gene contribute to Alzheimer's disease in Caucasians[END_REF][START_REF] Burguete | GGGGCC microsatellite RNA is neuritically localized, induces branching defects, and perturbs transport granule function[END_REF][START_REF] Harms | C9orf72 hexanucleotide repeat expansions in clinical alzheimer disease[END_REF], corticobasal degeneration [START_REF] Lindquist | Corticobasal and ataxia syndromes widen the spectrum of C9ORF72 hexanucleotide expansion disease[END_REF][START_REF] Amlie-Wolf | Transcriptomic changes due to cytoplasmic TDP-43 expression reveal dysregulation of histone transcripts and nuclear chromatin[END_REF], schizophrenia [START_REF] Vázquez-Costa | Clinical and neuroimaging characterization of two C9orf72-positive siblings with amyotrophic lateral sclerosis and schizophrenia[END_REF] and bipolar disorder [START_REF] Floris | Bipolar affective disorder preceding frontotemporal dementia in a patient with C9ORF72 mutation: Is there a genetic link between these two disorders?[END_REF][START_REF] Meijer | Regulation of autophagy by amino acids and MTORdependent signal transduction[END_REF][START_REF] Galimberti | C9ORF72 hexanucleotide repeat expansion as a rare cause of bipolar disorder[END_REF]. Another example is SQSTM1 gene, which is related to Paget's disease of the bone (PDB) (Rea et al, 2014a), inclusion body myositis (IBM) [START_REF] Gang | Rare variants in SQSTM1 and VCP genes and risk of sporadic inclusion body myositis[END_REF], ALS and FTLD (Fecto et al, 2011a) (Table 1).

The notion that ALS and FTD are related and represent two ends of a same spectrum is supported by histopathological and genetic evidence. Indeed, both diseases are characterized by TDP-43 pathology and several genes have been identified in both conditions (Neurochemistry, 2016;Ling et al, 2013a;[START_REF] Couratier | ALS and frontotemporal dementia belong to a common disease spectrum[END_REF]Lattante et al, 2015c) (Table1).

As mentioned above, genetic testing in ALS is controversial. Indeed, while some mutations appear to be directly pathogenic, this has not been demonstrated for many reported variants.

Beside variability in penetrance and pleiotropy of ALS genes, there are growing evidences implying that ALS is an oligogenic disease (in which a phenotypic trait is determined by more than one gene), which is consistent with the incomplete penetrance in many ALS pedigrees (Loh et al, 2015;Van Rheenen et al, 2016). For example, regarding C9ORF72 mutation, several SNPs have been shown to contribute to the length of the HRE [START_REF] Ng | Intermediate C9orf72 alleles in neurological disorders: Does size really matter[END_REF]. While great discoveries were achieved in genetic knowledge of ALS, these features nevertheless point out the need for new advances to decrypt the overall genetic complexity of ALS.

Cellular mechanisms

Unraveling the genetic etiology of ALS has provided fundamental insights into the pathophysiology of ALS and the related cellular mechanisms surrounding neurodegeneration.

In Fig. 3, are shown the monogenic causes of ALS and their related mechanisms. In patients and in animal models, several pathophysiological mechanisms have been identified, including proteostasis defects, RNA metabolism alteration, impaired axonal transport, mitochondria damage, oxidative stress and neuroinflammation.

Mitochondrial function

Oxydative stress

Cytoskeletal function

Autophagy

Myelination

Endoplasmic reticulum function

Axonal transport

Endocytosis

RNA metabolism

Proteasome

DNA metabolism

Stress granules

Axonal guidance

Inflammation

Nucleocytoplasmic transport

Proteostasis defects

Proteostasis defects

Maintaining intracellular protein homeostasis ("proteostasis") by balancing protein biogenesis, folding, trafficking and degradation, is paramount to protect the functionality of the cell [START_REF] Kurtishi | Cellular Proteostasis in Neurodegeneration[END_REF]. As previously mentioned, a pathological hallmark of ALS is the propensity of certain proteins to accumulate into neurotoxic aggregates. Remarkably, several genes affected by mutations related to ALS encode for proteins directly involved in protein degradation, which is maintained by a large number of mechanisms, including autophagy, the ubiquitin proteasome system (UPS), ER stress, and stress granules (SG), whose malfunctionings have been associated with neurodegeneration [START_REF] Medinas | Proteostasis disturbance in amyotrophic lateral sclerosis[END_REF] (Fig. 3).

Two main protein clearance pathways, the UPS and autophagy, can be central components of the disease mechanisms in ALS. Autophagy is a self-degradative process whose function is the removal of misfolded or aggregated proteins, and also damaged organelles [START_REF] Mizushima | Autophagy: Process and function[END_REF][START_REF] Lee | Neuronal Autophagy: A Housekeeper or a Fighter in Neuronal Cell Survival?[END_REF]. Autophagy defects contribution to ALS is a main subject of this thesis and a discussed thoroughly in section 2.2. In particular, mutations in SQSTM1 [START_REF] Komatsu | P62/SQSTM1/A170: Physiology and pathology[END_REF],

Valosin-containing protein (VCP) [START_REF] Jones | Valosin-containing protein (VCP) is required for autophagy and is disrupted in VCP disease[END_REF], C9ORF72 [START_REF] Almeida | Lost & found: C9ORF72 and the autophagy pathway in ALS/FTD[END_REF], Ubiquilin-2 (UBQLN2) [START_REF] Okada | The neuron-specific kinesin superfamily protein KIF1A is a uniqye monomeric motor for anterograde axonal transport of synaptic vesicle precursors[END_REF], Optineurin (OPTN) [START_REF] Ying | Optineurin: The autophagy connection[END_REF], Tankbinding kinase (TBK1) [START_REF] Maruyama | TBK1 controls autophagosomal engulfment of polyubiquitinated mitochondria through p62/SQSTM1 phosphorylation[END_REF], and TARDBP [START_REF] Bose | Regulation of autophagy by neuropathological protein TDP-43[END_REF] genes have been linked to both ALS and autophagy mechanism [START_REF] Ramesh | Autophagy Dysregulation in ALS: When Protein Aggregates Get Out of Hand[END_REF]Majcher et al, 2015a;[START_REF] Ghavami | Autophagy and apoptosis dysfunction in neurodegenerative disorders[END_REF].

The UPS is a mechanism of protein catabolism that involves the conjugation of the substrate protein by the covalent attachment of multiple ubiquitin molecules, and the subsequent degradation of the tagged protein by the proteasome [START_REF] Tanaka | The proteasome: From basic mechanisms to emerging roles[END_REF]. Several genes associated with familial ALS encode proteins that can promote dysfunction of the UPS (Ruegsegger & Saxena, 2016). As an example, mutant SOD1 is associated with reduced expression of components of the UPS [START_REF] Barthelme | An ALS disease mutation in Cdc48/p97 impairs 20S proteasome binding and proteolytic communication[END_REF]. The activity of the UPS has been demonstrated to be decreased in neuronal cultures [START_REF] Urushitani | Proteasomal inhibition by misfolded mutant superoxide dismutase 1 induces selective motor neuron death in familial amyotrophic lateral sclerosis[END_REF] or in mice expressing mutant SOD1 [START_REF] Jung | Multiplex image-based autophagy RNAi screening identifies SMCR8 as ULK1 kinase activity and gene expression regulator[END_REF]. Also, UBQLN2 and VCP have a role in substrate delivery to the proteasome, which is disrupted in the presence of ALS-associated mutations [START_REF] Ko | Ubiquilin interacts with ubiquitylated proteins and proteasome through its ubiquitin-associated and ubiquitin-like domains[END_REF]Deng et al, 2011a;[START_REF] Le | Motor neuron disease, TDP-43 pathology, and memory deficits in mice expressing ALS-FTD-linked UBQLN2 mutations[END_REF][START_REF] Nalbandian | The Homozygote VCPR155H/R155HMouse Model Exhibits Accelerated Human VCP-Associated Disease Pathology[END_REF][START_REF] Barthelme | An ALS disease mutation in Cdc48/p97 impairs 20S proteasome binding and proteolytic communication[END_REF].

ER stress is the cellular condition involving the accumulation of misfolded/unfolded proteins at the endoplasmic reticulum (ER). ER stress activates mechanisms to adapt to stress or trigger apoptosis of irreversibly-damages cells [START_REF] Lee | ER Stress and Autophagy[END_REF]. ER stress has been implicated broadly in ALS [START_REF] Matus | ER dysfunction and protein folding stress in ALS[END_REF]. Initial evidence arose from studies of mutant SOD1 that binds to the cytoplasmic surface of the ER membrane, indirectly inhibiting the ER-associated degradation [START_REF] Nishitoh | ALS-linked mutant SOD1 induces ER stress-and ASK1-dependent motor neuron death by targeting Derlin-1[END_REF]. Moreover, reducing ER stress delays the progression of the disease in an ALS animal model [START_REF] Saxena | A role for motoneuron subtype-selective ER stress in disease manifestations of FALS mice[END_REF].

SG are a form of highy dynamic ribonucleoprotein granules that rapidly assemble in response to a range of stressors [START_REF] Lin | Formation and Maturation of Phase-Separated Liquid Droplets by RNA-Binding Proteins[END_REF]. In ALS, the aberrant accumulation of SG represents a mechanistic combination of proteostatic inefficiency and aberrant RNA metabolism (Taylor et al, 2016;[START_REF] Webster | The C9orf72 protein interacts with Rab1a and the ULK1 complex to regulate initiation of autophagy[END_REF]. SG dynamics relies on phase separation biophysical properties of low-complexity domains of cytosolic RNA-binding proteins RBPs [START_REF] Lin | Formation and Maturation of Phase-Separated Liquid Droplets by RNA-Binding Proteins[END_REF]. TDP-43, FUS and the heterogeneous nuclear ribonucleoproteins HNRNPA1 and HNRNPA2B1 are RBPs, that can be incorporated into SG and whose ALS-causative mutations have been identified in low-complexity domains [START_REF] Lagier-Tourenne | TDP-43 and FUS/TLS: Emerging roles in RNA processing and neurodegeneration[END_REF]Kim et al, 2013a;[START_REF] Murakami | ALS/FTD Mutation-Induced Phase Transition of FUS Liquid Droplets and Reversible Hydrogels into Irreversible Hydrogels Impairs RNP Granule Function[END_REF][START_REF] Shang | Mechanisms of FUS mutations in familial amyotrophic lateral sclerosis[END_REF]. Moreover, ALS-related VCP mutations have been shown to disrupt SG dynamics and clearance [START_REF] Buchan | XEukaryotic stress granules are cleared by autophagy and Cdc48/VCP function[END_REF][START_REF] Seguin | Inhibition of autophagy, lysosome and VCP function impairs stress granule assembly[END_REF] (Fig. 3).

RNA metabolism alteration

The discovery of mutations in TARDBP and FUS has drawn much attention to altered RNA metabolism as they both encode for RBPs and function in various aspects of RNA processing, including transcription, splicing, RNA transport, miRNA processing, and translation [START_REF] Polson | Mammalian Atg18 (WIPI2) localizes to omegasome-anchored phagophores and positively regulates LC3 lipidation[END_REF][START_REF] Lattante | TARDBP and FUS Mutations Associated with Amyotrophic Lateral Sclerosis: Summary and Update[END_REF]. Mutated TDP-43 and FUS mislocalization from the nuclear to the cytoplasmic compartment is known to result in the loss of the normal processing of their numerous target RNAs (Amlie- Wolf et al, 2015;[START_REF] Zhou | FUS-regulated RNA metabolism and DNA damage repair[END_REF], but also in an acquired cytotoxic effect of the mutant proteins, which form SG in the cytoplasm, as previously outlined [START_REF] Moisse | Divergent patterns of cytosolic TDP-43 and neuronal progranulin expression following axotomy: Implications for TDP-43 in the physiological response to neuronal injury[END_REF][START_REF] Sato | Axonal ligation induces transient redistribution of TDP-43 in brainstem motor neurons[END_REF].

Besides TDP-43 and FUS proteins, numerous other DNA/RNA interacting proteins have been associated with ALS, including HNRNPA1 and HRNPA2B1 (Kim et al, 2013a), ANG (Angiogenin) [START_REF] Greenway | ANG mutations segregate with familial and 'sporadic' amyotrophic lateral sclerosis[END_REF], STEX (Senataxin) [START_REF] Chance | Linkage of the Gene for an Autosomal Dominant Form of Juvenile Amyotrophic Lateral Sclerosis to Chromosome 9q34[END_REF], ELP3 (Elongator complex protein 3) [START_REF] Simonsen | Promoting basal levels of autophagy in the nervous system enhances longevity and oxidant resistance in adult Drosophila[END_REF], TAF15 (TATA-binding protein-associated factor 15) [START_REF] Ticozzi | Mutational analysis reveals the FUS homolog TAF15 as a candidate gene for familial amyotrophic lateral sclerosis[END_REF] and MATR3 (Matrin 3) [START_REF] Johnson | Mutations in the Matrin 3 gene cause familial amyotrophic lateral sclerosis[END_REF] and are also involved in the RNA metabolism (Fig. 3).

In addition, both sense and antisense RNA strands from the ALS-related GGGGCC repeat

Impaired axonal transport

Axonal transport insures the efficient and correct spatiotemporal distribution of cell body synthesized cargoes, such as proteins, mRNAs, vesicles or organelles, to their specific destination. By doing so, it mediates the long-distance communication between cell body and synaptic terminals that allows neurons to react to their surroundings.

Evidence for axonal transport defects in ALS came from electronic microscopy and neuropathological studies of post-mortem cases that revealed abnormal accumulation of phosphorylated neurofilaments, mitochondria and lysosomes in motor neurons [START_REF] Okada | The neuron-specific kinesin superfamily protein KIF1A is a uniqye monomeric motor for anterograde axonal transport of synaptic vesicle precursors[END_REF]Hirano et al, 1984aHirano et al, , 1984b;;[START_REF] Rooke | Analysis of the KSP repeat of the neurofilament heavy subunit in familial amyotrophic lateral sclerosis[END_REF].

Mutations in ALS2 (encoding Alsin) as well as mutations in CHM2PB (encoding Charged multivesicular body protein 2B) and DCTN1 (encoding Dynactin subunit 1) have been shown

to disturb motor neurons trafficking [START_REF] Puls | Mutant dynactin in motor neuron disease[END_REF][START_REF] Hadano | Mice deficient in the Rab5 guanine nucleotide exchange factor ALS2/alsin exhibit age-dependent neurological deficits and altered endosome trafficking[END_REF][START_REF] Devon | Als2-deficient mice exhibit disturbances in endosome trafficking associated with motor behavioral abnormalities[END_REF][START_REF] Urwin | Disruption of endocytic trafficking in frontotemporal dementia with CHMP2B mutations[END_REF]) (Fig. 3). Furthermore, several SOD1 transgenic mice have shown motor neuron degeneration due to defects in anterograde and/or retrograde transport [START_REF] Bilsland | Deficits in axonal transport precede ALS symptoms in vivo[END_REF][START_REF] Ligon | Mutant superoxide dismutase disrupts cytoplasmic dynein in motor neurons[END_REF], as well as disturbed mitochondrial transport [START_REF] Magrané | Abnormal mitochondrial transport and morphology are common pathological denominators in SOD1 and TDP43 ALS mouse models[END_REF] (Fig. 3). Also, mutations in VAPB (encoding Vesicle-associated membrane protein-associated protein B), TARDBP and FUS ALS-genes cause reduced axonal transport of mitochondria in various ALS models [START_REF] Mórotz | Amyotrophic lateral sclerosisassociated mutant VAPBP56s perturbs calcium homeostasis to disrupt axonal transport of mitochondria[END_REF][START_REF] Wang | The ALS disease-associated mutant TDP-43 impairs mitochondrial dynamics and function in motor neurons[END_REF][START_REF] Xu | Expression of mutant TDP-43 induces neuronal dysfunction in transgenic mice[END_REF][START_REF] Baldwin | Axonal transport defects are a common phenotype in Drosophila models of ALS[END_REF] (Fig. 3).

Mitochondria damage

Motor neurons are large cells with high-energy requirement making it essential to maintain a healthy pool of correctly localized mitochondria for neuronal function and survival. ALS patients display mitochondrial dysfunctions, including defective oxidative phosphorylation, production of reactive oxygen species (ROS), impaired calcium buffering capacity, defective mitochondrial shifting and inappropriate activation of apoptosis [START_REF] Pasinelli | Caspase-1 and -3 are sequentially activated in motor neuron death in Cu , Zn superoxide dismutase-mediated familial amyotrophic lateral sclerosis[END_REF][START_REF] Wiedemann | Impairment of mitochondrial function in skeletal muscle of patients with amyotrophic lateral sclerosis[END_REF][START_REF] Li | Functional role of caspase-1 and caspase-3 in an ALS transgenic mouse model[END_REF][START_REF] Pasinelli | Caspase-1 and -3 are sequentially activated in motor neuron death in Cu , Zn superoxide dismutase-mediated familial amyotrophic lateral sclerosis[END_REF][START_REF] Grone | Epilepsy, behavioral abnormalities, and physiological comorbidities in syntaxin-binding protein 1 (STXBP1) mutant zebrafish[END_REF][START_REF] Wiedemann | Mitochondrial DNA and respiratory chain function in spinal cords of ALS patients[END_REF][START_REF] Parone | Enhancing Mitochondrial Calcium Buffering Capacity Reduces Aggregation of Misfolded SOD1 and Motor Neuron Cell Death without Extending Survival in Mouse Models of Inherited Amyotrophic Lateral Sclerosis[END_REF]. In patients and mice harboring mutations in SOD1, mitochondria display abnormal biochemical and morphological properties [START_REF] Velde | Misfolded SOD1 associated with motor neuron mitochondria alters mitochondrial shape and distribution prior to clinical onset[END_REF][START_REF] Magrané | Abnormal mitochondrial transport and morphology are common pathological denominators in SOD1 and TDP43 ALS mouse models[END_REF]) (Fig. 3). Interestingly, TDP-43 accumulates in the mitochondria of ALS patients, where it preferentially binds to mRNAs encoding respiratory chain subunits [START_REF] Wang | The inhibition of TDP-43 mitochondrial localization blocks its neuronal toxicity[END_REF]) (Fig. 3). Also, CHCHD10 mutations can compromise mitochondria structure in ALS patients [START_REF] Genin | CHCHD10 mutations promote loss of mitochondrial cristae junctions with impaired mitochondrial genome maintenance and inhibition of apoptosis[END_REF]) (Fig. 3).

Oxidative stress

Oxidative stress results from the imbalance between the production of ROS and the mitochondrial capacity to remove them or repair their damages. Prior to the identification of the repeat expansion in C9ORF72, the most common known mutation associated with ALS was in SOD1 that is involved in the elimination of ROS [START_REF] Hayashi | SOD1 in neurotoxicity and its controversial roles in SOD1 mutationnegative ALS[END_REF]) (Fig. 3). It is widely accepted that accumulation of ROS may lead to excessive oxidative stress and motor neuron death, due to destruction of cellular structures, lipids, proteins, and genetic materials such as DNA and RNA (McCord & Fridovich, 1969;[START_REF] Rosen | Nuclear accumulation of mRNAs underlies G4C2-repeatinduced translational repression in a cellular model of C9orf72 ALS[END_REF][START_REF] Rao | Effects of Cu/Zn Superoxide Dismutase on Estrogen Responsiveness and Oxidative Stress in Human Breast Cancer Cells[END_REF][START_REF] Tsang Ck Wa | Superoxide dismutase 1 acts as a nuclear transcription factor to regulate oxidative stress resistance[END_REF].

In addition, the analysis of CSF and serum from fALS and sALS patients showed increased concentrations of oxidative stress-induced damage products [START_REF] Lyras | Oxidative damage and motor neurone disease difficulties in the measurement of protein carbonyls in human brain tissue[END_REF][START_REF] Mitsumoto | Oxidative stress biomarkers in sporadic ALS[END_REF][START_REF] Simpson | Increased lipid peroxidation in sera of ALS patients: A potential biomarker of disease burden[END_REF][START_REF] Smith | Presence of 4-hydroxynonenal in cerebrospinal fluid of patients with sporadic amyotrophic lateral sclerosis[END_REF]. Oxidative damage has also been documented in cellular and rodents models of ALS [START_REF] Barber | Oxidative stress in ALS: Key role in motor neuron injury and therapeutic target[END_REF][START_REF] Parakh | Redox regulation in amyotrophic lateral sclerosis[END_REF].

Neuroinflammation

Early studies on ALS patient autopsies revealed that activated astrocytes, microglia, and infiltrating T cells are found at sites of motor neuron injury, in the earliest stages of the disease, suggesting that neuroinflammation might have a role to play in ALS [START_REF] Engelhardt | Igg Reactivity in the Spinal Cord and Motor Cortex in Amyotrophic Lateral Sclerosis[END_REF][START_REF] Engelhardt | Lymphocytic Infiltrates in the Spinal Cord in Amyotrophic Lateral Sclerosis[END_REF][START_REF] Henkel | Presence of Dendritic Cells, MCP-1, and Activated Microglia/Macrophages in Amyotrophic Lateral Sclerosis Spinal Cord Tissue[END_REF]. Indeed, non-cell-autonomous mechanisms have been demonstrated to be associated with neuronal loss in both ALS patients and animal models, even during the presymptomatic phase of the disease [START_REF] Endo | Neuroinflammation in motor neuron disease[END_REF].

In mutant SOD1G93A mice, activation of astrocytes and microglia markedly exacerbates the disease progression while motor neuron specific expression of mutant SOD1 has little influence on the progression of ALS [START_REF] Yamanaka | Astrocytes as determinants of disease progression in inherited amyotrophic lateral sclerosis[END_REF][START_REF] Haeusler | Astrocytes from familial and sporadic ALS patients are toxic to motor neurons[END_REF]) (Fig. 3).

C9ORF72-related ALS patients display microglial activation that correlates with the disease progression [START_REF] Brettschneider | Microglial activation correlates with disease progression and upper motor neuron clinical symptoms in amyotrophic lateral sclerosis[END_REF]. In C9orf72-deficient mice, the loss of the gene led to lysosomal accumulation and hyperactive immune responses in bone marrow macrophages (O'Rourke et al, 2016). These mice also developed progressive splenomegaly and lymphadenopathy, altered splenic myeloid cell populations and increased levels of circulating proinflammatory cytokines (Atanasio et al, 2016;Burberry et al, 2016;O'Rourke et al, 2016;Sudria-Lopez et al, 2016) (please refer to section 4.2.1.2. for more details about C9ORF72 ALS models).

Likewise, the recent identification of loss of function mutations in TBK1, a well-characterized regulator of innate immunity, as a cause of ALS [START_REF] Freischmidt | Haploinsufficiency of TBK1 causes familial ALS and fronto-temporal dementia[END_REF][START_REF] Cirulli | Loss of function of C9orf72 causes motor deficits in a zebrafish model of amyotrophic lateral sclerosis[END_REF] further reinforces the concept that immune dysregulation might be a characteristic feature of the disease. TBK1 is known to bind to and phosphorylate a number of proteins involved in innate immunity and autophagy, including SQSTM1/p62 and OPTN, which are both mutated in some ALS cases (Fig. 3).

It is likely that the underlying cause of motor neuron degeneration in ALS is a complex interplay between these multiple pathogenic, not mutually exclusive, cellular mechanisms (see section 2.2.3.2.) Furthermore, the causative relations between these processes and the disease aetiology remain unknown. The fact of the matter is that some of them could be secondary phenomena resulting from disturbances within upstream mechanisms. Among the 40 identified ALS-related genes, 21 have direct functions in proteostasis (Fig. 3), supporting the major role of this process in ALS pathogenesis, and the importance of proteostasis-related strategies for future therapeutic interventions. Above all, in proteostasis-associated ALS genes, more than half has been linked

with autophagy process (Fig. 3).

Autophagy deficiency in ALS

Autophagy in normal conditions

Overview

Autophagy was discovered in 1963 as an intracellular lysosome-mediated degradation process for nonessential or damaged cellular constituents, by electron microscopists who recognized the unique properties of a vesicle containing cytoplasmic organelles (de Duve, 1963).

Since then, work pioneered in yeast has revealed this widely conserved catabolic process as a crucial homeostasis pathway [START_REF] Takeshige | Autophagy in yeast demonstrated with proteinasedeficient mutants and conditions for its induction[END_REF][START_REF] Tsukada | Isolation and characterization of autophagy-defective mutants of Saccharomyces cerevisiae[END_REF]. Genetic studies have identified a class of autophagy-related, or ATG, proteins that are essential for the process and coordinate its specific steps [START_REF] Mizushima | The Role of Atg Proteins in Autophagosome Formation[END_REF].

The term "autophagy" refers to a range of processes, including chaperone-mediated autophagy (CMA), microautophagy and macroautophagy, the latter being by far the major and best characterized subtype of autophagy [START_REF] Klionsky | Guidelines for the use and interpretation of assays for monitoring autophagy. Autophagy Klionsky DJ[END_REF]. Macroautophagy occurs when a doublemembrane vesicle envelops cytosolic materials, such as proteins and organelles, forming a structure called the autophagosome that fuses with the lysosome, thereby transferring its luminal content for degradation (Fig. 4). Macroautophagy is the subject of this thesis and the one we refer to as "autophagy" [START_REF] Mizushima | Autophagy: Process and function[END_REF]. Microautophagy is a process where cytosolic proteins are directly engulfed by the lysosome [START_REF] Li | Microautophagy: Lesser-known self-eating[END_REF]. CMA utilizes cytosolic chaperones to deliver proteins to the surface of the lysosomes whereupon they unfold and cross the lysosomal membrane [START_REF] Kaushik | Chaperone-mediated autophagy: A unique way to enter the lysosome world[END_REF].

Autophagy is a tightly-regulated cellular process involved both in the basal turnover of cellular components, and in the response to chronic nutrient starvation or organelle damage. It involves the degradation of bulk cytoplasm, long-lived proteins, entire organelles, oligomers and protein aggregates, through their sequestration by autophagosome, their trafficking, to finally reach and fuse with the lysosome, where they are hydrolyzed [START_REF] Mizushima | Autophagy: Process and function[END_REF][START_REF] Ravikumar | Mammalian macroautophagy at a glance[END_REF]Rubinsztein et al, 2012).

Autophagy is essential for the development, the tumorigenesis and infection (Choi, Ryter & Levine, 2013). The first link between autophagy and human disease was made in 1999 with the discovery that the tumor suppressor BECN1 (Beclin 1, the mammalian orthologue of ATG6) is an autophagy gene [START_REF] Liang | Induction of autophagy and inhibition of tumorigenesis by beclin 1[END_REF]. Subsequently, in 2004, the generation of the first ATGknockout mouse, for ATG5, demonstrated that autophagy was essential for survival [START_REF] Kuma | Zebrafish as a model for epilepsy-induced cognitive dysfunction: A pharmacological, biochemical and behavioral approach[END_REF].

Autophagy has long been considered as a non-selective process responsible for the bulk sequestration of cytoplasmic components as a response to starvation, but it is now clear that selective autophagy ensures specific recognition and degradation of various cargoes such as aggregated protein and damaged organelles [START_REF] Noda | Mechanisms of Autophagy[END_REF].

Autophagy progress

The autophagic pathway proceeds through several phases, including initiation and nucleation, which result in the formation of an isolation membrane called "phagophore", vesicle elongation and autophagosome maturation and fusion with the lysosome. In the final stage, autophagosomal contents are degraded by lysosomal acid hydrolases and the contents of the autolysosome are released for metabolic recycling (Fig. 4).

Initiation and nucleation

The initiation of autophagy requires the membrane-associated ULK complex that contains the UNC51-like Ser/Thr kinases ULK1 (ATG1 orthologues), ATG13 and FIP200 (Focal Adhesion Kinase-family-interacting protein of 200 kDa, ATG 17 orthologue), and ATG101, forming the autophagy initiation complex [START_REF] Wirth | Autophagosome formation-The role of ULK1 and Beclin1-PI3KC3 complexes in setting the stage[END_REF]) (Fig. 5).

The activity of the ULK complex has been biochemically characterized following the inactivation of its negative regulator mTORC1 (mammalian target of rapamycin complex 1, see section 2.1.2.5.) [START_REF] Noda | Mechanisms of Autophagy[END_REF] and other regulatory signaling pathways, such as its positive regulator AMPK (AMP-activated protein kinase) [START_REF] Alers | Role of AMPK-mTOR-Ulk1/2 in the Regulation of Autophagy: Cross Talk, Shortcuts, and Feedbacks[END_REF]. Typically, under nutrient-rich conditions, mTORC1 inhibits autophagy through inactivation of the ULK complex [START_REF] Noda | Mechanisms of Autophagy[END_REF]. Upon amino acid starvation or rapamycin treatment, mTORC1 is inactivated and dissociates from the ULK complex, which results in increased ULK1 kinase activity and in the formation of the initiation complex.

The ULK complex recruits and acts in conjugation with the class III phosphatidylinositol 3kinase complex 3 (PI3KC3) that is composed of p150/VPS15, VPS34 (vacuolar proteins sorting 15 and 34), BECN1, ATG14 and AMBRA1 (activating molecule in Beclin-1 related autophagy 1 protein) (Fig. 5).

Once autophagy is initiated, ULK complex phosphorylates AMBRA1, thus inducing PI3KC3 activation. BECN1, upon release from the anti-apoptotic protein Bcl-2, activates VPS34 that is required for initiating the nucleation: the process of autophagosome formation, by generating isolation membrane (Di [START_REF] Bartolomeo | The dynamic interaction of AMBRA1 with the dynein motor complex regulates mammalian autophagy[END_REF][START_REF] He | The Beclin 1 interactome[END_REF]) (Fig. 5). The omegasome is the site in the ER where the phagophore comes from [START_REF] Roberts | Omegasomes: PI3P platforms that manufacture autophagosomes[END_REF]. It consists of a lipid bilayer membrane enriched for PI(3)P (phosphatidylinositol 3phosphate) and in proteins with FYVE (PI(3)P-binding) and PX (Phox homology) domains, such as DFCP1 (double FYVE domain-containing protein 1) (Fig. 5).

Electronic microscopy observations have indicated that while the endoplasmic reticulum (ER) is the most plausible candidate for the initial membrane source and the platform of autophagosome formation, contributions from the Golgi, endosomes, plasma membrane and mitochondria remain important [START_REF] Dunn | Studies on the mechanisms of autophagy: Formation of the autophagic vacuole[END_REF][START_REF] Ueno | Membrane markers of endoplasmic reticulum preserved in autophagic vacuolar membranes isolated from leupeptin-administered rat liver[END_REF][START_REF] Axe | Autophagosome formation from membrane compartments enriched in phosphatidylinositol 3-phosphate and dynamically connected to the endoplasmic reticulum[END_REF][START_REF] Vaccaro | Three families with amyotrophic lateral sclerosis and frontotemporal dementia with evidence of linkage to chromosome 9p[END_REF].

Elongation

After the nucleation, the phagophore expands to form the isolation membrane and engulf cytoplasmic contents requiring two ubiquitin-like conjugation systems [START_REF] Polson | Mammalian Atg18 (WIPI2) localizes to omegasome-anchored phagophores and positively regulates LC3 lipidation[END_REF].

The first one is formed by ATG12-ATG5-ATG16L1, and is bound to the phagophore [START_REF] Itakura | Characterization of autophagosome formation site by a hierarchical analysis of mammalian Atg proteins[END_REF][START_REF] Walczak | Next-generation mTOR inhibitors in clinical oncology: How pathway complexity informs therapeutic strategy[END_REF]. The formation of this complex is dependent on the activity of ATG7 and ATG10 [START_REF] Hamasaki | The risk to relatives of patients with sporadic amyotrophic lateral sclerosis[END_REF]) (Fig. 5).

The second ubiquitin-like system results from the ATG4B-dependent C-terminus cleavage of ATG8 (LC3, GABARAP or GATE16) and its conjugation with a lipid phosphatidylethanolamine (PE), which is mediated by ATG7 and ATG3 [START_REF] Tanida | HsAtg4B/HsApg4B/autophagin-1 cleaves the carboxyl termini of three human Atg8 homologues and delipidates microtubule-associated protein light chain 3-and GABAA receptor-associated proteinphospholipid conjugates[END_REF][START_REF] Katahira | Nuclear RNA export factor 7 is localized in processing bodies and neuronal RNA granules through interactions with shuttling hnRNPs[END_REF]) (Fig. 5).

LC3 (microtubule-associated protein 1A/1B light chain 3) is the most characterized ATG8 family member. Its lipidation with PE generates LC3-II [START_REF] Otomo | Structure of the human ATG12∼ATG5 conjugate required for LC3 lipidation in autophagy[END_REF], which is specifically bound and incorporated within the autophagic vesicle. Despite allowing it to associate with the autophagosomes membrane, LC3 lipidation also aids in cargo sequestration by associating with various autophagy receptors [START_REF] Mizushima | The Role of Atg Proteins in Autophagosome Formation[END_REF]) (Fig. 5,6).

Autophagy receptors

The most prevalent autophagy targeting signal in mammals is the modification of cargos with ubiquitin. Substrates recognized by the autophagy pathway are usually modified by lysine 63linked chains [START_REF] Shaid | Ubiquitination and selective autophagy[END_REF][START_REF] Stolz | Cargo recognition and trafficking in selective autophagy[END_REF]. The ubiquitylated substrates are recognized by autophagy receptors. More than 20 autophagy receptors have been identified but only a few have been characterized in detail so far, such as SQSTM1/p62, NBR1, NDP52, OPTN and UBQLN2. These proteins selectively recognize autophagic cargo via their ubiquitin-binding (UBA/UBAN) domain and mediate its engulfment into autophagosomes by binding to the molecules of the ATG8 family, LC3 and GABARAPs proteins (Fig. 6). The interaction of autophagic receptors with LC3-II is made possible by a short LC3-interacting region (LIR)

Autophagy measurement.

