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Les hétérotopies nodulaires périventriculaires (HNP) correspondent aux malformations cérébrales les plus fréquemment découvertes à l'âge adulte. Survenant au cours de la migration, elles consistent en l'apparition de nodules de neurones ectopiques positionnés le long de la paroi des ventricules latéraux. Sur le plan clinique, les HNP associent une épilepsie et/ou un retard mental. Les mutations dans le gène FLNA (Xq28) représentent la cause majeure des HNP. Une forme récessive rare d'HNP liée à des mutations du gène ARFGEF2 (20q13) et des réarrangements chromosomiques identifiés chez des patients présentant une HNP ont également été rapportés. Alors que le lien entre les HNP associées à des mutations du gène FLNA et leurs manifestations cliniques a été clairement établi, les mécanismes physiopathologiques sous-jacents restent à ce jour inconnus. Deux lignées de souris knockout pour FlnA ont été développées mais aucune de ces deux lignées n'a développé d'HNP. Nous avons donc choisi de créer un nouveau modèle, chez le rat, par inactivation in utero du gène FlnA en utilisant la technique de l'ARN interférence (RNAi). Par cette approche, nous avons reproduit avec succès un phénotype d'HNP chez le rat comparable à celui observé chez les patients. Sur ce modèle, nous avons montré que l'HNP est associée à une désorganisation de la glie radiaire et à une incapacité des progéniteurs neuronaux de progresser dans le cycle cellulaire. En accord avec ces observations, une désorganisation de la glie radiaire a été également observée dans des cerveaux post-mortem de deux patientes présentant une HNP associée à une mutation de FLNA. / Periventricular nodular heterotopia (PNH) is a brain malformation caused by defective neuronal migration resulting in ectopic neuronal nodules lining the lateral ventricles. Most patients have epilepsy, with normal to borderline cognitive function. Mutations in FLNA are the main cause of PH. A rare recessive form caused by mutations in the ARFGEF2 gene (20q13) and chromosomal rearrangements identified in patients with PNH have been reported. The link between FLNA-trelated PH and clinical manifestattions has been wee established but the underlying pathological mechanism remains unknown. Though two FlnA knockout mice strains have been developed, progress has been hindered by the fact that none of them showed the presence of ectopic nodules. Therefore, to recapitulate the loss of FlnA function in the developing rat brain, we used an in utero RNA interference (RNAi)-mediated knockdown approach and successfully reproduced a PNH phenotype in rats comparable to that observed in patients. Using this FlnA knockdown rodent model, we demonstrated that PNH is associated with a disruption in radial glial scaffold integrity in the ventricular zone and also an inability for neuroprogenitor cells to progress adequately through the cell cycle.Consistent with the observations made in rodents, we found similar alterations of radial glia in postmortem brains of two PNH patients harboring distinct FLNA mutations. These data highlights the complexity of the pathogenesis of PNH, the likelihood that several mechanisms are coalescing to lead to disrupted neuronal migration.

