Background: Mutations in the mitochondrial genome (mtDNA) can cause devastating maternally inherited diseases, while the accumulation of somatic mtDNA mutations is linked to common diseases of aging. Although mtDNA mutations impact human health, the process(es) that give rise to these mutations are unclear and are under considerable debate. We analyzed the distribution of naturally occurring somatic mutations across the mouse and human mtDNA obtained by Duplex Sequencing to provide clues to the mechanism by which de novo mutations arise as well as how the genome is replicated. Results: We observe two distinct mutational gradients in G→A and T→C transitions, but not their complements, that are delimited by the light-strand origin and the control region (CR). The gradients increase with age and are lost in the absence of DNA polymerase γ proofreading activity. A nearly identical pattern is present in human mtDNA somatic mutations. The distribution of mtDNA SNPs in the human population and genome base composition across >3,000 vertebrate species mirror this gradient pattern, pointing to evolutionary conservation of this phenomenon. Lastly, high-resolution analysis of the mtDNA control region highlights mutational hot-spots and cold-spots that strongly align with important regulatory regions. Conclusions: Collectively, these patterns support an asymmetric strand-displacement mechanism with key regulatory structures in the CR and argue against alternative replication models. The mutational gradient is a fundamental consequence of mtDNA replication that drives somatic mutation accumulation and influences inherited polymorphisms and, over evolutionary timescales, genome composition.

PurposeCharacterize how metabolic function in the murine retina and retinal pigment epithelium-choroid-sclera (eyecup) complex is impacted by natural aging.MethodsWe examined scotopic and photopic visual function of young (3-6 months) and aged (23-26 months) C57Bl/6J mice using electroretinograms (ERGs). Metabolic changes in retina and eyecup explants were characterized by measuring uptake and usage of U-13C-glucose or U-13C-glutamine at different timepoints by gas chromatography-mass spectrometry (GC-MS), measuring oxygen consumption rate (OCR) using a perifusion apparatus, and determining ATP levels with a bioluminescence assay.ResultsScotopic and photopic ERG responses declined in aged mice. Glucose metabolism, glutamine metabolism, OCR, and ATP pools in retinal explants were mostly unaffected by the age of the mouse. In eyecups, glutamine usage in the Krebs Cycle decreased while glucose metabolism, OCR, and ATP pools remained stable.ConclusionsThe ex vivo approach in our study to examine aging glucose and glutamine metabolism in retina and RPE showed negligible impact of age on retina and an impairment of glutamine anaplerosis in eyecups. The surprising metabolic stability of these tissues ex vivo suggests age-related metabolic alterations in these tissues may not be intrinsic. Future experiments should focus on determining whether external factors including nutrient supply, oxygen availability, or other structural changes influence ocular metabolism in vivo.