Friedreich ataxia (FRDA) is an inherited autosomal recessive neurodegenerative disorder caused by a GAA trinucleotide repeat expansion mutation within the first intron of the FXN gene. Normal individuals have 5 to 30 GAA repeats, whereas affected individuals have from approximately 70 to more than 1,000 GAA triplets. In addition to progressive neurological disability, FRDA is associated with cardiomyopathy and an increased risk of diabetes mellitus. Currently there is no effective therapy for FRDA and this is perhaps due to the lack of an effective system to test potential drugs. Therefore, the main aim of this thesis is to develop a novel cell culture system, to aid in rapid drug screening for FRDA. Firstly, I have demonstrated the establishment of novel cell culture systems, including primary fibroblasts, neural stem cells (NSC) and splenocytes, from FRDA YAC transgenic mouse models (YG8 and YG22). Then, I have shown the differentiation of NSCs into neurons, oligodendrocytes and astrocytes. The presence of these cells was confirmed by using cell specific immunofluorescence assays. I have also shown that both YG8 and YG22 rescue mice have less tolerance to hydrogen peroxide induced oxidative stress than WT mice, as similarly seen in FRDA patient fibroblasts. Recent findings indicate that FRDA is associated with heterochromatin-mediated silencing of the FXN gene accompanied by histone changes, flanking the GAA repeats. This suggested potential therapeutic use of compounds which can reduce the methylation and increase the acetylation of histone proteins. Therefore, using human and mouse primary fibroblast cell lines I have investigated the efficacy and tolerability of various DNA demethylating agents, GAA interacting compounds and class III histone deacetylase (HDAC) inhibitors. Although DNA demethylating agents showed increased FXN expression, no correlation between the level of DNA methylation and FXN expression was identified. Nevertheless, the use of GAA interacting compounds, particularly DB221, and the HDAC inhibitor, nicotinamide, have shown encouraging results, provoking us to use such compounds in future long-term in vivo studies. In addition, I have also investigated the long-term efficacy of two benzamide-type HDAC inhibitors, RGFA 136 and RGFP 109, on the FRDA YAC transgenic mice. No overt toxicity was identified with either drug, indicating a safe administration of these compounds. Both compounds produced improved functional analysis together with significantly reduced DRG neurodegeneration. However, neither of these compounds was shown to significantly increase the FXN mRNA expression. Nevertheless, elevated levels of frataxin protein in the brain tissues were obtained with RGFP 109, suggesting that RGFP 109 is capable of crossing the blood-brain barrier. I have also found increased levels of global acetylated H3 and H4 histone proteins in brain tissues, along with significant increase in aconitase enzyme activity, particularly with RGFP 109 treatments. Overall, these results support future clinical trial development with such compounds.

Le complexe EJC (Exon Junction Complex) joue un rôle central dans le couplage des processus post-transcriptionnels chez les métazoaires. Ce complexe multi-protéique est assemblé sur les ARN messagers (ARNm) par la machinerie d’épissage. Organisé autour d’un complexe cœur servant de plate-forme à de nombreux facteurs, les EJCs accompagnent les ARNm dans le cytoplasme et participent à leur transport, leur traduction et leur stabilité. L’importance physiologique de l’EJC est supportée par les nombreux défauts développementaux et les maladies génétiques associées aux composants de l’EJC. Les analyses transcriptomiques révélant un assemblage hétérogène des EJCs renforcent l’hypothèse que les EJCs participent à la régulation de l’expression des gènes. Cependant, malgré une connaissance précise de la structure de ce complexe, les liens fonctionnels entre l’assemblage de l’EJC et la régulation de transcrits spécifiques dans des conditions physiologiques doivent être établis puis caractérisés.Durant cette thèse, j’ai étudié l’expression d’eIF4A3, Y14 et MLN51, trois protéines du cœur de l’EJC, dans des cultures primaires de cellules souches neurales murines (CSN). Les CSN peuvent être différenciées en cellules épendymaires multi-ciliées qui tapissent les ventricules cérébraux et ont un rôle important dans le développement du cerveau. J’ai observé par immunofluorescence dans des CSN quiescentes que les 3 protéines sont concentrées autour du centrosome à la base du cil primaire. Ces localisations reflètent la présence d’EJC assemblés comme le prouve l’étude d’un mutant d’Y14 incapable de former l’EJC. / The Exon Junction Complex (EJC) plays a central role in coupling post-transcriptional processes in metazoans. This multi-protein complex is assembled onto messengers RNAs (mRNAs) by the splicing machinery. Organized around a core complex serving as a platform for numerous factors, EJCs accompany mRNAs to the cytoplasm and is involved in mRNA transport, translation and stability. The physiological importance of the EJC is supported by observations associating defects in EJC component expression to developmental defects and human genetic disorders. Transcriptomic studies revealing the non-ubiquitous deposition of EJCs strengthened the hypothesis that EJCs could participate to gene expression regulation. However, despite a precise picture of the structure of the EJC, functional links between EJC assembly and regulation of specific transcripts under physiological conditions is yet to be established.During this thesis, I studied the expression of eIF4A3, Y14 and MLN51 three core proteins of the EJC in primary cultures of mouse neural stem cells (NSCs). NSCs can be differentiated into multiciliated ependymal cells that line all brain ventricles and have important physiological functions in brain development. We observed by immunofluorescence that in quiescent NSCs, all three proteins are concentrated in the vicinity of the centrosome at the base of the primary cilia. This localization reflects the presence of fully assembled EJCs as proved by the study of Y14 mutant that prevent EJC core mounting.

