LRRK2 and Parkinson's Disease Cell Biology

描述(申请人提供)：LRRK2和帕金森病细胞生物学帕金森病是一种运动、认知和情绪障碍，神经病理学特征是神经元变性和蛋白质聚集体沉积，称为路易小体。虽然大多数病例是零星的，但由α-突触核蛋白、parkin、DJ-1和PINK1突变引起的罕见遗传形式的疾病有助于阐明发病机制。在以前的研究中，我们已经确定了α-突触核蛋白相互作用(包括与突触素-1和Parkin的相互作用)在帕金森病相关细胞生物学中的作用。富含亮氨酸的重复蛋白激酶2(LRRK2)基因突变是导致常染色体显性遗传性帕金森病的原因之一。我们的总体假设是，LRRK2蛋白相互作用的鉴定将有助于阐明LRRK2相关帕金森病的发病机制，可能还包括散发性帕金森病。我们已经确定了LRRK2与其他几种蛋白质之间的相互作用，包括Parkin、SynPhilin-1、WSB-1和CARD7。我们已经发现突变的LRRK2会引起直接的细胞毒性，并且有初步数据表明，至少对于一些LRRK2突变来说，GTP结合和激酶活性是毒性所必需的。在特定目标1中，我们将利用酵母双杂交系统和免疫共沉淀法从转基因细胞中鉴定LRRK2相互作用蛋白，并确定这些蛋白的相互作用结构域。在特定目标2中，我们将研究LRRK2在培养细胞、小鼠和人类组织中的表达研究中的相互作用，包括死后人类散发性和突变的LRRK2 PD组织。我们将通过使用siRNA和通过修改相互作用结构域来研究这些相互作用因子在LRRK2细胞毒性中的作用。在具体目标3中，我们将确定LRRK2激酶活性是否对细胞毒性至关重要，并确定LRRK2是否能使相互作用物磷酸化--如果是，我们将确定这是否在毒性中起作用。这些研究将有助于确定LRRK2及其相互作用蛋白在与PD相关的细胞发病机制中的作用，并有可能确定未来治疗干预的靶点。

DESCRIPTION (provided by applicant): LRRK2 and Parkinson's disease cell biology Parkinson's disease is a disorder of movement, cognition and emotion, characterized neuropathologically by neuronal degeneration and deposits of protein aggregates termed Lewy bodies. While most cases are sporadic, rare genetic forms of the disease, caused by mutations in alpha-synuclein, parkin, DJ-1 and PINK1, are helping to elucidate pathogenesis. In previous studies, we have defined the role of alpha- synuclein protein interactions (including interactions with synphilin-1 and parkin) in PD-related cell biology. Mutations in leucine-rich repeat kinase 2 (LRRK2) have recently been found to cause autosomal dominant PD. Our overall hypothesis is that identifications of LRRK2 protein interactions will help elucidate pathogeneses of LRRK2 related PD, and possibly sporadic PD. We have identified interactions between LRRK2 and several other proteins, including parkin, synphilin-1, WSB-1 and CARD7. We have found that that mutant LRRK2 causes direct cellular toxicity, and have initial data that for at least some of the LRRK2 mutations, GTP binding and kinase activity are necessary for toxicity. In Specific Aim 1 we will identify LRRK2 interacting proteins using the yeast two-hybrid system and co-immunopreciptation from transfected cells, and define interaction domains of these proteins. In Specific Aim 2 we will study the LRRK2 interactions in expression studies in cells in culture, and in mouse and human tissue, including postmortem human sporadic and mutant LRRK2 PD tissue. We will study the role of these interactors in LRRK2 cellular toxicity, by using siRNA, and by modifying the interaction domains. In Specific Aim 3 we will determine whether LRRK2 kinase activity is critical for cell toxicity, and determine whether LRRK2 can phosphorylate the interactors-and if so, we will determine whether this has a role in toxicity. These studies will help define the role of LRRK2 and its interacting proteins in cellular pathogenesis related to PD, and potentially identify targets for future therapeutic interventions.