La sclérose latérale amyotrophique (SLA) est une pathologie caractérisée par une perte progressive des neurones moteurs. Ces neurones spécialisés conduisent l'information du cerveau vers les muscles permettant ainsi les mouvements. La perte de ces cellules induit donc une paralysie musculaire conduisant au décès des patients par défaillance respiratoire.

A ce jour, aucun traitement efficace permettant le rétablissement des patients n'a été développé. Parmi les facteurs de risques génétiques associés au développement de la SLA chez les patients, ont été identifiées des mutations dans la séquence du gène TBK1. Le travail présenté ici est basé sur le développement d'un modèle d'étude chez le poisson zèbre associant une perte d'activité de la protéine Tbk1 avec les différentes marques cellulaires de la SLA. Ce travail a donc pour but d'identifier de nouvelles cibles thérapeutiques et par conséquent d'amener vers le développement de nouveaux traitements améliorant le pronostic des patients. Amyotrophic lateral sclerosis (ALS) is a pathology characterized by the progressive loss of motor neurons. These specialized neurons conduct the information from the brain to the muscles in order to induce the movements. The loss of these cells therefore induces a muscular paralysis leading to patients' decease via respiratory failure. At this time, no treatments allowing patients recovering have been developed. Mutations in the sequence of the tbk1 gene have been identified as risk factors in the development of ALS. The work presented in the following thesis is based on the development of a zebrafish model of study recapitulating the different cellular hallmarks of ALS associated to the loss of activity of TBK1 protein. This work is therefore aiming to identify new therapeutic target and consequently developed new treatments ameliorating patients' prognostic.

La sclérose latérale amyotrophique (SLA) est une pathologie caractérisée par une neurodégénérescence progressive des neurones moteurs (NM) corticaux et spinaux induisant une paralysie musculaire conduisant au décès des patients par défaillance respiratoire. TBK1 a été associé pour la première fois à la SLA en 2015. Etroitement lié à l'autophagie et à l'inflammation, deux mécanismes cellulaires associés à la pathophysiologie de la SLA, il est important de comprendre le rôle fonctionnel de TBK1 dans le développement de cette pathologie. Due à sa ressemblance anatomique et neurochimique avec le système nerveux humain et à l'homologie élevée avec son orthologue TBK1 humain, le poisson zèbre est aujourd'hui considéré comme un des meilleurs modèles pour l'étude de la SLA associée à TBK1. Afin d'identifier les mécanismes sous-jacents à la perte d'expression de TBK1 dans la dégénérescence des NM in vivo, nous avons généré deux nouveaux modèles de perte d'expression de tbk1 chez le poisson zèbre en utilisant deux approches : la réduction de l'expression via morpholinos (perte partielle d'expression -KD) et l'induction de mutations via la technologie CRISPR (perte totale d'expression -KO). Dans les 2 modèles nous avons identifié une diminution de la mobilité liée à la perte d'expression de tbk1, cependant le stade de développement du phénotype observé est différent en fonction du modèle : 2 jours post fécondation (jpf) dans le modèle KD et 5 jpf dans le modèle KO. De plus dans le modèle KD, la perte de capacité motrice est associée à une diminution dans le nombre de NM spinaux. Cette observation n'est pas reproduite dans le modèle KO et n'explique donc pas l'apparition d'un phénotype moteur. Enfin, à un stade plus avancé (9 jpf), nous avons observé une mortalité chez le modèle KO, mettant en évidence le rôle crucial de TBK1 dans le développement du poisson zèbre. Afin d'identifier les dérèglements moléculaires responsables des phénotypes observés chez le modèle KO, nous avons effectué une analyse métabolique aux deux stades de développement d'intérêt (6 et 8 jpf). L'analyse métabolomique a révélé que la perte d'expression de tbk1 était associée à une dérégulation du métabolisme du nicotinamide. En effet, l'incubation des larves avec un précurseur du NAD+ (métabolite central du métabolisme du nicotinamide) : le nicotinamide riboside (NR), a permis de restaurer la locomotion. Cette observation met en évidence que l'absence d'expression de tbk1 chez le poisson zèbre altère les niveaux physiologiques de NAD+ ce qui induit à une plus grande échelle une perte de mobilité. Cependant l'incubation avec le NR n'a eu aucun impact sur la survie des larves. J'ai donc cherché quelle pourrait être la cause de la mort de ces larves en combinant analyses protéomique et métabolomique. Ces données ont montré une augmentation de marqueurs inflammatoires ainsi qu'une modification de l'expression des protéines responsables de la régulation de la mort cellulaire active (MCA) (apoptose ou nécroptose). Par conséquent, nous avons cherché à identifier laquelle de la MCA ou de l'inflammation était la cause primaire de ce phénotype. Pour répondre à cette question, j'ai incubé les larves avec un inhibiteur de la neuroinflammation : l'acide caféique. Cependant, cette prévention de la réaction neuroinflammatoire n'a eu aucune conséquence sur la survie des larves. Bien que cela n'élimine pas totalement le rôle de l'inflammation, cela suggère que le facteur induisant la mortalité dans notre population de larves pourrait être l'activation d'une MCA. Un futur criblage moléculaire permettant de prouver cette hypothèse et d'identifier quel mécanisme de MCA est responsable de la mortalité observée sera effectué dans la lignée de cette étude. Pour autant, ce travail apporte une meilleure compréhension de la cascade d'évènements menant de la perte d'expression de TBK1 au développement de la SLA.

Amyotrophic lateral sclerosis (ALS) is a neurological disease characterized by the progressive degeneration of both spinal and cortical motor neurons that leads to muscle paralysis and the subsequent death by respiratory failure. TBK1 mutations potentially leading to haploinsufficiency were first described in ALS patients in 2015. Tightly linked with autophagy and inflammation, two cellular mechanisms reported to be dysregulated in ALS patients, it remains of major interest to understand the functional role of TBK1 in the development of this neurological condition. Due to its high anatomical and neurochemical resemblance with the human brain and the important homology with its orthologous TBK1 human gene, the zebrafish is today considered as an excellent vertebrate model suitable for the study of tbk1related ALS. In order to identify the subjacent mechanisms linked with tbk1 loss of function and the subsequent degeneration of motor neurons in vivo, we have generated and characterized two new zebrafish models of TBK1 loss of function using two approaches; the first was via antisense morpholino oligonucleotide to generate a zebrafish model with partial loss of expression referred to as knockdown (KD). The second approach was to induce genomic mutations via the utilization of the CRISPR technology and to generate deletion mutant with loss of expression referred as the tbk1 knockout KO) model. In these two models, I report an impaired locomotor activity associated with the loss of tbk1 expression.

However, the motor phenotype occurs at various stages of development according to the approach used: 2 days post fertilization (dpf) in the KD model and 5 dpf in the KO model. Moreover, we associated the impaired locomotion in the KD model with a decrease in the number of spinal motor neurons indicating neurodegenerative features in this model. On the other hand, we did not observe any increased mortality of motor neurons in the KO model but we reported a high lethality upon tbk1 complete deletion at 9dpf suggesting a crucial role for Tbk1 in the zebrafish development. In order to identify the subjacent molecular dysregulations responsible for the phenotypes observed in the KO model, we performed a metabolomics analysis at the two developmental stages of interest (6 and 8 dpf). The metabolomics analysis unveiled an association between tbk1 loss and a severe dysregulation of the nicotinamide metabolism. The incubation of the larvae with a precursor of NAD+ (central metabolite of the nicotinamide metabolism): the nicotinamide riboside, permitted to restore the locomotion defects in the tbk1 KO larvae. This observation revealed that tbk1 reduced expression provoke an alteration of the physiological level of NAD+ which could correlate with the loss of mobility. However, NR incubation was not sufficient to delay or to prolong increased mortality observed in the tbk1 KO larvae.

To determine the cause of increased mortality observed in the tbk1 KO model at 9 dpf, we combined the metabolomic analysis with proteomics to unravel altered pathways associated with tbk1 KO. Increased inflammation markers and dysregulation of two programmed cell death (PCD) mechanisms, namely the necroptosis and the apoptosis were observed. To separate between these two hypotheses, we incubated the larvae with an inhibitor of the neuroinflammation process: caffeic acid. Caffeic acid treatment failed to reduce mortality in the tbk1 KO model, thus suggesting that neuroinflammation may not play a crucial role in increased mortality observed. This suggests that the activation of programmed cell death pathways could be mainly responsible for the lethality observed in our KO model. A future pharmacological approach to reduce necroptosis and apoptosis activation will allow us to support this hypothesis, and to further identify which of the cell death pathways was involved in the tbk1 KO model; thus, increasing our understanding of the cascade of events leading from the loss of tbk1 expression to the development of the ALS and propose therapeutic avenues for this neurological disease.

INTRODUCTION 14

In this introduction, I will review the recent literature in the field of Amyotrophic Lateral Sclerosis (ALS) and one of the genetic causes for this neurodegenerative disorder, TBK1 on which this thesis will focus. First, I will introduce the clinical features of ALS before discussing about the cellular hallmarks of this pathology. I will also review major cellular and animal disease models and their importance to a better understanding of pathogenic mechanisms of ALS. Secondly, I will review the recent studies on TBK1 functional role and its involvement in the pathogenicity of ALS. Finally, as part of the present work will be focusing on the metabolomics characterization of the novel tbk1 KO zebrafish model that I developed during my doctoral work, I will review the emerging field of metabolic alterations both in patients and in genetic models of ALS.

Part I: Introduction to ALS

I.

History and description of the disease:

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive degeneration of both the upper and the lower motor neurons (MN). This is translated at a macroscopic scale by the progressive denervation and the atrophy of the skeletal muscles (except oculomotor and sphincter motor neurons) leading from paresis to complete paralysis and eventually death within 3 to 5 years after diagnosis, predominantly through respiratory failures (A. Chio et al., 2009).

The term ALS was first mentioned in 1874 by the French neurologist Jean- Martin Charcot (1825-1893). However, it is important to mention that this first description was made possible by the work of many scientists prior to Charcot:

-Charles Bell (1774-1842), a Scottish anatomist and physiologist, published in 1824

"Exposition of the natural system of the nerves" where he describes for the first time two distinct roots in the spinal cord: 1) the anterior roots involved in the motor abilities 2) the posterior roots involved in the sensory abilities. He is therefore the first to mention that neurological disease could be purely motor related.

-François Amilcar-Arran (1817-1861), a French doctor, is the first to describe ALS in 1850 without being able to distinguish it from other neurodegenerative disease associated with a loss of motor functions. However, according to Arran, this disease was only related to an alteration of the muscular system, without any neuronal involvement (F.A. Aran, 1850).

-Jean Cruvelier (1791-1874), a French doctor and anatomist, in 1852, after performing a series of autopsies on three patients, observed an atrophy of the anterior roots of the cervical region. He therefore proposes to switch Aran's paradigm of an exclusively muscle related disease to a model reflecting a primary alteration of the spinal cord's anterior roots leading to a secondary muscle atrophy (J. Cruveilhier, 1852Cruveilhier, -1853)).

-Jean-Martin Charcot was the first to observe an association between lateral sclerosis and grey matter of the spinal horn in 1868 (J. M Charcot, 1872M Charcot, -1873)). He proposed in 1872 to split the heterogenous group of muscular atrophies into two sub-classes: 1)

the 'true spinal' muscular atrophies caused by the loss of anterior horn cells (corresponding to the progressive muscular atrophies described by Aran and

Cruveilhier); 2) the 'false spinal' muscular atrophies caused by a secondary loss of anterior horn cells associated with the degeneration of the lateral fasciculi of the spinal cord (i.e the ALS). It is in 1874 that Charcot formalized the description of ALS in his lecture. This discovery was made possible for Charcot through the introduction of the microscopic analysis in opposition to the previous macroscopic observations performed by its precursors.

In addition, it is important to mention that although Charcot's name will always be associated with the disease, his discovery was only made possible by the findings of his two pupils and close collaborators Charles Bouchard (1837Bouchard ( -1915) ) and Albert Gombault (1844Gombault ( -1904) ) whose name have been forgotten.

II. Clinical features of ALS-FTD A. Epidemiology

Quantifying the worldwide incidence of ALS is complicated as the environment and the genetic background are greatly impacting the risk of contracting the pathology (B. Marin et al., 2016). Therefore, although it is now accepted that the global incidence of ALS is approximatively 1 to 2 cases per 100 000 persons per year and the prevalence is of 4-6 per 100 000 persons (L. [START_REF] Xu | Global variation in prevalence and incidence of amyotrophic lateral sclerosis: a systematic review and meta-analysis[END_REF][START_REF] Talbott | The epidemiology of amyotrophic lateral sclerosis[END_REF]B. Marin et al., 2016), it is more relevant to study ALS epidemiology in a specific sub-population. Moreover, this bias is accentuated by the overrepresentation of populations with a European genetic background in the epidemiology studies published (more than 75% are focusing on European or North American populations) (L. [START_REF] Xu | Global variation in prevalence and incidence of amyotrophic lateral sclerosis: a systematic review and meta-analysis[END_REF].

Several risk factors have been identified to explain the influence of geography on the heterogeneity of ALS. Interestingly, it seems that the incidence is highly linked to the wealth of the population (Fig. 1) (4.94 in the western world in opposition to 2.15 in developing territories) (L. [START_REF] Xu | Global variation in prevalence and incidence of amyotrophic lateral sclerosis: a systematic review and meta-analysis[END_REF]. This is possibly explained by a higher life expectancy, ALS being a late onset pathology. Still in this direction, although the onset of the disease occurs prevalently during adulthood, the median age of onset is highly variable according to the populations (E. [START_REF] Longinetti | Epidemiology of amyotrophic lateral sclerosis: an update of recent literature[END_REF]. For instance, a comparative study between

Chinese and German population have shown a 10 years difference in the median age of onset (51 years in China vs 61 years in Germany) (J. [START_REF] Dorst | Prognostic factors in ALS: a comparison between Germany and China[END_REF]. Moreover, the survival time to ALS is also highly different according to the population studied (24 months in Europe in opposite to 48 months in central Asia) (L. [START_REF] Xu | Global variation in prevalence and incidence of amyotrophic lateral sclerosis: a systematic review and meta-analysis[END_REF]B. Marin et al., 2016).

However, the age of onset has been showed to directly impact the prognosis of the patients (W-M. Su et al., 2022). Altogether, these observations suggest that population comparative epidemiology studies will always suffer from the lack of relevance of comparing various population.

Another risk factor is gender, with males more likely to develop ALS (Fig1) (Z. R. [START_REF] Manjaly | The sex ratio in amyotrophic lateral sclerosis: A population based study[END_REF]C. Ingre et al., 2014) and exhibit both a higher incidence and prevalence (L. Xu et al., 2020) with a male/female ratio located in between 1 and 2 (E. [START_REF] Longinetti | Epidemiology of amyotrophic lateral sclerosis: an update of recent literature[END_REF]; Z. R. [START_REF] Manjaly | The sex ratio in amyotrophic lateral sclerosis: A population based study[END_REF]. This sex ratio is inversely proportional to the age of onset (Z. R. [START_REF] Manjaly | The sex ratio in amyotrophic lateral sclerosis: A population based study[END_REF]. Therefore, it could be interesting to determine whether this ratio is increased in population characterized with an earlier onset of disease. In addition, it seems that gender could also impact the clinical subtypes of pathology, a study in a European population have reported a higher propensity for spinal-onset disease in male patients in opposition to female cases which are more associated with bulbar-onset pathologies (G. [START_REF] Logroscino | Incidence of amyotrophic lateral sclerosis in Europe[END_REF]. Due to a lack of comprehension in the pathogenicity of ALS it is difficult to understand biologically the origin of this sex-related difference of incidence.

B. Risk factors 1. Environmental risk factors

All these geographical/gender-based risk factors are even more difficult to elucidate as in most of the ALS cases (more than 80%), clinical features are thought to appear spontaneously within a generation without any first-degree relatives of the patients being affected. This feature leads to the creation of an over-simplified dichotomization of the pathology between the sporadic and the familial cases (which are hereditary passed through generations). The reality is different and ALS, similarly to other neurodegenerative diseases, is characterized by a multifactorial inheritance, thus triggered by a combination of dysregulated genes and environmental factors (O. Hardiman et al., 2017). So far, 30 genes have been associated with a higher risk of contracting ALS but the real number is probably higher [START_REF] Mathis | Genetics of amyotrophic lateral sclerosis: A review[END_REF].

Several environmental factors have been associated with a higher risk of triggering the disease, further explaining the heterogeneity of the epidemiology. These risk factors are very diverse and can be linked to the lifestyle: dietary factors, exercise, body mass index, or smoking; to the professional occupation: exposition to metal or pesticides, electric shock or proximity with electromagnetic fields (B. [START_REF] Oskarsson | Potential Environmental Factors in Amyotrophic Lateral Sclerosis[END_REF]. Moreover, the risk of ALS was shown to be associated to specific health conditions related to viruses, metabolism alterations, or innate immune system dysregulation (A. [START_REF] Jawaid | Disease-modifying effects of metabolic perturbations in ALS/FTLD[END_REF][START_REF] Oskarsson | Potential Environmental Factors in Amyotrophic Lateral Sclerosis[END_REF]. Interestingly, early life exposure to environmental cues has been reported to directly impact the innate immune response (M. [START_REF] Garand | Environment impacts innate immune ontogeny[END_REF]; D.M. MacGillivray and T.R. Kollmann, 2014), further complexifying the role of environmental risks on the development of the disease.

Because investigating the impact of the risk factors on the development of the pathology is challenging and to this point there is no direct evidence linking an environmental trigger to ALS, the vast majority of the research has focused on the genetic part of the disease as discussed in the next section.

Genetic risk factors

Although most of the ALS cases appeared sporadically within a generation without a first [START_REF] Morita | A locus on chromosome 9p confers susceptibility to ALS and frontotemporal dementia[END_REF][START_REF] Vance | Familial amyotrophic lateral sclerosis with frontotemporal dementia is linked to a locus on chromosome 9p13.2-21[END_REF]. However, it is in only 5 years later, in 2011, that the presence of a massive GGGGCC hexanucleotide repeat expansion (G4C2) was identified within the first intron of the C9ORF72 gene (M. [START_REF] Dejesus-Hernandez | Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS[END_REF][START_REF] Renton | A hexanucleotide repeat expansion in C9ORF72 is the cause of chromosome 9p21-linked ALS-FTD[END_REF]. Interestingly, healthy individuals present these G4C2 repeats, but at a lesser extend (under 30 copies) than patients with C9ORF72 associated ALS that carry from 45 to several thousand G4C2 repeats (I. [START_REF] Gijselinck | The C9orf72 repeat size correlates with onset age of disease, DNA methylation and transcriptional downregulation of the promoter[END_REF]. This discovery was a real breakthrough in the field of ALS, as it is the most frequent (around 30% of the cases) mutations found in ALS patients with a European genetic background (E. [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]. The role of C9ORF72 G4C2 repeats in the development of C9ORF72 related disease is still highly discussed as there is evidence of two deleterious consequences. First, this mutation induced a decrease in C9ORF72 expression and the subsequent loss of function of its encoded protein (I. [START_REF] Gijselinck | A C9orf72 promoter repeat expansion in a Flanders-Belgian cohort with disorders of the frontotemporal lobar degeneration-amyotrophic lateral sclerosis spectrum: a gene identification study[END_REF]. A decreased expression of C9ORF72 has been associated with an impaired autophagic process and the presence of cytoplasmic aggregates (C.J. [START_REF] Donnelly | RNA toxicity from the ALS/FTD C9ORF72 expansion is mitigated by antisense intervention[END_REF][START_REF] Sellier | Loss of C9ORF72 impairs autophagy and synergizes with polyQ Ataxin-2 to induce motor neuron dysfunction and cell death[END_REF]. This loss of function hypothesis is further confirmed in the zebrafish model where the reduced expression of the C9ORF72 ortholog results in locomotor deficits [START_REF] Ciura | Loss of function of C9orf72 causes motor deficits in a zebrafish model of amyotrophic lateral sclerosis[END_REF]. Secondly, the hexanucleotide repeats were shown to undergo a non-AUG translation leading to the expression of dipeptide repeat proteins (DPR) which are forming cytoplasmic aggregates (K. [START_REF] Mori | The C9orf72 GGGGCC repeat is translated into aggregating dipeptide-repeat proteins in FTLD/ALS[END_REF]. Interestingly, the accumulation of these DPRs does not appear to impact the efficiency of the autophagy or the proteasome (S. Leskela et al., 2021). Therefore, the actual paradigm concerning C9ORF72 associated disease due to expanded G4C2 hexanucleotide repeats is both a loss and a gain of function mutation, inducing the generation of cytoplasmic aggregates and preventing their clearance (Y. [START_REF] Shi | Haploinsufficiency leads to neurodegeneration in C9ORF72 ALS/FTD human induced motor neurons[END_REF][START_REF] Pal | Concomitant gain and loss of function pathomechanisms in C9ORF72 amyotrophic lateral sclerosis[END_REF][START_REF] Smeyers | C9ORF72: What It Is, What It Does, and Why It Matters[END_REF].

b. SOD1

Mutations in the SOD1 gene were the first identified mutations associated with the development of ALS in 1993 (D.R. [START_REF] Rosen | Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis[END_REF]. This discovery still remains as one of the biggest breakthroughs in the study of the ALS as it allowed the comprehension of many features of the disease through the design and generation of many in vivo model of the pathology. The murine model SOD1G93A is still the most used in-vivo model in the ALS research field (M.E. [START_REF] Gurney | Motor neuron degeneration in mice that express a human Cu,Zn superoxide dismutase mutation[END_REF]. Since 1993, more than 180 variants have been identified along the sequence of SOD1 in the context of ALS, and SOD1 mutations are still the most common known genetic cause of ALS in the Asian population (approximatively 30%) and the second in the European genetic background population (Z-Y. [START_REF] Zou | Genetic epidemiology of amyotrophic lateral sclerosis: a systematic review and meta-analysis[END_REF]. SOD1

protein is involved in the clearance of deleterious reactive oxygen species (ROS) through their conversion into oxygen and hydrogen peroxide species (J. [START_REF] Azadmanesh | A Review of the Catalytic Mechanism of Human Manganese Superoxide Dismutase[END_REF]. Although SOD1 mutations have been associated with oxidative stress (R.T. [START_REF] Ferrante | Evidence of increased oxidative damage in both sporadic and familial amyotrophic lateral sclerosis[END_REF], the cascade of events linking SOD1 mutations to MN degeneration is unclear. Large body of evidence indicates that it is not the actual loss of SOD1 activity that is responsible for these alterations but rather a gain of toxicity induced by a conformational change in the protein and subsequent cytoplasmic aggregation (Y. [START_REF] Hayashi | SOD1 in neurotoxicity and its controversial roles in SOD1 mutation-negative ALS[END_REF].

Evidences from in vitro and in vivo model support the hypothesis of a prion-like propagation mechanism in which a misfolded mutant or wild-type SOD1 protein transmits its aberrant misfolding properties to native proteins inducing the formation of toxic cytoplasmic aggregates (C. [START_REF] Münch | Prion-like propagation of mutant superoxide dismutase-1 misfolding in neuronal cells[END_REF][START_REF] Ayers | Prion-like propagation of mutant SOD1 misfolding and motor neuron disease spread along neuroanatomical pathways[END_REF][START_REF] Polymenidou | The seeds of neurodegeneration: prion-like spreading in ALS[END_REF].

c. TARDBP

TARDBP mutations were first associated with ALS in 2008 (E. [START_REF] Kabashi | TARDBP mutations in individuals with sporadic and familial amyotrophic lateral sclerosis[END_REF]. TARDBP is the third most common known genomic mutation associated with ALS in European genetic background population with 4.2% of the cases concerned (and the fourth in the Asian population: 1.5% of the cases) [START_REF] Mathis | Genetics of amyotrophic lateral sclerosis: A review[END_REF]. TARDBP encodes a nuclear ribonucleoprotein, TAR DNA binding protein 43 (TDP-43), which is involved in the regulation of RNA processing and gene transcription (T.J. [START_REF] Cohen | TDP-43 functions and pathogenic mechanisms implicated in TDP-43 proteinopathies[END_REF]. Mutation in the TARDBP sequence is known to induce misfolding of TDP-43 inducing its cytoplasmic mislocalization and its aggregation (T. [START_REF] Arai | TDP-43 is a component of ubiquitinpositive 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], which is the main pathological hallmark of ALS. Indeed, neuronal TDP-43 aggregates are found in approximatively 97% of the cases (E.M. [START_REF] Scotter | TDP-43 Proteinopathy and ALS: Insights into Disease Mechanisms and Therapeutic Targets[END_REF], with these ubiquinated inclusion bodies representing the most prevalent marker of the disease. However, TDP-43 involvement on the pathophysiology is not restricted to the aggregation of the misfolded proteins. Indeed, severe dysregulations in the RNA metabolism have been associated with the development of ALS. This includes: RNA mis-localization, altered splicing, and defect in transcription [START_REF] Reber | Minor intron splicing is regulated by FUS and affected by ALS-associated FUS mutants[END_REF][START_REF] Lagier-Tourenne | Divergent roles of ALS-linked proteins FUS/TLS and TDP-43 intersect in processing long pre-mRNAs[END_REF][START_REF] Arnold | ALS-linked TDP-43 mutations produce aberrant RNA splicing and adult-onset motor neuron disease without aggregation or loss of nuclear TDP-43[END_REF][START_REF] Ratti | Physiological functions and pathobiology of TDP-43 and FUS/TLS proteins[END_REF]. As TDP-43 is involved in the regulation of RNA processing and gene transcription (T.J. [START_REF] Cohen | TDP-43 functions and pathogenic mechanisms implicated in TDP-43 proteinopathies[END_REF], this suggests a two-step pathology induced by both the loss of function of TDP-43 in the nucleus and a toxic gain function in the cytoplasm through their aggregation. Moreover, the aggregated form of TDP43 is characterized by an abnormal phosphorylation and it has been proved that the TDP-43 phosphorylated form disseminates in a prion-like behaviour along the motor pathways similarly to what observed in SOD1 misfolded proteins (H. [START_REF] Braak | Amyotrophic lateral sclerosis-a model of corticofugal axonal spread[END_REF][START_REF] Hock | Prion-like propagation as a pathogenic principle in frontotemporal dementia[END_REF].

d. FUS

FUS was first associated with ALS in 2009 (T.J. [START_REF] Kwiatkowski | Mutations in the FUS/TLS gene on chromosome 16 cause familial amyotrophic lateral sclerosis[END_REF]C. Vance et al., 2009). FUS mutations are the fourth most common known genomic alterations associated with ALS in European genetic background population with around 2.8% of the ALS patients being concerned (and the third in the Asian population: 6.4% of the cases) [START_REF] Mathis | Genetics of amyotrophic lateral sclerosis: A review[END_REF]. FUS, the encoded protein, is a nuclear RNA-binding protein involved in the RNA biogenesis and function [START_REF] Dini Modigliani | An ALSassociated mutation in the FUS 3'-UTR disrupts a microRNA-FUS regulatory circuitry[END_REF], a transcription factor (L. [START_REF] Yang | Self-assembled FUS binds active chromatin and regulates gene transcription[END_REF], and is involved in the response to DNA damage (W-Y. [START_REF] Wang | Interaction of FUS and HDAC1 regulates DNA damage response and repair in neurons[END_REF]. Most of the FUS ALS-associated mutations are located in its nuclear localization sequence, therefore inducing its cytoplasmic mislocalization and aggregation (J. [START_REF] Gal | Nuclear localization sequence of FUS and induction of stress granules by ALS mutants[END_REF]. Once more, the FUS-induced pathology could be both induced by the loss of function of the encoded protein and by a toxic gain of function through its aggregation. Similarly, to SOD1 and TDP-43

proteins, there's evidence of a prion-like behaviour spreading of misfolded FUS along the motor tracts (J. [START_REF] Shorter | Liquidizing FUS via prion?like domain phosphorylation[END_REF][START_REF] Silverman | CNS-derived extracellular vesicles from superoxide dismutase 1 (SOD1)G93A ALS mice originate from astrocytes and neurons and carry misfolded SOD1[END_REF][START_REF] Polymenidou | The seeds of neurodegeneration: prion-like spreading in ALS[END_REF].

The identification of the genetic causes of this pathology has constituted the foundation of the majority of the current ALS research as it allowed the generation of a variety of cellular and animal models.

C. Symptoms and diagnosis:

1. Motor symptoms ALS is characterized by the degeneration of both the upper and the lower MN. At the clinical scale, this is translated by a progressive muscular weakness accompanied by cramps, fasciculations and muscle stiffness. Every voluntary muscle appears to be affected with the exception of the ocular and the sphincter muscles (M. [START_REF] Ahmadi | Human extraocular muscles in ALS[END_REF][START_REF] Carvalho | Involvement of the external anal sphincter in amyotrophic lateral sclerosis[END_REF]. However, the clinical features of ALS are rather heterogenous and differ from one patient to another.

These differences have led to the creation of two subclasses: spinal onset in upper and lower limbs observed in roughly two-thirds of the patients, and on the other hand, bulbar onset observed in patients presenting first dysarthria or dysphagia sometimes accompanied with dysphonia, or reduced mouth closure or chewing problems (P. [START_REF] Masrori | Amyotrophic lateral sclerosis: a clinical review[END_REF].

