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的机制 用于迟发性神经退行性疾病的缓慢进展。这 了解这些机制可能有助于找到实现这些过程的方法 甚至更慢，从而将这些使人衰弱的疾病的发作转移到体外 人类正常寿命。具体来说，我们建议检验以下假设： 大脑中关键细胞类型 mtDNA 体细胞突变的积累 是至少某些进展的必要条件之一 神经退行性过程。其中一种可能性是，一旦分数 特定细胞中突变的线粒体DNA超过一定阈值，这些细胞 对与某些疾病相关的生化损伤变得敏感。这 该假设源于初步发现，个体色素 黑质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 第一个在帕金森脑中死亡。也有可能是事态的进展 疾病加速突变的积累，从而产生积极的影响 反馈。这些努力将首先集中在帕金森病（PD）上 患者和黑质色素神经元。然后研究将 扩展到阿尔茨海默病（AD）、亨廷顿病（HD）和各种 相应的大脑区域和关键细胞类型。具体目标 建议是： 1）开发和优化必要的方法库 单细胞线粒体DNA突变的精确定量和表征 大脑的细胞。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 单细胞、单细胞竞争性 PCR 和单细胞限制 稀释PCR。 2) 识别线粒体DNA存在的大脑区域和细胞类型 突变最有可能导致神经退行性变。这将完成 通过测量黑质、皮质和单个细胞的突变负荷 壳核富含 mtDNA 缺失，对 PD 至关重要， 分别是AD和HD。 3) 检验克隆扩增的假设 单个细胞的 mtDNA 突变会导致线粒体缺陷并 神经变性和神经元死亡。这将通过比较来完成 各种标记物染色呈阳性的细胞的突变负荷 线粒体功能障碍、细胞变性和非染色控制死亡 细胞。我们还将研究突变随年龄的分布 以及疾病的存在和严重程度。