Exploring mechanisms of Parkinson's disease-linked D620N VPS35 in rat models

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。VPS35(PARK17)基因突变已被鉴定为一种 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 不知道。我们开发了一种新的病毒介导的成年大鼠帕金森病基因转移模型，其中 人D620N VPS35过表达诱导黑质多巴胺能神经元变性 正式确立了D620N突变的致病作用。在本应用程序中，我们建议利用 这一疾病模型阐明了帕金森病相关神经退行性变表型的完整谱系 D620N VPS35，包括多巴胺能神经元和轴突变性的发展和进展， 纹状体儿茶酚胺和运动缺陷、神经病理学、蛋白质聚集和超微结构 随着转基因剂量的增加，细胞病理学改变和巨细胞自噬改变(目标1.1)。我们会 用脑组织定量蛋白质组学分析评估逆转录蛋白质相互作用中的扰动 该模型和原代神经元以确定D620N突变的可能分子机制，以及 我们将确定选定的蛋白质相互作用因子是否对神经退行性变重要(目标1.2)。我们的 研究还将阐明这种啮齿动物帕金森病模型中显性D620N突变的机制，并将解决 D620N VPS35是否可能以显性-负性方式起作用。因此，我们将使用病毒载体来传递 短发夹状RNA沉默大鼠黑质多巴胺能神经元VPS35的表达 对人类VPS35变种进行功能模型和抢救实验，以确定D620N 突变是正常的或受损的(目标2.1)。类似的救援研究将在新的有条件的VPS35中进行 神经元选择性缺失的基因敲除小鼠(目标2.2)。最后，我们将评估VPS35的交互作用 在这种帕金森病大鼠模型中使用LRRK2来确定LRRK2激酶激活是否是 D620N VPS35诱导的神经变性。我们将确定LRRK2缺失(目标3.1)或药理学 在这种大鼠模型中，抑制激酶(AIM 3.2)具有神经保护作用。我们的建议新颖、创新、及时， 将使用转基因啮齿动物模型为PD相关VPS35突变的机制提供关键的见解。

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 have been 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 developed a novel viral-mediated gene transfer model of PD in adult rats where the overexpression of human D620N VPS35 induces the degeneration of nigral dopaminergic neurons, thereby formally establishing a pathogenic role for the D620N mutation. In the present application, we propose to exploit this disease model to elucidate the full repertoire of PD-related neurodegenerative phenotypes induced by D620N VPS35, including the development and progression of dopaminergic neuronal and axonal degeneration, striatal catecholamine and motoric deficits, neuropathology, protein aggregation and ultrastructural cytopathology, and altered macroautophagy, as a function of increasing transgene dosage (Aim 1.1). We will evaluate perturbations in retromer protein interactions by quantitative proteomic analyses of brain tissue from this model and primary neurons to identify putative molecular mechanisms underlying the D620N mutation, and we will determine whether selected protein interactors are important for neurodegeneration (Aim 1.2). Our studies will also clarify the mechanism of the dominant D620N mutation in this rodent PD model and will address whether D620N VPS35 may act in a dominant-negative manner. Accordingly, we will use viral vectors to deliver short hairpin RNAs to silence VPS35 expression in nigral dopaminergic neurons of rats to create a VPS35 loss- of-function model and conduct rescue experiments with human VPS35 variants to determine whether the D620N mutation is functional or impaired (Aim 2.1). Similar rescue studies will be conducted in new conditional VPS35 knockout mice with selective deletion in neurons (Aim 2.2). Finally, we will evaluate the interaction of VPS35 with LRRK2 in this rat model of PD to determine whether LRRK2 kinase activation is critically required for neurodegeneration induced by D620N VPS35. We will determine if LRRK2 deletion (Aim 3.1) or pharmacological kinase inhibition (Aim 3.2) in this rat model is neuroprotective. Our proposal is novel, innovative and timely and will provide critical insight into the mechanisms of PD-linked VPS35 mutations using transgenic rodent models.