Des mutations du gène ARX (aristaless-related homeobox gene) ont été identifiées dans un large spectre de désordres neurologiques précoces, incluant ou non des malformations cérébrales, le plus souvent associés à des épilepsies. Il est proposé que le gène ARX, codant pour un facteur de transcription, joue un rôle primordial au cours du développement cérébral, notamment sur la migration des neurones GABAergiques, mais son implication au cours de la mise en place du système nerveux central reste cependant encore mal connue. L’objectif de ce travail a été d’étudier le rôle du gène ARX et les conséquences de ses mutations sur le développement cérébral dans le but de mieux comprendre ces pathologies. Dans un premier temps, nous avons étudié l’effet d’une mutation particulière du gène, la mutation ARX(CGC)7, une expansion polyalanine retrouvée principalement dans des pathologies sans malformation cérébrale mais avec des épilepsies, tels que les syndromes de West ou d’Ohtahara. Des analyses réalisées sur une lignée de souris knock-in pour cette mutation (GCG)7 et sur des rats après électroporation in utero ont montré que la migration neuronale des neurones glutamatergiques et GABAergiques ainsi que la maturation des neurones GABAergiques ne sont pas altérées par cette mutation. De façon intéressante, nos données suggèrent que les épilepsies observées chez les souris knock-in résulteraient plutôt d’une réorganisation du réseau glutamatergique. Etant donné que le gène ARX n’est pas exprimé dans les neurones glutamatergiques, l’ensemble de ce travail suggère donc que les épilepsies chez les souris knock-in pour la mutation (GCG)7 sont la conséquence d’une altération développementale secondaire à la mutation initiale du gène, et ceci aurait d’importantes répercussions thérapeutiques qui requièrent d’avantages d’études. Des expériences nous ont ensuite permis d’étudier l’effet de plusieurs mutations du gène ARX sur la morphologie des interneurones in vitro. Celles-ci ont montré que les mutations d’ARX n’engendrent pas une localisation subcellulaire anormale de la protéine dans les interneurones en culture. De façon intéressante, ces expériences suggèrent que la morphologie des interneurones est altérée seulement par certaines mutations, notamment les mutations P353R et Dup24. Ces données soulignent ainsi l’importance d’étudier de façon spécifique chaque mutation du gène pour expliquer les mécanismes engendrant l’hétérogénéité phénotypique liée aux mutations d’ARX. L’ensemble de ces travaux contribuent à une meilleure compréhension du rôle du gène ARX dans le développement cortical et à une meilleure caractérisation des mécanismes physiopathologiques des désordres neurologiques précoces liés aux mutations de ce gène. / Several mutations in ARX gene (aristaless-related homeobox gene) have been found in a large spectrum of infantile neurological disorders, with or without cerebral malformation, but frequently linked to epilepsy. It has been proposed that ARX, coding for a transcription factor, plays a crucial role in brain development, especially in migrating interneurons, but its involvement in nervous system development still remains to be clarified. The aim of this work has been to study the role of ARX gene and the consequences of ARX mutations on cerebral development in order to better understand these pathologies.We have first investigated the effects of an ARX polyalanine expansion, the mutation (GCG)7, which was found in pathologies without brain malformation but associated to epilepsy, such as West and Ohtahara syndromes. Analysis performed on knock-in mice for this mutation and in utero electroporated rat brains have shown that this mutation doesn’t alter neither glutamatergic and GABAergic neuronal migration, nor GABAergic neuron maturation. Interestingly, our data suggest that epilepsy observed in knock-in mice would result rather from a reorganization of glutamatergic networks. Since ARX gene is not expressed in excitatory neurons, our work suggests that epilepsy observed in knock-in mice is the consequence of developmental alterations secondary to the initial mutation, and this would have crucial therapeutic implications that require additional investigations. In vitro experiments have then allowed us to study the effect of several ARX mutations on interneurons morphology. These experiments have shown no abnormal subcellular localization of ARX protein following transfection of these different mutations in cultured interneurons. Interestingly, our data show that interneuron morphology is altered only by some mutations, particularly the P353R and the Dup24 ARX mutations. Our data underline the importance to study specifically each mutation in order to explain mechanisms generating phenotypic heterogeneity linked to ARX mutations.Taken together, this study contributes to a better understanding of ARX involvement in cerebral development and to a better characterization of pathophysiological mechanisms linked to ARX mutations.

