MtDNA mutations in brain aging: a single-cell approach

大脑衰老中的线粒体 DNA 突变：单细胞方法

• 批准号: 6368947
• 负责人: Konstantin Khrapko
• 金额: $ 15.24万
• 依托单位: BETH ISRAEL DEACONESS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-30 至 2006-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6368947
• 关键词: aging; brain cell; brain metabolism; cardiac myocytes; cell age; cell death; cell type; diaphragm; dissection; free radical oxygen; gene deletion mutation; gene mutation; human tissue; method development; mitochondrial DNA; mitochondrial disease /disorder; neural degeneration; neurogenetics; neurons; neurophysiology; oxidative stress; polymerase chain reaction; putamen; single cell analysis; striated muscles; substantia nigra

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.

描述（由申请人提供）：拟议的长期目标 研究是研究负责年龄相关退行性的机制 人脑的过程。对这些机制的理解可能有助于 找到减慢相应过程的方法，从而移动 正常人类寿命以外的恶化。我们建议探索是否 大脑关键细胞类型的mtDNA中体突变的积累 是大脑与年龄相关的恶化的原因之一。 提出了两个假设。首先，mtDNA中的突变可以作为 通过破坏细胞的某些大脑区域功能障碍的主要原因 代谢，促进细胞死亡，增加反应性的产生 氧自由基，可能是其他机制。其次，突变的存在 mtDNA可能使老化的细胞对相关的各种生化侮辱敏感 患有特定的晚期神经退行性疾病。这些假设是 在我们的初步发现的支持下，大多数个人有色 旧的黑质中的神经元，但不在年轻人的大脑中 累积了非常高的克隆扩展的mtDNA缺失。观察到 色素神经元缺失水平高于生理阈值 因此，很可能会干扰细胞功能以及 细胞对各种应力做出反应的能力。测试的关键 这些假设是对mtDNA突变的分布的分析和 单个大脑各个区域中细胞的生理状态 细胞水平，代表拟议研究的核心。 应用程序的具体目的是：（1）开发和优化 精确量化和表征所需的方法 大脑单细胞中的mtDNA突变。这些方法将包括激光 捕获微分解以进行单细胞分离，扩增全长 来自单细胞，单细胞竞争性PCR和的线粒体基因组和 单细胞限制稀释PCR。 （2）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命作用 过程。这将通过测量在单个单元中的突变负荷来完成 黑质尼格拉，皮质和put虫。这些区域已知富含mtDNA 删除并与随着年龄的增长而下降的大脑功能相关，并且 在主要发作神经退行性疾病中受到影响。 （3）测试 假设单个细胞中mtDNA突变的克隆膨胀 有助于线粒体缺陷，神经功能障碍和变性 正常衰老和晚期神经退行性疾病。这将由 比较各种细胞的突变负荷 线粒体功能障碍，氧化应激和细胞变性的标记 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功能。