Alterações morfológicas e funcionais ocorrem durante o envelhecimento, período da vida com maior incidência de doenças neurodegenerativas. No presente trabalho acompanhou-se a evolução dos grânulos de lipofucsina durante o envelhecimento para investigar alterações sinápticas, assim como proteínas associadas com doenças neurodegenerativas (alfa-sinucleína) e com o sistema ubiquitina-proteossoma em indivíduos de diferentes idades. No córtex temporal humano e de ratos determinou-se, nos diferentes grupos etários, seguindo a área, o número e as características dos grânulos de lipofucsina, o número de sinapses excitatórias, inibitórias e elétricas, os locais de contatos pós-sinápticos, o número de vesículas sinápticas por terminal e a expressão das proteínas alfa-sinucleína e ubiquitina. Amostras de córtex temporal humano de indivíduos com diferentes idades (20 - 28, 37 - 41 e 50 - 55 anos) foram coletadas de pacientes com epilepsia submetidos à lobectomia do lobo temporal. Amostras de ratos de 2, 6, 10 e 12 meses também foram coletadas. Foram utilizadas técnicas de microscopia de luz, eletrônica, confocal e western blots. Os dados obtidos de grânulos de lipofucsina são consistentes com outros estudos que observaram aumento dessa estrutura em mamíferos de maior idade. No entanto, os grânulos parecem crescer em volume, mas não em número, com aumento considerável da fração elétron lúcida (lipídica). Não houve diferença na expressão das proteínas alfa-sinucleína e ubiquitina entre os grupos das idades estudadas. A densidade sináptica foi similar entre os grupos experimentais, assim como o local de contato pós-sináptico. O aumento de vesículas elétron densas em sinapses inibitórias deve estar associado à demanda de neurotransmissores catecolaminérgicos. Estes resultados não expressam totalmente o processo de envelhecimento, pois as faixas etárias de humanos e ratos correspondem a uma idade ainda jovem. A coleta de material humano mais idoso foi impossibilitada pela faixa etária dos doentes submetidos à lobectomia. Os ratos do biotério da FMRP, não sobrevivem mais do que 12 meses em no nosso ambiente, incluindo manutenção dos animais isolados em racks. / Morphological and functional changes occur during the aging, period of life with increased incidence of neurodegenerative diseases. Following the evolution of the lipofuscin granules along three periods of life in humans and rats, the present work investigated synaptic changes, as well as proteins associated with neurodegenerative diseases (alpha-synuclein) and the ubiquitin-proteossoma system in individuals of different ages. The objectives of the study were to analyze the temporal cortex of humans and rats: the number of excitatory, inhibitory, and electric synapses, the site of postsynaptic contacts, the number of synaptic vesicles per terminal, and the expression of the proteins alpha-synuclein and ubiquitin following the size and features of the lipofuscin granules. Samples of temporal cortex of human subjects with different ages (20-28, 37-41 and 50-55 years) were collected from patients with epilepsy who underwent temporal lobectomy. Samples from rats of 2, 6, 10 and 12 months were also collected. Light, confocal, and electron microscopy, and western blots techniques were used as procedures. The data obtained on lipofuscin granules were coincident with other studies that observed a higher area occupied by this structure in older mammals. However, the granules seem to grow in volume, but not in number, with considerable increase of the electron lucid fraction (lipidic). There was no difference in the alpha-synuclein and ubiquitin expressions between the experimental groups. The synaptic densities were similar between the groups, as well as the postsynaptic contacts. Increase of the electron dense vesicles in inhibitory synapses, appeared to be associated with the demand of catecholamines. These results do not express totally the aging process, because the range of age used in humans, and rats belong to a young age. The human samples from older ages was difficult because, in general, of the age of the patients submitted to lobotomy. The rats of the FMRP bioterium do not survived more that 12 months in our environment, even in controlled conditions.