Wnt signalling is indispensible for proper development of organisms and maintaining of adult tissue homeostasis. Its disruption often leads to disease. In nematode Caenorhabditis elegans, Wnt signalling governs vast array of developmental processes, among others also migration of the Q neuroblasts and their descendants. The sole Wnt acting in this process, EGL-20, triggers the canonical β-catenin Wnt signal transduction pathway in QL but not in QR which leads to QL remaining in the posterior while the QR migrates anteriorly. This represents a useful tool for studying Wnt signalling. Recently, mutation of gene cup-4 was found to disrupt migration of the QL neuroblast in a small proportion of the mutant population. cup-4 encodes a ligand-gated ion channel family homologue and it was shown to participate in endocytosis by coelomocytes, specialized phagocytic cells in the C. elegans body cavity. Here, I present the results of my effort to determine the place of CUP-4 action in Wnt signalling and to elucidate the mechanism of its function. I found that CUP-4 acts upstream of PRY- 1/Axin, which is involved in signal transduction in signal receiving cells, and most probably downstream of adaptin AP2, which is important for recycling of Wnt cargo receptor Wntless (Wls) in Wnt producing cell. cup-4 also...

Intellectual disability is a prevalent developmental disorder for which no effective treatments are available. Mutations of the X-linked protein PHF6 cause the Börjeson-Forssman-Lehmann syndrome (BFLS) that is characterized by intellectual disability and epilepsy. However, the biological role of PHF6 relevant to BFLS pathogenesis has remained unknown. Here, I present my dissertation research demonstrating that knockdown of PHF6 profoundly impairs neuronal migration in the mouse cerebral cortex in vivo, leading to the formation of white matter heterotopias that harbor aberrant patterns of neuronal activity. Importantly, BFLS patient specific mutation of PHF6 blocks its ability to promote neuronal migration. I also elucidate the mechanism by which PHF6 drives neuronal migration in the cerebral cortex. PHF6 physically associates with the PAF1 transcription elongation complex, and inhibition of PAF1 phenocopies the PHF6 knockdown-induced migration phenotype in vivo. I further identify Neuroglycan C (NGC), a susceptibility gene for schizophrenia, as a critical downstream target of PHF6 and the PAF1 complex, and I demonstrate that NGC mediates PHF6-dependent neuronal migration. These findings define PHF6, the PAF1 transcription elongation complex, and NGC as components of a novel cell-intrinsic transcriptional pathway that orchestrates neuronal migration in the brain, with important implications for the pathogenesis of intellectual disability and potentially other neuropsychiatric disorders.

<p>Neurons, with their enormous membrane contents, depend heavily on regulated membrane trafficking processes to maintain their morphology and function. The phosphatidylinositol 3-kinase class III, or PIK3C3, plays a critical role in various membrane trafficking processes including both the endocytic and autophagic pathways. The functions of PIK3C3 in the nervous system in vivo are un-characterized. We reasoned that studying PIK3C3 in neurons would provide us an entry point into understanding the regulations and functions of the neuronal membrane trafficking processes and their roles in neuronal morphogenesis and homeostasis. </p><p>We generated a conditional allele of Pik3c3 and first deleted it specifically in the peripheral sensory neurons. Mutant large-diameter myelinated sensory neurons accumulated numerous enlarged vacuoles and ubiquitin-positive aggregates and underwent rapid degeneration. By contrast, Pik3c3-deficient small-diameter unmyelinated neurons accumulated excessive numbers of lysosome-like organelles and degenerated slower than large-diameter neurons. These differential degenerative phenotypes are unlikely caused by a disruption of the autophagy pathway, because inhibiting autophagy alone by conditional deletion of Atg7 results in a completely distinct subcellular phenotypes and very slow degenerations of all sensory neurons. More surprisingly, a noncanonical PIK3C3-independent LC3-positive autophagosome formation pathway was activated in Pik3c3-deficient small-diameter neurons. This work uncovered unexpected differences of the endo-lysosomal systems in different types of neurons and discovered a novel autophagy initiation pathway in vivo in neurons. </p><p>To examine the role of PIK3C3 in the central nervous system (CNS), we next deleted Pik3c3 in CNS neural progenitor cells using the Nestin-Cre transgenic line. The resulting conditional knockout mice displayed a severe cortical lamination abnormality caused by defective cortical neuron migration. This finding uncovered