Age-associated accumulation of somatic mutations in mitochondrial DNA (mtDNA) has been proposed to be responsible for the age-associated mitochondrial respiration defects found in elderly human subjects. We carried out reprogramming of human fibroblast lines derived from elderly subjects by generating their induced pluripotent stem cells (iPSCs), and examined another possibility, namely that these aging phenotypes are controlled not by mutations but by epigenetic regulation. Here, we show that reprogramming of elderly fibroblasts restores age-associated mitochondrial respiration defects, indicating that these aging phenotypes are reversible and are similar to differentiation phenotypes in that both are controlled by epigenetic regulation, not by mutations in either the nuclear or the mitochondrial genome. Microarray screening revealed that epigenetic downregulation of the nuclear-coded GCAT gene, which is involved in glycine production in mitochondria, is partly responsible for these aging phenotypes. Treatment of elderly fibroblasts with glycine effectively prevented the expression of these aging phenotypes.
Mitochondrial dysfunction causes increased oxidative stress and depletion of ATP, which are involved in the etiology of a variety of renal diseases, such as CKD, AKI, and steroidresistant nephrotic syndrome. Antioxidant therapies are being investigated, but clinical outcomes have yet to be determined. Recently, we reported that a newly synthesized indole derivative, mitochonic acid 5 (MA-5), increases cellular ATP level and survival of fibroblasts from patients with mitochondrial disease. MA-5 modulates mitochondrial ATP synthesis independently of oxidative phosphorylation and the electron transport chain. Here, we further investigated the mechanism of action for MA-5. Administration of MA-5 to an ischemia-reperfusion injury model and a cisplatin-induced nephropathy model improved renal function. In in vitro bioenergetic studies, MA-5 facilitated ATP production and reduced the level of mitochondrial reactive oxygen species (ROS) without affecting activity of mitochondrial complexes I-IV. Additional assays revealed that MA-5 targets the mitochondrial protein mitofilin at the crista junction of the inner membrane. In Hep3B cells, overexpression of mitofilin increased the basal ATP level, and treatment with MA-5 amplified this effect. In a unique mitochondrial disease model (Mitomice with mitochondrial DNA deletion that mimics typical human mitochondrial disease phenotype), MA-5 improved the reduced cardiac and renal mitochondrial respiration and seemed to prolong survival, although statistical analysis of survival times could not be conducted. These results suggest that MA-5 functions in a manner differing from that of antioxidant therapy and could be a novel therapeutic drug for the treatment of cardiac and renal diseases associated with mitochondrial dysfunction.
It has been hypothesized that respiration defects caused by accumulation of pathogenic mitochondrial DNA (mtDNA) mutations and the resultant overproduction of reactive oxygen species (ROS) or lactates are responsible for aging and age-associated disorders, including diabetes and tumor development. However, there is no direct evidence to prove the involvement of mtDNA mutations in these processes, because it is difficult to exclude the possible involvement of nuclear DNA mutations. Our previous studies resolved this issue by using an mtDNA exchange technology and showed that a G13997A mtDNA mutation found in mouse tumor cells induces metastasis via ROS overproduction. Here, using transmitochondrial mice (mitomice), which we had generated previously by introducing G13997A mtDNA from mouse tumor cells into mouse embryonic stem cells, we provide convincing evidence supporting part of the abovementioned hypothesis by showing that G13997A mtDNA regulates diabetes development, lymphoma formation, and metastasis-but not aging-in this model. reactive oxygen species generating mutation | Warburg effect | chromosome aberration | lactic acidosis | hyperglycemia P athogenic mitochondrial DNA (mtDNA) mutations that induce significant mitochondrial respiration defects are responsible for mitochondrial diseases (1, 2) and could also be involved in aging and age-associated disorders, including tumor development (1-5). On the other hand, mitochondrial respiration defects caused by nuclear DNA mutations and the resultant enhanced glycolysis under normoxic conditions, i.e., the Warburg effect, are proposed to be involved in tumor development (6-9). Because pathogenic mtDNA mutations also induce mitochondrial respiration defects and up-regulation of aerobic glycolysis, accumulation of these mtDNA mutations with age could also be responsible for tumor development. In addition, it is possible that mtDNA mutations regulate tumor development as a consequence of overproduction of reactive oxygen species (ROS) and the resultant induction of genetic instability (1, 2, 10). In fact, somatic mtDNA mutations are preferentially accumulated in human tumor cells (11-13), and a germline mtDNA mutation (A8344G) in the tRNA Lys gene has been found in lipomas (benign adipose tissue tumors) (14). However, there is as yet no direct evidence for the contribution of mtDNA mutations to aging and tumor development, because of the difficulty of excluding the possible contribution of nuclear DNA mutations, including copy-number variants (CNVs), to these processes (2, 15).Our previous study (16) resolved this issue by using complete mtDNA exchange technology between mouse normal and tumor cells, and we showed that exogenously introduced mtDNA does not affect tumorigenicity. In contrast, our recent study (17) performed exchange of mtDNA between poorly and highly metastatic mouse tumor cells, providing convincing evidence that the somatic mtDNA mutation G13997A in the ND6 gene, which encodes one of the subunits of respiration complex I (NADH dehydrogenase), re...
Accumulation of somatic mutations in mitochondrial DNA (mtDNA) has been proposed to be responsible for human aging and age-associated mitochondrial respiration defects. However, our previous findings suggested an alternative hypothesis of human aging—that epigenetic changes but not mutations regulate age-associated mitochondrial respiration defects, and that epigenetic downregulation of nuclear-coded genes responsible for mitochondrial translation [e.g., glycine C-acetyltransferase (GCAT), serine hydroxymethyltransferase 2 (SHMT2)] is related to age-associated respiration defects. To examine our hypothesis, here we generated mice deficient in Gcat or Shmt2 and investigated whether they have respiration defects and premature aging phenotypes. Gcat-deficient mice showed no macroscopic abnormalities including premature aging phenotypes for up to 9 months after birth. In contrast, Shmt2-deficient mice showed embryonic lethality after 13.5 days post coitum (dpc), and fibroblasts obtained from 12.5-dpc Shmt2-deficient embryos had respiration defects and retardation of cell growth. Because Shmt2 substantially controls production of N-formylmethionine-tRNA (fMet-tRNA) in mitochondria, its suppression would reduce mitochondrial translation, resulting in expression of the respiration defects in fibroblasts from Shmt2-deficient embryos. These findings support our hypothesis that age-associated respiration defects in fibroblasts of elderly humans are caused not by mtDNA mutations but by epigenetic regulation of nuclear genes including SHMT2.
a b s t r a c tTo investigate the effects of respiration defects on the disease phenotypes, we generated trans-mitochondrial mice (mito-mice) by introducing a mutated G13997A mtDNA, which specifically induces respiratory complex I defects and metastatic potentials in mouse tumor cells. First, we obtained ES cells and chimeric mice containing the G13997A mtDNA, and then we generated mito-mice carrying the G13997A mtDNA via its female germ line transmission. The three-month-old mito-mice showed complex I defects and lactate overproduction, but showed no other phenotypes related to mitochondrial diseases or tumor formation, suggesting that aging or additional nuclear abnormalities are required for expression of other phenotypes.
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