The ratio LC3-II/LC3 correlates with the amount of produced autophagosomes making its level widely used as a marker of autophagy activity [START_REF] Klionsky | Guidelines for the use and interpretation of assays for monitoring autophagy. Autophagy Klionsky DJ[END_REF]. However, autophagosomes are bilayer, then while luminal LC3-II is finally degraded by the lysosome, external proteins are not, making interpretation of the results of LC3-II/LC3 quantification problematic. Another issue with this method is that LC3-II tends to be much more sensitive to be detected by immunoblotting than LC3 (Mizushima & Yoshimori, 2007). An alternative method for detecting the autophagy flux is measuring SQSTM1/p62 degradation by western blot. Indeed, SQSTM1/p62 is a receptor and a substrate of autophagy, which directly binds to luminal LC3-II, and is engulfed in the autophagosome to be degraded by the lysosome with its cargo (Bjørkøy et al, 2005;[START_REF] Pankiv | 62/SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of ubiquitinated protein aggregates by autophagy[END_REF], its level thus correlating with autophagy efficiency (Mizushima & Yoshimori, 2007; sequence [START_REF] Rogov | Interactions between Autophagy Receptors and Ubiquitin-like Proteins Form the Molecular Basis for Selective Autophagy[END_REF][START_REF] Stolz | Cargo recognition and trafficking in selective autophagy[END_REF][START_REF] Lippai | The role of the selective adaptor p62 and ubiquitin-like proteins in autophagy[END_REF][START_REF] Hensman | C9orf72 expansions are the most common genetic cause of Huntington disease phenocopies[END_REF].

Interestingly, mutations in SQSTM1/p62, OPTN and UBQLN2 autophagy receptors have been associated with ALS (Fecto et al, 2011b;Maruyama et al, 2010;Deng et al, 2011a).

Maturation (lysosomal degradation)

Following cargo sequestration, autophagosomes are trafficked along microtubule tracks and fuse with lysosomes, forming a structure termed "the autolysosome", where hydrolytic lysosomal enzymes are activated and degrade both the luminal content of the autophagosomes and its inner membrane [START_REF] Mizushima | Autophagy: Process and function[END_REF]. This process of degradation allows the formation of new metabolites that recycle back to the cytoplasm for being reused [START_REF] Lamb | The autophagosome: Origins unknown, biogenesis complex[END_REF].

Autophagosomes fuse with lysosome by the action of several SNARE proteins (soluble Nethyl-maleimide-sensitive fusion attachment protein receptors) [START_REF] Moreau | Connections between SNAREs and autophagy[END_REF].

mTORC1 regulation of the autophagic process

As previously mentioned, in the regulation of autophagic process, the most characterized protein regulator is mTORC1, a cellular nutritional sensor able to modulate different signaling pathways. mTORC1 is a kinase complex composed of five subunits: mTOR, RAPTOR (regulatory-associated protein of mTOR), mLST8 (mammalian lethal with SEC13 protein), DEPTOR (DEP domain-containing mTOR-protein) and PRAS40 (40 kDa Pro-rich AKT substrate) [START_REF] Saxton | mTOR Signaling in Growth, Metabolism, and Disease[END_REF]. In basal conditions, the kinase activity of mTOR renders ULK complex inactive, thus inhibiting the autophagy initiation [START_REF] Kim | Mutations in prion-like domains in hnRNPA2B1 and hnRNPA1 cause multisystem proteinopathy and ALS[END_REF][START_REF] Meijer | Regulation of autophagy by amino acids and MTORdependent signal transduction[END_REF]. In conditions of nutrient deprivation, a low ATP/AMP ratio activates AMPK, which upregulates ULK complex activity and allows its release from mTORC1 and its relocation to phagophore assembly site where it can activate PI3KC3 [START_REF] Wong | Optineurin is an autophagy receptor for damaged mitochondria in parkinmediated mitophagy that is disrupted by an ALS-linked mutation[END_REF].

Of interest, mTORC1 is negatively regulated by the GATOR1 complex (GAP Activity Toward Rags complex 1) that is composed of three proteins, including DEPDC5 (Bar-Peled et al, 2013a;

Panchaud et al, 2013a), whose mutations in focal epilepsies is a subject of this thesis (refer to section 6.).

Autophagy in ALS

Basal autophagy in neurons

Adult neurons, and especially motor neurons, are long-lived, highly specialized cells that do not undergo renewal. Due to their extreme polarization, size and post-mitotic nature, they are uniquely sensitive to the accumulation of misfolded proteins, dysfunctional organelles and protein aggregates, because they cannot rely on the dilution of cellular waste occurring during cell division [START_REF] Damme | Autophagy in neuronal cells: general principles and physiological and pathological functions[END_REF][START_REF] Tooze | Liaisons dangereuses: autophagy, neuronal survival and neurodegeneration[END_REF][START_REF] Nixon | The role of autophagy in neurodegenerative disease[END_REF]. In neurons, basal autophagy is relatively active and observations of the dynamics of autophagosomes have shown that they form anywhere within a neuron where there are toxic substrates that need to be degraded, including the distal tip of the axon and the neuromuscular junction (NMJ), while the majority of the lysosomes are located within the soma [START_REF] Lee | Lysosomal Proteolysis Inhibition Selectively Disrupts Axonal Transport of Degradative Organelles and Causes an Alzheimer's-Like Axonal Dystrophy[END_REF][START_REF] Maday | Autophagosomes initiate distally and mature during transport toward the cell soma in primary neurons[END_REF]Cheng et al, 2015b;[START_REF] Maday | Mechanisms of neuronal homeostasis: Autophagy in the axon[END_REF]. Electronic microscopy analysis demonstrated that there is extensive ER along the axon allowing the formation of the omegasome in the furthest part of the neurons [START_REF] Maday | Autophagosome biogenesis in primary neurons follows an ordered and spatially regulated pathway[END_REF]. Thus, neurons use active trafficking machinery that transport autophagosomes from the distal ends of the neurons to the soma, including microtubule-associated dynein motor proteins (Cheng et al, 2015b;[START_REF] Maday | Mechanisms of neuronal homeostasis: Autophagy in the axon[END_REF]. Many lines of evidence indicate that dynein drives the robust retrograde motility of autophagosomes along the axon [START_REF] Ravikumar | Dynein mutations impair autophagic clearance of aggregate-prone proteins[END_REF][START_REF] Stainier | Axonal autophagy: Mini-review for autophagy in the CNS[END_REF].

Autophagy, a guardian against neurodegeneration

Defects in autophagy are associated with the pathogenesis of many neurodegenerative diseases [START_REF] Klionsky | Guidelines for the use and interpretation of assays for monitoring autophagy. Autophagy Klionsky DJ[END_REF][START_REF] Menzies | Compromised autophagy and neurodegenerative diseases[END_REF][START_REF] Wong | Autophagy gone awry in neurodegenerative diseases[END_REF]. These pathological conditions share the common feature of accumulation of proteins in toxic aggregates, with proteins, cell type and cellular localization varying among the conditions [START_REF] Levine | Autophagy in the Pathogenesis of Disease[END_REF][START_REF] Nixon | The role of autophagy in neurodegenerative disease[END_REF][START_REF] Menzies | Compromised autophagy and neurodegenerative diseases[END_REF]. For example, tau and beta-amyloid aggregates are well-known features of Alzheimer's disease [START_REF] Sperling | Biomarkers of alzheimer disease: Current and future applications to diagnostic criteria[END_REF][START_REF] Calderon-Garcidueñas | Alzheimer disease[END_REF][START_REF] Jones | Valosin-containing protein (VCP) is required for autophagy and is disrupted in VCP disease[END_REF], while cytoplasmic aggregates of mutant alpha-synuclein (Lewy bodies) are characteristic of Parkinson's disease [START_REF] Spillantini | Neurodegeneration and the ordered assembly of α-synuclein[END_REF][START_REF] Mochizuki | A refined concept: α-synuclein dysregulation disease[END_REF][START_REF] Zeng | Cellular and molecular basis of neurodegeneration in Parkinson disease[END_REF] and are targeted by autophagy process (Lee et al, 2013a;[START_REF] Xilouri | Autophagy and Alpha-Synuclein: Relevance to Parkinson's Disease and Related Synucleopathies[END_REF][START_REF] Zare-Shahabadi | Autophagy in Alzheimer's disease[END_REF].

Consistently, in neurons, deletion of ATG5 and ATG7 inhibits autophagy and promotes neurodegeneration, characterized by axonal degeneration and accumulation of cytoplasmic inclusions with ubiquitinated proteins in neurons, without altering UPS function (Mizushima & Hara, 2006;[START_REF] Komatsu | Ubiquitinindependent function of optineurin in autophagic clearance of protein aggregates[END_REF]. In addition, upregulation of UPS cannot rescue the blockade of autophagy (Mizushima & Hara, 2006;[START_REF] Komatsu | Ubiquitinindependent function of optineurin in autophagic clearance of protein aggregates[END_REF].

Overall, these observations suggest that basal autophagy is crucial for neurons survival, by preventing the accumulation of toxic material and related neurodegeneration. In this sense, controlled modifications of autophagy may prove very useful in the development of possible therapeutic intervention strategies for neurodegenerative diseases [START_REF] Vidal | Targeting autophagy in neurodegenerative diseases[END_REF].

2.2.3.

Is ALS an autophagy disorder?

Autophagy genes and related pathological features in ALS

As previously exposed, there are a multitude of ALS genes with function related to autophagy process (Deng et al, 2017;Nguyen et al, 2018;[START_REF] Ramesh | Autophagy Dysregulation in ALS: When Protein Aggregates Get Out of Hand[END_REF]) (Fig. 3). Overall, these mutations play a critical role in ALS characteristic pathological inclusions and degradation mechanisms. The involvement of SQSTM1 and C9ORF72 ALS genes within autophagy will be discussed thoroughly in sections 3. and 4., respectively. Here is a review of some of others ALS genes with function related to autophagy pathway (Fig. 7).

TDP-43 has been detected as a major component of the ubiquitin-positive inclusions in ALS, [START_REF] Neumann | Ubiquitinated TDP-43 in Frontotemporal Lobar Degeneration and Amyotrophic Lateral Sclerosis[END_REF] and aggregated TDP-43 is cleared by autophagy [START_REF] Brady | Regulation of TDP-43 aggregation by phosphorylation andp62/SQSTM1[END_REF][START_REF] Scotter | Differential roles of the ubiquitin proteasome system and autophagy in the clearance of soluble and aggregated TDP-43 species[END_REF][START_REF] Wang | Degradation of TDP-43 and its pathogenic form by autophagy and the ubiquitin-proteasome system[END_REF][START_REF] Wang | Autophagy activators rescue and alleviate pathogenesis of a mouse model with proteinopathies of the TAR DNA-binding protein 43[END_REF]. Finally, ATG7 mRNA has been reported to be a target of TDP-43 [START_REF] Bose | Regulation of autophagy by neuropathological protein TDP-43[END_REF]. Furthermore, TDP-43 depletion causes an impairment of polyubiquitinated proteins and SQSTM1/p62 in neuroblastoma cells [START_REF] Bose | Regulation of autophagy by neuropathological protein TDP-43[END_REF].

Mutations in the initiator of autophagy TBK1 has been identified in ALS patients [START_REF] Cirulli | Loss of function of C9orf72 causes motor deficits in a zebrafish model of amyotrophic lateral sclerosis[END_REF][START_REF] Freischmidt | Haploinsufficiency of TBK1 causes familial ALS and fronto-temporal dementia[END_REF]. Patients with TBK1 mutations display decreased mRNA and protein expression, thus suggesting haploinsufficiency mechanisms [START_REF] Cirulli | Loss of function of C9orf72 causes motor deficits in a zebrafish model of amyotrophic lateral sclerosis[END_REF][START_REF] Freischmidt | Haploinsufficiency of TBK1 causes familial ALS and fronto-temporal dementia[END_REF]. TBK1 is essential for the phosphorylation and the activation of the autophagy receptors OPTN and SQSTM1/p62, to promote autophagy initiation [START_REF] Richter | Phosphorylation of OPTN by TBK1 enhances its binding to Ub chains and promotes selective autophagy of damaged mitochondria[END_REF][START_REF] Maruyama | TBK1 controls autophagosomal engulfment of polyubiquitinated mitochondria through p62/SQSTM1 phosphorylation[END_REF]. More recently, a collaborative study from our lab and Dr Charlet-Berguerand's team has shown that TBK1 phosphorylates SMCR8, which forms a complex with C9ORF72 and WDR41, participating to autophagy intiation (Sellier et al, 2016) (Fig. 7).

Mutations in the autophagy receptor OPTN have been identified in ALS cases (Maruyama et al, 2010). An important role of OPTN protein has been particularly demonstrated in mitophagy (the selective degradation of mitochondria by autophagy) [START_REF] Wong | Temporal dynamics of PARK2/parkin and OPTN/optineurin recruitment during the mitophagy of damaged mitochondria[END_REF]. This function is inhibited in several ALS-related mutations in OPTN that are found in the UBA region of the protein, whose role is to bind to ubiquitin [START_REF] Wong | Temporal dynamics of PARK2/parkin and OPTN/optineurin recruitment during the mitophagy of damaged mitochondria[END_REF][START_REF] Slowicka | Optineurin deficiency in mice is associated with increased sensitivity to Salmonella but does not affect proinflammatory NF-κB signaling[END_REF]) (Fig. 6 &7).

UBQLN2 is another autophagy receptor that is mutated in ALS (Deng et al, 2011a). In animal models, ALS-related UBQLN2 mutations lead to impairment of protein degradation, abnormal protein aggregation and neurodegeneration [START_REF] Ceballos-Diaz | Viral expression of ALS-linked ubiquilin-2 mutants causes inclusion pathology and behavioral deficits in mice[END_REF][START_REF] Hjerpe | UBQLN2 Mediates Autophagy-Independent Protein Aggregate Clearance by the Proteasome[END_REF][START_REF] Picher-Martel | Neuronal Expression of UBQLN2P497H Exacerbates TDP-43 Pathology in TDP-43G348C Mice through Interaction with Ubiquitin[END_REF]. Consistently with its role in protein degradation, UBQLN2 protein was also found to be a component of cytoplasmic inclusions in the brain and spinal cord of ALS patients (Deng et al, 2011a) (Fig. 6 &7).

1-2% of fALS and 1% of sALS cases have been attributed to mutations in VCP, another contributor of autophagy (Johnson et al, 2010). VCP is an AAA ATPase, whose role in autophagy is known from the findings that loss of VCP leads to ubiquitin-positive vacuole accumulation due to its maturation failure, while autophagy initiation is effective [START_REF] Jones | Valosin-containing protein (VCP) is required for autophagy and is disrupted in VCP disease[END_REF][START_REF] Tresse | An MND/ALS phenotype associated with C9orf72 repeat expansion: Abundant p62-positive, TDP-43-negative inclusions in cerebral cortex, hippocampus and cerebellum but without associated cognitive decline[END_REF]. VCP is essential for establishing the fusion of the autophagosomes [START_REF] Jones | Valosin-containing protein (VCP) is required for autophagy and is disrupted in VCP disease[END_REF][START_REF] Tresse | An MND/ALS phenotype associated with C9orf72 repeat expansion: Abundant p62-positive, TDP-43-negative inclusions in cerebral cortex, hippocampus and cerebellum but without associated cognitive decline[END_REF] and the lysosome (Fig. 7), as well as for the autophagy-mediated degradation of SG [START_REF] Buchan | XEukaryotic stress granules are cleared by autophagy and Cdc48/VCP function[END_REF]. Consequently, histopathological analysis of VCPrelated diseases, i. e. IBM, PDB, and FTLD, display TDP-43, ubiquitin and p62-positive inclusions [START_REF] Hübbers | Polymerase chain reaction and Southern blot-based analysis of the C9orf72 hexanucleotide repeat in different motor neuron diseases[END_REF][START_REF] Schröder | Mutant valosin-containing protein causes a novel type of frontotemporal dementia[END_REF][START_REF] Custer | Transgenic mice expressing mutant forms VCP/p97 recapitulate the full spectrum of IBMPFD including degeneration in muscle, brain and bone[END_REF][START_REF] Jones | Valosin-containing protein (VCP) is required for autophagy and is disrupted in VCP disease[END_REF].

Another ALS-related gene with a function in autophagy is Dynactin 1 (DCTN1) [START_REF] Münch | Point mutations of the p150 subunit of dynactin (DCTN1) gene ALS[END_REF] that is involved in the transport of autophagosomes by microtubules via its interaction with the motor protein, dynein [START_REF] Laird | Motor Neuron Disease Occurring in a Mutant Dynactin Mouse Model Is Characterized by Defects in Vesicular Trafficking[END_REF]) (Fig. 7). Mice with ALS-associated mutation in Dctn1 display accumulation of autophagosomes in motor neurons [START_REF] Laird | Motor Neuron Disease Occurring in a Mutant Dynactin Mouse Model Is Characterized by Defects in Vesicular Trafficking[END_REF].

Similarly, worms deficient for Dctn1 exhibit motor neuronal defects with axonal accumulation of autophagosomes in axons, neurodegeneration and decreased life span [START_REF] Ikenaka | dnc-1/dynactin 1 Knockdown Disrupts Transport of Autophagosomes and Induces Motor Neuron Degeneration. PLoS One Iłżecka J[END_REF].

In this model, induction of autophagy with the mTOR inhibitor rapamycin is able to restore autophagosomes degradation and to rescue the motor neuron features [START_REF] Ikenaka | dnc-1/dynactin 1 Knockdown Disrupts Transport of Autophagosomes and Induces Motor Neuron Degeneration. PLoS One Iłżecka J[END_REF].

Autophagy as a crossroad mechanism in ALS

As previously described, one of the key pathological hallmarks in ALS is the accumulation of cytoplasmic aggregates, composed of disease-associated proteins and their interactors, within affected neurons (Kabashi & Durham, 2006;[START_REF] Blokhuis | Protein aggregation in amyotrophic lateral sclerosis[END_REF][START_REF] Götzl | Impaired protein degradation in FTLD and related disorders[END_REF]Ruegsegger & Saxena, 2016;[START_REF] Medinas | Proteostasis disturbance in amyotrophic lateral sclerosis[END_REF]. However, not only has the proteinopathy of the abnormal proteins been identified as a major pathological feature in ALS, but the abnormal accumulation of the wild-type proteins, such as TDP-43 [START_REF] Kwong | TDP-43 proteinopathy: The neuropathology underlying major forms of sporadic and familial frontotemporal lobar degeneration and motor neuron disease[END_REF][START_REF] Arai | TDP-43 is a component of ubiquitin-positive tau-negative inclusions in frontotemporal lobar degeneration and amyotrophic lateral sclerosis[END_REF][START_REF] Neumann | Molecular neuropathology of TDP-43 proteinopathies[END_REF], SOD1 [START_REF] Watanabe | Histological evidence of protein aggregation in mutant SOD1 transgenic mice and in amyotrophic lateral sclerosis neural tissues[END_REF][START_REF] Forsberg | Novel antibodies reveal inclusions containing non-native SOD1 in sporadic ALS patients[END_REF][START_REF] Paré | Misfolded SOD1 pathology in sporadic Amyotrophic Lateral Sclerosis[END_REF] and SQSTM1/p62 [START_REF] Mizuno | Immunoreactivities of p62, an ubiqutin-binding protein, in the spinal anterior horn cells of patients with amyotrophic lateral sclerosis[END_REF][START_REF] Arai | Neuronal and glial inclusions in frontotemporal dementia with or without motor neuron disease are immunopositive for p62[END_REF][START_REF] Masanori | White matter lesions in the brain with frontotemporal lobar degeneration with motor neuron disease: TDP-43immunopositive inclusions co-localize with p62, but not ubiquitin[END_REF] have been detected in ALS or ALS/FTLD patients, highlighting the preponderance of defective protein quality control mechanisms in the aetiology of the disease. In this condition, inclusion formation may be an event secondary to a failure of control protein turnover, such as defects of autophagy machinery [START_REF] Levine | Autophagy in the Pathogenesis of Disease[END_REF][START_REF] Menzies | Compromised autophagy and neurodegenerative diseases[END_REF][START_REF] Tooze | Liaisons dangereuses: autophagy, neuronal survival and neurodegeneration[END_REF]. In the other hand, via a gain of toxic function mechanism, misfolded mutant proteins have been shown

to sequester functional proteins important for the autophagy process, thus inducing their loss of function [START_REF] Winklhofer | The two faces of protein misfolding: Gain-and loss-of-function in neurodegenerative diseases[END_REF][START_REF] Powers | Biological and Chemical Approaches to Diseases of Proteostasis Deficiency[END_REF][START_REF] Menzies | Protein misfolding disorders and macroautophagy[END_REF].

Regarding ALS pathogenesis, it seems important to consider autophagy and other ALS-related cellular mechanisms (section 1.6.) as being able to interact [START_REF] Blokhuis | Comparative interactomics analysis of different ALSassociated proteins identifies converging molecular pathways[END_REF]. Typically, concerted activity of UPS and autophagy pathways have been widely reported. Indeed, upregulation of autophagy occurs as a compensatory mechanism when the UPS is overloaded, possibly due to aggregation of proteins or mitochondrial dysfunction [START_REF] Bence | Impairment of the ubiquitin-proteasome system by protein aggregation[END_REF]Rubinsztein, 2006;Lamark & Johansen, 2010). Conversely, inhibition of autophagy lead to decrease UPS flux and compromised degradation of UPS substrates in different types of cells, including neurons [START_REF] Korolchuk | Mechanisms of cross-talk between the ubiquitin-proteasome and autophagy-lysosome systems[END_REF]Rubinsztein, 2006). In addition, ER stress has been shown to activate both UPS and autophagy initiation in the event of pathogenic accumulation of misfolded proteins [START_REF] Ogata | Autophagy Is Activated for Cell Survival after Endoplasmic Reticulum Stress[END_REF][START_REF] Cai | Interplay of endoplasmic reticulum stress and autophagy in neurodegenerative disorders. Autophagy de Calbiac H[END_REF]. Another example placing autophagy as a converging point in ALS pathogenesis, is the link between dysfunctional mitochondria and autophagy, within the crosstalk mechanism mitophagy [START_REF] Ding | Mitophagy: Mechanisms, pathophysiological roles, and analysis[END_REF][START_REF] Zimmermann | How to get rid of mitochondria: Crosstalk and regulation of multiple mitophagy pathways[END_REF]Chu, 2018;[START_REF] Youle | Mechanisms of mitophagy[END_REF][START_REF] Smith | Title: The Role of Mitochondria in Amyotrophic Lateral Sclerosis The Role of Mitochondria in Amyotrophic Lateral Sclerosis[END_REF]. Dysfunctional mitochondria are ubiquitinated by the autophagy receptors OPTN [START_REF] Ying | Optineurin: The autophagy connection[END_REF]Wong & Holzbaur, 2014;Majcher et al, 2015b) and UBQLN2 [START_REF] Itakura | Characterization of autophagosome formation site by a hierarchical analysis of mammalian Atg proteins[END_REF]Majcher et al, 2015b), to be degraded by autophagy process. Mitophagy also implies the contribution of two other autophagy proteins, whose mutations have been linked to ALS, namely VCP [START_REF] Guo | VCP recruitment to mitochondria causes mitophagy impairment and neurodegeneration in models of Huntington's disease[END_REF][START_REF] Evans | Autophagy and mitophagy in ALS[END_REF]) and TBK1 [START_REF] Moore | Dynamic recruitment and activation of ALS-associated TBK1 with its target optineurin are required for efficient mitophagy[END_REF]. Mitochondria dysfunction is the prominent source of ROS overproduction that lead to oxidative stress in ALS [START_REF] Emerit | Neurodegenerative diseases and oxidative stress[END_REF][START_REF] Federico | Mitochondria, oxidative stress and neurodegeneration[END_REF][START_REF] Lin | Formation and Maturation of Phase-Separated Liquid Droplets by RNA-Binding Proteins[END_REF][START_REF] Islam | Oxidative stress and mitochondrial dysfunction-linked neurodegenerative disorders[END_REF]. Interestingly, oxidative stress has the potential to change the conformation of proteins by increasing their propensity to aggregate [START_REF] Carija | Protein aggregation into insoluble deposits protects from oxidative stress[END_REF][START_REF] Squier | Oxidative stress and protein aggregation during biological aging[END_REF][START_REF] Grimsrud | Oxidative stress and covalent modification of protein with bioactive aldehydes[END_REF][START_REF] Cumming | Protein disulfide bond formation in the cytoplasm during oxidative stress[END_REF], thus providing a new potential connection with autophagy pathway. Concurrently, the newest drug to be approved for ALS patients, edaravone, has anti-oxidant properties [START_REF] Nagase | Increased oxidative stress in patients with amyotrophic lateral sclerosis and the effect of edaravone administration[END_REF][START_REF] Sawada | Clinical efficacy of edaravone for the treatment of amyotrophic lateral sclerosis[END_REF]. In addition, SG have been shown to be degraded by autophagy, particularly, through the involvement of VCP [START_REF] Buchan | XEukaryotic stress granules are cleared by autophagy and Cdc48/VCP function[END_REF][START_REF] Seguin | Inhibition of autophagy, lysosome and VCP function impairs stress granule assembly[END_REF]Chitiprolu et al, 2018). Another link between autophagy and RNA metabolism in ALS is reflected by the fact that the RBP protein TDP-43 is able to sequester ATG7 mRNA, surrounding the loss of the ATG7 protein and causing impairment in autophagy process [START_REF] Bose | Regulation of autophagy by neuropathological protein TDP-43[END_REF].

All these observations are non-exhaustive and relative, but they highlight that autophagy is a point of crosstalk between potential pathogenic mechanisms in ALS. Moreover, they also emphasize that, in ALS, autophagy can be affected both directly, through specific defects in its constitutive elements, or indirectly because of excessive cargo due to spill-over effect of deficient upstream mechanisms.

Autophagy as a therapeutic target in ALS

There is still no effective treatment for ALS but, based on the previous observations, autophagy is an attractive therapeutic target for this disease. However, the difficulty in targeting autophagy resides in the fact that autophagy is a complex pathway, which is tightly regulated and integrates various signals and effectors. Targeting autophagy in a compensatory strategy thus requests to characterize properly the structure, protein or complex in question, in the impaired system.

Autophagy may go awry when degradative capacity is insufficient to adequately respond to its induction. It can be halted due to impairment that could arise at one or more points along the pathway, from the initiation to the lysosomal degradation; thus giving rise to distinct neuropathological patterns. As an example, promoting autophagy induction when lysosomal function is less efficient may be not beneficial, with an increase in autophagosomes synthesis that could overwhelms the clearance capacity of the lysosome. However, if properly targeted, the expected benefits of such modulation might include reduced amount of toxic protein aggregates and incompletely digested autolysosomal structures.

The example of rapamycin

Rapamycin is a well-known inhibitor of mTOR, and accordingly, an activator of autophagy [START_REF] Li | Rapamycin: One drug, many effects[END_REF]) (Fig. 5). Rapamycin has been shown to upregulate autophagy and ameliorates neuropathology and neurodegeneration in cells [START_REF] Barmada | Autophagy induction enhances TDP43 turnover and survival in neuronal ALS models[END_REF][START_REF] Caccamo | Rapamycin rescues TDP-43 mislocalization and the associated low molecular mass neurofilament instability[END_REF][START_REF] Madill | Amyotrophic lateral sclerosis patient iPSC-derived astrocytes impair autophagy via non-cell autonomous mechanisms[END_REF] and animals [START_REF] Wang | Autophagy activators rescue and alleviate pathogenesis of a mouse model with proteinopathies of the TAR DNA-binding protein 43[END_REF]Cheng et al, 2015a;[START_REF] Deivasigamani | A genetic screen identifies Tor as an interactor of VAPB in a Drosophila model of amyotrophic lateral sclerosis[END_REF][START_REF] Nalbandian | Rapamycin and chloroquine: The In Vitro and In Vivo effects of autophagy-modifying drugs show promising results in Valosin containing protein multisystem proteinopathy[END_REF][START_REF] Staats | Rapamycin increases survival in ALS mice lacking mature lymphocytes[END_REF] ALS models. As a matter of fact, rapamycin is currently under a phase II in a clinical trial for ALS [START_REF] Mandrioli | Rapamycin treatment for amyotrophic lateral sclerosis[END_REF]. This is especially interesting since rapamycin is an already approved drug (sirolimus) with known pharmacokinetic, therefore offering a possibility of rapid translation to clinics [START_REF] Walczak | Next-generation mTOR inhibitors in clinical oncology: How pathway complexity informs therapeutic strategy[END_REF][START_REF] Li | Rapamycin: One drug, many effects[END_REF]. However, rapamycin has pleiotropic effects through the inhibition of mTOR, which can result in added toxicity. For example, in a SOD1 mouse model of ALS, rapamycin was shown to augment motor neuron degeneration [START_REF] Zhang | Pharmacological characterization of an antisense knockdown zebrafish model of Dravet syndrome: Inhibition of epileptic seizures by the serotonin agonist fenfluramine[END_REF]. This supports the importance to refine the understanding of autophagy's contribution to ALS, for more targeted therapies in the future.

The contribution of SQSTM1 to ALS

SQSTM1/p62 role in autophagy

The SQSTM1/p62 protein, encoded by the SQSTM1 gene, is a multi-functional adapter protein that regulates a variety of processes, including NF-κB signaling, caspase-mediated apoptosis, Nrf2 activation and degradation of ubiquitinated substrates, by autophagy and the UPS [START_REF] Komatsu | P62/SQSTM1/A170: Physiology and pathology[END_REF][START_REF] Rea | New insights into the role of sequestosome 1/p62 mutant proteins in the pathogenesis of paget's disease of bone[END_REF][START_REF] Bitto | 62/SQSTM1 at the interface of aging, autophagy, and disease[END_REF]. SQSTM1/p62 consists of 440 amino acids and different domains.

The N-terminal PB1 domain mediates SQSTM1/p62 polymerization and coordinates interactions with the other autophagy receptors OPTN and NBR1, in order to assemble an autophagy receptor complex [START_REF] Liu | Ubiquitylation of Autophagy Receptor Optineurin by HACE1 Activates Selective Autophagy for Tumor Suppression[END_REF][START_REF] Johansen | Exome Sequencing Reveals VCP Mutations as a Cause of Familial ALS[END_REF]. The TRAFI-binding sequence (TBS) activates NF-κB signal [START_REF] Rea | New insights into the role of sequestosome 1/p62 mutant proteins in the pathogenesis of paget's disease of bone[END_REF]. In autophagy, SQSTM1/p62 functions as an autophagy receptor (Fig. 6), interacting with ubiquitin-modified substrates via its C-terminal UBA domain and with autophagosomes-associated LC3 homologues via its LIR domain [START_REF] Johansen | Exome Sequencing Reveals VCP Mutations as a Cause of Familial ALS[END_REF]. SQSTM1/p62 also interacts with mTOR to regulate its translocation to the lysosomes, which defines its inhibitor activity in autophagy [START_REF] Duran | P62 Is a Key Regulator of Nutrient Sensing in the mTORC1 Pathway[END_REF]. Importantly, SQSTM1/p62 is abundantly expressed in spinal motor neurons, consistently with the requirement of basal autophagy in these cells [START_REF] Arai | Neuronal and glial inclusions in frontotemporal dementia with or without motor neuron disease are immunopositive for p62[END_REF].

SQSTM1 pathogenicity in ALS

Neuropathological features of SQSTM1/p62 inclusions

SQSTM1/p62 has originally been linked to neurodegeneration due to its enrichment in cytoplasmic proteinaceous inclusions in many neurological disorders such as Alzheimer's disease, Parkinson's disease [START_REF] Zatloukal | p62 is a common component of cytoplasmic inclusions in protein aggregation diseases[END_REF] and in ALS, with basophilic inclusions positive for SQSTM1/p62 being described in the anterior horn of patients [START_REF] Mizuno | Immunoreactivities of p62, an ubiqutin-binding protein, in the spinal anterior horn cells of patients with amyotrophic lateral sclerosis[END_REF] (Fig. 8). In ALS/FTLD, neuronal and glial inclusions positive for SQSTM1/p62 are associated with immunoreactivity for TDP-43 in ALS/FTLD patients (Hiji [START_REF] Masanori | White matter lesions in the brain with frontotemporal lobar degeneration with motor neuron disease: TDP-43immunopositive inclusions co-localize with p62, but not ubiquitin[END_REF][START_REF] Mizuno | Immunoreactivities of p62, an ubiqutin-binding protein, in the spinal anterior horn cells of patients with amyotrophic lateral sclerosis[END_REF][START_REF] Arai | Neuronal and glial inclusions in frontotemporal dementia with or without motor neuron disease are immunopositive for p62[END_REF][START_REF] Al-Sarraj | P62 positive, TDP-43 negative, neuronal cytoplasmic and intranuclear inclusions in the cerebellum and hippocampus define the pathology of C9orf72-linked FTLD and MND/ALS[END_REF][START_REF] Durán | The atypical PKC-interacting protein p62 is an important mediator of RANK-activated osteoclastogenesis[END_REF]. SQSTM1/p62-positive inclusions are also described in spinal cord ubiquitin-positive inclusions of patients with

SQSTM1 mutations in ALS

Mutations in SQSTM1 account for approximately 1-3.5% of ALS/FTLD cases with or without family history (Fecto et al, 2011b). SQSTM1 mutations have also been associated with PDB, a chronic skeletal disorder, such as other ALS genes, VCP [START_REF] Watts | Inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia is caused by mutant valosin-containing protein[END_REF] and OPTN [START_REF] Albagha | Genome-wide association study identifies variants at CSF1, OPTN and TNFRSF11A as genetic risk factors for Paget's disease of bone[END_REF]. Interestingly, patients with ALS/FTLD can show signs of other conditions: PDB, but also IBM. The spectrum of phenotypes characterized by the overlap of clinical signs of these diseases is called "multisystem proteinopathy" and has been associated

with mutations in VCP, SQSTM1 and heterogeneous nuclear ribonucleoproteins hnRNPA2B1 and hnRNPA1 (Kim et al, 2013b).

