Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal.

Amyotrophic lateral sclerosis (ALS) is a rapidly progressing neurodegenerative disorder characterized by loss of upper and lower motor neurons. TDP-43 was identified as a major protein component of the characteristic neuronal inclusions and it has been detected in 90% of ALS cases. Furthermore, pathogenic mutations in the gene encoding TDP-43, TARDBP, were found in both sporadic and familial ALS cases. The aim of this study is to investigate the molecular mechanisms of cellular dysfunction and ultimately death caused by TDP-43 mutations in human cells using established cell lines and human motor neurons derived from induced pluripotent stem cells (iPSCs). We generated a novel in vitro cellular model using a fluorescently tagged human genomic TARDBP locus carrying three ALS-specific mutations, A382T, M337V or Y374X. In site specific bacterial artificial chromosome (BAC) human stable cell lines, TDP M337V mislocalized to the cytoplasm more frequently than wild-type TDP-43 (TDP Ypet) and TDP-A382T, an effect potentiated by oxidative stress. Cytoplasmic mislocalization was significantly higher in TDP M337V cells compared to TDP-Ypet and correlated with cell death. Cells expressing the mislocalized TDP M337V mutant spontaneously developed cytoplasmic punctae, while for TDP-A382T punctae were only revealed after endoplasmic reticulum (ER) stress induced by the calcium-modifying drug thapsigargin (TG). Lowering Ca2+ concentration in the ER of TDP-Ypet cells partially recapitulated the effect of pathogenic mutations by increasing TDP-43 cytoplasmic mislocalization, suggesting Ca²⁺ dysregulation as a potential mediator of pathology. Ca²⁺ signaling from the ER was impaired in cells carrying TDP-43 mutations, with a 50% reduction in the levels of luminal ER Ca²⁺ stores content and delayed Ca²⁺ release induced by carbachol compared to TDP-Ypet cells. The deficits in Ca²⁺ release correlated with upregulation of Bcl-2 and siRNA-mediated knockdown of Bcl-2 restored amplitude of Ca²⁺ oscillations in TDP-M337V cells. These results suggest that TDP-43 pathogenic mutations elicit cytoplasmic mislocalization of TDP-43 through Bcl-2 regulation of ER Ca²⁺ signalling. Preliminary work in iPSC-derived motor neurons transduced with genomic DNA expression TDP-43 vectors using Herpes Simplex Virus type 1 (HSV-1) amplicons showed cytoplasmic redistribution of TDP-43 under high oxidative stress, without significant differences between mutations and wild-type. TDP-43 mutations delivered by HSV-1 amplicons also did not affect survival of iPSC-derived motor neurons. In ALS patient-derived motor neurons carrying C9orf72 expansions, TDP-43 pathology was not detected. However, preliminary data indicate that C9orf72 MNs present ER Ca²⁺ dysregulation with significantly high intracellular Ca²⁺ concentration, which correlates with high protein levels of ER stress markers and low levels of Bcl 2. This work highlights a potentially pathogenic role for TDP-43 mutations in the dysregulation of Ca²⁺ homeostasis and explores the use of iPS technology to investigate the effects of ALS-associated mutations in healthy and patient-derived motor neurons.

In der vorliegenden Dissertation wurden Neurone untersucht, die von einer speziellen Form der extrazellulären Matrix, dem perineuronalen Netz (PN), umgeben sind. Neurone mit einem PN zeichnen sich durch eine geringe Vulnerabilität bei neurodegenerativen Erkrankungen aus. Da das PN mit hoher Affinität Eisen bindet, war zu klären, ob das PN den Eisenhaushalt der Neurone beeinflusst und diese mit einer protektiven Eigenschaft gegenüber Eisen-induzierten oxidativen Stress ausstattet. Es wurde die Eisenkonzentration und der Gehalt an Eisentransport- und Eisenspeicherproteinen von Neuronen mit PN und Neuronen ohne PN in der Ratte untersucht. Dabei kamen quantitative Methoden wie die ortsaufgelöste Ionenstrahlmikroskopie und die Objektträger-basierte Laser Scanning Zytometrie sowie Western Blot Analysen und quantitative Real-Time-PCR zum Einsatz. Die Untersuchungen zeigen, dass Neurone, die mit einem PN umgeben sind, eine höhere Konzentration an Eisen sowie Eisentransport- und Eisenspeicherproteinen besitzen als Neurone ohne ein PN. Das PN könnte so den Eisenhaushalt der Neurone beeinflussen und diese mit einer protektiven Eigenschaft gegenüber Eisen-induziertem oxidativen Stress ausstatten.