a previously under-appreciated role of endocytic trafficking in neural migration, which was further confirmed by electron microscopic analyses of the developing cortex. Moreover, overexpressing the dominant negative forms of Dynamin2 or Rab5, two regulators of endocytosis, caused similar migration defects as Pik3c3-deletion. Mechanistically, Pik3c3-deficient cortical neurons drastically reduced surface Reelin binding sites, and showed significantly decreased levels of Dab1 phosphorylation, despite expressing normal total amount of Reelin receptor ApoER2. This work suggests endocytosis and recycling of Reelin receptors are likely to play an important role in cortical migration regulated by the Reelin signaling pathway. </p><p>These studies represent the first in vivo characterization of PIK3C3 functions in mammals, and provide insight into the complexity and functional importance of neuronal endo-lysosomal and autophagic pathways.</p> / Dissertation

The Distal-less homeobox (Dlx) genes encode a group of transcription factors that are involved in various developmental processes including forebrain development. Dlx genes are arranged in convergently transcribed bigene clusters with enhancer sequences located in the intergenic region of each cluster. The expression patterns of Dlx1/Dlx2 and of Dlx5/Dlx6 are attributed in part to the activity of I12a/I12b and I56i/I56ii intergenic enhancers, respectively. In an effort to determine how Dlx intergenic enhancers interact with the promoter regions of each cluster, I employed the Chromosome Conformation Capture (3C) technique on developing forebrain at E13.5 and E15.5. My 3C analysis provided potential enhancer-promoter interaction, in cis, that are consistent with previously known regulatory mechanisms. Furthermore, trans interactions may exist between Dlx1/Dlx2 and Dlx5/Dlx6 clusters in the developing forebrain at E13.5, thus providing a possible novel cross-regulatory mechanism between these two loci. I have also investigated the phenotypic consequences of Dlx enhancer deletion(s) on forebrain development by characterizing mice with I56ii and I56ii/I12b enhancer deletions. Enhancer deletions significantly impair Dlx expression as well as that of Evf2, Gad2 and of the striatal markers Islet1 and Meis2. Enhancer deletion(s) also reduce the expression of ISLET1 and CTIP2 proteins and Semaphorin 3A, Slit1 and Ephrin A5 that are thought to provide guidance cues in the corridor cells. Overall, these changes may disrupt the guidance of the thalamocortical axons. The data presented here further our understanding of the interactions between Dlx intergenic enhancers and promoter regions. Enhancer deletion(s) furthers our understanding of Dlx regulatory networks necessary that ensure proper Dlx expression, which, in turn may be involved in a genetic pathway underlying the synthesis of GABA, which may be further essential in maintaining the GABAergic phenotype.

Lymphocytic choriomeningitis virus (LCMV) infection during pregnancy severely injures the human fetal brain. Neonatal rats inoculated with LCMV are an excellent model of congenital LCMV infection, as they develop neuropathology, including cerebellar injuries, similar to those seen in humans. The goal of this thesis was to determine what underlies brain injury and the differential immune response and to determine the role of T-cells in LCMV induced pathology. First, I examined whether cytokine and chemokine expression after LCMV infection was higher in the cerebellum and olfactory bulbs, which undergo destruction, compared to the hippocampus and septum, which undergo no acute destruction. Second, I used T-cell deficient and T-cell competent animals to evaluate the role of T-lymphocytes in LCMV-induced cerebellar and hippocampus pathology. Finally, I characterized the migration abnormality that develops in the cerebellum after LCMV infection. My results showed that cytokine and chemokine expression is higher in the cerebellum and olfactory bulb than in the hippocampus and septum. Using astrocyte cultures, I determined that astrocytes isolated from the cerebellum have a more robust cytokine response to infection compared to astrocytes from the hippocampus. Furthermore, inoculation of congenitally athymic (rnu/rnu) rats, which are deficient in T-lymphocytes, demonstrated that cerebellar hypoplasia is T-cell independent while cerebellar destruction and abnormal neuron migration is T-cell dependent. In the hippocampus, T-cells protect against loss of dentate granule cells. A study of the migration abnormality determined that LCMV infection disrupts radial glia fibers and extends proliferation of granule cells in a T-cell dependent manner. The findings reported here support a pivotal role of the immune system in regional brain pathology as well as in the disruption of migration.