In contrast to the PDB-associated mutations that affect mainly the UBA domain (Rea et al, 2014b), SQSTM1 mutations identified in ALS/FTLD encompass several different regions, although their frequency is higher in the UBA and LIR domains, and in the TBS sequence that are critical for its role in autophagy (Fig. 9). Most of them are missense mutations but a few truncating mutations have also been identified (Fecto et al, 2011b;[START_REF] Elisa | SQSTM1 mutations in frontotemporal lobar degeneration and amyotrophic lateral sclerosis[END_REF][START_REF] Hirano | Mutations in the gene encoding p62 in Japanese patients with amyotrophic lateral sclerosis[END_REF]Le Ber et al, 2013;[START_REF] Teyssou | Mutations in SQSTM1 encoding p62 in amyotrophic lateral sclerosis: Genetics and neuropathology[END_REF][START_REF] Shimizu | Sporadic ALS with compound heterozygous mutations in the SQSTM1 gene[END_REF][START_REF] Chen | SQSTM1 mutations in Han Chinese populations with sporadic amyotrophic lateral sclerosis[END_REF][START_REF] Kwok | Sequestosome-1 (SQSTM1) sequence variants in ALS cases in the UK: Prevalence and coexistence of SQSTM1 mutations in ALS kindred with PDB[END_REF][START_REF] Van Der Zee | Rare mutations in SQSTM1 modify susceptibility to frontotemporal lobar degeneration[END_REF]. SQSTM1/p62 mutations in the UBA domain are associated with defective ubiquitin-recognition [START_REF] Rea | New insights into the role of sequestosome 1/p62 mutant proteins in the pathogenesis of paget's disease of bone[END_REF]. Mutations within the LIR domain of SQSTM1/p62 have the potential to impact LC3 recognition and substrate delivery to the autophagosomes [START_REF] Pankiv | 62/SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of ubiquitinated protein aggregates by autophagy[END_REF][START_REF] Ichimura | Structural basis for sorting mechanism of p62 in selective autophagy[END_REF]) (Fig. 9). Mutations within the TBS domain are also relevant in the context of autophagy dysregulation, as inhibition of TRAF6 blocks autophagosomes formation in skeletal muscle through its interaction with SQSTM1/p62 [START_REF] Paul | The E3 Ubiquitin Ligase TRAF6 Intercedes in Starvation-Induced Skeletal Muscle Atrophy through Multiple Mechanisms[END_REF]) (Fig. 9). Furthermore, mutations detected in the promoter region of SQSTM1 of ALS/FTLD patients have been associated with reduced SQSTM1/p62 levels [START_REF] Du | Oxidative damage to the promoter region of SQSTM1/p62 is common to neurodegenerative disease[END_REF][START_REF] Elisa | SQSTM1 mutations in frontotemporal lobar degeneration and amyotrophic lateral sclerosis[END_REF].

At the beginning of my PhD thesis, the only available models for SQSTM1 pathogenicity analysis were SQSTM1 knock-out mice [START_REF] Durán | The atypical PKC-interacting protein p62 is an important mediator of RANK-activated osteoclastogenesis[END_REF][START_REF] Rodriguez | Mature-onset obesity and insulin resistance in mice deficient in the signaling adapter p62[END_REF][START_REF] Babu | Genetic inactivation of p62 leads to accumulation of hyperphosphorylated tau and neurodegeneration[END_REF], or mice overexpressing SQSTM1 mutations [START_REF] Kurihara | Mutation of the sequestosome 1 (p62) gene increases osteoclastogenesis but does not induce Paget disease[END_REF][START_REF] Daroszewska | A point mutation in the ubiquitin-associated domain of SQSMT1 is sufficient to cause a Paget's disease-like disorder in mice[END_REF][START_REF] Seibenhener | Behavioral effects of SQSTM1/p62 overexpression in mice: Support for a mitochondrial role in depression and anxiety[END_REF], which have been developed to study PDB.

SQSTM1 knock-out mice display reduced osteoclastogenesis, decreased IL6 expression [START_REF] Durán | The atypical PKC-interacting protein p62 is an important mediator of RANK-activated osteoclastogenesis[END_REF]) and insulin resistance [START_REF] Rodriguez | Mature-onset obesity and insulin resistance in mice deficient in the signaling adapter p62[END_REF]. Interestingly, they also develop an Alzheimer-like phenotype, consisting in cognitive impairment and anxiety, associated with hyperphosphorylated tau and neurofibrillary tangles [START_REF] Babu | Genetic inactivation of p62 leads to accumulation of hyperphosphorylated tau and neurodegeneration[END_REF]. Mice overexpressing SQSTM1 variants carrying mutations in the UBA domain (equivalent of p.P932L) have a PDB-like skeletal disorder [START_REF] Kurihara | Mutation of the sequestosome 1 (p62) gene increases osteoclastogenesis but does not induce Paget disease[END_REF][START_REF] Daroszewska | A point mutation in the ubiquitin-associated domain of SQSMT1 is sufficient to cause a Paget's disease-like disorder in mice[END_REF] and affected spatial learning and long-term memory deficits [START_REF] Seibenhener | Behavioral effects of SQSTM1/p62 overexpression in mice: Support for a mitochondrial role in depression and anxiety[END_REF].

A381V P387L P392L G411S G425R PB1 LIR N-term C-term A33V A53T M87V V90M I99L K102E TBS R110C V153I E155K P228L V234V K238E/del V259L H261H E274D P296P R312G S318P E319K R321C/H D337E L341V K344E UBA V346V P348L S370P P438L P439L

Hypotheses and objectives

As described in this introduction, evidence implicates impairment of autophagy in the spectrum of ALS/FTLD disorders [START_REF] Navone | Autophagy and Neurodegeneration: Insights from a Cultured Cell Model of ALS[END_REF][START_REF] Mis | Autophagy in motor neuron disease: Key pathogenetic mechanisms and therapeutic targets[END_REF][START_REF] Budini | Autophagy and Its Impact on Neurodegenerative Diseases: New Roles for TDP-43 and C9orf72[END_REF]Nguyen et al, 2018). SQSTM1/p62 is a key protein of autophagy-mediated degradation, since it takes part in the selective recognition of autophagy substrates, as well as in their targeting to the elongated autophagosomes. Mutations of SQSTM1 gene have been identified in patients

with ALS/FTLD. The majority of these mutations are missense variants, but truncating mutations have also been identified, suggesting that haploinsufficiency could mediate the pathogenicity of these mutations. Moreover, as pointed out above, half of mutations identified in ALS/FTLD patients in SQSTM1 affect protein domains that are directly involved in autophagy, leading to the hypothesis that loss of function of SQSTM1 could underlie neurodegeneration in ALS/FTLD due to defects in autophagy process. To test this hypothesis, we aimed to generate a new model to investigate SQSTM1 pathogenicity and autophagy implication in ALS. This was the first objective of this work.

4.

The contribution of C9ORF72 to ALS/FTLD

The C9ORF72 hexanucleotide repeat expansion (HRE) in ALS/FTLD

In 2011, the seminal discovery of a mutation within C9ORF72 represented a major advance in ALS research by revealing the most common genetic insult responsible for the development of ALS/FTLD, accounting for 23-47 % of fALS and 4-21 % of sALS (DeJesus-Hernandez et al, 2011a;Renton et al, 2011a). Prior to that, linkage analysis of ALS/FTLD cases had suggested that there was an important locus for the disease on the short arm of the chromosome 9 [START_REF] Boxer | Clinical, neuroimaging and neuropathological features of a new chromosome 9plinked FTD-ALS family[END_REF][START_REF] Morita | A locus on chromosome 9p confers susceptibility to ALS and frontotemporal dementia[END_REF][START_REF] Pearson | Familial frontotemporal dementia with amyotrophic lateral sclerosis and a shared haplotype on chromosome 9p[END_REF][START_REF] Vance | Familial amyotrophic lateral sclerosis with frontotemporal dementia is linked to a locus on chromosome 9p13[END_REF][START_REF] Laaksovirta | Chromosome 9p21 in amyotrophic lateral sclerosis in Finland: A genome-wide association study[END_REF][START_REF] Shatunov | Chromosome 9p21 in sporadic amyotrophic lateral sclerosis in the UK and seven other countries: A genome-wide association study[END_REF][START_REF] Van Deerlin | Common variants at 7p21 are associated with frontotemporal lobar degeneration with TDP-43 inclusions[END_REF][START_REF] Gijselinck | Identification of 2 loci at chromosomes 9 and 14 in a multiplex family with frontotemporal lobar degeneration and amyotrophic lateral sclerosis[END_REF][START_REF] Ber I Le | Chromosome 9p-linked families with frontotemporal dementia associated with motor neuron disease[END_REF][START_REF] Luty | Pedigree with frontotemporal lobar degeneration -Motor neuron disease and Tar DNA binding protein-43 positive neuropathology: Genetic linkage to chromosome 9[END_REF][START_REF] Vaccaro | Three families with amyotrophic lateral sclerosis and frontotemporal dementia with evidence of linkage to chromosome 9p[END_REF][START_REF] Van Es | 2 as susceptibility loci for sporadic amyotrophic lateral sclerosis[END_REF]) (Fig. 10). Finally, by using repeat-primed PCR method, DeJesus-Hernandez et al and Renton et al studies reported originally that the C9ORF72 mutation consists of the expansion of a GGGGCCC HRE that is located within the first intron of the gene (DeJesus-Hernandez et al, 2011a; Renton et al, 2011a) (Fig. 10).

Although the normal number of GGGGCC repeats range from 2 to 30 repeats [START_REF] Benussi | C9ORF72 hexanucleotide repeat number in frontotemporal lobar degeneration: A genotype-phenotype correlation study[END_REF][START_REF] Gómez-Tortosa | C9ORF72 hexanucleotide expansions of 20-22 repeats are associated with frontotemporal deterioration[END_REF][START_REF] Majounie | Frequency of the C9orf72 hexanucleotide repeat expansion in patients with amyotrophic lateral sclerosis and frontotemporal dementia: A cross-sectional study[END_REF][START_REF] Rutherford | Length of normal alleles of C9ORF72 GGGGCC repeat do not influence disease phenotype[END_REF]van der Zee et al, 2013;[START_REF] Xi | Investigation of C9orf72 in 4 neurodegenerative disorders[END_REF], C9ORF72 ALS/FTLD patients have several hundred to several thousands [START_REF] Beck | Large C9orf72 hexanucleotide repeat expansions are seen in multiple neurodegenerative syndromes and are more frequent than expected in the UK population[END_REF][START_REF] Buchman | Simultaneous and independent detection of C9ORF72 alleles with low and high number of GGGGCC repeats using an optimised protocol of Southern blot hybridisation[END_REF]DeJesus-Hernandez et al, 2011a;[START_REF] Dobson-Stone | C9ORF72 Repeat Expansion in Australian and Spanish Frontotemporal Dementia Patients[END_REF][START_REF] Hübbers | Polymerase chain reaction and Southern blot-based analysis of the C9orf72 hexanucleotide repeat in different motor neuron diseases[END_REF][START_REF] Ishiura | C9ORF72 repeat expansion in amyotrophic lateral sclerosis in the Kii peninsula of Japan[END_REF][START_REF] Van Blitterswijk | Association between repeat sizes and clinical and pathological characteristics in carriers of C9ORF72 repeat expansions (Xpansize-72): A cross-sectional cohort study[END_REF]) (Fig. 10).

Several analysis have tried to correlate the size of the expansion to clinical features, such as type of syndrome, age at symptom onset, or disease duration. However, to date, no clear correlation have emerged due to discrepancies between studies [START_REF] Beck | Large C9orf72 hexanucleotide repeat expansions are seen in multiple neurodegenerative syndromes and are more frequent than expected in the UK population[END_REF][START_REF] Benussi | C9ORF72 hexanucleotide repeat number in frontotemporal lobar degeneration: A genotype-phenotype correlation study[END_REF][START_REF] Dols-Icardo | Characterization of the repeat expansion size in C9orf72 in amyotrophic lateral sclerosis and frontotemporal dementia[END_REF][START_REF] Gijselinck | The C9orf72 repeat size correlates with onset age of disease, DNA methylation and transcriptional downregulation of the promoter[END_REF][START_REF] Nordin | Extensive size variability of the GGGGCC expansion in C9orf72 in both neuronal and non-neuronal tissues in 18 patients with ALS or FTD[END_REF][START_REF] Suh | Semi-automated quantification of C9orf72 expansion size reveals inverse correlation between hexanucleotide repeat number and disease duration in frontotemporal degeneration[END_REF][START_REF] Van Blitterswijk | Association between repeat sizes and clinical and pathological characteristics in carriers of C9ORF72 repeat expansions (Xpansize-72): A cross-sectional cohort study[END_REF]. One possible explanation for these differences might be that defining the exact repeat size is challenging. One confounding factor for accurate sizing of the HRE is somatic mosaicism. Indeed, individuals have been identified with different repeats length between the CNS and the blood [START_REF] Gijselinck | The C9orf72 repeat size correlates with onset age of disease, DNA methylation and transcriptional downregulation of the promoter[END_REF][START_REF] Nordin | Extensive size variability of the GGGGCC expansion in C9orf72 in both neuronal and non-neuronal tissues in 18 patients with ALS or FTD[END_REF][START_REF] Fratta | Screening a UK amyotrophic lateral sclerosis cohort provides evidence of multiple origins of the C9orf72 expansion[END_REF], and several studies even reported different length of repeats within the brain itself [START_REF] Beck | Large C9orf72 hexanucleotide repeat expansions are seen in multiple neurodegenerative syndromes and are more frequent than expected in the UK population[END_REF][START_REF] Suh | Semi-automated quantification of C9orf72 expansion size reveals inverse correlation between hexanucleotide repeat number and disease duration in frontotemporal degeneration[END_REF][START_REF] Van Blitterswijk | Association between repeat sizes and clinical and pathological characteristics in carriers of C9ORF72 repeat expansions (Xpansize-72): A cross-sectional cohort study[END_REF].

Haploinsufficiency of C9ORF72 in patients

The reduction in C9ORF72 expression has been originally described in the studies that disclosed the mutation (DeJesus-Hernandez et al, 2011a;Renton et al, 2011a), and it is now widely reported that expansion carriers show reduced C9ORF72 mRNA (DeJesus-Hernandez et al, 2011a;Renton et al, 2011a;Ciura et al, 2013a;Donnelly et al, 2013;Gijselinck et al, 2012;Haeusler et al, 2014), but also, more recently confirmed, protein expression (Frick et al, 2018;[START_REF] Waite | Reduced C9orf72 protein levels in frontal cortex of amyotrophic lateral sclerosis and frontotemporal degeneration brain with the C9ORF72 hexanucleotide repeat expansion[END_REF][START_REF] Xiao | Isoform-specific antibodies reveal distinct subcellular localizations of C9orf72 in amyotrophic lateral sclerosis[END_REF] (Fig. 11). Humans express two C9ORF72 isoforms, a 222 amino acids short isoform, "C9-S" (exons 2-5) and a 481 amino acids long isoform "C9-L"(exons [2][3][4][5][6][7][8][9][10][11]. The recent study from Frick et al, gave new prospects as they presented new and thoroughly characterized antibodies directed toward C9-S and C9-L, in a context where such tools were lacking. As a matter of fact, they observed that C9-L is from far the predominant form in human brain, and that its level is reduced in C9ORF72 ALS/FTLD patients cerebellum (Frick et al, 2018).

However, haploinsufficiency of C9ORF72 in ALS/FTD spectrum has been highly contested because no deleterious mutations were reported within cohorts. Only one patient with ALS/FTLD features was shown to carry a premature stop codon in C9ORF72, that is associated with reduced mRNA transcript levels (Liu et al, 2016a). This patient lacked a repeat expansion, suggesting that the neurodegenerative syndrome could have been driven by a haploinsufficiency of C9ORF72, most likely than a sporadic origin coinciding with a C9ORF72 non-pathogenic variant (Liu et al, 2016a).

Nevertheless, elevated C9ORF72 expression has been shown to be associated with a survival advantage in ALS, supporting the notion that C9ORF72 transcript levels and disease penetrance could correlate (van Blitterswijk et al, 2015). Moreover, a recent study has shown that CNS areas with reduced C9ORF72 expression correlate with the extent of disease pathology in patients carrying the C9ORF72 expansion (Saberi et al, 2018).

One hypothesis explaining the loss of function of C9ORF72 that occurs in ALS/FTLD patients carrying this mutation is the hypermethylation of CpG islands at the 5' end of the HRE (Xi et al, 2013(Xi et al, , 2014)), as well as abnormal binding of tri-methylated histones onto the HRE (Belzil et al, 2013); both of which could result in the silencing of the expression of C9ORF72.

Animal models of C9ORF72 loss of function

The first vertebrate model of C9ORF72 loss of function was developed in our team and consists of the knockdown of the c9orf72 orthologue expression in zebrafish (Ciura et al, 2013a).

C9orf72 inhibition triggered behavioral and cellular deficits related to locomotion, that are rescued upon overexpression of human C9ORF72 mRNA (Ciura et al, 2013a). Consistently with these findings, the deletion of the c9orf72 orthologue in C.elegans has been shown to be associated with major motor neuron deficits (Therrien et al, 2013). In addition, a recent report showed that reduced expression of C9ORF72 mRNA was a necessary and sufficient condition to recapitulate the neurotoxic phenotype associated with the presence of the HRE in iPSCderived motor neurons (iMNs) (Shi et al, 2018).

However, in mouse models, C9orf72 (3110043O21Rik orthologue) deletion leads to major defects in the myeloid tissue (Atanasio et al, 2016;Burberry et al, 2016;O'Rourke et al, 2016;Sudria-Lopez et al, 2016), which is involved in innate and adaptative immunity [START_REF] Rizzu | C9orf72 is differentially expressed in the central nervous system and myeloid cells and consistently reduced in C9orf72, MAPT and GRN mutation carriers[END_REF]. While the viability is affected in several of these models (Burberry et al, 2016;Sudria-Lopez et al, 2016;Atanasio et al, 2016), no specific neuronal defects could be identified. This variability between models suggests that loss of function of C9ORF72 alone is not sufficient for initiating motor neuron disease in higher vertebrates, and that a second injury could be necessary for triggering ALS (Ciura et al, 2016;Nassif et al, 2017;Balendra & Isaacs, 2018a). et al, 2013;Gendron et al, 2013a;Lee et al, 2013b;Mizielinska et al, 2013;Renton et al, 2011b;Zu et al, 2013a;DeJesus-Hernandez et al, 2011b), as well as in lymphoblasts, fibroblasts and induced pluripotent stem cell-differentiated neurons from C9ORF72 expanded cases (Almeida et al, 2013a;Donnelly et al, 2013;Lagier-Tourenne et al, 2013).

Gain of function mechanisms

In vitro studies have shown that GGGGCC RNA form length-dependent G-quadruplex structures [START_REF] Fratta | C9orf72 hexanucleotide repeat associated with amyotrophic lateral sclerosis and frontotemporal dementia forms RNA Gquadruplexes[END_REF][START_REF] Reddy | The disease-associated r(GGGGCC)nrepeat from the C9orf72 gene forms tract length-dependent uni-and multimolecular RNA Gquadruplex structures[END_REF]. Such secondary structures have been proposed

to exert toxicity by sequestering RBPs, and thus mediating their loss of function. The most frequently identified RBPs that interact with C9ORF72 repeat RNA, in human tissues, are the heterogeneous nuclear ribonucleoproteins (hnRNPs), in particular hnRNPAH, hnRNPA1 and hnRNPA3; but also, ADARB2 (double-stranded RNA-specific editase B2), Pur-α, SRSF1

(serine/arginine-rich splicing factor 1), SRSF2 (serine/arginine-rich splicing factor 2) and ALYREF (THO complex sunbunit 4) RBPs have been shown to colocalize with C9ORF72

RNA foci (Donnelly et al, 2013;Lee et al, 2013b;Xu et al, 2013;Cooper-Knock et al, 2014, 2015;[START_REF] Conlon | The C9ORF72 GGGGCC expansion forms RNA G-quadruplex inclusions and sequesters hnRNP H to disrupt splicing in ALS brains[END_REF][START_REF] Davidson | Heterogeneous ribonuclear protein A3 (hnRNP A3) is present in dipeptide repeat protein containing inclusions in Frontotemporal Lobar Degeneration and Motor Neurone disease associated with expansions in C9orf72 gene[END_REF]Hautbergue et al, 2017).

RAN-translation and DPRs in C9ORF72 patients

The third mechanism associated with C9ORF72-mediated pathogenicity in ALS/FTLD is based on the non-ATG dependent (RAN) translation of the aberrant expanded mRNA into polypeptide chains (Ash et al, 2013a;Mori et al, 2013c;Wen et al, 2014;Kwon et al, 2014) (Fig. 11). All the frames in the sense and antisense strands are translated, giving rise to five species of dipeptide repeat (DPR): Glycine-Alanine -poly(GA), Glycine-Proline -poly(GP), Glycine-Arginine -poly(GR), Proline-Alanine -poly(PA) and Proline-Arginine -poly(PR) (Ash et al, 2013a), which commonly appear in neuronal cytoplasmic inclusions (Gendron et al, 2013b;Mori et al, 2013c;Ash et al, 2013b;MacKenzie et al, 2013) (please refer to section 4.3.1. for neuropathological features of C9ORF72 ALS/FTLD patients).

Poly(GP) is the only DPR that is synthesized from both sense and antisense RNA strands (Mori et al, 2013a;Zu et al, 2013a) and represents one of the most abundant DPRs identified in patients (Mackenzie et al, 2015;Balendra & Isaacs, 2018a). Thanks to the availability of excellent antibodies to label poly(GP) in patients, it is currently considered as a potential pharmacodynamic marker in ALS (Su et al, 2014;Gendron et al, 2017). Using a specific immunoassay, researchers have been able to quantitatively detect poly(GP) and poly(GA) in the CNS and in the CSF, but not any of the others DPRs (Su et al, 2014;Gendron et al, 2017).

Interestingly, a recent study demonstrated that poly(GP) levels in the cerebrospinal fluid (CSF) discriminate presymptomatic and symptomatic expansion carriers [START_REF] Meeter | Poly(GP), neurofilament and grey matter deficits in C9orf72 expansion carriers[END_REF], thus

showing the significance of its accumulation within C9ORF72 condition in ALS/FTLD.

Insights from gain of function models

In vitro (Donnelly et C9ORF72 repeat RNA interact with RBPs, supporting the notion that the sequestration of the concerned RBPs has the potential to mediate their loss of function, as well as those of their targets. This could consequently affect the neuronal integrity, due to associated major RNA processing alterations. However, the relative contribution of RNA foci to neurodegeneration is still under debate due to notable inconsistencies regarding their toxicity in animal models.

In vitro, expression of expanded repeats have been associated with nuclear RNA foci and reduced survival, without any detection of DPRs, in primary cortical and motor neurons (Wen et al, 2014). In Drosophila model, overexpression of repeats led to eye and motor neuron degeneration (Xu et al, 2013). Similarly, in zebrafish model, both sense and antisense RNA repeats have been associated with a motor axonopathy (Swinnen et al, 2018). However, in another Drosophila model, neither sense nor antisense repeat RNA led to neurodegeneration (Moens et al, 2018). This last study was conducted in adult Drosophila, with similar repeat lengths to those found in C9ORF72 ALS/FTLD patients, suggesting that differential susceptibility to RNA toxicity could appear according to neuronal maturity status, and/or that the length of the repeat RNA could influence its toxicity.

Also, we cannot exclude that differences between these studies could be due to variation in the sensibility of detection assays for DPRs. Indeed, in Drosophila model, two studies have shown that neurotoxicity of the HRE was attributable to DPRs rather than repeat RNA (Mizielinska et al, 2014;Tran et al, 2015). They observed that the neurotoxicity associated with the repeats was not present when the translation into DPRs was prevented, suggesting that repeat RNA is not toxic by itself. Consistently with these studies, overexpression of DPRs has been associated

with generalized cellular toxicity, in other Drosophila models (Wen et al, 2014;Freibaum et al, 2015;Zhang et al, 2015a;Lee et al, 2016), as well as in vitro (Mizielinska et al, 2014;Wen et al, 2014;Freibaum et al, 2015;Zhang et al, 2015a;Lee et al, 2016;Zhang et al, 2016), and in zebrafish models (Ohki et al, 2017;Swaminathan et al, 2018a).

Among all the DPRs, in vitro and in vivo studies have shown that poly(GR) and poly(PR) are the most toxic (Wen et al, 2014;[START_REF] Yamakawa | Characterization of the dipeptide repeat protein in the molecular pathogenesis of c9FTD/ALS[END_REF][START_REF] Tao | Nucleolar stress and impaired stress granule formation contribute to C9orf72 RAN translation-induced cytotoxicity[END_REF][START_REF] Kanekura | Polydipeptides encoded by the C9ORF72 repeats block global protein translation[END_REF][START_REF] Moens | Genetic models of C9orf72: what is toxic?[END_REF]. Indeed, in zebrafish, poly(GR) expression is associated with developmental and locomotor abnormalities, as well as reduced survival (Swinnen et al, 2018). In Drosophila, these arginine-rich DPRs were associated with a locomotor phenotype and reduced survival due to neurotoxicity (Wen et al, 2014;Mizielinska et al, 2014;Lee et al, 2016;Freibaum et al, 2015;[START_REF] Boeynaems | Drosophila screen connects nuclear transport genes to DPR pathology in c9ALS/FTD[END_REF][START_REF] Yang | FTD/ALS-associated poly(GR) protein impairs the Notch pathway and is recruited by poly(GA) into cytoplasmic inclusions[END_REF][START_REF] Baldwin | Axonal transport defects are a common phenotype in Drosophila models of ALS[END_REF]. [START_REF] Brady | Regulation of TDP-43 aggregation by phosphorylation andp62/SQSTM1[END_REF][START_REF] Scotter | Differential roles of the ubiquitin proteasome system and autophagy in the clearance of soluble and aggregated TDP-43 species[END_REF][START_REF] Wang | Degradation of TDP-43 and its pathogenic form by autophagy and the ubiquitin-proteasome system[END_REF][START_REF] Wang | Autophagy activators rescue and alleviate pathogenesis of a mouse model with proteinopathies of the TAR DNA-binding protein 43[END_REF].

In addition to TDP-43 pathology, a unique hallmark of C9ORF72-mediated disorder is the presence of cytoplasmic star-like inclusions that are positive for SQSTM1/p62 and ubiquitin, and negative for TDP-43, in neurons of cerebellar granule cell layer, hippocampus, neocortex

(p62+ inclusions)

RNA foci Haploinsufficiency

C9ORF72 as an autophagy player

In primary neurons and in human cell lines, knockdown of C9ORF72 induces SQSTM1/p62 (Webster et al, 2016a;Sellier et al, 2016) and TDP-43 accumulation (Sellier et al, 2016), thus recapitulating the neuropathological features presented in the previous section. Consistently with these findings, in induced pluripotent stem cell derived neurons from C9ORF72-related ALS/FTLD patients, basal level of autophagy is reduced (Almeida et al, 2013b;Webster et al, 2016a), and sensitivity to autophagy inhibitors is increased (Almeida et al, 2013b).

Bioinformatic analysis have shown that C9ORF72 has a DENN (Differentially expressed in Normal and Neoplasia) domain that is found in several proteins that are involved in the membrane trafficking machinery, such as the GEF proteins (GDP/GTP exchange factors) (Zhang et al, 2012;Levine et al, 2013a). GEF proteins are able to activate small-GTPases like the Rab family of proteins. Interestingly, in primary neuronal cultures, C9ORF72 has been shown to colocalize with several Rab proteins involved in autophagy trafficking (Farg et al, 2014): Rab1 and Rab5, which participate in autophagy initiation, as well as Rab7 and Rab11, which participate in closure and maturation of autophagosomes [START_REF] Ao | C9orf72 and RAB7L1 regulate vesicle trafficking in amyotrophic lateral sclerosis and frontotemporal dementia[END_REF].

C9ORF72 was also shown to interact with another DENN protein, SMCR8 (Smith-Magenis syndrome chromosome region candidate 8) and with WDR41 (WD-40 repeat 41) (Sellier et al, 2016;Sullivan et al, 2016;Yang et al, 2016a;[START_REF] Amick | C9orf72 binds SMCR8, localizes to lysosomes, and regulates mTORC1 signaling[END_REF]. As a result of this interaction, C9ORF72 acts as a GEF complex for two other Rab proteins, Rab8a and Rab39b (Sellier et al, 2016;Sullivan et al, 2016;Yang et al, 2016a). Interestingly, in the complex C9ORF72/SMCR8/WDR41, SMCR8 is activated through a phosphorylation mediated by TBK1, which is mutated in some ALS/FTLD cases [START_REF] Freischmidt | Haploinsufficiency of TBK1 causes familial ALS and fronto-temporal dementia[END_REF][START_REF] Cirulli | Loss of function of C9orf72 causes motor deficits in a zebrafish model of amyotrophic lateral sclerosis[END_REF], as described above (Fig. 7). The effect of these interactions on autophagy is mediated through an interaction with the ULK1 initiation complex (Webster et al, 2016a;Sellier et al, 2016;Sullivan et al, 2016;Yang et al, 2016a;Jung et al, 2017). In addition, C9ORF72 has been detected in neuronal cytoplasmic puncta that often co-localizes with lysosomes (Frick et al, 2018;[START_REF] Xiao | Isoform-specific antibodies reveal distinct subcellular localizations of C9orf72 in amyotrophic lateral sclerosis[END_REF]Farg et al, 2014;[START_REF] Amick | C9orf72 binds SMCR8, localizes to lysosomes, and regulates mTORC1 signaling[END_REF]Shi et al, 2018).

Hypotheses and objectives

At the beginning of my thesis, C9ORF72 HRE -mediated haploinsufficiency was still very controversial and the role of C9ORF72 in autophagy was still only suspected: basal autophagy had been shown to be reduced in C9ORF72-expanded carriers with ALS/FTLD (Almeida et al, 2013a), and C9ORF72 had just been described as a DENN domain-containing protein (Zhang et al, 2012;Levine et al, 2013a), interacting with some autophagy-related Rab proteins (Farg et al, 2014). We hypothesized that the loss of function of C9ORF72 could impair autophagy pathway in ALS/FTLD pathogenesis. Then, we investigated C9ORF72 function in autophagy.

This was the second objective of my thesis.

We also pointed out the need of new models to study C9ORF72 pathogenicity in ALS. Indeed, a model that integrates both gain and loss of function properties of the C9ORF72 HRE could have the potential to unravel the controversies surrounding this mutation. We hypothesized that if C9ORF72 has actually a role in autophagy pathway, its loss of function could induce defects in the clearance of aggregates, including peptide inclusions from the translation of the HRE (Fig. 13). To test this hypothesis, we developed a new model where both inhibition of C9ORF72

and DPRs expression co-occur. This was the third objective of my thesis.

Peptide inclusions

C9orf72 gDNA

Haploinsufficiency

Gain of function

5.

Beyond ALS: focal epilepsies 5.1. Epilepsies

Clinical features and diagnosis

Epilepsy is the most common chronic neurological disorder as its affects nearly 65 million people worldwide [START_REF] Thurman | Standards for epidemiologic studies and surveillance of epilepsy[END_REF]. It is a very heterogeneous disorder that is characterized by recurrent seizures due to excessive and synchronous electrical discharges in one or several groups of brain cells [START_REF] Newton | The Epilepsies: Seizures, Syndromes and Management[END_REF]. These spontaneous events disrupt the cerebral function and trigger the transient appearance of a wide range of clinical manifestations, depending on the involved region of the brain.

Epilepsy can result from a tumor, a cerebrovascular accident, a severe brain injury or a cerebral infection. Generally, these symptomatic epilepsies respond well to drug or surgical therapies [START_REF] Nevalainen | Epilepsy-related clinical characteristics and mortality: A systematic review and meta-analysis[END_REF].

However, epilepsy may also result from cerebral dysfunctions without any structural lesions but associated with genetic mutations. For these genetic epilepsies, the diagnosis relies on the type of seizures that are identified by electroencephalography, and on molecular genetic testing.

According to International League Against Epilepsy (ILAE), seizures are divided into three categories: generalized, focal and epileptic spasms. Generalized seizures begin in bilateral distributed neuronal networks, whereas focal seizures originate within one cerebral hemisphere [START_REF] Scheffer | ILAE classification of the epilepsies: Position paper of the ILAE Commission for Classification and Terminology[END_REF].

Characteristics of focal epilepsies

Focal seizures, which originate within spatial limited neuronal network, are the most frequent epilepsies [START_REF] Scheffer | ILAE classification of the epilepsies: Position paper of the ILAE Commission for Classification and Terminology[END_REF][START_REF] Chen | Genetic and epigenetic mechanisms of epilepsy: A review[END_REF]. Among familial focal epilepsies, three main

Genetics of focal epilepsies

Focal epilepsies have been proven to be genetically heterogeneous monogenic disorders [START_REF] Picard | DEPDC5 mutations in families presenting as autosomal dominant nocturnal frontal lobe epilepsy[END_REF]. Mutations in the genes CHRNA4 [START_REF] Steinlein | A missense mutation in the neuronal nicotinic acetylcholine receptor α4 subunit is associated with autosomal dominant nocturnal frontal lobe epilepsy[END_REF], CHRNB2 [START_REF] Aridon | Increased Sensitivity of the Neuronal Nicotinic Receptor α2 Subunit Causes Familial Epilepsy with Nocturnal Wandering and Ictal Fear[END_REF] and CHRNA2 [START_REF] Fusco M De | The nicotinic receptor β2 subunit is mutant in nocturnal frontal lobe epilepsy[END_REF], encoding respectively the α4, α2 and β2 subunits of the nicotinic acetylcholine receptor (nAChR) and in the gene encoding for the potassium channel KCNT1 (potassium sodium-activated channel subfamily T member 1) [START_REF] Heron | Missense mutations in the sodium-gated potassium channel gene KCNT1 cause severe autosomal dominant nocturnal frontal lobe epilepsy[END_REF] have linked ion channel subunit genes to ADNFLE. Another gene identified in focal epilepsy is LGI1 that encodes for the neuronal-secreted protein epitempin and has been linked

to ADEAF. The first monogenic cause that has been associated with FFEVF is DEPDC5, encoding for Dishevelled, Egl-10 and Pleckstring (DEP) domain-containing protein 5, whose mutations have been first reported in 2013.

DEPDC5 in focal epilepsies

DEPDC5 mutations

In 2013, two studies revealed that mutations in DEPDC5, were a major cause of FFEVF [START_REF] Ishida | Mutations of DEPDC5 cause autosomal dominant focal epilepsies[END_REF][START_REF] Dibbens | Mutations in DEPDC5 cause familial focal epilepsy with variable foci[END_REF]. These discoveries have opened novel perspectives as DEPDC5

does not encode a membrane ion channel, unlike most other epilepsy genes [START_REF] Picard | DEPDC5 mutations in families presenting as autosomal dominant nocturnal frontal lobe epilepsy[END_REF].