Les adénovirus ont une double nature, soit comme pathogène omniprésent qui peuvent occasionnellement causer des maladies soit comme vecteurs utilisés de transfert de gène. À nos connaissances, les 30 premières minutes depuis la liaison au récepteur jusqu'à l'arrivée au pore nucléaire sont identiques pour le pathogène comme pour le vecteur. L'objectif de ma thèse était de comprendre les mécanismes impliqués dans la liaison au récepteur, l'internalisation, l'échappement et le trafic endosomal vers le MTOC. J'ai d'abord étudié le mécanisme impliqué dans l'hémagglutination des virus à tropisme pour CAR et à tropisme pour SA. J'ai identifié la présence de CAR sur les érythrocytes humains et montré qu'il était le principal responsable de l'agglutination induite par les virus à tropisme pour CAR. De plus, j'ai montré que la présence de CAR sur les érythrocytes pouvait piéger le virus dans le sang et ainsi empêcher l'infection au niveau du foie. Dans un deuxième temps, j'ai participé à la caractérisation du rôle de la protéine VI et la translocation du virus au MTOC. Nous avons montré que Nedd4 était impliqué dans le ciblage du virus au MTOC via l'ubiquitination de la protéine VI. Enfin, j'ai travaillé sur le neurotropisme de CAV-2 et caractérisé sa localisa tion subcellulaire au niveau des synapses. J'ai montré qu'une partie de CAR était localisée dans des radeaux lipidiques à la synapse et que CAV-2 entrait via la voie de recyclage des vésicules synaptiques. / Adenoviruses have a dual nature as ubiquitous pathogens that occasionally cause life-threatening disease and their use as gene transfer vectors. To the best of our current knowledge, the first 30 min from binding to nuclear pore docking of both wild-type virus and vector are identical. The goal of my thesis is to understand different mechanisms involved in receptor binding, internalization, endosomal escape and trafficking to the MTOC. First I studied the mechanism involved in hemagglutination of CAR-tropic and SA-tropic viruses. I identified the presence of CAR on human erythrocytes and showed that it was the main responsible for the agglutination mediated by CAR-tropic viruses. Moreover, I show that CAR on erythrocytes can sequester virus in the bloodstream and block liver infection. In a second part I participated to the characterization of the role of the protein VI and the translocation of HAd to the MTOC. We showed that Nedd4 was involved in the targeting of the virus to MTOC through ubiquitination of this protein VI. Finally, I worked on the neurotropism of CAV-2 and characterize its subcellular localization at the synapse. I showed that a part of CAR was localized in lipid raft at the synapse and enter through the synaptic vesicle-recycling pathway.

L’effet neuroprotecteur des agents anesthésiques est maintenant établi depuis plus de 30ans. Cependant, les mécanismes impliqués restent imparfaitement élucidés. A cours de cetravail nous avons étudié deux volets de leur protection :La première partie porte sur l’implication de la transmission glutaminergique dans leurseffets neuroprotecteurs directs, c'est-à-dire lorsqu’ils sont utilisés au cours d’une l’ischémiecérébrale. Nous avons étudié deux agents anesthésiques de classe distincte: le propofol et lesévoflurane sur des co-cultures de neurones et d’astrocytes corticaux de rat soumis à uneprivation en oxygène et en glucose transitoire (POG). Nous avons ainsi observé que laprésence de propofol ou de sévoflurane pendant la POG prévenait la mort neuronale,l’accumulation de glutamate extracellulaire et la diminution de la capture du glutamateinduites par l’ischémie. Nous avons également montré que cette restauration partielle del’activité de capture du glutamate impliquait des transporteurs distincts entre le propofol et lesévoflurane.La deuxième partie a porté sur la neuroprotection obtenue par un préconditionnement (PC)pharmacologique liée à l’utilisation avant l’ischémie d’agents anesthésique volatils. Nousavons tout d’abord confirmé in vitro l’existence d’une telle protection avec le sévoflurane etmis en évidence le rôle primordial, au cours de cette protection, des canaux potassiques ATPdépendantset des radicaux libres. Puis sur un modèle in vivo d’occlusion transitoire del’artère cérébrale moyenne, le PC par sévoflurane a induit une neuroprotection supérieure àcelle obtenue avec l’utilisation de sévoflurane uniquement pendant l’ischémie. Cependantcette protection est transitoire et ne perdure pas dans le temps. Le sévoflurane ne fait queretarder, sans l’empêcher, la mort neuronale liée à l’apoptose. Il offre cependant une fenêtrethérapeutique intéressante. / The neuroprotective effect of anesthetic agents is now established for over 30 years.However, the mechanisms involved remains to be fully explored. This work focuses on twoneuroprotective strategies:The first part is on the involvement of glutamatergic transmission in their directneuroprotective effects. We studied the effect of two separate classes of anesthetic agents:propofol and sevoflurane on co-cultures of cortical neurons and astrocytes from rats subjectedto a transient oxygen and glucose deprivation (OGD) mimicking cerebral ischemia. Weobserved that the presence of propofol or sevoflurane during OGD prevented neuronal death,accumulation of extracellular glutamate and decreased uptake of glutamate induced byischemia. We also demonstrated that this partial restoration of glutamate uptake mediated bypropofol and sevoflurane involved differential transporters.The second part deals with the neuroprotection achieved by pharmacologicalpreconditioning with regard to the use of volatile anesthetic agents before ischemia. We firstconfirmed in vitro the existence of such protection with sevoflurane. We also highlighted therole of ATP-dependent potassium channels and reactive oxygen species in sevofluranepreconditioning-induced neuroprotection. Then, using an in vivo model of focal transientischemia, we showed that sevoflurane preconditioning significantly improved functionaloutcome and reduced infarct volume. However, this protection was transient. Sevofluraneonly delayed the neuronal death associated with apoptosis but offers an interesting therapeuticwindow.

Amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA) are the commonest motor neuron diseases of adult- and childhood onset. Alterations of the RNA binding protein TDP-43 are associated with most cases of ALS, while SMA is caused by deletion of the Survival Motor Neuron (SMN1) gene. SMN has been well characterised in its role in the assembly of the cellular machinery that carries out splicing of pre-mRNA, but is thought to have other functions in RNA metabolism unrelated to pre-mRNA splicing. It is conceivable that specific aspects of RNA handling are disrupted in both SMA and ALS. A variety of genetic, molecular and neuropathological approaches were applied to investigate a potential common pathway in these diseases. The spectrum of genetic mutations underlying motor neuron disorders were explored by screening patient DNA. Cell culture and mouse models were used to test the hypothesis that altered pre-mRNA splicing causes motor neuron death. Human neuropathological specimens were examined for changes in proteins involved in RNA metabolism. The results indicate that altered pre-mRNA splicing is a late occurrence in disease and more likely to be a consequence rather than the cause of motor neuron degeneration. However, the notion that RNA metabolism is highly relevant to motor neuron diseases was strengthened by the discovery of mutations in another RNA binding protein, FUS, in cases of ALS without TDP-43 pathology. Overall the findings highlight the need to consider disruption of mRNA transport and regulation of mRNA translation in future motor neuron disease research.

Motor neuron disorders are a heterogeneous group of diseases characterized by the selective degeneration of motor neurons leading to muscle wasting and atrophy. Amyotrophic Lateral Sclerosis (ALS) is the most common amongst these disorders and is characterized by the selective loss of both upper and lower motor neurons in the brain and spinal cord. 20% of familial cases of ALS are caused by mutations in the Cu, Zn-superoxide dismutase gene (SOD1), a ubiquitously expressed enzyme responsible for scavenging superoxide radicals. The exact mechanisms underlying mutant SOD1-mediated neurotoxicity are unknown. Misfolded mutant SOD1 accumulates in the cytosol and mitochondrial intermembrane space (IMS) indicating the involvement of unfolded protein responses in ALS pathogenesis. Unfolded protein responses (UPRs) are complex signal transduction cascades which detect perturbations in protein folding and couple them to the expression of protein quality control machinery thereby allowing individual compartments to adapt to stress. In the cytosol, this study has shown that HspB8 was upregulated by SOD1 mutants, where it induced the clearance of aggregates by macroautophagy. This is a protective mechanism, as overexpression of HspB8 suppressed mutant-SOD1 mediated toxicity. In contrast, HspB8 mutants were impaired in macroautophagy and are toxic to NSC-34 cells. The mechanisms for the IMS-UPR have not been previously identified. To address this issue, a model for the accumulation of misfolded mutant SOD1 within the IMS was created and candidate proteins involved in protein quality control within the IMS were explored at the transcriptional level and at the level of protein expression. Preliminary results revealed some possible candidates that may have a role in the adaptation to mitochondrial stress. Interestingly, increased mitophagy was also found in IMS-G93A expressing cells, advocating the central role of macroautophagy in eliminating protein aggregates and damaged mitochondria in SOD1-FALS.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.