Aging Aging

246 Taylor et al.is also highly vulnerable to nucleolytic attack by free radicals, a naturalbyproduct of oxidative phosphorylation. Moreover, because mtDNA has nointrons and little redundancy, any mutational event within the genome is likelyto affect a coding sequence, and therefore biochemical function.An increasing body of scientific evidence has arisen supporting a mitochondrialinvolvement in the aging process, in particular the finding of pathogenicmtDNA mutations in healthy, elderly subjects. Numerous mtDNA deletions(4–15) have been shown to appear and accumulate with age in a variety ofhuman tissues. Point mutations have also been detected (16), although whetherthey increase with age is debatable (17). The accumulation of age-relatedmtDNA deletions has also been shown to correlate with increased cellular levelsof 8-hydroxy-2-deoxyguanosine, an indicator of free radical inducedmtDNA damage. Furthermore, cytochrome c oxidase (COX)-deficient musclefibers, a pathological hallmark of mitochondrial disease, have also been shownto accumulate in an age-related manner (18,19). Although an apparent declinein the activity of several respiratory chain enzymes has been reported with age,these changes (as much as a 50% decline in activity in a tissue homogenate) arehard to equate with the low levels of mtDNA mutations (85% mutant mtDNA)required before any biochemical dysfunction is apparent (20,21), a more subtlemolecular mechanism must exist to enable the low levels of mutations reportedin various tissues to impair biochemical function.Recent studies in single muscle fibers from patients with autosomal dominantprogressive external ophthalmoplegia (adPEO) (22) and healthy elderlyindividuals (23) have shown that the clonal expansion of somatic mtDNA deletionsin individual cells occurs to the extent that it results in a biochemical(COX activity) defect. It has been proposed that a replicative advantage (eitherat the level of the genome or for the mitochondria itself) allows mutant mtDNAto accumulate within a cell over time to such a level that it surpasses the criticalthreshold required to express a biochemical defect. Because somatic mtDNAmutations may occur anywhere within the genome, one would expect to findindividual COX-deficient fibers harboring high levels of different deletedmtDNA species, which has been shown to be the case (22,23).In view of these findings, the molecular genetic techniques that are appliedto the study of somatic mtDNA mutations in aging must allow either the accuratequantification of these mutations at very low levels within tissuehomogenates, or be applicable to single-cell studies in which the level of mutant