Extra-motor symptoms:

In addition to muscular symptoms, up to 50% of the patients can present extra-motor cognitive and behavioural impairments. Fronto-temporal dementia (FTD) designed a group of neurodegenerative diseases characterized by the progressive loss of neurons in the frontal and temporal lobes, inducing a change in the behaviour, a decrease in the language proficiency and the executive function (J. [START_REF] Bang | Frontotemporal dementia[END_REF]. FTD is considered as the third cause of dementia worldwide (J.J. [START_REF] Young | Frontotemporal dementia: latest evidence and clinical implications[END_REF][START_REF] Bang | Frontotemporal dementia[END_REF]. In the last two decades, more and more patients that were primary diagnosed with ALS have been exhibiting FTD signs suggesting clinical overlap between these two disorders. It is now considered that 13% of the ALS patients will develop a form of FTD in the course of the disease (G.M. [START_REF] Ringholz | Prevalence and patterns of cognitive impairment in sporadic ALS[END_REF][START_REF] Phukan | The syndrome of cognitive impairment in amyotrophic lateral sclerosis: a population-based study[END_REF]. Furthermore, both of these pathologies are characterized by the presence of cellular inclusions in neurons prior to their death, with TDP-43 positive inclusions, a pathological hallmark for an important percentage of FTD cases (I.R.A Mackenzie and R. Rademakers, 2010; S-C. [START_REF] Ling | Converging mechanisms in ALS and FTD: Disrupted RNA and protein homeostasis[END_REF]. The development of genomic screening further strengthened the link between these two diseases. Indeed, several genes associated with ALS, such as FUS, TARDBP, C9orf72, or TBK1 (R. Ferrari et al., 2013;[START_REF] Borroni | Mutation within TARDBP leads to frontotemporal dementia without motor neuron disease[END_REF][START_REF] Sha | Frontotemporal dementia due to C9ORF72 mutations: clinical and imaging features[END_REF]I. Gijselinck et al., 2015) were found mutated in FTD patients. However, it is unclear why certain genes seem to be more associated with FTD and others with ALS. For instance, TBK1 mutations were shown to be associated with approximately 0.4-4% of the total ALS cases whereas they are much more frequent in patients presenting an ALS-FTD comorbidity (I. [START_REF] Lattante | Sqstm1 knock-down causes a locomotor phenotype ameliorated by rapamycin in a zebrafish model of ALS/FTLD[END_REF]. Nowadays, due to their genetic and pathological overlap, these two diseases are considered to represent the two ends of a clinical, neuropathological and genetic single spectrum disorder, often termed ALS-FTD (F.B. [START_REF] Gao | Dysregulated molecular pathways in amyotrophic lateral sclerosis-frontotemporal dementia spectrum disorder[END_REF]S-C. Ling et al., 2013;[START_REF] De Silva | Motor function and behaviour across the ALS-FTD spectrum[END_REF].

Diagnosis

Due to the heterogeneity of symptoms, identifying ALS can be challenging and misdiagnosis often occurs. Because of the urgent need of a precise diagnosis criteria, the World Federation of Neurology Subcommittee on Motor Neuron Disease gathered in 1990 in El Escorial, Spain, to design and write a list of universal criteria that would help the diagnosis of the disease: The El Escorial criteria (B.R. [START_REF] Brooks | El Escorial World Federation of Neurology criteria for the diagnosis of amyotrophic lateral sclerosis. Subcommittee on Motor Neuron Diseases/Amyotrophic Lateral Sclerosis of the World Federation of Neurology Research Group on Neuromuscular Diseases and the El Escorial "Clinical limits of amyotrophic lateral sclerosis" workshop contributors[END_REF]. These criteria are based on a clinical, electrophysiological and neuropathologic surveillance to assess the evidence of lower or upper MN degeneration and the monitoring of the course of the disease with the spreading of neurodegeneration to other areas of the central nervous system (CNS).

Together with these diagnostic inclusion criteria, exclusion criteria were also designed to distinguish the pathology from others.

As the El Escorial criteria based on the identification of neurodegeneration, when a patient is diagnosed with ALS, the disease course is already well advanced (B.R. [START_REF] Brooks | El Escorial World Federation of Neurology criteria for the diagnosis of amyotrophic lateral sclerosis. Subcommittee on Motor Neuron Diseases/Amyotrophic Lateral Sclerosis of the World Federation of Neurology Research Group on Neuromuscular Diseases and the El Escorial "Clinical limits of amyotrophic lateral sclerosis" workshop contributors[END_REF].

Therefore, a change of paradigm and a revised set El Escorial criteria in the close future is conceivable. With the great advances in the technology of metabolites measurement and the identification of dysregulated biomarkers, diagnosis could occur a lot earlier. The most promising biomarker is the presence of neurofilaments (NF) (both light and heavy chains), quantifying their levels in the cerebro-spinal liquid (CSF) or in the blood could help diagnosed patients at earlier stages of the disease (Z. [START_REF] Xu | Neurofilaments as Biomarkers for Amyotrophic Lateral Sclerosis: A Systematic Review and Meta-Analysis[END_REF]. In addition to help diagnosis, NF have also been reported to reflect accurately the disease progression (Y-N. [START_REF] Zhou | Role of Blood Neurofilaments in the Prognosis of Amyotrophic Lateral Sclerosis: A Meta-Analysis[END_REF], and are now used in clinical trials to investigate the impact of a treatment on the course of the pathology (T. [START_REF] Miller | Trial of Antisense Oligonucleotide Tofersen for SOD1 ALS[END_REF].

Amongst these biomarkers under investigation we can also cite, the elevated level in glucose originated from a hypermetabolism associated with the disease (T.W. [START_REF] Tefera | CNS glucose metabolism in Amyotrophic Lateral Sclerosis: a therapeutic target?[END_REF] in the brain, spinal cord, muscles or in circulating fluids. Unfortunately, the quest for metabolites that are prevalent in ALS patients has not been entirely successful with no real specific metabolic signature for this disease as of yet (see Part III -metabolites dysregulation in ALS).

D. Treatment:

Despite approximately 200 clinical trials, only a few compounds have been identified to have a positive impact on ALS patients.

Riluzole:

Riluzole is a glutamate inhibitor that has shown modest but beneficial effect in the late stages of disease, prolonging the survival by 2-3 months (J. [START_REF] Wokke | Riluzole[END_REF][START_REF] Fang | Stage at which riluzole treatment prolongs survival in patients with amyotrophic lateral sclerosis: a retrospective analysis of data from a dose-ranging study[END_REF] U. [START_REF] Saitoh | Riluzole for the treatment of amyotrophic lateral sclerosis[END_REF]. Glutamate excitotoxicity has been shown to play an important role in ALS pathogenicity (E. [START_REF] Foran | Glutamate transporters and the excitotoxic path to motor neuron degeneration in amyotrophic lateral sclerosis[END_REF]. Indeed, patients exhibit higher level of glutamate in the CNS (O. [START_REF] Spreux-Varoquaux | Glutamate levels in cerebrospinal fluid in amyotrophic lateral sclerosis: a reappraisal using a new HPLC method with coulometric detection in a large cohort of patients[END_REF]. Glutamate is used in the brain as a neurotransmitter, transmitting excitatory signals between neurons, however, it is known that an increased firing rate is detrimental for neurons (Y. [START_REF] Zhou | Glutamate as a neurotransmitter in the healthy brain[END_REF]. It is still unclear how riluzole blocks the glutamatergic neurotransmission, and three hypotheses have been proposed: the blockade of the glutamate release in the synaptic compartment, the inactivation of the glutamate sodium channels of the receiving neuron, or the interference with the cellular response to glutamate signal. Despite its modest impact on the disease, riluzole have remained the only approved drug in the treatment of ALS for almost 30 years after its approval by the United-states Food and Drug Administration (FDA) in 1995.

Edaravone

Evidence of high oxidative stress has been put in evidence in ALS patients (T. [START_REF] Cunha-Oliveira | Oxidative Stress in Amyotrophic Lateral Sclerosis: Pathophysiology and Opportunities for Pharmacological Intervention[END_REF] and is suspected to play an important role in the degeneration of motor neurons. Therefore, it was hypothesized that reducing the level of ROS could show beneficial impact on patient health and, to this extend, several antioxidant compounds have been used. Edaravone is a molecule that has been shown to protect nerve cells in the brain by reducing the level of ROS and inhibiting the oxidative stress induced apoptosis (M-S. [START_REF] Jami | Edaravone leads to proteome changes indicative of neuronal cell protection in response to oxidative stress[END_REF]. It is the sole tested antioxidant molecule that has been shown to have a beneficial effect on the course of the disease (H. Cho and S. Shukla, 2021). However, similarly to riluzole, it only has a modest impact on the course of the disease, prolonging the survival by a few months. Edaravone was approved by the FDA in 2017 following a clinical trial on Japanese patients (K. [START_REF] Abe | Safety and efficacy of edaravone in well defined patients with amyotrophic lateral sclerosis: a randomised, double-blind, placebo-controlled trial[END_REF]. However, the pool of patients selected for this trial was criticized for not being representative of the ALS population (O. Hardiman and L.H. van den Berg, 2017). Based on these doubts, the European Medicines Agency (EMA) has not approved yet the usage of this drug on European territory and its availability in France is segregated to a temporary authorisation of usage created by the Agence National de Sécurité du Médicament.

Relyvrio

Relvyrio (formerly known as AMX0035) is a treatment based on two compounds: taurursodiol and phenylbutyrate. Both of these molecules act respectively on two cellular components and mechanisms altered in the context of ALS: mitochondria and elevated oxidative stress (C.M.P. [START_REF] Rodrigues | Tauroursodeoxycholic acid prevents Bax-induced membrane perturbation and cytochrome C release in isolated mitochondria[END_REF] and unfolded protein aggregates (C.T. [START_REF] Mai | 4-Phenylbutyrate suppresses the unfolded protein response without restoring protein folding in Saccharomyces cerevisiae[END_REF]. A recent clinical trial using the combination of these two molecules has shown some beneficial effect, prolonging survival and reducing hospitalisation risk of ALS patients [START_REF] Paganoni | Trial of Sodium Phenylbutyrate-Taurursodiol for Amyotrophic Lateral Sclerosis[END_REF][START_REF] Paganoni | Long-term survival of participants in the CENTAUR trial of sodium phenylbutyrate-taurursodiol in amyotrophic lateral sclerosis[END_REF]. Based on this trial, the FDA has approved the usage of relvyrio this year (2022), however, the EMA has still not approved its utilization on European territory.

Non-pharmacological treatments:

Besides these disease-modifying pharmacological treatments, the patients can beneficiate of symptomatic treatments, which do not impact the course of the disease but are based on the improvement of the quality of life, including tracheostomy. Tracheostomy consist of the incision of the trachea to open a direct airway for long-term mechanical ventilation in a context of respiratory failure. However, this procedure is rarely used due to the heavy consequences on the patient's life as it provokes a total loss of oral communication.

Overall the treatments are limited to symptomatic management and palliative care by the lack of early diagnosis of the disease. Indeed, the diagnosis based on El Escorial criteria is based on the identification of neurodegeneration, therefore, when a patient is diagnosed with ALS, the course of the disease is already irreversible. The solution to switch from a palliative to a curative treatment would be to identify early onset biomarkers prior to neurodegeneration in order to update and revise the actual criteria paradigm and allow early diagnosis where the disease course could still be reversed.

As a future perspective, we can mention increased importance of genetic therapies illustrated by a major clinical trial ongoing that is testing whether intrathecal injections directly in the cerebrospinal fluid of antisense oligonucleotide targeting the expression of SOD1 could prolong ALS disease duration (T. [START_REF] Miller | Trial of Antisense Oligonucleotide Tofersen for SOD1 ALS[END_REF].

III. Physiopathology of ALS-FTD -cellular dysregulations:

ALS pathophysiology complexity is proportional to the number of genes associated with its development. Many alterations of cellular processes are involved and the identification of the primary dysregulations from their consequences can be challenging. In fact, each of the pathways identified as impacted are considered in a hypothesis as the putative cause of the disease. In the following section we will review the cellular pathways identified in the development of the pathology.

A. Impaired protein homeostasis:

One universal hallmark of the ALS pathology is the presence of cellular inclusions in the cytoplasm of neurons and skeletal muscles (A.M. [START_REF] Blokhuis | Protein aggregation in amyotrophic lateral sclerosis[END_REF][START_REF] Cykowski | Dipeptide repeat (DPR) pathology in the skeletal muscle of ALS patients with C9ORF72 repeat expansion[END_REF]. However, how these inclusions are formed remains obscure and the mechanisms responsible for their apparition are still unknown. From their presence we can suspect an imbalance in the protein's synthesis and their degradation. (derived from M.C. [START_REF] Kiernan | Amyotrophic lateral sclerosis[END_REF] The majority of the aggregates in both sporadic and familial forms are found to be positive for TDP-43 (M. [START_REF] Neumann | Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis[END_REF]. However, the presence of aggregates in ALS patients of other pathological markers has been observed, including FUS (C. [START_REF] Vance | Mutations in FUS, an RNA processing protein, cause familial amyotrophic lateral sclerosis type 6[END_REF], SOD1 (Y. [START_REF] Hayashi | SOD1 in neurotoxicity and its controversial roles in SOD1 mutation-negative ALS[END_REF], DPRs (K. [START_REF] Mori | The C9orf72 GGGGCC repeat is translated into aggregating dipeptide-repeat proteins in FTLD/ALS[END_REF] or NF (A. [START_REF] Hirano | Fine structural observations of neurofilamentous changes in amyotrophic lateral sclerosis[END_REF] according to the form of ALS.

Autophagic dysregulation:

Autophagy is a highly conserved process that is involved in protein homeostasis maintenance through their degradation. Autophagy is a term englobing 3 distinct mechanisms: macroautophagy, microautophagy and chaperone mediate autophagy (reviewed in the following section).

Macroautophagy is the major autophagic process, it relies on the targeting and engulfment of cellular components into a double membrane vacuole called autophagosome which later fused with the high acidic vacuoles lysosomes to induce the degradation and the reuse of the target molecules. Microphagy, as opposed to macroautophagy, is not selective, it only relies on the digestion of small volumes of cytosolic substrates by the lysosomes.

Among the 30 genes associated with the development of ALS, 6 are directly involved in the autophagic processes: C9ORF72, TBK1 and the less ALS represented VCP, OPTN, SQSTM1 and UBQLN2 [START_REF] Mathis | Genetics of amyotrophic lateral sclerosis: A review[END_REF]. At the clinical level, the most obvious sign of the autophagic involvement in the development of the ALS disease is the accumulation of autophagosomes in the cytoplasm of patients' MN [START_REF] Sasaki | Autophagy in spinal cord motor neurons in sporadic amyotrophic lateral sclerosis[END_REF]. This would suggest an increased autophagic activity in a physiological context. However, this activity is not reflected in the degradation of the typical aggregates during the course of the disease questioning about the efficiency of this system. Moreover, autophagy cargo proteins p62 are found to colocalize with the cytoplasmic inclusions of ALS patients suggesting proteostasis defects (J. [START_REF] Gal | p62 accumulates and enhances aggregate formation in model systems of familial amyotrophic lateral sclerosis[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]. Stimulation of the autophagy system has beneficial effect in ALS in vivo and in vitro models (L. [START_REF] Marrone | FUS pathology in ALS is linked to alterations in multiple ALS-associated proteins and rescued by drugs stimulating autophagy[END_REF][START_REF] Lattante | Sqstm1 knock-down causes a locomotor phenotype ameliorated by rapamycin in a zebrafish model of ALS/FTLD[END_REF]. Moreover, macroautophagy efficiency have been shown to be inversely proportional to the age, and a decreased in its activity is observed in ageing models (C. [START_REF] Ott | Macroautophagy is impaired in old murine brain tissue as well as in senescent human fibroblasts[END_REF][START_REF] Aman | Autophagy in healthy aging and disease[END_REF]. These observations link the reduced autophagic activity with the late onset development of the disease.

TBK1, the gene studied in the present study has been extensively implicated in autophagic process. Therefore, we will review more in depth its function in the context of autophagy in the following section (Part II -II.B. TBK1 as a regulator of the autophagic process).

Proteasomal impairment:

In response to misfolded proteins as observed in ALS, the cell can initiate a process called the unfolded-protein response (UPR) (R. [START_REF] Bravo | Endoplasmic reticulum and the unfolded protein response: dynamics and metabolic integration[END_REF]. It results in the transport of the defective proteins toward the endoplasmic reticulum (ER) where chaperones correct the folding of the proteins. However, when the level of misfolded proteins exceeds the UPR capacity or when the chaperones are unable to restore the native conformation, a new emergency mechanism activates: the endoplasmic reticulum-associated protein degradation (ERAD) (A. [START_REF] Stolz | Endoplasmic reticulum associated protein degradation: a chaperone assisted journey to hell[END_REF]. The misfolded proteins are then released in the cytosol where they undergo ubiquitination and proteasomal degradation. Mutated SOD1

proteins have been shown to interfere with ERAD proteins, preventing the correct ER stress response pathway (H. [START_REF] Nishitoh | ALS-linked mutant SOD1 induces ER stress-and ASK1-dependent motor neuron death by targeting Derlin-1[END_REF]. Moreover, DPR derived from C9ORF72 G4C2 repeats can reduce proteasomal activity resulting in increased ER stress and accumulation of misfolded proteins (Y-J. [START_REF] Zhang | Aggregation-prone c9FTD/ALS poly(GA) RAN-translated proteins cause neurotoxicity by inducing ER stress[END_REF]. Importantly, dysregulation of the proteasomal degradation of misfolded proteins induced the formation of aggregates, which in turn activate a cascade of events that ultimately results in cell death (R. [START_REF] Sano | ER stress-induced cell death mechanisms[END_REF]. Finally, the hypothesis of an altered proteasomal pathway is underlined by the presence of ER stress marker in the motor neurons of ALS patients suggesting that this dysregulation could be responsible for their death in patients (K. [START_REF] Kanekura | ER stress and unfolded protein response in amyotrophic lateral sclerosis[END_REF][START_REF] Sasaki | Endoplasmic reticulum stress in motor neurons of the spinal cord in sporadic amyotrophic lateral sclerosis[END_REF]. In addition, there are evidences of an age-dependent reduction in the level of the ER stress chaperones (A. [START_REF] Apostolopoulos | Nuclear Accumulation of HSP70 in Mouse Skeletal Muscles in Response to Heat Stress, Aging, and Unloading With or Without Reloading[END_REF][START_REF] Hall | Aging reduces adaptive capacity and stress protein expression in the liver after heat stress[END_REF], suggesting a reduced UPR and connecting the proteasomal impairment theory to the late-onset of the disease. Studies assessed the impact of chaperones supplementation on ALS SOD1 mutant murine model with limited success, suggesting that the sole reduction in UPR is not responsible for all the subsequent neuronal cell death (Y.J. K Patel et al., 2005;[START_REF] Krishnan | Over-expression of Hsp27 does not influence disease in the mutant SOD1G93A mouse model of amyotrophic lateral sclerosis[END_REF][START_REF] Sharp | Protective effects of heat shock protein 27 in a model of ALS occur in the early stages of disease progression[END_REF].

B. Extracellular trafficking:

Physiologically, exosomes are key players of the cell-to-cell communication and rapid interaction between the different cell types of the central nervous system is crucial for a correct activity (L.R Lizarraga-Valderramaet and G.K. Sheridan 2021). In the ALS context, it is suggested that exosomes carry neurotoxic elements such as aggregates or proteins with a prion-like behaviour (such as described in the previous section) subsequently inducing motor neuron death (J. [START_REF] Silverman | CNS-derived extracellular vesicles from superoxide dismutase 1 (SOD1)G93A ALS mice originate from astrocytes and neurons and carry misfolded SOD1[END_REF][START_REF] Westergard | Cell-to-Cell Transmission of Dipeptide Repeat Proteins Linked to C9orf72-ALS/FTD[END_REF].

C. Intracellular trafficking

MNs are highly specialized neurons, bearing very long axonal processes that allow muscle innervation. They are the longest cells of the body, their axons reaching up to 1 meter long.

Due to their unique anatomical characteristic, motor neurons highly depend on intracellular trafficking to convey organelles or vesicles (containing neuronal receptors or RNA), to their synapses compartment. Axonal transport dysregulation is one of the earliest events in the development of ALS (T.L. [START_REF] Williamson | Slowing of axonal transport is a very early event in the toxicity of ALS-linked SOD1 mutants to motor neurons[END_REF][START_REF] Bilsland | Deficits in axonal transport precede ALS symptoms in vivo[END_REF].

For this reason, it has been considered by a part of the scientific community as causative for the development of the disease. Indeed, several mutations associated with the pathology, impacting genes not necessarily involved in the axonal transport, have been shown to impair transport dynamics early in the development of the pathology. Amongst these genes we can cite the well-studied C9orf72, FUS, SOD1, TARDBP but also genes that are more rarely mutated in the context of ALS such as DCTN-1, KIF5A, ALS2, TUBA4A, SPG11, CHMP2B or VAPB (D.M. Baron et al., 2020). The most obvious consequence of these alterations is a defective transport of mitochondria. Indeed, lower mitochondrial axonal concentration was reported in several in vitro and in vivo experimental models (C. Vande [START_REF] Velde | Misfolded SOD1 Associated with Motor Neuron Mitochondria Alters Mitochondrial Shape and Distribution Prior to Clinical Onset[END_REF] G.M. [START_REF] Mórotz | Amyotrophic lateral sclerosis-associated mutant VAPBP56S perturbs calcium homeostasis to 167 disrupt axonal transport of mitochondria[END_REF][START_REF] Magrané | Abnormal mitochondrial transport and morphology are common pathological denominators in SOD1 and TDP43 ALS mouse models[END_REF][START_REF] De Vos | Neurobiology of axonal transport defects in motor neuron diseases: Opportunities for translational research?[END_REF]. These defects are thought to be mainly due to weakening of the cytoskeleton structures or impaired vesicles formation. Alterations of the neurofilament, actin or microtubules integrity have been

shown to be associated with some form of ALS (D. [START_REF] Fil | Mutant Profilin1 transgenic mice recapitulate cardinal features of motor neuron disease[END_REF][START_REF] Figlewicz | Variants of the heavy neurofilament subunit are associated with the 142 development of amyotrophic lateral sclerosis[END_REF], M.H. LaMonte et al., 2002). As mentioned before, intracellular vesicle formation is altered in the context of ALS with traffic between the ER and the Golgi was reported to be inhibited (J. [START_REF] Atkin | Mutant SOD1 inhibits ER-Golgi transport in amyotrophic lateral sclerosis[END_REF]K. Soo et al., 2015). Altogether, these results suggest that alteration of the intracellular trafficking, which is a central pathway in the neuronal physiology, could be an early event of the disease leading to the dysregulation of other cellular pathways.

Moreover, it seems that altered vesicles production is not restricted to the nervous system, as muscles cells of ALS patients present an accumulation of cytoplasmic vacuoles (A. [START_REF] Scaramozza | Skeletal muscle satellite cells in amyotrophic lateral sclerosis[END_REF].

D. Mitochondrial dysfunction and oxidative stress:

Mitochondria are commonly described as the engine of the cell due to their involvement in adenosine triphosphate (ATP) generation, but their function is much more diverse, for instance they are major regulator of cell apoptosis (S.W.G. Tait and D.R. Green, 2013) or of calcium homeostasis and therefore involved in many neuronal functions (C. [START_REF] Giorgi | The machineries, regulation and cellular functions of mitochondrial calcium[END_REF].

Mitochondrial damaged was one of the first cellular hallmarks of ALS reported (T. Astumi, 1981). Since then, many studied have confirmed these observations and evidences of mitochondrial damages during the course of the disease accumulated in patients (K.

Otamoto [START_REF] Okamoto | Axonal swellings in the corticospinal tracts in amyotrophic lateral sclerosis[END_REF][START_REF] Sasaki | Mitochondrial alterations in the spinal cord of patients with sporadic amyotrophic lateral sclerosis[END_REF] and in in vivo models (C.M.J. [START_REF] Higgins | ALS-associated mutant SOD1G93A causes mitochondrial vacuolation by expansion of the intermembrane space and by involvement of SOD1 aggregation and peroxisomes[END_REF][START_REF] Mattiazzi | Mutated human SOD1 causes dysfunction of oxidative phosphorylation in mitochondria of transgenic mice[END_REF][START_REF] Magrané | Abnormal mitochondrial transport and morphology are common pathological denominators in SOD1 and TDP43 ALS mouse models[END_REF].

Interestingly, these mitochondrial alterations have been reported to occur early in the development of the disease (J. [START_REF] Magrané | Abnormal mitochondrial transport and morphology are common pathological denominators in SOD1 and TDP43 ALS mouse models[END_REF]C. Vande Velde et al., 2011, N. Damiano et al., 2006), suggesting that mitochondria loss of integrity could be a preventive therapeutic target.

It is suspected that ALS related mitochondrial damages is due to structural alteration through either formation of vacuoles within both the inner-and the outer-mitochondrial membranes (P. [START_REF] Pasinelli | Amyotrophic lateral sclerosis-associated SOD1 mutant proteins bind and aggregate with Bcl-2 in spinal cord mitochondria[END_REF]; C.M.J. [START_REF] Higgins | ALS-associated mutant SOD1G93A causes mitochondrial vacuolation by expansion of the intermembrane space and by involvement of SOD1 aggregation and peroxisomes[END_REF] or sequestering of mitochondrial protein in cytoplasmic aggregates (J. [START_REF] Deng | FUS Interacts with HSP60 to Promote Mitochondrial Damage[END_REF][START_REF] Deng | FUS interacts with ATP synthase beta subunit and induces mitochondrial unfolded protein response in cellular and animal models[END_REF][START_REF] Pasinelli | Amyotrophic lateral sclerosis-associated SOD1 mutant proteins bind and aggregate with Bcl-2 in spinal cord mitochondria[END_REF][START_REF] Choi | C9ORF72-ALS/FTD-associated poly(GR) binds Atp5a1 and compromises mitochondrial function in vivo[END_REF]. The association between ALS and mutation in CHCHD10 [START_REF] Bannwarth | A mitochondrial origin for frontotemporal dementia and amyotrophic lateral sclerosis through CHCHD10 involvement[END_REF], a gene coding for a protein located between the innerand outer-mitochondrial membrane, is a further evidence of the link between structural alterations of the mitochondria and the development of ALS. The most obvious consequences of these mitochondrial damages in the context of ALS is the increased level of toxic cytoplasmic ROS which can lead to neurodegeneration (J. [START_REF] Deng | FUS Interacts with HSP60 to Promote Mitochondrial Damage[END_REF][START_REF] Cunha-Oliveira | Oxidative Stress in Amyotrophic Lateral Sclerosis: Pathophysiology and Opportunities for Pharmacological Intervention[END_REF]. However, mitochondria could also lead to neuronal death through proapoptotic signalling (S.W.G. Tait and D.R. Green, 2013). Finally, a disturbed mitochondrial activity is suspected to weaken the cells through an impaired calcium homeostasis subsequently increasing the sensitivity to glutamate toxicity (N. [START_REF] Damiano | Neural mitochondrial Ca2+ capacity impairment precedes the onset of motor symptoms in G93A Cu/Zn-superoxide dismutase mutant mice[END_REF],

another ALS features that we'll discuss in the next section.

E. Glutamate excitotoxicity:

Glutamate is the most abundant excitatory neurotransmitter in the human CNS. It is released in the synaptic compartment by the pre-synaptic neuron, when in the synapse, glutamate binds to NMDA and AMDA glutamate receptor of the post-synaptic cell in order to induce a Ca 2+ and Na + influx and the subsequent membrane depolarization. An excessive level of glutamate in the extracellular medium is deleterious for the neurons, leading to extreme firing rate. Evidences of dysregulated glutamate excitatory response have been reported on two levels in the context of ALS.

First, it is suspected that an increased permeability of the glutamate receptors induces an excessive Ca 2+ influx leading to mitochondrial dysfunction and subsequent ROS production.

Indeed, ADAR2, a protein regulating the permeability of AMDA have been reported to be dysregulated in the context of ALS [START_REF] Moore | ADAR2 mislocalization and widespread RNA editing aberrations in C9orf72-mediated ALS/FTD[END_REF]. Moreover, evidence of a physiological higher sensibility of motor neurons would link the selective loss of motor neurons in the context of ALS to the dysregulated glutamate excitability. Indeed, specific overactivation of AMDA in a murine model lead to motor neuron degeneration and the subsequent paralysis similarly to what was observed in ALS patients (J.C. [START_REF] Corona | AMPA receptor activation, but not the accumulation of endogenous extracellular glutamate, induces paralysis and motor neuron death in rat spinal cord in vivo[END_REF]. The hypothesis of a higher calcium sensitivity of the motor neurons is supported by evidence of physiologically lower expression of the GRIA2 gene coding for the AMPA subunit responsible of the permeability compared to their neighbouring neuronal cells (P.R. [START_REF] Heath | Quantitative assessment of AMPA receptor mRNA in human spinal motor neurons isolated by laser capture microdissection[END_REF].

Secondly, it is suggested that glutamate accumulates in the synapse compartment because of an impaired astrocytes clearance leading to continuous excitatory signal. Indeed, astrocytes express on their synaptic process, GLT1, which maintains a low extracellular glutamate concentration and therefore prevents an excessive neuronal intake of positive ions (K. [START_REF] Kim | Role of Excitatory Amino Acid Transporter-2 (EAAT2) and glutamate in neurodegeneration: Opportunities for developing novel therapeutics[END_REF]. Supporting this hypothesis, GLT1 levels have been reported to be severely depleted, leading to a decreased activity and extracellular glutamate accumulation (L.A. [START_REF] Bristol | Glutamate transporter gene expression in amyotrophic lateral sclerosis motor cortex[END_REF][START_REF] Sasaki | Excitatory amino acid transporter 1 and 2 immunoreactivity in the spinal cord in amyotrophic lateral sclerosis[END_REF][START_REF] Vanoni | Increased internalisation and degradation of GLT-1 glial glutamate transporter in a cell model for familial amyotrophic lateral sclerosis (ALS)[END_REF][START_REF] Peterson | Post-translational Regulation of GLT-1 in Neurological Diseases and Its Potential as an Effective Therapeutic Target[END_REF]. This hypothesis is supported by two other studies respectively inhibiting and boosting the expression and the activity of GLT1. Inhibiting the expression of GLT1 has been shown to increase glutamate levels extracellularly and causing neuron degeneration and a progressive motor deficit (J.D. [START_REF] Rothstein | Beta-lactam antibiotics offer neuroprotection by increasing glutamate transporter expression[END_REF] whereas enhancing the activity of GLT1 was reported to be beneficial in the SOD1 G93A murine model (J.D. [START_REF] Rothstein | Beta-lactam antibiotics offer neuroprotection by increasing glutamate transporter expression[END_REF].

F. Inflammation:

Upon tissue lesion or pathogen invasion, damaged cells components or motifs shared by pathogens are recognized by surrounding cells. The response to these signals, resulting in tissue repairing is called the inflammation. Once these signals are integrated, a cascade of events lead to the activation of two main pathways: the nuclear factor-kappa B (NFκB) complex and the interferon regulatory factors (IRFs) responsible for the expression of proinflammatory genes and the respective synthesis of cytokines/chemokines and interferons.

The release of these endogenous molecules alerts the host of a danger and allow recruitment of cells of the immune system. As this process is so important, the synthesis and response to inflammatory signals is precisely tuned. When a dysregulation occurs along the synthesis of these endogenous molecules, it leads to hypo-or hyper-stimulation of the inflammatory system and provokes different types of pathologies. In ALS patients and animal model, inflammation was found to be up-regulated (K.A. [START_REF] Staats | Blood-based biomarkers of inflammation in amyotrophic lateral sclerosis[END_REF][START_REF] Tortelli | Plasma Inflammatory Cytokines Are Elevated in ALS[END_REF][START_REF] Mccombe | The Role of immune and inflammatory mechanisms in ALS[END_REF]. However, it is still obscure whether it is a consequence of the death of MN or if the hyperstimulation of the inflammatory response leads to MN loss.

