Gene Environment Interactions in Parkinson's Disease

帕金森病的基因环境相互作用

基本信息

批准号：
8074236
负责人：
KIM TIEU
金额：
$ 1.17万
依托单位：
UNIVERSITY OF ROCHESTER
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-05-28 至 2010-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8074236
关键词：
1-Methyl-4-phenylpyridinium Attenuated Biological Assay Cell Death Cell Survival Cells Cessation of life Chemicals Chimeric Proteins Data Dominant-Negative Mutation Dopamine Dose Electron Transport Experimental Models Exposure to Gene Mutation Genetic Goals Herbicides High Pressure Liquid Chromatography Human Immunoblotting Induced Mutation Intervention Lead Measures Mediating Mitochondria Molecular Mutation Nerve Degeneration Neurodegenerative Disorders Neurotoxins PTEN-induced putative kinase Paraquat Parkinson Disease Pathogenesis Pathway interactions Patients Phosphotransferases Play Polarography Predisposition Process Production Proteins Reporting Research Role Shapes Small Interfering RNA System Testing Time Toxic Environmental Substances Toxic effect Translating base design functional outcomes gene environment interaction genetic manipulation inhibitor/antagonist insight mitochondrial dysfunction mutant neuroprotection neurotoxicity novel overexpression prevent public health relevance research study response small molecule stable cell line toxicant vector vector control

项目摘要

DESCRIPTION (provided by applicant): Our long term goal is to study the mechanism of neurodegeneration induced by environmental neurotoxicants. This proposal is submitted to investigate gene-environment interactions in the pathogenesis of Parkinson's disease mediated through the mitochondrial pathway. Mitochondrial dysfunction has been proposed as a major mechanism of neurodegeneration in PD for years, but direct evidence in humans was inadequate until the recent discoveries of mutations in PTEN-induced putative kinase 1 (PINK1), which encodes a mitochondrial kinase. Although PINK1 mutations are associated with autosomal recessive PD, interestingly, an increasing number of PD patients carrying single heterozygous mutations have been reported. These observations suggest that a heterozygous PINK1 mutation may act as a susceptibility factor that interplays with environmental insults. To determine the interactions between PINK1 mutations and environmental toxicants, we have created stable cell lines with inducible over-expression of various PINK1 mutants. Based on our preliminary results, we hypothesize that PINK1 mutations increase cell susceptibility to environmental toxicants such as paraquat (PQ) through a novel mechanism: mitochondrial fragmentation via the mitochondrial fission / fusion machinery. In the first specific aim, we will characterize neurotoxicity induced by PQ and MPP+ (two toxicants representing different mechanisms of toxicity) through the mitochondrial fission and fusion pathway in N27 cells with a PINK1 mutation (L347P) and empty vector control, as well as in N27 cells with PINK1 knockdown mediated by siRNA. We will assess cell viability, functional outcomes (ATP production, dopamine release and electron transport chain activity), as well as mitochondrial fragmentation (size/shape) and alterations in mitochondrial fission and fusion proteins. In specific aim 2, we will perform neuroprotective experiments against PQ and MPP+ toxicity in mutant PINK1 cells by targeting the mitochondrial fission and fusion pathway, through genetic manipulations and a small molecule. We will transfect cells with relevant constructs and use a chemical inhibitor to attenuate mitochondrial fragmentation. The neuroprotective effects of these two strategies against MPP+ and PQ toxicity will be determined using cell viability and functional assays as described in specific aim 1. PUBLIC HEALTH RELEVANCE This research may provide insights into the complexity of gene-environment interactions in the cause of cell death as seen in Parkinson's disease by unraveling a still unrecognized molecular pathway. Furthermore, this novel mechanism may offer an additional avenue to develop neuroprotective therapy for patients with Parkinson's disease.

描述（由申请人提供）：我们的长期目标是研究环境神经毒物引起的神经变性的机制。提交该提案是为了研究通过线粒体途径介导的帕金森病发病机制中的基因与环境相互作用。多年来，线粒体功能障碍一直被认为是帕金森病神经退行性疾病的主要机制，但人类的直接证据并不充分，直到最近发现 PTEN 诱导的推定激酶 1 (PINK1)（编码线粒体激酶）发生突变。尽管 PINK1 突变与常染色体隐性遗传 PD 相关，但有趣的是，据报道越来越多的 PD 患者携带单一杂合突变。这些观察结果表明，杂合的 PINK1 突变可能作为与环境损害相互作用的易感因素。为了确定 PINK1 突变与环境毒物之间的相互作用，我们创建了可诱导过度表达各种 PINK1 突变体的稳定细胞系。根据我们的初步结果，我们假设 PINK1 突变通过一种新机制增加细胞对环境毒物（例如百草枯 (PQ)）的敏感性：通过线粒体裂变/融合机制进行线粒体断裂。在第一个具体目标中，我们将表征 PQ 和 MPP+（代表不同毒性机制的两种毒物）在具有 PINK1 突变 (L347P) 和空载体控制的 N27 细胞中通过线粒体裂变和融合途径诱导的神经毒性，以及通过 siRNA 介导 PINK1 敲低的 N27 细胞。我们将评估细胞活力、功能结果（ATP 产生、多巴胺释放和电子传递链活性），以及线粒体碎片（大小/形状）以及线粒体裂变和融合蛋白的改变。在具体目标 2 中，我们将通过基因操作和小分子，针对线粒体裂变和融合途径，在突变 PINK1 细胞中进行针对 PQ 和 MPP+ 毒性的神经保护实验。我们将用相关构建体转染细胞，并使用化学抑制剂来减弱线粒体断裂。这两种针对 MPP+ 和 PQ 毒性的策略的神经保护作用将通过具体目标 1 中所述的细胞活力和功能测定来确定。公共健康相关性这项研究可能会为细胞死亡原因中基因-环境相互作用的复杂性提供见解。正如在帕金森病中所看到的那样，通过解开仍然未被识别的分子途径。此外，这种新机制可能为开发帕金森病患者的神经保护疗法提供额外的途径。