Genetic mosaic analysis of a deleterious mitochondrial DNA mutation in Drosophila reveals novel aspects of mitochondrial regulation and function.
Bottom Line: In the present study, we found that the decrease in cytochrome c oxidase (COX) activity was ascribable to a temperature-dependent destabilization of cytochrome a heme.Using a genetic scheme that expresses a mitochondrially targeted restriction enzyme to induce tissue-specific homoplasmy in heteroplasmic flies, we found that mt:CoI(T300I) homoplasmy in the eye caused severe neurodegeneration at 29°C.Our results demonstrate a novel approach for Drosophila mtDNA genetics and its application in modeling mtDNA diseases.
Affiliation: Laboratory of Molecular Genetics, National Institutes of Health, Bethesda, MD 20892.Show MeSH
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Mentions: Disruption of respiratory chain complexes could lead to overproduction of damaging ROS. However, we found no difference in H2O2 level between wt and mutant flies, although there were clearly higher levels of H2O2 in old flies than in young flies (Supplemental Figure S3B). We also assayed the level of protein carbonylation, an alternative approach to evaluate accumulative oxidative damage caused by ROS. Consistent with a previous report (Wehr and Levine, 2012), the levels of protein carbonylation were higher in old flies than in young flies in both total cellular extracts and mitochondrial preparations. However, there was no obvious difference between wt and mutant flies at either age (Supplemental Figure S3C). In addition, overexpression of SOD2 and catalase, two scavenging enzymes that have been shown to successfully suppress phenotypes caused by excess ROS in Drosophila (Anderson et al., 2005), had no effect on the lifespan of mt:CoIT300I flies (Supplemental Figure S3A). Blockage of electron flow could also reduce mitochondrial membrane potential. Indeed, we found that mt:CoIT300I mitochondria had much lower tetramethylrhodamine methylester (TMRM) fluorescence than wt mitochondria (Figure 5, A and B). It is possible that effects of electron blockage on ROS production are offset by the reduction in membrane potential associated with mt:CoIT300I (Lambert and Brand, 2004).
Affiliation: Laboratory of Molecular Genetics, National Institutes of Health, Bethesda, MD 20892.