Since then, DEPDC5 mutations have been reported in a wide range of focal epileptic syndromes, including ADNFLE, FMTLE and ADEAF [START_REF] Martin | A recurrent mutation in DEPDC5 predisposes to focal epilepsies in the French-Canadian population[END_REF][START_REF] Picard | DEPDC5 mutations in families presenting as autosomal dominant nocturnal frontal lobe epilepsy[END_REF][START_REF] Ishida | Mutations of DEPDC5 cause autosomal dominant focal epilepsies[END_REF]. While most individuals with DEPDC5-related epilepsy have a normal brain MRI (Magnetic resonance imaging), some have DEPDC5 mutation associated with a cortical malformation, usually focal cortical dysplasia [START_REF] Scheffer | Mutations in mammalian target of rapamycin regulator DEPDC5 cause focal epilepsy with brain malformations[END_REF][START_REF] Baulac | Familial focal epilepsy with focal cortical dysplasia due to DEPDC5 mutations[END_REF][START_REF] Gama | Mammalian target of rapamycin pathway mutations cause hemimegalencephaly and focal cortical dysplasia[END_REF][START_REF] Scerri | Familial cortical dysplasia type IIA caused by a germline mutation in DEPDC5[END_REF], which is characterized by malformation of cortical development and represent the most common cause of severe, drug-resistant childhood epilepsy [START_REF] Blümcke | The clinicopathologic spectrum of focal cortical dysplasias: A consensus classification proposed by an ad hoc Task Force of the ILAE Diagnostic Methods Commission[END_REF]. To date, DEPDC5 mutations are estimated to account for 12 % of cases of familial focal epilepsy [START_REF] Dibbens | Mutations in DEPDC5 cause familial focal epilepsy with variable foci[END_REF].

DEPDC5-related epilepsy is generally inherited in an autosomal dominant manner, although de novo DEPDC5 pathogenic variants have been reported [START_REF] Chen | Genetic and epigenetic mechanisms of epilepsy: A review[END_REF]. Thirty variants of DEPDC5 have been identified and encompass several different regions of the gene; most of them are located in coding exons, except for some mutations that concern splice sites [START_REF] Picard | DEPDC5 mutations in families presenting as autosomal dominant nocturnal frontal lobe epilepsy[END_REF]. The majority (two-thirds) of the mutations in DEPDC5 causes premature codon termination, while the others are missense mutations [START_REF] Dibbens | Mutations in DEPDC5 cause familial focal epilepsy with variable foci[END_REF][START_REF] Ishida | Mutations of DEPDC5 cause autosomal dominant focal epilepsies[END_REF][START_REF] Martin | A recurrent mutation in DEPDC5 predisposes to focal epilepsies in the French-Canadian population[END_REF][START_REF] Picard | DEPDC5 mutations in families presenting as autosomal dominant nocturnal frontal lobe epilepsy[END_REF][START_REF] Scheffer | Mutations in mammalian target of rapamycin regulator DEPDC5 cause focal epilepsy with brain malformations[END_REF], suggesting that DEPDC5 mutations could mediate focal epilepsy through the haploinsufficiency of the gene.

DEPDC5 function and mTOR pathway

DEPDC5 gene is located in chromosome 22q12.2-q12.3 and its transcript is ubiquitously and strongly expressed at rather constant levels in both developing and adult human brain [START_REF] Dibbens | Mutations in DEPDC5 cause familial focal epilepsy with variable foci[END_REF][START_REF] Chen | Genetic and epigenetic mechanisms of epilepsy: A review[END_REF]. It encodes a protein of 1603 amino acids, containing two functional domains involved in protein-protein interaction: DEP and DUF 3964 [START_REF] Ishida | Mutations of DEPDC5 cause autosomal dominant focal epilepsies[END_REF][START_REF] Dibbens | Mutations in DEPDC5 cause familial focal epilepsy with variable foci[END_REF][START_REF] Picard | DEPDC5 mutations in families presenting as autosomal dominant nocturnal frontal lobe epilepsy[END_REF].

DEPDC5 is part of a complex along with NPRL2 (nitrogen permease regulator-like 2) and NPRL3 (nitrogen permease regulator-like 3) [START_REF] Ishida | Mutations of DEPDC5 cause autosomal dominant focal epilepsies[END_REF][START_REF] Dibbens | Mutations in DEPDC5 cause familial focal epilepsy with variable foci[END_REF]. All three proteins form the GATOR1 complex (GAP Activity Toward Rags complex 1), which is a mTORC1 inhibitor (Bar-Peled et al, 2013b;Panchaud et al, 2013a). Similarly, mutations of the other two GATOR1 genes, NPRL2 [START_REF] Scheffer | Mutations in mammalian target of rapamycin regulator DEPDC5 cause focal epilepsy with brain malformations[END_REF] and NPRL3 [START_REF] Sim | Familial cortical dysplasia caused by mutation in the mammalian target of rapamycin regulator NPRL3[END_REF][START_REF] Korenke | Nocturnal frontal lobe epilepsy caused by a mutation in the GATOR1 complex gene NPRL3[END_REF] have been associated with familial focal epilepsies [START_REF] Ricos | Mutations in the mammalian target of rapamycin pathway regulators NPRL2 and NPRL3 cause focal epilepsy[END_REF].

These two genes are estimated to account for 1-2% of focal epilepsy cases, highlighting the GATOR1 complex as a potent genetic actor in focal epilepsy [START_REF] Ricos | Mutations in the mammalian target of rapamycin pathway regulators NPRL2 and NPRL3 cause focal epilepsy[END_REF].

GATOR1 complex acts as an inhibitor of mTORC1 (Bar-Peled et al, 2013b) that is known to regulate numerous functions, including cellular proliferation and survival, transcriptional regulation to environmental cues and proteostasis (also refer to section 2.1.2.4.).

Furthermore, mTORC1 is upregulated in several neurological conditions associated with cortical malformations and seizures (Baulac, 2016). These disorders include focal cortical dysplasia, hemimegalencephaly and tuberous sclerosis complex (TSC). TSC is associated with mutations in the TSC1 and TSC2 genes that encode for inhibitors of mTORC1 (Baulac, 2016;Tee et al, 2002;Thiele, 2004;[START_REF] Webb | On the incidence of fits and mental retardation in tuberous sclerosis[END_REF][START_REF] Osborne | Epidemiology of Tuberous Sclerosis[END_REF]. Overall, these mTORopathies features demonstrate that increased mTORC1 signaling can trigger neuronal hyperexcitability.

Hypothesis and objectives

With one-third of patients with epilepsy showing no significant improvement in current therapeutic treatments, there remains a substantial need for better understanding and treatments for this disease [START_REF] Schmidt | Drug treatment of epilepsy: Options and limitations[END_REF]. DEPDC5 mutations are the most common known cause of familial focal epilepsies [START_REF] Dibbens | Mutations in DEPDC5 cause familial focal epilepsy with variable foci[END_REF][START_REF] Ishida | Mutations of DEPDC5 cause autosomal dominant focal epilepsies[END_REF]. This gene does not encode a membrane ion channel, unlike most of other epilepsy genes. Instead, DEPDC5 forms part of the GATOR1 complex, which functions as a negative regulator of mTOR pathway activity (Bar-Peled et al, 2013a;Panchaud et al, 2013a). Interestingly, hyperactivation of mTOR pathway is dysregulated in several neurological disorders associated with seizures syndromes (Baulac, 2016).

The majority of DEPDC5 mutations have been shown to be associated with haploinsufficiency of the gene, suggesting that loss of function mechanisms could trigger the disease, possibly leading to the hyperactivation of mTOR pathway [START_REF] Dibbens | Mutations in DEPDC5 cause familial focal epilepsy with variable foci[END_REF][START_REF] Ishida | Mutations of DEPDC5 cause autosomal dominant focal epilepsies[END_REF][START_REF] Martin | A recurrent mutation in DEPDC5 predisposes to focal epilepsies in the French-Canadian population[END_REF][START_REF] Picard | DEPDC5 mutations in families presenting as autosomal dominant nocturnal frontal lobe epilepsy[END_REF][START_REF] Scheffer | Mutations in mammalian target of rapamycin regulator DEPDC5 cause focal epilepsy with brain malformations[END_REF]. To test this hypothesis, we therefore aimed to generate and characterized a new vertebrate model and investigate DEPDC5 pathogenicity in epilepsy. This was the fourth objective of my thesis.

Zebrafish model in neurological disorders

General properties of zebrafish model

Zebrafish (Danio rerio) is a vertebrate specie that is increasingly used to model human diseases [START_REF] Seth | The emerging use of zebrafish to model metabolic disease[END_REF] as it exhibits several advantages lending them to be amenable to biomedical research. This model has rapid organ development and tissue formation, with the majority of systems being functional within the two days post fertilization (dpf), including the motor system (Kimmel et al, 1995a;[START_REF] Schmidt | Neurogenesis in zebrafish -from embryo to adult[END_REF]Drapeau et al, 2002). In addition, zebrafish produce large clutches of externally fertilized eggs that develop rapidly and in synchrony, giving rise to large size experimental samples. In addition, the translucent body of zebrafish embryos facilitates in vivo tracking and imaging of fluorescently labeled components or cells (Kimmel et al, 1995a;Kabashi et al, 2010Kabashi et al, , 2011b)).

Over the last decades, genome-wide association studies (GWAS) and sequencing have identified a lot of loci and genes associated with human diseases, thus raising new need for the fast characterization of the role of these genes, and of the pathogenic mechanisms of their dysfunction. A fundamental advantage of zebrafish model in the study of human disorders is that it shares a considerable amount of genetic identity with humans (Howe et al, 2013). The genome of zebrafish is fully sequenced and available, and 84 % of genes known to be associated with human diseases have zebrafish homologues [START_REF] Hjerpe | UBQLN2 Mediates Autophagy-Independent Protein Aggregate Clearance by the Proteasome[END_REF].

To test experimentally the functional relationship between an underlying human mutation and a disease phenotype, many genetic tools are available in zebrafish (with prior verification of sequence and expression pattern similarities between human and zebrafish species): antisense morpholino oligonucleotides (AMOs) that mediates transitory knockdowns (Stainier et al, 2017a;Rossi et al, 2015a;[START_REF] Stainier | Axonal autophagy: Mini-review for autophagy in the CNS[END_REF][START_REF] El-Brolosy | Cognitive changes predict functional decline in ALS: A population-based longitudinal study[END_REF]; but also genome-editing methods (Schmid & Haass, 2013), with TALEN [START_REF] Bedell | In vivo genome editing using a high-efficiency TALEN system[END_REF][START_REF] Sander | Targeted gene disruption in somatic zebrafish cells using engineered TALENs[END_REF][START_REF] Zu | hpf: Hours post fertilization HRE: Hexanucleotide repeat expansion IBM: Inclusion body myopathy ILAE: International League Against Epilepsy LC3: Microtubule-associated protein 1A/1B light chain 3 LIR: LC3-interacting region LMN: Lower motor neurons mTORC1: mammalian target of rapamycin complex 1 Nefl: Neurofilament light chain NMJ: Neuromuscular junction NPRL2: Nitrogen permease regulator-like 2 NPRL3: Nitrogen permease regulator-like 3 OPTN: Optineurin PA: Proline -Alanine PDB: Paget of disease of bone PE: Phosphatidylethanolamine PI(3)P: Phosphatidylinositol 3-phosphate PI3KC3: Class III phosphatidylinositol 3-kinase complex 3 PR: Proline -Arginine PRAS40: 40 kDa Pro-rich AKT substrate RAN-translation: Non-ATG dependant translation RAPTOR: Regulatory-associated protein of mTOR ROS: Reactive oxygen species sALS: Sporadic ALS SG: Stress granules SMCR8: Smith-Magenis syndrome chromosome region candidate 8 SOD1: Superoxide dismutase 1 SQSTM1: Sequestosome 1/p62 STEX: Senataxin TARDBP: TAR DNA binding-protein TBK1: Tank binding Kinase 1 TDP-43: TAR DNA-binding protein 43 TEER: Touched evoked escape response TSC: Tuberous sclerosis complex UBA(N): Ubiquitin binding domain UBQLN2: Ubiquilin ULK1: UNC51-like Ser/Thr kinases 1 UMN: Upper motor neurons UPS: Ubiquitin proteasome system VCP: Valosin-containing protein WDR41: WD-40 repeat 41[END_REF] and CRISPR-Cas systems [START_REF] Hjerpe | UBQLN2 Mediates Autophagy-Independent Protein Aggregate Clearance by the Proteasome[END_REF][START_REF] Hwang | Efficient genome editing in zebrafish using a CRISPR-Cas system[END_REF][START_REF] Jao | Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system[END_REF], as the most recent.

Another major advantage of zebrafish embryos is that it is the only vertebrate organism amenable for high-throughput screening for toxic or therapeutic drugs library, as it is permeable for small molecules in its medium during early stages of development, and small enough to fit in 96-well plates [START_REF] Zon | In vivo drug discovery in the zebrafish[END_REF].

In addition, behavioral analysis in genetic or chemical models are easily accessible in zebrafish embryos. Zebrafish embryos exhibit early stereotyped movement patterns, as soon as 17 hpf, consisting of repetitive tail flicks and complete rotations in their chorion. Later, at 48 hpf, embryos have tactile response abilities that can be used to quantify motor parameters (Drapeau et al, 2002).

Zebrafish as a vertebrate model of ALS

Over the last two decades, zebrafish has been used to model genetic aspects of several major neurodegenerative diseases, including ALS (Kabashi et al, 2011c;Xi et al, 2011;Baxendale et al, 2017). There are now several studies using the zebrafish as an in vivo model for investigating the pathogenesis of ALS (Kabashi et al, 2010(Kabashi et al, , 2011c;;[START_REF] Babin | Zebrafish models of human motor neuron diseases: Advantages and limitations[END_REF].

Knockdown of the zebrafish c9orf72 led to axonopathy of the motor neurons and specific alteration of the locomotion (Ciura et al, 2013b). Mutations in zebrafish sod1 have been shown

to cause motor neuron loss, and transgenic zebrafish overexpressing human mutant SOD1 display hallmarks of ALS, including loss of motor neurons and defective motor ability [START_REF] Lemmens | Overexpression of mutant superoxide dismutase 1 causes a motor axonopathy in the zebrafish[END_REF][START_REF] Sakowski | Neuromuscular effects of G93A-SOD1 expression in zebrafish[END_REF]. Both overexpression of human variant of TARDBP and knockdown of zebrafish tardbp produce motor neuronal axonopathy in zebrafish embryos [START_REF] Kabashi | Gain and loss of function of ALS-related mutations of TARDBP (TDP-43) cause motor deficits in vivo[END_REF]. Similarly, both gain and loss of function mechanisms of FUS yielded a specific phenotype, and WT human FUS is able to rescue tardbp knockdown phenotype in zebrafish, thus suggesting a common pathogenic pathway for these genes [START_REF] Armstrong | Loss and gain of FUS function impair neuromuscular synaptic transmission in a genetic model of ALS[END_REF]Kabashi et al, 2011a).

Moreover, zebrafish represents a suitable model for the screening of chemicals, in order to identify those that specifically affect motor neurons. Similarities in motor neurons phenotypes between genetic and chemical models is a good opportunity for understanding how genetic factors interact with the environment. This is particularly interesting in ALS research since 90% of sporadic cases are idiopathic [START_REF] Chia | Novel genes associated with amyotrophic lateral sclerosis: diagnostic and clinical implications[END_REF][START_REF] Ji | Genetics insight into the amyotrophic lateral 200 sclerosis/frontotemporal dementia spectrum[END_REF]. Drug screening in characterized genetic zebrafish models is also a relevant tool to identify new therapeutic drugs. For example, methylene blue has been shown to be able to rescue the ALS-like phenotype associated with mutant TARDBP and FUS overexpression, through decreased ER stress response [START_REF] Vaccaro | Three families with amyotrophic lateral sclerosis and frontotemporal dementia with evidence of linkage to chromosome 9p[END_REF][START_REF] Vaccaro | Pharmacological reduction of ER stress protects against TDP-43 neuronal toxicity in vivo[END_REF]).

An advantage of studying ALS in zebrafish consists in the rapid development of the spinal cord, with motor neurons starting their axogenesis as early as 20 hpf. By 2 dpf, motor neurons have undergone their axogenesis and reached their muscle targets [START_REF] Levine | The product of C9orf72, a gene strongly implicated in neurodegeneration, is structurally related to DENN Rab-GEFs[END_REF]Myers et al, 1986a). A good tool to study ALS pathogenic mechanisms, at motor neuron level in zebrafish, is a transgenic line expressing a fluorescent reporter in spinal motor neurons, thus allowing the observation of cell bodies and axonal arborization of single motor neurons in alive animals (Knafo et al, 2017) (Fig. 14).

As previously described, autophagy has a central role in ALS pathogenicity. Methods to study and monitoring this pathway have been described in zebrafish [START_REF] Klionsky | Guidelines for the use and interpretation of assays for monitoring autophagy. Autophagy Klionsky DJ[END_REF][START_REF] Fodor | Methods to Study Autophagy in Zebrafish[END_REF]. The detection of autophagy activity can be achieved at multiple levels, including both molecular and cellular levels, since the majority of the genes and the structures involved in autophagy are highly conserved from zebrafish to humans [START_REF] Varga | Autophagy in zebrafish[END_REF] (Table 2).

Different strategies to study autophagy in zebrafish have been validated, including quantification of autophagy markers by western blot, microscopy of fluorescent autophagy reporters, electron microscopy, silencing of autophagy-related genes and drug screening of autophagy activators and/or inhibitors [START_REF] Mostowy | The Zebrafish as a New Model for the In Vivo Study of Shigella flexneri Interaction with Phagocytes and Bacterial Autophagy[END_REF]He & Klionsky, 2010;[START_REF] Fodor | Methods to Study Autophagy in Zebrafish[END_REF]. midbrain, hindbrain and spinal cord have formed and connected (Kimmel et al, 1995a;[START_REF] Schmidt | Neurogenesis in zebrafish -from embryo to adult[END_REF].

In zebrafish, epilepsy can manifest by general hyperactivity, with convulsive seizure-like behavior, which has been first characterized in PTZ-treated embryos, PTZ (pentylenetetrazol) being a pro-convulsive compound (Baraban et al, 2005). This behavior is characterized, since early stages of development, by a whirl pool-like circling around the well, and possibly more severe behavior, with embryos exhibiting high-speed head-to-tail convulsions (Baraban et al, 2005;Griffin et al, 2016). Furthermore, zebrafish genetic models of epilepsy, as any animal model of epilepsy, must be validated by identification of abnormal electrical discharges by electrophysiological recordings (Baraban, 2013).

As an example, zebrafish embryos of a mutant line carrying a mutation in the gene scn1

(encoding for sodium voltage-gated channel alpha subunit 1), which is associated with an epileptic disorder called Dravet syndrome (DS), recapitulate these typical electrophysiological and motor characteristics of epilepsy (Baraban et al, 2013). These seizure-like behaviors have also been reported from other genetic zebrafish models of epilepsy, using AMO-based knockdown approaches of the scn1 gene (Zhang et al, 2015b), and the lgi1a gene that has been associated with TLE [START_REF] Teng | Knockdown of zebrafish lgi1a results in abnormal development, brain defects and a seizure-like behavioral phenotype[END_REF]. Thanks to the possibility of high-throughput screening in zebrafish embryos, several compounds have been tested in the scn1 mutant line, leading to the identification of clemizole as a new antiepileptic drug (AED) (Baraban et al, 2013).

1.

Development of a Sqstm1 zebrafish model of ALS 1.1.

Objectives and overview

Mutations in SQSTM1, encoding for the protein SQSTM1/p62, have been reported in 1-3.5%

of patients with ALS/FTLD (Fecto et al, 2011b). SQSTM1/p62 is a key player of autophagy pathway, as it is involved in the selective recognition and the targeting of autophagy substrates toward autophagy degradation mechanisms (Majcher et al, 2015b;[START_REF] Pankiv | 62/SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of ubiquitinated protein aggregates by autophagy[END_REF]. This pathway has been shown to be critical for neuronal survival and is particularly involved in ALS pathogenesis [START_REF] Navone | Autophagy and Neurodegeneration: Insights from a Cultured Cell Model of ALS[END_REF][START_REF] Mis | Autophagy in motor neuron disease: Key pathogenetic mechanisms and therapeutic targets[END_REF][START_REF] Budini | Autophagy and Its Impact on Neurodegenerative Diseases: New Roles for TDP-43 and C9orf72[END_REF]Nguyen et al, 2018). We hypothesized that SQSTM1 mutations in ALS could mediate ALS/FTLD phenotype through the haploinsufficiency of the gene and its loss of function in autophagy. As previously discussed, zebrafish is a relevant model to study ALS pathogenicity, as well as to monitor autophagy flux.

In order to investigate the pathogenic mechanisms induced by SQSTM1 mutations in ALS/FTLD, we developed a zebrafish model that implied SQSTM1 loss of function.

Knockdown of the sqstm1 zebrafish orthologue, as well as impairment of its splicing, led to an ALS-like phenotype, typically consisting of behavioral and axonal abnormalities. We reported swimming deficits associated with shorter motor neuronal axons that could be rescued by the overexpression of wild-type human SQSTM1. Interestingly, no rescue of the loss of function phenotype was observed when overexpressing human SQSTM1 constructs carrying ALS/FTLD-related mutations.

Consistent with its role in autophagy regulation, we found increased mTOR levels upon knockdown of sqstm1. Furthermore, treatment of zebrafish embryos with rapamycin, a known inhibitor of the mTOR pathway, and therefore an activator of autophagy, yielded an amelioration of the locomotor phenotype in the sqstm1 knockdown model. Overall, our results imply that SQSTM1 pathogenicity in ALS is associated with its loss of function that mediates a misregulation of autophagic process.

1.2.

Article 1: Sqstm1 knockdown causes a locomotor phenotype ameliorated by rapamycin in a zebrafish model of ALS/FTLD.

2.

Exploring the role of C9ORF72 in autophagy

Objectives

At the time of these experiments (in 2015), the role of C9ORF72 in autophagy was still only suspected. Basal autophagy had been shown to be reduced in C9ORF72-expanded carriers with ALS/FTLD (Almeida et al, 2013a), and C9ORF72 had just been described as a DENN domaincontaining protein (Zhang et al, 2012;Levine et al, 2013a) that interacts with some autophagyrelated Rab proteins (Farg et al, 2014). Conversely, the function of SQSTM1 in autophagy initiation was well established [START_REF] Duran | P62 Is a Key Regulator of Nutrient Sensing in the mTORC1 Pathway[END_REF][START_REF] Johansen | Exome Sequencing Reveals VCP Mutations as a Cause of Familial ALS[END_REF][START_REF] Rea | New insights into the role of sequestosome 1/p62 mutant proteins in the pathogenesis of paget's disease of bone[END_REF]. As described in the previous part, the knockdown of sqstm1 orthologue in zebrafish led to locomotor defects associated with shorter motor neuronal axons (Lattante et al, 2015a). In another ALS zebrafish model that was developed in the team, a similar phenotype was obtained under c9orf72 orthologue inhibition (Ciura et al, 2013a). Thus, we get interested in what happens, at motor neurons levels specifically, when C9orf72 and Sqstm1 are depleted in these cells, both in vivo and in vitro. For this purpose, I had the great opportunity to do a three months internship in the team of Professor Heather Durham, at the Montreal Neurological Institute of

McGill University, where I learnt a unique technique of microinjection in cultured murine motor neurons, that has been established by this lab [START_REF] Tradewell | Calcium dysregulation, mitochondrial pathology and protein aggregation in a culture model of amyotrophic lateral sclerosis: Mechanistic relationship and differential sensitivity to intervention[END_REF][START_REF] Tradewell | Calpastatin reduces toxicity of SOD1G93A in a culture model of amyotrophic lateral sclerosis[END_REF][START_REF] Tibshirani | Cytoplasmic sequestration of FUS/TLS associated with ALS alters histone marks through loss of nuclear protein arginine methyltransferase 1[END_REF][START_REF] Tibshirani | Dysregulation of chromatin remodelling complexes in amyotrophic lateral sclerosis[END_REF]. I used this cellular model to examine the functional impacts of ALS-associated C9orf72 and Sqstm1 haploinsufficiency on basal autophagy, in motor neurons.

2.2.

C9orf72 and sqstm1 epistatic interactions in zebrafish 2.2.1.

C9orf72 and sqstm1 belong to the same pathway

We hypothesized that SQSTM1 and C9ORF72 operate through a common genetic pathway. To examine this possibility, we used a classical epistatic interaction strategy, testing whether partial knockdown of sqstm1 and c9orf72 can have additive effect in zebrafish.

In that purpose, we used subphenotypic doses of specific atg-AMOs for each of sqstm1 and c9orf72 genes. In practice, we injected half the effective doses of AMOs that were previously identified in sqstm1 and c9orf72 loss of function models (Lattante et al, 2015b;Ciura et al, 2013a), within zebrafish one cell-stage embryos. We used two specific control AMOs containing mismatch nucleotides with the respective c9orf72-atg and sqstm1-atg AMOs sequences, that does not bind anywhere in the zebrafish genome. As expected, none of c9orf72 In addition, the distribution of phenotypes per condition represented in Fig. 2 shows the additive effect of sqstm1 and c9orf72 partial knockdowns on zebrafish motor behavior. Overall, these features are specific of ALS zebrafish models, as previously reported (Ciura et al, 2013a;Lattante et al, 2015b;[START_REF] Kabashi | Gain and loss of function of ALS-related mutations of TARDBP (TDP-43) cause motor deficits in vivo[END_REF]Kabashi et al, , 2011aKabashi et al, , 2011b)).

C9orf72 acts downstream than sqstm1

The additive effect of subtoxic doses indicates that c9orf72 and sqstm1 could be acting on a common pathway. If this is the case, then overexpressing the gene that is more downstream in this shared pathway would restore a phenotype caused by the loss of the upstream gene. We tested this by investigating whether overexpressing either C9ORF72 or SQSTM1 could rescue the knockdown of the other gene in the zebrafish model. As previously described, c9orf72 knockdown, with c9orf72-atg AMO, and sqstm1 knockdown, with sqstm1-atg AMO, are able to induce specific motor phenotype in zebrafish (Ciura et al, 2013a;Lattante et al, 2015b). As a matter of fact, by injecting the effective dose of c9orf72-atg AMO or the effective dose of sqstm1-atg AMO, TEER results of 48 hpf embryos show typical decreased distance (Fig. 3 a) and velocity (Fig. 3 b), as well as shortened swimming projections (Fig. 3 c). These motor phenotypes have been shown to be specifically rescued by the respective human orthologue mRNAs: both C9ORF72 long (C9-L) and short (C9-S) isoforms human mRNAs (hRNAs) are able to rescue c9orf72 loss of function in zebrafish, and SQSTM1 hRNA is able to rescue sqstm1 loss of function in zebrafish (Ciura et al, 2013a;Lattante et al, 2015b).

Then, we investigated whether SQSTM1 hRNA overexpression was able to rescue c9orf72 loss of function. In that purpose, we co-injected c9orf72-atg AMO along with SQSTM1 hRNA, but we were unable to rescue the motor phenotype, as shown by TEER results (Fig. Representations of swimming trajectories (Fig. 3 c), as well as phenotype distribution among conditions (Fig. 4), also illustrate the potential of C9ORF72 isoforms to rescue sqstm1 loss of function, while the opposite is not true.

These results reveal that a genetic interaction between c9orf72 and sqstm1 exists, and that c9orf72 is a downstream effector of sqstm1 in their common pathway, in ALS pathogenesis.

2.3.

Insights from primary murine motor neurons 2.3.1.

C9orf72 and Sqstm1 knockdowns induce motor neuron degeneration

We used specific shRNAs to knockdown C9orf72 and Sqstm1 mouse orthologues in motor neurons of spinal cord -dorsal root ganglion (DRG) cultures. To determine the impact of C9orf72 and Sqstm1 knockdowns on motor neurons survival, we co-injected shRNA-containing plasmid along with a fluorescent dye. Injected motor neurons were counted from 1 day-postinjection (dpi) until 5 dpi. We counted fluorescent motor neurons and we observed early and drastic decreases in the survival of motor neurons where C9orf72 or Sqstm1 were inhibited, starting on the first day after the injection, when compared to the scramble control shRNA (Fig. 5). These results are in line with the hypotheses of loss of function mechanisms that induce motor neuronal degeneration for C9ORF72 and SQSTM1-mediated ALS/FTLD pathogenesis.

Furthermore, we wondered if C9orf72 or Sqstm1 depletion is associated with any defects in motor neuron morphology. We quantified different parameters in the surviving motor neurons, such as soma area (Fig. 6), but we did not observe any difference when compared to the scramble shRNA condition, when C9orf72 or Sqstm1 were depleted.

Loss of function of C9orf72 affects autophagy

Our results in zebrafish indicate that C9ORF72 shares a common pathway with SQSTM1. We hypothesized that this pathway could be autophagy. Accordingly, C9ORF72-related haploinsufficiency may contribute to motor neuron degeneration by the loss of function of the role of C9ORF72 in autophagy.

To test this directly, we reduced the expression of C9orf72 in motor neurons of mice spinal cord -DRG cultures and determined the effect of C9orf72 depletion on autophagy by monitoring microtubule-associated protein 1A/1B light chain (LC3), thanks to a plasmid containing a LC3-eGFP construct. We quantified autophagy flux by counting LC3-eGFPpositive puncta per region of interest, thus reflecting the number of autophagosomes [START_REF] Klionsky | Guidelines for the use and interpretation of assays for monitoring autophagy. Autophagy Klionsky DJ[END_REF]. In parallel, we also co-injected this LC3-eGFP plasmid along with Sqstm1 shRNA that was used as a positive control, since its role in autophagy initiation is established [START_REF] Duran | P62 Is a Key Regulator of Nutrient Sensing in the mTORC1 Pathway[END_REF][START_REF] Johansen | Exome Sequencing Reveals VCP Mutations as a Cause of Familial ALS[END_REF][START_REF] Rea | New insights into the role of sequestosome 1/p62 mutant proteins in the pathogenesis of paget's disease of bone[END_REF].

In control condition, LC3-eGFP puncta were visible in almost all injected motor neurons (data not shown), which is consistent with the fact that these specific cells undergo basal autophagy Overall, these results show that C9ORF72 and SQSTM1 are required to maintain basal autophagy, which is known to be essential to maintain survival in neurons (Hara et

Material and methods

Zebrafish Maintenance

Adult and larval zebrafish (Danio rerio) were maintained at the ICM (Institut du Cerveau et de la Moelle épinière, Paris) fish facility and bred according to the National and European

Guidelines for Animal Welfare. Experiments were performed on wild type and transgenic embryos from AB and TL strains. Zebrafish were raised in embryo medium: 0.06 g/L aquarium salt (Instant Ocean, Blacksburg, VA) in reverse osmosis water +0.01 mg/L methylene blue.

Experimental procedures were approved by the National and Institutional Ethical Committees.

Embryos were staged in terms of hours post fertilization (hpf) based on morphological criteria (Kimmel et al, 1995) and manually dechorionated using fine forceps at 24 hpf.

Microinjection of oligonucleotides and RNA into zebrafish embryos

The microinjections were carried out at one cell stage. Antisense morpholino oligonucleotides (AMOs) were used to knockdown the expression of the c9orf72 and sqstm1 genes. The AMOs were designed to bind with complementary to the ATG of the C9orf72 and the Sqstm1 orthologues in zebrafish, and synthetized from GeneTools (Philomath, USA). The sequences of the C9orf72-atg AMO and of the Sqstm1-atg AMOs are respectively: 5'-ATTGTGGAGGACAGGCTGAAGACAAT-3' and 5'-ATGAAGAGACGGAAAGTGTCATCCT-3'. Control AMOs (mismatch), containing five mismatch nucleotides with the respective C9orf72 and Sqstm1 -atg AMO sequences, and not binding anywhere in the zebrafish genome, were used to assess the specificity of the observed phenotypes (respectively 5'-ATTcTcGAGcACAGcCTcAAGACAT-3' and 5'-ATcAAcAGACGGAAAcTcTCATCCT-3'). We used the phenotypic dose of 0.4 mM or a subphenotypic dose of 0.2 mM for the C9orf72-atg AMO and its respective mismatch AMO (Sellier et al, 2016), and the phenotypic dose of 0.6 mM or a subphenotypic dose of 0.3 mM for Sqstm1-atg AMO and its respective mismatch AMO (Lattante et al, 2015a). For rescue experiments, mRNA encoding human long C9-L and short C9-S variants were microinjected at a concentration of 100 ng/µl, as previously described (Ciura et al, 2013a), and mRNA encoding human Sqstm1 variant was microinjected at a concentration of 100 ng/µl. Optimal mRNA concentrations were determined as the point on the toxicity curve where there was no significant increase in the percentage of dead and morphologically deformed larvae. Human wild-type SQSTM1, C9-L and C9-S RNAs were cloned into a pCS2 + and Not1-linearized. RNA purification was performed using the Ambion kit (Invitrogen).

Locomotion analysis of zebrafish embryos

48 hpf zebrafish embryos were touched slightly at the tail with a pipette tip to perform the touched evoked escape response (TEER) test, as previously described (Ciura et al, 2013a;Lattante et al, 2015a;Sellier et al, 2016;de Calbiac et al, 2018a). Their behavior was sorted between dead, developmentally abnormal, having a specific motor phenotype and normal, in order to analyze the phenotype distribution of each set of experiment. In morphologically normal embryos, TEER responses were recorded using a Grasshopper 2 camera (Point Grey Research, Canada), at 30 frames per second, and the trajectories were traced using the videotracking plugin of ImageJ 1.47 (Sun Microsystems, USA) software. The distance and the velocity were quantified frame by frame from the path projections.

