Mutations in the mitochondrial genome (mtDNA) are implicated in a wide range of metabolic and age-related diseases. A single animal cell can contain hundreds to thousands of mtDNA copies, so when a mutation arises, the mutant genome (?mtDNA) coexists with wildtype mtDNA in a state known as heteroplasmy. At low levels, deleterious mutations may exert no discernible phenotypic effects due to the high levels of wildtype mtDNA. However, mtDNA replication occurs independently of the cell cycle, hence the frequency of ?mtDNA can vary over time and between cells, and if ?mtDNA levels become high enough, the mutation can become pathogenic. The mechanisms underlying this rise in ?mtDNA levels, despite their disruptive effects on cell fitness, remain largely elusive. A major challenge to understanding the proliferative success of ?mtDNA is the limited ability to quantitatively track heteroplasmies across generations. This proposed research will take advantage of key innovations to overcome these challenges, including the powerful genetic toolkit available with Caenorhabditis elegans, the largest collection of heteroplasmic strains available in any model system, and the ability to quantify mtDNA levels at single-molecule resolution. This study will build on previous work?which showed that activation of the mitochondrial unfolded protein response (UPRmt) can facilitate the propagation of the mtDNAuaDf5 mutant variant?by assaying UPRmt activation across a collection of heteroplasmies. Mutations that are most disruptive to protein quality control in the mitochondria are predicted to elicit the most robust UPRmt activation. This work will test the hypothesis that such mutations can exploit UPRmt to propagate at high levels. The proposed study will also investigate nuclear-encoded factors that modulate the propagation of ?mtDNA independently of UPRmt activation. Specifically, RNA sequencing will be used to identify non-UPRmt genes activated in C. elegans in the presence of mtDNAuaDf5. These genes will be used to compile a list of candidate pathways, which will subsequently be interrogated for their effect on ?mtDNA propagation using targeted gene knockdown. Finally, this research will also explore the possibility that bioenergetics, particularly fat metabolism, can modulate the transmission of ?mtDNA. Oxidation of fatty acids occurs in the mitochondria and provides the electron transport chain with a glycolysis-independent oxidizable carbon source. Previous studies have shown that genes which regulate fatty acid oxidation exert a downstream effect on mitochondria by modulating organelle biogenesis and turnover, which are separately known to affect ?mtDNA levels. The research proposed here will employ a combination of genetic, molecular and pharmacological approaches to test the hypothesis that by modulating organelle dynamics, altering fat metabolism can affect the propagation of mitochondrial mutations. Understanding the role of altered fat metabolism in ?mtDNA dynamics will provide valuable insights on the risks of developing mitochondrial disorders especially in the context of obesity.
Although mutations in the mitochondrial genome are implicated in a wide range of human diseases, little is known about the mechanisms that govern the propagation of mutant mitochondrial genomes. The focus of this proposal is to identify nuclear-encoded mechanisms by which mutant mitochondrial genomes reach pathogenically high levels within the host. This work will yield valuable insights on the basic molecular principles underlying the risk of developing mitochondrial diseases.
