PD Cell Models: Alpha-Synuclein and Interacting Proteins

家族性帕金森病发病机制的研究可能有助于理解各种形式的帕金森病的发病机制。家族性帕金森病可由α-突触核蛋白、parkin或DJ-1基因突变引起。我们已经鉴定出SynPhilin-1是一种α-突触核蛋白相互作用蛋白，并发现它是帕金森氏病死后脑内泛素化的路易小体的组成部分，并且可以作为Parkin泛素化的底物。在一项合作研究中，我们有提示证据表明，SynPhilin-1的R621C突变可能是帕金森病的罕见原因。我们已经发现S 129的磷酸化 α-突触核蛋白在细胞培养中强烈调控包涵体的形成。我们正在建立突变α-突触核蛋白毒性的可诱导细胞模型，并发现A53T突变体α-突触核蛋白的表达会导致直接的细胞毒性。我们建议研究突变型α-突触核蛋白导致细胞死亡的机制，以及这些蛋白在帕金森病发病机制中的相互作用。在具体目标1中，我们将研究α-突触核蛋白诱导的PC 12细胞系和细胞毒性的机制，包括α-突触核蛋白的磷酸化和裂解的作用(与项目3合作)，以及DJ-1对毒性的可能调节(与项目4合作)。在具体目标2中，我们将描述α-突触核蛋白、突触核蛋白-1和Parkin之间的相互作用(与项目1和神经病理学核心合作)。我们假设包涵体优先通过K63连接而不是K48连接泛素化，这与在细胞信号和蛋白质降解中的潜在作用一致。在……里面 具体目标3我们将研究α-突触核蛋白的磷酸化，并与项目1和3以及神经病理学核心合作，确定磷酸化是否调节与突触核蛋白-1的相互作用，并促进聚集或切割。最后，在特定的目标4中，我们将创造SynPhilin-1转基因小鼠。我们将把它们与过度表达α-突触核蛋白的小鼠杂交，并与 项目3和转基因小鼠核心。我们还将把这些双转基因小鼠与Parkin基因敲除小鼠杂交。如果这看起来很有希望，我们可以培育出带有R621C突变的高表达SynPhilin-1的小鼠，这可能与PD有关，并表征它们的表型。我们还将通过S129A突变产生过表达α-突触核蛋白的小鼠，并对其表型进行鉴定。这些研究加在一起应该有助于揭示 与帕金森病相关的蛋白质，以及帕金森病发病机制。他们可能会改进这种疾病的细胞和小鼠模型，这将有助于寻找合理的治疗方法。

Studies of the pathogenesis of familial PD will likely contribute to understanding the pathogenesis of all forms of PD. Familial PD can be caused by mutations in alpha-synuclein, parkin or DJ-1. We have identified synphilin-1 as an alpha-synuclein interacting protein, and found that it is a component of the ubiquitinated Lewy bodies in PD postmortem brain, and can be a substrate for ubiquitination by parkin. In a collaborative study, we have suggestive evidence that an R621C mutation in synphilin-1 may be a rare cause of PD. We have found that S 129 phosphorylation of alpha-synuclein strongly modulates inclusion formation in cell culture. We are developing inducible cell models of mutant alpha-synuclein toxicity, and have found that expression of A53T mutant alpha-synuclein causes direct cell toxicity. We propose to characterize the mechanisms of cell death caused by mutant alpha-synuclein, and the interactions of these proteins in the pathogenesis of PD. In Specific Aim 1 we will study alpha-synuclein inducible PC 12 cell lines and mechanisms of cell toxicity, including the role of phosphorylation and cleavage of alpha-synuclein (in collaboration with Project 3), and possible modulation of toxicity by DJ-1 (in collaboration with Project 4). In Specific Aim 2 we will characterize the interactions among alpha-synuclein, synphilin-1 and parkin (in collaboration with Project 1 and the Neuropathology Core). We hypothesize that inclusions are preferentially ubiquitinated via K63 linkage rather than K48 linkage, consistent with a potential role in cell signaling as well as protein degradation. In Specific Aim 3 we will study the phosphorylation of alpha-synuclein, and determine whether phosphorylation modulates interactions with synphilin-1, and promotes aggregation or cleavage, in collaboration with Project 1 and 3, and the Neuropathology Core. Finally in Specific Aim 4 we will create synphilin-1 transgenic mice. We will cross them with mice overexpressing alpha-synuclein, and characterize their behavior and pathology, in collaboration with Project 3 and the Transgenic Mouse Core. We will also cross these double transgenic mice with parkin knockout mice. If this looks promising, we can generate mice overexpressing synphilin-1 with an R621C mutation which may be linked to PD, and characterize their phenotype. We will also generate mice overexpressing alpha-synuclein with the S129A mutation, and characterize their phenotype. These studies taken together should shed light on interactions among proteins implicated in PD, and on the mechanisms of PD pathogenesis. They are likely to lead to improved cell and mouse models of the disease, which will facilitate the search for rational therapeutics.