Investigating the use of the uaDf5 C. elegans strain as a potential model of mitochondrial DNA deletion disease

Abstract

Mutations in mitochondrial DNA (mtDNA) result in primary mitochondrial disease (MD), which presents with a wide range of clinical phenotypes. There is currently no cure for MD, this can be attributed to the major phenotypical discrepancies that exist between MD patients. A single mutation can lead to different clinical phenotypes while different mutations can lead to the same phenotype, which makes diagnosis difficult. Heteroplasmy, a state where more than one mtDNA genotype coexist within the same cell, is thought to be one of the confounding factors influencing the differences in clinical phenotypes. Although the effect of heteroplasmy on MD severity has been widely studied in vitro, not much is known about the effect of heteroplasmy in vivo. This is due to a lack of animal models that harbour functional heteroplasmic mutations. This study employed the LB138 (uaDf5) C. elegans strain to investigate the characteristics that influence the suitability of this strain as a disease model for harbouring a range of mtDNA deletion heteroplasmy levels and ii) elucidate the cellular responses to increasing heteroplasmy levels in this model. To achieve the aim of this study, a combination of investigative approaches including genetic, biochemical, and metabolomic investigations were carried out in succession. First, genetic characterization was carried out to determine changes in mtDNA copy number and uaDf5 mtDNA heteroplasmy levels during larval development. While significant changes in mtDNA copy number were observed in the larval stages, alterations in heteroplasmy levels were not significant. Furthermore, comparing uaDf5 mtDNA heteroplasmy levels between parent and progeny, as well as among progeny from the same parent, showed considerable variation in heteroplasmy levels in individual nematodes. This suggests that it is difficult to predict the heteroplasmy levels of a large number of worms from the same sample (i.e., plate). Second, biochemical investigation indicated that the uaDf5 C. elegans strain presents with a mitochondrial disease phenotype, as evident by the significant reduction in basal respiration and complex (CIII) activity. Third, one of the main objectives of this study was to standardize a metabolite extraction method for metabolomics investigation on C. elegans. A method utilizing iv glass beads, to ensure efficient disruption of the outer protective cuticle of C. elegans, was successfully standardized. Using the standardized method, metabolomics investigations were performed on thirty C. elegans samples, harbouring different levels of uaDf5 mtDNA deletion across an established but narrow range. Metabolomics revealed no significant correlations between metabolite concentrations and the different levels of uaDf5 mtDNA. In conclusion, the uaDf5 C. elegans strain was deemed to be not suitable for investigating the effect of an mtDNA deletion along the heteroplasmy range, thus future studies should consider using model organisms that harbour predictable heteroplasmy levels.