Somatic mtDNA mutations in brain Aging: a single-cell approach

描述（由申请人提供）：拟议研究的长期目标是研究与年龄相关的退行性过程的机制。对这些机制的理解可能有助于找到减缓相应过程的方法，从而将恶化的开始时间推迟到正常人类寿命之外。长期以来，mtDNA突变的积累一直被假设为各种衰老症状的可能原因，然而，缺乏证据证明这种参与。关于哪些组织和/或细胞类型（如果有的话）最有可能受到影响，尚未达成共识。我们最近的研究表明，在老年人大脑的黑质中，线粒体DNA缺失前所未有地积累，这导致色素神经元的呼吸链缺陷。这一证明在概念上是重要的，作为一个例子，体细胞mtDNA突变在与年龄相关的退行性过程中最重要的参与，但它也提出了关于这一过程的健康后果的问题，以及它是否仅限于s。黑鬼因此，具体的目的是：（1）检验mtDNA突变的克隆扩增（无论是缺失还是点突变）导致脑特定细胞类型的功能缺陷和/或导致变性或使细胞易于变性的假设。(2)验证黑质色素神经元线粒体DNA缺失的累积与老年人轻度帕金森综合征的发生有关的假设。我们将比较线粒体DNA缺失的细胞在一组特征明确的大脑中的患病率，以确定是否存在帕金森症状。(3)发展新的方法学用于单个细胞中mtDNA突变的定量。这些方法包括用于定量mtDNA缺失的mtDNA荧光原位杂交（FISH），以及用于研究mtDNA点突变的新型大规模平行454 DNA测序方法。简而言之，我们发现了一种新的、常见的、与年龄相关的大脑退化过程，涉及线粒体的DNA，线粒体是细胞的发电厂。这个过程影响帕金森病的一个区域。因此，我们正在探讨这一过程是否是老年人普遍存在的行动问题的原因。我们还在测试类似的过程是否会影响衰老大脑的其他部分。

DESCRIPTION (provided by applicant): The long-term goal of the proposed research is to study the mechanisms responsible for age-related degenerative processes. The understanding of these mechanisms may help to find ways to slow down the corresponding processes thus moving the onset of deterioration outside the normal human lifespan. Accumulation of mtDNA mutations has been long hypothesized as a probable cause of the various symptoms of aging, however, proof of such involvement was lacking. There is no consensus regarding which tissues and/or cell types if any are most likely to be affected. Our recent research has demonstrated unprecedented accumulation of mtDNA deletions in the substantia nigra of the aged human brain, which results in respiratory chain defects in pigmented neurons. This demonstration is important conceptually as an example the most significant involvement of somatic mtDNA mutations in an age-related degenerative process, but it also raises questions regarding the health consequences of this process, and whether it is limited to s. nigra. The specific aims therefore are: (1) To test the hypothesis that clonal expansions of mtDNA mutations, either deletions or point mutations, cause functional defects in specific cell types of the brain, and/or cause degeneration or predispose cells to degeneration. (2) To test the hypothesis that accumulation of mtDNA deletions in pigmented neurons of substantia nigra contributes to the development of Mild Parkinsonian Signs in seniors. We will compare the prevalence of cells with mtDNA deletions in a collection of well- characterized brains with respect to the presence of parkinsonian signs. (3) To develop new methodologies for mtDNA mutation quantification in individual cells. The methods include mtDNA Fluorescent In Situ Hybridization (FISH) for quantification of mtDNA deletions, and a novel massive parallel 454 DNA sequencing approach for studying mtDNA point mutations. In short, we have discovered a novel, common, age-related degenerative process in the brain involving the DNA of mitochondria, the power plants of the cell. This process affects an area involved in Parkinson's disease. We are therefore exploring if the process is responsible for movement problems widespread among the senior population. We are also testing whether similar processes affect other parts of the aging brain.