Ce travail porte sur des approches translationnelles dans les synucléinopathies notamment l’atrophie multisystématisée (AMS). Au-delà de leur rôle dans la régulation du glucose, l’insulin et l’insulin like growth factor-1 (IGF-1) ont des propriétés neurotrophiques. Des études ont montrées que la signalisation de l’insuline/IGF-1 est altérée dans la maladie d'Alzheimer et des données suggèrent l’altération de l’insuline/IGF-1 dans la maladie de Parkinson (MP) et l’AMS. Nous avons mis en évidence une résistance à l’insuline dans les neurones des patients MP et AMS ainsi que dans les oligodendrocytes chez les patients AMS.Mon travail a également consisté à cibler la troncation de l’α-synuclein (α-syn) comme cible thérapeutique. Nous avons démontré dans un modèle murin d’AMS que la diminution de l’α-syn tronquée permettait de réduire l’agrégation d’α-syn et la dégénérescence des neurones dopaminergiques.Enfin, nous avons étudié l’implication dans l’AMS des métalloprotéinases matricielles (MMP), des enzymes impliquées dans remodelage de la matrice, la démyélinisation, la troncation de l’α-syn et la perméabilité de la barrière hémato-encéphalique. Ce travail nous a permis de montrer une augmentation de l’expression et de l’activité de MMPs chez les patients AMS. Nous avons également montré que les cellules gliales sont la source de cette augmentation et que la MMP-2 est retrouvée dans les agrégats des patients AMS.Nous montrons ici de caractéristiques distinctes de l’AMS comme des altérations qui se produisent dans les oligodendrocytes. Nous présentons aussi VX-765 comme un candidat prometteur pour ralentir la progression de la pathologie dans un contexte de synucléinopathie. / This work focused on translational approaches in synucleinopathies and more specifically in multiple system atrophy (MSA). Beyond their role in glucose homeostasis, insulin/IGF-1 are neurotrophic factors in the brain. Studies have shown altered insulin/IGF-1 signalling in Alzheimer’s disease and data suggest impaired insulin signaling/IGF-1 in Parkinson's disease (PD) and MSA. The aim of my work was to characterize insulin/IGF-1 signalling in MSA and PD brain tissue. Both groups showed neuronal insulin resistance. Oligodendrocytes in MSA patients were also insulin resistant.In line with the translational approach, we also targeted α-synuclein (α-syn) truncation pharmacologically in MSA transgenic mice, which led to reduced α-syn aggregation and the protection of dopaminergic neurons.We also assessed the activity and distribution of matrix metalloproteinases (MMPs) in the brain of MSA patients compared to healthy controls. MMPs are involved in the remodelling of the extracellular matrix, demyelination, α-syn truncation and blood brain barrier permeability. We showed altered expression and activity of MMPs in two distinct structures in MSA brains. We were also able to show that glial cells were the source of increased MMPs and show a unique expression of MMPs in α-syn aggregates of MSA patients compared to PD, evidence that might hint at a mechanism that is differently altered between PD and MSA.We here show distinct pathological features of MSA such as key alterations occurring in oligodendrocytes, further supporting MSA as a primary oligodendrogliopathy. We also present VX-765 as a candidate drug for disease modification in synucleinopathies.

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The formation and deposition of α-Synuclein fibrils in the human brain is at the origin of Parkinson's disease. The objective of my thesis was to document the role of two molecular chaperones on the assembly of α-Syn into fibrils: Hsc70, a constitutively expressed human heat shock protein, and Ssa1p, its yeast equivalent. The aim was to expand the catalogue of known effects of molecular chaperones on the PD implicated protein, which could have therapeutic significance. We showed that Hsc70 inhibits the assembly of α-Syn into fibrils, by binding with high affinity to the soluble form of α-Syn. We documented that Hsc70 binds preferentially to α-Syn fibrils and that this binding has a cytoprotective effect, as it renders the fibrils less toxic to cultured mammalian cells. Similarly to Hsc70, Ssa1p inhibits the assembly of α-Syn into fibrils, and has a higher affinity for fibrils than for the soluble form of α-Syn. On the other hand, binding of Ssa1p to α-Syn fibrils does not have a cytoprotective effect, almost certainly due to differences in the amino acid sequences of the peptide binding sites of the two molecular chaperones, which mean that Ssa1p has a lower affinity than Hsc70 for α-Syn fibrils. We stabilized the complex between Ssa1p and α-Syn using chemical cross-linkers, to then map the interaction site between the two proteins. This is indispensable if a "mini" Ssa1p, comprised of only what is necessary and sufficient of Ssa1p, is to be used as a therapeutic agent to decrease the toxicity of α-Syn fibrils. A therapeutic agent based on exogenous protein Ssa1p is less likely to trigger an autoimmune response than for example the endogenous protein Hsc70.

