Mechanisms of VPS35-Dependent Neurodegeneration in Parkinson's Disease

项目摘要 帕金森氏病（PD）是一种进行性神经退行性运动障碍，主要由帕金森氏病引起。 黑质多巴胺能神经元变性。目前的PD治疗是姑息性的，但没有 目前存在改善疾病的疗法。VPS 35（PARK 17）基因的突变最近被鉴定为一种新的突变。 晚发型常染色体显性PD的病因，在PD个体中检测到单一突变（D 620 N）， 全世界的家庭。VPS 35突变如何在PD中促进多巴胺能神经变性仍然存在 晦涩难懂。关键是要确定导致神经变性的分子和细胞机制， VPS 35突变，以便了解PD的病理生理学并开发新的治疗策略。 VPS 35是逆转录酶复合物的核心组分，负责识别和分选 跨膜蛋白货物从内体到高尔基体网络或质膜用于再循环。如何 家族性突变影响PD相关神经元群体和动物模型中VPS 35逆转录功能 不知道。我们最近开发了一种新的病毒介导的大鼠PD基因转移模型， 人D 620 N VPS 35过表达诱导黑质纹状体通路多巴胺能神经元变性 神经元，从而正式建立体内D 620 N突变的致病作用。本 应用，我们现在建议将我们的研究扩展到新的D 620 N VPS 35敲入小鼠，其具有生理学上的 VPS 35表达水平作为VPS 35相关PD的新相关模型（目的1）。我们建议确定 D 620 N VPS 35敲入小鼠中的神经退行性表型，包括发育和 多巴胺能神经元和轴突变性的进展，纹状体儿茶酚胺和运动缺陷， 神经病理学和蛋白质聚集。我们将评估异常逆转录病毒货物分选和VPS 35蛋白 这些小鼠脑组织中的相互作用，以确定D 620 N突变的分子机制。 我们还将在PD啮齿动物模型中评估VPS 35与α-突触核蛋白和LRRK 2的新型相互作用， 确定这些蛋白质是否在神经变性的共同致病途径中会聚， PD（目标2-3）。我们将确定VPS 35过表达或致病性突变是否可以保护或 在两种充分表征的啮齿类PD模型中， (Aim 2）。我们还将提供家族性LRRK 2突变是否能诱导retromer缺陷的证据 使用两种不同的LRRK 2啮齿动物PD模型，我们将评估VPS 35突变或 在小鼠中的LRRK 2缺乏可加剧突变体LRRK 2诱导的多巴胺能神经变性（Aim 3）。我们 全面的建议是新颖的，创新的和及时的，将提供重要的洞察致病 VPS 35突变在PD中的作用和机制。

Project Summary Parkinson's disease (PD) is a progressive neurodegenerative movement disorder caused primarily by the degeneration of dopaminergic neurons in the substantia nigra. Current therapies for PD are palliative but no disease-modifying therapies exist today. Mutations in the VPS35 (PARK17) gene were recently identified as a cause of late-onset, autosomal dominant PD, with a single mutation (D620N) detected in PD individuals and families worldwide. How mutations in VPS35 precipitate dopaminergic neurodegeneration in PD remains obscure. It is critical to identify the molecular and cellular mechanisms that lead to neurodegeneration due to VPS35 mutations in order to understand the pathophysiology of PD and develop new therapeutic strategies. VPS35 is a core component of the retromer complex responsible for the recognition and sorting of transmembrane protein cargo from endosomes to the Golgi network or plasma membrane for recycling. How familial mutations influence VPS35 retromer function in PD-relevant neuronal populations and animal models is not known. We have recently developed a novel viral-mediated gene transfer model of PD in rats where the overexpression of human D620N VPS35 induces the degeneration of nigrostriatal pathway dopaminergic neurons, thereby formally establishing a pathogenic role for the D620N mutation in vivo. In the present application, we now propose to extend our studies to novel D620N VPS35 knockin mice with physiological levels of VPS35 expression as a new relevant model of VPS35-linked PD (Aim 1). We propose to identify neurodegenerative phenotypes in the D620N VPS35 knockin mice, including the development and progression of dopaminergic neuronal and axonal degeneration, striatal catecholamine and motoric deficits, neuropathology and protein aggregation. We will evaluate abnormal retromer cargo sorting and VPS35 protein interactions in brain tissue from these mice to identify molecular mechanisms underlying the D620N mutation. We will also evaluate a novel interaction of VPS35 with α-synuclein and LRRK2 in rodent models of PD to determine whether these proteins converge in common pathogenic pathways underlying neurodegeneration in PD (Aims 2-3). We will determine whether VPS35 overexpression or pathogenic mutations can protect or exacerbate αSyn-dependent neurodegeneration, respectively, in two well-characterized rodent models of PD (Aim 2). We will also provide evidence of whether familial LRRK2 mutations act to induce a retromer deficiency in the brain using two distinct LRRK2 rodent models of PD, and we will evaluate whether VPS35 mutations or deficiency in mice can exacerbate mutant LRRK2-induced dopaminergic neurodegeneration (Aim 3). Our comprehensive proposal is novel, innovative and timely and will provide important insight into the pathogenic actions and mechanisms of VPS35 mutations in PD by using a novel knockin mouse model.