LRRK2 and cellular pathways of Parkinson's Disease

描述（由申请人提供）：帕金森病（PD）是一种进行性神经退行性运动障碍，其特征是多巴胺能神经元的选择性丧失，以及被称为路易小体的细胞内蛋白聚集体的存在。PD的发病机制尚不完全清楚，但它似乎涉及遗传易感性和环境因素。目前已经确定了5种主要的遗传原因，包括α -突触核蛋白、parkin、PINK1、DJ-1和LRRK2的突变，这些突变为研究遗传性帕金森病的发病机制提供了模型，对散发性帕金森病具有指导意义。LRRK2（或dardarin）突变最近被发现导致常染色体显性晚发，临床典型的多形性病理PD。LRRK2是一种大型复杂蛋白，其包含的结构域可能与蛋白质相互作用、激酶活性和ras相关信号传导有关。我们在研究神经退行性疾病中的细胞毒性、信号通路和蛋白质相互作用方面有丰富的经验。我们之前对PD的研究已经阐明了α -突触核蛋白毒性的作用，以及蛋白质与突触蛋白-1和帕金的相互作用在路易体样聚集体形成中的作用。我们现在建议使用类似的方法来研究LRRK2。我们有证据表明LRRK2与其他PD相关基因产物相互作用，并且有很有希望的数据表明突变的LRRK2可以直接引起神经元毒性，为研究LRRK2相关PD的发病机制提供了一个独特的模型。在Specific Aim 1中，我们将研究LRRK2的细胞表达和毒性，并确定LRRK2诱导神经元变性的信号通路。我们将使用野生型和突变型LRRK2进行细胞转染研究，检查LRRK2的分布模式，并确认LRRK2的毒性。我们将产生稳定的PC12细胞系，诱导表达野生型或突变型LRRK2，以研究LRRK2毒性的分子机制。在Specific Aim 2中，我们将使用Aim 1中描述的细胞模型，测试LRRK2与synphilin-1和parkin相互作用对细胞毒性和细胞聚集途径的功能影响。这些研究将阐明LRRK2在细胞毒性中的功能作用及其在细胞培养中与synphilin-1和parkin相互作用的功能后果，将有助于阐明LRRK2相关PD的发病机制，并将确定PD干预的潜在治疗靶点。

DESCRIPTION (provided by applicant): Parkinson's disease (PD) is a progressive neurodegenerative movement disorder characterized by selective loss of dopaminergic neurons, and the presence of intracellular protein aggregates termed Lewy bodies. The pathogenesis of PD remains incompletely understood, but it appears to involve both genetic susceptibility and environmental factors. Five major genetic causes have been identified, including mutations in alpha-synuclein, parkin, PINK1, DJ-1 and LRRK2, and these mutations have provided models for study of the pathogenesis of genetic PD, with implications for sporadic PD. Mutations in LRRK2 (or dardarin) have been recently identified to cause autosomal dominant late onset, clinically typical PD with pleomorphic pathology. LRRK2 is a large complex protein, and contains domains that suggest roles for protein interactions, kinase activity, and Ras-related signaling. We have extensive experience in studying cell toxicity, signaling pathways, and protein interactions in neurodegenerative diseases. Our previous studies of PD have elucidated the role of alpha-synuclein toxicity, and protein interactions with synphilin-1 and parkin in the formation of Lewy body-like aggregates. We now propose to use similar methods to study LRRK2. We have evidence that LRRK2 interacts with other PD-related gene products and have promising data indicating that mutant LRRK2 can cause direct neuronal toxicity, providing a unique model for studying pathogenesis of LRRK2-related PD. In Specific Aim 1, we will study LRRK2 cellular expression and toxicity, and determine the signaling pathways by which LRRK2 induces neuronal degeneration. We will conduct cell transfection studies using wild type and mutant LRRK2, examine the LRRK2 distribution patterns, and confirm the LRRK2 toxicity. We will generate stable PC12 cell lines which inducibly express either wild type or mutant LRRK2 to study the molecular mechanisms underlying LRRK2 toxicity. In Specific Aim 2, we will test the functional effects of LRRK2 interaction with synphilin-1 and parkin on the pathways of cell toxicity and cellular aggregation using the cell models described in Aim 1. These studies will elucidate the functional role of LRRK2 in cell toxicity and the functional consequences of its interaction with synphilin-1 and parkin in cell culture, will help elucidate the pathogenesis of LRRK2-associated PD, and will identify potential therapeutic targets for intervention in PD.