Mitophagy: mechanisms, pathophysiological roles, and analysis

Mitophagy in yeast

It should be noted that although mitophagy was first observed in rat hepatocytes, the molecular machinery and pathways regulating mitophagy were best documented in yeast. The first evidence that mitophagy is genetically controlled in yeast was reported by Kissova et al. (2004), who identified that Uth1p is required for the autophagic degradation of mitochondria. Uth1p is a member of the so-called SUN family, which was initially found in a genetic screen aiming to identify proteins involved in the regulation of the yeast life span (Austriaco, 1996). Uth1p is mainly localized in the mitochondrial outer membrane and is required for the removal of excessive mitochondria during starvation (Kissova et al., 2004). In addition to Uth1p, it was reported that Aup1p, one of a family of protein phosphatase homologs that localizes to the mitochondrial intermembrane space, is also required for efficient mitophagy in stationary-phase cells (Tal et al., 2007). Interestingly, neither Uth1p nor Aup1p is required for starvation-induced autophagy, suggesting their specificity for mitophagy. Furthermore, deletion of Mdm38, a mitochondrial inner membrane protein that regulates the mitochondrial K⁺/H⁺ exchange system, leads to mitochondrial depolarization and fragmentation followed by degradation in the vacuole (Nowikovsky et al., 2007). Blocking fission by the deletion of a profission gene dnm1 prevented mitochondrial fragmentation and also mitophagy. Thus, it appears that mitochondrial fragmentation facilitates mitophagy, a phenomenon that is also observed in mammalian cells (see below). Because there were no externally added drugs or physiological stresses placed on the mdm38Δ cells, this study also suggests that damaged mitochondria themselves could be sufficient to trigger mitophagy. It should also be noted that deletion of Mdm38 leads to mitochondrial depolarization followed by mitophagy. However, whether depolarization is truly required for mitophagy may require further investigation, as treatment of a mitochondria uncoupler, carbonyl cyanide m-chlorophenylhydrazone (CCCP), did not induce mitophagy in wild-type yeast (Wang and Klionsky, 2011).

More recently, two independent groups using genome-wide screening reported that Atg32 is a mitochondrial receptor for mitophagy in the yeast (Kanki et al., 2009; Okamoto et al., 2009). Similar to Uth1p and Aup1p, Atg32 is necessary for mitophagy but not for non-specific autophagy. Atg32 is a 59-kDa protein that has an intramitochondrial domain at its C terminus and a tetrapeptide sequence WQAI in its cytoplasmic domain, which is critical for its interaction with Atg8. The tetrapeptide sequence WXXL is also known to be present in several other Atg8- or LC3-binding partners important for selective autophagy. Atg32 also binds to Atg11, a selective autophagy receptor important for cargo selection (Yorimitsu and Klionsky, 2005) and autophagosome formation by acting as a scaffold protein, which further recruits other autophagy proteins to form the autophagosome. Indeed, these studies found that Atg11 is also required for mitophagy (Kanki et al., 2009; Okamoto et al., 2009). In contrast, Atg19, which is specifically involved in the Cvt pathway, is not required for mitophagy.

It was found that Atg32 is phosphorylated after nitrogen starvation, and the phosphorylation of Atg32 is required for mitophagy (Aoki et al., 2011). Phosphorylation of Atg32 promotes Atg32-Atg11 interaction but does not affect Atg32-Atg8 interaction (Aoki et al., 2011). In yeast, Hog1 is a mitogen-activated protein kinase (MAPK), whose activation is dependent on its phosphorylation by Pbs2, an MAPK kinase. Deletion of either Hog1 or Pbs2 leads to inhibition of Atg32 phosphorylation as well as mitophagy. Unlike Hog1 and Pbs2, which function specifically for mitophagy, Slt2, another MAPK kinase involved in the protein kinase C-cell wall integrity kinase signaling pathway in yeast, is required for the degradation of both mitochondria and peroxisomes (Mao et al., 2011). Slt2, but not Hog1, affects the recruitment of mitochondria to the phagophore assembly site, a critical step in the packaging of cargo for selective degradation (Mao et al., 2011).

