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

在大鼠模型中探索帕金森病相关 D620N VPS35 的机制

基本信息

批准号：
10656398
负责人：
Darren John Moore
金额：
$ 44.65万
依托单位：
VAN ANDEL RESEARCH INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-07-01 至 2025-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10656398
关键词：
Adult Animal Model Biological Process Brain Catecholamines Cell membrane Cells Clinical Complex Corpus striatum structure Cytopathology Development Diet Disease Disease Progression Disease model Dominant-Negative Mutation Dopamine Dose Endosomes Family Functional disorder Future Gene Dosage Gene Silencing Gene Transfer Genes Golgi Apparatus Human Impairment Individual Integral Membrane Protein Knockout Mice LRRK2 gene Lead Link Mediating Missense Mutation Modeling Molecular Movement Disorders Mus Mutation Nerve Degeneration Neurons Orthologous Gene Outcome Parkinson Disease Pathogenicity Pathologic Pathway interactions Phenotype Phosphotransferases Population Proteins Proteomics Rat Transgene Rattus Recycling Retrieval Rodent Rodent Model Role Sorting Substantia nigra structure Tissue Model Tissues Transgenes Transgenic Organisms Vacuolar Protein Sorting Variant Viral Viral Vector autosome axonal degeneration brain tissue dominant genetic mutation dopaminergic neuron drug development experimental study gain of function innovation insight kinase inhibitor loss of function mutant neurodegenerative phenotype neuron loss neuropathology neuroprotection nigrostriatal dopaminergic pathway novel novel therapeutic intervention overexpression palliative pharmacologic postnatal protein aggregation segregation small hairpin RNA trans-Golgi Network

项目摘要

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.

项目概要帕金森病（PD）是一种进行性神经退行性运动障碍，主要由黑质多巴胺能神经元变性。目前的帕金森病治疗方法只是姑息治疗，但没有效果如今已有疾病缓解疗法。 VPS35 (PARK17) 基因突变已被确定为迟发性常染色体显性 PD 的病因，在 PD 个体中检测到单突变 (D620N)，世界各地的家庭。 VPS35 突变如何促进 PD 多巴胺能神经变性朦胧。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 PD 的病理生理学并开发新的治疗策略。 VPS35是逆转录酶复合物的核心成分，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进行回收。如何家族突变影响 PD 相关神经元群体和动物模型中的 VPS35 逆转录酶功能不知道。我们在成年大鼠中开发了一种新型病毒介导的 PD 基因转移模型，其中人 D620N VPS35 的过度表达会诱导黑质多巴胺能神经元的变性，从而正式确定 D620N 突变的致病作用。在本申请中，我们建议利用该疾病模型旨在阐明 PD 相关神经退行性表型的全部内容 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细胞病理学和巨自噬的改变，作为增加转基因剂量的函数（目标 1.1）。我们将通过对脑组织进行定量蛋白质组分析来评估逆转录酶蛋白质相互作用的扰动该模型和原代神经元可识别 D620N 突变背后的假定分子机制，以及我们将确定选定的蛋白质相互作用因子是否对神经变性很重要（目标 1.2）。我们的研究还将阐明该啮齿动物 PD 模型中显性 D620N 突变的机制，并将解决 D620N VPS35 是否可以以显性负向方式起作用。因此，我们将使用病毒载体来传递短发夹 RNA 沉默大鼠黑质多巴胺能神经元中的 VPS35 表达，导致 VPS35 缺失功能模型并使用人类 VPS35 变体进行救援实验，以确定 D620N 是否突变是功能性的还是受损的（目标 2.1）。类似的救援研究将在新的有条件 VPS35 中进行选择性删除神经元的基因敲除小鼠（目标 2.2）。最后我们来评估一下VPS35的交互在这种 PD 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）或药理学在此大鼠模型中，激酶抑制（目标 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提供对 PD 相关 VPS35 突变机制的重要见解。

项目成果

期刊论文数量（5）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Understanding the contributions of VPS35 and the retromer in neurodegenerative disease.

DOI：
10.1016/j.nbd.2022.105768
发表时间：
2022-08
期刊：
NEUROBIOLOGY OF DISEASE
影响因子：
6.1
作者：
Williams, Erin T.;Chen, Xi;Otero, P. Anthony;Moore, Darren J.
通讯作者：
Moore, Darren J.

LRRK2 and the Endolysosomal System in Parkinson's Disease.

DOI：
10.3233/jpd-202138
发表时间：
2020
期刊：
Journal of Parkinson's disease
影响因子：
0
作者：
Erb ML;Moore DJ
通讯作者：
Moore DJ

Neuronal VPS35 deletion induces spinal cord motor neuron degeneration and early post-natal lethality.

DOI：
10.1093/braincomms/fcab208
发表时间：
2021
期刊：
Brain communications
影响因子：
4.8
作者：
Sargent D;Cunningham LA;Dues DJ;Ma Y;Kordich JJ;Mercado G;Brundin P;Cowell RM;Moore DJ
通讯作者：
Moore DJ

Mechanisms of VPS35-Mediated Neurodegeneration in Parkinson's Disease.