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AUTOPHAGY IN HEALTH AND DISEASE: V. Mitophagy as a Way of Life1Roberta A. Gottlieb, Raquel S. Carreira2

34

Abstract56

Our understanding of autophagy has expanded greatly in recent years, largely due to the identification of the7many genes involved in the process, and to the development of better methods to monitor the process, such8as GFP-LC3 to visualize autophagosomes in vivo. A number of groups have demonstrated a tight connection9between autophagy and mitochondrial turnover. Mitochondrial quality control is the process whereby10mitochondria undergo successive rounds of fusion and fission with a dynamic exchange of components in11order to segregate functional and damaged elements. Removal of the mitochondrion that contains damaged12components is accomplished via autophagy (mitophagy). Mitophagy also serves to eliminate the subset of13mitochondria producing the most reactive oxygen species, and episodic removal of mitochondria will reduce14the oxidative burden, thus linking the mitochondrial free radical theory of aging with longevity achieved through15caloric restriction. Mitophagy must be balanced by biogenesis to meet tissue energy needs, but the system is16tunable and highly dynamic. This process is of greatest importance in long-lived cells such as cardiomyocytes,17neurons, and memory T cells. Autophagy is known to decrease with age, and the failure to maintain18mitochondrial quality control through mitophagy may explain why the heart, brain, and components of the19immune system are most vulnerable to dysfunction as organisms age.20

21Autophagy machinery and regulation22

23Many reviews have covered the details of autophagy in recent years (32, 72); the key elements are depicted in24Fig. 1. For the purpose of this discussion it is important to recognize that there are a growing number of25adaptor proteins that serve to recruit the autophagosome to its target. For instance, p62 binds to ubiquitinated26proteins and links autophagosome membranes through an interaction with LC3 (44). Ubiquitin ligases27restricted to specific subcellular locations, such as MULAN and Parkin on mitochondria, may serve to target28select organelles for autophagic destruction, although whether this is a selective process, and how selection is29accomplished, remain to be elucidated (28, 58). Chaperone-mediated autophagy, cytoplasm to vacuole30targeting, and macroautophagy are examples of selective forms of autophagy. Macroautophagy (hereafter31

referred to as autophagy) includes the selective elimination of mitochondria (mitophagy), endoplasmic32reticulum (reticulophagy), peroxisomes (pexophagy), ribosomes (ribophagy), granules (crinophagy), and33pathogens (xenophagy) (1). Cytosol, cytoskeleton, nuclei (nucleophagy), and protein aggregates (aggrephagy)34can also be removed by autophagy (85)35

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can also be removed by autophagy (85).35

binding domain appears to be necessary for autophagy (5). Given the importance of Bcl-2 family members in57regulating mitochondrial integrity, it seems reasonable to hypothesize that Bcl-2 proteins might also govern58selective mitophagy.59

60Mitochondria and their dark side61

62While mitochondria perform essential functions for the cell, notably ATP production via oxidative63phosphorylation, heme biosynthesis, and calcium homeostasis, their continued existence within the cell may64sometimes seem like a pact with the devil. Damage to the mitochondrial outer membrane leads to release of65

cytochrome c, triggering caspase activation and apoptosis. More catastrophic stresses can lead to pathologic66 opening of the mitochondrial permeability transition pore (MPTP), accompanied by transient but massive67release of ROS and calcium (4, 112). This can trigger neighboring mitochondria to do the same, culminating in68activation of calcium-dependent proteases (calpains) and lipases (cPLA2), as well as ROS-activated iPLA2,69which together ensure the necrotic destruction of the cell (64). However, even when mitochondria are70functionally normal, 1-2% of the oxygen they consume is converted to superoxide and then to hydrogen71peroxide. Akin to cars that emit smog even when idling, mitochondria produce small amounts of superoxide72even when ATP production is minimal. Damaged but still functional mitochondria might release up to ten-fold73more hydrogen peroxide (30). In a mitochondria-rich, metabolically active organ like the heart, this will impose74a substantial oxidative burden over time. Given the mitochondrial half-life of two weeks and a cardiomyocyte75lifespan of many decades, the organism is confronted with a sizable challenge to deal with the cumulative76

