外泌体介导的α-突触核蛋白异常传播的炎症机制研究

The pathological aggregation and propagation ofα-synuclein (α-syn) is the core mechanism implied in the process of Parkinson’s Disease (PD) onset and progression. In our previous study, thedata demonstratedthat α-syn oligomers were obviously abundant in PD patients’ serum-derivedexosomes compared with controls.Innovatively, we intravenously and intrastriatallyinjected those exosomes into the C57 mice, inducing PD-like behaviors aswell as pathological alteration in these animals. Moreover, the microglia activation and pro-inflammatory cytokines overexpression were observed in the substantianigra of these mice. All theseevidence strongly indicate that exosomes areable to mediate pathogenic ingredients (α-syn oligomers) transmission from PD patients to mice dopaminergic neurons, and microglia activation and subsequent inflammatory mechanisms may be invloved in the transmission. However, the exact underlying mechanisms of how inflammatory microglia have been activated, which signaling pathways have been involved, and how dopaminergic neurons have been affected remain to be elucidated.In this project,we aim to reveal the effects of PD patients-derived exosomes on dopaminergic neuron and microglia both in vitro and in vivo. Moreover, we will explore the upstream and downstream pathways of microglia activation in this process, in order to stop α-syn transmission via prohabit these pathways. The proposed project will provide a novelinsight into the pathogenesis of PD from a perspective of neuroinflammatory mechanismand bringinspiration to develop new strategies to postpone and even halt PD progression.

α-突触核蛋白（α-syn）的聚集和传播是帕金森病（PD）发生与发展的核心环节。本课题组前期发现患者血清外泌体中α-syn寡聚体明显增多，将这些外泌体经尾静脉及纹状体注射给小鼠可诱发其PD样行为和病理改变，且小鼠黑质区可观察到小胶质细胞激活和炎症介质表达，提示PD患者血清来源的外泌体介导了致病性α-syn从PD患者向小鼠黑质多巴胺能神经元的“传播”，小胶质细胞激活和后续的炎症机制则可能是α-syn传播的重要途径。然而，小胶质细胞如何被激活，哪些信号途径参与其中，又是通过何种方式进一步影响多巴胺能神经元等关键问题尚不清楚。本项目拟在动物和细胞模型中分别观察PD患者来源的外泌体对神经元和小胶质细胞的影响，探寻可能的相关机制，尤其是小胶质细胞激活的具体途径和其下游通路，并探讨阻断小胶质细胞激活和炎症反应是否能阻止α-syn的传播，为从神经炎症角度阐明PD发病机制及开展神经保护研究提供科学依据。

本课题组前期发现PD患者血清外泌体中α-syn寡聚体明显增多，将这些外泌体经尾静脉及纹状体注射给小鼠可诱发其PD样行为和病理改变，且小鼠黑质区可观察到小胶质细胞激活和炎症介质表达，提示PD患者血清来源的外泌体介导了致病性α-syn从PD患者向小鼠黑质多巴胺能神经元的“传播”。小胶质细胞激活和后续的炎症机制则可能是α-syn传播的重要途径，探究这一机制，将有助于阐明PD的发病机制，为PD的治疗研究提供新的科学依据。本课题的第一部分，在细胞和动物水平分别观察 PD 患者来源的外泌体对多巴胺能细胞和小胶质细胞的影响。我们发现，PD 病人来源的外泌体在神经元和小胶质细胞中有明显的吸收，提示外泌体的靶细胞为神经元和小胶质细胞。且在经尾静脉及纹状体注射PD患者外泌体的小鼠的纹状体和黑质区，荧光检测到PKH-26标记的外泌体与IBA-1标记的小胶质细胞大量共存，提示小胶质细胞激活可能在外泌体向神经元的传递过程中起重要作用。本课题的第二部分，探索PD患者来源的外泌体诱导小胶质细胞激活的主要途径。我们发现TLR2在PD外泌体注射的小鼠模型脑组织中表达明显增多，且沉默TLR2基因可减弱小胶质细胞吞噬能力和神经炎症，从而减轻α-syn病理。本课题的第三部分，进一步探索TLR2受体被抑制后小胶质细胞的改变情况，以及后续对黑质区α-syn异常聚集和传播的影响。我们用TLR2 siRNA或Control siRNA转染的BV2细胞注射到小鼠的单侧纹状体，我们发现与对照组小鼠相比，注射TLR2 siRNA处理的BV2细胞的小鼠，SNpc的多巴胺能神经元中的磷酸化α-syn减少，结果表明TLR2是小胶质细胞识别、吞噬外泌体α-syn，从而发生炎性激活的主要靶点。本课题的第四部分，探索褪黑素在α-syn PFF的PD模型中，减轻神经炎症，阻止α-syn的异常聚集和传播的分子机制。我们发现褪黑素通过下调TLR2，抑制小胶质细胞的激活和后续的NLRP3炎性通路，从而减轻α-syn的异常聚集，保护多巴胺能神经元，这表明TLR2是延缓PD病理进展的一个重要治疗靶点。