In the CNS, inflammation response is triggered by the glial cells (the myeloid precursor derived cells in the brain) composed of microglia and the astrocytes. These cells remain in a silent state physiologically but activates when exposed to immune challenge or injury.

Evidence of activated myeloid cells have been reported in patients (M.R. [START_REF] Turner | Evidence of widespread cerebral microglial activation in amyotrophic lateral sclerosis: an [11C](R)-PK11195 positron emission tomography study[END_REF] B.E. Clarke and R. Patani, 2020) and animal models (E.D. [START_REF] Hall | Relationship of microglial and astrocytic activation to disease onset and progression in a transgenic model of familial ALS[END_REF][START_REF] Clarke | The microglial component of amyotrophic lateral sclerosis[END_REF]. The microglia population can be divided into two subtypes: the M1 subgroup is activated by pro-inflammatory signals and is responsible for the increased inflammation whereas the M2 subgroup is stimulated by anti-inflammatory signals and suppresses the inflammation and therefore neuron survival (Y. [START_REF] Tang | Differential Roles of M1 and M2 Microglia in Neurodegenerative Diseases[END_REF]. It has been reported

in the SOD1 G93A murine model that the ratio M1/M2 is proportional to the age (J.M. [START_REF] Crain | Microglia express distinct M1 and M2 phenotypic markers in the postnatal and adult central nervous system in male and female mice[END_REF][START_REF] Liao | Transformation from a neuroprotective to a neurotoxic microglial phenotype in a mouse model of ALS[END_REF], this difference could be linked to the late onset of the disease, an older brain being more sensitive to pro-inflammatory signals.

Astrocytes involvement in the development of the disease is complicated to apprehend. It is suspected that they have a minor impact on the course of the disease and deletion of the NFκB pathway doesn't improve the phenotype in the SOD1 G93A murine model (C. [START_REF] Crosio | Astroglial inhibition of NF-?B does not ameliorate disease onset and progression in a mouse model for amyotrophic lateral sclerosis (ALS)[END_REF] suggesting that they are not involved in the inflammatory state. However, despite the suggested minimal involvement in the inflammatory state of ALS models, astrocytes' impaired clearance of synaptic glutamate molecules have been suggested as an important factor in the ALS pathogenicity (K.A. [START_REF] Staats | Astrocytes in amyotrophic lateral sclerosis: direct effects on motor neuron survival[END_REF][START_REF] Yamanaka | Astrocytes as determinants of disease progression in inherited ALS[END_REF].

G. Aberrant RNA metabolism:

As mentioned previously, the main cellular hallmarks of the disease is the presence of TDP43

and at a lesser extend FUS positive cytoplasmic inclusions (T. [START_REF] Arai | TDP-43 is a component of ubiquitinpositive 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]. These proteins are RNA binding proteins that are involved throughout all the RNA maturation. Therefore, the sequestration of these two proteins in the cytoplasm impact the RNA metabolism transcription defects, alternate splicing changes, miRNA biogenesis, stress granule formation, and RNA nucleocytoplasmic transport (L. Le Gall et al., 2020).

First, the most obvious consequence is the loss of function of these two proteins. It is unclear what are their precise roles as they are interacting with a large number of proteins (A. [START_REF] Ratti | Physiological functions and pathobiology of TDP-43 and FUS/TLS proteins[END_REF][START_REF] Kamelgarn | Proteomic analysis of FUS interacting proteins provides insights into FUS function and its role 154 in ALS[END_REF]. However, when they are inhibited as well as in the context of ALS, there is evidence of several RNA metabolism alterations:

RNA mis-localization, unvalid splicing, defect in transcription [START_REF] Reber | Minor intron splicing is regulated by FUS and affected by ALS-associated FUS mutants[END_REF][START_REF] Lagier-Tourenne | Divergent roles of ALS-linked proteins FUS/TLS and TDP-43 intersect in processing long pre-mRNAs[END_REF][START_REF] Arnold | ALS-linked TDP-43 mutations produce aberrant RNA splicing and adult-onset motor neuron disease without aggregation or loss of nuclear TDP-43[END_REF][START_REF] Ratti | Physiological functions and pathobiology of TDP-43 and FUS/TLS proteins[END_REF].

Besides TDP43 and FUS loss of function's impacts on RNA metabolism, their cytoplasmic aggregation has severe consequences. Indeed, highly dense RNA foci are observed in ALS patients colocalizing with TDP43, which suggests an RNAs mis-localization also being responsible for the observed altered RNA metabolism (O. Aladesuyi [START_REF] Aladesuyi Arogundade | Antisense RNA foci are associated with nucleoli and TDP-43 mislocalization in C9orf72-ALS/FTD: a quantitative study[END_REF]. Moreover, it was reported that when forming aggregates FUS proteins still bind and sequester important actors of RNA splicing in the cytoplasm (Y. Yu et al., 2015;[START_REF] Reber | Minor intron splicing is regulated by FUS and affected by ALS-associated FUS mutants[END_REF][START_REF] Kamelgarn | Proteomic analysis of FUS interacting proteins provides insights into FUS function and its role 154 in ALS[END_REF], further impacting the physiological RNA metabolism.

H. Programmed cell death:

Cell death is part of the normal cellular development. It can occur in a physiologically and genetically controlled manner: we therefore talk of a programmed cell death (PCD), or in an alternative uncontrolled cell death induced by environmental causes, the necrosis. Necrosis is induced by an external injury, for instance hypoxia or inflammation and is usually considered as an accidental death when the cell cannot maintain its homeostasis with its environment. At the cellular level, necrosis results in the swelling and subsequent rupture of the plasma membrane (D. [START_REF] Moujalled | Molecular mechanisms of cell death in neurological diseases[END_REF]. In the context of ALS, the characteristic MN degeneration has been suspected to be induced through a PCD-related mechanism (C. [START_REF] Guégan | Programmed cell death in amyotrophic lateral sclerosis[END_REF]. There are several types of PCD: the well-known apoptosis but also necroptosis, pyroptosis, or ferroptosis (D. [START_REF] Moujalled | Molecular mechanisms of cell death in neurological diseases[END_REF]. Regarding to ALS, two of these pathways have been suggested to be involved in neurodegeneration (J.R. Morris et al., 2017): apoptosis (L.J. [START_REF] Martin | Neuronal death in amyotrophic lateral sclerosis is apoptosis: possible contribution of a programmed cell death mechanism[END_REF]; K.Y. [START_REF] Kim | Downregulated miR-18b-5p triggers apoptosis by inhibition of calcium signaling and neuronal cell differentiation in transgenic SOD1 (G93A) mice and SOD1 (G17S and G86S) ALS patients[END_REF] and necroptosis (D.B. [START_REF] Re | Necroptosis drives motor neuron death in models of both sporadic and familial ALS[END_REF][START_REF] Ito | RIPK1 mediates axonal degeneration by promoting inflammation and necroptosis in ALS[END_REF]. As TBK1 has been reported to be involved in the induction of these two cell processes, we will review their mechanistic insights and the functional role of TBK1 in these cellular pathways later in the introduction (Part II -section II functional roles of TBK1).

IV. Modelling the ALS-FTD -state of the art A. In vitro models

Although they suffer from the lack of tissue connectivity or functional connectivity, cellular models are powerful tools to study the role of genes involved in ALS through the expression of WT and mutant ALS associated proteins or with their respective gene deletion. They also allow genetic and compound screening which are great tools to find drug that could lead to therapeutic avenues.

The first use of unicellular model in the ALS research was the expression of a SOD1 human wild type or mutant plasmid in NSC34 cells (a hybrid mouse cell line produced to mimic motor neurons) (M.R. [START_REF] Cookson | Cu/Zn superoxide dismutase (SOD1) mutations associated with familial amyotrophic lateral sclerosis (ALS) affect cellular free radical release in the presence of oxidative stress[END_REF]. These experiments have reported the production of intracellular ROS upon expression of mutant form of SOD1. However, this mouse cultured cell type has been put aside with the apparition of primary culture of mouse MN in which various factors have been tested to investigate toxicity: for instance, glutamate (P. [START_REF] Van Damme | Astrocytes regulate GluR2 expression in motor neurons and their vulnerability to excitotoxicity[END_REF], astrocytes released factors (M. [START_REF] Nagai | Astrocytes expressing ALS-linked mutated SOD1 release factors selectively toxic to motor neurons[END_REF] or DPR synthesis (X; [START_REF] Wen | Antisense Proline-Arginine RAN Dipeptides Linked to C9ORF72-ALS/FTD Form Toxic Nuclear Aggregates that Initiate In Vitro and In Vivo Neuronal Death[END_REF].

Since the use of NSC34 cells, various types of cell lines have been used, and many ALS genes have been investigated. One model that has been extensively used to perform genetic screen in the context of ALS is the Saccharomyces cerevisiae budding yeast. In the context of TDP-43 overexpression, genetic screening in yeast have allowed the identification of several genes modulating the induced toxicity: ATAXIN2 (ATXN2) (A.C. [START_REF] Elden | Ataxin-2 intermediate-length polyglutamine expansions are associated with increased risk for ALS[END_REF], CNC1, DNM1, YBH3 (V. [START_REF] Bharathi | Role of CNC1 gene in TDP-43 aggregationinduced oxidative stress-mediated cell death in S. cerevisiae model of ALS[END_REF], and DBR1 (M. [START_REF] Elden | Ataxin-2 intermediate-length polyglutamine expansions are associated with increased risk for ALS[END_REF].

Furthermore, a real advance in modelling of ALS disease was made with the introduction of induced pluripotent stem cells (iPSCs) directly derived from differentiated cells of ALS patients (K. [START_REF] Takahashi | Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors[END_REF]. This model represents enormous advantages. First, they remove the need of artificially expressing mutant transgenes to study their impact. The development of tools allowing their differentiation into differentiated cells allow to study the impact of ALS associated mutants in specific cell types such as spinal MN (Y. [START_REF] Maury | Combinatorial analysis of developmental cues efficiently converts human pluripotent stem cells into multiple neuronal subtypes[END_REF][START_REF] Lenzi | Differentiation of control and ALS mutant human iPSCs into functional skeletal muscle cells, a tool for the study of neuromuscolar diseases[END_REF]). Finally, this is the only experimental model that can allow the study of sporadic ALS through the induction of iPSCs derived from sporadic ALS patients.

However, these models still have limits and they suffer from the lack of tissue connectivity.

The development of iPSCs co-cultures could bypass these issues as cultured of mature MN innervating muscle cells have been developed recently (N.R.W. [START_REF] Martin | Neuromuscular Junction Formation in Tissue-Engineered Skeletal Muscle Augments Contractile Function and Improves Cytoskeletal Organization[END_REF][START_REF] Santhanam | Stem cell derived phenotypic human neuromuscular junction model for dose response evaluation of therapeutics[END_REF]M.A. Bakooshli et al., 2019). Nevertheless, these cellular models have allowed the recapitulation of characteristic hallmark of the disease such as the TDP-43/FUS/SOD1/DPRs (I.V. [START_REF] Chestkov | Patient-Specific Induced Pluripotent Stem Cells for SOD1-Associated Amyotrophic Lateral Sclerosis Pathogenesis Studies[END_REF][START_REF] Lenzi | ALS mutant FUS proteins are recruited into stress granules in induced pluripotent stem cell-derived motoneurons[END_REF][START_REF] Bilican | Mutant induced pluripotent stem cell lines recapitulate aspects of TDP-43 proteinopathies and reveal cell-specific vulnerability[END_REF]S. Almeida et al., 2019) aggregations, NF disorganization (H. [START_REF] Chen | Modeling ALS with iPSCs reveals that mutant SOD1 misregulates neurofilament balance in motor neurons[END_REF], defects in the intracellular transport (K. [START_REF] Zhang | The C9orf72 repeat expansion disrupts nucleocytoplasmic transport[END_REF], and changes in excitability (B.J. [START_REF] Wainger | Intrinsic membrane hyperexcitability of amyotrophic lateral sclerosis patient-derived motor neurons[END_REF].

Overall, although many aspects of the disease cannot be studied in these unicellular models, the can offer mechanistical insights of the cytotoxicity and the proteinopathy. Moreover, these cellular models are optimal to identify possible therapeutic target to be further tested in more complex in vivo model.

B. In vivo models 1. Mouse models

Murine models are the most widely studied in vivo models for ALS. It's a popular model as it is a mammalian species with a high genetic and physiology homology compared to human.

Since the identification of the causative genes of "familial" ALS, a large number of murine models have been developed. For instance, the sole study of the toxicity of mutated SOD1

has led to the design of more than 10 models each expressing a different mutant protein (P.C. [START_REF] Wong | An adverse property of a familial ALS-linked SOD1 mutation causes motor neuron disease characterized by vacuolar degeneration of mitochondria[END_REF]L.I. Bruijn et al., 1997;[START_REF] Ripps | Transgenic mice expressing an altered murine superoxide dismutase gene provide an animal model of amyotrophic lateral sclerosis[END_REF]. The first and still most used model developed is the transgenic SOD1 G93A overexpression line (M.E. [START_REF] Gurney | Motor neuron degeneration in mice that express a human Cu,Zn superoxide dismutase mutation[END_REF]. Murine models for the major genetic causes of ALS have been key to better establish the pathogenicity of these major genetic factors and the consequences of these mutations upon MN degeneration (Y.F. [START_REF] Xu | Wild-type human TDP-43 expression causes TDP-43 phosphorylation, mitochondrial aggregation, motor deficits, and early mortality in transgenic mice[END_REF][START_REF] Wegorzewska | TDP-43 mutant transgenic mice develop features of ALS and frontotemporal lobar degeneration[END_REF][START_REF] Sharma | ALS-associated mutant FUS induces selective motor neuron degeneration through toxic gain of function[END_REF][START_REF] Scekic-Zahirovic | Toxic gain of function from mutant FUS protein is crucial to trigger cell autonomous motor neuron loss[END_REF][START_REF] Jiang | Gain of Toxicity from ALS/FTD-Linked Repeat Expansions in C9ORF72 Is Alleviated by Antisense Oligonucleotides Targeting GGGGCC-Containing RNAs[END_REF][START_REF] O'rourke | C9orf72 is required for proper macrophage and microglial function in mice[END_REF]. Although these models have led to a great advance in the comprehension of the disease, most of them are based on either the deletion of a target gene or an overexpression of a mutant. The arrival of new genetic engineering technologies is shifting the standard models with knock-in murine mutants already developed for FUS and TDP-43 mutations (S-H. [START_REF] Huang | A robust TDP-43 knock-in mouse model of ALS[END_REF]J. Scekic-Zahirovis et al., 2016). Furthermore, to define therapeutic strategies for ALS patients, an array of simpler animal models has been emerging over the last two decades as reviewed below.

Drosophila

Besides the mouse standard in vivo model, a wide number of small animal models have been developed. Due to their fast development and cheap maintenance, they are widely used.

The most widely used of these small in-vivo models for ALS study is the Drosophila melanogaster. Due to its quite developed nervous system (U.B. [START_REF] Pandey | Human disease models in Drosophila melanogaster and the role of the fly in therapeutic drug discovery[END_REF], the large repertoire of genetics tools design for its study (B. [START_REF] Ugur | Drosophila tools and assays for the study of human diseases[END_REF] and the 40% of homology between its functional gene and their respective human orthologs (L. [START_REF] Mcgurk | Drosophila as an In Vivo Model for Human Neurodegenerative Disease[END_REF], the Drosophila is an excellent model to study the complex genetics of ALS. Many Drosophila lines expressing human ALS associated mutant transgenes have been developed

for instance for SOD1 (M.R. [START_REF] Watson | A drosophila model for amyotrophic lateral sclerosis reveals motor neuron damage by human SOD1[END_REF], TARDBP (P.S. [START_REF] Estes | Wildtype and A315T mutant TDP-43 exert differential neurotoxicity in a Drosophila model of ALS[END_REF], FUS (Y. Chen et al., 2011), C9orf72 induced DPRs [START_REF] Mizielinska | C9orf72 repeat expansions cause neurodegeneration in Drosophila through argininerich proteins[END_REF]. For instance, mutant SOD1 has been extensively studied in the Drosophila and several antioxidant compounds have been identified to ameliorate the phenotypes of their respective mutant SOD1 models (C. Zhang et al., 2019 (1); C. Zhang et al., 2019 (2); T. [START_REF] Wang | Fisetin Exerts Antioxidant and Neuroprotective Effects in Multiple Mutant hSOD1 Models of Amyotrophic Lateral Sclerosis by Activating ERK[END_REF]. Through the use of these models, many advances have been made in the field of ALS, for instance, the identification of Pur-α protein as a regulator of the toxicity of C9orf72-DPRs expression (Z. [START_REF] Xu | Expanded GGGGCC repeat RNA associated with amyotrophic lateral sclerosis and frontotemporal dementia causes neurodegeneration[END_REF] and of the mutant FUS expression (J.G. [START_REF] Daigle | Pur-alpha regulates cytoplasmic stress granule dynamics and ameliorates FUS toxicity[END_REF]. Moreover, Drosophila is also a great model to confirm observation made in unicellular models: for instance, ATXN2 after being identified in the yeast through genetic screens, was confirmed as a modifier of TDP-43 toxicity in the drosophila (A.C. [START_REF] Elden | Ataxin-2 intermediate-length polyglutamine expansions are associated with increased risk for ALS[END_REF]. Recent genetic screening in FUS and TARDBP Drosophila models identified an array of enhancers and suppressors, which were able to impact the pathogenic induced phenotype (M.K. [START_REF] Kankel | Amyotrophic Lateral Sclerosis Modifiers in Drosophila Reveal the Phospholipase D Pathway as a Potential Therapeutic Target[END_REF].

C.elegans

Another invertebrate in vivo model used in the context of ALS research is the nematode Caenorhabditis elegans (C. elegans). This model presents a simplified but highly characterized nervous system and although it has a lower genomic homology with humans than other models, cellular stress pathways are still well conserved. Transgenic expression of the four main ALS-gene has been developed in C. elegans (T. [START_REF] Oeda | Oxidative stress causes abnormal accumulation of familial amyotrophic lateral sclerosis-related mutant SOD1 in transgenic Caenorhabditis elegans[END_REF][START_REF] Ash | Neurotoxic effects of TDP-43 overexpression in C. elegans[END_REF][START_REF] Murakami | ALS mutations in FUS cause neuronal dysfunction and death in Caenorhabditis elegans by a dominant gain-of-function mechanism[END_REF][START_REF] Therrien | Deletion of C9ORF72 results in motor neuron degeneration and stress sensitivity in C. elegans[END_REF]. The use of these models has contributed to the identification of several cellular alterations linked to the expression of mutant ALS proteins such as ER stress (A. [START_REF] Jablonski | Loss of RAD-23 Protects Against Models of Motor Neuron Disease by Enhancing Mutant Protein Clearance[END_REF] or Ca 2+ homeostasis disruption (D. [START_REF] Aggad | TDP-43 toxicity proceeds via calcium dysregulation and necrosis in aging Caenorhabditis elegans motor neurons[END_REF]. These models have also led to the identification of calcineurin as a regulator of TDP-43 toxicity (N.F. [START_REF] Liachko | The phosphatase calcineurin regulates pathological TDP-43 phosphorylation[END_REF]. More recently, this model was used to report the differential impact of SOD1 mutant knock in expression in cholinergic vs glutamatergic neurons (N. [START_REF] Baskoylu | Single copy/knock-in models of ALS SOD1 in C. elegans suggest loss and gain of function have different contributions to cholinergic and glutamatergic neurodegeneration[END_REF].

Modelling ALS in the zebrafish:

The Danio Rerio model, or zebrafish, is in many aspects an optimal model for modelling the ALS physiopathology.

Zebrafish is a vertebrate that is part of the Cyprinidae fish family. The use of this model was at first limited to the understanding of the genetic basis of vertebrate development (G. [START_REF] Streisinger | Production of clones of homozygous diploid zebra fish (Brachydanio rerio)[END_REF]. However, due to the high degree of conservation between its cellular mechanism and the human's, this model has grown in popularity over the years.

Technological advances led to the sequencing of its entire genome (K. Howe et al., 2013), unveiling a high conservation of the genes between the zebrafish and the human (up to 80%

of the human genes have a known orthologue in the zebrafish) [START_REF] Patten | Fishing for causes and cures of motor neuron disorders[END_REF].

Combined with this high homology with the human, the zebrafish also present some practical advantages due to their very fast development: 2 days post fertilization (dpf) larvae already present organs formation and function (C.B. [START_REF] Kimmel | Stages of embryonic development of the zebrafish[END_REF][START_REF] Drapeau | Development of the locomotor network in zebrafish[END_REF], with a sexually maturation up to 3 to 5 months after birth (C. [START_REF] Singleman | Growth and maturation in the zebrafish, Danio rerio: a staging tool for teaching and research[END_REF]. Moreover, zebrafish also have the advantages to give large clutches of extra-utero fertilized and transparent eggs which facilitates the observation and the in vivo or ex vivo fluorescence imaging (E. Kabashi et al., 2010;[START_REF] Kabashi | Zebrafish models for the functional genomics of neurogenetic disorders[END_REF]. The extrautero fertilization also facilitates genetic manipulation by antisense morpholino oligonucleotide (AMO) mediating transitory knockdown (KD) of a target transcript (B.R. [START_REF] Bill | A Primer for Morpholino Use in Zebrafish[END_REF] or by genome editing techniques such as CRISPR cas9 system to develop knockouts (KO) (B. [START_REF] Schmid | Genomic editing opens new avenues for zebrafish as a model for neurodegeneration[END_REF][START_REF] Hwang | Efficient genome editing in zebrafish using a CRISPR-Cas system[END_REF]. Finally, one of the biggest advantages of the zebrafish is the experimental facility to perform drug screening due to their permeability to small molecules at the different stages of development.

Moreover, due to its small size, a larva can fit in a single well of an up to 384 wells plate.

Therefore, high scale drug screening can be easily performed in this model (L.I. Zon and R.T. [START_REF] Zon | In vivo drug discovery in the zebrafish[END_REF].

In the context of ALS, numerous studies have used the zebrafish as a model to study genetic dysregulation associated with the development of the disease. Overexpression of mutant SOD1 for instance recapitulates several hallmarks of the pathology such as locomotion defects, loss of MNs, or loss of neuromuscular connectivity and subsequent degeneration (T. [START_REF] Ramesh | A genetic model of amyotrophic lateral sclerosis in zebrafish displays phenotypic hallmarks of motoneuron disease[END_REF][START_REF] Sakowski | Neuromuscular effects of G93A-SOD1 expression in zebrafish[END_REF]. A zebrafish model of C9orf72 reduced expression has also been designed, displaying axonopathy and locomotion impairment [START_REF] Ciura | Loss of function of C9orf72 causes motor deficits in a zebrafish model of amyotrophic lateral sclerosis[END_REF]. These defects were subsequently rescued via expression of the human C9orf72 orthologue. Finally, both decreased expressions of tardbp and fus have been investigated in the zebrafish resulting in severe motor deficits and alteration of the MNs integrity (E. Kabashi et al., 2009: A-R. [START_REF] Bourefis | Functional characterization of a FUS mutant zebrafish line as a novel genetic model for ALS[END_REF]. Interestingly, both the tardbp and fus downregulation have been shown to be rescued via the expression of human TDP-43 protein (E. [START_REF] Kabashi | Zebrafish models for the functional genomics of neurogenetic disorders[END_REF]. More recently, a tardbp KD zebrafish model has led to important understanding in the field by confirming the "distal axonopathy" theory, a hypothesis that claimed that pathological alterations appear during the presymptomatic phases of the disease at the neuromuscular junctions (M.L. [START_REF] Campanari | TDP-43 Regulation of AChE Expression Can Mediate ALS-Like Phenotype in Zebrafish[END_REF].

Part II: TBK1 as a novel genetic factor in ALS

I. Introduction to TBK1 A. Discovery and initial characterization

Tumor necrosis factor (TNF) receptor associated factor NFκB activator (or TANK) is a protein thought to be involved in both the stimulation and the inhibition of NFκB (G. [START_REF] Cheng | TANK, a co-inducer with TRAF2 of TNF-and CD 40Lmediated NF-kappaB activation[END_REF][START_REF] Rothe | I-TRAF is a novel TRAF-interacting protein that regulates TRAF-mediated signal transduction[END_REF]. In an article of 1999, J.L Pomerantz and D. Baltimore were investigating the mechanisms involved in the stimulation of NFκB activity by TANK (J.L. [START_REF] Pomerantz | NF-kappaB activation by a signaling complex containing TRAF2, TANK and TBK1, a novel IKK-related kinase[END_REF]. The lack of homology in the signatory region of TANK led these researchers to seek for other proteins interacting with TANK: one of the identified proteins was called TANK binding kinase 1 (TBK1 also called NAK or T2K). Due to the fact that Tbk1 mutant mice lacked the TANK-mediated stimulation of NFκB (J.L. [START_REF] Pomerantz | NF-kappaB activation by a signaling complex containing TRAF2, TANK and TBK1, a novel IKK-related kinase[END_REF]D. Baltimore, 1999, Y. Tojima et al., 2000) and died from a massive liver degeneration at E14.5 (M. [START_REF] Bonnard | Deficiency of T2K leads to apoptotic liver degeneration and impaired NF-kappaB-dependent gene transcription[END_REF], similarly to NFκB mutant mice (A. [START_REF] Beg | Constitutive NF-kappa B activation, enhanced granulopoiesis, and neonatal lethality in I kappa B alpha-deficient mice[END_REF][START_REF] Tanaka | Embryonic lethality, liver degeneration, and impaired NF-kappa B activation in IKK-beta-deficient mice[END_REF][START_REF] Rudolph | Severe liver degeneration and lack of NF-?B activation in NEMO/IKK?deficient mice[END_REF][START_REF] Li | IKK1-deficient mice exhibit abnormal development of skin and skeleton[END_REF], TBK1 was first considered as a NFκB activating kinase.

NFκB is activated through the inactivation via phosphorylation of IkBα, its repressor (A. [START_REF] Oeckinghaus | The NF-kappaB family of transcription factors and its regulation[END_REF]. This post-transcriptional modification is performed by a family of homologous proteins called the IκB kinases (IKK) composed of IKKα, IKKβ, and IKKγ (or NEMO). Due to its presumed role in the NFκB activation and its structural resemblance to this family of gene, TBK1 along with IKKε (another highly structurally identical kinase protein)

were first linked to IKK (J.L. Prochiants andD. Baltimore, 1999, Y. Tojima et al., 2000).

However, despite their homology with IKKs, the TBK1 and IKKε kinases were found to play distinct role in cell physiology (K.A. [START_REF] Fitzgerald | IKKepsilon and TBK1 are essential components of the IRF3 signaling pathway[END_REF][START_REF] Hemmi | The roles of two IkappaB kinase-related kinases in lipopolysaccharide and double stranded RNA signaling and viral infection[END_REF][START_REF] Mcwhirter | IFN-regulatory factor 3-dependent gene expression is defective in Tbk1-deficient mouse embryonic fibroblasts[END_REF] and therefore a new classification was made separating the canonical IKK (α, β, γ) from the non-canonical IKK related TBK1 and IKKε.

TBK1 is constitutively expressed throughout all the organism. Despite its low tissue specificity, its expression level is higher in some specific tissues as the nervous system, the endocrinal system, the digestive and respiratory tracts, the skin and the female tissue (M.

Ulhen et al., 2015 -The human protein atlas). Importantly, TBK1 expression is enriched in the nervous system within the cerebral cortex, the hippocampus and the basal ganglia. • Ubiquitin like domain (ULD):

B. Structure and properties

It is a 80 AA long domain shared within the IKK family. Ubiquitin is a small molecule of 76 AA that can be considered as a post-transcriptional modification: one or several ubiquitin molecules can be attached to a substrate protein targeting it for proteasomal degradation (C. M. [START_REF] Pickart | Mechanisms underlying ubiquitination[END_REF] or altering their activity or localization (S. E. [START_REF] Dougherty | Expanding Role of Ubiquitin in Translational Control[END_REF].

Ubiquitin is highly conserved throughout the evolution as is its structure: a unique fold formed by a β-sheet with five antiparallel β-strands and a single helical segment (S. Martin-Villanueva et al., 2021). Interestingly, some proteins, as TBK1 and the IKKs, bear this motif as part of their larger structures, called a ubiquitin like domain. These domains are not processed to produce a ubiquitin molecule, but in the vast majority of these proteins, they only function as protein-protein interaction region and whether they have an additional role linked to their resemblance to ubiquitin remains obscure (M. [START_REF] Hochstrasser | Origin and function of ubiquitin-like proteins[END_REF]. In TBK1, this motif does not bind to any known ubiquitin-binding domains but only to its own kinase domain and to the IRF3 protein (F. Iketa et al., 2007). However, it has been shown that this domain is crucial for TBK1 activation and its phosphorylation capacity (F. Iketa et al., 2007) suggesting a more important role in the dimerization or the autophosphorylation of the protein.

• Scaffold dimerization domain (SDD):

The SDD has a structural function and it allows TBK1 activation by interacting with the other subunit of the homodimer. It is a 250 AA long domain characterized by four helices that form three helical antiparallel coiled coils, which runs along the entire length of the molecule. This dimerization is allowed by 4 dimerization interfaces, each involving interactions in between the SSD domain and respectively the KD and ULD domains of the other sub-unit (A. [START_REF] Larabi | Crystal structure and mechanism of activation of TANK-binding kinase 1[END_REF]. The importance of the SDD domain is underlined as the sole mutation of 3 residues in the first SSD helix leads to accumulation of monomeric TBK1 (A. [START_REF] Larabi | Crystal structure and mechanism of activation of TANK-binding kinase 1[END_REF]. Moreover, mutant protein lacking the interaction between ULD-SDD domains results in the inhibition of TBK1's function without preventing its activation (A. [START_REF] Larabi | Crystal structure and mechanism of activation of TANK-binding kinase 1[END_REF].

These results suggest that the SDD is also important in the functional role of the protein besides its importance in its activation.

• C terminal domain (CTD) or TANK binding domain:

It is a 100 AA long domain involved in the interaction with adaptor proteins such as TANK, NAP1, SINTBAD (A. [START_REF] Goncalves | Functional dissection of the TBK1 molecular network[END_REF]. The truncation of the CTD does not alter the kinase activity (A. [START_REF] Goncalves | Functional dissection of the TBK1 molecular network[END_REF] and the similar abolishment of the dimerization does not prevent binding of TANK or NAP1 to the CTD (A. [START_REF] Larabi | Crystal structure and mechanism of activation of TANK-binding kinase 1[END_REF]. These adaptors proteins reside at different sub-cellular location (A. [START_REF] Goncalves | Functional dissection of the TBK1 molecular network[END_REF] while they are competing for a TBK1 interaction and as the CTD function seems to be restricted to the interaction with these binding partners. This domain could be then used for the protein as a localization signal.

