No description available.

No description available.

Congenital myasthenic syndrome (CMS) and myasthenia gravis (MG) are, respectively, inherited or autoimmunological disorders caused by aberrant neuromuscular transmission, which manifests as fatiguable muscle weakness. A novel subtype of CMS, resulting from mutations in GFPT1 and characterised by a limb girdle pattern of muscle weakness, has been described. The gene encodes L glutamine:D fructose-6-phosphate amidotransferase 1 (GFAT1) – a key rate limiting enzyme in the hexosamine biosynthetic pathway, providing building blocks for glycosylation of proteins and lipids. The research focused on the molecular bases of the CMS resulting from mutations in the ubiquitously expressed gene, but with symptoms largely restricted to the neuromuscular junction (NMJ). The work has established a link between the NMJ and GFPT1 CMS by demonstrating that the AChR cell surface is decreased in GFPT1 patient muscle cells and in GFPT1-silenced cell lines. The decrease is likely to be caused by reduced steady-state levels of individual AChR α, δ and ε, but not β, subunits. To optimise treatment for myasthenic disorders, a comparative in vivo trial of therapy with pyridostigmine bromide and salbutamol sulphate, and pyridostigmine bromide alone, was conducted. Supplementation of the AChE inhibitor-based therapy with the β2-adrenergic receptor agonist had a beneficial effect. This offers promise for more effective treatments for CMS and MG affected individuals. Molecular causes of MG were also investigated. The search for novel antibody targets was conducted with the use of a designed cell-based assay for the detection of anti COLQ autoimmunoglobulins in MG patient sera. The antibodies were detected in 24 out of 418 analysed samples, but their pathogenicity has not been determined.

No description available.

Durant le vieillissement, l’ensemble des fonctions de l’organisme se détériore, que ce soit aussi bien au niveau moteur que cognitif. Le vieillissement s’accompagne d’une diminution de la force, ainsi que de la masse musculaire. Des études récentes tendent à montrer que cette perte de masse musculaire que l’on appelle sarcopénie aurait pour origine un dérèglement de la jonction neuromusculaire. Les changements au niveau du présynaptique et du post synaptiques lors du vieillissement normal font l’objet de plusieurs études, mais les changements relatifs aux cellules de Schwann périsynaptique sont très peu connus. Le but de cette étude est donc d’analyser les modifications des interactions neurone-glie à la jonction neuromusculaire. Dans cette étude, nous montrons que certaines fonctions des cellules gliales de la synapse âgée sont déréglées, en particulier, le type de récepteurs activés par une stimulation nerveuse à haute fréquence. D’autre part, nos résultats montrent que les mécanismes responsables de l’augmentation de la transmission synaptique suite à cette stimulation nerveuse à haute fréquence sont altérés à la synapse âgée. Enfin, outre les modifications de la terminaison axonale et de la fibre musculaire, les cellules gliales montrent des signes de réorganisation structurelle propre à une synapse en réparation. Ces résultats montrent que le fonctionnement de la jonction neuromusculaire et a fortiori les interactions neurones-glie sont altérées lors du vieillissement normal. / Aging comes with an alteration and organism functions including cognitive and motor functions. Major weakening of the neuromuscular system occurs which includes muscle weight loss, difficulties in initiating voluntary movement and reduced muscle strength. The possible role of the alteration of the neuromuscular junction has been examined but always only considering the pre- and postsynaptic elements. However, perisynaptic Schwann cells (PSCs), glial cells at the neuromuscular junction (NMJ), play fundamental roles in the regulation of the synaptic efficacy of the NMJ as well as in its maintenance and stability. Hence, we analysed NMJ properties and their glial cells in aging. This study shows that PSCs function at the old NMJ are dysregulated. Indeed, PSCs ability to detect synaptic transmission, determined using imaging of intracellular Ca2+, was maintained in PSCs at NMJs from old mice, but the contribution of the muscarinic component was greatly reduced. On the other hand, our results using synaptic recordings are showing that a number of synaptic plasticity events known to be regulated by PSCs are reduced at NMJs of old mice. Finally, morphological NMJ reorganisation and sprouting of PSCs were also observed. These data suggest that PSC properties are consistent with the repair of the NMJ that may also result in their reduced ability in regulating synaptic efficacy.

