Somatic Mitochondiral DNA Mutations in Neurons and Glia

描述（由申请人提供）：来自转基因小鼠研究的最新数据支持衰老的线粒体理论，该理论提出线粒体DNA的氧化损伤引起突变，随着年龄的增长而积累，导致线粒体功能障碍并导致与年龄相关的疾病。在人脑中，我们发现氧化诱导的体细胞线粒体DNA （mtDNA）突变随着年龄的增长而积累，达到很高的水平。线粒体复合体I功能障碍在帕金森病（PD）中起着关键作用，间接数据表明mtDNA突变是导致这种线粒体功能障碍的原因。然而，我们和其他人无法在大多数PD患者中明确识别致病的遗传mtDNA突变。总之，这些观察结果提出了一种可能性，即大脑中体细胞mtDNA突变的积累在PD的发病机制中起着关键作用。然而，关于大脑中积累这些突变的细胞类型，或者这些突变的积累是否在线粒体功能障碍和神经变性中起作用，我们知之甚少。我们假设体细胞mtDNA突变随着单个神经元和神经胶质细胞的衰老而积累，神经元中的体细胞mtDNA突变水平，可能在星形胶质细胞中，与年龄匹配的对照组相比，PD患者的体细胞mtDNA突变水平更高，并且这些突变导致线粒体复合体I功能障碍。我们将使用激光捕获显微解剖（LCM）从年轻和老年对照受试者以及早期和晚期PD患者的黑质和控制区分离单个神经元（有和没有路易小体）、星形胶质细胞和小胶质细胞。我们已经开发并验证了一种高度敏感的克隆测序策略，我们将使用该策略来分析这些细胞的体细胞mtDNA突变水平和模式，并将评估这些突变与线粒体复合体I功能障碍之间的关系。公共卫生相关性：帕金森病（PD）是一种常见的与年龄相关的神经退行性疾病，可导致进行性残疾。氧化应激和线粒体功能障碍在帕金森病的发病机制中起关键作用。对人脑单细胞体细胞mtDNA突变的研究将有助于深入了解衰老、线粒体DNA氧化损伤和线粒体功能障碍之间的关系，并可能有助于确定新的治疗策略，以延缓或预防帕金森病的残疾。

DESCRIPTION (provided by applicant): Recent data from studies of transgenic mice lend support to the mitochondrial theory of aging, which proposes that oxidative damage to mitochondrial DNA gives rise to mutations that accumulate with age, resulting in mitochondrial dysfunction and contributing to age-related disorders. In the human brain, we have found that oxidatively-induced somatic mitochondrial DNA (mtDNA) mutations accumulate with aging to reach high levels. Mitochondrial complex I dysfunction plays a key role in Parkinson's disease (PD), and indirect data implicates mtDNA mutations as the cause of this mitochondrial dysfunction. Yet, we and others have been unable to identify clearly pathogenic inherited mtDNA mutations in most PD patients. Together, these observations raise the possibility that the accumulation of somatic mtDNA mutations in the brain plays a key role in the pathogenesis of PD. However, little is known regarding the cell types in the brain that accumulate these mutations, or whether or not the accumulation of these mutations plays a role in mitochondrial dysfunction and neurodegeneration. We hypothesize that somatic mtDNA mutations accumulate with aging in single neurons and glia, that the levels of somatic mtDNA mutations in neurons, and possibly in astrocytes, are greater in PD compared to age-matched controls, and that these mutations contribute to mitochondrial complex I dysfunction. We will use laser capture microdissection (LCM) to isolate single neurons (with and without Lewy bodies), astrocytes, and microglia from the substantia nigra and control regions of young and old human control subjects, and from early and late stage PD patients. We have developed and validated a highly sensitive cloning-sequencing strategy that we will use to analyze these cells for levels and patterns of somatic mtDNA mutations, and will assess the relationship between these mutations and mitochondrial complex I dysfunction. PUBLIC HEALTH RELEVANCE: Parkinson's disease (PD) is a common age-related neurodegenerative disorder that leads to progressive disability. Oxidative stress and mitochondrial dysfunction play key roles in the pathogenesis of PD. The proposed studies of somatic mtDNA mutations in single cells in the human brain will provide insights into the relationship between aging, oxidative injury to mitochondrial DNA and mitochondrial dysfunction, and might help to identify new treatment strategies to delay or prevent the disability of PD.