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，从而将退化的开始移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，缺乏这种参与的证据。对于哪些组织和/或细胞类型（如果有的话）最有可能受到影响，目前还没有达成共识。我们最近的研究表明，在老年人的大脑黑质中，mtDNA缺失的积累是前所未有的，这导致了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. nigra。因此，具体目的是：(1)验证mtDNA突变的克隆扩增，无论是缺失还是点突变，都会导致大脑特定细胞类型的功能缺陷，和/或导致变性或使细胞易变性的假设。(2)验证老年人黑质色素神经元mtDNA缺失的积累与轻度帕金森症状的发生有关的假说。我们将比较mtDNA缺失的细胞在一组具有良好特征的大脑中与帕金森症状相关的患病率。(3)发展单细胞mtDNA突变定量的新方法。这些方法包括用于量化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，我们在大脑中发现了一种新的、常见的、与年龄相关的退化过程，涉及细胞的发电厂——线粒体的DNA。这个过程影响与帕金森病有关的区域。因此，我们正在探索这一过程是否导致了老年人普遍存在的行动问题。我们还在测试类似的过程是否会影响大脑老化的其他部分。

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.