Alterações morfológicas e funcionais ocorrem durante o envelhecimento, período da vida com maior incidência de doenças neurodegenerativas. No presente trabalho acompanhou-se a evolução dos grânulos de lipofucsina durante o envelhecimento para investigar alterações sinápticas, assim como proteínas associadas com doenças neurodegenerativas (alfa-sinucleína) e com o sistema ubiquitina-proteossoma em indivíduos de diferentes idades. No córtex temporal humano e de ratos determinou-se, nos diferentes grupos etários, seguindo a área, o número e as características dos grânulos de lipofucsina, o número de sinapses excitatórias, inibitórias e elétricas, os locais de contatos pós-sinápticos, o número de vesículas sinápticas por terminal e a expressão das proteínas alfa-sinucleína e ubiquitina. Amostras de córtex temporal humano de indivíduos com diferentes idades (20 - 28, 37 - 41 e 50 - 55 anos) foram coletadas de pacientes com epilepsia submetidos à lobectomia do lobo temporal. Amostras de ratos de 2, 6, 10 e 12 meses também foram coletadas. Foram utilizadas técnicas de microscopia de luz, eletrônica, confocal e western blots. Os dados obtidos de grânulos de lipofucsina são consistentes com outros estudos que observaram aumento dessa estrutura em mamíferos de maior idade. No entanto, os grânulos parecem crescer em volume, mas não em número, com aumento considerável da fração elétron lúcida (lipídica). Não houve diferença na expressão das proteínas alfa-sinucleína e ubiquitina entre os grupos das idades estudadas. A densidade sináptica foi similar entre os grupos experimentais, assim como o local de contato pós-sináptico. O aumento de vesículas elétron densas em sinapses inibitórias deve estar associado à demanda de neurotransmissores catecolaminérgicos. Estes resultados não expressam totalmente o processo de envelhecimento, pois as faixas etárias de humanos e ratos correspondem a uma idade ainda jovem. A coleta de material humano mais idoso foi impossibilitada pela faixa etária dos doentes submetidos à lobectomia. Os ratos do biotério da FMRP, não sobrevivem mais do que 12 meses em no nosso ambiente, incluindo manutenção dos animais isolados em racks. / Morphological and functional changes occur during the aging, period of life with increased incidence of neurodegenerative diseases. Following the evolution of the lipofuscin granules along three periods of life in humans and rats, the present work investigated synaptic changes, as well as proteins associated with neurodegenerative diseases (alpha-synuclein) and the ubiquitin-proteossoma system in individuals of different ages. The objectives of the study were to analyze the temporal cortex of humans and rats: the number of excitatory, inhibitory, and electric synapses, the site of postsynaptic contacts, the number of synaptic vesicles per terminal, and the expression of the proteins alpha-synuclein and ubiquitin following the size and features of the lipofuscin granules. Samples of temporal cortex of human subjects with different ages (20-28, 37-41 and 50-55 years) were collected from patients with epilepsy who underwent temporal lobectomy. Samples from rats of 2, 6, 10 and 12 months were also collected. Light, confocal, and electron microscopy, and western blots techniques were used as procedures. The data obtained on lipofuscin granules were coincident with other studies that observed a higher area occupied by this structure in older mammals. However, the granules seem to grow in volume, but not in number, with considerable increase of the electron lucid fraction (lipidic). There was no difference in the alpha-synuclein and ubiquitin expressions between the experimental groups. The synaptic densities were similar between the groups, as well as the postsynaptic contacts. Increase of the electron dense vesicles in inhibitory synapses, appeared to be associated with the demand of catecholamines. These results do not express totally the aging process, because the range of age used in humans, and rats belong to a young age. The human samples from older ages was difficult because, in general, of the age of the patients submitted to lobotomy. The rats of the FMRP bioterium do not survived more that 12 months in our environment, even in controlled conditions.

