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: 6344295
• 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/6344295
• 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（根据申请人提供的摘要改编）： 拟议的研究的长期目标是研究负责的机制 对于晚期神经退行性疾病的缓慢进展。这 理解这些机制可能有助于找到制定这些过程的方法 甚至较慢，从而将这些令人衰弱的疾病的发作移到 正常的人类寿命。具体而言，我们建议检验以下假设 大脑关键细胞类型的mtDNA中体突变的积累 是至少某些进展所必需的条件之一 神经退行性过程。一种可能性之一是一旦分数 特定细胞中突变的mtDNA超过一定阈值，这些细胞 对与某些疾病相关的生化侮辱敏感。这 假设是由初步发现，即单个有色的发现 黑质中的神经元积累了非常高的mtDNA缺失， 这很可能会损害细胞对各种应力的抗性。 此外，有迹象表明，突变负荷重的细胞是 首先死在帕金森氏症的大脑中。也有可能 疾病加速突变的积累，从而产生阳性 反馈。这些努力将首先关注帕金森氏病（PD） 黑质的患者和色素神经元。然后研究将是 扩展到阿尔茨海默氏病（AD），亨廷顿氏病（HD）和各种 相应的大脑区域和关键细胞类型。特定目标 提案是：1）开发和优化所需方法的武库 单个中mtDNA突变的精确定量和表征 大脑细胞。这些方法将包括激光捕获微截断 对于单细胞分离，扩增全长线粒体基因组 来自单细胞，单细胞竞争性PCR和单细胞限制 稀释pcr。 2）识别mtDNA的大脑区域和细胞类型 突变最有可能导致神经变性。这将完成 通过测量黑质，皮质和皮质和 已知富含mtDNA缺失并且对PD至关重要的壳壳 广告和高清。 3）测试克隆扩张的假设 单个细胞中的mtDNA突变有助于线粒体缺陷和 神经变性和神经元死亡。这将通过比较 对各种标记的染色阳性的细胞的突变负荷 线粒体功能障碍，细胞变性和对非染色控制的死亡 细胞。我们还将研究突变的分布随着年龄的函数 以及疾病的存在和严重程度。