Characterization of Mitochondrial DNA Mutations in Drosophila melanogaster

Abstract

Mitochondria are vital organelles as they produce the majority of cellular energy in the form of ATP through oxidative phosphorylation (OXPHOS). Mitochondria contain their own genome (mtDNA), which encodes essential respiratory chain proteins that function in OXPHOS along with RNAs that are necessary for the translation of the OXPHOS genes. Mutations in mtDNA cause maternally inherited mitochondrial diseases and somatic mutations are implicated in aging and age-related diseases, such as Parkinson's disease. To explore the pathogenesis of mitochondrial disease due to an mtDNA mutation, we characterized a mutation in NADH dehydrogenase subunit 2 (mt:ND2). We found that ND2 mutants model mitochondrial disease as they exhibit a shortened lifespan, neurodegeneration, and seizures. Additionally, ND2 mutants display complex I specific defects along with decreased mitochondrial membrane potential and ATP levels. Importantly, we show that the ND2 mutation uncouples complex I dependent proton pumping from electron transport and demonstrate a role for ND2 in proton pumping. These studies support the use of Drosophila as a model to study complex I disease and potential complex I therapeutics. In addition to studying mitochondrial disease pathogenesis, I adapted techniques to measure mtDNA mutations in Drosophila, including the Random Mutation Capture Assay and Duplex Sequencing. Utilizing the Random Mutation Capture Assay, I found that the somatic mtDNA mutation patterns in flies are conserved with vertebrates, including the mutation frequency (~10-5) and the age-dependent accumulation of mutations. I also explored factors that influence somatic mtDNA mutations, including reactive oxygen species (ROS), which are thought to be an important contributor to mtDNA mutations. Using the distribution of mutations along with loss of function studies in oxidative stress defense factors, I concluded that oxidative stress is only a minor contributor to the mutation frequency. Instead, errors created during replication of mtDNA are the primary source of somatic mtDNA mutations. After completing these studies, I established the Duplex Sequencing method in flies, which is a next-generation sequencing approach as a more comprehensive and high-throughput technique to measure mtDNA mutations. These studies extend our knowledge of sources that contribute to the mtDNA mutation frequency and support Drosophila as a model to study genetic factors that influence mtDNA mutations.