To note, the expression of Atg32 is increased in respiratory growth conditions (not fermentable conditions), and N-acetylcysteine (NAC), a precursor of the antioxidant glutathione and a scavenger for free radicals, suppresses Atg32 expression and mitophagy (Okamoto et al., 2009). However, it is not clear whether Atg32-dependent mitophagy is directly mediated by oxidative stress under respiratory conditions because reactive oxygen species (ROS) levels were not determined in this study (Okamoto et al., 2009). Interestingly, it was reported in another study that NAC suppresses mitophagy by fueling the cellular glutathione (GSH) pool rather than scavenging for free radicals (Deffieu et al., 2009). Deprivation of nitrogen in yeast was associated with an early decrease of GSH before mitophagy. How exactly GSH can modulate mitophagy in yeast is not clear, but it is possible a decreased GSH level may alter mitochondrial function and subsequently lead to mitochondrial damage and mitophagy. Moreover, it is still unknown what increased the expression of Atg32 under respiratory conditions. It would be interesting to determine whether there is also a depletion of GSH levels under respiratory conditions or whether other antioxidants in addition to the GSH precursor NAC would also suppress Atg32 expression and mitophagy.

Mitophagy in mammalian cells

Mammalian homologues of Atg32, Uth1p, and Aup1p have not been found. In addition, mammalian mitophagy presents some unique features that may also be specific to mammals. The molecular mechanisms and signaling pathways behind mitophagy regulation only begin to be revealed in mammalian cells. In general, it seems that multiple molecules could be involved and there are both autophagy-dependent and -independent pathways that regulate mitochondrial homeostasis. The possible mitophagy pathways are summarized in Figure 1 and discussed in the following sections.

Mitochondrial fragmentation

Mitochondria are dynamic organelles constantly undergoing fusion and fission (Westermann, 2010). Mitochondrial fusion is mediated by the profusion genes mitofusin (Mfn) 1 and 2, and optic atrophy 1 (OPA1). Mfn 1 and 2 are large GTPases located on the outer mitochondrial membrane, whereas OPA1 is a dynamin-related GTPase that is essential for inner membrane fusion. Mammalian OPA1 has eight isoforms that are generated by alternative splicing and alternative processing at two cleavage sites located between the N-terminal transmembrane domain and the first heptad repeat. It has been suggested that the rhomboid-related protease, presenilins-associated rhomboid-like (PARL) (Cipolat et al., 2006), or the AAA protease in the matrix, paraplegin, may be responsible for OPA1 processing (Ishihara et al., 2006). However, their roles in regulating OPA1 processing have been challenged because normal OPA1 processing was found in mouse embryonic fibroblasts (MEFs) lacking either PARL or paraplegin (Duvezin-Caubet et al., 2007). OPA1 isoforms are constitutively cleaved by the intermembrane space AAA protease Yme1 to generate the short and long forms of OPA1 (S- and L-OPA1) under normal conditions (Griparic et al., 2007). When cells were treated with CCCP, a mitochondria uncoupler that depolarizes mitochondria, L-OPA1 was further cleaved by an inducible protease OMA1, which is a metalloprotease with a zinc-binding motif. This cleavage resulted in mitochondrial fragmentation by preventing mitochondrial fusion (Ehses et al., 2009; Head et al., 2009).

Similar to mitochondrial fusion, mitochondrial fission requires a dynamin-related GTPase (Drp1) in mammals. Drp1 is a cytosolic protein, but it can be recruited to the surface of mitochondria where it interacts with a partner protein, mitochondrial fission 1 (Fis1), through the adaptor proteins Mdv1 and Caf4 to trigger mitochondrial fission in yeast (Griffin et al., 2005; Westermann, 2010). The mammalian homologue of yeast Fis1 has been identified, which suggests an evolutionarily conserved mechanism of mitochondrial fission (Smirnova et al., 2001; Yoon and Koob, 2003); however, homologues of Mdv1 and Caf4 have not yet been determined. More recent evidence suggests that mitochondria fission factor, a tail-anchored mitochondrial outer membrane protein that physically interacts with Drp1 but not Fis1, is an essential factor for Drp1-mediated mitochondria fission in mammalian cells (Gandre-Babbe and van der Bliek 2008; Otera et al., 2010). Moreover, mitochondria and endoplasmic reticulum are often tightly associated and in physical contact. A recent study reported that mitochondrial fission dynamics, such as Drp1, are localized at the endoplasmic reticulummitochondria contact sites and that the endoplasmic reticulum may play a direct role in the process of mitochondrial fission (Friedman et al., 2011).

