We recently showed that the Wiskott-Aldrich syndrome protein (WASP) family member,WASH, localizes to endosomal subdomains and regulates endocytic vesicle scission in an Arp2/3-dependent manner. Mechanisms regulating WASH activity are unknown. Here we show that WASH functions in cells within a 500 kDa core complex containing Strumpellin, FAM21, KIAA1033 (SWIP), and CCDC53. Although recombinant WASH is constitutively active toward the Arp2/3 complex, the reconstituted core assembly is inhibited, suggesting that it functions in cells to regulate actin dynamics through WASH. FAM21 interacts directly with CAPZ and inhibits its actin-capping activity. Four of the five core components show distant (approximately 15% amino acid sequence identify) but significant structural homology to components of a complex that negatively regulates the WASP family member, WAVE. Moreover, biochemical and electron microscopic analyses show that the WASH and WAVE complexes are structurally similar. Thus, these two distantly related WASP family members are controlled by analogous structurally related mechanisms. Strumpellin is mutated in the human disease hereditary spastic paraplegia, and its link to WASH suggests that misregulation of actin dynamics on endosomes may play a role in this disorder.

No description available.

Au cours des dernières années, la génétique a subi une progression phénoménale suite au développement de nouvelles technologies de séquençage. En effet, le séquençage de l’exome entier chez des familles a permis l’identification de nouveaux gènes impliqués pour plusieurs maladies. La neurologie a d’ailleurs bénéficié de ces avancées et plusieurs gènes ont été mis en évidence comme causatifs pour différents désordres neurologiques. Dans ce travail il sera question de deux désordres du mouvement pour lequel nous avons utilisés des technologies de séquençage traditionnelles, en l’occurrence le séquençage par Sanger, ainsi que de nouvelles technologies pour le séquençage de l’exome entier afin d’identifier de nouveaux gènes causatifs. Le premier désordre du mouvement qui sera décrit est l’ataxie, où ne seront abordées que les ataxies de cause génétiques, à transmission récessive. Le premier chapitre relatera les nouvelles mutations qui ont été trouvées chez des canadiens-français souffrant de l’ataxie de Beauce. Il sera aussi question de nouvelles mutations retrouvées dans deux autres populations, confirmant l’implication du gène SYNE1 dans les cas d’ataxie cérébelleuse à travers le monde. Le second chapitre fera la démonstration qu’il est souhaitable d’utiliser le séquençage de l’exome entier dans le but de poser un diagnostic clinique. En effet, il a été possible de trouver la cause génétique d’une famille comportant deux membres atteints d’atrophie congénitale du cervelet, où le symptôme prédominant est l’ataxie. Le séquençage de l’exome a permis la mise en évidence de mutations dans le gène PMM2, déjà connues pour cause le syndrome des glycoprotéines déficientes en hydrates de carbone. Dans un second temps, il sera question d’un autre désordre du mouvement la paraplégie spastique familiale (PSF). Le chapitre 3 relatera les mutations trouvées dans le gène CYP7B1 dans notre cohorte de patients PSF. / Over the past years, genetics has undergone a phenomenal growth due to the development of new sequencing technologies. Indeed, whole exome sequencing in families led to the identification of new genes involved in many diseases. Neurosciences were also able to benefit from these discoveries, where several new genes have been identified in several neurological diseases. This thesis will covered two different movement disorders for which we have used traditional sequencing technology, in this case by Sanger sequencing, combined with whole exome sequencing for new gene discovery. The first movement disorder that is described is ataxia, which will be focused on autosomal recessive mode of inheritance. The first chapter will relate the new mutations found in French-Canadian with ataxia of Beauce. We will also discuss new mutations found in two other populations, confirming the involvement of the gene SYNE1 in worldwide cases of cerebellar ataxia. The second chapter will demonstrate that it is desirable to use whole exome sequencing in order to make a clinical diagnosis. Indeed, it has been possible to find the genetic cause for a family with two members with congenital cerebellar atrophy, which the predominant symptom is ataxia. Exome sequencing allowed the identification of mutations in the PMM2 gene, already known to cause a syndrome leading to glycosylation deficit of glycoprotein. For the second part, we will cover another movement disorder call hereditary spastic paraplegia (HSP). Chapter 3 relates the mutations found in the CYP7B1 gene in our cohort of HSP patients.

As doenças do neurônio motor (DNM) apresentam grande variabilidade clínica e genética. A Esclerose Lateral Amiotrófica (ELA) é a forma mais comum de DNM de início tardio. Sua manifestação devastadora e incurável leva a uma profunda perda da qualidade de vida do paciente. A ELA8 é uma forma autossômica dominante de ELA familial causada por mutações no gene VAPB. A proteína VAPB está envolvida com diversos processos celulares. Nossos dados sugerem que a mutação P56S na VAPB diminui a interação dela com outras duas outras proteínas: Tubulina e GAPDH. Por terem sido previamente relacionadas a outras formas de doenças neurodegenerativas, essas proteínas são potenciais pontos-chave para desvendar a patogênese da ELA8 e de outras formas de DNM. Um número significativo de drogas testadas com sucesso em modelos animais de ELA não pôde ser transferido para os pacientes, trazendo à tona a necessidade de novos sistemas de estudo da doença. A tecnologia das iPSC torna possível a reprogramação celular para um estágio de pluripotência, permitindo a modelagem de doenças in vitro. Apresentamos um novo modelo de ELA baseado em células iPSC derivadas de pacientes com ELA8. Este modelo apresenta uma redução dos níveis de VAPB quando comparado com os controles. Contudo, não identificamos os agregados intracitoplasmáticos, característicos dos modelos de super-expressão. Mostramos pela primeira vez que células-tronco embrionárias humanas normais expressam VAPB. Adicionalmente, nossos resultados sugerem que os pacientes com ELA8 teriam uma redução dos níveis de VAPB desde o início de suas vidas e revelam a importância de modelar a ELA num background humano. Dessa forma, sugerimos a hipótese de que quantidades específicas de VAPB sejam cruciais para a viabilidade dos neurônios motores. A busca por alterações no número de cópias levou à identificação do primeiro caso de uma duplicação multi-exônica (exon10_12dup) no gene SPG4, ampliando o espectro de mutações encontradas nesse gene. Esta mutação leva à formação de um códon de parada prematuro, sugerindo que a proteína produzida não é funcional. A análise de 30 indivíduos portadores desta mutação mostrou que os homens possuem, na média, idade de início antecipada e quadro clínico mais severo. Esses dados sugerem que certas doenças neuromusculares podem ser moduladas por fatores relacionados ao background individual e ao gênero. Em suma, apoiamos a idéia de que os microtúbulos estariam envolvidos na patogênese das doenças neuromusculares, visto que tanto a VAPB quanto a Espastina interagem com estes elementos do citoesqueleto. Ainda, apresentamos um novo modelo in vitro de análise da ELA e além de justificar os estudos num background humano. / The motor neuron diseases (MND) show a huge clinical and genetic variability. Amyotrophic Lateral Sclerosis (ALS) is the most common late-onset form of MND. ALSs devastating and incurable manifestation leads to a profound loss of life quality. ALS8 is an autosomal dominant form of ALS caused by mutations in the VAPB gene. The VAPB protein is involved with many cellular processes and our data suggest that P56S mutation in this protein reduces its interaction with two other proteins: Tubulin and GAPDH. Since these proteins were previously related to other types of neurodegenerative diseases they potentially are key points to reveal the processes responsible for ALS8 and other MND. A substantial number of successful drug tests in ALS animal models could not be translated to humans, showing the need of novel ALS systems. iPSC technology made possible cellular reprogramming. The iPSC technology brings new hope in this area since it can be used to model diseases in vitro. Here we present a new ALS model based on ALS8-iPSC. Compared to control samples, this model shows a reduction of VAPB levels. However, we could not identify intracytoplasmic aggregates, which characterize overexpression models. We show for the first time that human embryonic stem cells express VAPB. Combined with results showing a VAPB reduction in ALS8 samples, it suggests that ALS8 patients present diminished protein levels since the beginning of their lives and reveal the importance of modeling ALS in a human background. The search for copy number variations has led to the identification of the first multiexonic duplication (exon10_12dup) in SPG4 gene, expanding the mutation spectrum in this gene. This mutation leads to a premature stop codon, which suggests that the expressed protein is not functional. The analysis of 30 individuals who carry the mutation showed that males have on average an earlier AAO and are more severely affected. These data suggest specific neuromuscular diseases can be modulated by factors related to individual background and gender. In sum, we support the idea that microtubules can be involved with neuromuscular disorders pathogenesis, since both VAPB and Spastin interact with these cytoskeleton components. Additionally, we present a new in vitro model to ALS analysis and we justify the studies in a human background.

As doenças do neurônio motor (DNM) apresentam grande variabilidade clínica e genética. A Esclerose Lateral Amiotrófica (ELA) é a forma mais comum de DNM de início tardio. Sua manifestação devastadora e incurável leva a uma profunda perda da qualidade de vida do paciente. A ELA8 é uma forma autossômica dominante de ELA familial causada por mutações no gene VAPB. A proteína VAPB está envolvida com diversos processos celulares. Nossos dados sugerem que a mutação P56S na VAPB diminui a interação dela com outras duas outras proteínas: Tubulina e GAPDH. Por terem sido previamente relacionadas a outras formas de doenças neurodegenerativas, essas proteínas são potenciais pontos-chave para desvendar a patogênese da ELA8 e de outras formas de DNM. Um número significativo de drogas testadas com sucesso em modelos animais de ELA não pôde ser transferido para os pacientes, trazendo à tona a necessidade de novos sistemas de estudo da doença. A tecnologia das iPSC torna possível a reprogramação celular para um estágio de pluripotência, permitindo a modelagem de doenças in vitro. Apresentamos um novo modelo de ELA baseado em células iPSC derivadas de pacientes com ELA8. Este modelo apresenta uma redução dos níveis de VAPB quando comparado com os controles. Contudo, não identificamos os agregados intracitoplasmáticos, característicos dos modelos de super-expressão. Mostramos pela primeira vez que células-tronco embrionárias humanas normais expressam VAPB. Adicionalmente, nossos resultados sugerem que os pacientes com ELA8 teriam uma redução dos níveis de VAPB desde o início de suas vidas e revelam a importância de modelar a ELA num background humano. Dessa forma, sugerimos a hipótese de que quantidades específicas de VAPB sejam cruciais para a viabilidade dos neurônios motores. A busca por alterações no número de cópias levou à identificação do primeiro caso de uma duplicação multi-exônica (exon10_12dup) no gene SPG4, ampliando o espectro de mutações encontradas nesse gene. Esta mutação leva à formação de um códon de parada prematuro, sugerindo que a proteína produzida não é funcional. A análise de 30 indivíduos portadores desta mutação mostrou que os homens possuem, na média, idade de início antecipada e quadro clínico mais severo. Esses dados sugerem que certas doenças neuromusculares podem ser moduladas por fatores relacionados ao background individual e ao gênero. Em suma, apoiamos a idéia de que os microtúbulos estariam envolvidos na patogênese das doenças neuromusculares, visto que tanto a VAPB quanto a Espastina interagem com estes elementos do citoesqueleto. Ainda, apresentamos um novo modelo in vitro de análise da ELA e além de justificar os estudos num background humano. / The motor neuron diseases (MND) show a huge clinical and genetic variability. Amyotrophic Lateral Sclerosis (ALS) is the most common late-onset form of MND. ALSs devastating and incurable manifestation leads to a profound loss of life quality. ALS8 is an autosomal dominant form of ALS caused by mutations in the VAPB gene. The VAPB protein is involved with many cellular processes and our data suggest that P56S mutation in this protein reduces its interaction with two other proteins: Tubulin and GAPDH. Since these proteins were previously related to other types of neurodegenerative diseases they potentially are key points to reveal the processes responsible for ALS8 and other MND. A substantial number of successful drug tests in ALS animal models could not be translated to humans, showing the need of novel ALS systems. iPSC technology made possible cellular reprogramming. The iPSC technology brings new hope in this area since it can be used to model diseases in vitro. Here we present a new ALS model based on ALS8-iPSC. Compared to control samples, this model shows a reduction of VAPB levels. However, we could not identify intracytoplasmic aggregates, which characterize overexpression models. We show for the first time that human embryonic stem cells express VAPB. Combined with results showing a VAPB reduction in ALS8 samples, it suggests that ALS8 patients present diminished protein levels since the beginning of their lives and reveal the importance of modeling ALS in a human background. The search for copy number variations has led to the identification of the first multiexonic duplication (exon10_12dup) in SPG4 gene, expanding the mutation spectrum in this gene. This mutation leads to a premature stop codon, which suggests that the expressed protein is not functional. The analysis of 30 individuals who carry the mutation showed that males have on average an earlier AAO and are more severely affected. These data suggest specific neuromuscular diseases can be modulated by factors related to individual background and gender. In sum, we support the idea that microtubules can be involved with neuromuscular disorders pathogenesis, since both VAPB and Spastin interact with these cytoskeleton components. Additionally, we present a new in vitro model to ALS analysis and we justify the studies in a human background.

How organelles acquire their unique shapes is a fundamental question of cell biology. The peripheral endoplasmic reticulum (ER) consists of a vast network of membrane sheets and tubules, the formation of which requires homotypic membrane fusion. Previous studies suggest that the dynamin-like GTPase, atlastin (ATL), mediates ER fusion, but the mechanism by which this occurs is unclear. In this study, I investigate 1) the role of dimerization and conformational changes in the N-terminal domain of ATL, 2) how the C-terminal amphipathic helix and the transmembrane domain of ATL cooperate with the N-terminal domain, and 3) the formation of cis and trans ATL dimers in the fusion mechanism. ATL has a cytosolic N-terminal domain, consisting of a GTPase domain and three-helix bundle (3HB), followed by two transmembrane segments (TMs) and a cytosolic C-terminal tail (CT). Crystal structures of ATL and biochemical experiments suggest that nucleotide-dependent dimerization between ATL molecules sitting in different membranes can tether the membranes together. A subsequent conformational change triggered by GTP hydrolysis could pull the membranes toward one another for fusion. This mechanism is supported by in vitro membrane tethering and fusion assays using vesicles containing full-length Drosophila ATL. The CT and TMs of ATL are also required for efficient membrane fusion. A synthetic peptide corresponding to a conserved amphipathic helix in the CT can act in trans to restore the fusion activity of a tailless ATL mutant. We characterize CT mutants to show that the C-terminal helix promotes fusion by perturbing the lipid bilayer. The TMs of ATL also mediate nucleotide-independent oligomerization, which may allow ATL molecules in the same membrane to synchronously undergo the conformational change leading to fusion. Lastly, we show that continuous GTP hydrolysis is required for membrane tethering, occasionally resulting in fusion. The N-terminal cytosolic domain mediates trans dimer formation between ATL molecules on different membranes. GTP binding induces dimerization through the GTPase domains and 3HBs. We propose that GTP hydrolysis and phosphate release are required not just to drive fusion, but also to dissociate cis dimers that form on the same membrane, thus allowing ATL molecules to form trans dimers.

Les ataxies spastiques héréditaires forment une famille hétérogène de désordres qui ont des points communs avec les ataxies héréditaires et les paraplégies spastiques héréditaires. Un de ces éléments est une ataxie, soit une difficulté de coordination des membres souvent due à un dommage au cervelet. L’autre est une spasticité des membres inférieurs, souvent due à des dommages à la voie cortico-spinale. Une seule ataxie spastique à hérédité autosomique dominante a été rapportée dans la littérature, et il s’agit de SPAX1. À l’aide de trois familles de Terre-Neuve présentant ce phénotype, le locus a été identifié en 2002. Dans ce mémoire, c’est de la découverte du gène causal dont il est question. La mutation a été trouvée dans le gène VAMP1, qui encode la protéine synaptobrévine 1, une protéine synaptique impliquée dans l’exocytose des neurotransmetteurs. Il est aussi question de la caractérisation fonctionnelle de la mutation sur l’ARN et des conséquences possibles sur la protéine, concordant avec les symptômes de la maladie. / Hereditary spastic ataxias comprise a family of heterogeneous disorders resembling both hereditary ataxias and hereditary spastic paraplegias. The similar symptoms are ataxia, which is a problem with limb coordination due to cerebellar damage, and lower-limb spasticity due to corticospinal tract degeneration. Only one spastic ataxia inherited in an autosomal dominant fashion has been reported in the literature: SPAX1. The locus was identified in 2002 using three families from Newfoundland with the specific phenotype. This thesis reports the discovery of the causative mutation in the VAMP1 gene, which encodes VAMP1/synaptobrevin 1, a synaptic protein involved in neurotransmitter exocytosis. Experiments characterizing the effect of the mutation on RNA were conducted, leading to a possible molecular explanation of the symptoms.