TBK1's activation requires two features: the homodimerization of the protein and its polyubiquitinylation at two crucial residues (K30 and K401) (D. [START_REF] Tu | Structure and ubiquitination-dependent activation of TANK-binding kinase 1[END_REF]. Mutant proteins lacking one of those polyubiquitinalyted marks exhibit an impaired activation and signalling (D. [START_REF] Tu | Structure and ubiquitination-dependent activation of TANK-binding kinase 1[END_REF]. When in dimer, TBK1 undergo transphosphorylation on the Ser172 which leads to its activation via a change in its conformation: the C-loop of the KD rotates until formation of a salt-bridge between glutamate residue of the KD with a lysine residue of the SSD. Serine to alanine substitution at the location abolishes TBK1's function (C. [START_REF] Shu | Structural insights into the functions of TBK1 in innate antimicrobial immunity[END_REF] and phosphomimetic mutation S279E partially restores the activity of the wildtype TBK1.

II. Functional roles of TBK1

TBK1 functional relevance has been largely investigated, and its activity has been reported to be involved in inflammatory responses (T. Upon invasion of the host by microbial pathogens such as viruses, bacteria and parasites, the first line of defence of the organism is the innate immunity. As mentioned previously, the response to these stimuli is triggered by the pattern recognition receptors (PRRs).

Interestingly, some of these PRRs have been associated with ALS. For instance, toll-like receptors (TLRs) are PRRs that have been reported to be upregulated in glial cells of ALS patients (M. [START_REF] Casula | Toll-like receptor signaling in amyotrophic lateral sclerosis spinal cord tissue[END_REF]. Moreover the inhibition of some members of this family have been shown to be beneficial in some murine model (J.Y. [START_REF] Lee | Absence of toll-like receptor 4 (TLR4) extends survival in the hSOD1G93A mouse model of amyotrophic lateral sclerosis[END_REF][START_REF] Fellner | Toll-Like Receptor-4 Inhibitor TAK-242 Attenuates Motor Dysfunction and Spinal Cord Pathology in an Amyotrophic Lateral Sclerosis Mouse Model[END_REF]. Other PRRs subclasses have also been linked to the ALS pathology such as Nodlike receptors (NLRs) [START_REF] Johann | NLRP3 inflammasome is expressed by astrocytes in the SOD1 mouse model of ALS and in human sporadic ALS patients[END_REF][START_REF] Lehmann | Expression profile of pattern recognition receptors in skeletal muscle of SOD1(G93A) amyotrophic lateral sclerosis (ALS) mice and sporadic ALS patients[END_REF] or AIM2-like receptors (ALRs) [START_REF] Lehmann | Expression profile of pattern recognition receptors in skeletal muscle of SOD1(G93A) amyotrophic lateral sclerosis (ALS) mice and sporadic ALS patients[END_REF].

These receptors, upon recognition of PAMPs/DAMPs, are responsible for the cascade of event that leads to the activation of NFκB and IRF3 and the subsequent synthesis of inflammatory cytokines and interferons (IFN) (O. Takeushi and S. Akira, 2010).

a. Inflammatory cytokines

Cytokines is a term that describes small proteins released by cells in order to interact and communicate with other cells (J-M. Zhang and J. An, 2009). There are different types of cytokines according to the types of cells where these factors originated, for instance, lymphokines are synthesized by lymphocytes, monokines by monocytes, interleukins by leukocytes. Pro-inflammatory cytokines are mainly produced by activated macrophages, however, during cell injury, infection, invasion, and subsequent inflammation they can be synthesized by non-immune cells (J-M. Zhang and J. An, 2009).

Pro-inflammatory cytokines, upon binding, are stimulating several cellular processes mostly resulting in cellular death (ER stress, apoptosis, oxidative stress) (J.A. Smith et al., 2012).

Their aim is to slow down a possible infection or lesion. Amongst the most studied proinflammatory cytokines, we can mention IL-1β, IL-6, or TNF-α. The anti-inflammatory cytokines are secreted to reduce inflammation and promote healing via regulation of the pro-inflammatory cytokine production. Similarly, pro-inflammatory cytokines can be both produced by immune and non-immune cells, and for instance in the CNS they can be synthesized by neurons and glial cells (L. Vitkovic et al., 2001). Amongst the most studied anti-inflammatory cytokines, we can mention several interleukins such as IL-4,10,11, or 13.

One last set of specific cytokines, the chemokines are known to induce chemotaxis, a process in which chemokines are able to attract immune cells at a lesion site (C.E. [START_REF] Hughes | A guide to chemokines and their receptors[END_REF].

b. Interferons

Interferons, are a family of anti-viral cytokines named after their capacity to interfere with viral replication. Although more than 20 different IFNs have been described, only 3 IFNreceptors have been reported: IFNAR, IFNGR, IFNLR (E.V. [START_REF] Mesev | Decoding type I and III interferon signalling during viral infection[END_REF]. These receptors are composed by 2 unique heterodimers units, which are responsible for the specificity of the binding. Upon IFN-receptor binding, two receptors associated proteins transmit the signal and further induce STAT1 or STAT2 transcription factors' nuclear translocation and the subsequent expression of interferons stimulated genes (ISGs) (S.A. [START_REF] Qureshi | Tyrosine-phosphorylated Stat1 and Stat2 plus a 48-kDa protein all contact DNA in forming interferon-stimulated-gene factor 3[END_REF]. According to the recognized receptors, IFN are sorted into 3 subgroups (E.V. [START_REF] Mesev | Decoding type I and III interferon signalling during viral infection[END_REF]:

-the IFN-I: the largest group of IFN is composed by the IFNβ, IFNε, IFNω and 13 types of IFNα. These IFN are specifically binding to the IFNAR.

-The IFN-II: this class is only composed by the IFNγ. These IFN are specifically binding to the IFNGR and therefore follow the classical one ligant -one receptor schem. 2020). Moreover, interferons are often produced in a cascade, meaning that one interferon stimulates its target cells to make additional interferons.

TBK1 is a crucial regulator of the type I IFN as described in the following section.

TBK1 activates IRF3

TBK1 is involved in the cascade of signalisation induced by five of the PRRs: the toll-like receptor 3 (TLR3) and 4 (TLR4), two RIG-I like receptors (RLR): RIG-I and MDA5 and the DNA sensing receptor cGAS (S. Liu et al., 2015). Even if they are involved in the responses to different factors such as nucleic acid detection (X. Li et al., 2013;[START_REF] Shu | The mechanism of double-stranded DNA sensing through the cGAS-STING pathway[END_REF] or bacterial and viral signatures (T. [START_REF] Kawai | TLR signaling[END_REF][START_REF] Rehwinkel | RIG-I-like receptors: their regulation and roles in RNA sensing[END_REF], these 5 receptors all lead to the recruitment of TBK1/IKKε, however, the intermediate factors involved in these signalling mechanisms are different. Indeed, within these pathways TBK1 is known to be recruited and to interact respectively with TRIF, MAVS-TRAF3 and STING (S. Liu et al., 2015;[START_REF] Kawai | TLR signaling[END_REF][START_REF] Paz | A functional C-terminal TRAF3-binding site in MAVS participates in positive and negative regulation of the IFN antiviral response[END_REF][START_REF] Shu | The mechanism of double-stranded DNA sensing through the cGAS-STING pathway[END_REF]. When activated these two closely related kinases phosphorylate interferon regulatory factor 3 (IRF3). P-IRF3 forms homodimers leading to their nuclear translocation, which further induce the expression of type I IFN and the subsequent immune response.

Interestingly, upon activation, these receptors also lead to the activation of the NF-κB complex and the subsequent production of inflammatory cytokines by alternate pathways (Y. [START_REF] Liu | NF-?B signaling in inflammation[END_REF][START_REF] Kawai | TLR signaling[END_REF].

These receptors are therefore crucial for the immune response to pathogen invasion and their dysregulation can have severe impact on the organism defence. For this reason, several viruses directly target members of these pathways to bypass the host defence (A. [START_REF] Zotta | SARS-CoV-2 targets MAVS for immune evasion[END_REF][START_REF] Qu | Disruption of TLR3 Signaling Due to Cleavage of TRIF by the Hepatitis A Virus Protease-Polymerase Processing Intermediate, 3CD[END_REF][START_REF] Christensen | HSV-1 ICP27 targets the TBK1-activated STING signalsome to inhibit virus-induced type I IFN expression[END_REF].

TBK1 activates NFκB:

Although it was reported that its main function in the innate immune response was linked to the production of type I IFN, it is important not to forget its involvement in the production of inflammatory cytokines through the activation of NFκB. Indeed, TBK1 was first discovered as a TANK binding partner involved in the regulation of NFκB and repression of IκB (J.L.

Prochiants and D. Baltimore, 1999). However, TBK1 dependent regulation was reported to be limited compared to its crucial contribution in the IFN synthesis (K.A. [START_REF] Fitzgerald | IKKepsilon and TBK1 are essential components of the IRF3 signaling pathway[END_REF].

Nevertheless TBK1's contribution to NFκB, although secondary, exists and the report of a specific NFκB activation via activation of the cGAS-STING pathway supports this idea. Indeed, a recent study [START_REF] Yum | TBK1 recruitment to STING activates both IRF3 and NF-?B that mediate immune defense against tumors and viral infections[END_REF] has designed a STING mutant line deficient in the STING-IRF3 interaction. However, this mutant line was still able to induce an anti-viral response to HSV-1, through the activation of the NFκB response despite the impaired TBK1phosphorylation of IRF3. This alternate innate immune response was prevented in another mutant STING line inhibiting the STING-TBK1 pathway suggesting that TBK1 was responsible for the cGAS-STING-NFκB pathway.

It is unclear in what context the TBK1 dependent stimulation of NFκB occurs but it was reported that this function was partially shared with IKKε and that TBK1 loss could be compensated by IKKε activity in opposition to IRF3 stimulation in which TBK1 activity is indispensable (K.R. [START_REF] Balka | TBK1 and IKK? Act Redundantly to Mediate STING-Induced NF-?B Responses in Myeloid Cells[END_REF]. This further supports the theory of the unequilibrated dual role of TBK1 in IRF3 and NFκB regulation. In addition, TBK1 has been reported to be involved in the regulation of the NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome complex. NLRP3 is a NOD-like intracellular sensor that detects a broad range of DAMPs or PAMPs, resulting in the formation and activation of the NLRP3 inflammasome (K.V. [START_REF] Swanson | The NLRP3 inflammasome: molecular activation and regulation to therapeutics[END_REF]. Upon activation, NLRP3 leads to the caspase 1dependent release of the pro-inflammatory cytokines IL-1β and IL-18 via activation of the NFκB complex. NLRP3's activation is induced by the protein phosphatase 2A (PP2A) and is prevented by an inhibiting phosphorylation mark via TBK1 and IKKε activity (F.A. [START_REF] Fischer | TBK1 and IKK? act like an OFF switch to limit NLRP3 inflammasome pathway activation[END_REF]. Consequently, pharmacological and genetic inhibition of these two kinases has led to uncontrolled NLRP3 inflammasome response. However, the sole losses of either TBK1 or

IKKε were not sufficient to induce increased NLRP3 activation, suggesting redundant roles of the two kinases in its regulation (F. A. Fisher et al., 2021). Besides the implication of TBK1 in this inflammation process, this suggests a more complex role of TBK1 in the regulation of NFκB as it is in some context also preventing its activation.

B. TBK1 as a regulator of the autophagic process:

Because TBK1 is mainly involved in the selective macroautophagy process, I will use the term "autophagy" to specifically describe this process. Autophagy initiation is regulated by the activity of mammalian target of rapamycin (mTOR) complex 1 (mTORC1), a kinase complex involved in several cellular pathways. In the autophagy regulation, mTORC1 targets two kinases called autophagy activating kinase 1 and 2 (ULK1/2) for inhibitory phosphorylation.

Upon autophagy activation, AMP-activated protein kinase (AMPK) in turn inhibits via phosphorylation mTORC1 (Y. [START_REF] Kiriyama | The Function of Autophagy in Neurodegenerative Diseases[END_REF], subsequently, ULK1/2 without their regulatory marks are free to interact with ATG13 and FIP200. FIP200 is phosphorylated by the ULK proteins within the newly formed complex which induces the formation of an immature membrane structure, the phagophore (C.H. [START_REF] Jung | ULK-Atg13-FIP200 complexes mediate mTOR signaling to the autophagy machinery[END_REF]. Formation of the immature phagophore triggers the recruitment of a second complex the phosphatidylinositol 3-kinase class III (PI3K-cIII) complex, which mediates the elongation of the phagophore and the subsequent envelopment of the proteins or organelles marked for degradation (J.H.

Hurley and L.N. [START_REF] Hurley | Mechanisms of Autophagy Initiation[END_REF]). An additional complex contributing to the phagophore's maturation is the ATG12-ATG5-ATG16 complex, which allows production of LC3-II (N. [START_REF] Mizushima | A protein conjugation system essential for autophagy[END_REF][START_REF] Walczak | Dissecting the role of the Atg12-Atg5-Atg16 complex during autophagosome formation[END_REF]. The newly formed LC3-II molecules bind to the surface of the phagophore and act as anchor for the autophagy cargo proteins in addition to their role in the elongation of the phagophore membrane (Y. [START_REF] Kiriyama | The Function of Autophagy in Neurodegenerative Diseases[END_REF]. Once this process of maturation is done, the newly formed vacuole is called an autophagosome. Subsequently, the autophagosome is transported along microtubules to fuse with a low pH organelle called lysosome. The fusion of these structures is called an autophagolysosome, and the content of these structures digested by low-pH dependent enzymes (J.A. [START_REF] Oakes | TBK1: a new player in ALS linking autophagy and neuroinflammation[END_REF].

Protein or organelles that are targeted to autophagy dependent degradation undergo a ubiquitin posttranscriptional modification, which consists in the addition of a ubiquitin chain on a target lysine residue (M. Rape, 2017). These ubiquitin chains are later recognized by autophagy cargo proteins such as p62, OPTN, NDP52, (D. [START_REF] Gatica | Cargo recognition and degradation by selective autophagy[END_REF]; J.M. [START_REF] Heo | The PINK1-PARKIN Mitochondrial Ubiquitylation Pathway Drives a Program of OPTN/NDP52 Recruitment and TBK1 Activation to Promote Mitophagy[END_REF] which are phosphorylated by TBK1. Autophagy cargo proteins are binding to the anchored phagophore LC3-II proteins to sequester the ubiquitinated components for subsequent autophagosome engulfment (T. [START_REF] Johansen | Selective Autophagy: ATG8 Family Proteins, LIR Motifs and Cargo Receptors[END_REF].

TBK1 regulates the autophagic process at several levels. As described above, TBK1 is a known regulator of autophagy cargo proteins. Indeed, it has been reported that TBK1 dependent phosphorylation on the 3 cargo proteins p62, OPTN, and NDP52 (J.M. [START_REF] Heo | The PINK1-PARKIN Mitochondrial Ubiquitylation Pathway Drives a Program of OPTN/NDP52 Recruitment and TBK1 Activation to Promote Mitophagy[END_REF] regulates their activity and enhance their binding capacity to LC3-II and to ubiquitinated compounds. The importance of TBK1 in this crucial process is underlined by decreased p62 mRNA levels in ALS patients bearing a TBK1 mutation (C. [START_REF] Portera-Cailliau | Wholegenome sequencing reveals important role for TBK1 and OPTN mutations in frontotemporal lobar degeneration without motor neuron disease[END_REF] which could result in the impairment of the autophagy process and the accumulation of the cytosolic inclusions. TBK1 is also known to interact and phosphorylate SMCR8, a member of the C9orf72 complex along with WDR41. A direct interaction between the C9orf72-SMCR8-WDR41 complex and the FIP200/ULK1/ATG13 complex was reported and it was suggesting that the C9orf72 complex was a substrate downstream of ULK1 (P.M. [START_REF] Sullivan | The ALS/FTLD associated protein C9orf72 associates with SMCR8 and WDR41 to regulate the autophagy-lysosome pathway[END_REF].

Altogether the C9orf72 complex was suggested to regulate autophagy at 3 different levels:

on the autophagy initiation, on the autophagic flux (M. [START_REF] Yang | A C9ORF72/SMCR8-containing complex regulates ULK1 and plays a dual role in autophagy[END_REF], and on the lysosomal initiation (J.G. O' Rourke et al., 2016). TBK1 also regulates autophagy by interacting with mTORCs. Indeed, TBK1 has been reported to stimulate via direct phosphorylation both of the mTOR complexes mTORC1/2 (C. Bodur et al., 2017;[START_REF] Tooley | The innate immune kinase TBK1 directly increases mTORC2 activity and downstream signaling to Akt[END_REF]. This observation exhibits a dual role of TBK1 in both activating autophagy (via interaction with adaptors proteins and SMCR8 phosphorylation) and inhibiting autophagy (via stimulation of mTORC1 a known inhibitor of the autophagy initiation). Moreover, TBK1 has also been reported to activate RAPTOR, a known inhibitor of mTORC1 (R.J. [START_REF] Antonia | TBK1 Limits mTORC1 by Promoting Phosphorylation of Raptor Ser877[END_REF].

Altogether, this suggests that TBK1 is an important regulator of mTORC1 activity and subsequent autophagy, however, more investigations would be required in order to understand in which context each of the regulation is occurring.

Finally, TBK1 is also regulating autophagy process indirectly through IRF3 activation and subsequent type-I interferons production. Indeed, amongst the interferon-inducible genes, ISG15 have been linked to the activation of autophagy (D. [START_REF] Xu | Modification of BECN1 by ISG15 plays a crucial role in autophagy regulation by type I IFN/interferon[END_REF][START_REF] Nakashima | Interferon-stimulated Gene 15 (ISG15) and ISG15-linked Proteins Can Associate with Members of the Selective Autophagic Process, Histone Deacetylase 6 (HDAC6) and SQSTM1/p62 *[END_REF].

C. Cell death in ALS:

As previously introduced (cf: part I section B.9), programmed cell death (PCD) mechanisms have been suspected to be responsible for the motor neuron degeneration in ALS. I will discuss the evidence reported of the involvement of these pathways in ALS pathophysiology and provide evidence for the functional roles of TBK1 within these pathways.

Programmed cell death: a. Apoptosis

The discovery and definition of the apoptosis process can be attributed to John Kerr in 1972 (J.F.R [START_REF] Kerr | Apoptosis: A Basic Biological Phenomenon with Wide-ranging Implications in Tissue Kinetics[END_REF]. It was considered for a long time as the only cause of PCD.

Apoptosis can be triggered by 2 distinct pathways:

-The mitochondrial (or intrinsic) induction

In response to cellular stress, a protein family called the BH3-only proteins are being upregulated (H. [START_REF] Puthalakath | Keeping killers on a tight leash: transcriptional and post-translational control of the pro-apoptotic activity of BH3-only proteins[END_REF]. These crucial initiators of apoptosis -The death receptor (or extrinsic) induction:

Although it results in a similar cell death mechanism than the mitochondrial induction, the death receptor pathway is triggered by external stimuli. As mentioned before, cells bear PRRs at the surface of their plasma membrane to facilitate cell-cell communication. The TNF family of receptors are involved in the inflammatory process. Upon activation, TNF receptors induce the formation of an intracellular death-signalling complex (D. [START_REF] Moujalled | Molecular mechanisms of cell death in neurological diseases[END_REF].

The formation of this complex results in the activation of CASP8 and its downstream effector CASP 3/7 (H.R. [START_REF] Stennicke | Pro-caspase-3 is a major physiologic target of caspase-8[END_REF]M. Lamkanfiand and T-D. Kanneganti, 2010), which results in the subsequent trigger of apoptosis. Interestingly, these two receptors have been shown to connect as CASP8 activation can lead to the stabilisation of a BH3-only protein, BID which leads to mitochondrial outer membrane permeabilization (H. [START_REF] Li | Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis[END_REF][START_REF] Luo | Bid, a Bcl2 interacting protein, mediates cytochrome c release from mitochondria in response to activation of cell surface death receptors[END_REF].

Upon apoptotic death, cells appear smaller with tightly packed organelles and chromatin condensation. At the final stage of the process, an extensive plasma membrane blebbing and a nuclear fragmentation are reported, resulting in the formation of apoptotic bodies and their release in the extracellular environment, which are eliminated by neighbouring cells via phagocytosis (S. Elmore, 2007; R. [START_REF] Kakarla | Apoptotic cell-derived exosomes: messages from dying cells[END_REF].

b. Necroptosis

Historically, most researchers believed that cell death exists as a dichotomy between apoptosis and necrosis (L.J. [START_REF] Martin | Neurodegeneration in Excitotoxicity, Global Cerebral Ischemia, and Target Deprivation: A Perspective on the Contributions of Apoptosis and Necrosis[END_REF] until it became clear that some specific cases of cellular death were not fitting in either of these boxes (C. [START_REF] Portera-Cailliau | Wholegenome sequencing reveals important role for TBK1 and OPTN mutations in frontotemporal lobar degeneration without motor neuron disease[END_REF].

Necroptosis was only described in 2000 (N. [START_REF] Holler | Fas triggers an alternative, caspase-8-independent cell death pathway using the kinase RIP as effector molecule[END_REF][START_REF] Vanden Berghe | Regulated necrosis: the expanding network of non-apoptotic cell death pathways[END_REF] and is usually described as an active form of necrosis.

Importantly, necroptosis initiation is dependent of receptor-interacting serine/threonine protein kinase 1 (RIPK1) activity. RIPK1 itself is regulated by several post-transcriptional marks: phosphorylation by MAK2, TBK1, the IKK complex, TAK1 or by autophosphorylation and via polyubiquitination by cIAP2/5 and the LUPAC complex (T. [START_REF] Delanghe | RIPK1 kinase-dependent death?: a symphony of phosphorylation events[END_REF].

Necroptosis initiation is directly dependent on the deubiquitination process of RIPK1 by Lys63-deubiquitylating enzyme cylindromatosis (CYLD) (P. [START_REF] Vandenabeele | Molecular mechanisms of necroptosis: an ordered cellular explosion[END_REF]. A.L. [START_REF] Samson | MLKL trafficking and accumulation at the plasma membrane control the kinetics and threshold for necroptosis[END_REF]. This results in membrane disruption and rapid influx of Ca 2+ ions and water leading to the dissolution of cell.

Because this type of cell death is characterized by the disruption of the membrane, numerous cell components are released in the extracellular medium. The cellular debris resulting from necroptosis activation is recognized as DAMPs or PAMPs (in case of infected cells) further leading to the initiation of the inflammation response (M. Pasparakis and P.

Vandenabeele, 2015).

Evidence of programmed cell death in ALS:

a. Apoptosis in ALS:

Multiple reports have described apoptosis-mediated cell death involved in the massive motor neuron degeneration observed in ALS. The first suspicion of apoptosis involvement came from preliminary observations reporting altered expression of BCL-2 and increased levels of BAX in the spinal cord of ALS patients (X. [START_REF] Mu | Altered expression of bcl-2 and bax mRNA in amyotrophic lateral sclerosis spinal cord motor neurons[END_REF] and in a SOD1 mutant model (J.L. Gonzalez de [START_REF] González De Aguilar | Alteration of the Bcl-x/Bax ratio in a transgenic mouse model of amyotrophic lateral sclerosis: evidence for the implication of the p53 signaling pathway[END_REF]. Consecutively, sequestration of BCL-2 by SOD1 mutant proteins was reported in the murine SOD1G93A line suggesting an important role of SOD1 mutant in regulating BCL-2 protein and therefore possible trigger of the apoptosis process without active intrinsic signalling (P. [START_REF] Pasinelli | Amyotrophic lateral sclerosis-associated SOD1 mutant proteins bind and aggregate with Bcl-2 in spinal cord mitochondria[END_REF]. The involvement of BCL-2 was further exposed as an overexpression of this anti-apoptotic protein was reported to extend the survival of the mSOD1 [START_REF] Vukosavic | Delaying caspase activation by Bcl-2: A clue to disease retardation in a transgenic mouse model of amyotrophic lateral sclerosis[END_REF]. Dysregulation of the antiapoptotic machinery with neuronal apoptosis inhibitory protein (NAIP), a member of the anti-apoptosis IAP family (N. [START_REF] Roy | The gene for neuronal apoptosis inhibitory protein is partially deleted in individuals with spinal muscular atrophy[END_REF] shown to selectively suppress oxidative stressinduced cell death (P. [START_REF] Liston | Suppression of apoptosis in mammalian cells by NAIP and a related family of IAP genes[END_REF], was reported to be decreased in ALS patients (O. [START_REF] Kano | Neuronal apoptosis inhibitory protein is implicated in amyotrophic lateral sclerosis symptoms[END_REF]. These results were further confirmed as the use of bromocriptine, a positive regulator of NAIP and the antioxidant response, was reported to extend survival in a murine mSOD1 model (K. [START_REF] Tanaka | Bromocriptine methylate suppresses glial inflammation and moderates disease progression in a mouse model of amyotrophic lateral sclerosis[END_REF]. Similarly, evidence of increased levels of proapoptotic compounds have been reported with both of the BAX and BIM proteins upregulated respectively in patients and mSOD1 murine model (T. [START_REF] Ekegren | Upregulation of Bax protein and increased DNA degradation in ALS spinal cord motor neurons[END_REF][START_REF] Hetz | The proapoptotic BCL-2 family member BIM mediates motoneuron loss in a model of amyotrophic lateral sclerosis[END_REF]. Moreover, deficiencies in both of these proteins were reported to ameliorate mSOD1 mouse phenotype and extend their survival (N.A. [START_REF] Reyes | Blocking the mitochondrial apoptotic pathway preserves motor neuron viability and function in a mouse model of amyotrophic lateral sclerosis[END_REF][START_REF] Hetz | The proapoptotic BCL-2 family member BIM mediates motoneuron loss in a model of amyotrophic lateral sclerosis[END_REF]. Finally, caspase activation has also been reported to be involved in the development of the pathology (P. [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] Inoue | The crucial role of caspase-9 in the disease progression of a transgenic ALS mouse model[END_REF], which has been further confirmed by the use of a broad anti-caspase drug ameliorating survival still in the mSOD1 mouse model (M. [START_REF] Li | Functional role of caspase-1 and caspase-3 in an ALS transgenic mouse model[END_REF].

Overall, these results suggest an activation of the apoptotic pathway as the major cause of neurodegeneration in the context of ALS. However, it is important to underline that these studies mostly focused on SOD1 associated ALS models. Still in this direction, several studies have identified robust activation of the different members of the necrosome complex in an in vitro OPTN KD mouse model (Y. [START_REF] Ito | RIPK1 mediates axonal degeneration by promoting inflammation and necroptosis in ALS[END_REF], in mSOD1 mouse (G. [START_REF] Dermentzaki | Deletion of Ripk3 Prevents Motor Neuron Death In Vitro but not In Vivo[END_REF] or in ALS patients (M. Chevin and G. Sébire, 2020). Moreover, a double mutant TBK1-TAK1 KD murine model was reported to exhibit alleviated phenotype upon pharmacological deletion of RIPK1 (D. [START_REF] Xu | TBK1 Suppresses RIPK1-Driven Apoptosis and Inflammation during 195 Development and in Aging[END_REF].

However, a recent study described a minimal implication of necroptosis in ALS, via a lack of impact from the deficiency of MLKL in the mSOD1 murine model (T. [START_REF] Wang | Sting is a critical regulator of spinal cord injury by regulating microglial inflammation via interacting with TBK1 in mice[END_REF].

Results concerning RIPK1 upregulation have to be taken with caution as this kinase has also been reported to be involved in a specific type of apoptotic cell death pathway via the formation of a RIPK1-FADD-CASP8 complex (D. [START_REF] Ofengeim | Regulation of RIP1 kinase signalling at the crossroads of inflammation and cell death[END_REF]. Therefore, an increase in RIPK1 expression could leads to activation of either apoptosis or necroptosis.

TBK1 involvement in programmed cell death

TBK1 is a known endogenous RIPK1 regulator via an inhibitory phosphorylation mark on the T189 RIPK1 residue (D. [START_REF] Xu | TBK1 Suppresses RIPK1-Driven Apoptosis and Inflammation during 195 Development and in Aging[END_REF]T. Delanghe et al., 2020). This phosphorylation has been reported to be an important regulator of cell death in several reports (E. [START_REF] Lafont | TBK1 and IKK? prevent TNF-induced cell death by RIPK1 phosphorylation[END_REF]Y. Yang et al., 2022;[START_REF] Xu | TBK1 Suppresses RIPK1-Driven Apoptosis and Inflammation during 195 Development and in Aging[END_REF]. Interestingly, it was shown that TAK1, another kinase regulator of RIPK1, expression was decreased in aging human brain (D. [START_REF] Xu | TBK1 Suppresses RIPK1-Driven Apoptosis and Inflammation during 195 Development and in Aging[END_REF], therefore exhibiting the aged increased sensitivity to TBK1 dysregulation in the inhibition of RIPK1 activity and further giving a possible link with the late-onset ALS disease.

As a regulator of RIPK1 activity, a critical protein in the regulation of two critical PCD pathways, TBK1 appears as an important pro-survival protein.

In addition, TBK1 has been reported to directly regulate via phosphorylation the activity of the pro-survival AKT protein (Y-H [START_REF] Ou | TBK1 directly engages Akt/PKB survival signaling to support oncogenic transformation[END_REF]. AKT is an important member of the mTOR pathway, stimulating the formation of the mTORC1 complex and further cell proliferation.

These observations made a clear link between TBK1 activity and pro-survival signal.

