Role of Somatic mtDNA Mutations in Neurodegeneration

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 治疗迟发性神经退行性疾病的缓慢进展。这个 对这些机制的理解可能有助于找到使这些过程 甚至更慢，从而将这些令人衰弱的疾病的发病转移到 正常的人类寿命。具体地说，我们建议检验这一假设 脑内关键细胞类型线粒体DNA体细胞突变的积累 是至少有些人进步的必要条件之一 神经退行性变过程。其中一种可能性是，一旦分数 在特定细胞中突变的mtDNA的数量超过了一定的阈值，这些细胞 对与某些疾病相关的生化侮辱变得敏感。这 这一假说是从初步发现的个体色素 黑质神经元积累了非常高水平的mtDNA缺失， 这极有可能损害细胞对各种压力的抵抗力。 此外，有迹象表明，具有较大突变负荷的细胞是 第一个死于帕金森氏症大脑的人。也有可能是经济衰退的进程 疾病加速了突变的积累，从而产生了阳性 反馈。这些努力将首先集中在帕金森氏症(PD)上 患者和黑质着色神经元。那么研究将会是 扩展到阿尔茨海默病(AD)、亨廷顿病(HD)和各种 相应的脑区和关键细胞类型。《公约》的具体目标 建议是：1)开发和优化所需的各种方法 单个个体mtDNA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解剖 对于单细胞分离，全长线粒体基因组的扩增 从单细胞、单细胞竞争性聚合酶链式反应和单细胞限制 稀释聚合酶链式反应。2)确定mtDNA所在的脑区和细胞类型 突变最有可能导致神经退化。这件事会做到的 通过测量黑质、皮质和大脑皮质单个细胞的突变负荷 已知富含线粒体DNA缺失并对帕金森病至关重要的壳蛋白， AD和HD。3)检验克隆扩展的假设 单个细胞中的mtDNA突变会导致线粒体缺陷和 神经变性和神经元死亡。这将通过比较 不同标志物染色阳性的细胞的突变负荷 线粒体功能障碍、细胞变性和死亡与非染色对照 细胞。我们还将研究突变的分布与年龄的关系 以及疾病的存在和严重程度。

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.