Les maladies neurodégénératives constituent un enjeu humain, sociétal et économique majeur, dont l'importance croît avec le vieillissement des populations. Cette dénomination regroupe un grand nombre de maladies neurologiques, comme les maladies d'Alzheimer, de Parkinson ou la sclérose latérale amyotrophique (SLA). L'étiologie de ces maladies est complexe et reste encore mal comprise. Néanmoins, elles résultent vraisemblablement d'une combinaison de facteurs génétiques et environnementaux et se caractérisent toutes par la dégénérescence d'une population neuronale spécifique. Les mitochondries jouent un rôle primordial dans l'apport énergétique, la régulation calcique ou la gestion du stress cellulaire et sont directement impliquées dans le déclenchement de la mort cellulaire programmée (ou apoptose). L'implication centrale de cet organite dans les pathologies neurodégénératives a logiquement suscité un ciblage croissant des fonctions mitochondriales dans les nouvelles approches thérapeutiques envisagées ces dernières années. Des curcuminoïdes ont par exemple été testés pour contrer les effets dus au stress oxydatif, mais les résultats restent encore à être améliorés. L'utilisation de facteurs anti-apoptotiques dérivés de virus représente une piste très prometteuse. En effet, l'apoptose des cellules infectées représente la "première ligne" de défense de l'hôte contre une invasion virale. En réponse à ce mécanisme de défense, de nombreux virus expriment divers facteurs (protéines, ARN ...) dont la fonction est de bloquer le processus apoptotique, en particulier en ciblant la mitochondrie, favorisant ainsi la réplication virale au sein de la cellule. Dans ce contexte, notre équipe a établi que la protéine X du Bornavirus possède de remarquables propriétés neuroprotectrices. Si le potentiel thérapeutique de cette protéine virale est maintenant démontré, les mécanismes moléculaires à l'origine de cet effet neuroprotecteur étaient à ce jour inconnus. Au cours de ma thèse, nous avons contribué à une meilleure compréhension de la biologie de cette protéine au sein de la cellule et en particulier dans les mitochondries. Nous avons déterminé ses séquences d'adressages intracellulaires ainsi que son impact sur la dynamique mitochondriale et sur certains paramètres de la bioénergétique mitochondriale tels que le potentiel de membrane mitochondrial, la respiration cellulaire et la production d'ATP (énergie de nos cellules). L'ensemble de ces connaissances devrait nous permettre d'améliorer sa capacité neuroprotectrice. / Bornavirus, a non-cytolytic RNA virus establishes a long-lasting persistence in the central nervous system of infected animals. Viral persistence is facilitated by the expression of the non-structural X protein, which is addressed both to nucleus and mitochondria, where it interferes both with cellular antiviral responses and the initiation of apoptosis. Our team recently reported that the singled-out expression of the X protein could protect neurons against toxins of the mitochondrial respiratory chain, both in vitro and in a mouse model of Parkinson's disease (PD). During my Ph.D., we further demonstrated that the X protein triggered enhanced filamentation of the mitochondrial network, in physiological as well as in oxidative stress conditions. This effect is particularly interesting when considering the importance of mitochondrial dynamics in the pathophysiology of neurodegenerative diseases. Even if the therapeutic potential of this viral protein is now well established, the underlying molecular and cellular mechanisms are far from being elucidated. It is however clear that neuroprotection conferred by the X protein is strictly dependent on its mitochondrial localization. In this context, the goal of my Ph.D. project was to clarify the molecular mechanisms whereby the X protein is targeted to mitochondria and/or to the nucleus, in link with its protective capabilities. We focused on the amino terminal residues of X, by performing fusion proteins of various forms of these residues with GFP and by analyzing their cellular localization. We demonstrated that this region contains overlapping and interdependent signals for nuclear localization, nuclear export and mitochondrial targeting of the X protein. We also identified a point mutation or deletion leading to an almost exclusively mitochondrial localization of the X protein. As a consequence, these X mutants exhibited a better neuroprotective function. In order to get further insight into X-mediated neuroprotection, we also searched for the cellular partners of X in mitochondria. We revealed a direct and specific interaction of the X protein with the chaperone Hspa9, a protein that was recently shown to be involved in neurodegenerative diseases, notably in PD's patients. We observed that the down-regulation of Hspa9 triggered by mitochondrial toxins was attenuated by the coexpression of X, suggesting a functional link between these two proteins. We also demonstrated that Hspa9 overexpression could protect neurons from mitochondrial dysfunctions, similarly to the X protein. Altogether, these results have contributed to a better understanding of the mechanisms underlying the neuroprotective potential of the X protein, which may favor the development of novel therapeutic strategies.