Dissociated spinal cord -DRG cultures

Spinal cord-DRG cultures were prepared from embryonic day 13 (E13) CD1 mouse embryos (Charles River Laboratories, Wilmington, MA). Spinal cords with attached DRGs were collected in a 60 mm dish containing 1 mL of dissecting medium (40 g/L sucrose, 1 g/L dextrose, 2,4 g/L HEPES dissolved in 1X Dulbecco's PBS). Tissues were cut with a scalpel, followed by addition of 300 μL of 10X trypsin in 2 mL dissecting medium and 30 min incubation at 37°C. Then, samples were transferred to plating medium : "N3 medium" (composition : insulin 10 µg/mL (Sigma-Aldrich, Canada Ltd., Oakville, ON), apotransferrin 200 µg/mL (Sigma-Aldrich), bovine serum albumine 10 µg/mL (Invitrogen Life Technologies, Burlington, ON), putrescine 32 µg/mL (Sigma-Aldrich), selenium 26 ng/mL, triiodothyronine 20 ng/mL (Sigma-Aldrich), hydrocortisone 9.1 ng/mL (Sigma-Aldrich), progesterone 13 ng/mL (Sigma-Aldrich), 1,5% horse serum (Invitrogen) supplemented with 1% penicillin/streptomycin/neomycin (Invitrogen) and 1% fetal bovine serum (Invitrogen).

Microinjections of plasmids into motor neurons

Microinjections were performed on spinal cord-DRG cultures from 3 to 8 weeks after dissection. At these stages, motor neurons are distinguishable from other neuronal cell types as they have much larger cell bodies (> 20 µm) and highly-branched dendrites [START_REF] Roy | Glutamate potentiates the toxicity of mutant Cu/Zn-superoxide dismutase in motor neurons by postsynaptic calcium-dependent mechanisms[END_REF].

Injectate is prepared by diluting plasmids DNA in a solution of 20 mg/mL dextran in 50% Tris-EDTA (TE). For experiments with sole shRNAs plasmids (without LC3-eGFP), where injection markers are necessary, dextran conjugated to fluorescent isothiocyanate (FITC; Invitrogen Life Technologies, Burlington, ON) was used. Needles were prepared with a Narishige PN-3 microelectrode puller (Narishige International INC., NY, USA), using 1 mm diameter quick fill glass capillaries (World Precision Instruments, Sarasota, FL). Prior to microinjection, coverslips were placed in a dish containing pre-warmed "injection buffer" (composition:

Eagle's Minimum Essential Medium (EMEM) without bicarbonate (Invitrogen Life Technologies, Burlington, ON), supplemented with 5 g/L glucose, and pH adjusted to 7,4). The dish was placed on the stage of a Zeiss Axiovert 35 microscope (Carl Zeiss Microscopy, LLC, USA) and microinjection was accomplished at 400X magnification using an Eppendorf 5246 and an Eppendorf 5171 micromanipulator (Eppendorf, Hamburg, Germany). After injection, the coverslips were placed in a solution of 0,75% gentamicin in culture medium to prevent bacterial growth and cultures were incubated at 37°C in 4% CO2. In survival experiments, the number of motor neurons was expressed as a percentage of those injected.

Plasmids containing MISSION® shRNA directed toward coding regions of mouse orthologues of C9orf72 (3110043 O21 Rik) or Sqstm1 mRNAs were purchased from Sigma-Aldrich (Canada Ltd., Oakville, ON, respective references : SHCLND-NM_001081343 and SHCLND-NM_011018, the sequences are not disclosed by the supplier). A control plasmid containing a "scramble shRNA" that does not target any known genes from any species was used as a negative control (Sigma-Aldrich reference SHC002). Each MISSION® shRNA is constructed within the plasmid vector pLKO.1-pure and was microinjected at 20 ng/µL, diluted in dextran 20 mg/mL as described above. Plasmid encoding for LC3-eGFP was provided by Durham's lab (Montreal Neurological Institute, McGill University, Canada) and was injected at 20 ng/µL.

Survival analysis

Motor neurons counting was done from 1 day post-injection (dpi) to 5 dpi. Cells were analyzed a minimum of 24 hours after the injection to ensure that no motor neurons that were lethally damaged by the microinjection procedure were included in the experiment.

Autophagosomes counting and somas areas measurement

Coverslips were fixed with 4% paraformaldehyde at 1 dpi stage and all LC3-eGFP injected motor neurons were captured with an Apotome.2 system and an Imager.M2 stand (Carl Zeiss, Germany), with a 63x objective. Autophagosomes numbers were calculated using the ITCN plugin of ImageJ 1.47 (Sun Microsystems, USA). Images were reversed in order that LC3-eGFP puncta can be properly detected. For morphological analysis, somas outlines were traced and their area was calculated using ImageJ 1.47.

Data analysis

In zebrafish experiments Three to twelve clutches per condition were used and thirty to one hundred and seventy-three embryos were analyzed per condition. Microinjections, TEER test and video analysis were done randomly and blindly. In spinal cord -DRG cultures Cultures from three different dissections were used. Microinjections, surviving motor neurons counting and images analysis were carried out randomly and blindly. Fifty motor neurons were injected per coverslip. Survival analysis was done on nine coverslips per condition. Autophagosomes counting was done on three coverslips per condition. degradation process (Mackenzie et al, 2014a;[START_REF] Vatsavayai | C9ORF72-FTD/ALS pathogenesis: evidence from human neuropathological studies[END_REF]. In the previous part, we showed that C9ORF72 and SQSTM1 genetically interact and that C9ORF72, as well as SQSTM1, are essential to maintain basal autophagy and survival in motor neurons. In the introduction, we described that autophagy is a key mechanism in ALS pathogenesis [START_REF] Navone | Autophagy and Neurodegeneration: Insights from a Cultured Cell Model of ALS[END_REF][START_REF] Mis | Autophagy in motor neuron disease: Key pathogenetic mechanisms and therapeutic targets[END_REF][START_REF] Budini | Autophagy and Its Impact on Neurodegenerative Diseases: New Roles for TDP-43 and C9orf72[END_REF]Nguyen et al, 2018).

Loss and gain of function mechanisms synergize in the

To develop a vertebrate model of C9ORF72 HRE pathogenicity that would recapitulate both gain and loss of function properties, we simultaneously induced the partial knockdown of c9orf72 and expressed plasmids encoding for the DPRs in zebrafish embryos. We found out that the loss of function of c9orf72 is essential to trigger DPRs accumulation in zebrafish.

Focusing on poly(GP), we observed that its accumulation is associated with a robust motor phenotype with embryos losing their ability to swim. These embryos display severe motor neurons abnormalities with aborted axogenesis and motor neuron loss. These phenotypes can be rescued by the inhibition of caspase 9, an essential regulator of apoptosis. In addition, we were able to phenocopy c9orf72 loss of function by knocking-down sqstm1. Also, inducing autophagy in zebrafish embryos by rapamycin treatment leads to an improvement of the clearance of poly(GP), associated with a restored swim and motor neurons growth, thus confirming the role of c9orf72 in autophagy.

These results indicate that DPRs toxicity is related to lowered expression of c9orf72 suggesting that both gain and loss of function synergize in the C9ORF72 HRE pathogenicity, thus reconciling the two hypotheses for the first time in vivo.

Article 2: Synergistic gain and loss of function properties of the C9ORF72

mutation induce motor neuron degeneration in vivo.

Under review

Synergistic gain and loss of function properties of the C9ORF72 mutation induce motor neuron degeneration in vivo

Hortense de Calbiac 1 , Maria-Letizia Campanari 1 , Doris Lou Demy 1 , Anca Marian 1 , Sorana Ciura 1,2 and Edor Kabashi 1,2 .

INTRODUCTION

Amyotrophic Lateral Sclerosis (ALS) is the most common form of motor neuron disease, affecting 0.3-3.6:100000 worldwide [START_REF] Kabashi | In the swim of things: Recent insights to neurogenetic disorders from zebrafish[END_REF]2]. ALS is characterized by the progressive degeneration of spinal and cortical motor neurons, muscle wasting and paralysis. This disorder shares common clinical and pathological features with another major neurodegenerative disease, Frontotemporal Lobar Degeneration (FTLD), a progressive dementia resulting from the atrophy of the frontal and temporal lobes [3]. A major breakthrough in understanding the genetics of ALS and FTLD was the discovery of a GGGGCC hexanucleotide repeat expansion (HRE) within the non-coding region of chromosome 9 open reading frame 72 (C9ORF72) as the most common mutation in both familial and sporadic forms of ALS-FTLD [4,5].

The pathogenic processes associated with the HRE remain a hotly debated topic in the field, as both gain and loss of function mechanisms have been proposed as principal causes of disease.

A loss-of-function mechanism has been supported by numerous reports documenting the reduction of C9ORF72 transcript in heterozygous carriers of the mutation [4][5][6][7][8][9][10]. Increased methylation of the peri-promoter region of the C9ORF72 gene [11,12] or abnormal binding of try-methylated histones onto the HRE [13] in ALS and FTLD patients carrying the C9ORF72 mutation could be responsible for the reduction in transcript levels. Moreover, a recent study has shown that CNS areas with reduced C9ORF72 expression correlate with the extent of disease pathology [14]. In concordance with a direct correlation between C9ORF72 transcript levels and disease penetrance, elevated C9ORF72 expression was correlated to a survival advantage in ALS [15]. Knock-down of the c9orf72 orthologue expression in zebrafish [16], as well deletion of the c9orf72 orthologue in C.elegans [17] has been shown to be associated with major motor neuron deficits. However, in mouse models, C9orf72 deletion leads to major defects in the myeloid tissue [18][19][20][21]. While the viability is affected in several of these models [18,19,21], no specific motor phenotype or loss of motor neurons could be identified, suggesting that loss of function of c9orf72 alone is not sufficient for initiating motor neuron disease in higher vertebrates and that a second injury is necessary for triggering ALS [22,23].

Gain of function mechanisms engendered by the C9ORF72 HRE mutation have also been associated with its pathogenicity. Aberrantly expanded C9ORF72 hexanucleotide repeats are transcribed into RNA species which can accumulate in nuclear foci [4,9,24,25]. Indeed, sense and antisense RNA foci have been observed in the CNS of patients with C9FTLD/ALS [4,5,9,[26][27][28][29][30]. Although these RNA foci have been proposed to exert toxicity by sequestering RNA-binding proteins [9,10,24,26,28,[31][32][33][34][35], their relative contribution to neurodegeneration is still under debate due to notable discrepancies regarding their toxicity in animal models [START_REF] Xu | Expanded GGGGCC repeat RNA associated with amyotrophic lateral sclerosis and frontotemporal dementia causes neurodegeneration[END_REF][START_REF] Swinnen | A zebrafish model for C9orf72 ALS reveals RNA toxicity as a pathogenic mechanism[END_REF][START_REF] Moens | Sense and antisense RNA are not toxic in Drosophila models of C9orf72-associated ALS/FTD[END_REF][START_REF] Tran | Differential Toxicity of Nuclear RNA Foci versus Dipeptide Repeat Proteins in a Drosophila Model of C9ORF72 FTD/ALS[END_REF][START_REF] Burguete | GGGGCC microsatellite RNA is neuritically localized, induces branching defects, and perturbs transport granule function[END_REF]. C9ORF72 HRE-containing RNAs are further translated by non-canonical self-priming mechanisms into polypeptides chains [START_REF] Ash | Unconventional Translation of C9ORF72 GGGGCC Expansion Generates Insoluble Polypeptides Specific to c9FTD/ALS[END_REF][START_REF] Mori | The C9orf72 GGGGCC repeat is translated into aggregating dipeptide-repeat proteins in FTLD/ALS[END_REF][START_REF] Wen | Antisense proline-arginine RAN dipeptides linked to C9ORF72-ALS/FTD form toxic nuclear aggregates that initiate invitro and invivo neuronal death[END_REF][START_REF] Kwon | Poly-dipeptides encoded by the C9orf72 repeats bind nucleoli, impede RNA biogenesis, and kill cells[END_REF]. All the frames in the sense and antisense strands are translated, giving rise to five distinct dipeptide repeats (DPR): Glycine-Alanine -poly(GA), Glycine-Proline -poly(GP), Glycine-Arginine -poly(GR), Proline-Alanine -poly(PA) and Proline-Arginine -poly(PR) [START_REF] Ash | Unconventional Translation of C9ORF72 GGGGCC Expansion Generates Insoluble Polypeptides Specific to c9FTD/ALS[END_REF]. In patient tissue, DPRs proteins are the main components of SQSTM1/p62 and ubiquitin-positive, TDP43-negative aggregates, indicating a defect in proteostasis [START_REF] Swaminathan | Expression of C9orf72-related dipeptides impairs motor function in a vertebrate model[END_REF][START_REF] Esanov | A C9ORF72 BAC mouse model recapitulates key epigenetic perturbations of ALS/FTD[END_REF][START_REF] Levine | The product of C9orf72, a gene strongly implicated in neurodegeneration, is structurally related to DENN Rab-GEFs[END_REF]. Poly(GP) is the only DPR that is synthesized from both sense and antisense RNA strands [30,[START_REF] Mori | Bidirectional transcripts of the expanded C9orf72 hexanucleotide repeat are translated into aggregating dipeptide repeat proteins[END_REF], representing one of the most abundant DPR identified in patients [START_REF] Mackenzie | Quantitative analysis and clinico-pathological correlations of different dipeptide repeat protein pathologies in C9ORF72 mutation carriers[END_REF] and shown to accumulate in nuclear inclusions in the CNS [START_REF] Ash | Unconventional Translation of C9ORF72 GGGGCC Expansion Generates Insoluble Polypeptides Specific to c9FTD/ALS[END_REF][START_REF] Mori | The C9orf72 GGGGCC repeat is translated into aggregating dipeptide-repeat proteins in FTLD/ALS[END_REF]. Due to the availability of excellent antibodies to label poly(GP) in patients, it is currently considered as a potential pharmacodynamic marker in ALS [START_REF] Su | Discovery of a Biomarker and Lead Small Molecules to Target r(GGGGCC)-Associated Defects in c9FTD/ALS[END_REF][START_REF] Gendron | Poly(GP) proteins are a useful pharmacodynamic marker for C9ORF72-associated amyotrophic lateral sclerosis[END_REF]. Overexpression of DPRs has been associated with generalized cellular toxicity, both in vitro [START_REF] Wen | Antisense proline-arginine RAN dipeptides linked to C9ORF72-ALS/FTD form toxic nuclear aggregates that initiate invitro and invivo neuronal death[END_REF][START_REF] Mizielinska | C9orf72 repeat expansions cause neurodegeneration in Drosophila through arginine-rich proteins[END_REF][START_REF] Freibaum | GGGGCC repeat expansion in C9orf72 compromises nucleocytoplasmic transport[END_REF][START_REF] Zhang | The C9orf72 repeat expansion disrupts nucleocytoplasmic transport[END_REF][START_REF] Lee | C9orf72 Dipeptide Repeats Impair the Assembly, Dynamics, and Function of Membrane-Less Organelles[END_REF][START_REF] Zhang | C9ORF72 poly(GA) aggregates sequester and impair HR23 and nucleocytoplasmic transport proteins[END_REF], as well as in simple animal models such as zebrafish [START_REF] Ohki | Glycine-alanine dipeptide repeat protein contributes to toxicity in a zebrafish model of C9orf72 associated neurodegeneration[END_REF][START_REF] Swaminathan | Expression of C9orf72-related dipeptides impairs motor function in a vertebrate model[END_REF] and Drosophila [START_REF] Wen | Antisense proline-arginine RAN dipeptides linked to C9ORF72-ALS/FTD form toxic nuclear aggregates that initiate invitro and invivo neuronal death[END_REF][START_REF] Mizielinska | C9orf72 repeat expansions cause neurodegeneration in Drosophila through arginine-rich proteins[END_REF][START_REF] Freibaum | GGGGCC repeat expansion in C9orf72 compromises nucleocytoplasmic transport[END_REF][START_REF] Zhang | The C9orf72 repeat expansion disrupts nucleocytoplasmic transport[END_REF][START_REF] Lee | C9orf72 Dipeptide Repeats Impair the Assembly, Dynamics, and Function of Membrane-Less Organelles[END_REF]. Constitutive mouse models of DPR overexpression presented variable phenotypes: while RNA foci and DPR accumulation were evident in all of these models [START_REF] Zhang | C9ORF72 poly(GA) aggregates sequester and impair HR23 and nucleocytoplasmic transport proteins[END_REF][START_REF] O'rourke | C9orf72 BAC Transgenic Mice Display Typical Pathologic Features of ALS/FTD[END_REF][START_REF] Liu | C9orf72 BAC Mouse Model with Motor Deficits and Neurodegenerative Features of ALS/FTD[END_REF][START_REF] Esanov | A C9ORF72 BAC mouse model recapitulates key epigenetic perturbations of ALS/FTD[END_REF], only a fraction of the mice developed a motor phenotype [START_REF] Liu | C9orf72 BAC Mouse Model with Motor Deficits and Neurodegenerative Features of ALS/FTD[END_REF].

Novel ALS models where both gain and loss of function features of the C9ORF72 HRE are occurring simultaneously, as in the human condition, have the potential to unravel the controversies surrounding this mutation. Recent studies have shown that C9ORF72, a DENNdomain containing protein [START_REF] Levine | The product of C9orf72, a gene strongly implicated in neurodegeneration, is structurally related to DENN Rab-GEFs[END_REF][START_REF] Zhang | Discovery of novel DENN proteins: Implications for the evolution of eukaryotic intracellular membrane structures and human disease[END_REF] plays a function in the endosome/lysosome trafficking and autophagy pathways [7,[63][START_REF] Sellier | Loss of C9ORF72 impairs autophagy and synergizes with polyQ Ataxin-2 to induce motor neuron dysfunction and cell death[END_REF][START_REF] Webster | The C9orf72 protein interacts with Rab1a and the ULK1 complex to regulate initiation of autophagy[END_REF][START_REF] Yang | A C9ORF72/SMCR8-containing complex regulates ULK1 and plays a dual role in autophagy[END_REF][START_REF] Jung | Multiplex image-based autophagy RNAi screening identifies SMCR8 as ULK1 kinase activity and gene expression regulator[END_REF][START_REF] Chitiprolu | A complex of C9ORF72 and p62 uses arginine methylation to eliminate stress granules by autophagy[END_REF][START_REF] Shi | Haploinsufficiency leads to neurodegeneration in C9ORF72 ALS/FTD human induced motor neurons[END_REF], similarly to numerous other ALS-related genes [START_REF] Nguyen | Autophagy as a common pathway in amyotrophic lateral sclerosis[END_REF][START_REF] Deng | Is amyotrophic lateral sclerosis/frontotemporal dementia an autophagy disease?[END_REF][START_REF] Medinas | Proteostasis disturbance in amyotrophic lateral sclerosis[END_REF].

Indeed, neurons derived from inducible Pluripotent Stem Cells (iPSCs) carrying C9ORF72 HRE show defects of vesicle trafficking [20,[START_REF] Shi | Haploinsufficiency leads to neurodegeneration in C9ORF72 ALS/FTD human induced motor neurons[END_REF][START_REF] Webster | The C9orf72 protein interacts with Rab1a and the ULK1 complex to regulate initiation of autophagy[END_REF][START_REF] Aoki | C9orf72 and RAB7L1 regulate vesicle trafficking in amyotrophic lateral sclerosis and frontotemporal dementia[END_REF][START_REF] Farg | C9ORF72, implicated in amytrophic lateral sclerosis and frontotemporal dementia, regulates endosomal trafficking[END_REF], and functionally recapitulate some features of cellular pathology associated with ALS, notably hyperexcitability [START_REF] Shi | Haploinsufficiency leads to neurodegeneration in C9ORF72 ALS/FTD human induced motor neurons[END_REF][START_REF] Wainger | Intrinsic membrane hyperexcitability of amyotrophic lateral sclerosis patient-derived motor neurons[END_REF][START_REF] Devlin | Human iPSC-derived motoneurons harbouring TARDBP or C9ORF72 ALS mutations are dysfunctional despite maintaining viability[END_REF] and decreased viability when exposed to glutamate [9,[START_REF] Shi | Haploinsufficiency leads to neurodegeneration in C9ORF72 ALS/FTD human induced motor neurons[END_REF][START_REF] Selvaraj | C9ORF72 repeat expansion causes vulnerability of motor neurons to Ca2+-permeable AMPA receptor-mediated excitotoxicity[END_REF]. The mechanism of neurotoxicity associated with the C9ORF72 mutation in this in vitro model remains controversial. Previous studies have shown that treatment with antisense oligonucleotides (ASOs) targeting the C9ORF72 transcript can significantly reverse the transcriptional changes [24] and the increased susceptibility to glutamate-induced neurotoxicity [9] of iPSC-derived neurons issued from patients carrying the C9ORF72 HRE, supporting a gain-of-function hypothesis. In contrast, a recent report showed that reduced expression of C9ORF72 mRNA was a necessary and sufficient condition to recapitulate the neurotoxic phenotype associated with the presence of the HRE in iPSC-derived motor neurons (iMNs) [START_REF] Shi | Haploinsufficiency leads to neurodegeneration in C9ORF72 ALS/FTD human induced motor neurons[END_REF]. This study firmly implicated the loss of function of C9ORF72 as a trigger for disease onset through the sensitization of neurons to toxic insults, such as excess glutamate or DPR accumulation [START_REF] Shi | Haploinsufficiency leads to neurodegeneration in C9ORF72 ALS/FTD human induced motor neurons[END_REF]. While the diverging conclusions of these studies can be explained by methodological differences in iPSC derivation protocols or by variability introduced by clonal expansion [START_REF] Selvaraj | Modeling the C9ORF72 repeat expansion mutation using human induced pluripotent stem cells[END_REF][START_REF] Lee | Modeling ALS and FTD with iPSC-derived neurons[END_REF], they also highlight the importance of testing the proposed disease mechanisms in an in vivo setting.

Here, we use the vertebrate model, zebrafish, to evidence for the first time in vivo a synergistic mode of action of c9orf72 gain and loss-of-function, resulting in paralysis and motor neuron degeneration. Indeed, the loss of function of c9orf72 and the co-expression of DPRs converge on common pathways to impair cellular proteostasis implicating autophagy-mediated degradation, thus preventing the clearance of toxic DPRs and ultimately giving rise to motor neuron specific degeneration through caspase-9 activity. This toxicity can be mitigated by rapamycin, an inducer of autophagy. Furthermore, we found that the partial loss of function of the autophagy receptor sqstm1/p62 in zebrafish gives rise to a similar phenotype when combined with poly(GP) (GP100) expression, with embryos displaying polypeptide accumulation and paralysis. These combined results present a formal demonstration of a double hit engendered by the C9ORF72 hexanucleotide expansion in vivo, where autophagy impairment linked to the loss of function of the gene allows the accumulation of toxic DPRs, thus converging to trigger motor toxicity.

RESULTS

C9orf72 knockdown can synergize DPR toxicity to trigger a motor phenotype.

To generate a partial loss of function of c9orf72, we performed knockdown (KD) of the zebrafish orthologue (ENSDARG00000011837) using antisense morpholino oligonucleotides (AMOs) that specifically target the translation initiation codon. We have previously established a subphenotypic dose for this AMO that leads to a reduction of 50% of the c9orf72 transcript [START_REF] Sellier | Loss of C9ORF72 impairs autophagy and synergizes with polyQ Ataxin-2 to induce motor neuron dysfunction and cell death[END_REF]. At this level of KD, no motor or morphological deficits were observed in zebrafish embryos [START_REF] Sellier | Loss of C9ORF72 impairs autophagy and synergizes with polyQ Ataxin-2 to induce motor neuron dysfunction and cell death[END_REF]. Importantly, this level of KD reflects the haploinsufficiency of the c9orf72 transcript observed in ALS-FTLD patient tissue, including pathological brain samples and lymphoblastoid cell lines [START_REF] Conlon | Unexpected similarities between C9ORF72 and sporadic forms of ALS/FTD suggest a common disease mechanism[END_REF].

To To further investigate the pathogenic mechanisms induced by the combined gain and loss of function of c9orf72, we focused on expression of the poly(GP) as the representative DPR in this study. Poly(GP) is expressed from both the sense and the antisense frames of the hexanucleotide repeat expansion and is abundant in brain tissue of ALS-FTLD patients [START_REF] Mackenzie | Quantitative analysis and clinico-pathological correlations of different dipeptide repeat protein pathologies in C9ORF72 mutation carriers[END_REF].

Furthermore, GP are the only DPR that could have potential as a biomarker for ALS [START_REF] Su | Discovery of a Biomarker and Lead Small Molecules to Target r(GGGGCC)-Associated Defects in c9FTD/ALS[END_REF][START_REF] Gendron | Poly(GP) proteins are a useful pharmacodynamic marker for C9ORF72-associated amyotrophic lateral sclerosis[END_REF].

Individual swimming episodes were traced for zebrafish co-expressing GP100 or GFP-only control plasmid together with mismatch or c9orf72 AMO. Partial paralysis was observed in the c9orf72 KD + GP100 condition, but not in the GP100 or the c9orf72 KD conditions alone, as

shown

Concomitant DPR expression and c9orf72 depletion causes motor neuron degeneration

Next, we assessed whether the reduced locomotor capacity of c9orf72 KD embryos expressing GP100 are associated with motor neuron toxicity. We employed double transgenic Tg(mnx1:gal4)/Tg(UAS:RFP) embryos displaying RFP fluorescence in both primary and secondary spinal motor neurons, which allows the visualization of axonal growth in vivo.

Furthermore, visual identification of motor neuron cell bodies allows for the rapid evaluation of cellular survival. During normal zebrafish development, axonal projections from primary motor neurons initiate at 19 hpf, reaching the muscle targets and forming functional neuromuscular junctions starting at 27 hpf [START_REF] Drapeau | Development of the locomotor network in zebrafish[END_REF]. At 43 hpf, ventrally-projecting motor neurons such as the primary CaP neuron, reach their distant targets and start forming complex arborizations [START_REF] Myers | Development and axonal outgrowth of identified motoneurons in the zebrafish[END_REF][START_REF] Goulding | Circuits controlling vertebrate locomotion: Moving in a new direction[END_REF]. The underlying mechanism of cell death involved in motor neuron degeneration in ALS is not entirely understood; however, caspase-9 was suggested to play a role in this process [START_REF] Iłżecka | Serum caspase-9 levels are increased in patients with amyotrophic lateral sclerosis[END_REF][START_REF] Yamazaki | Is motoneuronal cell death in amyotrophic lateral sclerosis apoptosis? In[END_REF]. In order to determine whether the motor neuron degeneration observed in this model was linked to apoptosis, we employed a transgenic line Tg(mnx1:gal4)/Tg(UAS:RFP)/Tg(UAS:GFP-dncaspase9) where caspase 9 activity is specifically ablated in motor neurons [START_REF] Gerety | An inducible transgene expression system for zebrafish and chick[END_REF].

GP100 specifically accumulates upon c9orf72 depletion

Cellular apoptotic death can be triggered by toxicity of insoluble aggregates found in degenerating neurons of ALS patients [START_REF] Sathasivam | ALS-what is the evidence?[END_REF]. The function of C9ORF72 has been linked to impaired proteostasis leading to protein aggregation [START_REF] Ruegsegger | Proteostasis impairment in ALS[END_REF]. To determine if KD of the c9orf72 orthologue in zebrafish alters the kinetics of GP100 clearance, we have monitored the expression of the GFP-labeled DPR at 17 somites, 36 hpf and 50 hpf. At 17 somites, the expression of GP100 is ubiquitous and equivalent between mismatch and c9orf72 KD conditions. In control conditions, GP100 is rapidly cleared, with little fluorescence remaining at 50 hpf. In contrast, However, starting at 30 hpf to 50 hpf, embryos display significant differences in GP100 clearance between the mismatch and c9orf72 KD conditions (66.07 ± 5.54% for the mismatch vs. 91.83 ± 2.66% for the c9orf72 KD at 30 hpf; 57.14 ± 6.64% vs. 84.77 ± 3.68% at 36 hpf; 40.19 ± 10.56% vs. 71.89 ± 7.68% at 50 hpf). In order to define whether autophagy could participate in the GP100 clearance, we used rapamycin, an mTOR inhibitor, for activating autophagy in zebrafish [START_REF] He | Analyzing autophagy in zebrafish[END_REF]. In the c9orf72 KD condition, rapamycin treatment starting at 24 hpf mitigated the accumulation of the DPR, leading to a significant decrease in the expression of GP100 at 30 hpf, 36 hpf and 50 hpf stages when compared to the non-treated condition ( 91 Overall, these results indicate that activation of the autophagy pathway can boost the degradation of DPRs and thus reduce axonal and motor phenotypic features caused by a combination of c9orf72 KD with GP100 expression.

c9orf72
To further define the contribution of autophagy deficits to the pathogenicity of the C9ORF72 mutation, we explored the functional link between the autophagy receptor, SQSTM1/p62 and DPR-related toxicity. SQSTM1/p62 is known to be mutated in about 1% of ALS and FTLD cases [START_REF] Fecto | SQSTM1 mutations in familial and sporadic amyotrophic lateral sclerosis[END_REF][START_REF] Teyssou | Mutations in SQSTM1 encoding p62 in amyotrophic lateral sclerosis: Genetics and neuropathology[END_REF] and co-localizes with DPRs in inclusion bodies in patients carrying the C9ORF72 hexanucleotide repeat expansion [START_REF] Swaminathan | Expression of C9orf72-related dipeptides impairs motor function in a vertebrate model[END_REF][START_REF] Esanov | A C9ORF72 BAC mouse model recapitulates key epigenetic perturbations of ALS/FTD[END_REF][START_REF] Levine | The product of C9orf72, a gene strongly implicated in neurodegeneration, is structurally related to DENN Rab-GEFs[END_REF]. As an autophagy receptor and an integral component of the autophagosome, the degradation of SQSTM1/p62 correlates with the efficiency of the autophagy flux and the degradation of the autophagosome itself [START_REF] Mizushima | How to interpret LC3 immunoblotting[END_REF]. We hypothesized that a combination of c9orf72 KD and DPR expression leads to impaired autophagy through a double hit process: the loss of function of one of its components [22,[START_REF] Sellier | Loss of C9ORF72 impairs autophagy and synergizes with polyQ Ataxin-2 to induce motor neuron dysfunction and cell death[END_REF],

and the strain on cellular proteostatic pathways due to DPR accumulation. Indeed, we found that zebrafish with partial c9orf72 KD and concomitant DPR expression display increased sqstm1/p62 levels as compared to control conditions, whereas c9orf72 KD + GP100 embryos treated with rapamycin have similar sqstm1/p62 protein levels as the control conditions (Fig

DISCUSSION

Our results demonstrate for the first time in vivo, that reduced expression of C9ORF72 is able to specifically promote toxicity of RAN translated DPR proteins, thus exhibiting an innate pathogenic synergy within the C9ORF72 mutation. In fact, we found that the sole expression of DPR proteins does not cause motor deficits per se in zebrafish, whilst neurodegeneration occurs in a context of C9orf72 haploinsufficiency. Indeed, partial KD of c9orf72 associated with GP100 expression induces motor phenotypic features characterized by aborted axogenesis as well as death of motor neurons, thus leading to paralysis. In addition, we observed that c9orf72 depletion combined with GP100 expression triggers sqstm1/p62 accumulation. This suggests that targeting of GP100 cargoes by autophagy machinery remains effective but that one or several of the further steps toward lysosomal degradation is deficient. Our results are also in line with the notion that DPRs play a role in the pathogenicity of ALS to trigger neurodegeneration. However, we found that the sole expression of DPRs were not neurotoxic per se, but required a second hit to cause specific motor deficits. Consistent with our results, a recent study demonstrated that low c9orf72 activity combined with DPRs expression decreases survival of motor neurons, in vitro [START_REF] Shi | Haploinsufficiency leads to neurodegeneration in C9ORF72 ALS/FTD human induced motor neurons[END_REF], confirming that combined gain and loss of function properties could synergize to lead to motor neuron degeneration. Similarly, the loss of function of c9orf72 was shown to aggravate the toxicity of polyQ Ataxin-2 repeats of intermediate length, with the combined hits resulting in neurodegeneration in vivo [START_REF] Sellier | Loss of C9ORF72 impairs autophagy and synergizes with polyQ Ataxin-2 to induce motor neuron dysfunction and cell death[END_REF].

Many pathways have been suggested to play a role in ALS pathogenesis including proteostatic mechanisms [START_REF] Taylor | Decoding ALS: From genes to mechanism[END_REF][START_REF] Ling | Converging mechanisms in als and FTD: Disrupted RNA and protein homeostasis[END_REF]. Indeed, there are a multitude of ALS genes with functions related to autophagy and vesicle trafficking, including SQSTM1 [START_REF] Fecto | SQSTM1 mutations in familial and sporadic amyotrophic lateral sclerosis[END_REF][START_REF] Teyssou | Mutations in SQSTM1 encoding p62 in amyotrophic lateral sclerosis: Genetics and neuropathology[END_REF], OPTN [START_REF] Maruyama | Exome Sequencing Reveals VCP Mutations as a Cause of Familial ALS[END_REF],

VCP [98], TBK1 [99], UBQLN2 [100], PFN1 [101] or CHMP2B [102],
suggesting a key role of these pathways in the aetiology of ALS. Importantly, SQSTM1/p62-positive inclusions have also been identified in ALS patients' autopsy tissue, co-localizing with the DPR-positive aggregates in C9ORF72 ALS-FTLD [START_REF] Swaminathan | Expression of C9orf72-related dipeptides impairs motor function in a vertebrate model[END_REF][START_REF] Esanov | A C9ORF72 BAC mouse model recapitulates key epigenetic perturbations of ALS/FTD[END_REF][START_REF] Levine | The product of C9orf72, a gene strongly implicated in neurodegeneration, is structurally related to DENN Rab-GEFs[END_REF]. Furthermore, several recent in vitro studies have described that depletion of c9orf72 impairs autophagy [63][START_REF] Sellier | Loss of C9ORF72 impairs autophagy and synergizes with polyQ Ataxin-2 to induce motor neuron dysfunction and cell death[END_REF][START_REF] Webster | The C9orf72 protein interacts with Rab1a and the ULK1 complex to regulate initiation of autophagy[END_REF][START_REF] Yang | A C9ORF72/SMCR8-containing complex regulates ULK1 and plays a dual role in autophagy[END_REF]103] and leads to accumulation of aggregates of proteins, including Sqstm1/p62 [START_REF] Sellier | Loss of C9ORF72 impairs autophagy and synergizes with polyQ Ataxin-2 to induce motor neuron dysfunction and cell death[END_REF][START_REF] Webster | The C9orf72 protein interacts with Rab1a and the ULK1 complex to regulate initiation of autophagy[END_REF].