oxidative damage in its longest-lived tissues. Oxidative stress will cause DNA damage, and mitochondrial77 DNA repair enzymes are less efficient (55). A defective version of mitochondrial DNA polymerase gamma78(involved in synthesis and repair) introduces a high frequency of mutations in the mitochondrial genome. A79mouse model expressing the mutant polymerase gives rise to a phenotype of accelerated aging (104).80

81Mitophagy as a general feature of autophagy82

83Mitochondrial integrity is essential to cellular homeostasis. If the energetic demand is low, excess mitochondria84are unnecessary and may generate excessive reactive oxygen species. If they become uncoupled, they can85actually consume ATP (106). Therefore, their elimination by autophagy is an efficient cytoprotective response.86Furthermore, damaged or unstable mitochondria may release ROS, cytochrome c, AIF, SMAC/DIABLO, and87

other apoptosis-promoting factors which would promote damage to neighboring mitochondria and the entire88 cell (15). In this setting, one can imagine that elimination of a few unstable and potentially dangerous89mitochondria by autophagy will preserve the integrity of the remaining population and maintain cellular90homeostasis. In fact, autophagic removal of mitochondria has been shown to occur following both induction91

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mitochondrial outer membrane protein necessary for the selective autophagic degradation of mitochondria 13Atg32. Atg32 mutant cells are defective in mitophagy, but competent in bulk autophagy and the Cvt pathway,14which indicates that Atg32 is specific for mitophagy (47, 75). Atg32 serves as a mitochondrial surface receptor15and binds to Atg11 and Atg8 to direct selective mitophagy . The mammalian homologs for Atg32 and UTH116have not been identified. The selective removal of mitochondria also appears to occur in mammals, mediated17by the mitochondria permeability transition pore (8, 23, 87, 101) and apoptotic proteins, such as Bnip3 (Bcl-218and adenovirus E1B 19 kDa-interacting protein 3) and NIX (Bnip3-like protein X) (73, 89, 91, 110).19

20The mitochondrial permeability transition pore (MPTP)21

22 The pathological role of the MPTP is well known [for review see (33, 36, 84)]. Opening of the MPTP (this23phenomenon is termed the mitochondrial permeability transition or MPT) is a common response to ischemia-24reperfusion injury, particularly to stresses such as ROS and calcium overload. MPT makes the inner25mitochondrial membrane permeable to solutes of up to 1,500 Da (2), resulting in depolarization due to26dissipation of the electrochemical gradient, which in turn causes ATP synthase to operate in reverse,27consuming ATP (106). However, recent evidence suggests a physiological role for the MPTP in: i) calcium28homeostasis (2, 43); ii) cardioprotection against ischemia-reperfusion (IR) injury provided by ischemic and29pharmacological preconditioning and postconditioning (39, 59); and iii) mitochondrial removal by autophagy (23,3087, 101). Mitochondria are known to be degraded by the autophagosomal-lysosomal pathway, but the basis on31which individual mitochondria are selected for autophagy is unknown. Lemasters group (23, 87) demonstrated32

that induction of autophagy in rat hepatocytes by serum deprivation and glucagon causes an increase of33 spontaneously depolarizing mitochondria, and these mitochondria are sequestered by autophagosomes. The34authors concluded that the MPTP is responsible for mitochondrial depolarization and that it leads to35mitochondrial sequestration into autophagosomes. Similar results were obtained using nicotinamide-treated36human fibroblasts (45). Nicotinamide-activated autophagy selectively removes mitochondria with low37membrane potential, which is attenuated by treatment with cyclosporin A (45). Twig and colleagues (101)38showed that in pancreatic beta cells, fission generates asymmetric daughter mitochondria: one subpopulation39has increased membrane potential and high probability of fusion, while the other has decreased potential and40reduced probability of fusion (101). Dysfunctional mitochondria are excluded from subsequent rounds of fission41and fusion, and eventually are removed by autophagy. The authors argued against the MPTP as the cause of42the depolarization because the depolarization was observed even in the presence of 1 M cyclosporin A.43

