帕金森病中TREM2介导α-突触核蛋白清除和免疫炎症反应的机制研究

The pathological characteristics of Parkinson's disease (PD) include aggregation of α-synuclein (α-syn), loss of dopaminergic (DA) neurons and subsequent immune/inflammatory disorders. The emerging evidences have shown that mutations of Trem2 are correlated to the risk of PD in American and European, but it has not been confirmed in Chinese yet. In brain, TREM2 is only highly expressed on microglia, also the mechanism of TREM2 on α-syn aggregation is unclear. Our exciting preliminary findings demonstrated that TREM2 regulated the endocytosis of microglia. We propose that TREM2 may engage in the recognition and endocytosis of microglia on α-syn through Rac1 pathway, and inhibit the subsequent immuno-inflammatory response regulated by NF-κB pathway. Thus, the first aim of our plan is to analyze the polymorphisms of Trem2 in Chinese population, and evaluate the correlation between the potential SNPs on Trem2 and Chinese PD; secondly, we will use the microglia conditional knockout and Trem2 knockout mice to explore the signal network of TREM2-Rac1/NF-κB under the α-syn aggregation from the views of genomics and proteomics; finally, we will clarify the microglia functions on endocytosis and regulation of immune/inflammatory under the degeneration of DA neurons. This study will reveal the mechanism of TREM2 involved in the molecular mechanism of PD from the aspects of α-syn aggregation and immune/inflammatory disorders, and these findings may shed light on the possibility of developing the precise treatment for PD.

帕金森病(PD)的病理特征为α-突触核蛋白异常聚集(α-syn)、多巴胺能(DA)神经元丢失并继发免疫炎症紊乱。最近研究发现Trem2基因突变与欧美PD罹患风险相关，但在中国人群尚不明确。在脑组织，TREM2仅在小胶质细胞高表达；其与α-syn聚集的关系尚不清楚。预实验中我们发现TREM2可调控小胶质细胞的内吞功能，推测其可能通过Rac1途径影响小胶质细胞识别、内吞α-syn，并抑制NF-κB通路介导的继发免疫炎症紊乱。本研究拟分析Trem2基因多态性在中国人群的分布，评估其与PD风险的关系；构建条件性小胶质细胞和Trem2基因敲除小鼠，从基因组学和蛋白质组学角度，探索α-syn积聚下TREM2-Rac1/NF-κB信号网络；阐明DA能神经元变性过程中小胶质细胞内吞及炎症调控机制。本研究将从蛋白聚集和免疫炎症紊乱角度揭示TREM2参与PD的分子机制，为临床PD精准治疗提供参考。

髓系细胞2中表达触发受体(TREM2)作为小胶质细胞特有受体，被报道参与脑内异常蛋白沉积的清除。帕金森病（PD）的病理表现是脑内a-突触核蛋白（α-syn）的异常聚集，我们推测TREM2可能参与PD中的α-syn清除影响发病。据此，我们开展研究：（1）在PD患者DNA标本，选取Trem2基因进行PCR-直接测序，应用PCR-SBT技术进行等位基因分型，筛选和分析Trem2的SNPs和散发性PD的相关性。ELISA检测脑脊液α-syn水平，评估Trem2的SNPs与脑脊液中α-syn水平的相关性。基于 UPLC-MS/MS 检测平台，对脑脊液中的脂质代谢物进行定性定量检测，寻找作为TREM2受体的候选脂质配体。（2）通过rAAV- A53T转染构建的PD小鼠模型中，采用小胶质细胞条件性敲除技术进行干预，分析小胶质细胞与α-syn积聚的关系，评估小胶质细胞释放炎性介质和细胞因子的功能。（3）通过条件性小胶质细胞敲除技术，探索小胶质细胞参与调控神经元的再生和突触修饰过程。（4）构建Trem2 敲除鼠上，通过rAAV- A53T模型，观察Trem2 敲除对PD小鼠的影响；及Trem2敲除后对脑脊液α-syn水平的影响；探索TREM2信号通路调控神经免疫炎症的分子机制。（5）在六羟多巴构建的动物模型中，我们寻找参与调控黑质纹状体中小胶质细胞的活化的信号通路；分析与TREM2受体形成的调控网络。在研究中我们发现：（1）TREM2第二外显子的基因多态性与PD易感性无显著相关，但TREM2表达水平与PD罹患风险有关，可溶性TREM2有作为PD分子标志物的潜在价值；（2） 小胶质细胞参与神经元再生的调控；（3）免疫蛋白酶体参与抗原呈递可能影响小胶质细胞激活；（4）TREM2信号通路介导小胶质细胞内吞α-syn及释放炎症因子；（5）脂类分子可能是TREM2受体的配体，参与PD致病过程。上述研究结果有助了改进PD临床的早期诊断，并为靶向药物研发提供参考。