ASIC1a对a-synuclein自噬降解调控及其在帕金森病发病中的作用研究

Acid sensing ion channels (ASICs) are always activated when tissue acidosis occurs. A recent study demonstrated that MPTP-induced parkinson's disease (PD) mice is associated with cerebral lactic acidosis. Further, ASIC1a inhibitors significantly alleviated the dopaminergic(DA) neuron loss in the midbrain, implying a role of ASIC1a in the pathogenesis of PD. However, whether ASIC1a blocking could also regulate a-synuclein aggregation and DA neuron loss remains to be clarified. In the previous study, we found that ASICs inhibitors could upregulate the expression of LC3-II, reduce the intracellular a-synuclein level, and protect cells against injury. These findings led us to propose that ASICs inhibition may reduce a-synuclein aggregation by enhancing its autophagic degradation and thus protect neurons. This merits to be investigated further. Hence, this project is to examine the potential role of ASIC1a in reducing a-synuclein aggregation via autophagic clearance and its underlying mechanisms, and to identify its role in the progression of PD with both in vitro and in vivo studies, by combining the pharmacological and molecular techniques to inhibit or knockdown/knockout of ASIC1a. The findings in this work will make contributions to disclose the possible pathogenic factor in PD and also provide novel theoretical and experimental evidence in searching for promising target(s) for PD therapy.

组织酸化会引起酸敏感性离子通道（ASICs）的异常激活。最近研究报道，PD模型小鼠脑内也存在组织酸化，且ASIC1a抑制剂能减少多巴胺能神经元缺失，提示ASIC1a可能参与PD发病。但是，ASIC1a是否影响a-synuclein的病理性蓄积进而影响多巴胺能神经元存活，国内外鲜有报道。我们前期研究发现，抑制ASICs活化能上调LC3-II、降低a-synuclein的表达同时促进细胞存活，提示ASICs可能通过调节自噬活性抑制a-synuclein的病理性蓄积，从而保护神经元，但这仍缺乏足够的实验学依据。本课题将综合药理学及分子生物学等多项技术、分别在细胞和整体水平抑制ASIC1a功能或敲减其表达，研究它对a-synuclein降解的影响及机制，并进一步揭示其在PD发病中的作用。本课题成果将为阐明PD发病新机制和寻找治疗新靶标提供理论和实验依据。

组织酸化会引起酸敏感性离子通道（ASICs）的异常激活。近年来研究发现PD患者脑内存在乳酸含量增高的现象，且酸敏感离子通道抑制剂对PD 小鼠具有一定的保护作用，提示ASICs 的异常激活可能参与PD 发病机制，我们前期研究发现，抑制ASICs 活化能上调LC3-II、降低a-synuclein 的表达同时促进细胞存活，提示ASICs 可能通过调节自噬活性抑制a-synuclein的病理性蓄积，从而保护神经元。本课题运用免疫荧光，western blot，膜片钳等方法从细胞和整体水平研究发现：1，MES32.5细胞以及中脑多巴胺神经元上有功能性的ASIC1通道表达；2， ASIC1a的特异性抑制剂PCTX1和非特异性抑制剂阿米洛利（Amiloride，Ami），可下调由6-OHDA诱导的PC12细胞中LC3-II 和p62 蛋白水平，从而使a-synuclein降解。3，通过钙成像技术观察MES23.5细胞上ASIC1a通道在酸化条件下对钙离子的通透性。当胞外pH值迅速降至 6.0 后可明显增加胞内钙离子浓度（[Ca2+]i），达到峰值后回落，该反应可被ASICs通道非特异性抑制剂阿米洛利和特异性抑制剂PCTX1抑制。提示ASIC1a参与了胞外酸化引起的MES23.5细胞胞内Ca2+浓度升高；4， 6-OHDA诱导的PD大鼠模型中，ASIC1a的抑制剂PCTX1和阿米洛利对多巴胺能神经元具有神经保护作用，通过调控自噬途径对a-synuclein的异常聚集起到降解作用；5，在ASIC1 KO小鼠模型中，MPTP诱导的中脑黑质多巴胺神经元的丢失数量较WT小鼠少，提示，敲除ASIC1对多巴胺能神经元具有保护作用；同时P62及a-synuclein蛋白表达下调，提示敲除ASIC1促进a-synuclein降解。以上结果为全面评估ASIC1在PD发病中的作用提供了理论基础。