44 In cardiac cells, starvation-induced autophagy has been shown to cause mitochondrial depolarization which is45prevented by cyclosporin A (8), indicating the MPTP is involved in starvation-induced mitochondrial46depolarization. We have also shown that cyclophilin D, a component of the MPTP , is required for cardiac47

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remodeling and large amplitude swelling of the inner membrane, which leads to disassembly of OPA169complexes and release from the mitochondria (80). Given the wide array of consequences of Bnip3 activation,70it may induce autophagy by several mechanisms, including via mitochondrial depolarization and opening of the71MPTP or through interference with the fusion-fission machinery.72

73NIX is required for programmed mitophagy during reticulocyte maturation (89, 91, 110). The maturation74process includes elimination of membrane bound organelles, such as mitochondria (31, 52). Mitochondria are75cleared from reticulocytes through an autophagy-related process with the major difference that the contents of76the autophagic vacuole are not recycled but are eliminated by exocytosis (31, 40). Novak et al. (73) showed77

that in murine reticulocytes, Nix functions as a selective autophagy receptor that binds to LC3/GABARAP78 proteins. Autophagy machinery is still functional in Nix-deficient reticulocytes, but mitochondria are not79engulfed by the autophagosome. The authors speculate that Nix-dependent recruitment of autophagosome-80associated LC3/GABARAP proteins to mitochondria might mediate membrane tethering and/or hemifusion of81mitochondria with autophagosomes (73). This could be the reason why mitochondria align to autophagosomes,82but are not engulfed in Nix-deficient reticulocytes (73). NIX-deficient mice exhibit defective erythroid83development, mild-to-moderate anemia, reticulocytosis, and an increase in splenic erythropoiesis (21, 89, 91).84Interestingly,knockout of autophagy-specific genes (Ulk1, Atg5 and Atg7) does not prevent mitophagy during85reticulocyte maturation, suggesting there is an alternative pathway for mitochondrial removal (56, 65, 110).86

87Mitochondrial turnover and role of bouts of autophagy88

89 Mitochondrial biogenesis consists of the growth and division of pre-existing mitochondria. According to the90endosymbiotic theory, mitochondria descend from a proteobacteria endosymbiont that became established in a91eukaryotic host cell. Given their bacterial origin, mitochondria have their own genome, and mitochondrial92proteins are encoded by both nuclear and mitochondrial genomes (103). Mitochondrial biogenesis requires the93coordination of several distinct processes: i) inner and outer mitochondrial membrane synthesis; ii) synthesis of94mitochondrial proteins; iii) synthesis and import of proteins encoded by the nuclear genome; iv) replication of95mitochondrial DNA; and v) mitochondrial fusion and fission.96

97Mitochondrial turnover comprises mitophagy followed by biogenesis. Mitochondria were proposed to turn over98as a unit, since inner membrane proteins such as cytochrome aa3, b and c, the inner membrane lipid99cardiolipin, and mitochondrial DNA in the matrix all have the same half life (29, 81, 82). The mitochondrial half-

00 live values found in the literature are very disparate. Reports from the early 1970s suggest that under normal01conditions, mitochondria of non-proliferating tissues (e.g., brain, heart, kidney and liver) turn over with a half-02life of 1025 days (69, 77), while others suggest ~5-6 days for rat heart and liver (82). The methods used to03

