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

描述(申请人提供)：线粒体复合体I活性在帕金森病(PD)中受损，用MPTP或鱼藤酮抑制复合体I可在动物模型中再现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，提示mtDNA突变是复合体I缺陷的原因。但是，尽管人们试图识别它们，但导致这种缺陷的具体突变仍然未知。线粒体复合体I功能障碍会增加线粒体中自由基的产生，导致大分子损伤，尤其是对线粒体DNA的潜在突变损伤水平更高。这种对线粒体DNA的损伤随着年龄的增长而积累，在帕金森病患者中达到特别高的水平。我们推测，这种对线粒体DNA的氧化损伤导致体细胞线粒体DNA突变的积累，最终导致多巴胺能终末的丧失，并可能导致细胞死亡。因此，我们预测，在帕金森病的早期阶段，黑质(SN)神经元将拥有高水平的体细胞线粒体DNA突变。与这一预测一致，我们提供的初步数据表明，在帕金森氏病非常早期的SN神经元中，体细胞mtDNA点突变水平非常高，而高水平突变的神经元在帕金森氏病末期基本消失。此外，我们发现，与对照组或晚期帕金森病神经元相比，预测由氧化应激导致的线粒体DNA突变亚集的水平在早期帕金森病神经元中几乎是10倍。这些数据与我们的假设一致，即在帕金森氏病早期，体细胞mtDNA突变在黑质神经元中积累，这些突变导致帕金森病患者神经元丢失。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技术来分析早期帕金森病患者、晚期帕金森病患者和对照组的人死后黑质神经元和其他脑区神经元和神经胶质细胞的点突变和大量缺失。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中的关键机制，并可能导致新的神经保护策略。PUBLIC健康相关性：帕金森病(PD)是一种导致进行性残疾的常见疾病。尽管存在许多针对帕金森病的对症治疗，但每种疗法都有局限性，减缓帕金森病进展的策略可能会对帕金森病患者的生活质量产生巨大的积极影响。拟议中的实验将检验这一假设，即大脑中体细胞线粒体DNA突变的积累有助于帕金森病的发病，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.