III. TBK1 as an ALS-FTD associated gene:

A. Epidemiology and genetic features of TBK1 related ALS-FTD:

In 2015, two independent studies have exhibited a link between TBK1 mutations and ALS through a whole exome sequencing on a European background population (E.T. Cirulli et al., 2015) and a Germano-Swedish population (A. Freischmidt et al., 2015). Several studies have further confirmed the involvement of TBK1 in ALS-FTD (I. Gijselinck et al., 2015;L. Kelly et al., 2015). Approximately 0.5% of ALS cases are linked to a mutation in TBK1 (I. [START_REF] Lattante | Sqstm1 knock-down causes a locomotor phenotype ameliorated by rapamycin in a zebrafish model of ALS/FTLD[END_REF], even though this percentage is rather low, TBK1 remains one of the most common low-frequency mutations associated with ALS. Moreover, this percentage greatly increases when considering ALS patients presenting an FTD comorbidity with up to 10% of cases associated with TBK1 mutations (K. L Williams et al., 2015;[START_REF] Lattante | Sqstm1 knock-down causes a locomotor phenotype ameliorated by rapamycin in a zebrafish model of ALS/FTLD[END_REF]. TBK1

variants have also been associated with the sole development of FTD without any motor involvement (C.A.M. [START_REF] Koriath | The clinical, neuroanatomical, and neuropathologic phenotype of TBK1-associated frontotemporal dementia: A longitudinal case report[END_REF][START_REF] Portera-Cailliau | Wholegenome sequencing reveals important role for TBK1 and OPTN mutations in frontotemporal lobar degeneration without motor neuron disease[END_REF] Wilke et al., 2017;Koriath et al., 2016;[START_REF] Portera-Cailliau | Wholegenome sequencing reveals important role for TBK1 and OPTN mutations in frontotemporal lobar degeneration without motor neuron disease[END_REF]. Interestingly, these cases exhibit specific clinical features with mesencephalon and cerebellum injuries suggesting that TBK1 associated phenotypic spectrum could be extended beyond ALS and FTD and also include progressive supranuclear palsy-like and cerebellar syndromes (C. Wilke et al., 2017).

C. Modelling TBK1 related ALS-FTD:

In the context of ALS, 2 main in vitro models have been developed. First the generation of hiPSC derived TBK1-mutant motor neurons exhibiting a reduced viability and accumulation of p62 positive cytosolic inclusions due to the failure of the phagophore's elongation phase (A. [START_REF] Catanese | Retinoic acid worsens ATG10-dependent autophagy impairment in TBK1-mutant hiPSC-derived motoneurons through SQSTM1/p62 accumulation[END_REF]. The second in vitro model developed is a human embryonic stem cells (ESCs) derived motor neurons line characterized by the expression of a TBK1 loss-offunction mutant. This deficiency in TBK1 activity has been linked to an impaired autophagic flux with deficient endosomal maturation and lysosomal function associated with an increased cytoplasmic and insoluble TDP-43 accumulation (J. [START_REF] Hao | Loss of TBK1 activity leads to TDP-43 proteinopathy through lysosomal dysfunction in human motor neurons[END_REF]. Interestingly, this cellular model also revealed that the loss of activity of TBK1 in hMN was linked to an hyperexcitability state (J. [START_REF] Hao | Loss of TBK1 activity leads to TDP-43 proteinopathy through lysosomal dysfunction in human motor neurons[END_REF].

Murine models have been difficult to establish as Tbk1 deletion induces an early embryonic death at E14.5 due to massive liver degeneration and apoptosis (M. [START_REF] Bonnard | Deficiency of T2K leads to apoptotic liver degeneration and impaired NF-kappaB-dependent gene transcription[END_REF].

This suggests an important role of TBK1 in the development. However, loss of Tbk1 specifically in motor neurons did not lead to a neurodegenerative disease phenotype (V. [START_REF] Gerbino | The Loss of TBK1 Kinase Activity in Motor Neurons or in All Cell Types Differentially Impacts ALS Disease Progression in SOD1 Mice[END_REF]. Moreover, a sole hemizygous loss of function is insufficient to provoke any phenotype (D. [START_REF] Brenner | Heterozygous Tbk1 loss has opposing effects in early and late stages of ALS in mice[END_REF][START_REF] Gerbino | The Loss of TBK1 Kinase Activity in Motor Neurons or in All Cell Types Differentially Impacts ALS Disease Progression in SOD1 Mice[END_REF].

Surprisingly Tbk1 hemizygous loss in mice expressing ALS-linked SOD1 G93A mutant inversely modulates early and late already characterized phenotype: at the early stage, the heterozygous deletion impairs autophagy and accelerates the muscle denervation, whereas at later stages it alleviates microglial neuroinflammation and consequently decelerates the disease progression and extends the survival (D. [START_REF] Brenner | Heterozygous Tbk1 loss has opposing effects in early and late stages of ALS in mice[END_REF][START_REF] Gerbino | The Loss of TBK1 Kinase Activity in Motor Neurons or in All Cell Types Differentially Impacts ALS Disease Progression in SOD1 Mice[END_REF]. However, the conditional deletion of Tbk1 in SOD1 G93A motor neurons increases the SOD1 aggregation and accelerates early disease onset without affecting lifespan (V. [START_REF] Gerbino | The Loss of TBK1 Kinase Activity in Motor Neurons or in All Cell Types Differentially Impacts ALS Disease Progression in SOD1 Mice[END_REF]. These results unveil the importance of TBK1 in several pathways according to the cell types. More recently, a double mutant murine model bearing a heterozygous deletion of Tbk1 and expressing the ALS associated human mutant TDP-43 G298S has been developed. In this model, the monoallelic loss of Tbk1 aggravated the characterized muscle denervation but didn't impact the motor neuron loss or the reactive gliosis in the spinal cord (K. Sieverding et al., 2020). This further suggests a role of TBK1 in the neuromuscular synapse integrity.

In addition, two recent publications have developed two independent Tbk1 murine models which gave great insights on the understanding of TBK1 associated ALS pathology. In 2018, a study has implemented the already characterized and lethal Tbk1 KO murine model with a new kinase dead knock-in mutation in the Ripk1 gene (D. [START_REF] Xu | TBK1 Suppresses RIPK1-Driven Apoptosis and Inflammation during 195 Development and in Aging[END_REF]. These genetic manipulations resulted in the prevention of the lethality previously observed. This study therefore revealed an important role for TBK1 in the regulation of RIPK1 a crucial actor of the CASP8 apoptosis and necroptosis. Furthermore, D. Xu also reveals during his work that combined inhibition of both TBK1 and TAK1 (another kinase involved in TNF receptor response and NFκB's activation) were sufficient to induce RIPK1 activation and further neuroinflammation. Interestingly, this study has also reported that levels of TAK1 protein were decreased in an age-related manner. Therefore, these results describe a model for TBK1 associated development of ALS-FTD where a heterozygous loss of TBK1 is supported by the age-dependent decreased expression of TAK1 leading to the development of a late-onset disease such as ALS-FTD. The hypothesis of a double-hit model resulting in the induction of TBK1 associated ALS pathology was further supported by a second murine model published in 2022. In this recent landmark study, W. Shao has reported the interplay between two disease associated genes C9orf72 and Tbk1. Indeed, in a murine model bearing a GGGGCC repeat expansion within the C9orf72 gene, TBK1 was found to be sequestered in cytoplasmic inclusions induced by the expression of these repeats. Interestingly, the double knock-out of both C9orf72 and TBK1 worsens the condition of the mouse via endosomal disruption.

Therefore, these two studies lead to the hypothesis that the sole loss of TBK1 might not be sufficient to induce the pathology. Therefore, there is abundant emerging evidences that there is a combination of TBK1 dysregulation and other age-related factors or additional genetic alterations that could result in the development of motor neuron degeneration upon TBK1 mutations or reduced activity of this key factor.

Part III: metabolites dysregulation in ALS

Metabolites are an emerging topic in the ALS research as they could help the diagnosis of the patients and therefore apprehend early-dysregulations responsible for the development of the disease (H. [START_REF] Blasco | Understanding and managing metabolic dysfunction in Amyotrophic Lateral Sclerosis[END_REF].

In the following section, I will review some of the main observations that have been reported in the literature describing metabolomics alterations in ALS.

I. Hypermetabolism as a cardinal feature in ALS:

Alteration of the nutritional status and its subsequent loss of weight is a common symptom of ALS patients (S.T. [START_REF] Ngo | Loss of appetite is associated with a loss of weight and fat mass in patients with amyotrophic lateral sclerosis[END_REF]. The combination of two factors is suspected to be responsible for this feature: the loss of appetite and an increased resting energy expenditure (REE) (M. [START_REF] Kurihara | Factors Affecting Energy Metabolism and Prognosis in Patients with Amyotrophic Lateral Sclerosis[END_REF]; F.J. [START_REF] Steyn | Hypermetabolism in ALS is associated with greater functional decline and shorter survival[END_REF]. The REE represents the amount of energy required in a 24-hour period by the body in a resting state without activity. When an organism exhibits an increase in this metabolism indicator, we talk of the hypermetabolism state, which means that the body is in a higher demand of energy (L. [START_REF] Dupuis | Energy metabolism in amyotrophic lateral sclerosis[END_REF].

At the cellular level, hypermetabolic features are translated through dysregulated glucose levels, which are reported as reduced in both patients and animal models (T. Lerskiatiphanich et al., 2022;T.S. McDonald et al., 2020). Physiologically, glucose is transferred to the cells via the activity of the GLUT family receptors. Following glucose uptake, it is either stocked under the form of glycogen which constitute stocks for a later use or it undergoes a series of enzymatic reactions resulting in the production of ATP. Two hormones tightly regulate the glucose levels: insulin and glucagon. Insulin is secreted in the pancreas in response to high levels of glucose in the blood, promoting the glucose uptake by the peripheral tissues. In opposition, glucagon is secreted when plasma glucose levels are too low to induce the production of glucose via gluconeogenesis and glycogenolysis. Both the uptake of glucose and its regulation are suspected to induce the hypermetabolic state observed in ALS patients (T. Lerskiatiphanich et al., 2022).

Glucose uptake is reduced in CNS of both ALS patients and animal model (T. W Tefera et al., 2021). Supporting this observation, overexpression of the GLUT3 receptor in a drosophila TDP-43 ALS model, was reported to be beneficial and alleviates features of the TDP-43 proteinopathy (E. [START_REF] Manzo | Glycolysis upregulation is neuroprotective as a compensatory mechanism in ALS[END_REF]. In contrast, glucose uptake in peripheral tissue such as skeletal muscles, liver or adipose tissue was reported to be upregulated in the context of ALS (T. S. McDonald et al., 2021). Interestingly, muscle that relies mostly on anaerobic ATP production through glycolysis (glycolytic muscle fibres) have been reported to be decreased in murine SOD1 ALS model (L. [START_REF] Palamiuc | A metabolic switch toward lipid use in glycolytic muscle is an early pathologic event in a mouse model of amyotrophic lateral sclerosis[END_REF][START_REF] Scaricamazza | Skeletal-Muscle Metabolic Reprogramming in ALS-SOD1G93A Mice Predates Disease Onset and Is A Promising Therapeutic Target[END_REF]. This reduction in muscle anaerobic ATP production was linked to the impaired activity of phosphofructokinase, a key enzyme in the glucose metabolism (L. [START_REF] Palamiuc | A metabolic switch toward lipid use in glycolytic muscle is an early pathologic event in a mouse model of amyotrophic lateral sclerosis[END_REF].

With a combination of deficient glucose metabolism and mitochondrial damage, muscle could then switch to a lipidic aerobic ATP production (L. [START_REF] Palamiuc | A metabolic switch toward lipid use in glycolytic muscle is an early pathologic event in a mouse model of amyotrophic lateral sclerosis[END_REF][START_REF] Scaricamazza | Skeletal-Muscle Metabolic Reprogramming in ALS-SOD1G93A Mice Predates Disease Onset and Is A Promising Therapeutic Target[END_REF].

Moreover, glucose can be stored for later use under the form of glycogen by glycogenesis. In ALS patients and animal models, glycogen accumulation has been also reported (L. Palamiuc impairment in the glucose metabolism. Supporting this hypothesis, some enzymes of the TCA cycle were found to be downregulated, suggesting that a decreased utilization of the glycogen stocks could be linked to an impaired capacity of producing ATP via glucose.

Altogether, these data suggest an impaired ATP production via glucose degradation, leading to a metabolic switch towards ATP production via enhanced lipid degradation. This hypothesis is supported by the several dysregulations in lipid levels observed in the ALS context.

II. Lipidic dysregulation in ALS:

Lipids are involved in several cellular processes: they constitute a source of energy storage, they are involved in signalling, they constitute the structural components of every cell membranes, they can be used as protein recruitment platforms, and are substrates for posttranslational protein-lipid modifications (T. [START_REF] Harayama | Understanding the diversity of membrane lipid composition[END_REF]. Due to their high concentration in the CNS, lipids dysregulation has been associated with several neurodegenerative diseases and an alteration in their levels can be induced by multiple causes.

There are eight main families of lipids, however only four of those are associated with ALS, namely the sterols, the glycerolipids, the sphingolipids and the glycerophospholipids (J.M. [START_REF] Godoy-Corchuelo | Lipid Metabolic Alterations in the ALS-FTD Spectrum of Disorders[END_REF]. Although dysregulations are diverse amongst these pathways some of their members have been considered as potential biomarkers of ALS. The main representative of the sterol family is cholesterol. Cholesterol and its associated lipids have been extensively measured and reported to be increased in ALS patients (J.

Godoy

Abdel-Khalik et al., 2017; R.G. [START_REF] Cutler | Evidence that accumulation of ceramides and cholesterol esters mediates oxidative stressinduced death of motor neurons in amyotrophic lateral sclerosis[END_REF]. These observations were confirmed in ALS murine FUS and SOD1 mutant lines (T. [START_REF] Burg | Histone Deacetylase Inhibition Regulates Lipid Homeostasis in a Mouse Model of Amyotrophic Lateral Sclerosis[END_REF][START_REF] Chaves-Filho | Alterations in lipid metabolism of spinal cord linked to amyotrophic lateral sclerosis[END_REF].

Moreover, the reported dysregulation of enzymes from this metabolic pathway in patients (J.C. [START_REF] Dodge | Neutral Lipid Cacostasis Contributes to Disease Pathogenesis in Amyotrophic Lateral Sclerosis[END_REF] the increased level of one specific glycerolipid: arachidonic acid, seems to be shared in all types of ALS (H. [START_REF] Lee | Multi-omic analysis of selectively vulnerable motor neuron subtypes implicates altered lipid metabolism in ALS[END_REF][START_REF] Shibata | Increased expression and activation of cytosolic phospholipase A2 in the spinal cord of patients with sporadic amyotrophic lateral sclerosis[END_REF].

Sphingolipid metabolism has also been reported to be dysregulated in ALS patients' blood samples (J. [START_REF] Sol | Lipidomic traits of plasma and cerebrospinal fluid in amyotrophic lateral sclerosis correlate with disease progression[END_REF][START_REF] Area-Gomez | Lipidomics study of plasma from patients suggest that ALS and PLS are part of a continuum of motor neuron disorders[END_REF]. Interestingly, theses decreased levels are accentuated along the course of the disease, and therefore could constitute an optimal biomarker to supervise the disease progression. In opposition, brain and spinal cord tissues from ALS patients present increased levels of sphingolipids (R.G. what observed in the other lipid metabolisms, their levels are decreased in blood samples of ALS patients and increased in brain and spinal cord tissues. One specific glycerophospholipid in particular, phosphatidylcholine, has been described as a relevant biomarker of the disease as its dysregulation seems to be shared in all ALS subtypes (H. [START_REF] Blasco | Lipidomics Reveals Cerebrospinal-Fluid Signatures of ALS[END_REF][START_REF] Sol | Lipidomic traits of plasma and cerebrospinal fluid in amyotrophic lateral sclerosis correlate with disease progression[END_REF].

III. Kynurenine pathway and associate metabolisms:

A. The kynurenine pathway:

The kynurenine pathway is the main metabolism of tryptophan degradation. Products of the kynurenine pathway are further catabolized in several subsequent metabolites routes: the serotonin pathway, the immune related tetrahydrobiopterin (BH4) pathway, glycolysis, and the de novo nicotinamide adenine dinucleotide (NAD+) synthesis (V.X. Tan and G.J.

Guillemin, 2019). The kynurenine pathway is active in every cell but predominantly in liver and activated in monocytic cells during inflammation (M.P. [START_REF] Heyes | Different kynurenine pathway enzymes limit quinolinic acid formation by various human cell types[END_REF][START_REF] Jones | Expression of the Kynurenine Pathway in Human Peripheral Blood Mononuclear Cells: Implications for Inflammatory and Neurodegenerative Disease[END_REF]. Due to their capacity to cross the blood-brain barriers, some of the metabolites of this pathway such as kynurenine, 3-hydroxykynurenine, or tryptophane raised the interest as they could be used as a biomarker to supervise the progression of the disease [START_REF] Fukui | Blood-brain barrier transport of kynurenines: implications for brain synthesis and metabolism[END_REF].

Indeed, this metabolism pathway leads to the synthesis of several neuroactive toxic compounds such as quinolinic acid and 3-hydroxykynurenine (only in monocytes activated cells upon IFN-γ stimulation) (J.R. [START_REF] Moffett | Quinolinate as a Marker for Kynurenine Metabolite Formation and the Unresolved Question of NAD+ Synthesis During Inflammation and Infection[END_REF]. In vitro, these neurotoxic compounds have been shown to induce neurodegeneration and neuronal cell death through excitotoxicity, N-methyl-D-aspartate receptor antagonism, increased glutamate release, and the production of reactive oxygen species (L. [START_REF] Khaspekov | Neurotoxic effect induced by quinolinic acid in dissociated cell culture of mouse hippocampus[END_REF]; J.P. [START_REF] Kim | Quinolinate neurotoxicity in cortical cell culture[END_REF]S. Okuda et al., 2002;[START_REF] Leipnitz | In vitro evidence for an antioxidant role of 3-hydroxykynurenine and 3-hydroxyanthranilic acid in the brain[END_REF]P. Pierozan et al., 2014; J.-M. [START_REF] Lee | Involvement of quinolinic acid in the neuropathogenesis of amyotrophic lateral sclerosis[END_REF]. Interestingly, all these processes overlap with mechanisms of neurodegeneration in ALS. Moreover, this pathway also leads to the production of neuroprotective compounds such as respectively kynurenic acid and picolinic acid by astrocytes and neurons respectively (R.S. [START_REF] Grant | The physiological action of picolinic Acid in the human brain[END_REF][START_REF] Klein | The neuroprotector kynurenic acid increases neuronal cell survival through neprilysin induction[END_REF].

In the context of ALS, increased levels of quinolinic acid, tryptophan, kynurenin and decreased levels of picolinic acid were reported in both CSF and patients' plasma (Y. Chen et al., 2010; J.-M. [START_REF] Lee | Involvement of quinolinic acid in the neuropathogenesis of amyotrophic lateral sclerosis[END_REF]. These increased levels of kynurenine associated metabolites correlate with the increased neuroinflammation observed along the progression of the disease and are suspected to drive neurodegeneration. Kynurenine dysregulation has already been identified to regulate inflammation (G.J. Guillemin et al., 2003), therefore the modulation of this metabolism is considered as an interesting therapeutic target (K.R. Jacobs and D.B. Lovejoy, 2018). In the nervous system, dysregulation in kynurenine pathway and increased levels of quinolinic acid have been associated with the development of several pathologies such as schizophrenia (M.E. [START_REF] Kegel | Imbalanced kynurenine pathway in schizophrenia[END_REF][START_REF] Cathomas | Quinolinic acid is associated with cognitive deficits in schizophrenia but not major depressive disorder[END_REF],

Parkinson disease (P.A. Lewitt et al., 2013;[START_REF] Venkatesan | Kynurenine pathway in Parkinson's disease-An update[END_REF], multiple sclerosis (C.K. [START_REF] Lim | Kynurenine pathway metabolomics predicts and provides mechanistic insight into multiple sclerosis progression[END_REF], Alzheimer disease (Z. [START_REF] Hartai | Decreased serum and red blood cell kynurenic acid levels in Alzheimer's disease[END_REF]; G.J. Guillemin et al., 2013) and Huntington (N. [START_REF] Stoy | Tryptophan metabolism and oxidative stress in patients with Huntington's disease[END_REF]; P.R. [START_REF] Sanberg | The quinolinic acid model of Huntington's disease: locomotor abnormalities[END_REF]. Therefore, its link to a neurodegenerative disease such as ALS is not surprising. Although this confirmed the use of biomarkers derived from the kynurenine metabolism to accurately quantify the progression of the disease, it also prevents their use as diagnosis biomarkers as they are not specific to ALS pathophysiology.

Besides the kynurenine pathway, it is interesting to look at the impact of such dysregulation on metabolism deriving from the tryptophan catabolism, including serotonin and NAD+ metabolism.

B. Serotonin metabolism:

Due to its association with the regulation of motor neuron excitability and the subsequent global control of motor behaviour and its involvement in neuronal maturation and aging (A. [START_REF] Barreiro-Iglesias | Serotonin Promotes Development and Regeneration of Spinal Motor Neurons in Zebrafish[END_REF][START_REF] Sandyk | Serotonergic mechanisms in amyotrophic lateral sclerosis[END_REF], serotonin have been considered as a therapeutic target in the context of ALS for a long time. Moreover, it was observed that the levels of serotonin were reported to be reduced in ALS patients (L. [START_REF] Dupuis | Platelet serotonin level predicts survival in amyotrophic lateral sclerosis[END_REF].

Interestingly, MN that are the most affected in ALS pathology are also the most densely innervated by serotonin neurons. In opposition, the MN that are the most resistant to ALSrelated neurodegeneration receive smaller inputs from the serotonin neurons (R. [START_REF] Sandyk | Serotonergic mechanisms in amyotrophic lateral sclerosis[END_REF]. Physiologically, serotonin interacts at the synapse with 5-HT1 receptors which are involved in the inhibition of the glutamate excretion (A. [START_REF] Barreiro-Iglesias | Serotonin Promotes Development and Regeneration of Spinal Motor Neurons in Zebrafish[END_REF].

Therefore, a reduced level of serotonin could easily be linked to an increased synaptic glutamate release and its subsequent neurotoxicity. For these reasons, exogenous input of serotonin has been reported to be beneficial on in vivo animal ALS models (B. J Turner et al., 2003;[START_REF] He | Distribution Features and Potential Effects of Serotonin in the Cerebrum of SOD1 G93A Transgenic Mice[END_REF]. Furthermore, melatonin, the major downstream metabolite of serotonin metabolism, due to its ability to regulate glutamate-induced neurotoxicity, has been associated with neuroprotection in ALS patients and in ALS animal model. (J.H. [START_REF] Weishaupt | Reduced oxidative damage in ALS by high-dose enteral melatonin treatment[END_REF].

C. NAD+ metabolism:

Another downstream metabolism of the kynurenine pathway is the nicotinamide metabolism. Nicotinamide adenine dinucleotide (NAD), its central metabolite, is a molecule involved in many cellular reactions either as a proton receiver when oxidized (NAD+) or as a donor when reduced (NADH). Due to its central place in the cellular metabolism, NAD is involved in the clearance of the oxidative stress, the genomic stability and expression, RNA processing, and immunity/inflammation. Depletion of the NAD levels were observed in in vivo models and ALS patients (E. [START_REF] Blacher | Potential roles of gut microbiome and metabolites in modulating ALS in mice[END_REF][START_REF] Harlan | Evaluation of the NAD+ biosynthetic pathway in ALS patients and effect of modulating NAD+ levels in hSOD1-linked ALS mouse models[END_REF]. When looking at the nicotinamide metabolism (Fig. 12), NAD is synthesized from 3 distinct pathways: 1) the kynurenine pathway catabolizing the degradation of tryptophan into NAD; 2) the Preiss-Handler pathway catabolizing the degradation of nicotinic acid into NAD and 3) the salvage pathway. NAD is used in many physiologic reactions and in many of those result the production of NAM (Fig. 12). NAM can be then retransformed into NAD through the salvage pathway via an intermediate called nicotinamide mononucleotide (NMN). NMN can also be formed through the conversion of another molecule called nicotinamide riboside (NR) via the action of nicotinamide riboside kinase.

Several hypotheses have been emitted to explain these reduced levels. First as mentioned previously, in physiological conditions, quinolinic acid is degraded into nicotinic acid, and ultimately NAD+ (Fig. 10). However, in a neuroinflammatory state, quinolinic acid is degraded at a much lower rate and is instead released by activated migroglia (J.A. [START_REF] Fifita | Genetic Analysis of Tryptophan Metabolism Genes in Sporadic Amyotrophic Lateral Sclerosis[END_REF]. Therefore, the NAD levels could be depleted in ALS context due to impaired de novo synthesis associated with excessive release of quinolinic acid induced by the neuroinflammation. Besides the novo synthesis, the salvage pathway has also been suspected to be responsible for the depleted level of NAD in ALS context. Indeed, most NAD degradative reactions lead to the formation of nicotinamide, which is subsequently retransformed into nicotinamide mononucleotide (NMN), a direct NAD precursor, via the activity of nicotinamide phosphoribosyltransferase (NAMPT) (Fig 10). NAMPT exists in an intracellular and extracellular form (X. [START_REF] Wang | Deletion of Nampt in Projection Neurons of Adult Mice Leads to Motor Dysfunction, Neurodegeneration, and Death[END_REF]. Interestingly, intracellular NAMPT levels were reported to be downregulated in ALS patients in opposition to the extracellular form which exhibits increased levels (X. [START_REF] Wang | Deletion of Nampt in Projection Neurons of Adult Mice Leads to Motor Dysfunction, Neurodegeneration, and Death[END_REF]. Moreover, neuronal Nampt deletion was reported to induce motor reduced capacity and death in a murine model (X. [START_REF] Wang | Deletion of Nampt in Projection Neurons of Adult Mice Leads to Motor Dysfunction, Neurodegeneration, and Death[END_REF]. Supporting this hypothesis, nicotinamide riboside (NR) supplementation, a direct precursor of NMN without NAMPT involvement, was shown to be beneficial SOD1 G93A ALS mice model (B.A. [START_REF] Harlan | Evaluation of the NAD+ biosynthetic pathway in ALS patients and effect of modulating NAD+ levels in hSOD1-linked ALS mouse models[END_REF]E. Obrador et al., 2021). Moreover, a clinical trial involving NR supplementation in ALS patients is ongoing.

The final hypothesis concerns the indirect link of NAD with axonal degeneration via the sterile alpha and TIR motif-constraining 1 (SARM1) hydrolase activity. This protein is involved in the Wallerian axonal degeneration upon lesion through the degradation of NAD into nicotinamide (J. [START_REF] Gerdts | SARM1 activation triggers axon degeneration locally via NAD+ destruction[END_REF]. The involvement of this process in ALS pathophysiology was put in evidence in a TDP-43 mutant murine model where Sarm1 deletion prevents MN degeneration (M.A. [START_REF] White | Sarm1 deletion suppresses TDP-43-linked motor neuron degeneration and cortical spine loss[END_REF]. Therefore, NAD level could be depleted due SARM1 degradation upon axonal degradation. The results section describes the development of two zebrafish models targeting the expression of the TBK1 orthologue, Tbk1.

-

I. Background and state of the art-TBK1 in ALS

ALS is fatal neurodegenerative disease characterized by the progressive degeneration of both the upper and the lower motor neurons. The pathogenicity is characterized by the death of MN leading to the denervation of muscles. At the molecular level, multiple pathways are involved in the development of ALS including autophagy dysregulation, mitochondrial dysfunction, inflammation, axonal transport and oxidative stress (M.C. [START_REF] Kiernan | Amyotrophic lateral sclerosis[END_REF]. The main neuropathological hallmark of the disease is the presence and the accumulation of TDP-43 and ubiquitin positive inclusions within the motor neurons (M. [START_REF] Neumann | Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis[END_REF][START_REF] Blokhuis | Protein aggregation in amyotrophic lateral sclerosis[END_REF]. In 2015, two independent studies have demonstrated causative mutations of TBK1 in ALS patients through a whole exome sequencing (E.T. Cirulli et al., 2015) (A. Freischmidt et al., 2015). Subsequent studies have confirmed TBK1 mutations as a major cause of ALS as well as in another related neurodegenerative disease, FTD (K. L Williams et al., 2015;[START_REF] Portera-Cailliau | Wholegenome sequencing reveals important role for TBK1 and OPTN mutations in frontotemporal lobar degeneration without motor neuron disease[END_REF][START_REF] Lattante | Sqstm1 knock-down causes a locomotor phenotype ameliorated by rapamycin in a zebrafish model of ALS/FTLD[END_REF]. A study on a French population in particular have revealed that TBK1 genetic alterations occurred more frequently in patients presenting an ALS-FTD comorbidity (10.8%) than those with ALS alone (0.5%) (I. [START_REF] Lattante | Sqstm1 knock-down causes a locomotor phenotype ameliorated by rapamycin in a zebrafish model of ALS/FTLD[END_REF]. Nonsense and/or missenses mutations associated to ALS are located throughout the TBK1 genetic sequence, giving rise to a loss-of-function hypothesis thought to be responsible for the disease. Pathological studies of TBK1 mutation carriers show accumulation of ubiquitinated aggregates in their motor neurons and the glia in several area of the CNS (especially in the spinal cord and the motor cortex) as well as fronto-temporal lobar degeneration (S. Van Mossevelde et al., 2016;C.A.M Koriath et al., 2017;[START_REF] Weinreich | Neuropathological characterization of a novel TANK binding kinase (TBK1) gene loss of function mutation associated with amyotrophic lateral sclerosis[END_REF].

TBK1 is a serine-threonine kinase known to be an activator of the innate immunity through the induction of type I interferon (IFN-I) and the production of inflammatory cytokines via the regulation of NFκB [START_REF] Yum | TBK1 recruitment to STING activates both IRF3 and NF-?B that mediate immune defense against tumors and viral infections[END_REF][START_REF] Ahmad | Human TBK1: A Gatekeeper of Neuroinflammation[END_REF]. TBK1 has also been shown

to regulate the inflammatory TNF-mediated cell death through inactivation of the RIPK1 protein (D. [START_REF] Xu | TBK1 Suppresses RIPK1-Driven Apoptosis and Inflammation during 195 Development and in Aging[END_REF][START_REF] Lafont | TBK1 and IKK? prevent TNF-induced cell death by RIPK1 phosphorylation[END_REF]. In ALS patients, the loss of TBK1 results in lower but sufficient IFN-I induction via compensatory pathways, but high autoinflammation due to the TNF-induced cell death caused by dysregulated RIPK1 (J. [START_REF] Taft | Human TBK1 deficiency leads to autoinflammation driven by TNFinduced cell death[END_REF].