Here, our results provide additional evidence that misregulated autophagy plays a crucial role in the C9ORF72-related neurodegeneration. Indeed, we showed that concomitant expression of DPRs with c9orf72 KD leads to accumulation of GP100 and sqstm1/p62 proteins, in zebrafish.

Motor neurons are known to be particularly sensitive to proteotoxicity caused by protein misfolding or aggregation, as they are post mitotic cells, structurally complex, and highly polarized [START_REF] Ling | Converging mechanisms in als and FTD: Disrupted RNA and protein homeostasis[END_REF]104]. In this context, GP100-associated toxicity triggers an exceeding burden that leads to motor neuron degeneration and paralysis in our model. Interestingly, chemical activation of autophagy with rapamycin treatment, a specific inhibitor of the mTOR complex, compensates the loss of function of c9orf72 as demonstrated by the specific clearance of GP100 and sqstm1/p62 proteins. Associated to its molecular effect, rapamycin is also able to restore motor abilities and neuronal features.

In order to validate the specific involvement of autophagy, we developed another zebrafish model where we combined GP100 expression with the partial KD of the ALS/FTLD-related and autophagy receptor, sqstm1. Combined sqstm1 KD and GP100 expression resulted in GP100 protein accumulation and major motor deficits in zebrafish. Treatment with rapamycin accelerates the clearance of GP100 proteins, confirming the contribution of autophagy defects to the motor phenotype. These results are consistent with the well-known role of SQSTM1/p62 in autophagy. Phenotypes similarities of our two models suggest that c9orf72 and sqstm1 have overlapping molecular targets.

In this study, blocking caspase-9 activity was found to prevent motor phenotypic features and neurodegenerative processes related to combined c9orf72 KD and GP100 expression.

Interestingly, caspase-9 expression and activity was shown to be specifically increased in the serum [START_REF] Iłżecka | Serum caspase-9 levels are increased in patients with amyotrophic lateral sclerosis[END_REF] and in motor neurons [START_REF] Yamazaki | Is motoneuronal cell death in amyotrophic lateral sclerosis apoptosis? In[END_REF] of ALS patients. These results indicate that caspase-9mediated apoptosis could be an outcome of defective autophagy flux, as previously suggested [105,106]. Overall, the results presented here point to targeting autophagy as a central pathway of neurodegeneration in ALS-FTLD caused by the gain and loss of function derived by the C9ORF72 mutation.

MATERIALS AND METHODS

Zebrafish Maintenance

Adult and larval zebrafish (Danio rerio) were maintained at the ICM (Institut du Cerveau et de la Moelle épinière, Paris) fish facility and bred according to the National and European

Guidelines for Animal Welfare. Experiments were performed on wild type and transgenic embryos from AB and TL strains. Tg(mnx1:gal4)icm11 [107] zebrafish were crossed with Tg(UAS :RFP) zebrafish to generate double transgenic zebrafish line having a specific expression of RFP in spinal motor neurons allowing the observation of cell bodies and axonal arborization of single motor neurons within a somitic segment in fixed and live animals (hereinafter referred as Tg(mnx1:gal4)/Tg(UAS:RFP)). Tg(mnx1:gal4)/Tg(UAS:RFP) zebrafish were crossed with Tg(UAS:GFP-dnCaspase9) [START_REF] Gerety | An inducible transgene expression system for zebrafish and chick[END_REF] to generate triple transgenic embryos expressing a GFP-fluorescent dominant negative caspase-9 in RFP-fluorescent spinal motor neurons in order to inhibit and label apoptosis initiation within those specific cells (hereinafter referred as Tg(mnx1:gal4)/Tg(UAS:RFP)/Tg(UAS:GFP-dnCasp9)). Zebrafish were raised in embryo medium: 0.6 g/L aquarium salt (Instant Ocean, Blacksburg, VA) in reverse osmosis water +0.01 mg/L methylene blue. Experimental procedures were approved by the National and Institutional Ethical Committees. Embryos were staged in terms of hpf based on morphological criteria [108] and manually dechorionated using fine forceps at 24 hpf.

Microinjection of oligonucleotides and plasmids into zebrafish embryos

Morpholino antisense oligonucleotides (AMOs; GeneTools, Philomath, USA) were used to knock down the expression of the C9orf72 and Sqstm1 genes. The AMOs were designed to bind to the ATG of the C9orf72 and the Sqstm1 orthologues in zebrafish. The sequences of the c9orf72-atg AMO and of the sqstm1-atg AMO are respectively: 5'-ATTGTGGAGGACAGGCTGAAGACAAT-3' and 5'-ATGAAGAGACGGAAAGTGTCATCCT-3'. Control AMOs (mismatch), containing mismatch nucleotides with the respective ATG AMO sequences, and not binding anywhere in the zebrafish genome, were used to assess the specificity of the observed phenotypes (respectively 5'-ATTcTcGAGcACAGcCTcAAGACAT-3' and 5'-ATcAAcAGACGGAAAcTcTCATCCT-3'). We used subphenotypic doses (half the effective dose) of 0.2 mM for c9orf72-ATG and mismatch AMOs [START_REF] Sellier | Loss of C9ORF72 impairs autophagy and synergizes with polyQ Ataxin-2 to induce motor neuron dysfunction and cell death[END_REF] and 0.3 mM for sqstm1-ATG and mismatch AMOs [109]. For rescue experiments, mRNA encoding human C9ORF72 variant (long isoform) was microinjected at a concentration of 100 ng/µl as previously described [6].

The GFP plasmid encoded for 100 repeats of DPRs were provided by Charlet-Berguerand's lab with the DPR optimized cDNAs in which the sequence has been modified to evade pure GGGCC repeats and encode each only one DPR as described previously [110]. Each DPR optimized sequence has been cloned into the EcoR1 / BamH1 restriction sites of the pGFP vector (Clonetech). Each of the DPR plasmids and the GFP-only control were microinjected at the final DNA concentration of 100 ng/μl. Optimal plasmid concentrations were determined as the point on the toxicity curve where there was no significant increase in the percentage of morphologically deformed larvae, an indication of overall toxicity. The microinjections were carried out at one cell stage.

Locomotion analysis of zebrafish embryos

50 hpf zebrafish embryos of normal morphology were touched on the tail with a plastic pipette tip to trigger the touched evoked escape response (TEER), as previously described [6,[START_REF] Sellier | Loss of C9ORF72 impairs autophagy and synergizes with polyQ Ataxin-2 to induce motor neuron dysfunction and cell death[END_REF]109,111]. TEER responses were recorded using a Grasshopper 2 camera (Point Grey Research, Canada), at 30 frames per second, and the trajectories were traced using the video tracking plugin of ImageJ 1.47 (Sun Microsystems, USA) software. The distance and the velocity were quantified from the path projections. TEER experiments (corresponding to the clutch number) were repeated three to eight times. Analysis of locomotion parameters at later stages, when there is significant spontaneous movement, was performed using an automated imaging and analysis system (Zebralab, ViewPoint, France). Single 72 hpf zebrafish embryos were placed in individual wells of a 96 well plate. Distance and velocity parameters of spontaneous swimming were computed using the Live Tracking module in 20 minute intervals.

Live imaging in zebrafish embryos

Embryos were screened for GFP fluorescence and counted at 17 somites, 30 hpf, 36 hpf and 50

hpf stages with a fluorescent binocular microscope (Olympus, Japan). For live imaging of whole mount embryos, double transgenic Tg(mnx1:gal4)/Tg(UAS:RFP) 17 somites, 36 hpf and 50 hpf embryos were mounted in 1% low-melting-point agarose dissolved in embryo medium and supplemented with 0.16mg/mL tricaine (A-5040, Sigma, USA). Images were captured with a Spinning Disk system (Intelligent Imaging Innovations, USA), an Examiner.Z1 upright stand (Carl Zeiss, Germany), a CSU-W1 head (Yokogawa, Japan), and an ORCA-Flash 4.0 camera (Hamamatsu, Japan), with a 20x objective (NA1).

Western Blot

Zebrafish embryos (50 hpf, 15-30 per tube) were homogenized in lysis buffer: 50 mM Tris-HCl, pH 7.4/ 500 mM NaCl/ 5 mM EDTA/ 1% (w/v) Nonidet P-40/ 0.5% (w/v) Triton X-100 supplemented with a cocktail of protease inhibitors (Complete mini, Roche, Switzerland).

Whole tissue lysates were then centrifuged at 14000×g at 4 ºC for 20 minutes. Supernatants were collected and frozen at -80ºC until biochemical analysis. 30 µg of proteins from 50 hpf embryos homogenized lysates were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Samples were denatured at 98°C for 7 minutes. The separated proteins were transferred to nitrocellulose membranes (GE Healthcare, UK) and probed with the following primary antibodies: anti-GFP CHIP Grade (Abcam, UK), anti-C9RANT (Novus BioTechne, USA). Anti-GAPDH (Abcam, UK) and anti-α-tubulin (Sigma, USA) antibodies were used as loading controls. The blots were incubated with the corresponding fluorescent secondary antibody and the signal was detected using the ODYSSEY® CLx (Li-Cor, USA). The intensity of bands was measured by the open source image processing program ImageJ 1.47 (Sun Microsystems, USA). Western Blot (WB) experiments have been repeated three to seven times.

Rapamycin treatment

Zebrafish embryos at 24 hpf were manually dechorionated and placed in 12-well plates containing rapamycin solution at the final concentration of 0.5 µM as previously described [109,111]. They were incubated in the rapamycin containing solution for the time indicated in the results.

Motor neuron morphology and survival

Tg(mnx1:gal4)/Tg(UAS:RFP) and Tg(mnx1:gal4)/Tg(UAS:RFP)/Tg(UAS:GFP-dnCaspase9) embryos were used to image and quantify motor neurons features. For time-lapse fluorescence imaging of the primary motor neuron axonal growth, 19 hpf embryos were mounted as described above. Images were captured with a Spinning Disk system (Andor technology, UK;

Leica Microsystems, Germany), a DMI8 inverted stand (Leica Microsystems, Germany), a CSU-X head (Yokogawa, Japan) and a QE-180 camera (Hamamatsu, Japan), with a 20x objective (NA0.5) from 19 hpf to 33 hpf. Axonal lengths of motor neurons ventral projections were measured using ImageJ 1.47 (Sun Microsystems, USA) on images of growth periods of sixty minutes. Measures were taken from the ventral root to the ventral edge of the musculature within the fifteenth to the eighteenth segments. For primary and secondary axonal projections measurements and cell bodies counting, 50 hpf embryos were fixed in 4% paraformaldehyde and captured at the same defined location within the intersomitic segments with an Apotome.2

system and an Imager.M2 stand (Carl Zeiss, Germany), with a 20x objective (NA0.8). Motor neurons axonal length as well as spinal cord thickness were also measured with ImageJ 1.47

(Sun Microsystems, USA). Fluorescent cell bodies of primary and secondary motor neurons were counted within the same intersomitic segments region. Experiments have been repeated

at least five times with a total number of embryos between five and twenty-two per condition.

Objectives and overview

Mutations in DEPDC5 have emerged as the most common known cause of familial focal epilepsies, including FFEVF, ADNFLE and FTLE, where the majority of DEPDC5 variants have been associated with haploinsufficiency mechanisms, suggesting the loss of function of the protein in its pathogenicity (Baulac & Weckhuysen, 2016).

Together with NPRL2 and NPRL3, DEPDC5 forms part of the GATOR1 complex, which functions as a negative regulator of mTORC1 (Bar-Peled et al, 2013b;Panchaud et al, 2013a).

Mutations in NPRL2 [START_REF] Scheffer | Mutations in mammalian target of rapamycin regulator DEPDC5 cause focal epilepsy with brain malformations[END_REF] and NPRL3 [START_REF] Sim | Familial cortical dysplasia caused by mutation in the mammalian target of rapamycin regulator NPRL3[END_REF] have also been reported in familial focal epilepsies and disruptions of mTOR pathway activity has been implicating in the pathogenesis of tuberous sclerosis, with the majority of patients having seizures (Tee et al, 2002;Thiele, 2004;[START_REF] Webb | On the incidence of fits and mental retardation in tuberous sclerosis[END_REF][START_REF] Osborne | Epidemiology of Tuberous Sclerosis[END_REF]. Overall, these observations highlight the mTOR pathway as a major actor in epilepsy aetiology.

In this study, we generated a zebrafish model of depdc5 knockdown to study the pathogenesis related to this gene in epilepsy. We showed that depdc5 inhibition, using two different AMOs, induces motor hyperactivity that starts at 28 hpf and persists at 48 hpf. Quantification of total motor activity demonstrated that depdc5 knockdown embryos present a hyperactive phenotype, with increased number of bursts of activity at early developmental stage (28 hpf). At later stage (48 hpf), depdc5 knockdown was associated with aberrant locomotion characterized by tortuous ("cork-screw") swimming trajectories. This motor phenotype was associated with abnormal neuronal activity as shown by increased duration of spontaneous neuronal activity in electrophysiological recordings of depdc5 knockdown embryos.

On the other hand, we were able to rescue this phenotype with the overexpression of wild-type human DEPDC5 cDNA alongside the translation blocking AMO. Furthermore, the coexpression of DEPDC5 variants, identified in patients with focal epilepsy, failed to restore the trajectory of depdc5 knockdown embryos. In addition, we have found that treatment with rapamycin was effective at restoring the phenotype features associated with depdc5 knockdown.

Overall, our results support the hypothesis of epileptogenic loss of function mechanisms in DEPDC5-related diseases and confirm the role of depdc5 in mTOR negative regulation.

Moreover, our model brings new perspectives in the characterization of epilepsy-associated genes and in the identification of new antiepileptic drugs. Indeed, epileptic features were quantifiable automatically as soon as 28 hpf, while in previous genetic or chemical zebrafish models of epilepsy, typical phenotype is generally obtained at later stages.

Accepted in Journal of Visualized Experiments

INTRODUCTION

Due to its small size, oviparous development and transparency at early stages of development, zebrafish has emerged as a valuable vertebrate organism for modeling human diseases as diverse as cardiovascular, cancer or neurological disorders 1, 2 . Zebrafish combines the advantages of a vertebrate, including the high conservation of organ architecture and genetic code, with the small size and ease of genetic manipulation of simpler model organisms, therefore facilitating both fundamental studies and translational applications. In particular, its amenability to high-throughput automated screening of behavior and fluorescent markers of cellular processes has made zebrafish a particularly attractive model for epilepsy research. This has been demonstrated by a high increase in the last decade of the number of publications featuring chemically-induced and/or genetic models of epilepsy 3-5 and, more recently, reports of promising therapeutics obtained from chemical screens in these models 6- 8 .

DEPDC5 is a member of the GATOR1 complex, a negative regulator of mTOR signaling 9 . Mutations in the DEPDC5 gene have been first discovered in 2013 in probands suffering from autosomal dominant focal epilepsies 10,11 , and have since been reported in a number of clinical conditions associated with focal epileptic manifestations and focal cortical dysplasia 12 . The large majority of reported mutations are predicted to cause the loss-of-function of the gene 12 , and this was formally demonstrated for a number of DEPDC5 mutated transcripts which are targeted by nonsense mediated mRNA decay 12, 13 . In agreement, knock-down of the gene orthologue in zebrafish using antisense morpholino oligonucleotides (AMOs) results in a number of features that are common to epileptic models in this organism, including hyperactivity, turn wheel-like swimming, spontaneous seizures and enhanced neuronal activity 14-18 . Interestingly, treatment with rapamycin, an inhibitor of mTOR signaling, reversed the behavioral features of this model 18 , supporting the hypothesis that DEPDC5 loss-offunction can trigger epilepsy due to a misregulation of the mTOR pathway 9, 19 .

Transient knock-down of gene expression in vivo using antisense oligonucleotides carrying the morpholino modification has been an invaluable tool for studying the role of specific genes, on par with si/shRNA-based techniques. Recently, AMO-based strategies have also found clinical applications, with a first AMO therapy receiving the FDA approval for the treatment of Duchenne muscular atrophy in 2016 20 . While it was reported that in zebrafish the phenotype of acute AMO-based gene knock-down does not always correlate with the constitutive knockout models 21 , this can be due at least in some instances to compensatory mechanisms engendered by constitutive genetic modifications 22 . However, the issue of specificity of the AMO-induced phenotype is an undisputable concern that has to be diligently addressed in studies using this technology 23 . In order to ensure the specificity of the AMO-based knockdown phenotype, several key controls are necessary. These include a dose-response curve that allows the selection of the lowest dose of AMO effective for gene knock-down, avoiding overall toxicity due to the introduction of an excess of genetic material. The use of a Mismatch AMO that does not target any particular region in the genome is also required for establishing an appropriate dose and in identifying a specific phenotype. A second AMO which targets a different region of the same gene, such as a splice-blocking AMO, is necessary to confirm that the phenotype is due to the knock-down of the target gene. Rescue of the knock-down phenotype with the cDNA of the gene, either the human orthologue or a codon-modified version of the zebrafish gene that cannot be targeted by the AMO, provides a strong argument in favor of the phenotype specificity. Lack of rescue with the same cDNA containing loss-offunction mutations (such as the introduction of an early stop codons) is a further proof in this direction.

Here we present a method for generating a zebrafish depdc5 loss-of-function model and the protocol for behavioral phenotyping at 28 and 48 hours post fertilization (hpf). At 28 hpf, depdc5 loss-of-function causes overall hyperactivity, as evidenced by enhanced coiling and twitching movements of the embryos within the chorion. An automated motion detection system can be used at this stage to quantify the overall activity per embryo. At 48 hpf, zebrafish exhibit stereotyped escape swimming in response to touch. In zebrafish with downregulated expression of depdc5, the swimming trajectory is significantly more tortuous than in controls, the fish exhibiting a "cork-screw" or "turn-wheel" like pattern, similar to other reported epilepsy models in this organism 3,4 . Electrophysiological recordings were obtained in the optic tectum in zebrafish larvae between 4-6 days post fertilization (dpf) and show a baseline increase in neuronal activity in the depdc5 knock-down animals. The advantage of this model is that it presents several phenotypic features at different time points, which can be useful in monitoring and assessing the efficacy of drug therapies during development.

PROTOCOL

Experimental procedures were approved by the National and Institutional Ethical Committees. Typically, AMO working concentrations will be in a range of 0.2 mmol/L to 1 mmol/L (0.4 mmol/L was determined as the effective concentration for this study 18 ). Inject the control Mismatch morpholino at the same concentration as the effective AMO.

1.1.9. Vortex tubes and centrifuge them briefly to bring the droplets at bottom of the tubes.

1.1.10. For rescue experiments, prepare a 5 µL injection sample with the AMO diluted at the working concentration and the cDNA expression plasmid diluted in water to a final concentration to be determined empirically. For the expression of DEPDC5 and the negative control plasmid, 100 ng/µl final concentration was effective for phenotypic rescue.

Embryo preparation:

1.2.1. The day prior to microinjection, set up zebrafish mating tanks. The morning of the injection, remove the dividers to enable spawning. Collect the eggs in 100 mm Petri dishes filled with embryo water using a fine sieve. Inject within 20-30 min from collection, while the eggs are at the one cell stage.

1.2.2. Pick 60-80 eggs with a plastic Pasteur pipette and arrange them in the silicon-coated Petri dish for injection. The silicon surface will prevent the eggs from sliding during the injections. Remove most of the embryo water, leaving just enough to cover the eggs halfway. 1.3.4. To calculate the volume of the injected solution, place a drop of mineral oil on a microtome slide. Inject the dye-containing solution using the set pressure and time parameters. Measure the diameter of the injected fluid sphere and calculate total volume using the formula Volume=4/3*π*(d/2) 3 , where d=the measured diameter of the injected bolus.

1.3.5. Using a dissecting binocular microscope with a 4x magnification, inject the eggs at the single cell stage by passing through the chorion and the yolk, and projecting the solution directly within the cell.

1.3.6. Collect the injected embryos in a 100 mm Petri dish with embryo water, label the dish and incubate them at 28°C. 1.3.7. Ensure that the incubator temperature is stable over time, as embryos development rate is thermosensitive. For example, the growth would be accelerated at higher temperatures and the developmental stage is critical for properly assessing the phenotype 24 .

1.3.8. 6-8 hours after injection, check the quality of the eggs and remove dead and unfertilized embryos using a plastic Pasteur pipette.

1.3.9. The next morning count and remove dead embryos in each dish with a plastic Pasteur pipette. 2.1.4. Have another experimenter randomize the order of testing of the embryos and mask the names of the conditions to be tested.

Behavior Analysis

2.1.5. Make sure that the mortality rate does not change among conditions and compared to non-injected embryos to ensure the specificity of the phenotype. The percent of dead embryos in all conditions shall not exceed 10-13 % 18 .

2.1.6. Place 10-12 embryos still within their chorion on the plastic mesh using a plastic Pasteur pipette. Fill the test dish with enough embryo water to keep the embryos submerged but not floating. If needed, move the embryos with care using a plastic tip to position them on the grid.

2.1.7. Using a video camera (see table of materials) attached to a dissection microscope, record the spontaneous coiling activity for a defined length of time (10-20 min long videos are usually sufficient to obtain representative samples of activity bursts for the quantification)

2.1.8. Return embryos to their respective dish and put them back in the incubator. Repeat the experiment with as many embryos as needed for each condition (as determined by a 90% power analysis).

2.1.9. To analyze total spontaneous movement, use a ZebraLab system (see table of materials). Using the activity quantification module, upload the recorded video and design the tracking arenas around each embryo as appropriate. Set the freeze and burst threshold to 10 and 50, respectively.

2.1.10. Run the automated video analysis, which quantifies total activity inside each of the defined arenas, then recover the data set as a spreadsheet and perform the analysis using a data analysis software.

Touched

Depolarization event determination

3.4.1. Field recording events have very slow dynamics (frequencies of interest are in the range of 0.005-0.2 s -1 ). Therefore, filter the signal with a low pass (Butterworth 5th order LPF at 100 s -1 ) in order to avoid aliasing. Subsample the recorded voltage data from the acquisition frame rate (in our case, 1 ks -1 ) down to 250 s -1 (RAW SIGNAL).

3.4.2. To identify the timestamps for each depolarization event, use a DETECTION SIGNAL, which is a high-pass filtered version of the recorded signal (Butterworth 1st order HPF at 0.01 s -1 ).

3.4.3 By eliminating the low frequency components, the detection of depolarization events can be performed using a simple thresholding method. Use a fixed threshold for noise elimination and event detection (0.3 mV was used for this study).

3.4.4. The depolarization event is characterized by a series of thresholds crossings that occur at time intervals smaller than 4 s. Compute the start and the end of depolarization event as determined from consecutive sequences of threshold crossings. Events that are shorter than 40ms can be discarded as noise.

3.4.5. Compute the amplitude of the events in the unfiltered (RAW SIGNAL) to eliminate errors due to the effect of the low pass filtering on the peak of the event. Select the depolarization wavelet from the raw signal using the timestamps determined in the filtered signal. Measure the amplitude as the difference between the maximum and minimum values of the wavelet selected from the raw signal.

Note: The script files to perform step 3.4 -Depolarization event determination -and to obtain Fig. 1 are provided as supplementary materials attached to this article.

REPRESENTATIVE RESULTS

Fig. 1 shows representative voltage traces of 4-6 dpf zebrafish larva extracellular field recordings in the case of two genetic conditions: Mismatch control and depdc5 knock-down.

In the baseline period of the recording, depdc5 knock-down shows a higher occurrence of spontaneous events, while the Mismatch control displays very few fluctuations. These activity patterns are representative of the significant increase in neuronal activity due to loss-offunction of depdc5, as we have previously reported 18 . After PTZ application, both Mismatch control and depdc5 knock-down show an increased number of depolarization events. During the first period after PTZ application (10 -60 min), a rate of 0.8 events per min is observed in both Mismatch control and depdc5 knock-down, where the majority of events are of high amplitude (> 1 mV). During the latter response period (60 -120 min after PTZ application), the rate of depolarization events increases to around 1 event per min, and the majority of the events are of low amplitude (≤ 1 mV). High amplitude events during the first period after PTZ application, (E) Low amplitude events during the latter period after PTZ application. Note that the script files to obtain these figures are provided as supplementary material.

DISCUSSION

Epilepsy is a complex neurological disease, featuring a wide range of etiologies that are starting to be elucidated with the advent of genetic sequencing technologies 25-27 . Versatile animal models are essential for an efficient translational strategy that will yield both insights into the pathological mechanisms of genetically linked epilepsies, as well as targeted therapies for the distinct forms of this condition. Zebrafish models have been very effective at reproducing major features of epilepsy and providing reliable readouts for anti-epileptic drug screening 5, 28 . Spontaneous seizures can be detected in genetically modified zebrafish 15, 29-31 and neurophysiological analysis in these models 28 has confirmed the neuronal basis of the epileptic-like behavior 32, 33 . Small-sized zebrafish larvae are amenable to chemical screens in 96-well format using automated detection of simple behavior, such as spontaneous swimming, which allows for rapid detection of potential therapeutics. The depdc5 knock-down model presented here is obtained by injection of AMO into the zebrafish embryo to block gene expression during development. This model presents several keystone phenotypic features during different time points of larval development, which can be used as indicators of therapy efficiency during a chemical or genetic screening protocol.

The AMO-mediated gene knock-down is a powerful technique, displaying advantages over chemically-induced seizure models, as it specifically targets the expression of a gene of interest, thus allowing the identification of the underlying pathogenic mechanisms triggered by a genetic mutation. Chemical inducers, which are nevertheless potent tools for drug screenings, can act through multiple cellular pathways that might not be always relevant to the genetic mutation under study. While AMO injection is in itself a simple technique when mastered by the experimenter, it also presents a number of limitations. The injections have to be performed at the one cell stage embryo; in our hands, injections at later stages greatly increased the variability of the phenotype. This limits the time available for injection; therefore, a strategy of generating eggs for injection in a time sequence is useful. We routinely use 4-5 crosses that we open at 15-20 minute intervals, allowing the injection of one clutch before obtaining the next one. Further, care must be taken to assess the phenotype at the same time points between different experiments, as stereotyped behaviors evolve rapidly during the first days of development. The volume and concentration of AMOs must also be carefully controlled, as general toxicity due to injecting excessive amounts will mask the specific phenotype. The different controls presented in the introduction are essential for determining the right injection dose and the corresponding phenotype.

Field recordings of the larval zebrafish brain are a useful tool for investigating the deleterious effects of genetic mutations involved in different brain disorders on the global neuronal activity 34 . Depolarization events seen under these experimental conditions are an established method for assessing electrophysiological effects of drugs in different epileptic conditions 15, 35 . However, the assessment of these effects has mostly been done qualitatively rather than quantitatively, and having a subjective observer as an actor in the analysis. Here, we develop an automatic detection strategy that can objectively quantify the rate of depolarizations, their amplitude and duration, and can evaluate the progress of these parameters across time, or with different genetic or pharmacologic interventions.

The representative results presented here show the expected field activity of the depdc5 knock-down genetic model in comparison to a Mismatch control in 4-6 dpf zebrafish, before and after the application of PTZ to introduce epileptiform-like electrographic activity. Previously, we have shown a significant increase in the basal activity of the depdc5 knockdown condition 18 . Here we show that the response of these two conditions to PTZ, a chemical epileptiform activity inducer, has a similar trajectory in time, starting with a period of relatively low frequency, high amplitude depolarization events and continuing with a period of higher frequency, lower amplitude depolarization events. Field recording events have slow dynamics (frequencies of interest are in the range of 0.005-0.2 s -1 ), therefore both low-pass and highpass filters are used in this protocol to isolate the events of interest. After eliminating the low frequency noise, the detection of depolarization events is performed using a simple threshold. Since the statistics of the signal are greatly affected by the presence of depolarization events, we could not use the standard deviation of the total signal to determine this threshold. The variability of the value of the standard deviation across datasets was greater than the observed recording noise levels. Therefore, after visual inspection of the traces, we used a fixed value of the threshold of 0.3 mV, in order to avoid the biasing induced by different levels of depolarization activity.

The described protocol provides a standardized and simple method for evaluating the motor behavior and the neuronal field activity, via extracellular current clamp voltage recording coupled with automatic detection of depolarization events in the optic tectum, to characterize epileptiform-like phenotypes in zebrafish models.

DISCLOSURES

The authors have nothing to disclose. Neurodegenerative disorders are characterized by the progressive loss of selectively vulnerable populations of neurons that typically leads to severe functional impairment. To date, we do not know the underlying cause of neurodegeneration and have no treatments that can either prevent or reduce its progression. The pathogenesis of neurodegenerative diseases, such Alzheimer's, Parkinson's and Huntington's diseases and ALS/FTLD spectrum share several common features. One of these consists of the accumulation and aggregation of disease-specific proteins, which have been associated with neurodegeneration. Genetic mutations or environmental factors have been suggested to promote protein misfolding and aggregation in these neurodegenerative diseases, but the exact underlying mechanisms need to be elucidated [START_REF] Scheper | The unfolded protein response in neurodegenerative diseases: a neuropathological perspective[END_REF][START_REF] Smith | The unfolded protein response: Mechanisms and therapy of neurodegeneration[END_REF][START_REF] Soto | Protein misfolding and neurodegeneration[END_REF][START_REF] Yerbury | Walking the tightrope: Proteostasis and neurodegenerative disease[END_REF].

There is increasing evidence for the physiological importance of autophagy in neuronal health with the emerging possibility that autophagy dysfunction may play a role in neurodegeneration [START_REF] Menzies | Compromised autophagy and neurodegenerative diseases[END_REF]Nguyen et al, 2018;[START_REF] Nixon | The role of autophagy in neurodegenerative disease[END_REF]. Circumstantially, this is supported by the major pathological phenotype of most late onset neurodegenerative diseases, namely the presence of intraneuronal aggregates of misfolded proteins, which are substrates for autophagic degradation [START_REF] Hu | Mechanism and Regulation of Autophagy and Its Role in Neuronal Diseases[END_REF][START_REF] Kurtishi | Cellular Proteostasis in Neurodegeneration[END_REF][START_REF] Menzies | Compromised autophagy and neurodegenerative diseases[END_REF][START_REF] Ravikumar | Aggregate-prone proteins with polyglutamine and polyalanine expansions are degraded by autophagy[END_REF].

Ageing is a common character of most of neurodegenerative disorders and has been shown to underlie the overload of the two main proteolytic systems, UPS and autophagy, notably in the CNS [START_REF] Carroll | Autophagy and ageing: implications for age-related neurodegenerative diseases[END_REF][START_REF] Daniele | Brain ageing and neurodegenerative disease: the role of cellular waste management[END_REF][START_REF] Douglas | Protein homeostasis and aging in neurodegeneration[END_REF][START_REF] Mccray | Unaffected mosaic C9orf72 case: RNA foci, dipeptide proteins, but upregulated C9orf72 expression[END_REF][START_REF] Tanaka | Proteostasis and neurodegeneration: The roles of proteasomal degradation and autophagy[END_REF]. In addition, as previously discussed, upregulation of autophagy occurs as a compensatory mechanism when the UPS is saturated [START_REF] Bence | Impairment of the ubiquitin-proteasome system by protein aggregation[END_REF]Lamark & Johansen, 2010;Rubinsztein, 2006). Consequently, when examining age-related neurodegeneration, it is important to consider that ageing can work in conjunction with a failed autophagy system, which may push the cell over the edge of a toxicity threshold, and thus predispose to neurodegeneration.

Neurons, and especially motor neurons are particularly sensitive to defects in autophagy due to their extreme polarization and size, and because they cannot rely on waste dilution occurring during cell division [START_REF] Damme | Autophagy in neuronal cells: general principles and physiological and pathological functions[END_REF][START_REF] Nixon | The role of autophagy in neurodegenerative disease[END_REF][START_REF] Tooze | Liaisons dangereuses: autophagy, neuronal survival and neurodegeneration[END_REF]. Indeed, autophagy is constitutively activated in these cells [START_REF] Hara | Suppression of basal autophagy in neural cells causes neurodegenerative disease in mice[END_REF][START_REF] Komatsu | Ubiquitinindependent function of optineurin in autophagic clearance of protein aggregates[END_REF][START_REF] Son | Neuronal autophagy and neurodegenerative diseases[END_REF][START_REF] Yue | The cellular pathways of neuronal autophagy and their implication in neurodegenerative diseases[END_REF], and dysregulation in this pathway has emerged as a major pathogenic mechanism of motor neuron degeneration in ALS (Mizushima & Hara, 2006;[START_REF] Simonsen | Promoting basal levels of autophagy in the nervous system enhances longevity and oxidant resistance in adult Drosophila[END_REF], with evidence pointing to reduced autophagy in affected neurons, and the subsequent therapeutic effects of inducing autophagy.

Autophagy dysregulation could be triggered by several mechanisms, including: i). an overburden, notably resulting from its activation by upstream mechanisms; ii). a perturbed crosstalk with other mechanisms, resulting in excessive cargo or inadequate disposal; iii). or by a direct dysfunction of proteins involved in the autophagy process. The common purpose of such dysregulations may consist of the accumulation of mutated proteins or aggregation-prone proteins within the cytoplasm of the cells. This notion is strengthened by the discovery of mutations in autophagy-related genes, and in genes involved in proteostasis mechanisms working in conjuncture with autophagy, as monogenic causes of ALS.

Each step of autophagy is meticulously controlled and failure of any of them could lead to the accumulation of abnormal proteins. Of interest, mutations in the autophagy receptor, SQSTM1 have been reported in around 1-3.5% of ALS/FTLD patients (Fecto et al, 2011a).