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amino acid changes are tolerated, while mutations causing amino acid changes are strongly underrepresented25in the protein-coding genes, providing incontrovertible evidence that mtDNA is subject to strong purifying26selection in the maternal germ line, and hints at a fitness test of the mitochondria that express proteins27encoded by the mitochondrial genome. This purifying selection of functional mtDNA genomes will decrease the28frequency of transmission of mutated genomes to the offspring (93). While this has been clearly demonstrated29in the oocyte, it is unknown whether such a purge might take place in somatic cells to maintain fitness of the30mitochondrial genome. We recently showed that in cell culture, an episode of starvation results in significant31depletion of mitochondria (8). Whether this process is followed by mitochondrial repopulation with the most32functional mtDNA genomes and improved mitochondrial function remains to be determined. However, studies33

of fasting and refeeding can result in myocardial dysfunction (78); if mitochondrial purging occurs during the34 fasting episodes, it may be maladaptive.3536

The removal of mitochondria usually needs to be compensated by mitochondrial biogenesis or it will become37detrimental (11). For example, in the liver during subacute sepsis, mitochondrial function is impaired and38mitochondria are removed by autophagy. In this model, clearance of mitochondria is followed by mitochondrial39replenishment (16). Mitochondrial biogenesis in the presence of defective mitochondrial function is also40observed in type 1 diabetes (92). In hearts of OVE26 mice, a chronic model of type 1 diabetes, there is41upregulation of mitochondrial proteins, increase in mitochondrial area and number and mitochondrial DNA (92).42Despite the higher number of mitochondria, their function is impaired. Similar results are observed in insulin-43resistant mice (22). It is possible that increased biogenesis is a compensatory mechanism for defective44

mitochondrial function. Biogenesis in the absence of balanced mitophagy to remove defective mitochondria45 may be maladaptive. Further studies are warranted to clarify this.4647

In most situations, increased mitochondrial biogenesis is beneficial, such as in caloric restriction and aging,48exercise, amyotrophic lateral sclerosis (ALS) treatment, neonatal cardiomyocytes response to LPS, and renal49proximal tubular cells subjected to oxidative stress. In skeletal muscles, caloric restriction enhances50mitochondrial protein turnover by enhancing mitochondrial degradation via autophagy and by stimulating51mitochondrial biogenesis. Caloric restriction activates sirtuin 1 (SIRT1) which activates autophagy, via52deacetylating autophagy proteins (57), and mitochondrial biogenesis, via activation of peroxisome proliferator-53activated receptor gamma cofactor-1alpha (PGC-1alpha) (12, 35, 62). The net result is that caloric restriction54ensures good mitochondrial function with aging by promoting mitochondrial turnover. A similar mechanism is55observed with exercise (60, 61, 68, 105). Skeletal muscle biopsies of humans performing high-intensity interval

56 training showed an increase in SIRT1, nuclear PGC-1alpha and mitochondrial transcription factor A, which57lead to an increase in skeletal muscle mitochondria and improved exercise performance (60, 61). Biopsies58performed in older men show that even with aging, exercise enhances mitochondrial respiratory chain activity59

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deleterious, by increasing the formation of ROS and leading to oxidative stress. Autophagy and biogenesis81work as intertwined mechanisms that assure mitochondrial quality and cellular homeostasis.82

83What happens when mitochondrial turnover is inadequate?84

85Proliferating cells can remove biological wastes by cell division; however cardiac myocytes cannot because86they are terminally differentiated and maintain cellular homeostasis only by the activation of degrading87pathways, namely, macroautophagy, microautophagy and chaperone-mediated autophagy (97). Thus, the88accumulation of defective mitochondria and lysosomes in aged myocytes is a reflection of inefficient autophagy89

