α-Synuclein激活小胶质细胞分泌组织蛋白酶L在帕金森病发病中的机制研究

Abnormally activated microglia plays an important role in the pathogenesis of Parkinson’s disease (PD), while the mechanism of neurotoxicity caused by microglia remains unclear. Our preliminary studies showed that α-Synuclein（α-Syn）oligomer, the pathogenic protein of PD, activated microglia by interacting with P2X7R, leading to the secretion of some neurotoxic substance which caused the apoptosis of neurons. Meanwhile P2X7R is confirmed to regulate the secretion function of microglia. Recent studies demonstrated that cathepsin L (CTSL) is upregulated in the substantia nigra compact of patients suffering PD, and microglia can secrete CTSL to affect the growth and survival of neurons. All of these evidences strongly suggest that the extracellular CTSL may participate in the pathogenesis of PD. Therefore we propose that the stimulation of the microglial P2X7R by α-Syn oligomer may lead to the secretion of CTSL and thus target neurons. Hence, this project is to investigate the underlying molecular mechanism of neuron damage caused by the α-Syn mediated CTSL secretion and identify its role in the progression of PD by using various molecular technologies. We intend to use exosome as the entry point to explore the releasing pattern of CTSL from activated microglia, and further analyze the involved signaling pathway. The findings in this work will provide contributions to make CTSL become the potential biomarker and the new drug target of PD therapy.

帕金森病（PD）中小胶质细胞的异常激活导致神经毒性的机制尚未明了。我们的前期研究表明PD致病蛋白α-Synuclein寡聚体与P2X7R相互作用激活小胶质细胞，使其异常分泌诱导神经细胞凋亡的物质。而P2X7R被证实调控小胶质细胞的分泌谱。近期研究报道组织蛋白酶L（CTSL）在PD患者脑中表达异常升高，且小胶质细胞能胞外分泌CTSL并影响神经细胞的生长和存活，提示胞外CTSL可能参与PD发病。由此我们推测α-Synuclein寡聚体与膜表面受体P2X7R作用激活小胶质细胞分泌CTSL靶向神经元。本课题综合多种分子生物学技术，拟研究在原代小胶质细胞中证明α-Synuclein促进了CTSL的分泌并导致神经细胞损伤的机制，试图以外泌体为切入点研究CTSL的分泌形式，并进一步解析此过程中所牵涉的信号通路。该研究将为CTSL成为PD潜在的生物学标记物及治疗新靶标提供理论和实验依据。

在帕金森病(PD)患者中，不受控制的小胶质细胞激活可分泌组织蛋白酶L (Cathepsin L, CTSL)影响神经元的存活，是PD发病机制的关键;然而，确切的机制尚未确定。有报道称P2X7嘌呤能受体(P2X7R)可能增加CTSL的释放。之前，我们发现P2X7R可以通过α-Synuclein (α-Syn)低聚物介导小胶质细胞的激活。本研究旨在研究CTSL是如何通过外泌体从α- syn激活的小胶质细胞中释放出来，并对神经元产生不良影响，导致神经元变性的。采用Western blotting、透射电镜(TEM)、纳米粒子跟踪分析(NTA)和纳米流式细胞术(NTA)检测重组野生型(WT)和A53T α- syn激活的小鼠原代小胶质细胞CTSL的释放规律。之后，通过共免疫沉淀(co-IP)、RNA干扰和几种激动剂和抑制剂的应用来评估P2X7R/PI3K/AKT信号通路的参与。最后，通过免疫荧光检测含ctsl外泌体诱导α- syn激活的小胶质细胞的神经毒性，并对神经元凋亡和形态进行分析。我们首次证实CTSL主要由外泌体中α- syn激活的小胶质细胞释放，导致神经元损伤和死亡。外泌体释放的经典抑制剂GW4869阻断了CTSL活性的升高，表明外泌体在介导CTSL释放中发挥了关键作用。此外，P2X7R/PI3K/AKT信号通路被确定为潜在的分子机制，因为该信号通路的特异性拮抗剂、P2X7R敲除和外泌体释放抑制剂显著减轻了培养的小鼠皮层神经元的损伤。我们的研究提出，α- syn激活的小胶质细胞通过外泌体释放的CTSL增加，放大和加重了小胶质细胞的神经毒性作用。本研究结果提示CTSL可能作为一种潜在的PD生物标志物和新的药物靶点。