Analyser les modalités de l'interaction des virus neurotropes avec leurs cellules cibles représente un défi majeur, cela pourrait favoriser notre compréhension de la physiopathologie de certains troubles neurologiques dont l'origine virale est parfois suspectée. Le Bornavirus (BDV), un virus neurotrope, représente un modèle idéal pour analyser les mécanismes moléculaires de la persistance virale dans les neurones et en étudier les conséquences sur l'homéostasie neuronale. Au cours de ma thèse, nous avons découvert une nouvelle forme d'interaction virus/cellule dans laquelle une protéine du BDV perturbe la signalisation épigénétique associée à l'acétylation des histones. Cette perturbation de l'acétylation des histones cellulaires est accompagnée d'une modulation de la réplication virale qui permettrait de favoriser la persistance du BDV dans les neurones tout en limitant les dommages cellulaires associés, démontrant ainsi la parfaite adaptation du BDV à sa cellule hôte. / In response to various environmental stimuli, neurons undertake specific cognitive functions, such as learning and memory. Many studies have shown that epigenetics, notably histone acetylation, is crucial for the regulation of gene expression involved in these processes. Moreover, dysregulation of signaling pathways that regulate epigenetics is clearly recognized as a main actor in the pathogenesis of several neurological disorders. Even if the origin of these disorders is sometimes genetic, their etiology remains elusive and environmental factors are also suspected. In particular, infectious agents are considered as serious candidates, notably for viruses that persist in the central nervous system (CNS). The Bornavirus (BDV) constitutes an interesting model to assess the impact of viral infection on neuronal epigenetics. This non-cytolytic RNA virus infects neurons of the CNS and causes various cognitive disorders in animals. It has the remarkable property to replicate in the nucleus, in close association with chromatin, thus leading to our working hypothesis that it may modify the epigenetics of infected neurons. During my Ph.D., we used primary cultures of cortical neurons to demonstrate that BDV infection decreases H2B and H4 acetylation levels on selected lysine residues, through inhibition of histone acetyltransferase (HAT) activities. We also showed that the viral phosphoprotein was responsible for this perturbation, acting on histone acetylation and HAT activities in a protein kinase C dependent manner. Finally, using pharmacological agents, we observed that histone acetylation levels were correlated with replication of BDV. Thus, BDV action on histone acetylation pathway may represent an original mechanism to control viral replication, thereby favoring long-term persistence in neurons. I am currently examining the consequences of this virus-mediated perturbation on H2B/H4 acetylation at the genome scale, using ChIP-sequencing experiments.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19613号 / 医博第4120号 / 新制||医||1015(附属図書館) / 32649 / 京都大学大学院医学研究科医学専攻 / (主査)教授 小柳 義夫, 教授 岩田 想, 教授 萩原 正敏 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

As doenças virais são de extrema importância para a saúde das aves. Os vírus podem se espalhar através de fezes, secreções respiratórias ou exfoliação epitelial de penas e pele, dificultando ainda mais esse controle. A utilização do PCR permite detecção de pequenas concentrações do vírus além da possibilidade de diagnosticar a patologia antes de iniciar alterações histológicas. O bornavírus aviário é responsável pela doença da dilatação pró-ventricular (PDD) em psitacídeos e outras aves, uma doença neurológica letal, que foi descoberta no início da década de oitenta na Europa e América do Norte. A detecção do vírus em passeriformes e outras famílias já ocorreu em alguns países. A ocorrência de poliomavírus em outras espécies diferentes dos