L'α-synucléine est reconnue comme une protéine clé dans la physiopathologie de la maladie de Parkinson ainsi que d'autres troubles neurodégénératifs appelés synucléinopathies. Dans ces maladies, la surexpression de l’α-synucléine entraîne la formation d'agrégats toxiques dans les neurones dopaminergiques (DA). Dans cette thèse, nous avons exploré l’effet de la régulation de microARN (miARN) sur l’expression de l’α-synucléine. Pour se faire, des études ont été menées avec la lignée cellulaire humaine SH-SY5Y qui peut être différenciée pour créer un modèle de neurones DA et ensuite traitée avec une neurotoxine pour induire des caractéristiques cellulaires de la maladie de Parkinson. Des observations importantes ont été supportées dans des modèles cellulaires plus avancés, notamment les neurones induits par reprogrammation directe de fibroblastes humains (iNs) et les neurones DA primaires de souris purifiés. Le premier objectif était de mieux comprendre comment la surexpression aberrante de l'α synucléine dans les synucléinopathies pourrait être due à une dérégulation de la maturation des miARN qui ciblent son ARN messager. Tout d’abord, nous avons sélectionné les miARN les plus susceptibles d'avoir un effet régulateur sur l’expression de l’α-synucléine à partir de recherche de la littérature et d’analyse de bases de données spécialisées. Nous avons observé que l’augmentation de l'expression de l'α-synucléine associée à l’ajout de neurotoxine est accompagnée d’une diminution concomitante de l'expression de plusieurs miARN sélectionnés. Sur la base de ces résultats, l'impact de ces miARN sur l'expression de l'α-synucléine a été évalué dans plusieurs types de cellules humaines, notamment les HEK 293T, les SH-SY5Y différenciées et les iNs. À cette fin, nous avons utilisé des cibles de miARN exogènes pour réprimer l'activité régulatrice des miARN et avons mesuré leur effet sur l'expression de l'α synucléine. Ainsi, nous avons démontré que la répression de miR-7, miR-93, miR-140, miR 153 et miR 214 mène systématiquement à la surexpression de l’α-synucléine dans les différents types de cellules. De plus, nous avons démontré que certains miARN sont régulés de manière post-transcriptionnelle en mesurant les niveaux des formes immatures et matures des miARN dans différents contextes cellulaires. Le deuxième objectif était d’identifier des protéines potentiellement aptes à réguler la maturation post-transcriptionnelle de miARN. Des études de purification par affinité et de spectrométrie de masse ont permis d'identifier les protéines qui s’associent avec la tige-boucle des formes immatures des miARN et régulent potentiellement leur maturation. Quelques protéines candidates ont été sélectionnées sur la base d’analyse informatique pour examiner l’effet de leur surexpression dans différents essais cellulaires. À ce jour, nous avons identifié quatre protéines (MIF, PCBP2, Prohibitin-2, and Tfr1) qui, en plus de répondre à certains critères de bases (lient l’ARN, sont présentes dans le cerveau et impliquées dans des maladies associées au système nerveux), ont un effet sur l’activité et l’expression de miR-153 ainsi que sur l’expression de l’α-synucléine. Ces travaux ont permis d’établir de solides bases dans notre compréhension de la régulation de l'α-synucléine par les miARN et d’ouvrir la voie à des études plus élaborées qui permettront d’établir les mécanismes de régulation des niveaux de miARN qui ciblent l’α-synucléine. À plus long terme, cet axe de recherche pourrait fournir des pistes pour le développement d'outils diagnostiques et thérapeutiques pour les synucléinopathies. / Alpha-synuclein is a key protein in the pathophysiology of Parkinson's disease and other neurodegenerative disorders called synucleinopathies. In these diseases, overexpression of α-synuclein leads to the formation of toxic aggregates in dopaminergic (DA) neurons. In this thesis, we explored the effect of microRNA (miRNA) regulation on α-synuclein expression. To do so, studies were conducted with the human SH-SY5Y cell line, which can be differentiated to create a model of DA neurons and then treated with a neurotoxin to induce cellular features of Parkinson's disease. Important observations were supported in more advanced cell models, including neurons induced by direct reprogramming of human fibroblasts (iNs) and purified primary mouse DA neurons. The first objective was to better understand how aberrant overexpression of α-synuclein in synucleinopathies results in the deregulation of the maturation of miRNAs that target its messenger RNA. First, we selected the miRNAs most likely to have a regulatory effect on α-synuclein expression based on literature searches and specialized database analyses. We observed that the increase in α-synuclein expression associated with neurotoxin addition is accompanied by a concomitant decrease in the expression level of several selected miRNAs. Based on these results, the impact of these miRNAs on αsynuclein expression was evaluated in several human cell