Research Article: Oxidative Stress Is Not a Major Contributor to Somatic Mitochondrial DNA Mutations

Date Published: February 6, 2014

Publisher: Public Library of Science

Author(s): Leslie S. Itsara, Scott R. Kennedy, Edward J. Fox, Selina Yu, Joshua J. Hewitt, Monica Sanchez-Contreras, Fernando Cardozo-Pelaez, Leo J. Pallanck, Douglas M. Turnbull

Abstract: The accumulation of somatic mitochondrial DNA (mtDNA) mutations is implicated in aging and common diseases of the elderly, including cancer and neurodegenerative disease. However, the mechanisms that influence the frequency of somatic mtDNA mutations are poorly understood. To develop a simple invertebrate model system to address this matter, we used the Random Mutation Capture (RMC) assay to characterize the age-dependent frequency and distribution of mtDNA mutations in the fruit fly Drosophila melanogaster. Because oxidative stress is a major suspect in the age-dependent accumulation of somatic mtDNA mutations, we also used the RMC assay to explore the influence of oxidative stress on the somatic mtDNA mutation frequency. We found that many of the features associated with mtDNA mutations in vertebrates are conserved in Drosophila, including a comparable somatic mtDNA mutation frequency (∼10−5), an increased frequency of mtDNA mutations with age, and a prevalence of transition mutations. Only a small fraction of the mtDNA mutations detected in young or old animals were G∶C to T∶A transversions, a signature of oxidative damage, and loss-of-function mutations in the mitochondrial superoxide dismutase, Sod2, had no detectable influence on the somatic mtDNA mutation frequency. Moreover, a loss-of-function mutation in Ogg1, which encodes a DNA repair enzyme that removes oxidatively damaged deoxyguanosine residues (8-hydroxy-2′-deoxyguanosine), did not significantly influence the somatic mtDNA mutation frequency of Sod2 mutants. Together, these findings indicate that oxidative stress is not a major cause of somatic mtDNA mutations. Our data instead suggests that somatic mtDNA mutations arise primarily from errors that occur during mtDNA replication. Further studies using Drosophila should aid in the identification of factors that influence the frequency of somatic mtDNA mutations.

Partial Text: Mitochondria play crucial cellular roles in energy production, Ca2+ buffering, metabolite synthesis, and programmed cell death in metazoans [1]–[3]. While most mitochondrial proteins are encoded in the nuclear genome, mitochondria also contain a compact genome that generally encodes 37 genes [4], and germline mutations that disrupt the functions of mitochondrial DNA (mtDNA) encoded genes cause a number of devastating familial syndromes [5]. mtDNA mutations also occur in somatic tissues, and the accumulation of somatic mtDNA mutations is implicated in aging and common diseases of the elderly, including cancer, diabetes, and neurodegenerative disease [5]–[8]. Because there are multiple copies of mtDNA in any given cell, when mtDNA mutations occur, they frequently coexist with wild-type (WT) mtDNA, a condition known as heteroplasmy. For reasons that are not presently understood, many mtDNA mutations expand clonally within a cell, such that a single somatic mtDNA mutation can ultimately represent a large fraction of the mtDNA within a given cell or tissue [9]. Although the ratio of mutated to WT mtDNA is believed to play a critical pathological role in diseases associated with mtDNA mutations [10], the molecular mechanisms that influence this ratio are poorly understood.

The accumulation of somatic mtDNA mutations is thought to contribute to aging and common age-related diseases, including cancer and Parkinson’s disease. The causes of somatic mtDNA mutations are poorly understood, as are the factors that influence their cellular frequency following their occurrence. Given the powerful genetic tools available in Drosophila for studying fundamental biological processes, we explored the utility of this model system to investigate the mechanisms that influence the frequency of somatic mtDNA mutations. We show that features associated with mtDNA mutations in vertebrates are conserved in Drosophila, including the somatic mtDNA mutation frequency, an increased frequency of somatic mtDNA mutations with age, a prevalence of G∶C to A∶T transition mutations, and possibly, clonal expansion of somatic mtDNA mutations. These findings indicate that Drosophila will serve as a valuable model system in which to examine the mechanisms influencing somatic mtDNA mutation frequency.

Source:

http://doi.org/10.1371/journal.pgen.1003974