京都大学 / 新制・課程博士 / 博士(医学) / 甲第23757号 / 医博第4803号 / 新制||医||1056(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 井上 治久, 教授 岩田 想, 教授 上杉 志成 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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La maladie de Parkinson est l’une des plus répandue dans le monde. Des analyses post mortem ont mis en évidences des inclusions dans les cerveaux des patients, composées d’α-synucléine. Plusieurs études ont visé à identifier des composés capables d’inhiber l’agrégation de cette protéine, étant donné que cette agrégation est liée à sa toxicité. Néanmoins, de nombreux composés restent encore à être identifiés afin de mettre en évidence des structures moléculaires actives partagées qui pourrait mener à la synthèse d’un principe actif. Les stilbènes sont des composés phénoliques démontrant régulièrement des activités biologiques intéressantes pour la santé. Dans cette étude, nous étudions le comportement anti-agrégatif des stilbènes monomériques (resvératrol, picéatannol) et oligomèriques (ampélopsine, viniférine, et isohopeaphénol). Les résultats présentés mettent en évidence la capacité des stilbènes à inhiber l’agrégation de l’α-synucléine et fournissent des éléments pour comprendre ce mécanisme. / Parkinson’s disease is one of the most spread neurodegenerative diseases in the world. Post mortem analyses have put in evidence small inclusion bodies in patient’s brain, composed of α-synuclein fibrils. Several studies attempted then to identify compounds that could inhibit α-synuclein aggregation, as its aggregation is linked to its toxicity. Still, numerous active compounds need to be identified in order to put on relief shared active structures that could lead to a potent drug design. Stilbenes are phenolic compounds that often display interesting health-related biological activities. In this study, the anti-aggregative behavior of stilbenes monomers (resveratrol, piceatannol) and oligomers such as ampelopsin and isohopeaphenol was assessed. The results put on evidence stilbene propensity to inhibit α-synuclein aggregation and provide an insight into their inhibition mechanisms.

Les signatures histopathologiques de principales maladies neurodégénératives - maladie d'Alzheimer et la maladie de Parkinson - sont les enchevêtrements neurofibrillaires formés par la protéine tau et les corps de Lewy, formés par l'α-synucleine agrégée. Les mécanismes précis du repliement et de l'agrégation de ces protéines, pour la protéine tau comme pour l'α-synucleine, ne sont pas totalement compris à ce jour. Ici, nous nous sommes intéressés à cette question en utilisant des modèles in vitro et in vivo. En étudiant l'agrégation tau in vitro, nous avons mis en évidence un nouvel auto- assemblage réversible de tau, qui dépend de la température et de la présence d’ions zinc, et qui est a priori différent de l'agrégation de tau en présence d'inducteurs d'agrégation tels que l'héparine. Ce processus pourrait néanmoins être impliqué dans les premières étapes de l'agrégation pathologique de tau. Dans une deuxième partie nous avons développé des modèles murin pour étudier les dysfonctionnement de l’α-synucleine. Nous avons montré que l’α-synucleine est directement impliquée dans le développement embryonnaire de régions spécifiques du système nerveux, et qu'elle a des propriétés modulatrices seulement sur les neurones dopaminergiques de la substantia nigra, qui sont touchés dans la maladie de Parkinson.Les résultats obtenus dans nos études de deux protéines qui subissent une agrégation pathogène et forment des inclusions intracellulaires ont contribué à la compréhension des processus moléculaires et cellulaires associés à la dégénérescence neuronale, ce qui fournira de nouvelles pistes pour développer de nouvelles stratégies de thérapies de maladies neurodégénératives. / The histopathological hallmarks of the most common neurodegenerative diseases – Alzheimer’s disease and Parkinson’s disease are neurofibrillary tangles formed by tau protein and Lewy bodies inclusions formed by aggregated α-synuclein. The formation and accumulation of these proteins into inclusions cause functional disruptions of the cytoskeleton and leads to neuronal degeneration. The precise mechanisms of tau and synuclein misfolding and aggregation leading to those cellulare incluses, even though very studied, are not fully understood neither for tau protein nor for α-synuclein.Here we have addressed this question using both in vitro and in vivo models. Investigating tau aggregation in vitro, we have found a reversible self-assembly of tau, which depends on temperature and is induced by zinc ions, which is different from the tau aggregation in the presence of aggregation-inducers such as heparin. This process could be implicated in the first steps of tau pathological aggregation. In a second part, we have developed a mouse model for studying the α-synuclein dysfunction. We have shown that α- synuclein is directly involved in the embryonic development of the specific regions of the nervous system, and that it has modulating effect only on the populations of dopaminergic neurons of substantia nigra, which are affected in Parkinson’s disease.Results obtained in our studies of two proteins that undergo pathogenic aggregation and form intracellular inclusions contributed to understanding of molecular and cellular processes associated with neuronal degeneration, which is important for the development of new disease-modifying therapies of neurodegenerative disorders.

