MITOCHONDRIAL DNA MUTATIONS AND HUMAN AGING

线粒体是细胞中能量的主要来源。它们包含了 自身DNA（mtDNA），其基因编码呼吸系统的组成部分， 链/氧化磷酸化系统。它们对于正常的 身体中所有细胞的功能，并且绝对是关键的。 这些组织的功能高度依赖于有氧代谢， 包括肌肉和大脑。 线粒体DNA的巨大缺失（delta-mtDNA）以前只被观察到 线粒体脑肌病的患者。令人惊奇的是， 现在也发现了极低水平的delta-mtDNA（可观察到的 仅通过PCR）在正常个体中。这些A-mtDNA以指数方式积累 随着年龄的增长，在整个过程中增加了3-4个数量级。 正常的人类寿命然而，个别delta-mtDNA物种（其中 可能有数百或数千个）存在于老化的肌肉中， 占总mtDNA的0.1%。由于线粒体的修复系统很差 对于DNA，点突变可能也会在衰老过程中积累，但这些点突变 尚未被研究。 线粒体DNA突变在衰老中的表型相关性是 目前不清楚。关于其总量和类型的基本知识 mtDNA突变及其在细胞水平上的分布缺乏。 我们也不了解突变的mtDNA基因型之间的关系 以及组织或细胞中异常的线粒体表型 水平最后，我们几乎不知道涉及的机制（S）， 突变mtDNA的起源、维持和传播。 我们建议通过结合遗传学， 生物化学和形态学的方法，使用人类肌肉-一个可访问的 我们非常熟悉的组织-作为我们的测试系统。具体地说， 我们将：（I）在水平上寻找线粒体功能障碍的证据， 个体肌纤维形态学和生物化学在正常老化;（2） 定量mtDNA突变的总量-缺失和点突变 突变-在整个肌肉和孤立的肌肉纤维;（3）发展 通过“原位PCR”快速和全面检测delta-mtDNA的方法， 肌肉切片;（4）分析mtDNA突变之间的关系 通过研究线粒体DNA突变， 具有快速寿命的模式生物（果蝇），并通过研究 线粒体DNA突变的生殖系传播。

Mitochondria are the main sources of energy in the cell. They contain their own DNA (mtDNA), whose genes encode components of the respiratory chain/oxidative phosphorylation system. They are essential for the -normal functioning of all cells in the body, and are absolutely criticaI for the function of those tissues that are highly dependent on aerobic metabolism, including muscle and brain. Giant deletions of mtDNA (delta-mtDNAs) had previously been observed only in patients with specific mitochondrial encephalomyopathies. Surprisingly, delta-mtDNAs have now also been found at extremely low levels (observable only by PCR) in normal individuals. These A-mtDNAs accumulate exponentially with age, increasing by 3-4 orders of magnitude over the course of the normal human lifespan. However, individual delta-mtDNA species (of which there may be hundreds or thousands) are present in aged muscle at a level of <0.1% of total mtDNA. Since mitochondria have very poor repair systems for DNA, point mutations probably also accumulate in aging, but these have not yet been studied. The phenotypic relevance of the presence of mtDNA mutations in aging is unclear at present. Basic knowledge as to the total amount and types of mtDNA mutations, and their distribution at the cellular level, is lacking. We also do not understand the relationship between mutated mtDNA genotypes and abnormal mitochondrial phenotypes at either the tissue or cellular level. Finally, we know almost nothing about the mechanism(s) involved in the origin, maintenance, and transmission of mutated mtDNAs. We propose to address some of these questions by a combined genetic, biochemical, and morphologicaI approach, using human muscle - an accessible tissue with which we are quite familiar - as our test system. Specifically, we will: (I) search for evidence of mitochondrial dysfunction at the level of individual muscle fiber morphology and biochemistry in normal aging; (2) quantitate the total amount of mtDNA mutations - both deletions and point mutations - in whole muscle and in isolated muscle fibers; (3) develop methods to detect delta-mtDNAs rapidly and globally by "in-situ PCR" of muscle sections; and (4) analyze the relationship between mtDNA mutations and aging at a more fundamental level, by studying mtDNA mutations in a model organism having a rapid lifespan (Drosophila) and by investigating germline transmission of mtDNA mutations.