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.

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Damaged mitochondria can be eliminated by autophagy, i.e. mitophagy, which is important for cellular homeostasis and cell survival. Despite the fact that a number of factors have been found to be important for mitophagy in mammalian cells, their individual roles in the process had not been clearly defined. Parkin is a ubiquitin-protein isopeptide ligase able to translocate to the mitochondria that are to be removed. We showed here in a chemical hypoxia model of mitophagy induced by an uncoupler, carbonyl cyanide m-chlorophenylhydrazone (CCCP) that Parkin translocation resulted in mitochondrial ubiquitination and p62 recruitment to the mitochondria. Small inhibitory RNAmediated knockdown of p62 significantly diminished mitochondrial recognition by the autophagy machinery and the subsequent elimination. Thus Parkin, ubiquitin, and p62 function in preparing mitochondria for mitophagy, here referred to as mitochondrial priming. However, these molecules were not required for the induction of autophagy machinery. Neither Parkin nor p62 seemed to affect autophagy induction by CCCP. Instead, we found that Nix was required for the autophagy induction. Nix promoted CCCP-induced mitochondrial depolarization and reactive oxygen species generation, which inhibited mTOR signaling and activated autophagy. Nix also contributed to mitochondrial priming by controlling the mitochondrial translocation of Parkin, although reactive oxygen species generation was not involved in this step. Deletion of the C-terminal membrane targeting sequence but not mutations in the BH3 domain disabled Nix for these functions. Our work thus distinguished the molecular events responsible for the different phases of mitophagy and placed Nix upstream of the events.

Autophagy is a cellular response to adverse environment and stress, but its significance in cell survival is not always clear. Here we show that autophagy could be induced in the mammalian cells by chemicals, such as A23187, tunicamycin, thapsigargin, and brefeldin A, that cause endoplasmic reticulum stress. Endoplasmic reticulum stress-induced autophagy is important for clearing polyubiquitinated protein aggregates and for reducing cellular vacuolization in HCT116 colon cancer cells and DU145 prostate cancer cells, thus mitigating endoplasmic reticulum stress and protecting against cell death. In contrast, autophagy induced by the same chemicals does not confer protection in a normal human colon cell line and in the non-transformed murine embryonic fibroblasts but rather contributes to cell death. Thus the impact of autophagy on cell survival during endoplasmic reticulum stress is likely contingent on the status of cells, which could be explored for tumor-specific therapy. Endoplasmic reticulum (ER)3 is critically involved in protein metabolism. Normal ER function is required for the correct folding of many proteins and their post-translational modifications, such as glycosylation and disulfide bond formation. ER stress is induced by the disturbance of the environment in the ER lumen, such as the calcium homeostasis or the redox status, or by the disturbance of ER function, such as glycosylation and transportation to Golgi complex (1). The typical chemicals that induce ER stress include A23187 and thapsigargin, both of which disturb the calcium homeostasis; tunicamycin, which suppresses glycosylation; and brefeldin A, which inhibits transportation to the Golgi complex (1, 2). Thus these chemicals cause protein folding dysfunction, and the accumulated misfolded/unfolded proteins induce ER stress. ER stress is frequently observed in pathological conditions where protein misfolding is caused by genetic mutations either in the molecule to be processed or in the machinery processing the folding (3, 4).The major protective and compensatory mechanism during ER stress is the unfolded protein response (UPR) (1, 5), which leads to translational attenuation and selective up-regulation of a number of bZip transcription factors (1, 5). UPR serves multiple functions, including the assistance of protein folding via the up-regulated ER protein chaperones and the enhanced degradation of misfolded proteins via the up-regulation of molecules involved in the ER-associated degradation pathway (1, 5). However, if the stress is excessive, the compensatory mechanisms may not be able to fully sustain ER function, and ER decompensation could lead to cell death (2, 6). It is not clear whether there are other mechanisms that can regulate ER stress.Macroautophagy (referred as autophagy hereafter) is mainly responsible for the degradation of long-lived proteins and subcellular organelles (7-9). Autophagy is frequently activated in response to adverse environment or stress (10 -13) and has been shown to be involved in many physiological and ...