SQSTM1/p62 is an essential actor of the initiation of selective autophagy, due to its role in the specific recognition and targeting of autophagy substrates to the autophagosomes [START_REF] Johansen | Exome Sequencing Reveals VCP Mutations as a Cause of Familial ALS[END_REF][START_REF] Rea | New insights into the role of sequestosome 1/p62 mutant proteins in the pathogenesis of paget's disease of bone[END_REF]. In this thesis work, we developed a zebrafish model to address the loss of function hypothesis of SQSTM1 in ALS/FTLD. Knockdown of sqstm1 in zebrafish, by either the inhibition of its translation, or the alteration of its splicing, caused a specific locomotor phenotype with features similar to that obtained in previous zebrafish AMO-based ALS models [START_REF] Armstrong | Loss and gain of FUS function impair neuromuscular synaptic transmission in a genetic model of ALS[END_REF][START_REF] Kabashi | Gain and loss of function of ALS-related mutations of TARDBP (TDP-43) cause motor deficits in vivo[END_REF]Ciura et al, 2013a;[START_REF] Lemmens | Overexpression of mutant superoxide dismutase 1 causes a motor axonopathy in the zebrafish[END_REF][START_REF] Sakowski | Neuromuscular effects of G93A-SOD1 expression in zebrafish[END_REF]. This phenotype includes impaired response after stimulation with consequent decreased swimming parameters, associated with reduced motor neuronal axonal projections. The specificity of the phenotype obtained with the atg-AMO was assessed in two ways (Stainier et al, 2017a). Firstly, we used a secondary AMO that specifically affected the splicing of sqstm1 and gave rise to similar motor features i.e. reduced locomotor parameters and motor neuronal axonopathy. Secondly, we observed that the co-injection of AMO with human wild-type SQSTM1 mRNA was able to rescue the phenotype obtained under sqstm1 knockdown, which also supports a functional conservation of the gene between zebrafish and human. However, and consistently with the hypothesis of the loss of function of SQSTM1 as a pathogenic mechanism in ALS/FTLD, overexpression of mRNAs encoding for two SQSTM1 variants identified in ALS/FTLD patients were unable to rescue the motor phenotypic features of our model, neither to cause any toxicity when injected alone. Moreover, treatment of zebrafish embryos with the autophagy activator rapamycin was able to ameliorate spontaneous swimming defects resulting from the knockdown of sqstm1. This suggests that this motor phenotype was at least in part imputable to the loss of sqstm1 function in autophagy.

Since this study, it has been reported that SQSTM1 pathogenicity in ALS/FTLD was functionally associated with defects in LC3-II recognition, which surround impaired targeting of autophagy substrates to the elongating autophagosome [START_REF] Goode | Defective recognition of LC3B by mutant SQSTM1/p62 implicates impairment of autophagy as a pathogenic mechanism in ALS-FTLD[END_REF]. This corroborates that the loss of function of SQSTM1 causes neurodegeneration due to consequent impairment in autophagy process. Coherently, the depletion of OPTN, another autophagy receptor, also caused motor axonopathy in zebrafish and was shown to aggravate the motor phenotype of a zebrafish model overexpressing an ALS causative mutant form of SOD1 [START_REF] Komatsu | Ubiquitinindependent function of optineurin in autophagic clearance of protein aggregates[END_REF].

On the other hand, SQSTM1/p62-positive inclusions have been identified in ALS/FTLD patients carrying a mutation in SQSTM1 [START_REF] Arai | Neuronal and glial inclusions in frontotemporal dementia with or without motor neuron disease are immunopositive for p62[END_REF][START_REF] Mizuno | Immunoreactivities of p62, an ubiqutin-binding protein, in the spinal anterior horn cells of patients with amyotrophic lateral sclerosis[END_REF][START_REF] Masanori | White matter lesions in the brain with frontotemporal lobar degeneration with motor neuron disease: TDP-43immunopositive inclusions co-localize with p62, but not ubiquitin[END_REF][START_REF] Al-Sarraj | P62 positive, TDP-43 negative, neuronal cytoplasmic and intranuclear inclusions in the cerebellum and hippocampus define the pathology of C9orf72-linked FTLD and MND/ALS[END_REF]. Whether these inclusions are the results of a consequent inefficiency of autophagy, and/or of gain of function properties of the mutant proteins, still need to be determined. Nevertheless, theoretically, SQSTM1/p62 sequestration in inclusions could also underlie its loss of function [START_REF] Winklhofer | The two faces of protein misfolding: Gain-and loss-of-function in neurodegenerative diseases[END_REF].

SQSTM1/p62 is also a component of TDP-43 negative inclusions of patients carrying the HRE in C9ORF72, the most common genetic cause of ALS/FTLD [START_REF] Al-Sarraj | P62 positive, TDP-43 negative, neuronal cytoplasmic and intranuclear inclusions in the cerebellum and hippocampus define the pathology of C9orf72-linked FTLD and MND/ALS[END_REF]Mahoney et al, 2012;MacKenzie et al, 2013;Mann et al, 2013), suggesting a common purpose between C9ORF72 and SQSTM1. In zebrafish, we show for the first time that sqstm1 and c9orf72 genetically interact. Indeed, expressions of both C9-L and C9-S wild-type human RNAs are able to rescue the motor phenotype caused by sqstm1 knockdown On the other hand, expression of wild-type SQSTM1 is unable to rescue the phenotype caused by c9orf72 knockdown. These results, along with neuropathological features of C9ORF72 ALS/FTLD patients, suggest that C9ORF72 and SQSTM1 share a common pathogenic pathway, with C9ORF72 being downstream than SQSTM1. Moreover, in line with the loss of function process of C9ORF72 and SQSTM1 mutations in ALS, we observed that their depletion in motor neurons induce drastic decreased survival in spinal cord -DRG cultures. Consistently with these findings, the depletion of the c9orf72 orthologue in C.elegans has been shown to be associated with major motor neuron deficits (Therrien et al, 2013). In addition, a recent report showed that reduced expression of C9ORF72 mRNA was a necessary and sufficient condition to recapitulate the neurotoxic phenotype associated with the presence of the HRE, in iPSC-derived motor neurons (iMNs) from C9ORF72 ALS/FTLD patients (Shi et al, 2018). However, in mouse, C9orf72 depletion was associated with major defects in the myeloid tissue (Sudria-Lopez et al, 2016;Burberry et al, 2016;O'Rourke et al, 2015;Atanasio et al, 2016). But, while the viability was affected in several of these models (Atanasio et al, 2016;Burberry et al, 2016;Sudria-Lopez et al, 2016), no specific or loss of motor neurons could be identified, suggesting that loss of function of C9orf72 alone is not sufficient for initiating motor neuron disease in higher vertebrates, and that a second injury is necessary to trigger ALS (Ciura et al, 2016;Nassif et al, 2017;Balendra & Isaacs, 2018b).

We used the motor neuronal model to investigate if a common pathway of C9ORF72 and SQSTM1 in ALS pathogenicity could be autophagy. We observed that C9orf72 depletion in motor neurons was associated with a near-complete suppression of autophagosomes in these cells. Theoretically, loss of autophagosomes could be due to an accelerated autophagy flux, or to defects in the formation of the autophagosomes. However, SQSTM1/p62 accumulation in C9ORF72 ALS/FTLD patients rather suggests that C9ORF72 depletion may be associated with decreased autophagy efficiency. Since then, a study confirmed in primary neurons, that disappearance of autophagosomes in C9orf72 knockdown condition was actually the result of reduced basal autophagy (Webster et al, 2016a). Then, C9ORF72 is essential for a proper basal autophagy activity: i.e. with sufficient degradative capacity to adequately respond to its induction.

The role of C9ORF72 in autophagy is now well admitted with several converging results supporting that C9ORF72 acts at the initiation step of the pathway (Webster et al, 2016a;Sellier et al, 2016;Sullivan et al, 2016;Yang et al, 2016a;Jung et al, 2017). The pathogenic processes associated with the HRE in C9ORF72, which mediates 23-47 % of fALS and 3-21 % of sALS, remain a hotly debated topic in the field, as both gain and loss of function mechanisms have been proposed as principal causes of the disease.

We used the vertebrate model, zebrafish, to evidence, for the first time in vivo, a double-hit mode of action of C9ORF72 gain and loss of function, that leads to paralysis and motor neuron degeneration. In our model, reduced expression of c9orf72 is able to specifically promote toxicity of RAN-translated DPRs, due to related autophagy impairment; thus exhibiting an innate pathogenic synergy within the C9ORF72 HRE.

We observed that partial knockdown of c9orf72, associated with DPR expression, induces a motor phenotype, characterized by locomotor defects that can be rescued by the overexpression of the long variant of human wild-type C9ORF72 (C9-L). This motor phenotype is dependent of the combination of both c9orf72 knockdown and expression of DPR, since each factor was not triggering any phenotype when applied alone. Indeed, we used a subphenotypic dose of c9orf72-atg and, at our working concentration, none of the DPRs were toxic per se. We have tried higher concentrations of plasmids, but they only generated generalized toxicity, as we obtain for every plasmid at some point, including GFP control.

Contrary to what was reported in several studies [START_REF] Freibaum | GGGGCC repeat expansion in C9orf72 compromises nucleocytoplasmic transport. Nature Freibaum BD & Taylor JP[END_REF]Swinnen et al, 2018;Shi et al, 2018), the rich-arginine DPR poly(GR) was not toxic in our model. This could be explained by the fact that the dose that we used was insufficient to trigger aggregation of GR100 in zebrafish. Indeed, thanks to the GFP reporter, we were able to look at expression pattern of DPRs, and we observed that GR100 were expressed as very few and sparse puncta, whereas GP100 and GA100, for example, built big bunches of fluorescence. Supporting this possibility, a study reported that preventing poly(GA) aggregation could rescue its toxicity in neuronal cultures (Zhang et al, 2016). Another explanation would be that degradation of poly(GR) is mediated by a c9orf72-independent autophagy process in zebrafish. Indeed, while poly(GR) are a component of SQSTM1/p62 inclusions in C9ORF72 HRE ALS/FTLD patients (Mann et al, 2013;MacKenzie et al, 2013;[START_REF] Vatsavayai | C9ORF72-FTD/ALS pathogenesis: evidence from human neuropathological studies[END_REF], we didn't observed any difference in the clearance of GR100 between control and c9orf72 knockdown conditions, in our model. In order to answer this question, we propose to combine poly(GR) expression with sqstm1 knockdown to verify that this DPR can be targeted by autophagy in zebrafish. Poly(GR)

has been shown to generate developmental toxicity in a zebrafish model (Swinnen et al, 2018), thus we can exclude the possibility that this DPR was not toxic in our model because zebrafish would not express eventual poly(GR) targets (such as RBPs) and we hypothesize that differences in our studies are related to different levels of expression of DPRs.

Nevertheless, the point of this study was to develop a new model, that would be close to the pathological features of C9ORF72 ALS/FTLD patients, i.e. with a partial depletion of C9ORF72 and the concomitant expression of DPR, especially of one of the most frequently detected, poly(GP) (Mackenzie et al, 2015;Balendra & Isaacs, 2018b). This model allowed us to demonstrate that C9ORF72 loss of function could synergize some DPRs' toxicity, by inducing their accumulation. We did not intend to study each DPR differentially. Although each DPR has been shown to display proper physical properties in vitro, with arginine-rich DPRs being the more toxic (Wen et al, 2014;[START_REF] Yamakawa | Characterization of the dipeptide repeat protein in the molecular pathogenesis of c9FTD/ALS[END_REF][START_REF] Tao | Nucleolar stress and impaired stress granule formation contribute to C9orf72 RAN translation-induced cytotoxicity[END_REF][START_REF] Kanekura | Polydipeptides encoded by the C9ORF72 repeats block global protein translation[END_REF][START_REF] Moens | Genetic models of C9orf72: what is toxic?[END_REF], the results of post-mortem studies have raised suspicions that DPR inclusions are not the primary culprit in C9ORF72 ALS/FTLD. Indeed, DPR pathology does not coincide with neurodegeneration (Mann et al, 2013;[START_REF] Davidson | Neurodegeneration in frontotemporal lobar degeneration and motor neurone disease associated with expansions in C9orf72 is linked to TDP-43 pathology and not associated with aggregated forms of dipeptide repeat proteins[END_REF]Mackenzie et al, 2015;[START_REF] Gomez-Deza | Dipeptide repeat protein inclusions are rare in the spinal cord and almost absent from motor neurons in C9ORF72 mutant amyotrophic lateral sclerosis and are unlikely to cause their degeneration[END_REF][START_REF] Gendron | Cerebellar c9RAN proteins associate with clinical and neuropathological characteristics of C9ORF72 repeat expansion carriers[END_REF]. In this context, our results raise the notion that DPRs play a role in neurodegeneration, but as a second hit in a vulnerable motor neuron already undergoing C9ORF72 loss of function. Supporting this position, two studies reported asymptomatic C9ORF72 HRE carriers, showing DPR inclusions but normal C9ORF72 mRNA and protein levels, but no TDP-43 aggregation or neurodegenerative changes [START_REF] Gami | A 30-unit hexanucleotide repeat expansion in C9orf72 induces pathological lesions with dipeptide-repeat proteins and RNA foci, but not TDP-43 inclusions and clinical disease[END_REF][START_REF] Mccray | Unaffected mosaic C9orf72 case: RNA foci, dipeptide proteins, but upregulated C9orf72 expression[END_REF]. However, despite these observations, we must consider that post mortem studies might not reflect the early pathogenic mechanisms of DPR. Nevertheless, conversely to DPR pathology, TDP-43 and neurodegeneration co-occur in affected regions of the CNS in ALS/FTLD post mortem tissues (MacKenzie et al, 2013;Mann et al, 2013;[START_REF] Davidson | Brain distribution of dipeptide repeat proteins in frontotemporal lobar degeneration and motor neurone disease associated with expansions in C9ORF72[END_REF]Mackenzie et al, 2015;[START_REF] Davidson | Neurodegeneration in frontotemporal lobar degeneration and motor neurone disease associated with expansions in C9orf72 is linked to TDP-43 pathology and not associated with aggregated forms of dipeptide repeat proteins[END_REF][START_REF] Gendron | Cerebellar c9RAN proteins associate with clinical and neuropathological characteristics of C9ORF72 repeat expansion carriers[END_REF].

Therefore, a crucial perspective of this project would be to look at the subcellular localization of TDP-43 in our model.

Furthermore, two studies reported that RAN translation of the expansion is initiated by a CUG, acting as a start codon [START_REF] Green | RAN translation at C9orf72-associated repeat expansions is selectively enhanced by the integrated stress response[END_REF][START_REF] Tabet | CUG initiation and frameshifting enable production of dipeptide repeat proteins from ALS/FTD C9ORF72 transcripts[END_REF]. In our model, as well as in the other models reported here, DPR are encoded after an ATG codon, thus inducing DPR production with a level of expression that is likely to be relatively much higher than what occurs in patients. This matter reinforces the question of the importance of the expression levels that we discussed previously. Thus, we have tried to co-inject a GP100-encoding plasmid containing a CUG codon (and a GFP reporter) along with the c9orf72-atg AMO. We were able to see few fluorescence puncta, but we did not get any specific phenotype under c9orf72 knockdown. It is conceivable that time-related accumulation would have driven a phenotype, the main inconvenient of AMO being its transitory effect. In this sense, a very interesting perspective to study ALS neurodegeneration would be to develop a zebrafish transgenic line where this plasmid would be constitutively expressed under a c9orf72+/-heterozygous background, raising the possibility to study an eventual effect of age on DPR accumulation. Moreover, this model would allow us to analysis behavioral features of adult zebrafish, such as FTLD-like phenotype, for example.

Transient knockdown of gene expression in vivo using antisense oligonucleotides carrying the morpholino modification has been an invaluable tool for studying the role of specific genes, on par with si/shRNA-based techniques. Recently, AMO-based strategies have also found clinical To conclude, this thesis work demonstrates that -SQSTM1 pathogenicity in ALS is associated with its loss of function in autophagy process; -C9ORF72 and SQSTM1 genetically and functionally interact in ALS pathogenesis; -the role of C9ORF72 in autophagy is essential; and that its loss of function in motor neurons mediate neurodegeneration. Finally, we demonstrate that gain and loss of function act in common pathogenic mechanisms to synergize the C9ORF72 HRE pathogenicity in ALS and FTLD. Table 1. Genes associated with ALS, since 1993 to 2018.

Figures and table list

Table 2. The majority of genes encoding major players involved in the autophagy pathway are well conserved from zebrafish to human. Supplementary Figure 3. Co-injection of sqstm1-atg and the RNA carrying the truncating mutation does not rescue the motor phenotype. .

Exploring the role of C9ORF72 in autophagy

4.

Depdc5 knockdown induces phenotypic features of epilepsy

Article 3: Depdc5 knockdown causes mTOR-dependent motor hyperactivity in zebrafish.

Modified from Taylor et al, 2016 Figure 1 .

 20161 Figure 1. Clinical features of ALS. Degeneration of corticospinal motor neurons -the descending axons of which in the lateral spinal cord seem affected ("lateral sclerosis"), and of spinal motor neurons occurs, leading to denervation and muscle wasting ("amyotrophy").

  , and sequestosome 1 (SQSTM1/p62) -positive, ubiquitin-positive and TDP-43 negative inclusions associated with dipeptide repeat proteins (DPR) in Chromosome 9 open reading frame 72 (C9ORF72) -related patients (Al-Sarraj et al, 2011; Troakes et al, 2012; Mackenzie et al, 2014a) (please refer to section 4.3.1. for more details).

Figure 2 .

 2 Figure 2. Histopathology of ALS. Loss of motor neurons is shown in the anterior horn of the spinal cord (a) and motor cortex (c) of a sALS patient when compared to a control individual (b, d). (a-d) Hematoxylin and eosin. Ubiquitin skein-like inclusions (arrows) are shown in spinal motor neurons of the lumbar anterior horn of a sALS patient (e) but not in a control individual (f). TDP-43 inclusions are shown to be diffuse (arrow) and skein-like (arrowhead) and there is nuclear clearing in the spinal motor neurons of a sALS patient (g), compared to the normal nuclear TDP-43 in a control individual (h). Microglial activation is shown by IBA1 immunorectivity in the anterior horn of the spinal cord in a sALS patient (i) compared to a control individual (j). Pictures from Saberi et al, 2015

  -not elucidate, *see Chapter 3 Result, C9ORF72 and SQSTM1 interaction, §section modified from[START_REF] Chia | Novel genes associated with amyotrophic lateral sclerosis: diagnostic and clinical implications[END_REF]

Figure 3 .

 3 Figure 3. ALS-related genes and underlying affected mechanisms.In the left column are the monogenic causes of ALS (in grey are the genes whose linkage to ALS needs to be confirmed). In the right column are the associated cellular mechanisms involved in ALS.

Figure 4 .

 4 Figure 4. Overview of autophagy progress.

Figure 5 .

 5 Figure 5. The autophagy machinery.

Figure 6 .

 6 Figure 6. Schematic model of the roles of SQSTM1/p62, OPTN and UBQLN2 proteins as autophagy receptors.

Figure 7 .

 7 Figure 7. Schematic representation of ALS-related genes and their function in autophagy process. ALS genes are written in white on a colored background.…

SQSTM1 mutation (Fig. 8 )Figure 8 .

 88 Figure 8. Neuropathological features of SQSTM1 inclusions. SQSTM1/p62 immunoreactivity in neurofibrillary tangles in Alzheimer's disease brain (a) and in Lewy bodies in Parkinson's disease brain (b). Large round cytoplasmic inclusions in the spinal cord of ALS patients with SQSTM1 mutation. Immunoreactivity for ubiquitin (c), SQSTM1/p62 (d) and TDP-43 (e). (f-g) SQSTM1/p62 immunostaining in the lower layers of the temporal cortex from cases carrying the C9ORF72 HRE, showing frequent star-shaped neuronal cytoplasmic inclusions. a,b Pictures from Zatlouka et al, 2002; c-e Pictures from Teyssou et al, 2013, scale bar 10 µM; f,g Pictures from Al-Sarraj et al, 2011, scale bar 10 μm.

Figure 9 .

 9 Figure 9. Schema of SQSTM1/p62 protein. Representation of the autophagy-related domains PB1, LIR and UBA and the autophagy-related sequence TBS. The identified ALS/FTLD mutations (Rea et al, 2014a) are represented on the protein, showing the potential impact of autophagy related mutations (in red) in the pathogenicity of this gene.

Figure 10 .

 10 Figure 10. Identification of the C9ORF72 mutation associated with ALS/FTLD. (a) Physical map of the chromosome 9p21 ALS/FTLD locus showing the p-values for single nucleotide polymorphisms genotyped in a previous GWAS from Laaksovirta et al., 2010, with the location of the GWAS association signal within a 232 kb block of linkage disequilibrium. (b) Detection of C9ORF72 hexanucleotide repeat expansion. Sequence traces of the repeat-primed PCR. Orange lines indicate the size markers (bp), and the vertical axis represents fluorescence intensity. A typical saw tooth tail pattern that extends beyond the 300 bp marker with a 6 bp periodicity is observed in the case carrying the GGGGCC repeat expansion. a,b from Renton et al., 2011. (c) Representation of the GGGGCC repeat expansion within the first intron of the C9ORF72 gDNA.

  C9ORF72 patients Beside loss of function properties of C9ORF72 HRE, gain of function mechanisms engendered by the mutation have also been described. Aberrantly expanded C9ORF72 hexanucleotide repeats are transcribed into RNA species that have been shown to accumulate in nuclear foci (DeJesus-Hernandez et al, 2011b; Sareen et al, 2013; Donnelly et al, 2013; Lagier-Tourenne et al, 2013) (Fig. 11). As a matter of fact, sense and antisense RNA foci have been observed in the CNS in patients with C9ORF72-mediated FTLD/ALS (Cooper-Knock et al, 2015; Donnelly

Figure 11 .

 11 Figure 11. Proposed mechanisms to explain C9ORF72 HRE pathogenicity.

Figure 12 .

 12 Figure 12. Neuropathological features of C9ORF72 expansion carriers. (a) TDP-43 pathology in a patient with C9ORF72 ALS/FTLD. Arrow indicates a neuronal cytoplasmic TDP-43 inclusion in the frontal cortex, with concomitant depletion of nuclear TDP-43. (bc) Immunostaining of the hippocampus for poly-GA peptids shows neuronal cytoplasmic inclusions in dentate gyrus granule cells (b) and pyramidal cells of CA4 region (c). (d-e) Immunostaining for SQSTM1/p62 proteins in dentate gyrus granule cells (d) and pyramidal cells of CA4 region (e), similar to those seen on poly(GA) immunostaining. (f-i) Poly(GP) immunostaining in (f) cerebellum, (g) frontal cortex, (h) motor cortex and (i) hippocampus. a Picture from Balendra & Isaacs, 2018; b-e Pictures from Mann et al 2013 (Immunoperoxidasehaematoxylin,× 40 microscope objective magnification); f-h Pictures from Gendron et al, 2015, Scale bar 5 µM).

Figure 13 .

 13 Figure 13. Hypothesis of synergistic mechanisms in the C9ORF72 pathogenicity. DPR accumulation would be mediated by loss of function of C9ORF72 role in autophagy process.

  syndromes are distinguished: -autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE), which are characterized by clusters of nocturnal motor seizures with rare daytime seizures;familial temporal lobe epilepsy (FTLE), which are distinguished between familial mesial temporal lobe epilepsies (FMTLE) and autosomal dominant epilepsy with auditory features (ADEAF), depending on the region of onset; -and familial focal epilepsy with variable foci (FFEVF), which is characterized by focal epilepsies that arise from different regions of the brain between different members of a same family[START_REF] Scheffer | ILAE classification of the epilepsies: Position paper of the ILAE Commission for Classification and Terminology[END_REF].

Figure 14 .6. 3 .

 143 Figure 14. Expression pattern of a fluorescent reporter in motor neurons. Fluorescent image in a 4 dpf Tg(mnx1:gal4)/Tg(UAS:GFP-aequorin-opt) double transgenic zebrafish larva showing selective expression in spinal motor neurons (arrowhead: dorsal primary motor neurons, arrow: ventral secondary motor neurons), and strictly no expression in muscle fibers (white arrow in the upper panel). Images from Knafo et al, 2017

  SQSTM1/p62-positive inclusions are highly characteristic of C9ORF72 pathology[START_REF] Al-Sarraj | P62 positive, TDP-43 negative, neuronal cytoplasmic and intranuclear inclusions in the cerebellum and hippocampus define the pathology of C9orf72-linked FTLD and MND/ALS[END_REF] Mahoney et al, 2012; MacKenzie et al, 2013; Mann et al, 2013), suggesting a common purpose for SQSTM1/p62 and C9ORF72. Indeed, recent work has confirmed the role of C9ORF72 in an autophagy initiation complex together with SMCR8 and WDR41(Sellier et al, 2016; Sullivan et al, 2016; Yang et al, 2016a;[START_REF] Amick | C9orf72 binds SMCR8, localizes to lysosomes, and regulates mTORC1 signaling[END_REF]. However, the question of its functional relation with SQSTM1 remains open. In order to establish whether genetic interaction exists between C9ORF72 and SQSTM1 in ALS, and to characterize the function of C9ORF72, we investigated c9orf72 and sqstm1 functional relation in zebrafish.

and

  sqstm1 partial knockdowns led to any locomotor defects at 48 hpf, as shown by the TEER test that reveals normal distance (Fig. 1 a) and velocity (Fig. 1 b) parameters for both sqstmatg ½ and c9orf72-atg ½ conditions. Consistently with these quantifications, c9orf72-atg ½ and sqstm1-atg ½ 48 hpf embryos display normal swimming trajectories (Fig. 1 c). In contrast, combination of sqstm1 and c9orf72 partial inhibitions, mediated by the simultaneous injection of both sqstm1-atg ½ and c9orf72-atg ½ AMOs, leads to drastic defects in locomotor parameters, as shown by the TEER results of 48 hpf embryos, which display significantly reduced distance (Fig. 1 a, 18.23 ± 4.20 mm vs 72.82 ± 3.66 mm for non injected and 75.03 ± 11.38 mm for sqstm-mis ½ + c9orf72-mis ½) and velocity (Fig. 1 b, 21.76 ± 1.23 mm/s vs 53.09 ± 0.94 mm/s for non injected and 49.37 ± 1.99 mm/s for sqstm-mis ½ + c9orf7-mis ½) parameters, when compared to control conditions, as well as reduced swimming trajectories (Fig. 1 c).

Figure 1 .

 1 Figure 1. Swimming parameters of 48 hpf embryos showing an additive effect of sqstm1 and c9orf72 partial inhibitions. sqstm1 and c9orf72 partial knockdowns do not lead to any swimming defects, whereas combination of both c9orf72 and sqstm1 partial inhibitions produces an additive effect as shown by drastic decrease of distance (a), and velocity (b) parameters in the TEER test of 48 hpf embryos (K=254.934 and P<0.001 from Kruskal-Wallis analysis of variance for distance parameter ; K=449.540 and P<0.001 from Kruskal-Wallis analysis of variance for velocity parameter).*** p < 0.0001 from post hoc Dunn's method. Data are presented as mean ± standard error. (c) Traces are representative swimming trajectories from the TEER test showing a strong motor disability in the sqstmatg ½ + c9orf72-atg ½ condition only. For each condition, nine embryos were depicted.

Figure 2 .

 2 Figure 2. Phenotype distribution of 48 hpf embryos showing an additive effect of sqstm1 and c9orf72 partial inhibitions. Embryos are sorted between dead, having developmental abnormalities, displaying specific motor phenotype, or normal, according to the TEER test.

Figure 3 .

 3 Figure 3. Swimming parameters of 48 hpf embryos showing that c9orf72 acts downstream than sqstm1. SQSTM1 human RNA (hRNA) is unable to improve swimming parameters of c9orf72 loss of function whereas both long (C9-L) and short (C9-S) isoforms of C9ORF72 hRNA are able to rescue sqstm1 loss of function, as demonstrated by improved distance (a) and velocity (b) parameters in the TEER test of 48 hpf embryos (K=190.724 and P<0.0001 from Kruskal-Wallis analysis of variance for distance parameter ; K=348.958 and P<0.0001 from Kruskal-Wallis analysis of variance for velocity parameter). *** p < 0.0001 from post hoc Dunn's method. Data are presented as mean ± standard error. (c) Traces are representative swimming trajectories from the TEER test showing the rescue of motor parameters by C9ORF72 hRNAs in sqstm1 knockdown conditions. Nine embryos per condition were depicted.

Figure 4 .

 4 Figure 4. Phenotype distribution of 48 hpf embryos showing that c9orf72 acts downstream than sqstm1. Embryos are sorted between dead, having developmental abnormalities, displaying specific motor phenotype or normal behavior according to the TEER test.

Figure 5 .

 5 Figure 5. C9orf72 and Sqstm1 knockdowns affect survival in motor neurons. Percent of average surviving motor neurons over days post injections (dpi) illustrating deleterious effects of C9orf72 and Sqstm1 inhibitions (F=145.643 and P<0,001, from two-way ANOVA with repeated measures). ** p<0.001, *** p<0.0001 from post hoc Bonferroni's test. Data are presented as mean +/-standard error.

Figure 6 .

 6 Figure 6. Soma area of motor neurons at 1 dpi, displaying no morphological difference between conditions (F=0.047 and P=0.95 from one-way ANOVA). The red crosses represent the average values.

Figure 7 .

 7 Figure 7. C9orf72 and Sqstm1 depletions reduce number of autophagosomes. Images (a) and quantification (b) of LC3-eGFP puncta in motor neurons at 1 dpi showing the alteration of autophagosomes amount in C9orf72 and Sqstm1 depletions conditions (K=14.115 and P=0.001 from Kruskal-Wallis analysis of variance). * p<0.05, ** p<0.001 from post hoc Dun's method. Data are presented as mean ± standard error.

  Tissues were triturated with a Pasteur pipette to dissociate the cells and plate in 12 wells plates with 18 mm glass coverslips pre-coated with poly-D-lysine (Sigma-Aldrich) and Matrigel matrix (Corning Incorporated, NY, USA) with a density of 375000 cells/well. Cultures were maintained at 37°C in 5% CO2 in modified N3 medium. At 4-6 days after plating, cells should have reached ∼90% confluency and are treated with 1,4 μg/mL cytosine-b-D-arabinofuranoside (Calbiochem, La Jolla, CA) for 3 days to stop mitosis of non-neuronal cells. Cultures were fed twice a week by replacing half of the medium with antibiotic-free N3 medium.

  major genetic contribution, the most common genetic abnormality being the GGGGCC HRE in the first intron of the C9ORF72 gene(DeJesus-Hernandez et al, 2011b; Renton et al, 2011a). Proposed mechanisms concerning C9ORF72 mutation pathogenicity are loss and gain of function, including aggregation of DPRs translated through a non-ATGinitiated translation of the HRE[START_REF] Gitler | There has been an awakening: Emerging mechanisms of C9orf72 mutations in FTD/ALS[END_REF] Balendra & Isaacs, 2018a). Moreover, DPRs inclusions are the main components of SQSTM1/p62-positive inclusions of ALS/FTLD patients carrying the C9ORF72, which indicates that they are targeted by autophagy

  determine whether gain and loss of function mechanisms of C9ORF72 HRE can synergize to induce motor deficits, c9orf72 KD was performed in parallel with overexpression of one of the five GFP-tagged DPR species, or with GFP alone (Fig 1a). Injection of GFP-tagged repeats GA100, GP100, GR100, PA100 or PR100 repeats, at one cell stage zebrafish embryos produced detectable expression at 20 hours post fertilization (hpf) (Fig EV1). At 50 hpf, zebrafish embryos have stereotyped escape responses to touch, allowing for a rapid and robust assessment of motor behavior using the Touch-Evoked Escape Response (TEER) test (Fig 1a). Quantitative analysis of the TEER demonstrated that expression of DPRs alone does not trigger any swimming abnormalities, as the swimming distance and velocity of mismatch + DPR embryos was not significantly different from either non-injected or GFP-only control conditions (Fig 1 b,c). Similarly, partial C9orf72 downregulation in combination with the GFP-expressing plasmid did not result in any phenotoxic features (Fig 1 b,c). However, concomitant downregulation of c9orf72 and expression of GP100, PA100, PR100 or GA100, but not GR100, induced a significant and specific reduction in motor abilities, as quantified by the reduced total distance (Fig 1b) and velocity (Fig 1c) travelled by 50 hpf embryos when compared to respective mismatch and GFP-only control conditions.

  by the swimming trajectories in Fig 1d, in the absence of any gross morphological abnormalities (Fig 1e). To confirm the specificity of these motor phenotypic features, we performed functional rescue experiments using co-expression of human C9ORF72 mRNA variant 2 (hC9ORF72). Injection of hC9ORF72 at the one cell stage was able to restore the swimming parameters to control levels in c9orf72 KD + GP100 embryos, as shown for the total distance (Fig 1f) and the velocity (Fig EV2) of swimming episodes. Importantly, the percentage of zebrafish larvae with motor deficits was substantially reduced upon co-expression of hC9ORF72 (Fig 1g), confirming the functional contribution of c9orf72 expression levels to the motor phenotype.

  First, we monitored by in vivo imaging the ventral axogenesis in the intersomitic segments 15-18 from 19 hpf to 33 hpf, thus comprising the timeline for the complete axonal projection of the primary motor neuron (Fig2a). These experiments show an early axonopathy of motor neurons related to the combination of c9orf72 KD with expression of GP100 when compared to controls(Fig 2b). Axogenesis initiates at the correct time in all genetic conditions. However, there is a strong trend towards a shortened axonal projection in the c9orf72 KD + GP100 condition as compared with controls, with this difference becoming significant starting at 31 hpf and persisting at later stages of development. Motor axon measurements at a later stage (50 hpf), when both primary and secondary motor neurons have reached their final target, revealed a strong pathogenic effect of the combined loss and gain of function of c9orf72 on the axonal length as normalized to the spinal cord thickness (Fig2c, d), whereas the overall spinal cord thickness remained unaltered between these conditions (Fig EV3). To define whether motor neuron degeneration was occurring in this model, we quantified the number of primary and secondary motor neurons in the fifteenth to the nineteenth intersomitic segments of 50 hpf Tg(mnx1:gal4)/Tg(UAS:RFP) embryos. A significant decrease of neuronal cell bodies was observed in the c9orf72 KD + GP100 condition as compared to mismatch and GFP controls conditions (Fig 2e,f), suggesting that the toxic effect of the combined gain and loss of function affects the survival of motor neurons. Similarly to the swimming parameters and phenotype distribution described in Fig 1e-g, overexpression of hC9orf72 is able to rescue both axonal length and survival of motor neurons (Fig 2d,f).

  Cell specific inhibition of caspase-9 function was able to rescue the axonal length (Fig 2d) and increase the survival of motor neurons (Fig 2f) upon combined c9orf72 KD and GP100 expression. These results implicate apoptotic cell death as a target cellular process triggered by the synergistic action of c9orf72 KD and GP100 expression.

