BAG3在多巴胺能神经元自噬调控中的作用及机制研究

Increasing data indicate that autophagy play an important role in Parkinson's disease (PD). But how to choose autophagic regulation target and control the degree of autophagy activation have troubled researchers for many years and need to be solved urgently. Selectively activate autophagy to eliminate the misfolded and aggregate-prone proteins may provide new insights for the prevention and treatment of Parkinson's disease (PD) whose pathological feature is α-synuclein aggregation. Recently, it has been reported that Bcl-2-associated athanogene 3 (BAG3) mediates chaperone-based aggresome-targeting and selective autophagy of misfolded proteins. However, the mechanisms of it are not yet fully understood. And till now there's no data show the role of selective autophagy mediated by BAG3 in dopaminergic neuron. Our preliminary studies indicated that BAG3 involved in the autophagic regulation of dopaminergic neuron. In the present study, PC12 cells, over-expression of wild-type and A30P mutant α-synuclein PC12 cells and A30Pα-synuclein transgenic mice are used to investigate the effects of BAG3 in wild-type and mutant α-syncline's autophagic degradation. The underlying mechanisms of autophagy in the clearance of different α-synuclein are explored after overexpression or RNA interference (RNAi) of BAG3. Furthermore, the regulatory mechanisms of BAG3 in autophagy are also explored by activating autophagy and inhibiting autophagy or dynein.The finding of this project may provide a novel effective target for develop promising drugs against PD.

自噬在帕金森病（PD）中的作用已越来越受到重视，但是如何调控自噬和控制其激活程度是一直困扰研究人员的难题，若能选择性激活自噬而促进错误折叠和易聚集蛋白降解，将为以α-synuclein（α-syn）病理性蓄积为特征的PD的防治提供新思路。最新研究发现BAG3介导了分子伴侣为基础的aggresome靶向运输和错误折叠蛋白的选择性自噬，但其机制尚未阐明，尤其是BAG3在多巴胺（DA）能神经元中的作用尚未见报道。我们的前期研究提示BAG3可能参与DA能神经元的自噬调控。本课题将在此基础上，采用已建立的过表达野生型和A30P突变型α-syn的PC12细胞及A30P 突变α-syn转基因小鼠，系统地研究BAG3对不同α-syn自噬降解的作用，通过RNA干扰、过表达BAG3以及干预自噬和自噬溶酶体通路中的动力蛋白深入阐明BAG3在α-syn自噬降解中的调控机制，从而为研发理想的PD治疗药物提供新靶标。

自噬在帕金森病（PD）中的作用已越来越受到重视，若能选择性激活自噬促进错误折叠和易聚集蛋白降解，将为以α-synuclein（α-syn）病理性蓄积为特征的PD的防治提供新思路。有研究发现BAG3介导了分子伴侣为基础的aggresome靶向运输和错误折叠蛋白的选择性自噬，但其机制尚未阐明，尤其是BAG3在多巴胺（DA）能神经元中的作用尚未见报道。本项目的研究结果显示：MG132可呈浓度依赖性降低SNCAWT-PC12细胞活力，引起SNCAWT-PC12细胞内α-syn蛋白蓄积，但对α-syn的mRNA水平无明显影响；MG132呈浓度及时间依赖性上调SNCAWT-PC12细胞内自噬相关蛋白LC3Ⅱ、p62及BAG3的蛋白水平；并呈浓度依赖性增加SNCAWT-PC12细胞p62及Bag3 mRNA水平；敲减Bag3可减少SNCAWT-PC12细胞及MG132处理后SNCAWT-PC12细胞内LC3Ⅱ的蛋白表达，并加剧细胞内α-syn的蓄积；过表达BAG3可增加SNCAWT-PC12细胞及MG132处理后SNCAWT-PC12细胞内LC3Ⅱ的蛋白表达，并减少细胞内α-syn的蓄积；敲减Atg5基因后，过表达BAG3也不能促进SNCAWT-PC12细胞内α-syn蛋白的降解；MG132可呈浓度及时间依赖性增加SNCAWT-PC12细胞内Hsp70的蛋白水平，同时可呈浓度依赖性增加Hsp70的mRNA水平；SNCAWT-PC12细胞内p62、Hsp70可分别与BAG3结合，在MG132处理后或BAG3过表达的情况下，p62、Hsp70与BAG3的结合量均进一步增加。提示，蛋白酶体抑制剂MG132可导致SNCAWT-PC12细胞内α-syn降解障碍；BAG3可诱导自噬并减少α-syn蓄积，参与了SNCAWT-PC12细胞内自噬过程及α-syn的蛋白降解过程；BAG3蛋白可通过形成BAG3-Hsp70复合体与自噬底物蛋白p62相互作用，BAG3介导的这一选择性自噬促进了SNCAWT-PC12细胞内α-syn的降解。此外，整体水平的研究结果也显示，SNCAA53T 转基因小鼠中脑中BAG3蛋白水平增加并与SNCA蛋白共定位，且BAG3可与Hsp70和SQSTM1相互作用形成复合物。上述结果将为研发理想的PD治疗药物提供新靶标。