The Roles of a-Synuclein and Parkin in Parkinson Disease

α-突触核蛋白和 Parkin 在帕金森病中的作用

• 批准号: 6855705
• 负责人: MICHAEL GEBHARD SCHLOSSMACHER
• 金额: $ 15.3万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-30 至 2006-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6855705
• 关键词: 6 hydroxydopamine; Lewy body; Parkinson' s disease; affinity chromatography; alpha synuclein; brain cell; clinical research; disease /disorder model; dopamine; family genetics; gene mutation; glycosylation; human tissue; mass spectrometry; mesencephalon; neural degeneration; neuropathology; parkin gene /protein; postmortem; protein isoforms; protein structure function; transfection

DESCRIPTION (provided by applicant): The pathogenesis of Parkinson disease (PD) is unknown but dopamine-induced oxidative stress, proteasomal abnormalities and mitochondrial dysfunction are associated with its neurodegeneration. Rare heritable forms of PD are linked to an increasing number of gene loci. At the PARK1 locus, SNCA encodes a neuronal protein, alpha-synuclein (alpha-S), that is involved in the transition of synaptic vesicles from the reserve-resting pool to the readily releasable pool in vivo and in vitro. It is linked to sporadic PD by the formation of fibrillar inclusions that contain phosphorylated alpha-S, and to autosomal dominant PD by a likely gain-of-function effect of two infrequent point mutations. The PARK2 gene encodes parkin, an E3 ubiquitin ligase. It is mutated in <50% of all autosomal recessive PD cases by a probable loss-of-function phenomenon. In normal human brain (but not rat brain), a pool of alpha-S undergoes O-linked glycosylation, thereby generating alpha-Sp22. This glycoprotein is a substrate for parkin's E3 ligase function in vitro and accumulates in PARK2-mutant PD brain. The central hypotheses of this application state that 1) a shared pathogenetic pathway is encoded by PD-linked genes, 2) characterization of the alpha-S glycosylation in primate brain will provide insights into the pathogenesis of PD, 3) the normal function of the Parkin E3 complex is essential for the sustained survival of catecholaminergic neurons in adult human brain, and 4) the identification of the in vivo subunits of the assembled parkin E3 complex will validate reported binding partners and reveal potentially neurotoxic substrates. To this end, I have identified two Specific Aims: Aim 1: To characterize the glycosylation of alpha-S in human control brain as well as PARK1-linked PD brain and to model its biosynthesis in a cell model, and Aim 2: To biochemically purify the subunits of the Parkin E3 ligase complex from human brain, and verify them in vitro.

描述（申请人提供）：帕金森病（PD）的发病机制尚不清楚，但多巴胺诱导的氧化应激、蛋白酶体异常和线粒体功能障碍与其神经变性有关。罕见的遗传性 PD 与越来越多的基因位点有关。在 PARK1 基因座，SNCA 编码一种神经元蛋白 α-突触核蛋白 (alpha-S)，该蛋白参与体内和体外突触小泡从储备静止池到易于释放池的转变。它通过含有磷酸化α-S的纤维状包涵体的形成与散发性帕金森病有关，并且通过两个不常见的点突变可能产生的功能获得效应与常染色体显性帕金森病有关。 PARK2 基因编码 Parkin，一种 E3 泛素连接酶。在所有常染色体隐性遗传性 PD 病例中，<50% 的患者会因可能的功能丧失现象而发生突变。在正常人脑（但不是大鼠脑）中，一组 α-S 发生 O-连接糖基化，从而生成 α-Sp22。这种糖蛋白是 Parkin E3 连接酶体外功能的底物，并在 PARK2 突变的 PD 大脑中积累。该申请的中心假设指出：1) 共享的发病途径由 PD 相关基因编码，2) 灵长类动物大脑中 α-S 糖基化的表征将提供对 PD 发病机制的深入了解，3) Parkin E3 复合物的正常功能对于成人大脑中儿茶酚胺能神经元的持续存活至关重要，4) 体内亚基的鉴定 组装的parkin E3复合物的分析将验证报告的结合伙伴并揭示潜在的神经毒性底物。为此，我确定了两个具体目标：目标 1：表征人对照大脑以及 PARK1 连接的 PD 大脑中 α-S 的糖基化，并在细胞模型中模拟其生物合成；目标 2：从人脑中生化纯化 Parkin E3 连接酶复合物的亚基，并在体外进行验证。