The Distal-less homeobox (Dlx) genes encode a group of transcription factors that are involved in various developmental processes including forebrain development. Dlx genes are arranged in convergently transcribed bigene clusters with enhancer sequences located in the intergenic region of each cluster. The expression patterns of Dlx1/Dlx2 and of Dlx5/Dlx6 are attributed in part to the activity of I12a/I12b and I56i/I56ii intergenic enhancers, respectively. In an effort to determine how Dlx intergenic enhancers interact with the promoter regions of each cluster, I employed the Chromosome Conformation Capture (3C) technique on developing forebrain at E13.5 and E15.5. My 3C analysis provided potential enhancer-promoter interaction, in cis, that are consistent with previously known regulatory mechanisms. Furthermore, trans interactions may exist between Dlx1/Dlx2 and Dlx5/Dlx6 clusters in the developing forebrain at E13.5, thus providing a possible novel cross-regulatory mechanism between these two loci. I have also investigated the phenotypic consequences of Dlx enhancer deletion(s) on forebrain development by characterizing mice with I56ii and I56ii/I12b enhancer deletions. Enhancer deletions significantly impair Dlx expression as well as that of Evf2, Gad2 and of the striatal markers Islet1 and Meis2. Enhancer deletion(s) also reduce the expression of ISLET1 and CTIP2 proteins and Semaphorin 3A, Slit1 and Ephrin A5 that are thought to provide guidance cues in the corridor cells. Overall, these changes may disrupt the guidance of the thalamocortical axons. The data presented here further our understanding of the interactions between Dlx intergenic enhancers and promoter regions. Enhancer deletion(s) furthers our understanding of Dlx regulatory networks necessary that ensure proper Dlx expression, which, in turn may be involved in a genetic pathway underlying the synthesis of GABA, which may be further essential in maintaining the GABAergic phenotype.

Le développement du cortex cérébral se déroule selon des étapes bien définies qui sont essentielles à la formation d’un cerveau fonctionnel. La perturbation de l’une ou plusieurs de ces étapes peut conduire à des malformations neuro-développementales, responsables de différents troubles cognitifs, d’épilepsies ou encore de déficience intellectuelle. De nombreuses mutations dans des gènes codant pour les tubulines ou bien les kinésines, sont retrouvées chez des individus présentant diverses anomalies neuro-développementales. Bien que les kinésines soient impliquées dans le développement cortical, les mécanismes fonctionnels par lesquels elles conduisent aux malformations demeurent encore méconnus. Mon travail de thèse identifie la kinésine Kif21b, jusqu’alors peu connue, comme étant essentielle au développement cortical. Nous montrons que Kif21b régule la migration neuronale dans le cortex et identifions quatre variants chez des individus présentant des malformations neuro-développementales. Nous montrons que l’expression ectopique des variants chez la souris et le poisson zèbre récapitulent les phénotypes observés chez ces patients. / The development of the cerebral cortex is a highly regulated process that is crucial for the establishment of functional cortical networks. Disruption of one or several of these steps can lead severe neurodevelopmental disorders that are associated with intellectual disabilities, epilepsies and cognitive impairment. Over the past few years, several genetic mutations in genes encoding either tubulin or microtubule-associated motors such as kinesins, have been found in individuals with neurodevelopmental disorders. Although kinesins have been found to be essential for a proper cortical development, the exact functions of kinesins in these processes are still poorly understood. My work clearly identified Kif21b, a poorly-known kinesin, as a novel key regulator of cortical development both in mouse and human. We show that Kif21b regulates both radial and tangential migration of cortical neurons, and identify four KIF21B variants in individuals presenting neurodevelopmental disorders. We show that ectopic expression of variants recapitulate phenotypes both in mice and zebrafish.