It has been suggested that fragmented mitochondria are more readily taken up by autophagosomes due to their smaller size. In nitric oxide-treated cultured cortical neurons, Fis1 was found to be involved in mitophagy (Barsoum et al., 2006). Gomes and Scorrano (2008) found that overexpression of Fis1 itself can induce general autophagy in HeLa cells as demonstrated by increased YFP-LC3 puncta and p62 degradation. Overexpressing a dominant-negative form of Fis1, which had ablated the first α-helix, induced autophagy and large mitochondrial structures. Moreover, a conserved mutation in Fis1 (hFis1^K148R) induced mitochondrial fission but was unable to induce mitochondrial dysfunction and did not induce autophagy (Gomes and Scorrano, 2008). These observations suggest that mitochondrial dysfunction, rather than mitochondrial fragmentation, is responsible for the induction of autophagy. Two independent groups recently reported that during starvation, cellular cyclic AMP levels increase and protein kinase A is activated, which in turn phosphorylates Drp1 to prevent its translocation to mitochondria (Gomes et al., 2011; Rambold et al., 2011). As a result, mitochondria are highly elongated in these starved cells. More importantly, elongated mitochondria are spared from autophagic degradation, further supporting the notion that mitochondrial fragmentation can facilitate mitophagy. Using a photo-labeling approach, Twig et al. (2008) found that mitochondria went through constant cycles of fusion and fission, and fission events generated two subsets of daughter mitochondria with either increased or decreased membrane potential. They further found that the daughter mitochondria with higher membrane potential would proceed to fusion, whereas the depolarized daughter mitochondria were unable to proceed to the fusion process and were removed by mitophagy. Overexpression of OPA1 led to increased mitochondria fusion and decreased mitophagy. Therefore, it seems that mitochondrial fission is essential for mitophagy.

Pathophysiological role of mitophagy

As mitochondria are essential organelles that regulate cellular energy metabolism and cell death, mitochondrial homeostasis has been linked to many pathophysiological conditions and diseases.

Development

As discussed above, mitophagy plays an essential role in erythrocyte differentiation and maturation. Both the autophagy machinery (such as Atg7 and ULK1) and mitochondrial recognition signals for mitophagy (such as Nix) are important in this physiological process. As mentioned, autophagy-independent pathways are likely also involved in the elimination of mitochondria in this process (Schweers et al., 2007; Sandoval et al., 2008).

Perhaps the most intriguing is the role of mitophagy in the destruction of paternal mitochondria in the fertilized oocytes. In almost all eukaryotes, paternal mtDNA and mitochondria themselves are selectively eliminated or degraded from the embryonic cytoplasm; thus, mitochondrial genes are inherited mainly from the maternal parent (Ankel-Simons and Cummins, 1996). In mammals, the ubiquitin proteasome system has been suggested to specifically degrade paternal spermborne mitochondria (which are ubiquitin positive) after they enter the ooplasm during fertilization (Sutovsky et al., 2004). Prohibitin, an inner mitochondrial membrane protein, was further identified as one of the ubiquitinated substrates that make sperm mitochondria recognizable by the ubiquitin-proteasome system in the egg, and likely facilitates sperm mitochondrial degradation after fertilization (Thompson et al., 2003). The expression level of Parkin in the sperm is not known. It would be interesting to see whether the Pink1-Parkin pathway or other E3 ligases such as SMURF1 would be involved in such a process. It would also be interesting to see whether Nix plays a role during the selective removal of paternal mitochondria in mammals.