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Dans le système nerveux central adulte, la glycine est principalement connue pour son rôle de transmission d’un signal inhibiteur à l'intérieur des neurones matures, régulant ainsi l'activité du réseau neuronal. Paradoxalement, durant l'embryogenèse, ce même neurotransmetteur génère une transmission excitatrice produisant ainsi le premier signal électrique dans les neurones immatures. Le rôle et la signification fonctionnelle de ce changement d’activité durant le développement neurologique restent toujours inconnus. En utilisant l’embryon du poisson-zèbre comme modèle, nous avons exploré les mécanismes moléculaires et cellulaires dépendants de la signalisation de glycine dans les cellules souches neuronales (CSNs). En premier lieu, nous avons développé un outil d’analyse basé sur une combinaison de deux éléments: une lignée transgénique qui exprime du GFP dans les CSNs et la technique de séquençage de l’ARN total. Nous avons utilisé cette technique pour isoler et déterminer les mécanismes moléculaires régulés par la glycine dans les CSNs. Ceci a permis d’identifier plusieurs gènes candidats dont l’expression est modulée par l’activité glycinergique. Ces gènes appartiennent principalement à cinq différentes voies de signalisation canoniques incluant la voie de signalisation du calcium, TGF-bêta, Shh, Wnt et p53. Pour en apprendre davantage sur ces mécanismes moléculaires, nous avons exploré l’un d’entre eux soit la régulation de la signalisation p53 par l’activité glycinergique. En effet, nous avons démontré que l’activité glycinergique favorise la survie des CSNs par la régulation de la signalisation de p53 et agit spécifiquement sur la sous-population CSN-nestin+ durant la neurogenèse. Dans un autre projet, nous avons examiné la régulation de l’expression de lnx1 par l’activité glycinergique. Nous avons démontré que la signalisation de glycine/lnx1 régule la prolifération des CSNs via la modulation de l’activité de Notch durant la neurogenèse. En conclusion, dans ce projet de thèse, j’ai mis en lumière plusieurs mécanismes moléculaires et cellulaires modulés par l’activité glycinergique dans les CSNs. Ceci peut contribuer dans le futur à la compréhension de la physiopathologie liée au dysfonctionnement de cette dernière ainsi qu’à l’identification de nouvelles cibles thérapeutiques. / In the adult central nervous system, glycine is mainly known as an inhibitory neurotransmitter in mature neurons, thereby regulating the neural network activity. Paradoxically, during embryogenesis, the same neurotransmitter generates excitatory transmission and induces the first electrical signal in immature neurons. The role and functional significance of this change in glycinergic activity during neurogenesis are still unknown. In this study, we used zebrafish embryos as a model to explore the glycine-dependent molecular and cellular mechanisms in neural stem cells (NSCs). First, we developed an in vivo analysis method based on two main elements: a transgenic line that expresses GFP within NSCs and the RNA sequencing technique. This method of analysis was used to determine glycine-dependent molecular mechanisms in NSCs. We identified several candidate genes whose expression is modulated by the glycinergic activity. These genes participate in five different canonical signaling pathways including the calcium signaling pathway, TGF-beta, Shh, Wnt and p53. To further understand these molecular mechanisms, we focused our investigation on the regulation of p53 signaling by the glycinergic activity. Indeed, we have demonstrated that glycinergic activity promotes the survival of NSCs by regulating p53 signaling and more specifically acting on NSC-nestin + subpopulation during neurogenesis. Finally, we explored the regulation of lnx1 expression by glycinergic activity. We have demonstrated that glycine/lnx1 signaling regulates the proliferation of NSCs via the modulation of Notch activity during neurogenesis. In conclusion, during this thesis project, I highlighted several molecular and cellular mechanisms modulated by the glycinergic activity in NSCs. These relevant results may contribute in the future to the understanding of the physiopathology related to glycinergic activity dysfunctions and the identification of new therapeutic targets.