Evaluating Determinants of Mitochondrial Mutagenesis and their Intersections with Cell Fate
Valente, William Joseph
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Mitochondria are critical components of cellular metabolism as mediators of oxidative phosphorylation. As semi-autonomous organelles, mitochondria possess their own DNA (mtDNA) which encodes elements of the electron transport chain (ETC). Aberrations in the mitochondrial genome are associated with neurodegenerative diseases, metabolic syndromes, cancer, and pathologies of aging; although establishing direct links between cause and consequence of mtDNA mutagenesis has remained elusive. In this thesis, I sought to directly test two possible mechanisms of somatic mitochondria mutagenesis and to characterize the effects of a model of reduced mtDNA mutation upon cell fate. First, I evaluated extrinsic genotoxic exposures, in either the form of established nuclear mutagens benzo[a]pyrene or N-ethyl-nitrosourea, for their effects on mtDNA frequency with newly developed droplet digital PCR assays. Here I found that these classic nuclear mutagens damage mtDNA, but are not capable of significant mtDNA mutation induction. Next, I evaluated the cellular effects resulting from expression of a mitochondrial-targeted form of the antioxidant enzyme catalase, which has featured in a murine model of increased lifespan which also demonstrated attenuated mtDNA mutation frequency. Finally, I addressed the hypothesis that metabolic byproducts of oxidative metabolism, reactive oxygen species (ROS), can influence mtDNA mutagenesis. I demonstrate, using genetically-encoded proteins with spatiotemporal control of ROS production, that ROS induce mtDNA damage and mutation. The results of this work have potentially broad-ranging impacts upon mitochondrial mutagenesis research, translational efforts to characterize tumor progression, and models of cellular aging.