Several reports now suggest that autophagy or the lysosome is required for the elimination of paternal mitochondria in Caenorhabditis elegans (Al Rawi et al., 2011; Sato and Sato, 2011; Zhou et al., 2011a). In lgg1 (a homologue of yeast Atg8)-deficient zygotes, paternal mitochondria and other membrane structures remain in the first larval stage. In fertilized mouse embryos, many autophagy markers such as LC3, GABRAP, and p62 are found closely associated with paternal ubiquitin-positive mitochondria. Ubiquitin has been suggested to serve as an ‘eat-me tag’ for the selective autophagy of various cellular cargos, including protein aggregates, damaged or excessive organelles, or invading microbes. Strikingly, although sperm mitochondria are often found to be ubiquitin positive, in C. elegans, the paternal mitochondria are not ubiquitinated. Instead, there are many vesicular structures called membranous organelles (MOs) that are ubiquitinated and degraded together with the paternal mitochondria by autophagy (Al Rawi et al., 2011). Therefore, it was suggested that paternal mitochondria are incorporated into autophagosomes owing to their close association with the MOs. It is also possible that the paternal mitochondria in C. elegans may use other tags for selective removal through autophagy in addition to ubiquitin. Collectively, although mitophagy seems to play a role in development, more work is needed in the future to further support such a notion.

Aging

It has been known that autophagy declines during aging, and the expression levels of many autophagy genes or related proteins (such as Atg7, Atg5, Atg4B, and LAMP-2) decreases in the brains and liver of aging humans and mice (Zhang and Cuervo, 2008; Lipinski et al., 2010; Wang et al., 2011). Conditions that activate autophagy, such as caloric restriction, have shown beneficial effects on delaying the aging-related degeneration process. Stimulation of autophagy has been shown to extend the life span in multiple organisms, including yeast, C. elegans, mice, and primates (Kissova et al., 2004; Alberti et al., 2010; Madeo et al., 2010). For example, in yeast, deletion of the mitochondrial membrane protein Uth1p leads to a selective defect in mitophagy and a decreased lifespan (Kissova et al., 2004). In C. elegans, worms that carry the loss-of-function mutation in the insulin-like signaling pathway (daf-2) display extended lifespans and induction of autophagy (Melendez et al., 2003). Although the exact mechanisms by which autophagy may delay aging are not clear, it has been suggested that mitophagy may help reduce the production of mitochondria-derived ROS and remove dysfunctional mitochondria that would generate mtDNA mutations during aging (Lemasters, 2005).

Cancer

Autophagy can act as a tumor suppressor, and loss of autophagy promotes tumorigenesis. An essential autophagy gene, Beclin 1, was frequently found monoallelically deleted in many human cancers such as breast, prostate, and ovarian cancers (Aita et al., 1999). Mice with allelic loss of Beclin 1 are prone to hepatocellular carcinoma, lung adenocarcinoma, mammary hyperplasia, and lymphoma. Many known tumor suppressor genes such as Lkt, Ampk, and Pten are positive regulators of autophagy (Cully et al., 2006; Liang et al., 2007; Degtyarev et al., 2008; Hezel and Bardeesy, 2008), whereas many oncogenes, including the class I PI3K, Akt, and anti-apoptotic Bcl-2 family proteins, suppress autophagy (Maiuri et al., 2009), further supporting the notion that autophagy suppresses tumorigenesis. Mice that have liver-specific loss of Atg7 or Atg5 have accumulated damaged mitochondria and develop liver injury, steatophepatitis, and adenocarcinoma (Inami et al., 2011; Takamura et al., 2011). However, it is likely that autophagy has multiple roles in acting as a tumor suppressor in addition to removal of damaged mitochondria. For example, autophagy may act to buffer metabolic stress caused by limited nutrients or oxygen in the tumor tissues (Rosenfeldt and Ryan, 2011). Future works are needed to further determine the exact role of mitophagy in tumorigenesis.

Neurodegenerative diseases

As discussed above, some familial forms of Parkinson’s disease are provoked by mutations in Pink1 or Parkin, some of which can result in defects in mitophagy. In addition, mitophagy may also play a role in other neurodegenerative diseases including Alzheimer’s disease (AD) and Huntington’s disease (HD). β-Amyloid fragments can target mitochondria and cause mitochondrial dysfunction in AD (Casley et al., 2002). Indeed, there is evidence that mitochondrial cytochrome oxidase is defective in AD (Maurer et al., 2000). Mitochondrial dysfunction has also been associated with HD due to the dysregulation of PGC1-α, an important transcription factor for mitochondrial biogenesis (Cui et al., 2006). However, the mechanisms and the role of mitophagy in AD and HD are unclear and need to be further studied.