psitaciformes não é estudada no Brasil, logo não se sabe se há ocorrência da doença nem em aves de cativeiro como em aves de vida livre. O presente trabalho teve como objetivo a padronização das técnicas de PCR e RT-PCR e o estudo epidemiológico dos três tipos virais descritos acima em aves de cativeiro no Brasil. Foram coletadas amostras de passeriformes de vida livre e passeriformes de cativeiro (n=327), galiformes de criação extensiva e comercial (n=90). Nenhuma amostra foi positiva para circovírus e nenhuma amostras de galiformes foi positiva para os vírus testados. A caracterização das amostras biológicas obtidas de psitacídeos resultou na descoberta de um novo genótipo, denominado PaBv-8, atualmente descrito apenas no Brasil. Das amostras de passeriformes testadas, foram encontradas 3 amostras positivas para bornavírus de canário (CnBv-1) e uma para poliomavírus. Esta é a primeira descrição de bornavírus em canário no Brasil. Duas das três amostras positivas para CnBv-1 pertenciam a canários de cativeiro que apresentavam sinais clínicos compatíveis com a doença, enquanto a terceira amostra pertencia a um pula-pula de vida livre, aparentemente assintomático. A ave positiva para poliomavírus apresentou crescimento excessivo do bico, sinal clínico compatível com lesão hepática, que pode ser causada pela infecção viral. A descrição de um novo genótipo assim como a identificação do vírus e da doença em passeriformes indica uma necessidade de reforçar os estudos sobre o assunto no país, assim como rever a legislação a fim de prevenir a disseminação de patógenos. / Viral diseases are of extreme importance for the health of birds. Viruses can spread through feces, respiratory secretions or epithelial exfoliation of feathers and skin, making it even more difficult to control. The use of PCR allows the detection of small concentrations of the vírus, giving the possibility of diagnosing the pathology before initiating histological alterations. The avian bornavirus is responsible for pro-ventricular dilation (PDD) disease in psittacines and other birds, a lethal neurological disease that was discovered in the early 1980s in Europe and North America. Virus detection in passerines and other families has already occurred in some countries. The occurrence of polyomaviruses in species other than psitaciformes was never studied in Brazil. Ut is not known whether the disease occurs in poultry or in free-living birds as well. The present study aimed to standardize the PCR and RT-PCR techniques and the epidemiological study of the three viral types described above in captive birds in Brazil. We collected samples from wild-caught and captive passeriformes (n = 327), galiformes from breeding facilities (n = 90) and captive psitacines (n=10). No samples were positive for circovírus. None of the galiformes samples were positive for any of the virus tested. The characterization of the biological samples obtained from psittacines resulted in the discovery of a new genotype, called PaBv-8, currently described only in Brazil. Passeriformes samples resulted in 3 positive samples for bornavírus, that were further analyzed as canarian bornaviruses 1 (CnBv-1) and one bird was positive for poliomavirus. This is the first description of bornavirus in canary in Brazil. Two out of the three CnBv-1-positive samples belonged to captive canaries showing clinical signs compatible with the disease, whereas the third sample belonged to an apparently asymptomatic free-living pula. The positive bird for polyomavirus presented excessive growth of the beak, clinical sign compatible with hepatic damage, that can be caused by the viral infection. The description of a new genotype as well as the identification of the virus and the disease in passeriformes indicates a need to reinforce the studies on the subject in the country, as well as to review the legislation to prevent the spread of pathogens.

