We have previously suggested that differences in bioenergetics might account for the selection bias 11. In the case of the most common pathological variant, m.3243A>G, which produces dysfunctional mt-tRNA Leu(UUR), the mutant molecules are selected in most cell lines 4, 11, 12, whereas A549 adenocarcinoma can occasionally select the wild-type mtDNAs 5, 6. Several lines of evidence indicate that active selection can determine heteroplasmy levels: rapid segregation of deleterious mtDNA variants occurs in human cultured cells 4, 5, 6, 7, segregation bias is a feature of some mouse tissues 8, 9, and selection against nonsynonymous mutants occurs in the mouse germline 10 yet, the underlying mechanisms remain unclear. Thus, a longstanding goal in the field has been to identify a means of increasing the proportion of wild-type molecules, as this could be an effective therapy. Hence, it is not necessary to eradicate all the mutant mtDNA to restore mitochondrial function instead, a modest decrease in mutant load should be sufficient to transition from a disease to a healthy state. Crucially, most heteroplasmic pathological mtDNA variants produce biochemical and clinical phenotypes only at very high levels 1, 2, 3. Mitochondrial DNA (mtDNA) is essential for energy production and mutations in this molecule cause an energy crisis, with consequent disease. Hence, we demonstrate that mitochondrial fitness dictates metabolite preference for mtDNA replication consequently, interventions that restrict metabolite availability can suppress pathological mtDNAs, by coupling mitochondrial fitness and replication. Additionally, by restricting glucose utilization, 2DG supports functional mtDNAs, as glucose-fuelled respiration is critical for mtDNA replication in control cells, when glucose and glutamine are scarce. Mechanistically, 2DG selectively inhibits the replication of mutant mtDNA and glutamine is the key target metabolite, as its withdrawal, too, suppresses mtDNA synthesis in mutant cells. Here, we show that both compounds selected wild-type over mutant mtDNA, restoring mtDNA expression and respiration. Glucose and glutamine consumption are increased in mtDNA dysfunction, and so we targeted the use of both in cells carrying the pathogenic m.3243A>G variant with 2-Deoxy-D-glucose (2DG), or the related 5-thioglucose. Because mitochondrial fitness does not favour the propagation of functional mtDNAs in disease states, we sought to create conditions where it would be advantageous. Pathological variants of human mitochondrial DNA (mtDNA) typically co-exist with wild-type molecules, but the factors driving the selection of each are not understood.
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