乌头酸酶ACO2调控LRRK2介导的帕金森病机制研究

Parkinson’s disease (PD) is a common neurodegenerative disorder. Its mechanisms remain largely unresolved. Recently studies have established the genetic variation in LRRK2 gene as one of the most important susceptible factors. A mutation, G2019S, in this gene can be detected in nearly 5% of familial and 1-2% of sporadic PD patients in the Western populations, while G2385R and R1628P are detected as risk variants in Asians. However, these variants can also be detected in normal individuals, suggesting that modifiers may exist to promote the pathological roles of LRRK2. In a preliminary study, we have analyzed the genetic variations in PD patients and normal controls carrying the LRRK2 risk variants by next-generation of sequencing. We identified rs2006715, a coding variant in the gene encoding aconitate 2 (ACO2), significantly modified the risk of LRRK2 carriers for developing PD. Further, knockdown of ACO2 prevented the neuronal apoptosis induced by the G2019S mutant, indicating that ACO2 may be involved in PD pathogenesis via modulating LRRK2. In this project, we will further confirm the ACO2-LRRK2 interaction and its relation to PD at the clinical, animal, cellular and molecular levels, and investigate the modulation of ACO2 on the LRRK2-mediated oxidative stress damage, mitochondrial dysfunction and apoptosis of dopaminergic neurons, and the possible underlying mechanisms. These studies will be important for unraveling novel mechanisms of the disease.

帕金森病（Parkinson’s disease, PD）是常见的神经退行性疾病，其机制未完全阐明。近来发现LRRK2基因变异是其最重要的易感因素之一。约5%西方家族性和1-2%散发性患者可检测到G2019S突变，而亚洲人携带G2385R和R1628P风险变异，然而这些变异也可见于正常人。因此，存在促进LRRK2致病作用的修饰因子。我们前期对LRRK2变异的PD患者和正常人进行二代测序，发现编码乌头酸酶的ACO2基因的rs2006715变异可显著修饰LRRK2变异携带者的风险。进一步敲降ACO2可阻止G2019S突变诱导的神经元凋亡，提示ACO2可通过调控LRRK2而参与PD发病。本课题将从临床、动物、细胞、分子水平进一步证实其互作关系及与PD的联系，探讨ACO2调控LRRK2介导的多巴胺能神经元氧化应激、线粒体异常、凋亡中的作用及其可能的机制，这对于揭示其发病新机制具有重要意义。

线粒体在帕金森病（PD）的发生发展中扮演重要的角色。中脑黑质多巴胺能（DA）神经元对线粒体的损伤及三羧酸（TCA）循环产生的ATP水平极为敏感。在TCA循环中，将柠檬酸可逆性地催化为异柠檬酸的线粒体乌头酸酶（ACO2）在PD中受到关注，但其作用机制尚不清晰。我们首先通过全基因组测序和目标区域靶向测序，检出了ACO2基因的杂合变异，且酶活性分析显示部分变异会导致ACO2酶活性实验的功能缺失。同时，我们提取了PD患者及正常人外周血单核细胞（PBMC），测定其ACO2酶活性，发现ACO2的表达量在PD患者中显著升高。我们进一步利用PD的细胞及果蝇模型，发现ACO2的活性降低加重了Rot所致的多巴胺能神经元（MES23.5）的毒性，并导致细胞的耗氧率（OCR）、ATP产生和线粒体膜电位（MMP）的显著下降，从而损害了线粒体功能。进一步分析表明，ACO2活性降低可导致Parkin和PINK1的积累、LC3-II的表达显著降低和p62蛋白的表达升高。同时自噬小泡增多和靶向进入线粒体基质的mt-Keima蛋白下降，证实自噬流下降。而且，伴随这些变化，Bcl-2/Bax比值显著降低，表明发生了线粒体介导的凋亡。这些结果表明，ACO2功能缺失可能影响线粒体自噬过程，诱导线粒体相关细胞凋亡，增加对Rot等毒素的敏感性，从而加速PD的发生发展。总之，本课题从临床、动物、细胞及分子水平探讨了ACO2通过调控线粒体功能及自噬影响PD发病的分子机制，对于进一步阐明PD的发病机制具有重要意义。