Innate immunity

Accumulating evidence now support the idea that autophagy is closely associated with innate immunity. Autophagy plays an important role in defending against invading intra-cellular microbes, including Mycobacterium tuberculosis, Salmonella, Listeria, Shigella, HIV-1, and Sindbis viruses (Deretic, 2012). Autophagy can be regulated by the DAMP molecules such as HMGB1 and IL-1β; Toll-like receptors; Nod-like receptors, including NLRC4, NLRP3, and NLRP4; and RIG-I-like receptors (Deretic, 2012). Other innate immune signaling molecules such as TBK-1 and IKKα/β are also associated with autophagy (Deretic, 2012; Weidberg and Elazar, 2011). Recent evidence suggests that suppression of mitophagy/autophagy leads to the accumulation of damaged, ROS-generating mitochondria, and this in turn activates the NLRP3 inflammasome (Zhou et al., 2011b). In addition to ROS, depletion of LC3B or Beclin 1 promotes the accumulation of dysfunctional mitochondria and cytosolic translocation of mtDNA in macrophages. Cytosolic mtDNA promotes the secretion of IL-1β and IL-18 following stimulation with lipopolysaccharide or ATP (Nakahira et al., 2011). These studies suggest that mitophagy can regulate NALP3-dependent inflammation by preserving mitochondrial integrity.

Tissue injury

It has been well known that the mitochondrion is a central executioner for regulating cell death (Ding and Yin , 2004 ; Green et al., 2011). In response to apoptotic stimuli, apoptotic proteins target mitochondria to cause mitochondrial swelling or membranepermeability changes that result in the release of apoptotic factors such as cytochrome c and Smac. In addition to apoptosis, necrotic stimuli can also damage mitochondria, resulting in the depletion of cellular ATP levels. Moreover, mitochondria are also the major source of ROS that promote cellular damage. In ischemia-reperfusion-induced heart injury, Parkin-mediated mitophagy has a protective role against the cell death of cardiomyocytes (Huang et al., 2011). In contrast, TIGAR (TP53-induced glycolysis and apoptosis regulator), which was exclusively upregulated in ischemic myocardium, suppresses mitophagy and exacerbates cardiac damage after ischemia (Hoshino et al. , 2012). Acetaminophen overdose, one of the most prevalent poisonings worldwide, induces liver damage that features mitochondrial damage-mediated hepatic necrosis (Jaeschke and Bajt , 2006 ). We recently found that mitophagy plays a critical role against acetaminophen-induced liver damage by reducing ROS production (Ni et al., 2012). In addition to regulating cell death, mitochondria also play an important role in regulating lipid homeostasis by burning fat through fatty acid β-oxidation. Decreased autophagic activity is found in the livers of obese mice, and genetic deletion of Atg7 or pharmacological inhibition of autophagy is associated with hepatic steatosis (Singh et al., 2009; Yang et al., 2010; Mei et al., 2011). We also found that induction of autophagy leads to reduced alcohol-induced hepatic steatosis (Ding et al. , 2010a). Although autophagy may selectively remove lipid droplets (lipophagy), it is possible that removal of damaged mitochondria may also contribute to the attenuation of steatosis. Therefore, targeting damaged mitochondria by activating mitophagy may be a novel approach for treating damaged mitochondria-mediated tissue injury in the future.

Analysis of mitophagy

Although mitophagy was first studied in mammalian cells, reliable quantitative methods for specifically monitoring mitophagy in mammalian cells are not yet available. In contrast, several reliable quantitative mitophagy assays have been developed for yeast. Below, we will discuss the current techniques that have been used to monitor mitophagy in both yeast and mammalian cells.

Electron microscopy

Transmission electron microscopy (TEM) is still one of the best approaches to provide direct evidence for mitophagy since autophagy was first detected by TEM in the 1950s. The morphological hallmark of autophagy is the formation of double-membrane autophagosomes that contain cytoplasmic material and/or organelles at various stages of degradation. The autophagosomes eventually fuse with the lysosomes to form matured autolysosomes, which usually have only one limiting membrane. The early stage of mitophagy (autophagosome with engulfed mitochondria) can be readily detected through identification of unique mitochondrial structures such as the cristae (Figure 2A and C). The late stage of mitophagy may be reflected by the single-membrane autolysosomes with residue mitochondria (Figure 2B and D), on the basis of their similar electron density with mitochondria. It would be better to perform immuno-EM for specific mitochondrial markers, such as Tom20 or CypD, to confirm the nature of mitochondria in the late stage of mitophagy.

