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

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）基因的突变已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。我们在成年大鼠中开发了一种新的病毒介导的PD基因转移模型， 人D 620 NVPS 35的过表达诱导黑质多巴胺能神经元的变性，从而 正式确立了D 620 N突变的致病作用。在本申请中，我们提出利用 这种疾病模型，以阐明PD相关的神经退行性表型的全部剧目诱导 D 620 N VPS 35，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 细胞病理学和改变的大自噬，作为增加转基因剂量的函数（目的1.1）。我们将 通过定量蛋白质组学分析脑组织来评估逆转录蛋白相互作用的扰动， 该模型和原代神经元鉴定D 620 N突变潜在的推定分子机制，以及 我们将确定所选择的蛋白质相互作用物是否对神经变性重要（目标1.2）。我们 研究还将阐明这种啮齿动物PD模型中显性D 620 N突变的机制，并将解决 D 620 N VPS 35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 短发夹RNA沉默大鼠黑质多巴胺能神经元中的VPS 35表达，以产生VPS 35缺失- 的功能模型，并进行救援实验与人类VPS 35变异体，以确定是否D 620 N 突变是功能性的或受损的（目标2.1）。将在新的条件性VPS中进行类似的补救研究35 在神经元中具有选择性缺失的基因敲除小鼠（Aim 2.2）。最后，我们将评估VPS 35的相互作用 与LRRK 2在该大鼠PD模型中进行比较，以确定LRRK 2激酶活化是否是PD的关键所需。 D 620 N VPS 35诱导的神经变性。我们将确定LRRK 2缺失（目标3.1）或药理学 激酶抑制（目的3.2）在该大鼠模型中具有神经保护作用。我们的建议新颖、创新、及时， 将使用转基因啮齿动物模型对PD连锁VPS 35突变的机制提供重要见解。

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.