MtDNA mutations in brain aging: a single-cell approach

描述(由申请人提供)：建议的长期目标 研究是为了研究与年龄相关的退行性变的机制 人脑的过程。对这些机制的理解可能有助于 想方设法减缓相应的进程，从而推迟 恶化超出了人类的正常寿命。我们建议探索是否 脑内关键细胞类型线粒体DNA体细胞突变的积累 是与年龄相关的大脑退化的原因之一。 提出了两个假设。首先，线粒体DNA的突变可以作为 某些脑区功能障碍的主要原因是细胞受到干扰 新陈代谢，促进细胞死亡，增加反应的产生 氧自由基，以及可能的其他机制。第二，突变的存在 线粒体DNA可能使衰老的细胞对相关的各种生化损伤敏感 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大多数个体的色素沉着 老年人黑质中的神经元，而年轻脑中没有 积累了非常高水平的克隆性扩展的mtDNA缺失。被观察到的 着色神经元的缺失水平高于生理阈值 因此极有可能干扰细胞功能以及 细胞对各种压力做出反应的能力。测试的关键 这些假设是对线粒体DNA突变的分布和 单个脑区不同区域细胞的生理状态 细胞水平，这代表了拟议研究的核心。 应用程序的具体目标是：(1)开发和优化 用于精确量化和表征的各种必要方法 大脑单个细胞的线粒体DNA突变。这些方法将包括激光 捕获显微切割用于单细胞分离、全长扩增 单细胞线粒体基因组、单细胞竞争性聚合酶链式反应和 单细胞限制性稀释法。(2)识别大脑区域和细胞类型 其中线粒体DNA突变最有可能在衰老中起到致病作用 进程。这将通过测量单个细胞中的突变负载来完成 黑质、皮质和壳核。众所周知，这些地区含有丰富的线粒体DNA。 基因缺失与大脑功能随年龄增长而下降有关，以及 在主要的迟发性神经退行性疾病中受到影响。(3)测试 线粒体DNA突变在单个细胞中的克隆性扩张假说 导致线粒体缺陷、神经功能障碍和变性 正常衰老和迟发性神经退行性疾病。这将通过以下方式完成 比较不同染色阳性细胞的突变负荷 线粒体功能障碍、氧化应激和细胞退变的标志物 非染色对照细胞。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.