G蛋白偶联受体MC1R抑制α-Syn聚集及细胞间传递的分子机制研究

Recent studies have strongly shown that cell-to-cell transmission of α-synuclein (α-Syn) plays an important role in the development of Parkinson’s diseases. However, the underlying cause is complex and little is known. We identify G protein-coupled melanocortin 1 receptor (MC1R) is enriched in dopaminergic neurons, the activation of MC1R is not only preventing the aggregation and transmission α-Syn, but also increasing the activity of transcription factor EB (TFEB) by promoting TFEB gene transcription and its nuclear translocation. However, the knockdown of TFEB by shRNA nearly completely abolished the effect that induced by MC1R agonist, but its mechanism remains unclear. Based on the above, this project will use gene transfection, western blot, co-immunoprecipitation and confocal microscopy to further verify whether MC1R is a newly target in preventing of α-Syn aggregation and transmission. We will explore whether the activation of MC1R: ① inhibits α-Syn aggregation, secretion and transmission by TFEB-mediated autophagy lysosome pathway; ②promotes TFEB transcription and nuclear translocation via the G protein classic cAMP signal and non-classical β-arrestin 2 pathway. Thereafter we will confirm the inhibition of α-Syn aggregation by MC1R receptor agonist in α-Syn transgenic mice.

近年来发现帕金森病的异常聚集α-synuclein (α-Syn) 具有"朊蛋白"样传播作用，但其调控机制一直未明。我们发现：多巴胺神经元上具有G蛋白偶联受体MC1R，其激活可抑制α-Syn聚集及细胞间传递并促进TFEB转录及核转位，而沉默TFEB后几乎可完全阻断MC1R受体激动剂的这一效应，提示TFEB介导了这一过程，但其中具体机制尚不清楚。本项目拟在前期基础上，采用基因转染、蛋白免疫印迹、免疫共沉淀、激光共聚焦等技术手段，进一步证明MC1R受体是抑制α-Syn聚集及传递的新靶点分子，并阐明其详细机制。包括MC1R受体激活后：①是否通过TFEB介导的自噬溶酶体途径抑制α-Syn聚集、分泌及传递；②是否通过G蛋白经典的cAMP信号及非经典的β-arrestin 2 途径促进TFEB转录及核转位。同时我们还将在α-Syn转基因鼠上验证MC1R受体激动剂对α-Syn聚集的调控。

近年来发现帕金森病的异常聚集α-Syn具有"朊蛋白"样传播作用，但其调控机制一直未明。本项目通过细胞及动物实验，采用基因转染、蛋白免疫印迹、免疫共沉淀、激光共聚焦等技术手段，发现MC1R受体是抑制α-Syn聚集及传递的新靶点分子，并阐明了其详细机制。我们发现多巴胺神经元上有G蛋白偶联受体MC1R表达，其激活可抑制α-Syn聚集及细胞间传递并促进转录因子TFEB转录及核转位，与G蛋白经典的cAMP信号及非经典的β-arrestin 2途径相关。我们发现，沉默TFEB后，MC1R受体激动剂抑制α-Syn传递的效应几乎被全部阻断，提示TFEB介导了MC1R受体激活效应。我们还发现，cAMP作为第二信使分子将MC1R受体被激活的效应放大，并作用于其下游的PKA、CREB，使得细胞核内的TFEB合成增加；另一方面，β-arrestin 2被招募至激活的MC1R受体表面，作为支架蛋白活化下游的AKT，并负调控mTORC1、从而促进TFEB核转位。上述体外实验结果在动物模型上得到了验证，同时在MPTP帕金森病动物模型上发现MC1R受体激活后能促进多巴胺神经元生存。最后，我们还在帕金森病临床样本中围绕MC1R受体及其相关基因开展了相关研究，我们发现rs75072999及rs1378602等多个基因变异明显增加帕金森病易感风险。总之，本项目发现的MC1R受体调控多巴胺神经元间α-Syn的聚集和播散的机制以及新发现的易感基因位点为帕金森病的防治和进一步机制研究提供了新思路。