Fluorescence microscopy for mitochondria-autophagosome or mitochondria-lysosome colocalization

Two different approaches can be used to determine mitochondria-autophagosome or mitochondria-lysosome colocalization depending on whether the cell being imaged is live or fixed. For live-cell imaging microscopy, MitoTracker, but not membrane potential sensitive dyes such as tetramethylrhodamine, methyl ester (TMRM) dye, should be chosen. MitoTracker is taken up electrophoretically by mitochondria, dependent on the mitochondrial membrane potential. Unlike TMRM, it covalently binds to mitochondrial proteins and thus remains in mitochondria even if they subsequently depolarize (Elmore et al., 2001). By using a GFP-LC3 and MitoTracker Red cola-beling approach, one could examine the relation between mitochondria and the GFP-LC3-positive autophagosomal structures. For fixed cells, in addition to MitoTracker staining, mitochondria can also be visualized using immunostaining for mitochondrial proteins with antibodies against mitochondrial proteins such as Tom20, VDAC, or the complex IV subunit. Quantification of the colocalization of mitochondria and autophagosomes provide an indication of the degree of sequestration and could function as a marker for mitophagy. Similar to autophagic flux assay, the number of mitochondria and GFP-LC3 colocalized puncta would also be increased in the presence of lysosomal inhibitors such as chloroquine or E64 D plus pepstatin A (Figure 3).

Because autophagosomes eventually need to fuse with lysosomes, the colocalization of mitochondria with lysosomal markers could alternatively be used to monitor mitophagy. For live-cell imaging microscopy, MitoTracker-stained mitochondria and LysoTracker-stained lysosomes may be used to visualize the process. However, it should be cautioned that LysoTracker will stain all the acidic compartments present and is therefore not specific for autolysosomes. Alternatively, fixed cells can be stained using antibodies specific for mitochondrial proteins and an antibody against the lysosomal-associated membrane protein 1 (LAMP-1) or LAMP-2. The successful fusion of mitochondria-containing autophagosome with the lysosomal structures can be quantified by analyzing the colocalization signals as an indicator for mitophagy.

Mitochondrial mass

Analyzing the mitochondrial mass has also been used as a quantitative way to monitor the last step of the degradation process of mitophagy. Mitochondrial mass can be measured by a fluorescence-activated cell sorting technique using MitoTracker staining. This technique has been successfully used to monitor mitochondrial elimination during red blood cell maturation (Zhang et al., 2009). Immunostaining for mitochondrial proteins using specific antibodies has also been applied to monitor mitochondrial mass. For example, immunostaining for Tom20 (an outer mitochondrial membrane protein) in CCCP-treated Parkin-positive cells has initially been used as a marker for the loss of mitochondria resulting from mitophagy. However, more recent studies suggest that most outer mitochondrial membrane proteins are degraded by the ubiquitin proteasome system, whereas the matrix proteins are degraded through autophagy, although the exact mechanisms and reasons why different mitochondrial proteins are degraded through different mechanisms are not clear (Yoshii et al., 2011). Therefore, immunostaining for mitochondrial matrix proteins such as CypD and HSP60 would be more appropriate for monitoring mitophagy. In addition, quantitative PCR can be used to quantify mtDNA, such as 16S rRNA, and nuclear DNA, such as hexokinase 2. The mitochondrial-to-nuclear DNA ratio in each sample can consequently be calculated by dividing the value of 16S rRNA by that of hexokinase (Lagouge et al., 2006).

Immunoblot for mitochondrial proteins

Western blot analysis for mitochondrial proteins is another way to quantify mitochondrial mass. However, as discussed above, this should be conducted to cover all mitochondrial compartment proteins, including outer and inner mitochondrial proteins, intermembrane space, and matrix proteins. Only monitoring outer mitochondrial membrane proteins such as Tom20, VDAC, and Mfn 1/2 could be misleading because these proteins could be preferentially degraded by the proteasome. Western blot analysis for mitochondrial matrix proteins would be more appropriate and specific for mitophagy.