(99). Senescent myocytes are characterized by the existence of giant mitochondria originating from oxidative90 damage followed by inefficient mitochondrial DNA repair and autophagy (9, 14, 95, 98). Terman and Brunk91suggested that autophagic engulfment of large mitochondria requires more energy, and consequently, is less92efficient (96). In an ideal situation of mitochondrial turnover, mitochondrial recycling should provide for removal93of damaged mitochondria and their replacement by normal, replicating mitochondria (6, 95). However, this is94not always the case and the accumulation of defective mitochondria in post-mitotic cells is frequently observed95(6). de Grey (17, 18) suggested that the lower respiratory rate of defective mitochondria would be96accompanied by less oxidative damage than that caused by normal mitochondria, which might make them less97prone to autophagy. Nekhaeva and collaborators (71) showed that in humans, mitochondrial fission can be98increased by certain mutations in the mitochondrial DNA, resulting in the replacement of normal mitochondria99by mutated ones which are less susceptible to oxidative damage and potentially less vulnerable to autophagy.00

With increasing age, lysosomal activity decreases and autophagy is inefficient due to the accumulation of01 lipofuscin, a brown granular pigment that consists of cross-linked lipids and proteins produced during02lysosomal digestion (48, 97). In the aging heart, the gradual inhibition of autophagy is at least in part caused03by the intralysosomal accumulation of lipofuscin (96). The impairment or suppression of autophagy plays a04critical role in the development of aging-related disorders in the heart. Therapeutic approaches to increase05autophagy and mitophagy may prove to be cardioprotective.06

07Life in the balance, longevity the goal08

09Self-eating, recycling, cash-for-your-clunkers:10

Trade up to the mitochondrial equivalent Prius.11The road to rejuvenation is paved with destruction,

12 For clearing the rubble precedes reconstruction.13But remember that lifes circular dance14Depends on opposite forces in balance:15

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Figure Legends9900

Fig. 1. Overview of autophagy. Cellular stresses, including reactive oxygen or reactive nitrogen species (ROS,01RNS), or nutritional/energetic stress activate AMPK and inhibit mTOR to signal autophagy through Atg1. Once02initiated, Beclin-1 and VPS34, a Class I PI3 kinase, trigger downstream events leading to activation of the first03of two ubiquitin-like pathways. First, Atg12 is activated similarly to ubiquitin by Atg7, an E1-like enzyme, which04then transfers it to Atg10, an E2-like enzyme, which then conjugates it to Lysine 130 of Atg5. The Atg5/Atg1205conjugate then complexes with a homodimer of Atg16, and the complex assembles on a membrane structure06termed the phagophore. This event is a prerequisite for the second ubiquitin-like pathway, which involves the07

cleavage of the terminal Cys residue of Atg8 (LC3) by Atg4, a cysteine protease, which exposes a terminal08 glycine residue. Atg4 is redox-regulated and appears to upregulate autophagy in the face of oxidative stress.09Atg4 in conjunction with Atg7 facilitates conjugation of LC3 onto phosphatidylethanolamine in the lipid bilayer10of the membrane. The cup-shaped phagophore is recruited to engulf targets via adaptor proteins such as p62,11which binds ubiquitinated protein aggregates and LC3. The growing ends of the phagophore eventually meet12and fuse to enclose the target within the double-membrane structure of the autophagosome. At this point13Atg12/5/16L are released, and the autophagosome fuses with a lysosome; fusion and degradation of contents14requires acidification, which is mediated by the vacuolar proton ATPase (VPATPase); lysosomal enzymes15such as cathepsins, and lipases have pH optima well below 6.0. The entire process of formation and16destruction occurs on a time-scale of minutes.17

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19 Fig. 2. Mitophagy. The murine atrial myocyte-derived cell line, HL-1, was transfected with mitochondria-20targeted red fluorescent protein (mitoDsRed) and LC3 fused to green fluorescent protein (GFP-LC3). Cells21were induced to undergo autophagy and imaged by deconvolution fluorescence microscopy. The raw,22unprocessed image of a region of a cell is shown at top right. After deconvolution and thresholding, it can be23seen that mitochondria are present within autophagosomes.24

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