PLA2G6基因对α-synuclein蛋白病理性聚集的影响及机制研究

Mutations in the PLA2G6 gene have been reported in autosomal recessive early-onset parkinsonism (AREP). The main pathogenic mechanisms are mitochondrial impairment and iron accumulation. Previously, it was reported that widespread α-synuclein-positive Lewy bodies were found in the brain of both patients with PLA2G6 gene mutations and PLA2G6 knockout mouses, which is suggested that there is a link between PLA2G6 gene and α-synuclein aberrant accumulation. But so far the exact mechanisms remain unclear. In previous studies, We discovered a homozygousPLA2G6 p.D331Y mutation in AREP. Compared to the wild-type PLA2G6 protein, the mutated p.D331Y protein showed a 70% reduction in enzyme activity. At present, we have successfully constructed PLA2G6 gene knockout and D331Y knock in mouse model. In this study, we plan to verify the effects of reduction in PLA2G6 enzyme activity on aberrant accumulation of α-synuclein in vitro cell models and in vivo mouse models. And then, we will further clarify the possible mechanisms through mitochondrial impairment, increased the ion load and paralysed protein degradation. This project will be helpful to explore the common targets or substrates between different pathogenic genes of Parkinson's disease (PD), and to provide new ideas and new insights for prevention and therapeutic strategies in PD.

PLA2G6基因突变可导致常染色体隐性遗传早发帕金森综合征（AREP），主要致病机制涉及线粒体功能障碍和铁离子沉积。有研究发现在PLA2G6突变患者和PLA2G6基因敲除小鼠的脑组织中，均证实有广泛α-synuclein（+）路易小体存在，因此推测PLA2G6基因突变可导致α-synuclein病理性聚集，且具体机制不明。前期工作中，我们在一AREP家系中，发现PLA2G6基因错义突变（D331Y），功能研究显示该突变后的PLA2G6蛋白酶活性下降70%。目前我们已成功构建PLA2G6基因敲除和D331Y敲入小鼠模型。本研究拟在前期研究的基础上，通过离体细胞和在体小鼠模型，探讨PLA2G6基因突变对α-synuclein病理性聚集的影响，并从线粒体、铁和α-synuclein蛋白降解方面阐明作用机制。本项目有助于深入探究帕金森病不同致病基因之间共同的作用靶点，为防治提供新思路和新视角。

PLA2G6基因是PARK14帕金森综合征的致病基因，且与多种神经变性疾病密切相关，统称为PLA2G6相关神经变性疾病（PLAN）。既往有研究在PLA2G6基因突变小鼠脑区及PLAN患者尸解脑区发现α-synuclein（+）的路易小体。前期工作中，我们在一常染色体隐性遗传早发帕金森综合征（AREP）家系中，发现PLA2G6基因错义突变（D331Y），功能研究显示该突变可致PLA2G6蛋白酶活性下降70%。我们推测PLA2G6基因功能缺失可能导致α-synuclein蛋白病理性聚集，但具体机制不明。本课题在细胞水平，验证PLA2G6基因功能缺失可出现α-synuclein蛋白病理性聚集现象；机制探讨，发现PLA2G6基因敲减可通过抑制细胞内自噬溶酶体途径致使α-synuclein降解半衰期延长和线粒体功能障碍；反之，PLA2G6基因过表达可部分逆转鱼藤酮介导的α-synuclein蛋白病理性聚集和线粒体功能障碍。以上研究，从离体细胞水平初步阐明了PLA2G6与α-synuclein蛋白病理性聚集的可能分子机制，为帕金森病防治提供新思路。在体动物水平研究中，对前期构建的PLA2G6基因敲除（KO）小鼠和PLA2G6基因定点敲入p.D331Y（KI）小鼠模型分别从gDNA、RNA和蛋白水平进行验证。小鼠行为学实验考虑观察时间有限，基因敲除/敲入实验小鼠年纪尚轻（2M, 4M和6M），暂时未出现运动平衡功能受损临床表型，后续拟连续进行行为学实验追踪评估及组织化学、病理生理学研究。