Om45-GFP processing assay

To quantitatively detect mitophagy in the yeast, Kanki et al. (2009) developed the Om45-GFP processing assay. Om45 is a mitochondrial outer membrane protein, and a GFP-tagged Om45 (GFP at the C terminus of Om45) is correctly localized on this organelle. When mitophagy is induced, mitochondria, along with Om45-GFP, are delivered into the vacuole for degradation. Om45 is proteolytically removed or degraded, whereas the GFP moiety is relatively stable and accumulates in the vacuole. Thus, mitophagy can be monitored and quantified according to the appearance and intensity of free GFP by immunoblot. A similar approach was used in another study in which yeast cells were expressing a mitochondria-targeted dehydrofolate reductase-GFP (Okamoto et al., 2009). However, the free GFP fragments could be difficult to detect in mammalian cells because lysosomal pH is more acidic in mammalian cells than in yeast vacuoles (Ni et al., 2011). Therefore, it remains to be determined whether a similar GFP-tagged mitochondrial protein-processing assay would work in mammalian cells. In contrast to using Western blot analysis for GFP fragments, Yoshii et al. (2011) developed a flow cytometry-based assay using cell lines that are stably expressing GFP proteins fused to either the mitochondrial matrix (subunit 9 of F₀-ATPase, Su9-GFP) or an outer mitochondrial protein (GFP-Omp25). They found that the Su9-GFP and GFP-Omp25 signals were largely unaffected in Parkin-untransfected MEFs but significantly decreased in Parkin-transfected wild-type MEFs in a time-dependent manner following CCCP treatment. In either FIP200 KO or Atg5 KO MEFs, CCCP-induced decreases in Su9-GFP but not GFP-omp25 signals were markedly inhibited (Yoshii et al., 2011). These data confirmed the notion discussed above that degradation of matrix proteins but not outer mitochondrial membrane proteins relies on autophagy and also provides a novel reliable quantitative approach to monitor mitophagy in mammalian cells.

Citrate synthase activity assay

As an important metabolic power house, mitochondria contain many important enzymes for the citric acid cycle and oxidative phosphorylation. The decrease in mitochondrial content over time due to mitophagy can be measured by analyzing the activities of some mitochondrial enzymes. For example, citrate synthase, a citric acid cycle enzyme, has been used to measure mitochondrial mass in cells and tissue (Watts et al. , 2004 ; Hargreaves et al. , 2007). Disruption of mitochondrial functions such as the inhibition of the electron transport chain or the depletion of mtDNA does not generally affect citrate synthase activity. Therefore, changes of citrate synthase activity are good indicators for mitochondrial content (Watts et al., 2004 ; Hargreaves et al. , 2007 ). In Parkin-overexpressing SH-SY5Y cells, treatment with CCCP induced mitophagy and resulted in decreased mitochondrial mass and citrate synthase activity. The levels of the decreased mitochondrial proteins were strongly correlated with citrate synthase activity (Gegg et al., 2010).

Future perspectives

Although there have been significant advances recently in our knowledge of selective mitophagy, many questions remain unanswered. The Pink1-Parkin signaling pathways play critical roles in mitophagy in cells/tissues that have high expression levels of Parkin, such as in the mouse brain, thymus, kidney, and muscle (Figure 4 and Table 1). However, cells that have low or undetectable levels of Parkin (Table 1) may utilize Parkin-independent mitophagy pathways. In addition to hypoxia-induced Parkin-independent mitophagy, what are the other Parkin-independent mitophagy pathways? How do the ubiquitin proteasome system and the autophagy machinery complete the degradation of mitochondrial proteins in coordination? Is there a mitochondrial clearance mechanism independent of the canonic autophagic process that requires the Atg genes? Finally, reliable quantitative assays to monitor mitophagy still need to be developed. Answering these questions will not only improve our knowledge regarding general mitochondrial homeostasis, but it may also reveal new avenues for treatment of diseases with dysfunctional mitochondria.