PD小胶质细胞中突变型α-synuclein异常积聚的分子机制研究

Both accumulation of mutant α-synuclein in or outside of microglial can lead to the abnormal activation of microglia and the subsequent inflammatory injury is closely related to the occurrence of PD. However, the mechanism underlying the abnormal accumulation has not yet been elucidated. As we know, regular endosomal transportation can guarantee the degradation of α-synuclein without abnormal accumulation. Our previous study found that mutant α-synuclein in microglia cannot be efficiently transported to the late endosomes and the activity of Rab5, a protein controlling the structure and function of the early endosomes, increased abnormally. These results suggested that mutant α-synuclein may affect some Rabs, along with its upstream regulatory factors, resulting in endosomal transportation obstacles and abnormal protein degradation. Base on our preliminary results, we propose in this project that we are going to further explore the following: (1) the specific pathways which lead to the blockage of the transportation of mutant α-synuclein in endosome of microglia and the related molecular mechanism; (2) the relationship between the abnormal regulation factors of the transport and the exceptional activation of microglia and PD pathogenesis. This project will replenish to the PD inflammatory pathogenesis and provide help to explore new avenues for the treatment of PD.

突变型α-synuclein在小胶质细胞内或细胞间隙间的异常积聚可导致小胶质细胞的异常激活，由此介导的炎性损伤与PD的发生密切相关，但突变型α-synuclein发生异常积聚的机制尚未明确。小胶质细胞内正常的内体运输是保证α-synuclein降解，避免α-synuclein异常积聚的关键，我们前期研究发现小胶质细胞内突变型α-synuclein不能被有效地运输至晚期内体，且控制早期内体形态及功能的Rab5活性异常增高，提示突变型α-synuclein可能通过影响Rab及上游调节因子导致内体运输的障碍、蛋白降解的异常。本课题将在我们前期发现基础上，进一步探讨（1）导致小胶质细胞内突变型α-synuclein内体运输障碍的具体途径和相关分子机制；（2）上述相关内体运输调控因素的异常与小胶质细胞异常激活和PD发生的关系。课题的开展将帮助完善PD的炎性发病机制，为PD治疗新途径的探索提供帮助。

突变型α-synuclein在小胶质细胞内或细胞间隙间的异常积聚可导致小胶质细胞的异常激活，由此介导的炎性损伤与PD的发生密切相关，但突变型α-synuclein发生异常积聚的机制尚未明确。小胶质细胞内正常的内体运输是保证α-synuclein降解，避免α-synuclein异常积聚的关键，我们前期研究发现小胶质细胞内突变型α-synuclein不能被有效地运输至晚期内体，通过活细胞摄影、GTP pull-down等方法我们明确了突变型α-synuclein运输障碍的部位位于早期内体，并鉴定出α-synuclein早期内体运输障碍与Rab5过度激活相关，其中相比于WT α-synuclein，Rab5活化程度在突变型α-synuclein更为显著。为了明确导致Rab5过度激活的上游调控因子，我们筛选了调控Rab5活性的因子并发现GDP解离抑制因子（GDI）是导致Rab5活性上调及突变型α-synuclein内体运输障碍的重要中间因子。在质谱、点突变技术等帮助下，我们进步筛选出了α-synuclein导致GDI活性改变的活性位点以及其上游磷酸化激酶。据此我们已构建Rab5显性负性形式转基因小鼠及GDI活性位点点突变小鼠，计划将上述两种小鼠分别与突变型α-synuclein转基因小鼠杂交，观察杂交后子代小鼠中α-synuclein的内体运输障碍及表型是否得到了改善。此外，项目组前期的研究发现在AD中Rab5的异常激活导致了NGF信号内体逆向运输障碍和胆碱能神经元的变性死亡，我们在项目的资助下进一步探讨了Rab５异常激活的机制，并发现Rab5经典GEF Rin3在AD早期即出现异常升高，我们同时发现Rin3致病性突变能够导致Rin3 GEF活性上调。由于Rin3生物学特性的相关研究缺乏，我们计划明确Rin3生物学特性和活性调控方式，并利用冷冻电镜或结晶，探讨突变对Rin3蛋白构象改变及对Rab5激活作用增强的分子机制。