LRRK2 and Parkinson's Disease Cell Biology

描述（由申请人提供）：LRRK 2和帕金森病细胞生物学帕金森病是一种运动、认知和情感障碍，其神经病理学特征为神经元变性和称为Lewy小体的蛋白质聚集体沉积。虽然大多数病例是散发性的，但由α-突触核蛋白，parkin，DJ-1和PINK 1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-突触核蛋白蛋白相互作用（包括与synphilin-1和parkin的相互作用）在PD相关细胞生物学中的作用。最近发现富含亮氨酸重复序列激酶2（LRRK 2）的突变可导致常染色体显性PD。我们的总体假设是，LRRK 2蛋白相互作用的鉴定将有助于阐明LRRK 2相关PD的发病机制，并可能是散发性PD。我们已经确定了LRRK 2和其他几种蛋白质之间的相互作用，包括parkin，synphilin-1，WSB-1和CARD 7。我们已经发现，突变LRRK 2导致直接的细胞毒性，并有初步的数据，至少一些LRRK 2突变，GTP结合和激酶活性是必要的毒性。在具体目标1中，我们将使用酵母双杂交系统和共免疫沉淀从转染细胞中鉴定LRRK 2相互作用蛋白，并定义这些蛋白的相互作用结构域。在特定目标2中，我们将在培养细胞以及小鼠和人体组织（包括死后散发性和突变型LRRK 2 PD组织）中的表达研究中研究LRRK 2相互作用。我们将研究这些相互作用在LRRK 2细胞毒性的作用，通过使用siRNA，并通过修改的相互作用域。在具体目标3中，我们将确定LRRK 2激酶活性是否对细胞毒性至关重要，并确定LRRK 2是否可以磷酸化相互作用物，如果是这样，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LRRK 2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.