Mitophagy: mechanisms, pathophysiological roles, and analysis

Abstract

Abstract Mitochondria are essential organelles that regulate cellular energy homeostasis and cell death. The removal of damaged mitochondria through autophagy, a process called mitophagy, is thus critical for maintaining proper cellular functions. Indeed, mitophagy has been recently proposed to play critical roles in terminal differentiation of red blood cells, paternal mitochondrial degradation, neurodegenerative diseases, and ischemia or drug-induced tissue injury. Removal of damaged mitochondria through autophagy requires two steps: induction of general autophagy and priming of damaged mitochondria for selective autophagic recognition. Recent progress in mitophagy studies reveals that mitochondrial priming is mediated either by the Pink1-Parkin signaling pathway or the mitophagic receptors Nix and Bnip3. In this review, we summarize our current knowledge on the mechanisms of mitophagy. We also discuss the pathophysiological roles of mitophagy and current assays used to monitor mitophagy.

Figure 1. Proposed models for mitophagy in mammalian cells

We propose a two-step mitophagy model in mammalian cells: the induction of canonic Atg-dependent macroautophagy and mitochondrial priming. The induction of canonic autophagy requires Atg proteins and furthermore involves mTOR suppression mediated by mitochon-drial damage-generated ROS production and ATP depletion-mediated AMPK activation. Priming of mitochondria is mediated by multiple mechanisms that could be Parkin dependent or Parkin independent. In the presence of Parkin, one common mechanism is that mitochondrial depolarization (e.g., following CCCP treatment) results in impaired PARL-mediated Pink1 cleavage, leading to Pink1 stabilization and Parkin recruitment to the mitochondria. Mitochondria-localized Parkin promotes ubiquitination of outer membrane proteins, which may either be degraded through the proteasome or serve as binding partners for p62. p62 may in turn act as an adaptor molecule through direct interaction with LC3 to recruit autophagosomal membranes to the mitochondria. Parkin can also interact with Ambra1, which in turn activates the PI3K complex around mitochondria to facilitate selective mitophagy. For the Parkin-independent mechanism, damaged mitochondria (particularly under hypoxia conditions) may increase FUNDC1 and Nix expression, which may in turn recruit autophagosomes to mitochondria by direct interaction with LC3 through their LIR domains. Upon mitochondrial depolarization, Smurf1 also targets mitochondria to promote mitophagy, most likely through the ubiquitination of mitochondrial proteins. Hsp90-Cdc37 stabilizes and activates Ulk1, which further phosphorylates Atg13. Phosphorylated Atg13 is recruited to damaged mitochondria to promote mitophagy. Atg-independent mitophagy is less understood, but 15-lipoxygenase has been shown to promote mitochondrial degradation. Direct lysosomal invagination or interaction with damaged mitochon-dria (microautophagy) could also play a role.

Figure 2. EM for mitophagy

Wild-type C57BL/6 mice were treated with acetaminophen (i.p., 500 mg/kg) for 6 h (A, B) or ethanol (gavage, 4.5 g/kg) for 6 h (C, D). Thin liver sections were processed for EM studies. (A and C) An early double-membraned autophagosome envelops mitochondria. (B and D) A late autolysosome contains degrading mitochondria.

Figure 3. Confocal microscopy for the colocalization of autophagosomes with mitochondria

GFP-LC3-expressing MEFs were fi rst loaded with MitoTracker Red (50 nM) for 30 min and then treated with CCCP (30 μM) plus the lyso-somal protease inhibitors E64D (10 μM) and Pepstatin A (10 μM) for 6 h. The cells were fi xed with 4% paraformaldehyde followed by confocal microscopy. Arrows: yellow dots represent colocalized GFP-LC3-positive autophagosomes with red fluorescence-labeled mitochondria.

Figure 4. The expression level of Parkin in different mouse tissues

A 2-month-old Parkin wild-type mouse was sacrifi ced, and different tissues were harvested. Total cell lysates were extracted and subjected to Western blot analysis using an anti-Parkin antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Total liver lysate from a Parkin-knockout mouse was used as a negative control.