By using multidisciplinary analysis of CA inclusions in prostate glands of patients diagnosed with prostate cancer, we have revealed that their major components are the amyloid forms of S100A8 and S100A9 proteins associated with numerous inflammatory conditions and types of cancer. We have demonstrated that material closely resembling CA can be produced from S100A8/A9 in vitro and shows the characters of amyloids. This process is facilitated by calcium or zinc, both of which are abundant in ex vivo inclusions. These observations were supported by computational analysis of the S100A8/A9 calcium-dependent aggregation propensity profiles. We have found DNA and proteins from Escherichia coli in CA bodies, suggesting that their formation is likely to be associated with bacterial infection. CA inclusions were also accompanied by the activation of macrophages and by an increase in the concentration of S100A8/A9 in the surrounding tissues, indicating inflammatory reactions. These findings, taken together, suggest a link between bacterial infection, inflammation and amyloid deposition of pro-inflammatory proteins S100A8/A9 in the prostate gland, such that a self-perpetuating cycle can be triggered and may increase the risk of malignancy in the ageing prostate. We evaluated the autoimmune reactions to endocrine (insulin) and astrocytical (S100B) biomarkers in the blood sera of PD patients compared with healthy controls. Peripheral immune responses can be sensitive indicators of disease pathology. We found a statistically significant increase of the autoimmune responses to both antigens in patients compared with controls. Heterogeneity of the immune responses observed in patients may reflect the modulating effect of multiple variables associated with neurodegeneration and also changes in the basic mechanisms of individual autoimmune reactivity. We did not detect any pronounced immune reactions towards insulin amyloid fibrils and oligomers in patients, indicating that an amyloid-specific conformational epitope is not involved in immune recognition of this amyloid type. Immune reactions towards S100B and insulin may reflect the neurodegenerative brain damaging processes and impaired insulin homeostasis occurring in PD. Generated auto-antibodies towards the major amyloidogenic protein involved in PD Lewy bodies - a-synuclein and its amyloid oligomers and fibrils were measured in the blood sera of early and late PD patients and controls by using ELISA, Western blot and Biacore surface plasmon resonance analyses. We found significantly higher antibody levels towards monomeric a-synuclein in the blood sera of PD patients compared to controls, though the responses decreased with PD progression. There were no noticeable immune responses towards amyloid oligomers, but substantially increased levels of IgGs towards a-synuclein amyloid fibrils both in PD patients and controls, which subsided with the disease progression. Pooled IgGs from PD patients and controls interacted also with amyloid fibrils of Ab (1-40) and hen lysozyme, however the latter were recognized with lower affinity. This suggests that IgGs bind to amyloid conformational epitope, though displaying higher specificity towards human amyloid species associated with neurodegeneration. The findings suggest the protective role of autoimmunity in PD and therefore immune reactions towards PD major amyloid protein - a-synuclein can be used in treatment strategies and in diagnostics, especially in identifying early disease.

Parkinson’s disease is a neurodegenerative disease resulting primarily from loss of dopaminergic innervation in the striatum subsequent to cell loss in the substantia nigra pars compacta. The abnormal accumulation of the normal pre-synaptic protein α-synuclein (αsyn) forms intraneuronal inclusions known as Lewy neurites and Lewy bodies. The origins of central Lewy pathology have been suggested to lie in the enteric nervous system, ascending through the vagus nerve to the dorsal motor nucleus of the vagus. To ascertain gastrointestinal regions most likely to be the source of central Lewy pathology, αsyn expression was evaluated in the neural elements of gastrointestinal regions receiving the densest vagal innervation. The vermiform appendix was found to have the densest αsyn-immunoreactive innervation in all layers of the gut wall. In addition, macrophages in the appendiceal mucosa were laden with αsyn within lysosomes, consistent with attempts to prevent the spread of disease or to correct synaptic dysfunction.

Parkinson’s disease is a neurodegenerative disease resulting primarily from loss of dopaminergic innervation in the striatum subsequent to cell loss in the substantia nigra pars compacta. The abnormal accumulation of the normal pre-synaptic protein α-synuclein (αsyn) forms intraneuronal inclusions known as Lewy neurites and Lewy bodies. The origins of central Lewy pathology have been suggested to lie in the enteric nervous system, ascending through the vagus nerve to the dorsal motor nucleus of the vagus. To ascertain gastrointestinal regions most likely to be the source of central Lewy pathology, αsyn expression was evaluated in the neural elements of gastrointestinal regions receiving the densest vagal innervation. The vermiform appendix was found to have the densest αsyn-immunoreactive innervation in all layers of the gut wall. In addition, macrophages in the appendiceal mucosa were laden with αsyn within lysosomes, consistent with attempts to prevent the spread of disease or to correct synaptic dysfunction.