Le système nerveux est composé de milliards de connexions synaptiques qui forment des réseaux complexes à la base de la communication dans le cerveau. Dès lors, contrôler la localisation, le type et le nombre des synapses est un défi considérable au cours du développement du système nerveux. Étonnamment, la production de connexions synaptiques est démesurée de façon à ce que beaucoup plus de synapses soient formées au cours du développement que ce qui est maintenu chez l’adulte. Ces connexions surnuméraires sont en compétition pour l’innervation d’une même cellule cible ce qui mène au maintien de certaines terminaisons nerveuses et à l’élimination de d’autres. Ces processus de compétition et d’élimination sont grandement façonnés par l’activité du système nerveux et l’expérience sensorielle de manière à ce que les terminaisons qui montrent la meilleure activité sont favorisées alors que les synapses mal adaptées sont éliminées. Jusqu’à récemment, les mécanismes et les types cellulaires responsables de l’élimination synaptique étaient inconnus. Les études de la dernière décennie montrent que les cellules gliales jouent un rôle clé dans l’élimination de synapses. Cependant, il demeure inconnu si les cellules gliales peuvent décoder les niveaux d’activité des terminaisons en compétition, ce qui est un déterminant majeur de l’issue de la compétition synaptique. De plus, il n’est pas connu si les cellules gliales sont capables de réguler l’activité synaptique des terminaisons, ce qui pourrait influencer l’issue de l’élimination synaptique. Ceci est d’un intérêt particulier puisqu’il est connu que les cellules gliales interagissent activement avec les neurones, détectent et modulent leur activité dans plusieurs régions du système nerveux mature. Par conséquent, l'objectif de cette thèse était d'étudier la capacité des cellules gliales à interagir avec les terminaisons nerveuses en compétition pour l'innervation d’une même cellule cible. Nous avons donc analysé la capacité des cellules gliales à décoder l’activité des terminaisons, à réguler leur activité synaptique et à influencer le processus de l’élimination synaptique au cours du développement du système nerveux. Pour cette fin, nous avons profité de la jonction neuromusculaire, un modèle simple et le bien caractérisé, et nous avons combiné l’imagerie Ca2+ des cellules gliales, un rapporteur fiable de leur activité avec des enregistrements synaptiques de jonctions neuromusculaires poly-innervées de souriceaux. Dans la première étude, nous montrons que les cellules gliales détectent et décodent l'efficacité synaptique des terminaisons nerveuses en compétition. L’activité des cellules gliales reflète la force synaptique de chaque terminaison nerveuse et l'état de la compétition synaptique. Ce décodage est médié par des récepteurs purinergiques gliaux fonctionnellement distincts et les propriétés intrinsèques des cellules gliales. Nos résultats indiquent que les cellules gliales décodent la compétition synaptique et, par conséquent, sont favorablement positionnées pour influencer son issue. Dans la seconde étude, nous montrons que les cellules gliales régulent différemment la plasticité synaptique de terminaisons en compétition. De manière dépendante du Ca2+, les cellules gliales induisent une potentialisation persistante de l’activité de la terminaison forte alors qu’elles n’ont que peu d’effets sur la terminaison faible. Bloquer l'activité gliale altère la plasticité des terminaisons in situ et se traduit par un retard de l'élimination des synapses in vivo. Ainsi, nous décrivons un nouveau mécanisme par lequel les cellules gliales, non seulement renforcent activement la terminaison forte, mais influencent aussi la compétition et l'élimination. Dans l'ensemble, ces études sont les premières à démontrer que les cellules gliales sont activement impliquées dans la modulation de l'activité synaptique des terminaisons en compétition ainsi que dans la régulation de l'élimination synaptique et la connectivité neuronale. / The nervous system is composed of billions of synaptic connections forming complex networks that define the basis of neuronal communication in the brain. The control of the localization, type and number of synapses is a considerable challenge during development of the nervous system. Surprisingly, there is an excessive production of synaptic connections so that many more synapses are formed during developmental stages than what is maintained in the adult. A process of competition and elimination then occurs during which connections are in competition for the innervation of the same target cell. These processes of competition and elimination are greatly shaped by activity and sensory experience. Nerve terminals that show the best activity are favoured, while weak and poorly adapted synapses are eliminated. Until recently, the mechanisms and the cell types responsible for the elimination of supernumerary connections were unknown. Studies from the last decade identified glial cells as major players in synapse elimination. However, it remains unknown whether glial cells are able to decode the levels of synaptic activity of competing terminals, which is a major determinant of the outcome of synaptic competition. Moreover, it is unknown whether glial cells are able to regulate synaptic activity, which could influence the outcome of synapse elimination. This is especially relevant because it is known that glial cells actively interact with neurons, detect and modulate their activity in many regions of the nervous system. Therefore, the goal of this thesis was to study the ability of glial cells to interact with terminals competing for the innervation of the same target cell. We tested the ability of glial cells to decode the activity nerve terminals, regulate their synaptic activity and influence the process of synapse elimination during development of the nervous system. For this purpose, we took advantage of the neuromuscular junction, a simple and well-characterized model, and used simultaneous Ca2+-imaging of glial cells, a reliable reporter of their activity and synaptic recordings of dually-innervated neuromuscular junctions from newborn mice. In the first study, we report that single glial cells detect and decode the synaptic efficacy of competing nerve terminals. Activity of single glial cells reflects the synaptic strength of each competing nerve terminal and the state of synaptic competition. This deciphering is mediated by functionally segregated purinergic receptors and intrinsic properties of glial cells. Our results indicate that glial cells decode ongoing synaptic competition and, hence, are poised to influence its outcome. In the second study, we show that glial cells differentially regulate the synaptic plasticity of competing terminals. In a Ca2+-dependent manner, glial cells induce a long lasting synaptic potentiation of strong but not weak terminals. Preventing glial activity alters the plasticity of terminals in situ and delays synapse elimination in vivo. Thus, we describe a novel mechanism by which glial cells, not only actively reinforce the strong input but regulate synapse competition and elimination. As a whole, these studies are the first to demonstrate that glial cells are actively involved in the modulation of synaptic activity of competing terminals as well as in the regulation of synapse elimination and neuronal connectivity.

