DJ-1抑制α-突触核蛋白异常聚集的机制

Loss-of-function mutations of PARK7/DJ-1 gene cause early-onset familial Parkinson’s disease (PD). DJ-1 is involved in various cellular processes. Previous research showed that DJ-1 could inhibit α-synuclein aggregation, but the detailed mechanisms underlying involved remain unclear. In normal condition, α-synuclein is mainly degraded by chaperone-mediated autophagy (CMA). CMA dysfunction resulted in α-synuclein aggregation, which is closely related to the pathogenesis of PD. Levels of lysosomal associated membrane protein 2A (LAMP2A), the rate-limiting receptor for CMA substrate flux, are directly dependent on lipid composition of the lysosomal membrane microdomains. Our preliminary studies showed that DJ-1 deficiency could exacerbate α-synuclein aggregation and suppress the protective upregulation of LAMP2A, but the detailed mechanisms underlying involved remain unclear. In this proposed study, we will investigate whether DJ-1 could reduce α-synuclein aggregation by CMA and further explore the molecular mechanisms. The research will be focused on the specific aims as follow: 1. to observe the effect of DJ-1 deficiency on the CMA function; 2. to elucidate the effect of DJ-1 deficiency on the level of LAMP-2A and the mechanism; 3. to explore possible mechanisms of the effect of DJ-1 deficiency on lipid composition of lysosomal membranes and lipid metabolism. The research will provide further evidence for the interplay between neurodegenerative disease-associated proteins and will provide the useful theoretical and experimental information for the novel treatment of PD.

DJ-1缺失性突变可导致常染色体隐性家族性早发性PD。DJ-1参与体内多种生理过程，DJ-1可抑制α-突触核蛋白（α-syn）聚集，但机制不明。生理情况下，α-syn主要通过分子伴侣介导的自噬（CMA）降解，CMA损伤可导致α-syn聚集，与PD发生密切相关。溶酶体相关膜蛋白2A(LAMP2A)是CMA的限速蛋白，其含量取决于溶酶体膜脂质微区的脂质构成。我们前期研究显示DJ-1缺失可抑制LAMP2A的保护性上调，加剧α-syn堆积。基于已有初步结果，本项目将深入探讨DJ-1通过调节CMA影响α-syn的分子机制，我们将观察DJ-1缺失对CMA功能的影响；考察DJ-1缺失对CMA关键组分LAMP2A的表达水平的作用及其机制；探索DJ-1缺失对脂质微区中脂质构成的影响。本项目将有助于更加深入地揭示PD致病基因相关蛋白DJ-1抑制α-syn聚集的分子机制，为探索PD治疗新途径提供理论及实验依据。

Park7/DJ-1缺失性突变可导致常染色体隐性家族性早发性帕金森病（Parkinson disease, PD）。PD是最常见神经系统退行性疾病，病理上主要表现为中脑黑质致密部多巴胺能神经元进行性丢失和残存神经元内富含α-突触核蛋白（α-synuclein）路易小体（Lewy body, LB）的形成。α-Synuclein蛋白的错误折叠和聚集在PD的发病机制中发挥重要作用。有研究报道DJ-1可抑制α-synuclein聚集，并可能发生在聚集体形成的早期阶段。可溶性野生型（WT）α-synuclein单体主要通过分子伴侣介导的自噬（chaperone-mediated autophagy, CMA）降解，CMA损伤与PD的发生密切相关。基于上述研究，本研究将从CMA的角度进一步探讨DJ-1缺失突变导致α-synuclein堆积与聚集的发生机制。在本研究中，我们利用DJ-1基因敲除小鼠和siRNA沉默DJ-1表达的SH-SY5Y细胞系作为DJ-1缺失突变的动物和细胞模型来阐述DJ-1缺失导致α-synuclein聚集的发生机制。结果显示，在整体与细胞水平DJ-1缺失可导致α-synuclein堆积，而在细胞水平过表达DJ-1则抑制了α-synuclein堆积。其次，α-synuclein过表达上调了CMA的限速步骤——溶酶体相关膜蛋白2A（Lysosomal associated membrane protein 2A, LAMP-2A）的表达水平；而DJ-1缺失则取消了这一变化。在WT α-synuclein过表达组中，DJ-1缺失显著降低了溶酶体富集组分中热休克同源蛋白70（Lysosomal heat-shock cognate protein70 kDa, Lys-HSC70）含量，但对全细胞裂解液HSC70的水平无明显影响。进一步分析机制显示，DJ-1缺失可加速LAMP-2A的降解，这可能是导致LAMP-2A含量降低的原因。本研究提示，DJ-1缺失可通过增加LAMP-2A的降解，进而加剧了α-synuclein的堆积，这提示DJ-1对CMA是一个重要的调节因素。本研究对PD致病基因相关蛋白之间的作用机制提供了实验证据，对了解DJ-1在PD发病机制中的作用提供了新的机制。