Mitochondrial quality control, which is crucial for maintaining cellular homeostasis, has been considered to be achieved exclusively through mitophagy. Here we report an alternative mitochondrial quality control pathway mediated by extracellular mitochondria release. By performing time-lapse confocal imaging on a stable cell line with fluorescent-labeled mitochondria, we observed release of mitochondria from cells into the extracellular space. Correlative light-electron microscopy revealed that majority of the extracellular mitochondria are in free form and, on rare occasions, some are enclosed in membrane-surrounded vesicles. Rotenone-and carbonyl cyanide m-chlorophenylhydrazone-induced mitochondrial quality impairment promotes the extracellular release of depolarized mitochondria. Overexpression of PRKN (parkin RBR E3 ubiquitin protein ligase), which has a pivotal role in mitophagy regulation, suppresses the extracellular mitochondria release under basal and stress condition, whereas its knockdown exacerbates it. Correspondingly, overexpression of PRKNindependent mitophagy regulators, BNIP3 (BCL2 interacting protein 3) and BNIP3L/NIX (BCL2 interacting protein 3 like), suppress extracellular mitochondria release. Autophagy-deficient cell lines show elevated extracellular mitochondria release. These results imply that perturbation of mitophagy pathway prompts mitochondria expulsion. Presence of mitochondrial protein can also be detected in mouse sera. Sera of PRKN-deficient mice contain higher level of mitochondrial protein compared to that of wild-type mice. More importantly, fibroblasts and cerebrospinal fluid samples from Parkinson disease patients carrying loss-of-function PRKN mutations show increased extracellular mitochondria compared to control subjects, providing evidence in a clinical context. Taken together, our findings suggest that extracellular mitochondria release is a comparable yet distinct quality control pathway from conventional mitophagy.

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...

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.

Mitochondrial dysfunction caused by pathogenic mutations in mitochondrial tRNA genes emerges only when mutant mitochondrial DNA (mtDNA) proportions exceed intrinsic pathogenic thresholds; however, little is known about the actual proportions of mutant mtDNA that can affect particular cellular lineage-determining processes. Here, we mainly focused on the effects of mitochondrial respiratory dysfunction caused by m.3243A>G heteroplasmy in MT-TL1 gene on cellular reprogramming. We found that generation of induced pluripotent stem cells (iPSCs) was drastically depressed only by high proportions of mutant mtDNA (≥ 90% m.3243A>G), and these proportions were strongly associated with the degree of induced mitochondrial respiratory dysfunction. Nevertheless, all established iPSCs, even those carrying ∼ 100% m.3243A>G, exhibited an embryonic stem cell-like pluripotent state. Therefore, our findings clearly demonstrate that loss of physiological integrity in mitochondria triggered by mutant mtDNA constitute a roadblock to cellular rejuvenation, but do not affect the maintenance of the pluripotent state.