Mitochondrial autophagy in cells with mtDNA mutations results from synergistic loss of transmembrane potential and mTORC1 inhibition

Abstract

Autophagy has emerged as a key cellular process for organellar quality control, yet this pathway apparently fails to eliminate mitochondria containing pathogenic mutations in mitochondrial DNA (mtDNA) in patients with a variety of human diseases. In order to explore how mtDNA-mediated mitochondrial dysfunction interacts with endogenous autophagic pathways, we examined autophagic status in a panel of human cytoplasmic hybrid (cybrid) cell lines carrying a variety of pathogenic mtDNA mutations. We found that both genetic- and chemically induced loss of mitochondrial transmembrane potential (Δψ(m)) caused recruitment of the pro-mitophagic factor Parkin to mitochondria. Strikingly, however, the loss of Δψ(m) alone was insufficient to prompt delivery of mitochondria to the autophagosome (mitophagy). We found that mitophagy could be induced following treatment with the mTORC1 inhibitor rapamycin in cybrids carrying either large-scale partial deletions of mtDNA or complete depletion of mtDNA. Further, we found that the level of endogenous Parkin is a crucial determinant of mitophagy. These results suggest a two-hit model, in which the synergistic induction of both (i) mitochondrial recruitment of Parkin following the loss of Δψ(m) and (ii) mTORC1-controlled general macroautophagy is required for mitophagy. It appears that mitophagy can be accomplished by the endogenous autophagic machinery, but requires the full engagement of both of these pathways.

The pathways of mitophagy for quality control and clearance of mitochondria

“…Recently, it has been reported that mitochondria carrying deleterious mutations in mtDNA can be selectively eliminated through mitophagy, 37 although additional activation of macroautophagy, such as rapamycin-mediated inhibition of TORC1, may be required for efficient clearance. 38 Of course, mitochondria with DNA mutations are encountered in human patients 39 and so the clearance mechanism cannot be perfectly efficient; presumably, some threshold of damage must be crossed before mitophagy is likely to occur. Mitophagy also serves as a mechanism to regulate organelle number in response to developmental or physiological cues.…”

p62/SQSTM1 at the interface of aging, autophagy, and disease

“…Interestingly, p62/SQSTM1 may be dispensable for parkin mediated mitophagy, but its presence is necessary to cluster the damaged mitochondria away from the remainder of the mitochondrial network (Narendra et al 2010a) and protect it from further damage. This function becomes critical for cellular homeostasis, especially considering that constitutive autophagy is not particularly efficient in clearing damaged mitochondria and inhibiting mTOR is required for fast removal of depolarized mitochondria (Gilkerson et al 2011). …”

Quality matters: how does mitochondrial network dynamics and quality control impact on mtDNA integrity?

“…The first question is whether a bioenergetic deficiency as the consequence of mtDNA mutations is sufficient to elicit a mitophagy response? In a recent study, using 143B osteosarcoma cybrid cell lines carrying various pathogenic mtDNA mutations, it was shown that loss of DC m alone was, in fact, not enough to stimulate mitophagy, but that it could be induced by co-treatment with the mTORC1 inhibitor rapamycin that stimulates macroautophagy [93]. The cybrids in question were all homoplasmic for their respective mutation.…”

“…Based on these findings, it has been suggested that the sensing of deteriorated mitochondrial function is not sufficient to trigger mitophagy but that activation of macroautophagy is also required [95]. Interestingly, in the study by Gilkerson et al [93], it was shown that loss of DC m did result in recruitment of Parkin but without induction of selective mitophagy. Overall, levels of Parkin in cybrid lines carrying mtDNA mutations or in the mtDNAless parental cell line however were reduced compared with the wt mtDNA cybrid counterpart.…”