Les ataxies spastiques héréditaires forment une famille hétérogène de désordres qui ont des points communs avec les ataxies héréditaires et les paraplégies spastiques héréditaires. Un de ces éléments est une ataxie, soit une difficulté de coordination des membres souvent due à un dommage au cervelet. L’autre est une spasticité des membres inférieurs, souvent due à des dommages à la voie cortico-spinale. Une seule ataxie spastique à hérédité autosomique dominante a été rapportée dans la littérature, et il s’agit de SPAX1. À l’aide de trois familles de Terre-Neuve présentant ce phénotype, le locus a été identifié en 2002. Dans ce mémoire, c’est de la découverte du gène causal dont il est question. La mutation a été trouvée dans le gène VAMP1, qui encode la protéine synaptobrévine 1, une protéine synaptique impliquée dans l’exocytose des neurotransmetteurs. Il est aussi question de la caractérisation fonctionnelle de la mutation sur l’ARN et des conséquences possibles sur la protéine, concordant avec les symptômes de la maladie. / Hereditary spastic ataxias comprise a family of heterogeneous disorders resembling both hereditary ataxias and hereditary spastic paraplegias. The similar symptoms are ataxia, which is a problem with limb coordination due to cerebellar damage, and lower-limb spasticity due to corticospinal tract degeneration. Only one spastic ataxia inherited in an autosomal dominant fashion has been reported in the literature: SPAX1. The locus was identified in 2002 using three families from Newfoundland with the specific phenotype. This thesis reports the discovery of the causative mutation in the VAMP1 gene, which encodes VAMP1/synaptobrevin 1, a synaptic protein involved in neurotransmitter exocytosis. Experiments characterizing the effect of the mutation on RNA were conducted, leading to a possible molecular explanation of the symptoms.

No description available.

Les ataxies (HCA) et paraparésies spastiques héréditaires constituent les deux extrémités du spectre des entités neurodégénératives spinocérébelleuses (SCE). Elles sont marquées par une forte hétérogénéité clinique, avec des signes associés variés, et génétique. Elles peuvent se transmettre sur tous les modes d'hérédité, et des mutations ont été décrites dans une myriade de gènes. Les SCE sont donc une entité qui bénéficie particulièrement des avancées technologiques de la Nouvelle Génération de Séquençage. Ce travail décrit des résultats obtenus sur de grandes cohortes, par séquençage de panel de gènes ciblés ou de l'exome entier, ainsi que des études de familles. Celles-ci nous ont permis de décrire de nouveaux modes de transmission de mutations dans des gènes déjà connus en pathologie humaine, avec un dans un cas une dysfonction similaire, dans l'autre un gain versus une perte de fonction. Nous rapportons aussi deux gènes nouvellement impliqués, dans une forme autosomique dominante de HCA (CACNA1A), et dans un sous-type autosomique récessif de dystonie avec atrophie cérébelleuse (TOR1AIP1). Nos résultats illustrent bien la refonte nosologique en marche dans les maladies génétiques complexes, qui remettent en permanence les corrélations génotype-phénotype en question. Nous discutons du pourquoi et du comment du diagnostic moléculaire dans cette nouvelle ère du séquençage. / Hereditary cerebellar ataxias (HCA) and spastic paraplegias constitute both ends of the neurodegenerative spectrum of spinocerebellar entities (SCE). Theses diseases are marked by a pronounced heterogeneity, both clinically, with various additional neurological or extraneurological signs, and genetically. They can indeed follow all transmission modes, and mutations in a myriad of genes have been described. SCE is hence a group of diseases that benefit greatly from Next-Generation Sequencing technologies. This work reports both screenings of large cohorts of patients with either panel or whole exome sequencing, as well as family studies. The latter allowed us to describe new modes of transmission for genes previously involved in human pathology, with either similar protein dysfunction, or loss- versus gain-of-function. We also describe two new genes implicated in a form of autosomal dominant HCA (CACNA1A), and an autosomal recessive subtype of dystonia and cerebellar atrophy (TOR1AIP1). Our results are illustrative of the genetic remodelling underway in complex genetic diseases, with permanent questioning of genotype-phenotype correlations. We discuss the how and the why of molecular diagnosis in this new era of sequencing.