As doenças virais são de extrema importância para a saúde das aves. Os vírus podem se espalhar através de fezes, secreções respiratórias ou exfoliação epitelial de penas e pele, dificultando ainda mais esse controle. A utilização do PCR permite detecção de pequenas concentrações do vírus além da possibilidade de diagnosticar a patologia antes de iniciar alterações histológicas. O bornavírus aviário é responsável pela doença da dilatação pró-ventricular (PDD) em psitacídeos e outras aves, uma doença neurológica letal, que foi descoberta no início da década de oitenta na Europa e América do Norte. A detecção do vírus em passeriformes e outras famílias já ocorreu em alguns países. A ocorrência de poliomavírus em outras espécies diferentes dos psitaciformes não é estudada no Brasil, logo não se sabe se há ocorrência da doença nem em aves de cativeiro como em aves de vida livre. O presente trabalho teve como objetivo a padronização das técnicas de PCR e RT-PCR e o estudo epidemiológico dos três tipos virais descritos acima em aves de cativeiro no Brasil. Foram coletadas amostras de passeriformes de vida livre e passeriformes de cativeiro (n=327), galiformes de criação extensiva e comercial (n=90). Nenhuma amostra foi positiva para circovírus e nenhuma amostras de galiformes foi positiva para os vírus testados. A caracterização das amostras biológicas obtidas de psitacídeos resultou na descoberta de um novo genótipo, denominado PaBv-8, atualmente descrito apenas no Brasil. Das amostras de passeriformes testadas, foram encontradas 3 amostras positivas para bornavírus de canário (CnBv-1) e uma para poliomavírus. Esta é a primeira descrição de bornavírus em canário no Brasil. Duas das três amostras positivas para CnBv-1 pertenciam a canários de cativeiro que apresentavam sinais clínicos compatíveis com a doença, enquanto a terceira amostra pertencia a um pula-pula de vida livre, aparentemente assintomático. A ave positiva para poliomavírus apresentou crescimento excessivo do bico, sinal clínico compatível com lesão hepática, que pode ser causada pela infecção viral. A descrição de um novo genótipo assim como a identificação do vírus e da doença em passeriformes indica uma necessidade de reforçar os estudos sobre o assunto no país, assim como rever a legislação a fim de prevenir a disseminação de patógenos. / Viral diseases are of extreme importance for the health of birds. Viruses can spread through feces, respiratory secretions or epithelial exfoliation of feathers and skin, making it even more difficult to control. The use of PCR allows the detection of small concentrations of the vírus, giving the possibility of diagnosing the pathology before initiating histological alterations. The avian bornavirus is responsible for pro-ventricular dilation (PDD) disease in psittacines and other birds, a lethal neurological disease that was discovered in the early 1980s in Europe and North America. Virus detection in passerines and other families has already occurred in some countries. The occurrence of polyomaviruses in species other than psitaciformes was never studied in Brazil. Ut is not known whether the disease occurs in poultry or in free-living birds as well. The present study aimed to standardize the PCR and RT-PCR techniques and the epidemiological study of the three viral types described above in captive birds in Brazil. We collected samples from wild-caught and captive passeriformes (n = 327), galiformes from breeding facilities (n = 90) and captive psitacines (n=10). No samples were positive for circovírus. None of the galiformes samples were positive for any of the virus tested. The characterization of the biological samples obtained from psittacines resulted in the discovery of a new genotype, called PaBv-8, currently described only in Brazil. Passeriformes samples resulted in 3 positive samples for bornavírus, that were further analyzed as canarian bornaviruses 1 (CnBv-1) and one bird was positive for poliomavirus. This is the first description of bornavirus in canary in Brazil. Two out of the three CnBv-1-positive samples belonged to captive canaries showing clinical signs compatible with the disease, whereas the third sample belonged to an apparently asymptomatic free-living pula. The positive bird for polyomavirus presented excessive growth of the beak, clinical sign compatible with hepatic damage, that can be caused by the viral infection. The description of a new genotype as well as the identification of the virus and the disease in passeriformes indicates a need to reinforce the studies on the subject in the country, as well as to review the legislation to prevent the spread of pathogens.

京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第23555号 / 生博第466号 / 新制||生||62(附属図書館) / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 朝長 啓造, 教授 野田 岳志, 教授 千坂 修 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM