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.

我们实验室和其他人的最新数据表明，在黑质中，靶向α-核蛋白的过表达导致帕金森氏神经变性。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）的发病机理中的功能。 α突触核蛋白可能是多蛋白复合物的一部分，并理解 α突触核蛋白及其蛋白质结合伴侣之间的相互作用应有助于理解PD发病机理的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种群对α突触核蛋白的选择性脆弱性。我们建议将重组腺相关病毒（RAAV）作为基因递送载体，以产生黑质纹状体的区域特异性体细胞基因。这 提出了以下3个目标。第一个目的是检查调节与α突触核蛋白相互作用的调节或过表达基因的效果。磷脂酶D2（PLD2）和/或G偶联受体蛋白激酶2（GRK2）将过表达或与α-突触核蛋白结合使用，以确定这些蛋白质与α突触核蛋白之间的已知相互作用是否参与Pd的病原体。我们将遵循 这些组合引起的神经变性的进展是通过查看th阳性神经元和行为缺陷的数量而引起的。目标2是鉴定直接与α突触核蛋白相互作用的其他蛋白质。标记的大鼠和人αssnuclein将用作多巴胺能MN9D细胞系中体内的snmuclein-蛋白质蛋白复合物的亲和力 - 免疫沉淀。亲和力纯化的复合物将通过质谱法分析， 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。目的3是确定多巴胺相关的氧化应激的毒性是否是某些多巴胺能神经元对α-突触核蛋白诱导的帕金森样神经变性的选择性脆弱性的因素。 AAV将用于单独递送α突触蛋白基因，或与增加或减少黑质氧化应激的基因结合使用。其中包括通过前体L-DOPA的过表达以及SOD1和CETALASE来减少氧化应激，从而增加二神纹二胺的产生，以减少氧化应激。与AIM 1一样，我们将遵循由这些组合引起的神经变性的进展。