  KD causes the expression of GP100 to persist, as indicated by accumulated fluorescence at this stage (Fig 3a) suggesting a direct role of c9orf72 in the clearance of GP100. Interestingly, we observed that GP100 expression occurs in motor neurons (Fig EV4). Immunoblotting using a specific anti-poly(GP) "C9RANT" antibody (Fig 3b) or a GFP antibody (Fig 3c) confirm the significantly increased dipeptide levels in 50 hpf c9orf72 KD + GP100 condition, as compared with the mismatch controls (Fig 3b,c); whereas at an early stage, at 20 hpf, GP100 levels were similar between conditions (Fig EV5a,b). Furthermore, we observed that GP10 is also retained in c9orf72 KD condition, as shown in Fig EV6, using the C9RANT antibody (Fig EV6a) or the GFP antibody (Fig EV6b). In addition, we looked at GR100 expression at both 20 hpf and 50 hpf timepoints (Fig EV5c,d), and we report no difference in GR100 proteins levels between mismatch + GR100 and c9orf72 KD + GR100 conditions. Similarly, in control condition, no difference in accumulation of GFP protein level was observed in the c9orf72 KD + GFP condition, as compared to the mismatch + GFP injected embryos (Fig 3f,g), suggesting a specific role of c9orf72 in the clearance of GP100 peptide chains. Autophagy integrity is necessary to trigger GP100 clearance. C9ORF72 has been found to play an important role in autophagy-mediated molecular cascades in vitro [63-68]. The expression of GFP-labeled DPR have been monitored at 20 hpf, 30 hpf, 36 hpf and 50 hpf stages. The percentage of GP100-expressing embryos is comparable between mismatch and c9orf72 KD conditions at early stages of development, as shown at the 20 hpf stage in Fig 4a.

(

  Fig 4a). Associated to its effect on GP100 clearance, rapamycin treatment significantly rescued the axonopathic phenotype induced by c9orf72 KD + GP100 when compared to mismatch AMO + GP100 (Fig 4b). To test the effect of rapamycin on the motor phenotype, spontaneous swimming after 2 days of rapamycin treatment was analysed using an automated motion detection set-up (Viewpoint, France). Consistent with the TEER results at 50 hpf, concomitant c9orf72 KD and GP100 expression was associated with a reduction in spontaneous swimming parameters (Fig 4c,d,e). Treatment with rapamycin restored the motor abilities in c9orf72 KD + GP100 as compared to the non-treated condition, as shown by the traces of automated swimming recordings (Fig 4c) and the quantification of distance and velocity (Fig 4d,e).

  4f,g), suggestive of an impaired autophagic flux.To test whether impairing autophagy was sufficient to mimic the effect of c9orf72 KD, we used a subphenotypic dose of a sqstm1-ATG targeting AMO in combination with GP100 expression.TEER testing in 50 hpf zebrafish show that the swimming parameters of sqstm1 KD + GP100 embryos were strongly affected, as demonstrated by decreased distance (Fig 5a), velocity (Fig 5b) and individual evoked swimming traces (Fig 5c), when compared to non-injected and mismatch + GP100 control conditions. Similarly to results obtained in the c9orf72 KD zebrafish model, GP100 clearance is diminished upon sqstm1 KD (Fig 5d and Fig EV7). Furthermore, boosting autophagy with rapamycin alleviates GP100 accumulation upon sqstm1 KD (Fig 5d). Overall, this study demonstrates in vivo, that C9ORF72 mutation pathogenicity is synergistically dependent of its related gain and loss of function, where decreased expression of c9orf72 supplies the toxic accumulation of the DPRs through an impairment of the proteostatic mechanisms of autophagy (Fig 5e).

Fig 1 C9orf72

 1 Fig 1C9orf72 knock-down (KD) is essential to trigger DPR toxicity and an associated motor phenotype. a Embryos are injected at one-cell stage with c9orf72 AMO (or mismatch AMO as a control) and a GFP-tagged plasmid containing dipeptide protein repeats (DPRs) : GA100, GP100, GR100, PA100 or PR100 (or a GFP-only plasmid as a control). For rescue experiments, human C9ORF72 mRNA was also injected. Two days after the injection (at 50 hpf), the motor phenotype was assessed using the Touch Evoked Escape Response (TEER). b Results of the TEER test for individual DPR and for the GFP-only control, with or without c9orf72 KD, showing the effects on the average swimming distance in 50 hpf embryos. (F=0,284 and P=0,944 from one-way ANOVA for different mismatch + DPR and non-injected conditions; F=10,111 and P<0,001 from one-way ANOVA for mismatch + DPR and c9orf72 KD + DPR). c Results of the TEER test for individual DPRs and for the GFP-only control, with or without c9orf72 KD, showing the effects on average velocity in 50 hpf embryos. (F=2,073 and P=0,058 from one-way ANOVA for different mismatch + DPR and non-injected conditions; F=18,636 and P<0,001 from one-way ANOVA for mismatch + DPR and c9orf72 KD + DPR). d Representative swimming trajectories from the TEER test showing a strong motor disability only in the c9orf72 KD + GP100 condition. e Images of mismatch + GP100 and c9orf72 KD + GP100 whole embryos at 50 hpf showing no effect on global morphology. f Expression of the human C9ORF72 transcript rescues swimming parameters, including the average distance in the c9orf72 KD + GP100 condition. (F=14,378 and P<0,001 from one-way ANOVA). g Restoring c9orf72 RNA levels rescues the percentage of affected fish in the c9orf72 KD + GP100 condition. p-values from post-hoc Student-Newman-Keuls Method are indicated on the graph bar. All data are presented as mean + sem. N represents the number of embryos.

Fig 2 GP100Fig 3 Fig 4 Fig 5 Sqstm1bb

 2345 Fig 2 GP100 expression combined with the c9orf72 loss-of-function causes axonopathy and spinal motor neuron (MN) loss. a Double transgenic Tg(mnx1:gal4)/Tg(UAS:RFP) or triple transgenic Tg(mnx1:gal4)/Tg(UAS:RFP)/Tg(UAS:GFP-dnCasp9) embryos were used for in vivo monitoring of axogenesis and for the quantification of spinal motor neuron (MN) axonal length and number. b Axogenesis monitoring shows deleterious effects of c9orf72 KD + GP100 on the emerging motor axon. (F=16,886 and P=0,006 for genotype factor from two-way ANOVA with

FIGURE LEGENDS: Figure 1 .

 1 FIGURE LEGENDS: Figure 1. Example traces of field recordings in the zebrafish larvae brain. (A) Overview of 180 min recording for a Mismatch control larva and a depdc5 Knock-down. First, spontaneous baseline activity was recorded, then PTZ was applied in bath (red bar). (B) Peri-stimulus time histograms of the depolarization events for Mismatch control and depdc5 knock-down. The events were classified as high amplitude (> 1 mV -blue) and low amplitude (≤ 1 mV -black). (C-E) Example traces of the different periods of the recording: (C) spontaneous activity, (D)High amplitude events during the first period after PTZ application, (E) Low amplitude events during the latter period after PTZ application. Note that the script files to obtain these figures are provided as supplementary material.

(Figure 1 .

 1 Figure 1. Clinical features of ALS.

Figure 2 .

 2 Figure 2. Histopathology of ALS.

Figure 3 .

 3 Figure 3. ALS-related genes and surrounded affected mechanisms.

Figure 4 .

 4 Figure 4. Overview of autophagy progress.

Figure 5 .

 5 Figure 5. The autophagy machinery.

Figure 6 .

 6 Figure 6. Schematic model of the roles of SQSTM1/p62, OPTN and UBQLN2 proteins as autophagy receptors.

Figure 7 .

 7 Figure 7. Schematic representation of ALS-related genes and their function in autophagy process.

Figure 8 .

 8 Figure 8. Schema of SQSTM1/p62 protein.

Figure 9 .

 9 Figure 9. Neuropathological features of SQSTM1 inclusions.

Figure 10 .

 10 Figure 10. Identification of the C9ORF72 mutation associated with ALS/FTLD.

Figure 11 .

 11 Figure 11. Proposed mechanisms to explain C9ORF72 pathogenicity.

Figure 12 .

 12 Figure 12. Neuropathological features of C9ORF72 expansion carriers.

Figure 13 .

 13 Figure 13. Hypothesis of synergistic mechanisms in the C9ORF72 HRE pathogenicity.

Figure 14 .

 14 Figure 14. Expression pattern of a fluorescent reporter in motor neuron.

Article 1 :Figure 1 .

 11 Figure 1. Sqstm1 is expressed in zebrafish embryos and its knock-down leads to motor phenotype.

Figure 2 .

 2 Figure 2. Locomotor parameters are affected by Sqstm1 knock-down.

Figure 3 .

 3 Figure 3. Sqstm1 knock-down causes axonopathy in zebrafish.

Figure 4 .

 4 Figure 4. Alteration of Sqstm1 splicing leads to motor and axonal defects.

Figure 5 .Supplementary Figure 1 .Supplementary Figure 2 .

 512 Figure 5. Knock-down of Sqstm1 leads to autophagy alterations. Supplementary Figure 1. Peptide alignment of human and zebrafish SQSTM1/p62 proteins. Supplementary Figure 2. No major neuronal deficits are observed upon sqstm1 knockdown.

Figure 1 .

 1 Figure 1. Swimming parameters of 48 hpf embryos showing an additive effect of Sqstm1 and C9orf72 partial inhibitions

Figure 2 .

 2 Figure 2. Phenotype distribution of 48 hpf embryos showing an additive effect of Sqstm1 and C9orf72 partial inhibitions.

Figure 3 .

 3 Figure 3. Swimming parameters of 48 hpf embryos showing that C9orf72 acts downstream than Sqstm1.

Figure 4 .

 4 Figure 4. Phenotype distribution of 48 hpf embryos showing that C9orf72 acts downstream than Sqstm1.

Figure 5 .

 5 Figure 5. C9orf72 and Sqstm1 knockdowns affect survival in motor neurons.

Figure 6 .

 6 Figure 6. Soma area of motor neurons at 1 dpi.

Figure 7 .

 7 Figure 7. C9orf72 and Sqstm1 depletions reduce number of autophagosomes.

Article 2 :

 2 Synergistic gain and loss of function properties of the C9ORF72 mutation induce motor neuron degeneration in vivo.

Figure 1 .

 1 Figure 1. C9orf72 knock-down (KD) is essential to trigger DPR toxicity and an associated motor phenotype.

Figure 2 .

 2 Figure 2. GP100 expression combined with the c9orf72 loss-of-function causes axonopathy and spinal motor neuron (MN) loss.

Figure 3 .

 3 Figure 3. C9orf72 loss-of-function reduces GP100 clearance and leads to GP100 accumulation.

Figure 4 .Figure 5 .

 45 Figure 4. Autophagy induction promotes GP100 clearance Figure 5. sqstm1 KD synergizes with GP100 toxicity to trigger motor phenotypic features in zebrafish.

Figure EV1 .

 EV1 Figure EV1. GP100 degradation under normal conditions Figure EV2. Restoring C9ORF72 levels rescues velocity parameter.

Figure EV3 .

 EV3 Figure EV3. Spinal cord morphology is preserved under c9orf72 knockdown associated with GP100 expression.

Figure EV4 .

 EV4 Figure EV4. GP100 expression occurs within motor neuron single cells.

Figure EV5 .

 EV5 Figure EV5. C9orf72 KD alters GP100 but not GR100 clearance Figure EV6. GP10 and GP100 accumulates under c9orf72 KD Figure EV7. GP100 tends to accumulate under sqstm1 knockdown.

Figure 1 .

 1 Figure 1. Depdc5 expression in early development in the zebrafish larva.

Figure 2 .

 2 Figure 2. Depdc5 knockdown larvae show an early hyperactive motor phenotype.

Figure 3 .

 3 Figure 3. Depdc5 knockdown zebrafish exhibit a characteristic corkscrew-like swimming pattern.

Figure 4 .

 4 Figure 4. Electrophysiological extracellular recordings in the optic tectum of zebrafish larvae.

Figure 5 .

 5 Figure 5. Human WT, but not mutant, DEPDC5 can rescue the motor phenotype of Depdc5 knockdown zebrafish larvae.

Figure 6 .Article 4 :

 64 Figure 6. Rapamycin treatment rescues the motor phenotype of Depdc5 knockdown larvae.

Figure 1 .

 1 Figure 1. Example traces of field recordings in the zebrafish larvae brain

Table 1 . Genes associated with ALS, since 1993 to 2018.

 1

	Gene	Protein		Locus	Prevalence (%) FTLD related? familial sporadic	Interactors (DNA, RNA or proteins) §	References	Discovery Date
	ALS2	Alsin		2q33.1	-	-		C21orf2, VCP, NEK1	Hadano Yang et al	et	al
	ANG	Angiogenin		14q11.1-q11.2	<1	<1	x	-		Greenway et al
	ARHGEF28	Rho guanine nucleotide exchange factor (GEF) 28	5q13.2	-	-		-		Droppelmann et al
	ATX2	Ataxin 2		12q24.1	-	-	x	TARDBP, C9ORF72	Elden et al
	C21ORF2	Chromosome 21 open reading frame 2		21q22.3	<1	<1		ALS2, NEK1		Van Rheenen et al
								HNRNPA1, HNRNPA2B1,	Renton	et	al
	C9ORF72	Chromosome 9 open reading frame 72		9p21	40	7	x	UBQLN2, SQSTM1 *		DeJesus-Hernandez et al
	CCNF	Cyclin F		16p13.3	4	2	x	-		Williams et al
	CHCHD10	Coiled-coil-helix-coiled-coil-helix containing 10	domain	22q11.23 <1	<1	x	-		Bannwarth et al
	CHMP2B	Charged multivesicular body protein 2B	3p11.2	-	-	x	-		Parkinson et al
	DCTN1	Dynactin subunit 1		2p13	1	<1	x	-		Puls et al
	ELP3	Elongator acetyltransferase complex subunit 3 8p21.1	-	-		-		Simpson et al
	ERBB4	Erb-b2 receptor tyrosine kinase 4		2q34	-	-		-		Takahashi et al
	FIG4	Lipid phosphatase FIG4		6q21	-	-		-		Chow et al
	FUS	Fused in Sarcoma		16p11	4	1	x	MATR3, TARDBP, TAF15, VCP, SQSTM1, PFN1	Vance Kwiatkowski et al et al
	GLE1	GLE1 RNA export mediator		9q34.11	-	-		-		Kaneb et al
								HNRNPA2B1,	TARDBP,
	HNRNPA1 Heterogenous nuclear ribonucleoprotein A1 12q13.1	-	-		UBQLN2,		Kim et al
								MATR3, FUS, PFN1
	HNRNPA2B1	Heterogenous nuclear ribonucleoprotein A2/B1	7p15	-	-		HNRNPA1, TARDBP, FUS, MATR3, SQSTM1	Kim et al
								HNRNPA1, HNRNPA2B1,
	MATR3	Matrin 3		5q31.2	<1	<1	x	TARDBP,		Johnson et al
								FUS
	NEFH	Neurofilament, heavy polypeptide		22q12.12 -	-		-		Rooke et al
	NEK1	Serine/threonine kinase NIMA protein		4q33	2	2		ALS2, C21ORF2		Brenner et al
	OPTN	Optineurin		10p13	<1	<1		TBK1, SQSTM1, SOD1	Maruyama et al
	PFN1	Profilin-1		17p13	<1	<1		FUS, HNRNPA1		Wu et al
	SCFD1	Sec1 family domain containing 1		14q12	-	-		-		Van Rheenen et al
	SETX	Senataxin		9q34.13	-	-		-		Chance et al
	SIGMAR1	Sigma non-opioid intracellular receptor 1	9p13.3	-	-	x	-		Al-Saif et al
	SOD1	Cu/Zn superoxide dismutase		21q22.1	12	1-3		OPTN		Rosen et al
	SPG11	Spastic paraplegia 11		15q14	-	-		-		Daoud et al
								TBK1, TARDBP, OPTN,
	SQSTM1	Sequestosome 1, p62		5q35	1	<1	x	CHMP2B,	FUS,	Fecto et al
								HNRNPA2B1, NEFM
	TAF15	TATA-box binding protein associated factor 15	17q11.1-q11.2	-	-		-		Ticozzi et al
	TARDBP	TAR DNA binding protein 43		1p36	4	1	x	ATXN2, MATR3, UBQLN2, HNRNAPA2B1, HNRNPA1, SQSTM1, UBQLN2, VCP, FUS	Kabashi Sreedharan et al et Van Deerlin et al	al
	TBK1	TANK-binding kinase 1		12q14.2	<1	<1	x	-		Freischmidt et al Cirulli et al
	TUBA4A	α-tubulin 4A chain		2q35	<1	<1		-		Smith et al
	UBQLN2	Ubiquilin 2		Xp11.21	<1	<1	x	HNRNPA1, C9ORF72	TARDBP,	Deng et al
	VAPB	VAMP (vesicle-associated membrane protein)-associated protein B and C	20q13.33 <1	-		VCP		Nishimura et al
	VCP	Valosin-containing protein		9p13.3	1-2	1	x	FUS, TARDBP, VAPB, HNRNPA1	Johnson et al

  al, 2013; Lee et al, 2013b; Mori et al, 2013b;[START_REF] Reddy | The disease-associated r(GGGGCC)nrepeat from the C9orf72 gene forms tract length-dependent uni-and multimolecular RNA Gquadruplex structures[END_REF] Sareen et al, 2013; Xu et al, 2013; Cooper-Knock et al, 2014; Haeusler et al, 2014; Rossi et al, 2015c;[START_REF] Conlon | The C9ORF72 GGGGCC expansion forms RNA G-quadruplex inclusions and sequesters hnRNP H to disrupt splicing in ALS brains[END_REF] Hautbergue et al, 2017;[START_REF] Stopford | C9ORF72 hexanucleotide repeat exerts toxicity in a stable, inducible motor neuronal cell model, which is rescued by partial depletion of Pten[END_REF] and in vivo(Almeida et al,

2013b;

Xu et al, 2013; Rossi et al, 2015b; Lee et al, 2013b)

models have corroborated that

Table 2 . The majority of genes involved in the autophagy pathway are well conserved from zebrafish to human.

 2 Modified from de Calbiac et al, 2018.

	Human gene	Zebrafish orthologue(s)	Transcript Accession number(s)	Target % identity
	Akt/Protein kinase B (Akt1)	akt1	NM_001281801	83
	AMBRA1	Duplication Ambra1a and Ambra1b	NM_001281992 and FR846230	54 and
	ATG10	atg10	NM_001037124	48
	ATG12	atg12	NM_001246200	70
	ATG16L1	atg16l1	NM_001017854	71
	ATG3	apg3l, autophagy 3-like	NM_200022	83
	ATG4A	atg4a	NM_001024434	67
	ATG4B	atg4b	NM_001089352	72
	ATG4C	atg4c	NM_001002103	58
	ATG4D	Duplication atg4da and atg4db	XM_001333057 and GenBank:CK678774	47 and
	ATG5	atg5	NM_001009914	81
	ATG7	atg7	XM_002663680	69
	ATG9A	atg9a	NM_001083031	72
	ATG9B	atg9b	NM_001320078	45
	BCL2	Duplication bcl2a and bcl2b	NM_001030253 and XM_001341178	48 and
	BECN1	becn1	NM_200872	79
	C9ORF72	c9orf72	NM_205603	75,76
	GABARAP	Duplication gabarapa and gaparapb	NM_001013260 and XM_003199019	93 and
	MAP1LC3A	map1lc3a	NM_214739	86
	MAP1LC3B	map1lc3b map1lc3b-like	NM_199604	93
	MAP1LC3C	zgc:56565	NM_200298	65
	MLST8	mlst8	NM_199877	88
	MTOR	mTOR	NM_001077211	89
	NPRL2	nprl2	NM_001045847	82
	NPRL3	nprl3	NM_001025534	77
	PTEN	Duplication ptena and ptenb	NM_200708 and NM_001001822	77 and
	RAPTOR	Raptor	NM_001076748	86
	RICTOR	Duplication rictora and rictor1b	XM_009301197 and XM_009295386	74 and
	RPS6	rps6	NM_001003728	96
	SMCR8	smcr8a	NP_001340784	54,75
	SQSTM1	sqstm1	NM_001312913	47
	TBK1	tbk1	NM_001044748	71
	TSC1	Duplication tsc1a and tsc1b	NM_200052 and NM_001282392	42 and
	TSC2	tsc2	XM_009294973
	UBQLN2	no orthologue	-	-
	ULK1	Duplication ulk1a and ulk1b	NM_001130631 and XM_002665925	56 and
	ULK1	ulk1b	-	60,73
	ULK2	Ulk2	XM_002665582	75
	UVRAG	uvrag	NM_201069	61
	VCP	vcp	NM_201481	97
	VPS45	vps45	NM_001256656	82
	WIPI1	wipi1	NM_200391	68

1. Transient Knock-Down of Depdc5 Gene In Zebrafish Embryo 1.1. Preparation of tools:

  The day of the injection, prepare microinjection borosilicate glass needles using a puller (see table of materials). Establish appropriate temperature settings on the needle puller. Use a 10 cm long, 1/0.5 OD/ID mm borosilicate glass capillary to generate two ~5 cm capillaries with thin tips with a length of approximately 1 cm.1.1.6. If the tip of the needles is very fine, preventing the ejection of solution, break the very end of the tapered tip using forceps under a microscope.1.1.7. Just prior to injection, prepare the working solutions of AMOs. Always prepare fresh solution of injection to ensure the reproducibility of the results. Heat the AMO stock vials at 65 °C for 5 min. Prepare a 5 µL injection sample containing Fast Green dye (0.02% final concentration, see table of materials) and the AMO diluted at the working concentration in water.1.1.8. The working concentration of the AMO is determined empirically for each gene using a dose response curve, and represents a concentration where the AMO is effective in knocking down the gene without causing general toxicity, such as gross morphological defects.

	1.1.1. Prepare silicon elastomer-coated injection petri dishes: Mix the base and curing agent
	of the kit (see table of materials) in a 10:1 ratio. Fill a 35 mm Petri dish halfway with the
	mixture. Wait for the silicon to harden before using it (this can take several days).
	1.1.2. Prepare 1.2 mmol/L stock solutions of antisense morpholino oligonucleotides (AMO;
	see table of materials). Add 250 µL of sterile water to 300 nmole lyophilized AMO to obtain a
	1.2 mmol/L stock solution. For complete dissolution, heat the vials 5 min at 65°C. Vortex
	briefly. Seal tube cap with a plastic film (see table of materials).
	1.1.3. For the control rescue experiments, prepare an expression plasmid containing the
	human cDNA of DEPDC5 (see table of materials) cloned in the pCS2 backbone or a similar
	zebrafish-compatible expression plasmid. As a negative control, a mutation causing an early
	stop codon (p.Arg487*) was introduced in the cDNA.
	1.1.4. Prepare Embryo water: 0.06 g/L aquarium salt (see table of materials) in reverse osmosis
	water + 0.5 mg/L Methylene blue.
	1.1.5.

  Fill a glass needle with injection solution. Place the needle vertically in one of the tubes containing the injection solution, ensuring that the bottom end of the needle is touching the solution. Wait several min until the colored injection solution rises by capillarity and is visible at the tip of the needle.

	1.3. Microinjections:
	1.3.1. 1.3.2. Mount the filled needle on the injecting handle of the microinjector (see table of
	materials).

1.3.3

. Turn on the air compressor and adjust the pressure setting to generate an injection volume of ~2 nl.

2.1. Global activity analysis at 28 hours post fertilization (hpf): 2

  .1.1. Conduct the test the afternoon of the day after microinjection(28 hpf), ensuring that the time of day at which the test is conducted is consistent over experiments to perform valid statistical analysis as embryos development is very rapid.2.1.2. Fill a 35 mm dish (test dish) with embryo water and allow it to warm up in the incubator (28°C) for at least 15 min prior to starting the test.2.1.3. Place a plastic mesh grid (1.2x1.2 mm) cut to size, on the bottom of the test dish.

  Mount the camera (see table of materials) over the test dish making sure that the entire test dish is within the field of view. Placing a ruler within the field of view provides an internal calibration for distance.2.2.7. With a plastic Pasteur pipette, put an embryo in the center of the test dish and begin the recording using an acquisition rate of 30 fps.2.2.8. With a fine plastic tip, touch slightly the tail of the embryo with a flicking motion. 3.2.3. Add 2 mL of the recording solution containing 10 µM Pancuronium bromide (see table of materials) to block neuromuscular transmission. The addition of the paralyzer is necessary to eliminate artefacts due to small movements during the recordings. With the patch clamp amplifier (see table of materials) in voltage clamp configuration, measure electrode resistance in bath to confirm its correct value.3.3.3. Using a 20X objective, position the head of the larva in the central field of view and lower the micropipette to reach the recording position in the brain, within the optic tectum. 3.3.4. Switch the patch clamp amplifier to current clamp and fix the holding current to 0 mA. 3.3.5. Using a low-pass filter of 1 kHz, an acquisition rate of 1 kHz and a digital gain of 10, record spontaneous activity for 60 min for determining baseline activity levels.

	2.2.2 At least two hours prior to testing, dechorionate the embryos using fine forceps. Ensure that the time of day for the dechorionation and the behavior test is consistent over experiments. 2.2.3. Fill a 130 mm dish (test dish) with embryo water and allow it to warm up in the incubator (28°C) for at least 15 min prior to starting the test. 2.2.4. Count and remove dead and morphologically deformed larvae. Record the numbers for each condition. 2.2.5. Have another experimenter randomize the order and codify the names of the conditions to be tested. 3.3.1. Fill the micropipette with recording solution. 2.2.6. 3.3. Electrophysiological recording 3.3.2.

-Evoked-Escape-Response (TEER) at 48 hpf: 2.2.1. Conduct the test in the morning two days after the injection (48 hours after the fertilization). 3.3.6. After 1h of baseline recording, 200 µL pentylenetetrazol (PTZ, see table of materials) solution 300 mmol/L is added to the bath for a final concentration of 20 mmol/L PTZ. 3.3.7. Record the neuronal activity in PTZ for another 120 min.

Table 1 . Table of materials

 1

	Name of Material	Company	Catalog Number	Comments/Description
	Depdc5-atg antisense morpholino	GeneTools, OR, USA	N/A	sequence 5'-TGCCTTCATGGTGACCGTCATTTTA -3'
	Depdc5-mis antisense morpholino	GeneTools, OR, USA	N/A	sequence 5'-TGCgTTgATcGTGACCcTgATTTTA -3'
	Depdc5-splice antisense morpholino GeneTools, OR, USA	N/A	sequence 5'-ACATTCCTGTTTCACCATAGATGAT -3'
	Fast Green Dye	Sigma-Aldrich, France	F7258	Stock solution of 0.2%
	human wild-type DEPDC5 cDNA	Dharmacon, France	NM_001242897.1	Accession: BC144291 Clone ID 905
	Grasshopper 2 camera	FLIR, BC, Canada	GRAS-03K2M-C	formerly Point Grey Research
	ImageJ software	NIH, USA	N/A
	Pneumatic PicoPump	World Precision Instruments, France	PV 820
	Sylgard 184 kit	Sigma-Aldrich Intl.	761036
	Borosilicate glass with filament Sutter Instruments	BF100-50-10	OD: 1,5mm, ID: 0,5 mm
	Glass-bottom petri dishes	Ibidi, Germany	81218
	Transfer plastic pipettes	Sigma-Aldrich, France	Z350605
	Pentylenetetrazol (PTZ)	Sigma-Aldrich, France	P6500	Stock solution of 300mM (dissolved in recording solution)
	Pancuronium bromide	Alomone Labs	P-130	Stock solution of 60mM in water
	Agarose	Sigma-Aldrich, France	A9539
	NaCl	Sigma-Aldrich, France	S7653
	KCl	Sigma-Aldrich, France	P9333
	CaCl2	Sigma-Aldrich, France	C1016
	MgCl2	Sigma-Aldrich, France	M2670
	glucose	Sigma-Aldrich, France	68270
	HEPES	Sigma-Aldrich, France	H3375
	NaOH	Sigma-Aldrich, France	71687
	pClamp10 acquisition software	Molecular Devices	N/A
	Matlab software	MathWorks, MA, USA	N/A
	Pipette puller	Narishige, Japan	PC-10
	Patch clamp amplifier	Molecular Devices, CA, USA	MultiClamp 700B	Computer-controled patch clamp amplifier
	Digitizer	Molecular Devices, CA, USA	Digidata 1550
	Zebralab	Viewpoint, France	N/A
	Aquarium salt	Instant Ocean, Blacksburg, VA	SS15-10
	Parafilm	Sigma-Aldrich, France	P7793

Table 1 .

 1 Table of materials

Axonal lenght (µM)

ACKNOWLEDGEMENTS

We would like to thank the staff of the ICM electrophysiology platform where the neurophysiology experiments were performed. We also thank Anca Marian for technical help. SC was supported by the Trampoline Grant #21488. EK was supported by the AFM Grant #18469 and ERC Consolidator Grant (ALS-Networks). HC was supported by PhD awards from the Fondation pour la Recherche Médicale (PLP20141031462) and ARSLA. For AD and RM, this work was supported by three grants from the Romanian National Authority for Scientific Research and Innovation, CNCS-UEFISCDI (project numbers PN-III-P4-ID-PCE-2016-0010, PN-III-P2-2.1-PED-2016-0007, and COFUND-NEURON-NMDAR-PSY), a grant by the European Union's Horizon 2020 research and innovation programme -grant agreement no. 668863-SyBil-AA, and a National Science Foundation grant NSF-IOS-1656830 funded by the US Government.

Data were plotted and analyzed using the Excel software (Microsoft, USA). SigmaPlot 11.0 (Systat, USA) software was used for statistical analysis. Normality was determined by Shapiro-Wilke test. Homoscedasticity was tested with Levene's test. According to its results, ANOVA or Kruskal-Wallis analysis of variance was applied. Details of each test, including post-hoc analysis are described in related figures legend.

Note

The object of this thesis was to elucidate the mechanisms associated with the most common cause of ALS: C9ORF72 mutation in amyotrophic lateral sclerosis. In parallel, I also worked on the development and the characterization of a zebrafish model of focal epilepsy. This study was a side-project during these years and is, therefore, dealt within a much more concise part.

Data analysis

Data were plotted and analyzed using the Excel software (Microsoft, USA). SigmaStat 11.0 (Systat, USA) software was used for statistical analysis. Normality was determined by Shapiro-Wilke test. Homoscedasticity was tested with Levene's test before the analysis of variance test (ANOVA or Kruskal-Wallis, as required). Details of each test are described in related figures legend. The number of sets of experiments (n) is indicated in the legend. For TEER results and motor neurons features, at least five sets of experiments were conducted. Except mention express in the legends, the measurements were taken from distinct samples.

AKNOWLEDGEMENTS

CONFLICTS OF INTEREST

The authors declare no conflicts of interest.

Supplemental figure 1. Please refer to Introduction,

SUMMARY:

Here, we present a protocol for the development and the characterization of a zebrafish model of epilepsy resulting from the transient inhibition of the depdc5 gene.

ABSTRACT

Epilepsy represents one of the most common neurological disorders, affecting an estimated 50 million people worldwide. Recent advances in genetic research have uncovered a large spectrum of genes implicated in various forms of epilepsy, highlighting the heterogeneous nature of this disorder. Appropriate animal models are essential for investigating the pathological mechanisms triggered by genetic mutations implicated in epilepsy and for developing specialized, targeted therapies. In recent years, zebrafish has emerged as a valuable vertebrate organism for modeling epilepsies, with the use of both genetic manipulation and exposure to known epileptogenic drugs, such as pentylenetetrazole (PTZ), to identify novel anti-epileptic therapeutics. Deleterious mutations in the mTOR regulator DEPDC5 have been associated with various forms of focal epilepsies and knock-down of the zebrafish orthologue causes hyperactivity associated with spontaneous seizure-like episodes, as well as enhanced electrographic activity and characteristic turn wheel swimming. Here we described the method involved in generating the DEPDC5 loss-of-function model and illustrate the protocol for assessing motor activity at 28 and 48 hours post fertilization (hpf), as well as a method for recording field activity in the zebrafish optic tectum. An illustration of the effect of the epileptogenic drug PTZ on neuronal activity over time is also provided.

CHAPTER 3 -

DISCUSSION, PERSPECTIVES AND CONCLUSION

applications, with a first AMO therapy receiving the FDA approval for the treatment of Duchenne muscular atrophy in 2016 (Lim et al, 2017). While it was reported that in zebrafish the phenotype of acute AMO-based gene knockdown does not always correlate with the constitutive knockout models [START_REF] Kok | Reverse genetic screening reveals poor correlation between morpholino-induced and mutant phenotypes in zebrafish[END_REF], this can be due at least in some instances to compensatory mechanisms engendered by constitutive genetic modifications (Rossi et al, 2015a). However, the issue of specificity of the AMO-induced phenotype is an undisputable concern that has to be diligently addressed in studies using this technology (Stainier et al, 2017b). In order to ensure the specificity of our phenotype, we would develop a gain of function transgenic line of C9ORF72, where the DPR are expressed alone, as well as replicate the injection of the plasmid containing the DPR in a c9orf72 TALEN transgenic line, which is available in the lab. Moreover, stable such transgenic lines have less variations, thus allowing more optimized drug screenings and molecular analysis, particularly RNA sequencing.

In this thesis, we also report that rapamycin is able to rescue the defects mediated by the synergy of c9orf72 loss of function and poly(GP) expression. Interestingly, rapamycin was also able to reduce the aberrant features induced by the knockdown of depdc5 gene in our zebrafish model of focal epilepsy. In this model, we observed that DEPDC5 pathogenicity could be mediated by its loss of function. Consistent with DEPDC5 role in the negative regulation of mTOR pathway (Bar-Peled et al, 2013b;Panchaud et al, 2013a), rapamycin treatment reduces the apparent hyperexcitability features of depdc5 knockdown embryos. Of interest, a recent study has shown that C9ORF72-mediated toxicity in ALS/FTLD was associated with hyperexcitability (Shi et al, 2018). Thus, it would be interesting to investigate whether autophagy and hyperexcitability are crosstalking mechanisms in the pathogenesis of epilepsy and ALS.