LRRK2 and cellular pathways of Parkinson's Disease

描述(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，其特征是选择性地失去多巴胺能神经元，并存在称为路易小体的细胞内蛋白质聚集体。帕金森病的发病机制尚不完全清楚，但似乎与遗传易感性和环境因素有关。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，并对散发性帕金森病的发生具有一定的意义。LRRK2(或dardarin)突变最近被发现导致常染色体显性遗传性晚发型、临床典型的多形性帕金森病。LRRK2是一种大的复杂蛋白质，它包含的结构域表明了蛋白质相互作用、激酶活性和RAS相关信号的作用。我们在研究神经退行性疾病的细胞毒性、信号通路和蛋白质相互作用方面拥有丰富的经验。我们以前对帕金森病的研究已经阐明了α-突触核蛋白毒性的作用，以及蛋白质与合成酶-1和Parkin的相互作用在形成路易体样聚集体中的作用。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，并且有前景的数据表明突变的LRRK2可以引起直接的神经元毒性，为研究LRRK2相关PD的发病机制提供了一个独特的模型。在特定的目标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。我们将利用野生型和突变型LRRK2进行细胞转染研究，检测LRRK2的分布模式，并确认LRRK2的毒性。我们将建立稳定的可诱导表达野生型或突变型LRRK2的PC12细胞系，以研究LRRK2毒性的分子机制。在特定的目标2中，我们将利用目标1中描述的细胞模型来测试LRRK2与SynPhilin-1和Parkin相互作用对细胞毒性和细胞聚集通路的功能影响。这些研究将阐明LRRK2在细胞毒性中的功能作用以及它与SynPhilin-1和Parkin相互作用在细胞培养中的功能后果，有助于阐明LRRK2相关PD的发病机制，并将找到潜在的干预PD的治疗靶点。

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.