alpha synuclein function

α突触核蛋白功能

• 批准号: 6754262
• 负责人: NICHOLAS MUZYCZKA
• 金额: $ 34.98万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-12-01 至 2008-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6754262
• 关键词: G protein coupled receptor kinase; Parkinson' s disease; adeno associated virus group; alpha synuclein; behavior test; corpus striatum; dopamine; gene delivery system; gene expression; gene therapy; laboratory rat; levodopa; mass spectrometry; neural degeneration; oxidative stress; phospholipase D; protein protein interaction; substantia nigra; superoxide dismutase; transfection /expression vector; tyrosine 3 monooxygenase

Recent data from our lab and others have demonstrated that targeted alpha-synuclein overexpression in the substantia nigra pars compacta leads to Parkinson-like neurodegeneration. Yet, the function of alpha synuclein is still unknown. The present proposal attempts to define the function of alpha synuclein in both normal brain function and in the pathogenesis of Parkinson disease (PD). Alpha synuclein is likely to be part of a multiprotein complex, and understanding the interaction between alpha synuclein and its protein binding partners should help in understanding the mechanism underlying pathogenesis in PD, as well as its function in the normal brain. It might also help explain the selective vulnerability of certain neuronal populations to alpha synuclein. We propose to use recombinant Adeno-Associated Virus (rAAV) as a gene delivery vector to generate regionally specific somatic transgenics of the nigrostriatal tract. The following 3 aims are proposed. The first aim is to examine the effect of down regulating or overexpressing genes that are known to interact with alpha synuclein. Phospholipase D2 (PLD2) and/or G coupled Receptor protein Kinase 2 (GRK2) will be overexpressed or knocked down in combination with alpha-synuclein to determine whether the known interactions between these proteins and alpha synuclein are involved in the pathogenesis of PD. We will follow the progression of neurodegeneration caused by these combinations by looking at the number of TH- positive neurons and behavioral deficits. Aim 2 is to identify additional proteins that interact directly with alpha synuclein. Tagged rat and human alpha synuclein will be used as a bait to affinity-immunoprecipitate a synuclein-protein complexes in vivo in the dopaminergic MN9D cell line. The affinity purified complexes will be analyzed by mass spectrometry methods and antibody to known interaction partners to identify the multiprotein interactions for alpha synuclein. Aim 3 is to determine whether the toxicity of dopamine related oxidative stress is a factor in the selective vulnerability of certain dopaminergic neurons to Parkinson-like neurodegeneration induced by alpha synuclein. AAV will be used to deliver the alpha synuclein gene alone, or in combination with genes that increase or decrease oxidative stress in substantia nigra. These include the tyrosine hydroxylase (TH) and GTP cyclohydrolase genes to increase nigrostriatal dopamine production by overexpression of the precursor L-dopa, and SOD1 and catalase to reduce oxidative stress. As in aim 1 we will follow the progression of neurodegeneration caused by these combinations.

我们实验室和其他实验室的最新数据表明，黑质致密部中的靶向 α-突触核蛋白过度表达会导致帕金森样神经变性。然而，α突触核蛋白的功能仍然未知。本提案试图定义 α 突触核蛋白在正常脑功能和帕金森病 (PD) 发病机制中的功能。 α突触核蛋白可能是多蛋白复合物的一部分，并且了解 α 突触核蛋白与其蛋白结合伴侣之间的相互作用应有助于了解 PD 发病机制及其在正常大脑中的功能。它还可能有助于解释某些神经元群体对α突触核蛋白的选择性脆弱性。我们建议使用重组腺相关病毒（rAAV）作为基因递送载体来产生黑质纹状体区的区域特异性体细胞转基因。这 提出以下 3 个目标。第一个目的是检查已知与 α 突触核蛋白相互作用的基因下调或过度表达的影响。磷脂酶 D2 (PLD2) 和/或 G 偶联受体蛋白激酶 2 (GRK2) 将与 α-突触核蛋白结合过表达或敲低，以确定这些蛋白和 α 突触核蛋白之间的已知相互作用是否参与 PD 的发病机制。我们将遵循 通过观察 TH 阳性神经元的数量和行为缺陷来了解这些组合引起的神经变性的进展。目标 2 是鉴定与 α 突触核蛋白直接相互作用的其他蛋白质。标记的大鼠和人 α 突触核蛋白将用作诱饵，在多巴胺能 MN9D 细胞系中体内亲和免疫沉淀突触核蛋白-蛋白质复合物。亲和纯化的复合物将通过质谱方法进行分析， 已知相互作用伙伴的抗体，以鉴定 α 突触核蛋白的多蛋白相互作用。目标 3 是确定多巴胺相关氧化应激的毒性是否是某些多巴胺能神经元选择性易受 α 突触核蛋白诱导的帕金森样神经变性的因素。 AAV 将用于单独递送 α 突触核蛋白基因，或与增加或减少黑质氧化应激的基因组合。其中包括酪氨酸羟化酶 (TH) 和 GTP 环化水解酶基因，通过前体左旋多巴的过度表达来增加黑质纹状体多巴胺的产生，以及 SOD1 和过氧化氢酶，以减少氧化应激。与目标 1 一样，我们将跟踪这些组合引起的神经变性的进展。