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

描述（由申请人提供）：帕金森病（PD）患者线粒体复合体I活性受损，MPTP或鱼藤酮抑制复合体I在动物模型中再现了PD的许多特征。复合体I缺陷可以转移到PD患者表达线粒体DNA （mtDNA）的细胞系中，表明mtDNA突变是复合体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。线粒体复合体I功能障碍增加线粒体中自由基的产生，导致大分子损伤，特别是对mtDNA的潜在致突变损伤水平特别高。这种对mtDNA的损害随着年龄的增长而积累，在PD患者中达到特别高的水平。我们假设这种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端的丧失，并可能导致细胞死亡。因此，我们预测黑质（SN）神经元在帕金森病的早期阶段会有高水平的体细胞mtDNA突变。与这一预测一致，我们提供的初步数据表明，在PD的早期阶段，SN神经元的体细胞mtDNA点突变水平非常高，而在晚期PD中，高水平突变的神经元基本缺失。此外，我们发现，与对照组或晚期PD神经元相比，早期PD的SN神经元中由氧化应激引起的mtDNA突变亚群的水平高出近10倍。这些数据与我们的假设一致，即在PD的早期阶段，体细胞mtDNA突变在SN神经元中积累，这些突变导致PD的神经元丢失。我们进一步预测，实验加速与年龄相关的体细胞mtDNA突变积累将导致表达校对缺陷mtDNA聚合酶（POLG）的转基因小鼠发生类似的变化。我们建议使用激光捕获显微解剖来分析人类死后SN神经元和其他大脑区域的神经元和胶质细胞的点突变和大缺失，这些区域包括早期PD、晚期PD和对照组。我们进一步建议在表达突变型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PD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）是一种导致进行性残疾的常见疾病。尽管PD有许多对症治疗，但每种治疗方法都有局限性，减缓PD进展的策略可能对PD患者的生活质量产生巨大的积极影响。本实验将验证大脑中体细胞线粒体DNA突变的积累与帕金森病的发病机制有关的假设，并可能导致减缓帕金森病进展的新策略。

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.