Moreover, in the context of ALS, TBK1 role in the protein degradation pathway, autophagy, has been thoroughly investigated. Indeed, TBK1 is shown to control the early stages of autophagy at several levels (J.A. [START_REF] Oakes | TBK1: a new player in ALS linking autophagy and neuroinflammation[END_REF] and has been shown to be crucial in the clearance of aggregates (R.M. Hedge et al., 2020;[START_REF] Raya | A36 Neuropathological function of the TANK1-binding kinase 1 (TBK1) in mouse models of Huntington's disease[END_REF]. To better elucidate the functional relevance of TBK1 in ALS animal models have been developed, including murine model lacking the expression of Tbk1 (M. [START_REF] Bonnard | Deficiency of T2K leads to apoptotic liver degeneration and impaired NF-kappaB-dependent gene transcription[END_REF]. However, the study of this model was challenging because of a massive liver degeneration leading to an early embryonic death at E14.5. TBK1 was reported to control CASP8-related apoptosis and necroptosis activations via regulation of RIPK1 function (E. [START_REF] Lafont | TBK1 and IKK? prevent TNF-induced cell death by RIPK1 phosphorylation[END_REF]M. Delhase et al., 2011). This observation lead to hypothesis that the lethality observed in the TBK1-/murine model was associated with the activation of cellular death pathways. Indeed, in 2018, a study has implemented a novel model by combining the deletion of TBK1 with a RIPK1 kinase-dead knock-in mutation (D. [START_REF] Xu | TBK1 Suppresses RIPK1-Driven Apoptosis and Inflammation during 195 Development and in Aging[END_REF]. Importantly, the authors prevented lethality in these double knockouts as compared to TBK1 deletion mutant. Moreover, combined inhibition of both Tbk1 and Tak1 (a gene coding for another kinase involved in TNF receptor response and NFκB activation) were sufficient to induce RIPK1 activation (D. [START_REF] Xu | TBK1 Suppresses RIPK1-Driven Apoptosis and Inflammation during 195 Development and in Aging[END_REF]. Interestingly, this study has also reported that the levels of TAK1 protein were decreasing in an age-related manner, therefore suggesting a convincing model for TBK1 associated development of ALS-FTD where the heterozygous loss of TBK1 is supported by the age-dependent decreased expression of TAK1 leading to the development of a late-onset disease such as ALS-FTD.

Further defining the potential role of TBK1 in the development of ALS-FTD, a recent study (W. [START_REF] Shao | Two FTD-ALS genes converge on the endosomal pathway to induce TDP-43 pathology and degeneration[END_REF] has reported the functional connection between the disease associated C9orf72 and TBK1 genes. Indeed, TBK1 was found to be sequestered within the cytoplasmic inclusions containing DPRs translated via the expression of C9orf72 hexanucleotide repeats in a mouse model. Moreover, mutation in Tbk1 worsened the condition of the murine model via endosomal disruption (W. [START_REF] Shao | Two FTD-ALS genes converge on the endosomal pathway to induce TDP-43 pathology and degeneration[END_REF].

Using the powerful vertebrate model, zebrafish, I targeted the unique TBK1 orthologue, tbk1 in order to define the pathogenic consequences related to tbk1 loss of expression to better understand TBK1 in the context of ALS-FTD pathophysiology and to open therapeutic strategies for these disorders.

II. Phenotypic characterization of a new tbk1 KD zebrafish model 2.1 Generation of the tbk1 KD zebrafish model

In order to disturb tbk1 expression in zebrafish, we took advantages of the high homology with its human orthologs. Indeed, genomic sequences between the TBK1 genes of these two species are shared at 63.16%. Consequently, the two encoded proteins also share a high homology with up to 71.39% of nucleotide identity and a conserved four domains architecture.

In order to study the depletion of Tbk1 in the zebrafish model, I initially targeted the expression of tbk1, as a hemizygous loss of expression of tbk1 has been reported in mutant TBK1 ALS-FTD patients (A. Freischmidt et al., 2015;E.T. Cirulli et al., 2015). To induce this partial decrease in tbk1 expression, we generated an antisense morpholino oligonucleotide (AMO) complementary to the mRNA start codon sequence of tbk1. An AMO is a stabilized RNA complementary to a target mRNA which sterically prevent the binding of ribosome and the subsequent translation of the gene without noticeable degradation of the target mRNA.

However, this technique is limited by the progressive degradation of the AMO in the developing zebrafish embryo. Indeed, it is commonly accepted that 4 days post injection, the concentration in AMO is insufficient to alter the expression of the gene anymore (D.Y. [START_REF] Stainier | Guidelines for morpholino use in zebrafish[END_REF], restricting the analysis to the first 4 days of development. For these reasons, the analyses of the AMO injected fish have been restricted in this study mainly to the 2 nd day post fertilization (dpf). During this period, the development of the motor neurons and their migration has been completed as secondary motor neurons enter the myotome around 34 hours post fertilization (hpf) (P.Z. [START_REF] Myers | Development and axonal outgrowth of identified motoneurons in the zebrafish[END_REF]. As a control of injection, a control AMO -bearing a sequence that does not target any sequence in the zebrafish transcriptome -has been used. This control condition will be called further on the "standard control" or "mismatch condition".

After quantifying the decrease in the expression of the tbk1 gene by western blot (Fig. 1), I analysed the possible impact on the swimming capacity of the larvae, locomotor alterations being the main clinical hallmark of the loss of TBK1 expression in patients (I. J Swift et al., 2021).

tbk1 reduced expression leads to altered larvae swimming capacity

In order to assess the impact of Tbk1 depletion on the locomotor activity of the zebrafish larvae, we performed a touch escape evoked response test at 2dpf. This exercise consists in inducing an escape response through non-invasive stimulation of the larva's tail (T.E. [START_REF] Sztal | Using Touchevoked Response and Locomotion Assays to Assess Muscle Performance and Function in Zebrafish[END_REF][START_REF] Ciura | Loss of function of C9orf72 causes motor deficits in a zebrafish model of amyotrophic lateral sclerosis[END_REF]; A-R. [START_REF] Bourefis | Functional characterization of a FUS mutant zebrafish line as a novel genetic model for ALS[END_REF]. The trajectory of the larva's response is recorded, the distance of the trail and the velocity are calculated. Theoretically, we expect that a modification in the locomotor system's responses due to either alteration of the muscle fibres or spinal cord integrity could result in a reduced velocity or distance swum [START_REF] Li | Defective sarcomere organization and reduced larval locomotion and fish survival in slow muscle heavy chain 1 (smyhc1) mutants[END_REF][START_REF] Dehnisch Ellström | Spinal cord injury in zebrafish induced by near-infrared femtosecond laser 137 pulses[END_REF].

Using this approach, we observed that Tbk1 KD induces an alteration of the locomotor capacity of the larvae at 2dpf compared to the larvae injected with the control AMO (Fig. 2).

Indeed, the Tbk1 depleted larvae exhibit shorter and slower swimming tracts as compared to the control larvae. This alteration of the locomotor capacity was even more marked on the velocity as the tbk1 depleted fish swum two-fold slower than the controls (Fig. 2).

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To our knowledge, this is the first study demonstrating that the sole heterozygous loss of tbk1 leads to motor defects in an animal model.

B C D

After characterizing the impact of the partial loss of tbk1 in the 2dpf larva at the behavioural scale, I determined whether tbk1 KD leads to alterations in motor neurons as well as their innervating targets, through the neuromuscular junction (NMJ).

Defects in locomotion are associated with spinal cord alteration upon tbk1 KD

To define whether impaired mobility could be linked to deficits at the level of the neuromuscular junction, I performed a detailed analysis in the zebrafish larvae. As the death of motor neurons being the main hallmark of the ALS pathogenicity (M.C. [START_REF] Kiernan | Amyotrophic lateral sclerosis[END_REF], I investigated the integrity of the spinal cord using a transgenic zebrafish line bearing an RFP gene under the regulation of a specific motor neuron promoter: mnx (S. [START_REF] Knafo | Bioluminescence Monitoring of Neuronal Activity in Freely Moving Zebrafish Larvae[END_REF]. Therefore, I assessed the axonal projections of the motor neurons upon depletion of Tbk1 expression and measured the axonal length. As the size of the larvae can be variable, I rationalized the length of the axons with the thickness of the spinal cord. Interestingly, I did not observe any difference in the size of the Tbk1 depleted larvae compared to the controls (Fig. 3) and did not associate any axonal alterations from the spinal motor neuron projections due to the partial loss of Tbk1. This suggests that the axonal projections of MN in this model appear to develop correctly upon depletion of Tbk1.

Using the double transgenic Tg(mnx1:gal4)/Tg(UAS:RFP) zebrafish line, I also quantified the number of MN of the tbk1 KD larvae and compared it with the mismatch condition.

Interestingly, we quantified a severe reduction in the number of MN upon depletion of Tbk1, with in average 15 MN per somite in the Tbk1 depleted larvae in opposition to the average 20 MN observable in the control larvae (Fig. 3). In order to assess the impact on the muscles, we took advantage of the transparency of zebrafish larvae. Indeed, it is possible to observe and measure the muscular fibres without any labelling under polarized light. However, after quantification, we didn't observe any differences in the length of the muscular fibres upon depletion of Tbk1 compared to the mismatch condition (Fig. 5). This suggests that the reduced number of motor neurons observed in the tbk1 KD condition does not impact the integrity of the muscular fibres.

To determine the Tbk1 role upon motor neuron development, I mounted whole larvae at earlier developmental stages: 23 and 32 hpf and quantify the number of spinal MN using the double transgenic Tg(mnx1:gal4)/Tg(UAS:RFP) zebrafish line. I selected these specific times A B C 76 point as they correspond to both the initiation and the termination of the secondary MN migration (P.Z. [START_REF] Myers | Development and axonal outgrowth of identified motoneurons in the zebrafish[END_REF]. Interestingly, no significant differences in the number of MN was observed at these two developmental stages (Fig. 4). Therefore, spinal MN loss observed at 2dpf was likely not due to a developmental delay, but to the activation of neurodegenerative pathways. In order to study Tbk1 depletion in a more extended time frame and through another genetic methodology, we developed a new tbk1 knock out model using the CRISPR/Cas9 system allowing a longer and more complete phenotypic analysis.

III. Phenotypic characterization of a new tbk1 KO zebrafish model

The first results obtained using AMO shedding light on the importance of studying the total loss of tbk1 expression in the zebrafish, I designed the strategy to obtain a tbk1 KO zebrafish line.

Generation of the tbk1 KO zebrafish model

Generation of the tbk1 KO zebrafish line was performed using the CRISPR technology (M. [START_REF] Jinek | A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity[END_REF]. Our aim was to take advantage of the high percentage of identity between the human and the zebrafish genomic and protein sequences (respectively 60 and 70%) to induce a deletion within the tbk1 kinase domain sequence. We targeted this region for two reasons: 1) the kinase domain of Tbk1 bears its catalytic activity and therefore its deletion would suppress its functional role; 2) Tbk1's kinase domain is encoded by the 5 first exons of the gene, therefore inducing a deletion within increasing the chance of generating an early stop codon or a frame shift impacting the whole transcript. To optimize the cutting efficiency of the Cas9, we adopted a strategy combining two sgRNA at the same time to induce a double strand break at two different loci and therefore generating the deletion of a long and significant segment of the kinase domain-coding region. This technique has been very efficient in inducing deletion mutants [START_REF] Wu | A Rapid Method for Directed Gene Knockout for Screening in G0 Zebrafish[END_REF].

We designed the sgRNA using the CHOPCHOP software (K. [START_REF] Labun | CHOPCHOP v3: expanding the CRISPR web toolbox beyond genome editing[END_REF] and selected the top scored sequences to create 2 pairs of guides targeting respectively the exons 1 and 2 and the exons 4 and 5 of the tbk1 gene, both creating a 700bp deletion (Fig. 6). Targeting exon 1-2 and 4-5 was assessed by genotyping individual embryos using a set of primers in this region. Importantly, tbk1 deletion efficiency was quite high with up to 70% of tbk1 KO homozygous larvae when genotyped (Fig. 6). The efficiency was further confirmed by a control injection of sgRNA targeting the tyrosinase gene, which is responsible for the pigmentation of the zebrafish (E. [START_REF] Camp | Tyrosinase gene expression in zebrafish embryos[END_REF], assessing the efficacity of this procedure as early as the first day post fertilization.

However, as we are going to show later in this thesis, similarly to what is observed in the murine model, the homozygous deletion of tbk1 leads to lethality early in the development, around 9dpf. This lethality associated with the loss of tbk1 makes generating a KO fish line impossible. Considering the efficiency being high enough at the first generation, we decided to pursue the study with larvae directly injected with the Cas9 mix at one cell egg. This strategy doesn't allow to control the nature of the cut induced in tbk1 but as we are looking to silence the gene, the certainty of having 70% of homozygous tbk1 KO fish is appropriate for the following analyses performed in this study. Furthermore, this strategy allows to define the role of TBK1 early in motor neuron development.

tbk1 KO leads to altered larvae swimming capacity

Similarly to the characterization of the tbk1 AMO model, I initially assessed the locomotor capacity of the fish at 2dpf with a TEER test. Interestingly, the larvae carrying the tbk1 deletion did not exhibit any significant difference in the distance swam or their velocity upon stimulation of the evoked response (Fig. 7). Following this analysis, I assessed the spontaneous swimming capacity at later developmental stages by performing an analysis using the zebrabox module (viewpoint technology ©). This device automatically tracks the trajectory of several larvae in a 96 or 48 wells plate on a defined time period, and gives the opportunity to implement the analysis with a sound or a light stimulus to measure an escape response. The protocol used in this study was based on a 40 minutes period of freeswimming capacity analysis followed by 2 sound stimuli of 450Hz and 80 dB separated by a 10 minutes rest period where the escape responses were measured (Fig. 8). At 5 and 6 dpf, we quantified a significant decrease in the locomotor activity of the larvae associated with the loss of tbk1 expression (Fig. 8). Indeed, the distance swum in the escape response induced by a sound stimulus was decreased by half in both KO larvae condition compared with the non-injected ones. This decreased was even more striking when analysing the freeswimming capacity, the larvae were almost immobilized when not exposed to any stimuli (around 25cm swum during 40 minutes whereas the non-injected larvae were travelling almost 100cm).

81 From the difference in the timing of apparition of the locomotor phenotype between the tbk1 AMO and the tbk1 KO CRISPR models, we can generate two hypotheses:

First, one difference between the two models is the expression of tbk1 maternal mRNA in the first phases of development. Indeed, the tbk1 AMO is targeting mRNA, this mode of action prevents as well the expression of mRNA transmitted by the mother and deposited in the egg during oocytes maturation, however the CRISPR technology targeting the larvae DNA is not impacting the expression of maternal mRNA. Therefore, this change in the phenotype apparition is suggesting a possible role for Tbk1 during the 10 first hours of development.

This will be assessed by qPCR at various stages of development. Importantly, this period is critical for the correct development of the locomotor system as the three primary motor neurons are being generated during the ninth and the tenth hours post fertilization.

Secondly, this difference in the timing of apparition could be induced by phenomenon of genetic compensation induced by the complete deletion of tbk1. Indeed, it is known that some TBK1 functions can be compensated by other proteins, for instance, TBK1 dependent activation of NFκB is rescued by IKKε when TBK1 is silenced (K.R. [START_REF] Balka | TBK1 and IKK? Act Redundantly to Mediate STING-Induced NF-?B Responses in Myeloid Cells[END_REF]. This compensation could be dependent of a threshold in TBK1 expression, explaining why it doesn't happen in the AMO injected larvae.

To answer these two hypotheses, unbiased analysis of the metabolic dysregulations as well as proteomic analysis to define pathophysiological alterations upon Tbk1 depletion locomotor phenotype at 5 and 6 dpf were performed.

tbk1 KO is lethal early in embryogenesis

After assessing the impact on the locomotor capacity of the tbk1 KO larvae, we also assessed whether there were other metabolic features during larval development. Interestingly, gross morphological analysis of larvae at 8dpf did not reveal any reduced length in the tbk1 KO larvae as compared to control conditions (Fig. 9). Interestingly, at 9dpf, we quantified a mortality rate of 90% in the tbk1 KO larvae compared to the 30% rate in non-injected or larvae injected with a control sgRNA construction targeting the tyrosinase gene, responsible for the pigmentation (Fig. 10). Therefore, we performed an unbiased analysis to define altered pathways that lead to increased mortality in the Tbk1 depleted zebrafish larvae.

IV. Metabolomic characterization of the tbk1 KO zebrafish model

In order to investigate the molecular dysregulations responsible for the apparition of the two phenotypes observed upon deletion of tbk1, two metabolomic analyses were performed at 6dpf, a developmental stage characterized by the deficit in locomotion and at 8 dpf a day preceding the initiation of the larval mortality.

Metabolism is a term that encompasses every cellular reactions occurring in a living organism to sustain life. Metabolites are small molecules involved either as an intermediate or as an end product of the metabolism. Dysregulations in the level of these metabolites reflect alternations of specific cellular pathways, unveiling the mechanistic insight of a pathology. Moreover, metabolomics have been of great interest lately to develop new strategies of diagnosis based on the surveillance of metabolites levels used as biomarkers of the disease progression. Therefore, analysing the metabolite profile, or metabolomics, of our tbk1 KO model could help explain the apparition of these two phenotypes (locomotor deficits and lethality) and the presence of biomarkers observed in patients could also further confirm our ALS model.

Metabolomic profile of the tbk1 KO larvae at day 6

After a first round of analysis comparing the metabolites level between tbk1 KO, WT and tyrosinase KO larvae, it became clear that even though they don't present any phenotype in exception with the loss of their pigmentation, the tyrosinase KO larvae are characterized by a specific metabolomic profile, different from the non-injected, that could mask metabolic alterations that are aberrant in the tbk1 KO model. Therefore, we pursued the analysis using only WT larvae as a control condition, and included the analysis on both sgRNA exons 1-2 KO and exons 4-5 KO conditions to define metabolic alterations that are present upon tbk1 loss of function.

After measuring the metabolites through mass spectrometry, we start looking common dysregulated metabolites in the tbk1 1-2 exon KO and 4-5 exon KO models. We implemented a list of 131 identified metabolites and their respective concentration in the samples on the metaboanalyst © software (Z. [START_REF] Pang | MetaboAnalyst 5.0: narrowing the gap between raw spectra and functional insights[END_REF]. At 6dpf, we identified mainly two metabolites that were dysregulated with a q-value under 5% in both of the conditions: nicotinamide (NAM) and glycerol-3-phosphate (G3P) (Fig. 10).

As an intermediate of the glycolysis pathway, G3P depletion in our model could indicate an impaired glucose metabolism. However, the level of glucose or pyruvate don't seem to be impacted by the loss of tbk1 in our analysis so it is complicated to conclude anything through the sole loss of this compound.

Depletion of the NAD levels was observed in in vivo model and in ALS patients (E. [START_REF] Blacher | Potential roles of gut microbiome and metabolites in modulating ALS in mice[END_REF][START_REF] Harlan | Evaluation of the NAD+ biosynthetic pathway in ALS patients and effect of modulating NAD+ levels in hSOD1-linked ALS mouse models[END_REF]. Increased levels of NAM, its direct precursor, suggests a similar decreased pool of NAD in our model. Moreover, when performing the analysing while pooling the two tbk1 KO conditions versus the non-injected larvae, a new metabolite appears to be significantly increased upon deletion of tbk1, the quinolinic acid (QUIN);

another precursor of NAD. Interestingly, in the brain, QUIN is only synthesized by microglia or astrocytes, therefore, its increased levels unveiled a possible implication of the immune 87 cells of the brain in the development of the phenotype observed in the context of tbk1 deletion. An enrichment analysis on the metabolomics results was performed to identify dysregulated pathways. Out this analysis, we identified one common dysregulated metabolic pathway in both of our models: the nicotinate and nicotinamide metabolism (Fig. 12). In this study, five identified metabolites are involved in this metabolic pathway and out of these 5 compounds, 3 are characterized by a common significative change in their levels (pvalue > 0.05) upon tbk1 deletion in the two models compared with non-injected control larvae: NAM and QUIN present increased levels (as shown in the previous section) in opposition to NAD which exhibit lower levels. In addition, the tbk1 exon 1-2 KO model exhibit a significantly decreased level of L-aspartate (a precursor of QUIN) but this decrease is not shared with the tbk1 exon 4-5 condition. The last compound identified belonging to the NAD metabolism is NADP, the phosphorylated form of NAD, however its level is not impacted by tbk1 loss.

Incubation with nicotinamide riboside rescue tbk1 KO larvae's altered locomotion

As the NAD salvage pathway was significantly impacted, through increased NAM levels and the decreased NAD pool in our model, this suggests an impaired salvage of NAD. With this hypothesis, I initiated a treatment at early developmental stage through incubation of the larvae with a precursor of NAD: the nicotinamide riboside (NR). NR has been shown to reduce the degeneration of spinal MN and to reduce the characteristic neuroinflammation observed in the hSOD1 G93A mouse model (E. Obrador et al., 2021a). Moreover, NR in combination with pterostilbene, an antioxidant compound, has been proposed as a therapeutic treatment for ALS (E. Obrador et al., 2021a) and clinical trials of using this compound mix are ongoing.

Following a toxicity analysis of NR in non-injected larvae (data not shown) we used the concentration of 10µM diluted in dimethyl sulfoxide (DMSO). Our control condition was incubation with DMSO only. Because the 9 days long incubation in NR was too deleterious for the larvae (data not shown), we designed a protocol based on alternating incubation of 10µM NR incubation with periodic NR-free water. The larvae were kept in NR free water until 3dpf and then incubated for a day in 10µM NR. This first treatment was followed by a NR-free water incubation for a day and finally larvae were kept in 10µM NR until the end of the experiment (Fig. 14). During this period, I assessed the locomotor ability of the larvae at day 5 and 6. Importantly, I observed a striking rescue of the locomotor activity of the tbk1 KO larvae incubated with 10µM NR in comparison with those incubated in DMSO only (Fig. 13). Indeed, the total distance swum during the test was no longer significatively decreased compared to non-injected WT control larvae with NR supplementation. Strikingly, at 6dpf, the distance swum by the tbk1 KO larvae incubated in 10µM NR was even significantly increased when compared to the larvae incubated with DMSO only (Fig. 13). However, the incubation in 10µM of NR did not impact the lethality observed at 9dpf upon deletion of tbk1 (Fig. 13). Indeed, at 9dpf, a mortality rate of 90% was observed in the tbk1 KO larvae treated with NR or in control conditions.

These results suggest that restoring the levels of NAD is sufficient to rescue the locomotor ability of the model, highlighting the importance of NAD metabolism in the pathogenicity of tbk1 deletion. It is important to mention that this is the first report to link tbk1 function with the regulation of NAD pathway so far. Furthermore, these results also imply the involvement After demonstrating that a dysregulation in the metabolism of nicotinamide is responsible for the apparition of the locomotor phenotype, we next investigated which molecular mechanisms could be responsible for the lethality observed upon deletion of tbk1. We therefore perform a similar metabolomic analysis on Tbk1 depleted larvae at 8dpf.

Metabolomic profile of the tbk1 KO larvae at day 8

At 8dpf, we did not identify any dysregulated metabolites in the tbk1 exons 4 and 5 deletion (q-value inferior to 0.05). However, upon deletion of the exons 1 and 2 of tbk1, we identified 3 metabolites that were significantly dysregulated (Fig. 14): quinolinic acid (as observed at 6dpf), uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), and uridine monophosphate (UMP).

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An increase in quinolinic acid levels, could again suggest a dysregulation in the NAD pathway, however we saw in the previous part that an alteration of the nicotinamide metabolism does not appear to be involved in the lethality phenotype observed. QUIN has been reported to be a neurotoxic compound via several mechanisms such as protein dysfunction, oxidative stress stimulation, glutamate excitotoxicity induction, mitochondrial dysfunction, autophagy, apoptosis or neuroinflammation (J.-M. [START_REF] Lee | Involvement of quinolinic acid in the neuropathogenesis of amyotrophic lateral sclerosis[END_REF].

UDP-GlcNAc and UMP are two uridine-based compounds. Therefore, an increase in their level could suggest a dysregulation in the metabolism of uridine. Uridine is a pyrimidine nucleoside involved in several biological processes such as phospholipid synthesis, glycogen storage, or RNA synthesis (G.P [START_REF] Connolly | Uridine and its nucleotides: biological actions, therapeutic potentials[END_REF]. Uridine has also been suggested to present neuroprotective and neuroregenerative abilities notably due to its involvement in synapse formation (B.S. [START_REF] Baumel | Potential Neuroregenerative and Neuroprotective Effects of Uridine/Choline-Enriched Multinutrient Dietary Intervention for Mild Cognitive Impairment: A Narrative Review[END_REF][START_REF] Wurtman | Nutritional modifiers of aging brain function: use of uridine and other phosphatide precursors to increase formation of brain synapses[END_REF].

Moreover, uridine supplementation was reported to be beneficial in the context of several neurodegenerative disease such as Huntington disease (J.A. [START_REF] Saydoff | Oral uridine pro-drug PN401 decreases neurodegeneration, behavioral impairment, weight loss and mortality in the 3-nitropropionic acid mitochondrial toxin model of Huntington's disease[END_REF][START_REF] Saydoff | Oral uridine pro-drug PN401 is neuroprotective in the R6/2 and N171-82Q mouse models of Huntington's disease[END_REF], Parkinson disease (M. [START_REF] Cansev | Restorative effects of uridine plus docosahexaenoic acid in a rat model of Parkinson's disease[END_REF], and more importantly, ALS (D.J. [START_REF] Amante | Uridine 126 ameliorates the pathological phenotype in transgenic G93A-ALS mice[END_REF]. In a metabolic context, increased uridine levels are associated with an increased ATP production and an enhanced glycolytic energy production (D.J. [START_REF] Amante | Uridine 126 ameliorates the pathological phenotype in transgenic G93A-ALS mice[END_REF]. UDP-GlcNAc is a compound that is widely used in the cell metabolism as a substrate

for the O-GlcNAc protein modification (J.C. Chatham et al., 2021). Interestingly, Oglycosylation has been reported to be involved in neurodegenerative disease such as Alzheimer disease (A.C. [START_REF] Wang | Loss of O-GlcNAc glycosylation in forebrain excitatory neurons induces neurodegeneration[END_REF][START_REF] Yuzwa | Increasing O-GlcNAc slows neurodegeneration and stabilizes tau against aggregation[END_REF] and more importantly in the context of ALS in a rat SOD1 G93A model where decreased levels of O-glycosylation were reported (N. Lüdemann et al., 2005). Although it was not mentioned in this study, a decrease in O-glycosylation could suggest an increase in UDP-GlcNAc level similarly as what we observed in our analysis.

Similarly to the 6dpf analysis, it is impossible to identify a clear implication of a compound in the apparition of the lethality observed upon deletion of tbk1 in our larvae when considering the metabolites as independent from each other. Therefore, we performed an enrichment analysis on our 8dpf data. Four pathways are identified as significantly dysregulated (Fig. 15):

1) the nicotinamide pathway similarly to what was observed at 6dpf; 2) the 93 glycosylphosphatidylinositol (GPI) anchor biosynthesis; 3) the pyrimidine metabolism; 4) the glycerophospholipids metabolism. Apart from the NAD metabolism dysregulation, no significant association was able to pinpoint metabolic dysregulation at 8dpf to explain the increased mortality upon deletion of tbk1. A metabolomic-proteomic comparative analysis was performed to unravel altered factors and pathways that could be involved in motor neuron degeneration.

V. Proteomic characterization of the tbk1 KO zebrafish model 94

Proteomic profile of the tbk1 KO larvae at day 8

Similarly to the metabolomic analysis, we select larvae at 8dpf for lysis and subsequent mass spectrometry. We used again the two tbk1 KO conditions (exon 1-2 KO and exon 4-5 KO).

Out of the 8640 identified proteins, 211 were significantly dysregulated by an ANOVA analysis adjusted with a false discovery rate. To confirm our model, we first check the expression level of tbk1. Tbk1 expression appeared significantly decreased from the ANOVA (p-value=0.043), however when adjusted with a false discovery rate, its expression was no longer significantly dysregulated (q-value=0.073). When looking in details, it became clear that this was due to a lower efficiency of the tbk1 exon 4-5 KO approach. We therefore decided to focus exclusively on the tbk1 exon 1-2 KO condition as we could assess the correct decrease in expression of tbk1 in this pool of samples.

The new tbk1 exon 1-2 KO versus non-injected larvae analysis identified 115 new significantly dysregulated proteins bringing the total number to 326. We implemented the 326 compounds on the string © online software (L.J. [START_REF] Jensen | STRING 8--a global view on proteins and their functional interactions in 630 organisms[END_REF]. Out of this analysis, the software grouped proteins according to different features as the biological process, their molecular function, or the cellular pathway (using KEGG resources) they are involved in.

When looking at the biological processes identified, immune response was overrepresented.

To define whether dysregulated immune response was present in the tbk1 KO model, I tested anti-inflammatory compounds in the next section.

Inhibition of the inflammatory response via caffeic acid incubation doesn't rescue tbk1 KO induced lethality

Caffeic acid (CA), an anti-inflammation, anti-oxidant and anti-carcinomic compound has been shown to exhibit beneficial effect in the SOD1G93A model (C.V. [START_REF] Fontanilla | Caffeic acid phenethyl ester extends survival of a mouse model of amyotrophic lateral sclerosis[END_REF].

The anti-inflammatory feature of this compound relies on the inhibition of the arachidonic acid, an inflammatory compound that was significantly increased in ALS models (H. [START_REF] Lee | Multi-omic analysis of selectively vulnerable motor neuron subtypes implicates altered lipid metabolism in ALS[END_REF][START_REF] Trostchansky | Profile of Arachidonic Acid-Derived Inflammatory Markers and Its Modulation by Nitro-Oleic Acid in an Inherited Model of Amyotrophic Lateral Sclerosis[END_REF]. Arachidonic acid is degraded by the cyclooxygenase (COX) 1 and 2 enzymes into lipidic cytokines, (H. [START_REF] Tallima | Arachidonic acid: Physiological roles and potential health benefits -A review[END_REF] with COX1 orthologue upregulated in our proteomic analysis. 10µM of CA was selected as the optimal concentration and tested on the locomotor phenotype observed at 5dpf. No significant difference in the swimming capacity was observed in the tbk1 KO larvae treated with 10µM of CA (Fig. 16). This suggest that a dysregulated immune response might not be solely responsible for motor defects observed in the tbk1 KO model. Similarly, no alteration of the lethality upon CA treatment was observed (Fig. 16). However, it is important to point out that CA has limited targets and the impact of other anti-inflammatory compounds should be assessed on the phenotypes observed upon tbk1 deletion.

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VI. Combined metabolomics-proteomics analysis of the tbk1 KO zebrafish

In order to perform the combined metabolomic and proteomic analysis, we selected the significantly dysregulated molecules and identified three main cellular processes: the necroptosis, the purine metabolism and the apoptosis.

