LRRK2 G2385R变异对神经元内线粒体功能影响及其机制研究

The variants of LRRK2 gene is closely related to the incidence of Parkinson's disease (Parkinson 's disease, PD). G2385R is the Asian-specific risk variation, and in the Asian population of PD, its variation rate is as high as 5%, what’s more, it can significantly increase the risk of developing of PD in Asian population, especially in Chinese Han population. However, there is little study on the pathogenic mechanism of G2385R. At present, in the field of PD, the research on the specific pathogenesis of common risk variants has been paid more attention. To date, few studies have suggested that the pathogenic mechanism of G2385R is different from G2019S, which can not cause the cell toxicity by increasing kinase activity the same as G2019S, and G2385R is more likely to affect the function of WD40 domain of LRRK2, then cause the dysfunction of the interaction between LRRK2 and other proteins, finally, affecting the downstream pathways. In our previous study, we also found that G2385R did not increase the activity of LRRK2 kinase, and found that G2385R can induce SH-SY5Y damage. We also found that G2385R can reduce the interaction between LRRK2 and ULK1 protein (Uncoordinated family member 51-like kinase (UNC) 1) and change the LC3 level. ULK1 and LC3 are related to mitophagy and autophagy in neurons, which prompted us to hypothesize that LRRK2 G2385R may affect on mitochondrial function and then cause abnormal mitophagy and autophagy in SH-SY5Y. To this end, we design the related physical/chemical and cell experiments and carry out this project.

LRRK2与帕金森病（PD）发病密切相关。G2385R是亚洲特异性风险位点，变异率高达5%，并可明确、明显的提高亚洲患PD风险，但G2385R致病机制研究甚少。目前，在PD，对常见风险位点致病机制研究逐渐重视。至今，已有的少量研究提示G2385R致病机制与G2019S不同，其并不像后者通过增加激酶活性造成细胞毒性，G2385R更可能对LRRK2 WD40结构域造成影响，进而影响LRRK2与互作蛋白结合，影响下游通路。前期研究中，我们也发现G2385R并不增加LRRK2激酶活性，并发现G2385R可导致SH-SY5Y细胞损伤增加，与ULK1蛋白互作增加，并影响细胞内LC3水平，而ULK1、LC3与神经元线粒体自噬及细胞自噬密切相关，这促使我们提出假设：LRRK2 G2385R变异可能对线粒体功能造成影响，进而影响线粒体自噬调控及细胞自噬。为此，我们设计相关理化及细胞实验，开展本项目研究。

本研究构建了VECTOR、flag-LRRK2 WT、flag-LRRK2 G2385R相关质粒，利用HEK293T细胞系开展了相关研究。我们发现LRRK2 G2385R变异可导致细胞凋亡增加，运用Annexin V-FITC细胞凋亡检测试剂盒，对分别转染VECTOR、LRRK2 WT、LRRK2 G2385R的三组细胞行流式细胞检测，结果显示相较于WT组，G2385R组细胞凋亡比例增加，相较于WT组，G2385R组cleaved Caspase3蛋白相对定量增加。针对细胞凋亡，我们发现LRRK2 G2385R变异不改变细胞自噬水平，相较于WT组，G2385R组的Beclin1、P62、LC3I/LC3II蛋白相对定量没有统计学差异，提示LRRK2 G2385R变异导致细胞凋亡增加可能不是通过自噬途径。为此，我们进一步探索LRRK2 G2385R变异对细胞线粒体功能的影响，我们运用ROS检测试剂盒，对三组细胞行流式细胞检测，结果显示相较于WT组，G2385R组细胞ROS水平升高；运用MMP检测试剂盒，行流式细胞检测，结果显示相较于WT组，G2385R组细胞MMP降低；运用ATP检测试剂盒，对三组细胞进行检测，结果显示相较于WT组，G2385R组细胞ATP减少。同时，我们发现LRRK2 G2385R变异不改变细胞线粒体分裂/融合蛋白（OPA1、Mfn1、Mfn2、Fis1、Drp1）的动态平衡。进一步探究这其中的可能机制，我们发现LRRK2 G2385R变异使mtDNA的表达减少，分别对三组细胞行转录组测序，RNA-seq结果提示G2385R变异主要导致mtDNA的表达减少，qPCR进一步验证mtDNA的减少。我们设想LRRK2 G2385R与线粒体转录因子TFAM互作发生改变，但试验发现LRRK2与TFAM不存在互作，我们拟进一步寻找相关线粒体转录因子。针对线粒体功能受累这一现象，我们进一步探索线粒体保护剂-艾地苯醌的潜在保护作用，我们发现艾地苯醌可以挽救LRRK2 G2385R变异导致的ATP减少，这将为在临床实践中利用艾地苯醌对携带LRRK2 G2385R变异的这类帕金森病患者开展修饰治疗提供可能的新思路。本项目成功构建了LRRK2 G2385R及正常对照iPSCs以备后续试验。