Congenital myasthenic syndromes (CMS) are a group of heterogeneous disorders caused by mutations in genes encoding for proteins that are essential for neuromuscular transmission. All CMS share the clinical feature of fatigable muscle weakness. The differential diagnosis of CMS is wide, with a range of diseases going from autoimmune myasthenia gravis to muscle disorders. In this thesis, it was shown that measuring antibodies to clustered acetylcholine receptors (AChRs) by cell-based assay is helpful in the differential diagnosis of CMS. The findings of the current investigations showed that mutations in COL13A1, encoding the Collagen Type XIII α1 chain, were responsible for the symptoms of several patients with previously undefined myasthenias. In addition, this work described the clinical and complementary features of a novel CMS subtype due to mutations in the glycosylation pathway gene GMPPB. Investigations on a novel MUSK missense mutation (p.Ala617Val) uncovered previously unrecognised mechanisms of how levels of MuSK phosphorylation are critical to maintain synaptic structure, and guided suitable treatment for the patient. The study on the clinical and molecular basis of stridor, a novel clinical feature recently identified in patients with DOK7-CMS, prompted the identification of a novel DOK7 isoform, which warrants further investigation to elucidate its role in AChR clustering. Finally, the therapy of patients with severe AChR-deficiency was optimised thanks to a case series study that showed a robust improvement following the addition of β2-adrenergic agonists to their long-term treatment regime that included pyridostigmine.

Au cours du développement du système nerveux, l'activité des cibles post-synaptiques permet le raffinement du nombre et de la force des connexions neuronales. En employant la jonction neuromusculaire de Caenorhabditis elegans comme système modèle, nous avons étudié deux aspects de la mise en place de ces connexions. D'une part, nous montrons que le nombre de récepteurs présents à la jonction neuromusculaire est contrôlé par l'activité musculaire : une augmentation de l'activation synaptique entraîne une régulation différentielle des trois types de récepteurs présents à la jonction neuromusculaire. D'autre part, nous avons étudié les changements de la morphologie de certains motoneurones de la tête du ver, appelés neurones SAB, en fonction de l’activité musculaire. Une diminution de l’activité musculaire durant une période critique du développement entraîne une surcroissance axonale des neurones SAB. À travers différentes approches, nous avons pu identifier la suppression de la surcroissance axonale dans des mutants où la biosynthèse des neuropeptides est perturbée. Enfin, nous avons mis en évidence que la surcroissance axonale apparait également lors de perturbations plus générales de la physiologie cellulaire, telles qu'un choc thermique ou la surexpression d'un transgène, ce qui suggère que le système SAB est plastique et particulièrement sensible au cours du développement / Throughout nervous system development, activity of the post-synaptic targets can regulate the connectivity of neural networks, affecting both the number and strength of synapses. Using the neuromuscular junction of Caenorhabditis elegans as a model system, we studied two processes displaying such plasticity. First, we show that the number of receptors present at the neuromuscular synapse is regulated by muscle activity: an increase in synaptic activity can lead to a differential regulation of the three types of receptors present at the neuromuscular junction. Second, we studied the activity-dependent morphological changes of one type of motor neurons in the worm’s head, called the SAB neurons. A decrease of muscle activity during a critical developmental phase leads to SAB axonal overgrowth. Using several approaches, we were able to observe suppression of SAB axonal overgrowth in mutants with a disruption of neuropeptides biosynthesis. Finally, we give evidence that axonal overgrowth also occurs following more general disruptions of cell physiology, such as a heat-shock or transgene overexpression, which suggest that the SAB system is plastic and sensitive during development

