患者来源的外泌体诱导小鼠帕金森病样损伤的机制研究

Recently, the role of “prion like” propagation of pathogenic α-synuclein (α-syn) in the pathogenesis of Parkinson’s disease (PD) is widely accepted, but the exact underlying mechanisms remain to be elucidated. The idea that exosomes may be involved in the spreading of α-syn is attracting considerable attention. Exosomes are microvesicles secreted by the cells which contains specified proteins, then loaded proteins can be transmitted to distant area and even pass through the blood brain barrier, which may alter the function of the recipient cells in central nervous system. In the previous study, our group demonstrated that α-syn oligomers is obviously abundant in PD patients’ serum derived exosomes compared with controls. Innovatively, we intravenously and striatally injected those exosomes into the mouse, surprisingly induced PD-like behaviors as well as pathological alteration. All these strongly indicate that exosomes are able to mediate pathogenic ingredients (α-syn oligomers) transmission from PD patients to mouse. On this basis, we contrive to reveal the underlying mechanisms as follows: exosomes containing α-syn oligomers will be extracted and purified by high speed centrifugation and immune-capture. Afterwards, exosomes will be added to cells and animals to explore the mechanisms related to their selective delivery and the “seeding” effects in recipient cells. Meanwhile, intervention study will be conducted by inhibition of exosomes biogenesis and interruption of extracellular exosomes transportation utilizing α-syn oligomers specified monoclonal antibody. The fulfillment of the proposed project will provide a novel insight into the pathogenesis of PD and bring inspiration to develop new strategies to halt PD progression.

异常聚集的α-突触核蛋白（α-syn）的“朊蛋白样”传播在帕金森病（PD）中的核心作用渐被认可，但确切的机制仍未充分阐明。外泌体是细胞分泌的超微膜性囊泡，可将细胞内的蛋白质长距离递送到“接受细胞”，也能自由穿透血脑屏障，因而在PD的诊治研究中备受瞩目。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，随后创新性地将这些外泌体经尾静脉及纹状体注射给小鼠，诱发其PD样行为和病理改变，提示外泌体介导了致病性α-syn从PD患者向小鼠的“传播”。据此，我们拟采用超速离心法、免疫吸附等技术提取PD患者血清中含α-syn寡聚体的外泌体，在细胞及动物模型中观察其对“接受细胞”的选择性传播及其“种子效应”，同时探索抑制外泌体生成以及采用α-syn寡聚体特异性抗体选择性阻断其传输，建立新的干预措施以延缓PD的病理进程，为PD的发病机制探索和防治新策略研发提供科学依据。

异常聚集的α-突触核蛋白（α-syn）“朊蛋白样”传播假说是帕金森病（PD）领域的重要研究方向；外泌体是细胞分泌的超微膜性囊泡，拥有长距离传递能力和良好的血脑屏障穿透性，然而以外泌体为载体的传播机制研究尚处在初步阶段。探究外泌体在α-syn传播过程中扮演的作用，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。本课题首先（第一部分）比较PD患者、动物模型与对照组血清来源外泌体含量的差异及内含物的差别。我们发现与对照组相比，PD患者及动物模型的外周血清中外泌体含量增加，且含有更多的α-syn寡聚体。这表明外泌体的差异有望成为反映疾病进程及疾病诊断的标记物。随后（第二部分）我们提取PD患者外周血中的α-syn，然后加入不同的细胞模型，观察含有α-syn寡聚体的外泌体对“接受细胞”的选择性传播及其“种子效应”。我们发现体外实验外泌体的主要目的细胞为神经元细胞，而小胶质细胞也在其吸收传播中发挥一定作用，PD外泌体可以引起接受细胞发生寡聚化改变，最终导致α-syn聚集体形成。接下来（第三部分）我们经尾静脉及纹状体注射PD患者来源的外泌体建模，在动物模型上探究外泌体对“接受细胞”的选择性损伤机制。研究表明两种造模方法均能够引起黑质纹状体系统发生变性，导致小鼠模型出现PD病理学和行为学上的改变。且PD外泌体在小鼠脑内被小胶质细胞高度选择性摄取，而与神经元细胞和星形胶质细胞极少共定位。最后（第四部分）我们提取PD外泌体处理小胶质细胞，探究小胶质细胞在外泌体a-syn传播中的呈递机制。我们发现PD外泌体处理小胶质细胞后，其自噬受到明显抑制，胞内沉积及胞外分泌的a-syn含量均增加，并可引发与其共培养的神经元细胞内病理性a-syn含量增加，这表明小胶质细胞在外泌体途径α-syn的传播中起着至关重要的作用。