MITOCHONDRIAL DNA MUTATIONS AND HUMAN AGING

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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.