Kidere 2018 MiP2018
Mitochondrial diseases are heterogenous multisystem organ disorders which can be caused by alterations in both nuclear and mitochondrial DNA (mtDNA). Defects in the oxidative phosphorylation system (OXPHOS) are presented in many cases. Although mitochondrial disease diagnostics have been improved over the past few years, prediction of the mtDNA single nucleotide variant pathogenicity remains challenging. As the cytoplasmic hybrid (cybrid) cells have the same nuclear background they are a good model to investigate the influence of mtDNA alterations on cell function . The aim of this study was to characterize functionality of the OXPHOS in the cybrid cell lines harbouring mutations m.9185T>C and m.13513G>A that cause Leigh syndrome and mutation m.3243A>G associated with MELAS syndrome.
During our previous work we have revealed two mutations associated with Leigh syndrome i.e., m.9185T>C in the gene MT-ATP6 and m.13513G>A in the gene MT-ND5 , and our collaborators from Taiwan found mutation m.3243A>G in the MT-TL1 gene that cause MELAS syndrome. Three cybrid cell lines harbouring pathogenic mutations m.9185T>C, m.13513G>A and m.3243A>G and six control group cell lines were developed using osteosarcoma derived rho zero cells and platelets from the patients and healthy donors respectively. The established cell lines were used to determine the mitochondrial functionality by high resolution respirometry using Oroboros O2k. Respiratory chain complex I – IV and additionally complex I+III and II+III activities were measured spectrophotometrically in isolated mitochondria as previously described  with modifications.
We obtained only homoplasmic clones for the lines containing mutation m.9185T>C and m.3243A>G but for the line containing m.13513G>A mutation we achieved three clones with different amount of mutant molecules - 70% mutant, 50% mutant and 100% wild type (WT) clones. Cell lines containing 50% and 70% of mutation m.13513G>A showed decrease of the OXPHOS complex I, IV and I+III activities with increasing level of heteroplasmy compared to the WT line. Respirometry measurements revealed declined complex I dependent oxygen consumption in the clone with 70% heteroplasmy. Cells with mutation m.9185T>C showed normal OXPHOS enzyme I – IV activities compared to control group cells. Though they showed declined efficiency of the oxidative phosphorylation compared with control cell line with the same haplogroup in the respirometry assay. Only OXPHOS enzyme activity measurements were performed for the cell line harbouring mutation m.3243A>G that showed decreased complex I, IV and I+III activities compared to the control cell lines.
High resolution respirometry showed oxidative phosphorylation defects in both cell lines harbouring Leigh syndrome mutations, but only cells with the mutation in the MT-ND5 gene showed reduced OXPHOS enzyme activities. Cell line containing MELAS syndrome mutation also showed decreased respiratory chain enzyme activities.
Labels: MiParea: Respiration, mtDNA;mt-genetics Pathology: Neurodegenerative
Organism: Human Tissue;cell: Other cell lines, Platelet Preparation: Isolated mitochondria Enzyme: Complex I, Complex II;succinate dehydrogenase, Complex III, Complex IV;cytochrome c oxidase
Kidere D(1), Makrecka-Kuka M(2), Stavusis J(1), Lace B(3), Liou CW(4), Inashkina I(1)
- Latvian Biomedical Research Study Centre
- Latvian Inst Organic Synthesis; Riga, Latvia
- Centre Hospitalier Univ Québec, Canada
- Dept Neurology, Chang-Gung Univ Kaohsiung Chang-Gung Memorial Hospital, Kaohsiung, Taiwan. – firstname.lastname@example.org
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