The nervous system is made up of specialized cells which receive and respond to environmental stimuli. Intercellular communication in the nervous system is achieved predominantly through chemical synaptic transmission. Within the chemical synapse, the actin cytoskeleton plays a major role in regulating synaptic activities, although the extent and clarity in our understanding of these processes is still limited. Using the genetically pliable model, Drosophila melanogaster, this thesis begins to unravel contributions of actin binding proteins to synaptic development and physiology at the larval neuromuscular junction (NMJ). Two actin binding proteins, Moesin and Nonmuscle Myosin II (NMMII) were selected for study based on previous studies implicating them in synaptic development. Combining genetics, fluorescent imaging and electrophysiological recordings this thesis unveils previously unidentified functions for Moesin and NMMII in morphology and physiology of the Drosophila NMJ. Moesin was found to help restrain synaptic growth but did not affect synaptic physiology. By correlating morphological and electrophysiological measurements in Moesin mutants, it was determined that physiology and morphology can be independently regulated at the NMJ. NMMII was used to investigate a role for actin binding proteins in physiology at the Drosophila NMJ. By using the fluorescent imaging technique, FRAP, this becomes the first research to implicate NMMII in unstimulated synaptic vesicle mobility. FRAP indicated that vesicle mobility was highly dependent on the expression level of NMMII. Electrophysiological analysis of NMMII indicated distinct mechanisms for spontaneous and evoked vesicle release. NMMII expression exhibited a positive correlation with basal synaptic transmission and was important in mobilizing vesicles for synaptic potentiation. In addition, NMMII was found to be involved in a high frequency dependent low frequency depression. This work begins to identify how vesicles traverse within boutons and suggests differential mechanisms of synaptic release, both of which are partially dependent of NMMII expression. Studying Moesin and NMMII have revealed a complex interplay between the actin cytoskeleton and synaptic function and together this research furthers our understanding of how the actin cytoskeleton regulates synaptic activity.

The nervous system is made up of specialized cells which receive and respond to environmental stimuli. Intercellular communication in the nervous system is achieved predominantly through chemical synaptic transmission. Within the chemical synapse, the actin cytoskeleton plays a major role in regulating synaptic activities, although the extent and clarity in our understanding of these processes is still limited. Using the genetically pliable model, Drosophila melanogaster, this thesis begins to unravel contributions of actin binding proteins to synaptic development and physiology at the larval neuromuscular junction (NMJ). Two actin binding proteins, Moesin and Nonmuscle Myosin II (NMMII) were selected for study based on previous studies implicating them in synaptic development. Combining genetics, fluorescent imaging and electrophysiological recordings this thesis unveils previously unidentified functions for Moesin and NMMII in morphology and physiology of the Drosophila NMJ. Moesin was found to help restrain synaptic growth but did not affect synaptic physiology. By correlating morphological and electrophysiological measurements in Moesin mutants, it was determined that physiology and morphology can be independently regulated at the NMJ. NMMII was used to investigate a role for actin binding proteins in physiology at the Drosophila NMJ. By using the fluorescent imaging technique, FRAP, this becomes the first research to implicate NMMII in unstimulated synaptic vesicle mobility. FRAP indicated that vesicle mobility was highly dependent on the expression level of NMMII. Electrophysiological analysis of NMMII indicated distinct mechanisms for spontaneous and evoked vesicle release. NMMII expression exhibited a positive correlation with basal synaptic transmission and was important in mobilizing vesicles for synaptic potentiation. In addition, NMMII was found to be involved in a high frequency dependent low frequency depression. This work begins to identify how vesicles traverse within boutons and suggests differential mechanisms of synaptic release, both of which are partially dependent of NMMII expression. Studying Moesin and NMMII have revealed a complex interplay between the actin cytoskeleton and synaptic function and together this research furthers our understanding of how the actin cytoskeleton regulates synaptic activity.