alpha synuclein function

我们实验室和其他实验室的最新数据表明，黑质中的靶向α-突触核蛋白过表达导致帕金森样神经变性。然而，α突触核蛋白的功能仍然是未知的。本建议试图定义α突触核蛋白在正常脑功能和帕金森病（PD）的发病机制中的功能。α突触核蛋白可能是多蛋白复合物的一部分， α-突触核蛋白和它的蛋白质结合伙伴之间的相互作用应该有助于理解PD发病机制的基础，以及它在正常大脑中的功能。这也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）作为基因传递载体，以产生区域特异性的黑质纹状体道体细胞转基因。的 提出了以下三个目标。第一个目的是检查已知与α突触核蛋白相互作用的基因的下调或过表达的影响。磷脂酶D2（PLD 2）和/或G偶联受体蛋白激酶2（GRK 2）将与α-突触核蛋白组合过表达或敲低，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的发病机制。我们将按照 通过观察TH阳性神经元的数量和行为缺陷来观察由这些组合引起的神经变性的进展。目的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，以在多巴胺能MN 9D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法分析， 已知的相互作用伴侣的抗体，以确定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。腺相关病毒将用于单独递送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递送。这些包括酪氨酸羟化酶（TH）和GTP环水解酶基因，以增加黑质纹状体多巴胺生产的前体L-多巴，SOD 1和过氧化氢酶的过度表达，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退行性变的进展。

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.