types, including HEK 293T, differentiated SHSY5Y, and iNs. To this end, we used exogenous miRNA targets to repress miRNA regulatory activity and measured their effect on α-synuclein expression. Thus, we demonstrated that repression of miR-7, miR-93, miR-140, miR-153, and miR-214 consistently leads to overexpression of α-synuclein in different cell types. In addition, we demonstrated that some miRNAs are regulated in a posttranscriptional manner by measuring the levels of immature and mature forms of miRNAs in different cellular contexts. The second objective was to identify proteins potentially able to regulate the post-transcriptional maturation of miRNAs. Affinity purification and mass spectrometry studies were used to identify proteins that associate with the stem-loop of immature forms of miRNAs and potentially regulate their maturation. A few candidate proteins were selected based on computational analysis to examine the effect of their overexpression in different cell-based assays. To date, we have identified four proteins (MIF, PCBP2, Prohibitin-2, and Tfr1) that, in addition, to fitting basic criteria (known to bind RNA, are present in the brain and associated with nervous system-related diseases) affect miR-153 activity and expression as well as α-synuclein expression. This work has established a solid foundation in our understanding of the regulation of α-synuclein by miRNAs and has paved the way for more elaborate studies that will establish the mechanisms of regulation of miRNA levels that target α-synuclein. In the longer term, this line of research could provide avenues for the development of diagnostic and therapeutic tools for synucleinopathies.

Les maladies neurodégénératives liées à l’âge, telle celle de Parkinson, sont un problème majeur de santé publique. Cependant, la maladie de Parkinson reste incurable et les traitements sont très limités. En effet, les causes de la maladie restent encore mal comprises et la recherche se concentre sur ses mécanismes moléculaires. Dans cette étude, nous nous sommes intéressés à deux phénomènes anormaux se produisant dans la maladie de Parkinson : l’agrégation de l’α-synucléine et l’activation des cellules microgliales. Pour étudier la polymérisation de l’α-synucléine, nous avons établi de nouvelles méthodes permettant la production in vitro de différents types d’oligomères d’α-synucléine. Grâce à des méthodes biophysiques de pointe, nous avons caractérisé ces différents oligomères à l’échelle moléculaire. Puis nous avons étudié leurs effets toxiques sur les neurones. Ensuite, nous nous sommes intéressés à l’activation des microglies et en particulier à leurs canaux potassiques et aux changements liés au vieillissement. Nous avons identifié les canaux Kv1.3 et Kir2.1 et montré qu’ils étaient impliqués dans l’activation des microglies. En parallèle, nous avons établi une méthode originale qui permet l’isolation et la culture de microglies primaires issues de cerveaux adultes. En comparaison à celles de nouveaux-nés, les microglies adultes montrent des différences subtiles mais cruciales qui soutiennent l’hypothèse de changements liés au vieillissement. Globalement, nos résultats suggèrent qu’il est possible de développer de nouvelles approches thérapeutiques contre la maladie de Parkinson en modulant l’action des microglies ou en bloquant l’oligomérisation de l’ α-synucléine. / Age-related neurodegenerative disorders like Parkinson’s disease take an enormous toll on individuals and on society. Despite extensive efforts, Parkinson’s disease remains incurable and only very limited treatments exist. Indeed, Parkinson’s pathogenesis is still not clear and research on its molecular mechanisms is ongoing. In this study, we focused our interest on two abnormal events occurring in Parkinson’s patients, namely α-synuclein aggregation and microglial activation. We first investigated α-synuclein and its abnormal polymerisation. For this purpose, we developed novel methods, which allowed the in vitro production of different types of α-synuclein oligomers. Using highly sensitive biophysical methods, we characterised these different oligomers at a single-particle level. Then, we tested their biological effects on neurons. Afterwards, we studied microglial activation. We concentrated our efforts on two axes, namely age-related changes in microglial function and K+ channels in microglia. We showed that Kv1.3 and Kir2.1 K+ channels are involved in microglial activation. In parallel, we developed a new approach, which allows the effective isolation and culture of primary microglia from adult mouse brains. Adult primary microglia presented subtle but crucial differences in comparison to microglia from neo-natal mice, confirming the hypothesis of age-related changes of microglia. Taken together, our results support the hypotheses that microglial modulation or inhibition of α-synuclein oligomerisation are possible therapeutic strategies against Parkinson's disease.

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