Nearly fifty years ago, Harman proposed that aging-related loss of tissue integrity and function is caused by damage to cellular macromolecules by free radicals produced largely in mitochondria. Since this seminal theory was first proposed, extensive evidence has accumulated that supports a strong association between changes to mitochondria and age-related loss of tissue function. In particular, several lines of evidence have implicated mutations in mitochondrial DNA as a potential causative agent in aging. First, human diseases that are clearly caused by mutations in mitochondrial DNA display features reminiscent of aging. Second, some aging human tissues that rely heavily on the mitochondria for energy and display significant age-related dysfunction (brain and heart, for example) accumulate mutations in mitochondrial DNA. Third, some mouse models that accumulate mutations in mtDNA especially rapidly also age prematurely, and some that accumulate mutations more slowly also live longer. However, the central question remains unanswered: do mutations in mitochondrial DNA cause aging? This question remains unanswered in part because we don't understand how mutations in mitochondrial DNA accumulate to levels that impair cellular function. Because there are multiple mitochondria per cell and multiple mitochondrial DNA copies per mitochondrion, mutations must accumulate within individual mitochondria and individual cells before cellular function is compromised. This process is known as clonal expansion, and its mechanism is poorly understood. If mitochondrial DNA mutation is in fact causal to aging in some way, perturbation of clonal expansion might directly reveal this relationship. Such an understanding might even facilitate the development of therapeutics that could slow the accumulation of deleterious mitochondrial DNA mutations in aging humans. Unfortunately, existing experimental methods lack the throughput and sensitivity needed to test various hypotheses that could explain clonal expansion. The emergence of next-generation DNA sequencing technologies has the potential to revolutionize the study of mitochondrial DNA mutations, but these technologies are still severely limited in their ability to detect rare variants because of a high substitution error rate. We will adapt technologies recently developed in the Shendure lab that overcome these limitations in sensitivity to develop an assay that is capable of detecting any type of mutation in mitochondrial DNA with very high sensitivity and throughput. Using this assay, we will determine the mutation frequencies from three different human tissue types isolated from young, middle-aged, and elderly individuals. We can then compare the rate at which different types of mutations arise and expand during human aging, which will allow us to distinguish between proposed mechanisms. This mechanistic information can then guide future studies that seek to perturb clonal expansion and examine its effect on tissue function during aging. PUBLIC HEALTH RELEVANCE: Age is the most important risk factor for many diseases, and, even in the absence of overt disease, is invariably associated with eventual disability and death. Damage to the mitochondrial genetic code, which contains critical instructions for energy production within the cell, has long been implicated in aging, but we don't understand how this damage accumulates over time. By characterizing this process, we will reveal important aspects of aging biology and might even facilitate the development of new therapeutics to slow the aging process.