Dysregulation of necroptosis and apoptosis in our model can be directly linked to motor neuron degeneration observed in the tbk1 KD as well as the increased mortality observed at 9dpf in the tbk1 KO model. Importantly, these two pathways can be activated upon RIPK1 aberrant activation. This kinase is physiologically sequestered via phosphorylation and ubiquitination at the plasma membrane in a TNF-receptor interacting complex. In this complex, RIPK1 plays an important role in the transmission of inflammatory signals. It is important to note that one of the regulatory phosphate group that can control RIPK1 activation is added through the activity of TBK1 (T. [START_REF] Delanghe | RIPK1 kinase-dependent death?: a symphony of phosphorylation events[END_REF].

The mechanistic insight of these two programmed cell death pathways was reviewed in the introduction (Metabolites dysregulation in ALS -part II.C.1)

As TBK1 is an important regulator of RIPK1 activity, an induced cell death via the activation of these pathways is a convincing hypothesis. Moreover, Tbk1 KO induced lethality in a murine model has been rescued through the additional deletion of Ripk1 (D. [START_REF] Xu | TBK1 Suppresses RIPK1-Driven Apoptosis and Inflammation during 195 Development and in Aging[END_REF].

The analysis suggests contradictory hypotheses concerning these two pathways. First, the necroptosis pathway appeared as severely enriched upon tbk1 deletion (top hit when using the KEGG pathway data pool). However, when looking independently at the dysregulated proteins, casp8 appears upregulated suggesting an inhibition of necroptosis, but Cyld level as well suggests the opposite (Fig. 17). Moreover, Bax, a crucial activator of the mitochondrial apoptosis induction, is downregulated in our model whereas Cytochrome c, which is a caspase activator complex downstream of Bax, is upregulated (Fig. 17). It is important to mention that apoptosis process was reported to be activated in the liver of Tbk1 -/-mouse model (M. [START_REF] Bonnard | Deficiency of T2K leads to apoptotic liver degeneration and impaired NF-kappaB-dependent gene transcription[END_REF]. Altogether, these data highly suggest the involvement of programmed cell death mechanisms in our model with a detailed analysis of these pathways, needed at the molecular level. Importantly, necroptosis has been shown to be implicated in the development of neurodegenerative diseases (S. Zhang et al., 2017) and that upregulation of necroptotic cell death occurs upon TBK1 downregulation in the context of ALS (D. [START_REF] Xu | TBK1 Suppresses RIPK1-Driven Apoptosis and Inflammation during 195 Development and in Aging[END_REF]. [START_REF] Luo | Pathview Web: user friendly pathway visualization and data integration[END_REF].

99

DISCUSSION 100

Phenotypic features of tbk1 reduced expression

Two zebrafish models of tbk1 loss of expression were developed during this thesis. These two models rely on different genetic approaches. The first one is a transient and ubiquitous KD based on the prevention of the tbk1 transcript translation. The second model is based on the genomic excision of the kinase domain-coding region and the subsequent repression of tbk1 catalytic activity.

Upon partial deletion of tbk1 expression, we reported the reduction of the locomotor capacity of the zebrafish larvae at the developmental stage of 2 dpf. We further associate this impaired swimming ability to a decreased number of spinal MN without visible muscular alterations. Moreover, we report that the loss of these cells was not due to a developmental delay as their number is progressively decreasing when looking at different stages.

Therefore, the remaining hypothesis is that this loss of spinal motor neurons is linked to cell death. However, more evidences are needed to claim the presence of neurodegeneration in our models, such as immunostaining of specific cell death markers (B.A. Smitch and B.D. [START_REF] Smith | Herpesvirus Transport to the Nervous System and Back Again[END_REF]. Moreover, if cell death is proven to be involved in our model, it would be interesting to determine what cellular processes are responsible. Indeed, they are at least dozens of ways for neurons to die (M. [START_REF] Fricker | Neuronal Cell Death[END_REF] and identifying the cellular process associated with the possible death of motor neurons in our model would give us great insight concerning the mechanisms of tbk1 KD impact on the spinal cord integrity and the subsequent impaired locomotion characterising our model. Interestingly, MN loss was not associated with major muscle defects.

Interestingly, the phenotype observed in this model is slowly rescued over time. Although not surprising when using morpholinos as they are degraded over time, in our context it is complicated to explain how the organism can recover from neuronal cell loss. This could be due to regeneration of MN progenitors. Indeed, zebrafish possess the capacity throughout all their life of regenerating spinal MN via reactivation of quiescent progenitor cells from the spinal MN progenitor domain (S.R. Alper and R.I. Dorsky, 2022; M.J. [START_REF] Cardozo | Reduce, reuse, recycle -Developmental signals in spinal cord regeneration[END_REF].

This process was observed early in development, with 3dpf damaged spinal MN being regenerated 48h later (J. [START_REF] Ohnmacht | Spinal motor neurons are regenerated after mechanical lesion and genetic ablation in larval zebrafish[END_REF]. Preliminary results show that the number of spinal MN is no longer different in tbk1 KD larvae compared to the negative control condition (data not shown), further supporting the hypothesis of a regeneration of the lost MNs. Moreover, it is important to mention in a study implying tbk1, a crucial regulator of the inflammatory response, that inflammatory cytokines are been reported to play an important role in the initiation of the regenerative mechanism (T.M. [START_REF] Tsarouchas | Dynamic control of proinflammatory cytokines Il-1? and Tnf-? by macrophages in zebrafish spinal cord regeneration[END_REF][START_REF] Kyritsis | Acute Inflammation Initiates the Regenerative Response in the Adult Zebrafish Brain[END_REF]. Although this hypothesis seems, in our opinion, plausible, we still need to confirm it. One strategy would be to combine the decreased expression of tbk1 with the incubation of the larvae in an immunosuppressant compound such as dexamethasone, which has been shown to prevent the regeneration (N. [START_REF] Kyritsis | Acute Inflammation Initiates the Regenerative Response in the Adult Zebrafish Brain[END_REF]. Therefore, the characterisation of the apparition of the motor phenotype upon reduced expression of tbk1 still remains to be investigated more in depth: identity of the lost MN, confirmation of a cellular death involvement, identification of the mechanism involved in the phenotype rescue upon tbk1 KD.

Interestingly, the phenotype observed upon genomic deletion of sequence coding for Tbk1's kinase domain appears delayed on a developmental time-scale. Indeed, the decrease in the swimming capacity is observed at a later stage, 5dpf, in comparison with the KD model. We have emitted several hypotheses possibly explaining this delay in the apparition of a phenotype:

The first hypothesis states that maternal tbk1 mRNA could influence the apparition of the phenotypic features in these two models. Indeed, early embryonic development occurs in the absence of transcription and therefore relies essentially on the translation of maternal origin mRNA molecules deposited within the egg during oocyte maturation [START_REF] Harvey | Identification of the zebrafish maternal and paternal transcriptomes[END_REF]. The CRISPR technology impacts positively or negatively genic expression through direct alteration of the genomic sequence. Therefore, tbk1 expression through translation of maternal mRNA can still occur within the first hours of the embryonic development, even in a CRISPR induced KO context. In opposition, the tbk1 KD model relying on injection of AMOs and the subsequent blockade of the translation, tbk1 maternal mRNA is no longer expressed.

Therefore, we designed both a model in which the first stage of development occurs without expression of tbk1 -but with its expression being rapidly restored within the following days -; and a model in which tbk1 is still being expressed within the first developmental stagesbut no longer as soon as the embryo start relying on its own transcriptional machinery. It is considered that the transcription machinery of the embryo is able to totally maintain the cell physiology at the gastrula stage, therefore the usage of maternal mRNAs is restricted to the first 10 hours of development (H. [START_REF] Chang | Terminal Uridylyltransferases Execute Programmed Clearance of Maternal Transcriptome in Vertebrate Embryos[END_REF]. tbk1 has been shown to be expressed in the gastrula stage at 5.25 hpf (Y.W. [START_REF] Hu | TANK-Binding Kinase 1 (TBK1) Isoforms Negatively Regulate Type I Interferon Induction by Inhibiting TBK1-IRF3 Interaction and IRF3 Phosphorylation[END_REF], therefore its expression in early development is indeed permitted by the maternal mRNA stocks. However, this hypothesis implicates an impaired activity of tbk1 within the first hours of development reverberating on the locomotor function at 2dpf. The eventuality of raising a stable tbk1 KO zebrafish

homozygous line to answer this hypothesis is made impossible by the induced lethality.

Therefore, generating a model with complete repression of the tbk1 maternal mRNA's expression and a time lasting phenotype would require the combination of the two approaches: injecting tbk1 AMO in one cell eggs produced by a heterozygous tbk1 KO stable line. However, even such approach has its limitations. indeed, selecting the homozygous larvae would require to perform genotyping through the cut of the tail's tip which would influence the swimming ability of the larvae and therefore might biase the phenotypic analysis.

A second hypothesis could explain the delay in the apparition of the locomotor phenotype.

Three alternates splicing transcripts of tbk1 have been identified (Y.W. [START_REF] Hu | TANK-Binding Kinase 1 (TBK1) Isoforms Negatively Regulate Type I Interferon Induction by Inhibiting TBK1-IRF3 Interaction and IRF3 Phosphorylation[END_REF]J. Zhang et al., 2020) in the zebrafish. The products of these transcripts have been reported to interfere with Tbk1 physiological function by competing in the binding of Irf3 (L. Zhang et al., 2016) or by inducing the degradation of both full length Tbk1 and Irf3 (J. [START_REF] Zhang | A Novel Transcript Isoform of TBK1 Negatively Regulates Type I IFN Production by Promoting Proteasomal Degradation of TBK1 and Lysosomal Degradation of IRF3[END_REF].

tbk1 AMO by directly preventing the ribosomes to recognize the ATG codon also inhibits the expression of Tbk1's isoforms whereas the CRISPR approach only prevent the expression of a kinase active protein, and therefore does not prevent the expression of non-functionally active Tbk1's isoforms. Due to their involvement in the regulation of the IFN production via the inhibition of Tbk1-Irf3 interaction, we cannot exclude that the difference observed in the phenotype apparition is linked to their expression.

A last hypothesis would be the existence of compensatory mechanism. Indeed, it was reported that genetic compensatory mechanisms require the degradation of mRNA to activate (M.A. [START_REF] El-Brolosy | Genetic compensation triggered by mutant mRNA degradation[END_REF]. This would explain why a possible compensatory mechanism would only occur in the KO model as AMO's activity does not implicate the degradation of the target mRNA. Moreover, the genetic compensation hypothesis is A second hypothesis that could explain this situation is an impaired salvage pathway. Indeed, upon synthesis, NAM is reintroduced into the nicotinate metabolism by being reconverted into NMN, the direct precursor of NAD+. The NR being able to rescue the phenotype, it is unlucky that the reaction responsible for NMN conversion into NAD+ is impaired. Therefore, this hypothesis relies on a possible impaired activity of the enzyme responsible for the catabolic production of NMN from NAM. This enzyme is called nicotinamide phosphoribosyltransferase (NAMPT) and to confirm its possible increased activity, an expression assay should be performed either at the mRNA or the protein levels. et al., 2021). G3P being directly derived from glucose metabolism, its dysregulation suggests an hypermetabolism state as observed in patients (P. [START_REF] Fayemendy | Hypermetabolism is a reality in amyotrophic lateral sclerosis compared to healthy subjects[END_REF]. However, as the level of glucose or pyruvate do not seem to be impacted by the loss of tbk1 in our analysis, it suggests that G3P depletion in our model could be linked to another catabolic reaction. An investigation of the different reaction involving G3P would be required to understand this decreased level.

We also report a high lethality upon complete deletion of tbk1 in the zebrafish larvae model.

M. Bonnard in 2000 has published a study based on a Tbk1 KO mouse model where she reports the death of the embryo early in development (at E14.5) (M. [START_REF] Bonnard | Deficiency of T2K leads to apoptotic liver degeneration and impaired NF-kappaB-dependent gene transcription[END_REF].

This induced lethality, associated with the liver's degeneration and an impaired expression of the inflammatory related complex NFκB, unveiled a crucial role for TBK1 in the liver development. A link with the lethality observed in our zebrafish model could be easily done.

However, I have not assessed the liver integrity of the larvae and therefore we cannot affirm that our larvae die from a liver failure. It would be an interesting feature to analyse but as the hepatic system is spared in ALS patients, we decided to focus on understanding the apparition of ALS hallmark in our model. Moreover, I assessed the responsibility of an hyperinflammatory state in the tbk1 KO zebrafish's developmental death by inhibiting the crucial neuroinflammation actor arachidonic acid through incubation with caffeic acid without success. This suggests that a dysregulation in the arachidonic acid dependent inflammatory response is not involved in our model, however, we still need to investigate the responsibility of inflammation by inhibiting other aspect of this cellular process by using glucocorticoid compounds for instance (Y. [START_REF] Xie | Modeling Inflammation in Zebrafish for the Development of Anti-inflammatory Drugs[END_REF] or directly inhibits quinolinic acid

(which exhibit increased level in our model) via blockade of its upstream enzyme KMO (G.

Constantino, 2014). Moreover, to further confirm the link between our zebrafish and the murine tbk1 KO models, it could be interesting to assess the integrity of the zebrafish liver at 8dpf.

Since the subjacent molecular mechanisms associated with the Tbk1 KO induced lethality have never been extensively explored, we performed a combined proteomic and metabolomic characterisation of the zebrafish just prior to their death (8dpf). The increase in quinolinic acid confirms the involvement of inflammation in our model which deepen our will of pursuing the investigation of inflammation in our model. Furthermore, the combined proteomic-metabolomic analysis also suggested the dysregulation of two programmed cell death mechanisms in our model: necroptosis and apoptosis. Interestingly this correlates with a study perform in the murine model in which a combined deletion of Tbk1 and a kinase dead mutation in Ripk1 was sufficient to significantly delay the lethality induced by the loss of tbk1 (D. [START_REF] Xu | TBK1 Suppresses RIPK1-Driven Apoptosis and Inflammation during 195 Development and in Aging[END_REF]. However, as RIPK1 is involved in both the necroptosis mechanism and the CASP8-associated apoptosis, this study does not allow to identify the cell death mechanism involved in the model. In our case, although the identification of these two pathways as possible therapeutic targets is really exciting, we have not been able to confirm their involvement in the lethality induced by tbk1 KO in the zebrafish larvae because of a lack of time. Therefore, in my opinion, the first experiment to perform in the continuity of this work would be the assessment of a possible rescue of the lethality via incubation with different drugs targeting one or both of these mechanisms. Amongst the drug identified to perform this series of test, we are considering the use of the following drugs: 1) in the continuity of D. Xu study in the murine model an inhibitor of RIPK1 activity: necrostatin-1 therefore inhibiting both necroptosis and apoptosis (X. [START_REF] Gao | Necrostatin-1 Relieves Learning and Memory Deficits in a Zebrafish Model of Alzheimer's Disease Induced by Aluminum[END_REF], 2) an apoptotic specific inhibitor such as decylubiquinone (inhibitor of the mitochondrial induced apoptosis) (A.R. [START_REF] Bourefis | Novel FUS and CHCHD10 models to investigate pathogenic mechanisms in Amyotrophic Lateral Sclerosis 223[END_REF] or Z-IETD-FMK (Casp8 inhibitor) (Z. [START_REF] Qi | Zebrafish BID Exerts an Antibacterial Role by Negatively Regulating p53, but in a Caspase-8-Independent Manner[END_REF] 3) necroptosis specific inhibitors such as necrosulfonamide (inhibitor of the MLKL activity -even though not orthologs have been identified in the zebrafish) (D. [START_REF] Liao | Necrosulfonamide inhibits necroptosis by selectively targeting the mixed lineage kinase domain-like protein[END_REF] or GSK872 (an inhibitor of RIPK3 activity) (W. [START_REF] Wen | Loss of Ripk3 attenuated neutrophil accumulation in a lipopolysaccharide-induced zebrafish inflammatory model[END_REF]. This drug screening experiment will unveil the suggested responsibility of these death pathways in the apparition of death in our model.

Additionally, the last molecular features of our model are the dysregulations of the purine and the pyrimidine metabolisms. These were not reviewed in the results section as nothing can be concluded out of the data from our analyses. However, because of their involvement in the composition of DNA and RNA, purines and pyrimidines are ones of the most abundant metabolites in living organisms. These metabolisms rely on the production of (poly)nucleotides, which are the crucial actors of the metabolism as they are used as cofactors in most of the metabolic pathways. During these reactions, phosphate groups are cleaved from their structures which eventually lead to the formation of the purine (adenine, guanine and inosine) or pyrimidine (cytosine, uridine, and thymidine) nucleosides (A.M. [START_REF] Pedley | A New View into the Regulation of Purine Metabolism -The Purinosome[END_REF]. Nucleosides are the structural components of the nucleic acid that allows coding of the genetic information.

The end reaction of the purine pathway is the degradation of the purines into uric acid via the generation of hypoxanthine (Fig. 18). In our data set, we observed a disequilibrium between the levels of nucleotides, which are either unchanged or significantly increased (GMP) and the levels of nucleosides which are all decreased upon tbk1 deletion (Fig. 18).

This is supported by increased levels of cytosolic 5'-nucleotidases-I (cN-I) in our model. This enzyme catalyzes the degradation of nucleotides to nucleosides. Accumulation of cNs proteins has been exhibited in the context of neuromuscular pathologies as they are colocalizing with characteristic inclusions of these diseases (H. [START_REF] Pluk | Autoantibodies to cytosolic 5?-nucleotidase 1A in inclusion body myositis[END_REF]. It could be interesting to investigate the presence of inclusions in our model to further confirm this hypothesis. To our knowledge, this is the first study to suggest an association between ALS and a cN-I dysregulation. upon tbk1 deletion in comparison with non-injected WT larvae. Adapted from pathview © (W. Luo et al., 2017).

Abbreviations: [PDE and 3.1.4.17]: cGMP-inhibited 3 ',5'-cyclic phosphodiesterase B; [4.6.1.2]: atrial natriuretic peptide receptor 1; [1.17.4.1]: ribonucleoside-diphosphate reductase large subunit;

[3. 1.3.5]: cytosolic 5'-nucleotidase 1A-like isoform; [3. 1.4.53]: cAMP-specific phosphodiesterase 4; [2.7.1.25 and 2.7.7.4]: 3'-phosphoadenosine 5'-phosphosulfate synthase

Concerning the pyrimidine dysregulation, it can be directly linked to the alteration of the UDP-GlcNAc level as seen in the results section. In the analysis we observe a significant increase in some mono-phosphate pyrimidine (CMP and UMP) (Fig. 19). However, the levels of their respective di-phosphate forms remain unchanged upon loss of tbk1 (CDP and UDP) 108 (Fig. 19). This suggests that the catabolic steps of the pathway are not altered. Similarly, increased levels of CMP and UMP suggest that the de novo synthesis path is functionally active (Fig. 19). However, orotate, a precursor of UMP exhibited decreased levels, questioning about a possible overactivation of the de novo synthesis step (Fig. 19). In the proteomics data, none of the identified enzymes involved in this pathway are significantly dysregulated upon tbk1 loss. Therefore, the observed increased levels of CMP and UMP must be imputated to another catabolic reaction and further investigations would be needed in order to understand their involvement in our model.

Limitations and future directions of this study

It is important to mention that although it recapitulates some hallmarks of the disease, this model still lacks some strength. First, we have not been able to report the presence of TDP-43 aggregates, which is the main cellular hallmark of the disease. Similarly, major targets and pathways involved in neurodegeneration, including autophagy and immunity can be impacted by ageing. Therefore, studying a late onset disease in a larval and embryonic model can lack specific age-related cellular processes. Moreover, the sole heterozygous loss of TBK1 in mouse, in opposition to our model, has not been reported to be sufficient to provoke the apparition of a phenotype in other models (D. [START_REF] Brenner | Heterozygous Tbk1 loss has opposing effects in early and late stages of ALS in mice[END_REF][START_REF] Gerbino | The Loss of TBK1 Kinase Activity in Motor Neurons or in All Cell Types Differentially Impacts ALS Disease Progression in SOD1 Mice[END_REF]. Therefore, we cannot exclude the hypothesis that zebrafish exhibit some specific Tbk1 related features that are not shared with higher complexity organisms. For all these reasons, the model designed in this study should be more considered as a tool to understand Tbk1 functional role. However, this model could be improved to better mimic ALS if implemented with another "hit". Indeed, because its development only occurs in a fraction of individuals with a genetic predisposition with the majority individuals develop ALS with no known mutation or genetic risk factor, this disease is thought to be caused by multiple factors [START_REF] Al-Chalabi | Analysis of amyotrophic lateral sclerosis as a multistep process: a population-based modelling study[END_REF][START_REF] Chiò | The multistep hypothesis of ALS revisited[END_REF]S. Vucic et al., 2018). This "multiple hit" or "multiple step" hypothesis is further supported by its late onset-feature as its development is caused by ageing (T. [START_REF] Niccoli | Ageing as a risk factor for ALS/FTD[END_REF] as well and by the high number of genes involved in its development [START_REF] Mathis | Genetics of amyotrophic lateral sclerosis: A review[END_REF]. This hypothesis was further deepened in the context of TBK1 related ALS, D. Xu in its 2018 study shown that the sole Tbk1 heterozygous loss was sufficient to induce an ALS-like phenotype when combined with the age-related reduced levels of MAP3K7 (or TAK1), another regulator of RIPK1's activity (D. et al., 2018). Moreover, in the same study, they report that according to the age of the mouse, Ripk1 activation has different outcome. Therefore, one strategy in order to create a double hit model involving tbk1 loss in the zebrafish would be to generate a stable tbk1 KO heterozygous line and investigate the consequences of this impaired expression at later stages with protein levels modified by the ageing process (through spinal cord, muscle or brain dissection and subsequent metabolomics/proteomics analyses). Moreover, another strategy would be to perform a double heterozygous knock out of the tbk1 and map3k7 and grow a stable line. However, to generate these lines and to perform the analysis are time consuming and could be performed in the continuity of the project. In addition, the second strategy would also require to assess map3k's expression. This example is based on the sole observations report in D. Xu's study but several other strategies could be developed, for instance additional tbk1 heterozygous loss has also been reported to have an impact on SOD1, TDP-43 or C9orf72 mice models (W. [START_REF] Shao | Two FTD-ALS genes converge on the endosomal pathway to induce TDP-43 pathology and degeneration[END_REF][START_REF] Brenner | Heterozygous Tbk1 loss has opposing effects in early and late stages of ALS in mice[END_REF]K. Sieverding et al., 2020).

Xu

To conclude, this work was aiming to generate a zebrafish model of tbk1 loss in the context of better understanding TBK1 associated ALS-FTD pathology. Here we show that Tbk1 loss of function can lead to locomotor defects and neurodegeneration such as observed in the ALS or FTD pathologies. We have been able to characterize the phenotype induced by the loss of bearing a nucleotide sequence (mismatch 5'-cctcttacctcagttacaatttata-3') that does not bind any zebrafish mRNA was used to exclude any injection-related phenotype. Microinjections were performed at 0.45mM for both the tbk1-targetting morpholino and the mismatch control.

Target nucleotide sequence for guide RNA design were selected using the CHOPCHOP software and scoring method and obtained directly synthesized as RNA oligo nucleotides (Alt-R® CRISPR-Cas9 crRNA, IDT, USA). Sequences are listed below: sgRNA tbk1 exon 1: GCCACAGCUAACGUGUACCG sgRNA tbk1 exon 2: CGUCAAGCUGUUCGCCGUCG sgRNA tbk1 exon 4: AUGAAUCACCUGCGCGAGUA Locomotor activity was assessed at 2 days post fertilization through a touch-evoked-escaperesponse test. Larvae were placed at the centre of a 15cm Petri dish and their tails were lighty touched with a tip. This stimulus provokes an escape response that was recorded using a grasshopper digital camera (Point Grey) at the speed of 30 frames per second. Larvae were this way scored for the distance swam, time travel and their velocity using the fiji plugin manual tracking.

At later stage (>3dpf) locomotor activity was assessed using a spontaneous locomotion test.

Larvae were maintained in a 96 wells plate and their total locomotor activity was record using a Zebrabox (Viewpoint Life Sciences, Lyon, France). The protocol used was composed of 2 distinct periods: 1) 40 minutes of free-swimming without any stimulus (dark environment), 2) 2 stimuli separated by a 10 minutes period to avoid any habituation effect.

Each stimulus consists of a 1 second sound (450 Hz, 80 dB) and an exposition to bright light.

Total swim activity was analyzed using ZebraLab V3 software © (Viewpoint Life Sciences, Lyon, France). The escape response induced by the stimuli was quantified by measuring the distance swam 5 seconds after stimulation.

Whole-mounts zebrafish larvae imaging and muscle-axonal measurements:

At 50hpf Mnx-RFP larvae were anesthetized with tricaine and fixed in 4% FA during 30 minutes at 4°C and agitating. Larvae were then washed 3 times in PBS 1X at 4°C. The larvae were mount by removing the cephalic part in a 70% glycerol (G9012, Sigma Alrich) solution.

Image were taken with an apotome (Apotome.2, Zeiss) at 20x magnification and a laser of 561nm (HXP 120V, OSRAM) for spinal integrity investigation or bright light for muscle fibres measurements.

Immunoblotting:

30 µg of proteins from zebrafish lysates (equal amount of protein in each lane) were separated by sodium dodecylepolyacrylamide gel electrophoresis. Samples were denatured at 98°C for 7 minutes. The separated proteins were transferred to nitrocellulose membranes (0.45μm, Life Sciences) and probed with the following primary antibody: NAK1/TBK1 antibody (ab109735, Abcam), and α-tubulin antibody (T5168, Sigma-Aldrich) was used as a loading control. The blots were incubated with the corresponding fluorescent secondary antibody and the signal was detected using the ODYSSEY® CLx. The intensity of bands in each of the lanes from the Western blots were measured by ImageJ.

Drug incubation and survival measurement:

Group of 12-10 larvae were kept in 12 wells multi-dish in 3.5mL of either egg-water (0.06g/L aquarium salt (Instant Ocean, Blacksburg, VA) in Milli-Q water +0.01 mg/L methylene blue) at 28.5 °C from 1dpf to 5dpf or water after 5dpf. Larvae were feed daily with paramecia and media was changed every two days. When needed media was implemented with 10µM of caffeic acid (C0625, Sigma Alrich) or nicotinamide riboside (SMB00907, Sigma Alrich) and dimethyl sulfoxide was used as negative control (D2438, Sigma Alrich). Number of dead larvae was scored each day for 12 days.

Statistical analysis:

All data values for the zebrafish experiments are represented as average standard error of mean (SEM) with significance determined using one-way ANOVAs. Differences between groups were identified via post hoc comparisons, specified in the legend of each figure. All analyses were performed using Prism 5.0 (Graph Pad, CA). Significance level was set at p<0.05.

Metabolomics analysis:

Zebrafish larvae were collected respectively at 6 and 8dpf and stored at -80°C until analyses.

After addition of an extraction solution made of 50% methanol, 30% acetonitrile, and 20% water (G.M. [START_REF] Mackay | Analysis of Cell Metabolism Using LC-MS and Isotope Tracers[END_REF] in ratio of 1ml per 30mg of larvae, the samples were vortexed for 5 minutes at 4°C, then centrifuged at 16,000g for 15 minutes at 4°C. The supernatants were collected and separated by liquid chromatography-mass spectrometry (LC-MS) using SeQuant ZIC-pHilic column (MilliporeSigma). The aqueous mobile-phase solvent was 20 mM ammonium carbonate plus 0.1% ammonium hydroxide solution, and the organic mobile phase was acetonitrile. The metabolites were separated over a linear gradient from 80% organic to 80% aqueous for 15 minutes. The column temperature was 50°C, and the flow rate was 200 μL/min. The metabolites were detected across a mass range of 75 to 1,000 m/z using the Q Exactive Plus mass spectrometer (Thermo Fisher Scientific) at a resolution of 35,000 (at 200 m/z) with electrospray ionization and polarity switching mode.

Lock masses were used to ensure mass accuracy below 5 ppm. The peak areas of different metabolites were determined using Thermo Fisher Scientific TraceFinder software using the exact mass of the singly charged ion and known retention time on the HPLC column. and extraction blanks, c) system stability blanks, d) solvent blanks, e) long-term reference standard interlaboratory QC mix to ensure system stability, and f) samples that were blinded and loaded in randomized order. The analyses of pooled samples' QC showed no significant difference in metabolites' levels between QCs.

Data analysis was performed in the MetaboAnalyst 5.0 software (Z. [START_REF] Pang | MetaboAnalyst 5.0: narrowing the gap between raw spectra and functional insights[END_REF].

Samples were normalized by sum and each metabolite level was adjusted by autoscaling.

PCA was performed including all the metabolites identified. Hierarchical clustering was performed using the Ward method including all the metabolites identified.

Proteomics analysis:

Sample preparation: S-TrapTM micro spin column (Protifi, Hutington, USA) digestion was performed on 20µg of whole zebrafish larvae lysate according to manufacturer's instructions. Briefly, samples were reduced with 20mM TCEP and alkylated with 50mM CAA (chloracetamide) for 15min at room temperature. Aqueous phosphoric acid was then added to a final concentration of 1.2% following by the addition of S-Trap binding buffer (90% aqueous methanol, 100mM TEAB, pH7.1). Mixtures were then loaded on S-Trap columns.

Five extra washing steps were performed for thorough SDS elimination. Samples were digested with 1.5µg of trypsin (Promega) at 47°C for 1h30. After elution, peptides were vacuum dried.

LC MS/MS: The tryptic peptides were resuspended in 20µL of 2% ACN, 0.1% Formic acid in HPLC-grade water and an amount of 400ng was injected on a nanoElute (Bruker Daltonics, Germany) HPLC (high-performance liquid chromatography) system coupled to a timsTOF Pro (Bruker Daltonics, Germany) mass spectrometer. HPLC separation (Solvent A: 0.1% formic acid in water; Solvent B: 0.1% formic acid in acetonitrile) was carried out at 250nL/min using a packed emitter column (C18, 25 cm×75μm 1.6μm) (Ion Optics, Australia) using a gradient elution ( to 2 % solvent B during 1 min; 2 to 13% during 41min; 13% to 20% during 23min;

20% to 30% for 5min; 30% to 85% for 5min and finally 85% for 5min to wash the column).

Mass-spectrometric data were acquired using the parallel accumulation serial fragmentation (PASEF) acquisition method. The measurements were carried out over the m/z range from 100 to 1700 Th. The range of ion mobilities values from 0.75 to 1.25 V.s/cm2 (1/k0). The false discovery rates (FDRs) at the peptide and protein level were set to 1%. Scores were calculated in MaxQuant as described previously [START_REF] Tyanova | The Perseus computational platform for comprehensive analysis of (prote)omics data[END_REF]. Proteins were quantified according to the MaxQuant label-free algorithm using LFQ intensities and protein quantification was obtained using at least 1 peptide per protein. Finally, match between runs was allowed during the analysis.

