Mechanisms of VPS35-Dependent Neurodegeneration in Parkinson's Disease

项目摘要 帕金森病(PD)是一种进行性神经退行性运动障碍，主要由 黑质内多巴胺能神经元变性。目前治疗帕金森病的方法是姑息性的，但没有 如今，治疗疾病的疗法已经存在。最近发现VPS35(PARK17)基因突变是一种 晚发性常染色体显性遗传性帕金森病的原因，在帕金森病患者和 世界各地的家庭。VPS35基因突变如何导致帕金森病患者多巴胺能神经变性 默默无闻。确定导致神经退行性变的分子和细胞机制至关重要 VPS35突变，以了解帕金森病的病理生理机制，并开发新的治疗策略。 VPS35是逆转录复合体的核心组件，负责识别和分类 跨膜蛋白货物从内体到高尔基体网络或质膜进行循环利用。多么 家族性突变对帕金森病相关神经元群体和动物模型VPS35逆转录功能的影响 是未知的。我们最近开发了一种新的病毒介导的大鼠帕金森病基因转移模型，其中 人D620N VPS35过表达诱导黑质纹状体通路多巴胺能变性 从而正式确立了D620N突变在体内的致病作用。在现在 应用，我们现在建议将我们的研究扩展到新的D620N VPS35敲击小鼠，具有生理 VPS35表达水平作为VPS35连锁PD的新相关模型(目标1)。我们建议确定 D620N VPS35敲门小鼠神经退行性变表型的研究 多巴胺能神经元和轴突变性、纹状体儿茶酚胺和运动缺陷的研究进展 神经病理学和蛋白质聚集。我们将评估异常逆转聚体货物分选和VPS35蛋白 这些小鼠的脑组织之间的相互作用，以确定D620N突变的分子机制。 我们还将评估一种新的VPS35与α-突触核蛋白和LRRK2在PD啮齿动物模型中的相互作用 确定这些蛋白是否汇聚在导致神经变性的共同致病途径中 PD(目标2-3)。我们将确定VPS35的过度表达或致病突变是否可以保护或 在两种特征良好的帕金森病啮齿动物模型中，分别加剧α突触蛋白依赖性神经变性 (目标2)。我们还将提供证据，证明家族性LRRK2突变是否会导致逆转录酶缺乏症 在大脑中使用两种不同的PD LRRK2啮齿动物模型，我们将评估VPS35突变或 小鼠的缺乏可加剧突变的LRRK2诱导的多巴胺能神经变性(目标3)。我们的 全面的建议是新颖、创新和及时的，将为致病原因提供重要的见解 用一种新的敲击小鼠模型研究VPS35突变在帕金森病中的作用和机制。

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.