Oxidative Stress, alpha-Synuclein, and mtDNA Mutations in Parkinson's Disease

描述（由申请人提供）：帕金森病（PD）中线粒体复合物I活性受损，用MPTP或鱼藤酮抑制复合物I可在动物模型中重现PD的许多特征。复合物I缺陷可以转移到表达来自PD患者的线粒体DNA（mtDNA）的细胞系中，这表明mtDNA突变是复合物I缺陷的原因。但是，尽管试图识别它们，但导致这种缺陷的特定突变仍然未知。线粒体复合物I功能障碍增加线粒体中的自由基产生，导致大分子损伤，特别是对mtDNA的高水平潜在致突变损伤。这种对mtDNA的损伤随着年龄的增长而积累，并在PD中达到特别高的水平。我们推测，这种对mtDNA的氧化损伤导致体细胞mtDNA突变的积累，最终导致多巴胺能末梢的丧失，并可能导致细胞死亡。因此，我们预测，黑质（SN）神经元将在PD的早期阶段具有高水平的体细胞mtDNA突变。与这一预测相一致，我们目前的初步数据表明非常高水平的体细胞线粒体DNA点突变的SN神经元在非常早期的PD，而神经元与高水平的突变在很大程度上是不存在的末期PD。此外，我们还发现，与对照组或晚期PD神经元相比，预测由氧化应激引起的mtDNA突变亚组的水平在早期PD的SN神经元中的普遍性高出近10倍。这些数据与我们的假设相一致，即体细胞mtDNA突变在PD的早期阶段在SN神经元中积累，并且这些突变有助于PD中的神经元损失。我们进一步预测，与年龄相关的体细胞mtDNA突变积累的实验加速将导致类似的变化，在转基因小鼠表达的校正缺陷型mtDNA聚合酶（POLG）。我们建议使用激光捕获显微切割来分析点突变和大缺失的神经元和神经胶质细胞从人死后SN神经元和其他大脑区域在早期PD，晚期PD，和控制。我们进一步建议在表达突变POLG的转基因小鼠中进行平行实验。总之，这些研究有可能揭示帕金森病发病机制的关键机制，并可能导致新的神经保护策略。公共卫生相关性：帕金森病（PD）是一种常见的疾病，导致进行性残疾。虽然PD有许多对症治疗方法，但每种方法都有局限性，减缓PD进展的策略可能会对PD患者的生活质量产生巨大的积极影响。拟议的实验将测试的假设，体细胞线粒体DNA突变在大脑中的积累有助于PD的发病机制，并可能导致新的策略，以减缓PD的进展。

DESCRIPTION (provided by applicant): Mitochondrial complex I activity is impaired in Parkinson's disease (PD), and inhibition of complex I with MPTP or rotenone reproduces many features of PD in animal models. The complex I defect can be transferred to cell lines expressing mitochondrial DNA (mtDNA) from PD patients, suggesting that mtDNA mutations account for the complex I defect. But despite attempts to identify them, the specific mutations that account for this defect remain unknown. Mitochondrial complex I dysfunction increases free radical production in the mitochondria, resulting in damage to macromolecules, with particularly high levels of potentially mutagenic damage to mtDNA. This damage to mtDNA accumulates with age and reaches especially high levels in PD. We hypothesize that this oxidative damage to mtDNA leads to the accumulation of somatic mtDNA mutations, ultimately contributing to the loss of dopaminergic terminals and potentially to cell death. Therefore, we predict that substantia nigra (SN) neurons will harbor high levels of somatic mtDNA mutations at early stages of PD. Consistent with this prediction, we present preliminary data indicating remarkably high levels of somatic mtDNA point mutations in SN neurons at very early stages of PD, whereas neurons with high levels of mutations are largely absent by end stage PD. Furthermore, we find that levels of the subset of mtDNA mutations predicted to result from oxidative stress are nearly 10-fold more prevalent in SN neurons from early PD compared to controls or to late PD neurons. These data are consistent with our hypothesis that somatic mtDNA mutations accumulate in SN neurons at early stages of PD, and that these mutations contribute to neuronal loss in PD. We further predict that experimental acceleration of the age-related accumulation of somatic mtDNA mutations will lead to similar changes in transgenic mice expressing a proofreading deficient mtDNA polymerase (POLG). We propose to use laser capture microdissection to analyze point mutations and large deletions in neurons and glia from human postmortem SN neurons and other brain regions in early PD, late PD, and controls. We further propose to conduct parallel experiments in transgenic mice expressing mutant POLG. Together, these studies have the potential to reveal a key mechanism in the pathogenesis of PD, and may lead to novel neuroprotective strategies.PUBLIC HEALTH RELEVANCE: Parkinson's disease (PD) is a common disorder that leads to progressive disability. Though many symptomatic treatments exist for PD, each has limitations, and a strategy to slow the progression of PD could have an enormous positive impact on the quality of life of PD patients. The proposed experiments will test the hypothesis that the accumulation of somatic mitochondrial DNA mutations in the brain contributes to the pathogenesis of PD, and may lead to novel strategies to slow the progression of PD.