Mitophagy is an important type of selective autophagy for specific elimination of damaged mitochondria. PTEN-induced putative kinase protein 1 (PINK1)-catalyzed phosphorylation of ubiquitin (Ub) plays a critical role in the onset of PINK1-Parkin-mediated mitophagy. Phosphatase and tensin homolog (PTEN)-long (PTEN-L) is a newly identified isoform of PTEN, with addition of 173 amino acids to its N-terminus. Here we report that PTEN-L is a novel negative regulator of mitophagy via its protein phosphatase activity against phosphorylated ubiquitin. We found that PTEN-L localizes at the outer mitochondrial membrane (OMM) and overexpression of PTEN-L inhibits, whereas deletion of PTEN-L promotes, mitophagy induced by various mitochondria-damaging agents. Mechanistically, PTEN-L is capable of effectively preventing Parkin mitochondrial translocation, reducing Parkin phosphorylation, maintaining its closed inactive conformation, and inhibiting its E3 ligase activity. More importantly, PTEN-L reduces the level of phosphorylated ubiquitin (pSer65-Ub) in vivo, and in vitro phosphatase assay confirms that PTEN-L dephosphorylates pSer65-Ub via its protein phosphatase activity, independently of its lipid phosphatase function. Taken together, our findings demonstrate a novel function of PTEN-L as a protein phosphatase for ubiquitin, which counteracts PINK1-mediated ubiquitin phosphorylation leading to blockage of the feedforward mechanisms in mitophagy induction and eventual suppression of mitophagy. Thus, understanding this novel function of PTEN-L provides a key missing piece in the molecular puzzle controlling mitophagy, a critical process in many important human diseases including neurodegenerative disorders such as Parkinson's disease.

Reactive oxygen species (ROS) and mitophagy are profoundly implicated in the pathogenesis of neurodegenerative diseases, such as Parkinson’s disease (PD). Several studies have suggested that ROS are not involved in mitochondrial translocation of Parkin which primes mitochondria for autophagic elimination. However, whether ROS play a role in the execution of mitophagy is unknown. In the present study, we show that carbonyl cyanide m-chlorophenylhydrazone (CCCP) treatment induced both mitochondrial depolarization and generation of ROS that were needed for the mitophagy process. Cells failed to proceed to complete mitophagy if CCCP treatment was discontinued even after recruitment of Parkin and autophagy machinery to mitochondria. Notably, treatment of pro-oxidant was able to replace CCCP treatment to take mitophagy forward, while it alone was insufficient to induce translocation of Parkin to mitochondria or autophagic clearance of mitochondria. In addition, an SOD mimetic that attenuated the superoxide level suppressed mitophagy, while an SOD inhibitor accumulated cellular superoxide and promoted mitophagy. Furthermore, blockage of the p38 signaling pathway inhibited mitophagy induced by ROS, suggesting that it may contribute to the activation of ROS-mediated mitophagy. Together, our study sheds light on the link between ROS and mitophagy at a molecular level, and suggests the therapeutic potential of regulating mitophagy through the superoxide–p38–mitophagy axis.

Parkin/PINK1-mediated mitophagy is implicated in the pathogenesis of Parkinson's disease (PD). Prior to elimination of damaged mitochondria, Parkin translocates to mitochondria and induces mitochondrial clustering. While the mechanism of PINK1-dependent Parkin redistribution to mitochondria is now becoming clear, the role of mitochondrial clustering has been less well understood. In our study, we found that loss of p62 disrupted mitochondrial aggregation and specifically sensitized Parkin-expressing cells to apoptosis induced by mitochondrial depolarization. Notably, altering mitochondrial aggregation through regulating p62 or other methods was sufficient to affect such apoptosis. Moreover, disruption of mitochondrial aggregation promoted proteasome-dependent degradation of outer mitochondrial membrane (OMM) proteins. The accelerated degradation in turn facilitated cytochrome c release from mitochondria, leading to apoptosis. Together, our study demonstrates a protective role of mitochondrial clustering in mitophagy and helps in understanding how aggregation defends cells against stress.

We apologize for three errors that we just found in the paper published online on 22 June 2018.

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