Role of Somatic mtDNA Mutations in Neurodegeneration

体细胞 mtDNA 突变在神经退行性疾病中的作用

• 批准号: 6635535
• 负责人: Konstantin Khrapko
• 金额: $ 26.68万
• 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: 2001-07-01 至 2006-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6635535
• 关键词: Alzheimer's disease; Huntington's disease; Parkinson's disease; brain mapping; cell death; cell type; cerebral cortex; gene deletion mutation; mitochondrial DNA; neural degeneration; nucleic acid amplification techniques; pathologic process; polymerase chain reaction; putamen; substantia nigra

DESCRIPTION (Adapted from the abstract provided by the applicant): The long-term goal of the proposed research is to study the mechanisms responsible for the slow progression of late-onset neurodegenerative diseases. The understanding of these mechanisms may help to find ways to make these processes even slower, thus moving the onset of these debilitating diseases outside the normal human lifespan. Specifically, we propose to test the hypothesis that accumulation of somatic mutations in mtDNA of critical cell types in the brain is one of the conditions necessary for the progression of at least some neurodegenerative processes. One of the possibilities is that once the fraction of mutated mtDNA in specific cells exceeds a certain threshold, these cells become sensitive to biochemical insults associated with some diseases. This hypothesis has arisen from the preliminary finding that individual pigmented neurons in substantia nigra accumulate very high levels of mtDNA deletions, which are highly likely to compromise cell's resistance to various stresses. Moreover, there are indications that cells with a heavy mutational load are the first to die in Parkinson's brain. It is also possible that progression of the disease accelerates accumulation of mutations thus creating a positive feedback. The efforts will be focused first on Parkinson's Disease (PD) patients and pigmented neurons of substantia nigra. Then research will be extended to Alzheimer's Disease (AD), Huntington's Disease (HD) and the various corresponding brain areas and critical cell types. The Specific Aims of the proposal are: 1) To develop and optimize the arsenal of methods necessary for the precise quantification and characterization of mtDNA mutations in single cells of the brain. These methods will include laser capture micro-dissection 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 contribute to neurodegeneration. This will be done by measuring mutation load in individual cells of substantia nigra, cortex and putamen that are known to be rich in mtDNA deletions and are critical for PD, AD, and HD, respectively. 3) To test the hypothesis that clonal expansions of mtDNA mutations in individual cells contribute to mitochondrial defects and to neurodegeneration and death of neurons. This will be done by comparing the mutational load of cells that stained positive for various markers of mitochondrial dysfunction, cell degeneration and death to non-staining control cells. We will also study the distribution the mutations as a function of age and the presence and severity of the disease.

描述（改编自申请人提供的摘要）： 拟议研究的长期目标是研究负责机制 迟发性神经退行性疾病进展缓慢的原因的 了解这些机制可能有助于找到使这些过程 甚至更慢，从而将这些使人衰弱的疾病的发病转移到 正常的人类寿命具体来说，我们建议测试假设， 脑中关键细胞类型的mtDNA中体细胞突变的积累 是至少一些疾病进展的必要条件之一 神经退化过程其中一种可能性是， 特定细胞中突变mtDNA的含量超过一定阈值，这些细胞 变得对某些疾病相关的生化损伤敏感。这 一种假说是从初步发现中产生的， 黑质的神经元积累了非常高水平的mtDNA缺失， 这很可能会损害细胞对各种压力的抵抗力。 此外，有迹象表明，具有重突变负荷的细胞是 第一个死在帕金森的大脑里也有可能是因为 疾病加速了突变的积累，从而产生了积极的 反馈这些努力将首先集中在帕金森病（PD）上 患者和黑质的色素神经元。然后研究将是 扩展到阿尔茨海默病（AD）、亨廷顿病（HD）和各种 对应的大脑区域和关键细胞类型。的具体目标 建议是：1）开发和优化必要的方法库， 线粒体DNA突变的精确定量和表征 脑细胞。这些方法将包括激光捕获显微切割 用于单细胞分离、全长线粒体基因组的扩增 从单细胞，单细胞竞争PCR，和单细胞限制 稀释PCR。2)为了确定线粒体DNA在大脑中的区域和细胞类型， 突变最有可能导致神经退化。为此将 通过测量黑质、皮质和大脑皮层的单个细胞中的突变负荷， 已知富含线粒体DNA缺失的壳核对PD至关重要， AD和HD。3)为了验证这一假设， 单个细胞中的mtDNA突变导致线粒体缺陷， 神经变性和神经元死亡。这将通过比较 细胞的突变负荷，这些细胞对各种标记物染色呈阳性， 线粒体功能障碍、细胞变性和死亡至非染色对照 细胞我们还将研究突变的分布作为年龄的函数 以及疾病的存在和严重程度。