Protein groups output from MaxQuant was then processed in Perseus (S. Tyanova et al., 2016) (v1.6.15.0). LFQ intensities values were log2 transformed and the reverse and common contaminants hits were removed as well as the proteins only identified by site. For further statistical analysis, categorical annotation was then created in order to label the 12 samples as followed: 6 "non-infected" (NI) replicates were labelled as the NI group and 6

TBK12 replicates (TBK12) were labelled as the TBK12 group. This groups separation was then used to keep proteins with at least 5 valid values (over 6) in at least one group. Next, the data were imputed to fill missing data points by creating a Gaussian distribution of random numbers with a standard deviation of 33% relative to the standard deviation of the

Figure 1 :

 1 Figure1: Illustration of the variation in the incidence and prevalence of ALS depending on sex, geographical population sets and wealth (derived from L.[START_REF] Xu | Global variation in prevalence and incidence of amyotrophic lateral sclerosis: a systematic review and meta-analysis[END_REF]

  degree relative being affected, approximately 10% of the cases are characterized by a familial history of the disease. As a partially hereditary disease, these cases are linked to one or several genomic mutations. Approximately 70% of the hereditary cases are associated with 4 genes out of the 30 identified: Chromosome 9 open reading frame 72 (C9ORF72), Superoxide dismutase 1 (SOD1), Transactive response DNA-binding protein (TARDBP) and Fused in sarcoma (FUS) (Fig 2) (A.M.G Ragagnin et al., 2019; M. Ghasemi and R.H. Brown, 2018).

Figure 2 :

 2 Figure 2: Circle chart illustrating the frequency of mutated genes in familial ALS patients (derived from A.M.G Ragagnin et al., 2019)

Figure 3 :

 3 Figure 3: Illustration of the symptoms induced by the different forms of pathologies of the ALS-FTD spectrum (derived from M.[START_REF] Ghasemi | Glial Cell Dysfunction in C9orf72-Related Amyotrophic Lateral Sclerosis and Frontotemporal Dementia[END_REF]

Figure 4 :

 4 Figure 4: Illustration of the different cellular dysfunction observed in the context of ALS-FTD

Figure 5 :

 5 Figure 5: Advantages and limitations of the in vitro and in vivo models in the context of ALS (derived from[START_REF] Van Damme | Modelling amyotrophic lateral sclerosis: progress and possibilities[END_REF]

  Tbk1 is a 50kb long gene encoding for a 729 amino acids (AA) long serine/threonine kinase protein. Below, I will review the 4 functional domains of TBK1 (C.[START_REF] Shu | Structural insights into the functions of TBK1 in innate antimicrobial immunity[END_REF][START_REF] Larabi | Crystal structure and mechanism of activation of TANK-binding kinase 1[END_REF][START_REF] Tu | Structure and ubiquitination-dependent activation of TANK-binding kinase 1[END_REF]:• Kinase domain (KD):Located at the N-term sequence of the protein, it is 300 AA long. The kinase domain is the key functional domain of the TBK1 protein. It allows its activation through autophosphorylation and bear the catalytic ability of the protein: the phosphorylation. The kinase domain of TBK1 exhibited the conserved architecture of a kinase protein composed of two lobes: the C and N terminal loops with the active cleft at the intersection (D.[START_REF] Tu | Structure and ubiquitination-dependent activation of TANK-binding kinase 1[END_REF].

Figure 6 :

 6 Figure 6: Schematic representation of TBK1 involvement in inflammation induction and phagophore maturation. (Derived from L. Ahmad et al., 2016).

  subsequently bind and sequester the anti-apoptotic BCL-2 protein family (L.[START_REF] Chen | Differential targeting of prosurvival Bcl-2 proteins by their BH3-only ligands allows complementary apoptotic function[END_REF]; G.L.Kelly and A. Strasser, 2019). These last proteins, in a non-apoptotic context, are preventing cell death by sequestering the two essential effectors of apoptosis: BAX and BAK (G.L.Kelly and A. Strasser, 2019). When inhibited by the BH3-only proteins, BCL-2 proteins no longer sequester BAX and BAK, which are therefore free to form oligomers. The formation of these secondary structures causes a mitochondrial outer membrane permeabilization which induces the release of both cytochrome c and DIABLO resulting in the subsequent activation of the caspases cascade. Caspases (Casp) are a family of protease that induces cleavages of hundreds of proteins upon apoptosis activation. The degradations of these proteins eventually lead to cell death.

  However, CASP8 has been reported to cleave CYLD and subsequently prevent the activation of RIPK1 (M.A.[START_REF] O'donnell | Caspase 8 inhibits programmed necrosis by processing CYLD[END_REF]. Therefore, necroptosis initiation relies on the blockade of CASP8's activity by degradation via viral or pharmacological agent or by sequestration by cFLIPs proteins (T.[START_REF] Kataoka | The caspase-8 modulator c-FLIP[END_REF][START_REF] Tummers | Caspase-8: regulating life and death[END_REF]. Indeed, upon TNF receptor activation in a CASP8 deprived context, RIPK1, via deubiquitination by CYLD, is able to perform an activating autophosphorylation (L.[START_REF] Laurien | Autophosphorylation at serine 166 regulates RIP kinase 1-mediated cell death and inflammation[END_REF]. This post-transcriptional mark allows the formation of a complex called necrosome which leads to activation of RIPK3 and subsequent activating of the mixed lineage kinase domain like pseudokinases (MLKL). This is the terminal effector of the necroptosis pathway and after oligomerization, MLKL chains translocate to the plasma membrane where they dysregulate its integrity (L.[START_REF] Sun | Mixed lineage kinase domain-like protein mediates necrosis signaling downstream of RIP3 kinase[END_REF][START_REF] Murphy | The pseudokinase MLKL mediates necroptosis via a molecular switch mechanism[END_REF]

Figure 7 :

 7 Figure 7: Schematic comparative illustration of Casp8 dependent apoptosis and necroptosis induction

  To palliate this lethality, conditional deletion of Tbk1 in neurons have been developed. In 2014, a study performing a T-cell specific Tbk1 deletion in mice exhibited an over-activation of T-cell leading to a neuroinflammatory autoimmunity model due to the activation of the mTORC1 pathway (J.Yu et al., 2014). This suggests a role of TBK1 in the regulation of T-cell activation in the murine model. More recently, conditional neuronal Tbk1 deletion in mice has been shown to induce several neuropathological changes as reduced dendritic spine density, cortical spinal loss, abnormal dendritic projections and p62/ubiquitin positive cytoplasmic inclusions in the Purkinje cell of the cerebellum (W.[START_REF] Duan | Deletion of Tbk1 disrupts autophagy and reproduces behavioral and locomotor symptoms of FTD-ALS in mice[END_REF].

  et al., 2015; T.W Tefera et al., 2021;[START_REF] Mcdonald | Glucose clearance and uptake is increased in the SOD1G93A mouse model of amyotrophic lateral sclerosis through an insulin-independent mechanism[END_REF]. This accumulation has been associated with a reduced expression of glycogen phosphorylase and an enhanced activity of glycogen synthase (L.[START_REF] Palamiuc | A metabolic switch toward lipid use in glycolytic muscle is an early pathologic event in a mouse model of amyotrophic lateral sclerosis[END_REF] T.W Tefera et al., 2021) further suggesting

Figure 8 :

 8 Figure 8: Schematic illustration of CNS glucose metabolism involvement in a healthy nervous system and the commonly observed defects observed in ALS. (Derived from T.W Tefera et al., 2021)

Figure 9 :

 9 Figure 9: Schematic illustration of lipids involvement in a healthy nervous system and the observed consequences induced by lipidic dysregulation in ALS patients. (Derived from T.J.Tracey et al., 2020)

Figure 12 :

 12 Figure 12: Graphical representation of the NAD metabolism

Figure 1 :

 1 Figure 1: 2dpf tbk1 AMO injected larvae exhibit reduced expression of Tbk1:

Figure 2 :

 2 Figure 2: tbk1 AMO injected larvae exhibit locomotor deficits at 2dpf:

Figure 3 :

 3 Figure 3: tbk1 knock-down causes spinal motor neuron loss.

Figure 4 :

 4 Figure 4: tbk1 induced loss of spinal motor neurons is not linked to developmental delay:

Figure 6 :

 6 Figure 6: target tbk1 CRISPR induction of a 700 base pairs deletion

Figure 7 :Figure 8 :

 78 Figure 7: CRISPR tbk1 deleted larvae do not exhibit locomotor deficits at 2dpf

Figure 9 :

 9 Figure 9: deletion of tbk1 in zebrafish does not impact the size of the larvae at 8dpf.

Figure 10 :

 10 Figure 10: tbk1 deletion is lethal early in the development at 9dpf

Figure 11 :

 11 Figure 11: Nicotinamide and glycerol 3-phosphate exhibit dysregulated levels upon tbk1 deletion at 6dpf

Figure 12 :

 12 Figure 12: Nicotinate and nicotinamide metabolism are significantly dysregulated in both of the tbk1 KO conditions at 6dpf

Figure 14 :

 14 Figure 14: Quinolinic acid, UDP-GlcNac and UMP exhibit dysregulated levels upon tbk1 exons 1 and 2 deletion at 8dpf

Figure 15 :

 15 Figure 15: Nicotinate/nicotinamide, GPI anchor biosynthesis, pyrimidine and glycerophospholipid metabolisms are dysregulated in the pooled tbk1 KO conditions at 8dpf

Figure 16 :

 16 Figure 16: Supplementation with 10µM of caffeic acid did not rescue the development of phenotype induced by the deletion of tbk1.

Figure 17 :

 17 Figure 17: Schematic overview of the programmed cell death pathway:

Figure 18 :

 18 Figure 18: Schematic overview of the purine metabolism:

Figure 22 :

 22 Figure 22: Schematic overview of the pyrimidine metabolism:

  tbk1 expression. Interestingly, due to its fast development, zebrafish is the first in vivo model where the sole loss of tbk1 is associated with deficits in locomotor functions induced at least partly by alteration of the NAD+ level. Moreover, we have also been able to partially confirm what was observed in the murine tbk1 KO model with an increased lethality early in the development (9dpf). Furthermore, a metabolic analysis demonstrated that activating the NAD+ pathway through NR can rescue motor deficits in the tbk1 KO model. Importantly, a combined proteomics and metabolics analysis pointed out to the necroptosis and apoptosis cell death pathways that will be investigated in detail at the molecular and pharmacological level in this novel model of ALS-FTD.Experimental model maintenance and handling:Adult and larval AB wildtype zebrafish (Danio rerio) were maintained at the Institut Imagine, Paris, zebrafish facility and bred according to the National and European Guidelines for Animal Welfare. All procedures were approved by the Institutional Ethics Committees at the Research Centers of Imagine. Embryos were raised at 28°C and kept in egg-water (0.06g/L aquarium salt (Instant Ocean, Blacksburg, VA) in Milli-Q water +0.01 mg/L methylene blue) until 5 days post fertilization. After 5 days post fertilization embryos were transferred in water. Egg clutches were collected from a single cross and injected at the one-cell stage. In this study both male and female fishes were used. The following transgenic lines were used for imagery Mnx:RFP.Morpholino and sgRNA synthesis and injection:Microinjections of antisense morpholino oligonucleotide (AMO) were performed on wildtype and Mnx:RFP transgenic AB fishes at the one cell stage using a PV 820 Pneumatic PicoPump injector (World Precision Instruments, USA) and heat-pulled borosilicate glass needles. tbk1 morpholino (synthesized by Gene Tools, Philomath, USA) targeting the initial ATG codon of the tbk1 gene (AMO-tbk1: 3'-ggccgtactctgcatgatgactgta -5') were injected at the one cell stage to realize a transient knockdown of tbk1. Mismatch control morpholino

  As a part of the routine analytical pipeline, the recommendations of the metabolomics Quality Assurance & Quality Control Consortium were applied. The routine quality controls included regular equipment maintenance (Thermo Fisher Scientific) and the use of standard operating procedures for sample extraction, storage, and analyses. General practices also included weekly test runs to ensure system stability and quality of runs. Regarding the quality controls (QCs) in relation to this study, we used a) pooled interstudy QC, b) process

  total cycle time was set to 1.16s and the number of PASEF MS/MS scans was set to 10.MS Data Processing and Bioinformatics Analysis: Data analysis was performed usingMaxQuant (version 2.0.1.0) and searched with Andromeda search engine against the UniProtKB/Swiss-Prot Danio rerio database containing also the Trembl entries (release 01-2022, 46841 entries). To search parent mass and fragment ions, we set a mass deviation of 10 ppm for the main search and 40 ppm respectively. The minimum peptide length was set to 7 amino acids and strict specificity for trypsin cleavage was required, allowing up to 2 missed cleavage sites. Carbamidomethylation (Cys) was set as fixed modification, whereas oxidation (Met) and N-term acetylation (Prot N-term) were set as variable modifications. The

  [START_REF] Yu | The pivotal role of TBK1 in inflammatory responses mediated by macrophages[END_REF] Y-Y. Wang et al., 2019), autophagy(B. Ritcher et al., 2016;[START_REF] Oakes | TBK1: a new player in ALS linking autophagy and neuroinflammation[END_REF], the insulin signalling pathway (M.A.[START_REF] Muñoz | TANK-binding kinase 1 mediates phosphorylation of insulin receptor at serine residue 994: a potential link between inflammation and insulin resistance[END_REF] and innate immunity against bacterial and viral infections (R.[START_REF] Zhou | TBK1, a central kinase in innate immune sensing of nucleic acids and beyond[END_REF] K.

	Mosallanejad and J.C. Kagan, 2022).

A. TBK1 in innate immune response and inflammatory response: 1. Brief introduction to innate immunity -what are IFN and cytokines?

-

  The IFN-III: this group is composed by 4 types of IFNλ. These IFN are specifically binding to the IFNLR.Importantly, in opposition to IFN-I and -III, the IFNγ are only inducing the phosphorylation of STAT1 and therefore, are not able to stimulate the expression of every ISGs. Their field of action is limited to ISGs bearing a so-called gamma-activated sequence (GAS) (L.C.[START_REF] Platanias | Mechanisms of type-I-and type-II-interferon-mediated signalling[END_REF]. Furthermore, even though IFN-I and IFN-II are supposedly able to activate every ISGs, the reality is different and the pool of expressed genes is directly depending on the type of IFN and the cell type (W.M.[START_REF] Schneider | Interferon-stimulated genes: a complex web of host defenses[END_REF]. As a consequence of these various pools of activated genes, the binding of an IFN results in a different cellular response such as antiviral immunity, proliferation, autophagy or apoptosis. This allows the cell to eliminate the threat by targeting multiple stages of the infection, for instance every step of

the viral replication can be targeted (W.M.

[START_REF] Schneider | Interferon-stimulated genes: a complex web of host defenses[END_REF][START_REF] Yang | All About the RNA: Interferon-Stimulated Genes That Interfere With Viral RNA Processes[END_REF]

  . It is considered that variations in the sequence of TBK1 are responsible for approximately 3% of the total FTD cases[START_REF] Portera-Cailliau | Wholegenome sequencing reveals important role for TBK1 and OPTN mutations in frontotemporal lobar degeneration without motor neuron disease[END_REF] making it the fourth genetic cause of development of the FTD(S. Van Mossevelde et al., 2016). Studying the prognosis of TBK1 mutation carriers can be challenging due to the reduced numbers of patients compared to the other main genes associated with the disease. A study following 16 TBK1 mutation carriers reported a median age of onset of 62.1 with a mean disease duration of 4.7 years(S. Van Mossevelde et al., From this study, analysing 110 cases, TBK1 associated ALS patients seem to present a propensity of limb-onset with 63% of the cases concerned, in opposition to 32% with bulbaronset, and 5% with respiratory-onset (I.J.[START_REF] Swift | Variable clinical phenotype in TBK1 mutations: case report of a novel mutation causing primary progressive aphasia and review of the literature[END_REF]. In opposition to what could be expected due to TBK1 involvement in the development of FTD, only 19% of the total cases had a cognitive presentation (I.J.[START_REF] Swift | Variable clinical phenotype in TBK1 mutations: case report of a novel mutation causing primary progressive aphasia and review of the literature[END_REF]. Moreover, out of these cases presenting cognitive impairment, 29% were presenting an FTD-ALS comorbidity and 29% an unspecified Interestingly, another study has reported alteration of the metabolism characterized by a marked hypermetabolism, weight loss, reduced fat mass and reduced appetite in TBK1 mutation carriers patients (P.A.[START_REF] Mccombe | 165 Patient with ALS with a novel TBK1 mutation, widespread brain involvement, behaviour changes and metabolic dysfunction[END_REF]. Several clinical studies have focused on cases of FTD only pathology associated with a TBK1 mutation (C.

	2016).

Since the initial identification of TBK1 mutations in ALS, more than 50 non-sense or missense variants throughout the entire sequence of the TBK1 gene have been described (M.

[START_REF] De Majo | ALS-associated missense and nonsense TBK1 mutations can both cause loss of kinase function[END_REF]

. This suggests that the ALS-FTD pathology is more probably linked to a loss-offunction mechanism. So far, no gain-of-function mechanism induced by a TBK1 variant has been reported. Therefore, the actual paradigm concerning TBK1 associated ALS-FTD is an haploinsuffiency mechanism

(A. Freischmidt et al., 2015;[START_REF] De Majo | ALS-associated missense and nonsense TBK1 mutations can both cause loss of kinase function[END_REF]

.

B. TBK1 related ALS-FTD pathogenicity:

Overall, ALS patients bearing a TBK1 mutation show a median age of onset compared to other types of ALS (M.

Weinreich et al., 2019; P.A. Mccombe et al., 2019; S. Van Mossevelde et al., 2016;[START_REF] Swift | Variable clinical phenotype in TBK1 mutations: case report of a novel mutation causing primary progressive aphasia and review of the literature[END_REF]

. Only one study brings together data from different clinical studies concerning focusing on ALS patients bearing TBK1 variants (I.J.

[START_REF] Swift | Variable clinical phenotype in TBK1 mutations: case report of a novel mutation causing primary progressive aphasia and review of the literature[END_REF]

. dementia (I.J.

[START_REF] Swift | Variable clinical phenotype in TBK1 mutations: case report of a novel mutation causing primary progressive aphasia and review of the literature[END_REF]

. Additionally, out of the 110 patients of the study, 4% were characterized by an atypical parkinsonian presentation (I.J.

[START_REF] Swift | Variable clinical phenotype in TBK1 mutations: case report of a novel mutation causing primary progressive aphasia and review of the literature[END_REF]

suggesting that TBK1 could be implemented in the gene panel associated with development of atypical parkinsonism.

  [START_REF] Cutler | Evidence that accumulation of ceramides and cholesterol esters mediates oxidative stressinduced death of motor neurons in amyotrophic lateral sclerosis[END_REF][START_REF] Dodge | Glycosphingolipids are modulators of disease pathogenesis in amyotrophic lateral sclerosis[END_REF] A. Henriques et al., 2017), this observation was further confirmed in murine model (A.Henriques et al., 2018;[START_REF] Fernández-Beltrán | A Transcriptomic Meta-Analysis Shows Lipid Metabolism Dysregulation as an Early Pathological Mechanism in the Spinal Cord of SOD1 Mice[END_REF].

Finally, the last lipidic family identified as dysregulated in ALS patients are the glycerophospholipids (E.

[START_REF] Area-Gomez | Lipidomics study of plasma from patients suggest that ALS and PLS are part of a continuum of motor neuron disorders[END_REF][START_REF] Goutman | Metabolomics identifies shared lipid pathways in independent amyotrophic lateral sclerosis cohorts[END_REF]

. Similarly to

  Moreover, glucose metabolism defects are suggested as the level of glycerol-3-phosphate (G3P) is impaired in our model. G3P is an intermediate in the glycolysis pathway. As mentioned in the introduction, glucose metabolism is altered in ALS patients because of either impaired cellular glucose uptake (T.W Tefera et al., 2021), defective glucose synthesis

regulation (T.

S. McDonald et al., 2021)

, or altered expression of enzymes involved in the catabolic degradation of glucose (L.

[START_REF] Palamiuc | A metabolic switch toward lipid use in glycolytic muscle is an early pathologic event in a mouse model of amyotrophic lateral sclerosis[END_REF] T.W Tefera

  measured values and 2.5 standard deviation downshift of the mean to simulate the distribution of low signal values. Finally, the remaining 8484 proteins were submitted to 2groups (NI vs TBK12) Student's T-test (volcano plot, FDR=0.05 and S0=1). From this comparison, 904 proteins were found statistically significant.

	Abbreviation Full name DNM1 Dynamin 1 MN Motor neurons SQSTM1 Sequestosome 1
	5-HT1 dpf mRNA STAT1	Serotonin 1A receptor Days post fecundation messenger ribonucleic acid Signal transducer and activator of transcription 1
	AA DPR mTOR STAT2	Amino acid Dipeptide repeats Mammalian target of rapamycin Signal transducer and activator of transcription 2
	ADAR2 EMA mTORC STING	Adenosine Deaminase Acting on dsRNA European medicines agency Mammalian target of rapamycin complex Stimulator of interferon genes
	AKT ER NAD+ TAK1	Protein kinase B Endoplasmic reticulum Nicotinamide adenine dinucleotide Mitogen-activated protein kinase kinase kinase 7
	ALR ERAD NAIP TANK	AIM2-like receptors Endoplasmic reticulum associated degradation NLR Family Apoptosis Inhibitory Protein TRAF Family Member Associated NFKB Activator
	ALS FADD NAMPT TARDBP	Amyotrophic Lateral Sclerosis Fas Associated Via Death Domain Nicotinamide phosphoribosyltransferase TAR DNA binding protein
	ALS2 FDA NAP1 TBK1	Alsin Rho Guanine Nucleotide Exchange Factor US Food and drug agency Nucleosome Assembly Protein 1 TANK-binding kinase 1
	AMDA FIP200 NDP52 TCA cycle	γ -α-amino-3-hydroxy-5-methylisoxazol-4-propionate RB1-inducible coiled-coil protein 1 Calcium-binding and coiled-coil domain-containing protein 2 Tricarboxylic acid cycle
	AMO FTD NEMO TDP43	Antisense morpholino oligonucleotide Fronto-temporal dementia NFκB essential modulator TAR DNA-binding protein 43
	AMPK FUS NF TLR	AMP-activated protein kinase Fused in Sarcoma Neurofilament Toll-like receptors
	ATG13 G3P NFkB TNF	Autophagy Related 13 Glycerol-3-phosphate Nuclear factor-kappa B Tumor necrosis factor
	ATP G4C2 NLR TRAF3	Adenosine tri-phosphate GGGGCC hexanucleotide repeat Nod-like receptors TNF Receptor Associated Factor 3
	ATXN2 GABA NLRP TRIF	Ataxin 2 γ-Aminobutyric acid Nucleotide-binding oligomerization domain TIR-domain-containing adapter-inducing interferon-?
	BAK GAS NMDA TUBA4A	BCL2 Antagonist/Killer Gamma activated sequence Acide N-methyl-D-aspartique Tubulin Alpha 4a
	BAX GLT1 NMN Tyr	Bcl-2-associated X protein Glutamate transporter 1 Nicotinamide mononucleotide Tyrosinase
	BCL-2 GMP NR UBQLN2	B-cell lymphoma 2 Guanine monophosphate Nicotinate riboside Ubiquilin 2
	BH4 GPI NSC34 UDP-GlcNAc Uridine diphosphate-N-acetylglucosamine Tetrahydrobiopterin Glycosylphosphatidylinositol Neuroblastoma x Spinal Cord-34
	BID GPL O-GlcNac ULD	BH3 interacting-domain death agonist Glycerophospholipid O-linked β-N-acetylglucosamine Ubiquitin like domain
	BIM GRIA2 OGT ULK	Bcl-2 Interacting Mediator of cell death Glutamate Ionotropic Receptor AMPA Type Subunit 2 O-GlcNAc transferase Unc-51 Like Autophagy Activating Kinase
	C9orf72 hpf OPTN UMP	Chromosome 9 open reading frame 72 Hours post fecundation Optineurin Uridine monophosphate
	CA IAP p62 UPR	Caffeic acid Inhibitor of Apoptosis Sequestosome 1 Unfolded protein response
	Ca2+ IFN PAMP VAPB	Calcium Interferon Pathogen associated molecular pattern molecules VAMP Associated Protein B
	Cas9 IFNAR PARP VCP	CRISPR-associated protein 9 Interferon-α/βreceptor Poly (ADP-ribose) polymerases Valosin Containing Protein
	CASP IFNGR PCD WB	Caspase Interferon-γ receptor Programmed cell death Western blot
	CD38 IFNLR PCR WDR41	Cyclic ADP ribose hydrolase Interferon-λ receptor Polymerase chain reaction WD repeat domain 41
	cFLIP PI3K-cIII WT	Cellular FLICE-like inhibitory protein Nuclear factor of kappa light polypeptide gene enhancer in B-cells Phosphatidylinositol-4,5-Bisphosphate 3-Kinase class III complex wild type
	cGAS IKBa PP2A YBH3	Cyclic GMP-AMP Synthase inhibitor Protein phosphatase 2 Yeast BH3 domain-containing protein
	CHCHD10 IKK PRR	Coiled-Coil-Helix-Coiled-Coil-Helix Domain Containing 10 Nuclear factor kappa-B kinase Pattern recognition receptors
	CHMP2B IL PUFA	Charged Multivesicular Body Protein 2B Interleukine Polyunsaturated fatty acid
	cIAP2/5 iPSC PUR-a	Baculoviral IAP repeat-containing protein Induced Pluripotent stem cells Purine Rich Element Binding Protein A
	CMP IRF QUIN	Cytosine monophosphate Interferon regulatory factor Quinolinic acid
	cN IRF3 RAPTOR	Cytosolic 5'-nucleotidases Interferon Regulatory Factor 3 Regulatory-associated protein of mTOR
	CNC1 ISG REE	Carney complex type 1 Interferon-stimulated gene Resting energy expenditure
	CNS ISG15 RIG-I	Central nervous system Interferon-stimulated gene 15 Retinoic acid-inducible gene I
	COX KD RIPK	Cyclooxygenase Kinase domain Receptor-interacting serine/threonine-protein kinase
	CRISPR KIF5A RLR	Clustered Regularly Interspaced Short Palindromic Repeats Kinesin Family Member 5A RIG-I like receptor
	CSF KMO RNA	Cerebro-spinal fluid Kynurenine 3-Monooxygenase Ribonucleic acid
	CTD LC3 ROS	C-terminal domain Microtubule-associated protein 1A/1B-light chain 3 Reactive oxygen species
	CYLD LUPAC SARM1	CYLD Lysine 63 Deubiquitinase Linear ubiquitin chain assembly complex Sterile Alpha aand TIR Motif Containing 1
	DAMP MAK2 SDD	Damage-associated molecular pattern molecules Male Germ Cell Associated Kinase 2 Scaffold dimerization domain
	DBR1 MAP3K7 SINTBAD	Debranching RNA Lariats 1 Mitogen-activated protein kinase kinase kinase 7 TANK-binding kinase 1-binding protein 1
	DCTN-1 MAVS SIRT	Dynactin Subunit 1 Mitochondrial Antiviral Signaling Protein Sirtuin
	DIABLO MDA5 SMCR8	Diablo IAP-Binding Mitochondrial Protein Melanoma differentiation-associated protein 5 Smith-Magenis Syndrome Chromosome Region candidate 8
	DMSO Mis SOD1	Dimethylsulfoxide Mismatch Superoxide dismutase type 1
	DNA MLKL SPG11	Deoxyribonucleic acid Mixed Lineage Kinase Domain-Like Pseudokinase Spastic paraplegia 11

supported by the activity of Ikkε, a kinase presenting overlapping function with Tbk1 (K.R. [START_REF] Balka | TBK1 and IKK? Act Redundantly to Mediate STING-Induced NF-?B Responses in Myeloid Cells[END_REF]. The measurement of ikkε mRNA levels via qPCR could unshadow an increased expression traducing a compensatory effect of Ikkε upon Tbk1 loss.

Metabolomic and proteomic targets in the tbk1 deletion mutants

In order to further understand the apparition of this locomotor phenotype, we performed a metabolomics analysis. From this analysis we have been able to associate the apparition of locomotor defects with a decreased level of NAD+ in the tbk1 KO larvae. Indeed, incubation with the NR precursor of NAD+ was sufficient to rescue the locomotor activity of the tbk1 KO larvae. This is not the first-time that decreased level of NAD+ are reported in the context of ALS (E. [START_REF] Blacher | Potential roles of gut microbiome and metabolites in modulating ALS in mice[END_REF][START_REF] Harlan | Evaluation of the NAD+ biosynthetic pathway in ALS patients and effect of modulating NAD+ levels in hSOD1-linked ALS mouse models[END_REF]. However, to our knowledge this is the first association between TBK1's impaired function and NAD metabolism dysregulation. This suggests that through an unknown mechanism, TBK1 regulates, indirectly or not, NAD+ stock. It is not clear at which step of the NAD+ metabolism TBK1 is involved but the decrease in NAD+ stock combined with an increase in NAM level suggest several mechanisms. Several catabolic reactions are associated with the degradation of NAD+ into NAM. Therefore, one could think that in response to TBK1 loss of activity, the rate of one of these enzymes is increased and therefore lead to the accumulation of NAM at the detriment of NAD+ level. Indeed, through their activity, the enzymes responsible of these reactions are regulating the pool of NAD+. Amongst these enzymes we can mention the PARPs family of protein (involved in the post-transcriptional addition of poly-ADP-ribosyl marks), the SIRTs protein (involved in proteic deacetylation), and the SARM1 and CD38 hydrolases (generating adenosine diphosphate ribose from NAD+) (N. [START_REF] Xie | NAD+ metabolism: pathophysiologic mechanisms and therapeutic potential[END_REF]. Interestingly, some of these NAD+ regulatory enzymes have been associated with ALS. For instance, SARM1 is a physiologically inactive protein that is involved in the axonal degradation upon lesion [START_REF] Essuman | The SARM1 Toll/Interleukin-1 Receptor Domain Possesses Intrinsic NAD+ Cleavage Activity that Promotes Pathological Axonal Degeneration[END_REF]. Mutation in SARM1 leading to constitutive activation of the encoded SARM1 protein was reported to be associated with the development of the ALS pathology (A.J. [START_REF] Bloom | Constitutively active SARM1 variants that induce neuropathy are enriched in ALS patients[END_REF]. Therefore, such a scenario could also be plausible in our model. To further deepen this hypothesis and investigate a potential increased rate of activity of these enzymes, their respective expressions should be quantified either at the transcriptional or protein level respectively by qPCR and WB.