MtDNA mutations in brain aging: a single-cell approach

描述（由申请人提供）：拟议的长期目标 研究的目的是研究与年龄相关的退行性变的机制， 人类大脑的过程。了解这些机制可能有助于 找到减缓相应过程的方法， 超出正常人类寿命的退化。我们建议探讨， 脑中关键细胞类型的mtDNA中体细胞突变的积累 是导致大脑老化的因素之一。 提出了两个假设。首先，线粒体DNA的突变可以作为 通过破坏细胞功能而导致某些大脑区域功能障碍的主要原因 代谢，促进细胞死亡，增加反应性 氧自由基，以及可能的其他机制。第二，变异的 线粒体DNA可以使衰老细胞对各种生化损伤敏感， 患有特定的迟发性神经退行性疾病这些假设是 我们的初步发现支持了这一点， 老年人的黑质中的神经元，而不是年轻人的大脑 累积非常高水平的克隆扩增mtDNA缺失。观察到的 色素神经元中的缺失水平高于生理阈值 因此很可能干扰细胞功能以及 细胞对各种压力的反应能力。测试的关键 这些假设是对mtDNA突变分布的分析， 大脑各区域细胞的生理状态， 细胞水平，这是拟议的研究的核心。 具体目标是：（1）开发和优化 精确定量和定性所需的方法库 线粒体DNA突变在大脑的单个细胞。这些方法包括激光 用于单细胞分离的捕获显微切割，全长扩增 单细胞线粒体基因组，单细胞竞争性PCR， 单细胞有限稀释PCR。(2)来识别大脑区域和细胞类型 其中mtDNA突变最有可能在衰老中发挥致病作用， 过程这将通过测量单个细胞中的突变负荷来完成。 黑质、皮质和壳核。已知这些区域富含线粒体DNA 缺失，并与随着年龄增长而下降的大脑功能有关， 在主要的迟发性神经退行性疾病中受到影响。(3)测试 假设单个细胞中mtDNA突变的克隆扩增 导致线粒体缺陷，神经功能障碍和退化， 正常衰老和迟发性神经退行性疾病。会来做这项工作 比较各种染色阳性的细胞的突变负荷， 线粒体功能障碍、氧化应激和细胞变性的标志物， 未染色的对照细胞。我们还将研究突变的分布 作为年龄和疾病严重程度的函数。

DESCRIPTION (provided by applicant): The long-term goal of the proposed research is to study the mechanisms responsible for age-related degenerative processes of the human brain. The understanding of these mechanisms may help to find ways to slow the corresponding processes thus moving the onset of deterioration outside the normal human lifespan. We propose to explore if accumulation of somatic mutations in mtDNA of critical cell types in the brain is one of the causative factors in the age-related deterioration of the brain. Two hypotheses are proposed. First, mutations in mtDNA could work as the primary cause of the dysfunction of certain brain areas by disrupting cellular metabolism, facilitating cell death, increasing the generation of reactive oxygen radicals, and possibly other mechanisms. Second, the presence of mutated mtDNA may render aged cells sensitive to various biochemical insults associated with specific late-onset neurodegenerative diseases. These hypotheses are supported by our preliminary finding that a majority of individual pigmented neurons in the substantia nigra in the old but not in the young brain accumulates very high levels of clonally expanded mtDNA deletions. The observed levels of deletions in pigmented neurons are above the physiological threshold and thus are highly likely to interfere with cellular function as well as with the ability of the cell to respond to the various stresses. Key to testing of these hypotheses is the analysis of the distribution of mtDNA mutations and physiological states of the cells in various areas of the brain at the single cell level, which represents the core of the proposed research. The specific aims of the application are: (1) To develop and optimize the arsenal of methods necessary for precise quantification and characterization of mtDNA mutations in single cells of the brain. These methods will include laser capture microdissection for single cell isolation, amplification of full length mitochondrial genomes from single cells, single cell competitive PCR, and single cell limiting dilution PCR. (2) To identify brain areas and cell types in which mtDNA mutations are most likely to play a causative role in the aging process. This will be done by measuring mutation load in individual cells of substantia nigra, cortex and putamen. These areas are known to be rich in mtDNA deletions and are associated with brain functions that decline with age, and are affected in the major late onset neurodegenerative diseases. (3) To test the hypothesis that clonal expansions of mtDNA mutations in individual cells contribute to mitochondrial defects, neural dysfunction and degeneration in normal aging and late-onset neurodegenerative diseases. This will be done by comparing the mutational load of cells that stained positive for various markers of mitochondrial dysfunction, oxidative stress and cell degeneration to non-staining control cells. We will also study the distribution the mutations as a function of age and the severity of the disease.