Autophagy in the pathogenesis of disease

process in which the cell self-digests its own components. This transitions or to rid themselves of damaging cytoplasmic com- g h l s c self-digestion not only provides nutrients to maintain vital cellular functions during fasting but also can rid the cell of superfluous or damaged organelles, misfolded proteins, and invading micro- organisms. Interestingly, self-digestion by autophagy—a process that is potently triggered by fasting—is now emerging as a central biological pathway that functions to promote health and longevity. The Autophagic Pathway Autophagy (from the Greek, “auto” oneself, “phagy” to eat) refers to any cellular degradative pathway that involves the delivery of cytoplasmic cargo to the lysosome. At least three forms have been identified—chaperone-mediated autophagy, microau- tophagy, and macroautophagy—that differ with respect to their physiological functions and the mode of cargo delivery to the lyso- some. This Review will focus on macroautophagy (herein referred to as autophagy), the major regulated catabolic mechanism that eukaryotic cells use to degrade long-lived proteins and organelles. This form of autophagy involves the delivery of cytoplasmic cargo sequestered inside double-membrane vesicles to the lysosome (Figure 1). Initial steps include the formation (vesicle nucleation) and expansion (vesicle elongation) of an isolation membrane, which is also called a phagophore. The edges of the phagophore ponents, for example, during oxidative stress, infection, or pro- tein aggregate accumulation. Nutritional status, hormonal fac- tors, and other cues like temperature, oxygen concentrations, and cell density are important in the control of autophagy. The molecular cascade that regulates and executes autophagy has been the subject of recent, comprehensive reviews (Klion- sky, 2007; Maiuri et al., 2007a; Mizushima and Klionsky, 2007; Rubinsztein et al., 2007). One of the key regulators of autophagy is the target of rapamy- cin, TOR kinase, which is the major inhibitory signal that shuts off autophagy in the presence of growth factors and abundant nutrients. The class I PI3K/Akt signaling molecules link recep- tor tyrosine kinases to TOR activation and thereby repress autophagy in response to insulin-like and other growth factor signals (Lum et al., 2005). Some of the other regulatory mole- cules that control autophagy include 5′-AMP-activated protein kinase (AMPK), which responds to low energy; the eukaryotic initiation factor 2α (eIF2α), which responds to nutrient starvation, double-stranded RNA, and endoplasmic reticulum (ER) stress; BH3-only proteins that contain a Bcl-2 homology-3 (BH3) domain and disrupt Bcl-2/Bcl-XL inhibition of the Beclin 1/class III PI3K complex; the tumor suppressor protein, p53; death-associated Leading Edge Review Introduction Fasting has been an integral part of health and healing practices throughout the recorded history of mankind. This ancient tradition may be partially rooted in a cellular process we are now beginning to understand in modern scientific terms. One of the most evolu- tionarily conserved cellular responses to organismal fasting is the activation of the lysosomal degradation pathway of autophagy, a Autophagy in the Patho Beth Levine1,2,* and Guido Kroemer3,4,5,* 1Department of Internal Medicine 2Department of Microbiology University of Texas Southwestern Medical Center, 5323 Harry Hines B 3Institut Gustave Roussy 4Université Paris Sud, Paris 11 5INSERM, U848 F-94805 Villejuif, France *Correspondence: beth.levine@utsouthwestern.edu (B.L.), kroemer@i DOI 10.1016/j.cell.2007.12.018 Autophagy is a lysosomal degradation pathway t opment, and homeostasis. Autophagy principal against diverse pathologies, including infection disease. However, in certain experimental disease even the prosurvival functions of autophagy may advances in understanding the physiological fun causation and prevention of human diseases. then fuse (vesicle completion) to form the autophagosome, a dou- ble-membraned vesicle that sequesters the cytoplasmic material. This is followed by fusion of the autophagosome with a lysosome to form an autolysosome where the captured material, together with the inner membrane, is degraded (Figure 1). Autophagy occurs at low basal levels in virtually all cells to perform homeostatic functions such as protein and organelle turnover. It is rapidly upregulated when cells need to generate intracellular nutrients and energy, for example, during starva- tion, growth factor withdrawal, or high bioenergetic demands. Autophagy is also upregulated when cells are preparing to undergo structural remodeling such as during developmental genesis of Disease oulevard, Dallas, TX 75390, USA r.fr (G.K.) at is essential for survival, differentiation, devel- y serves an adaptive role to protect organisms , cancer, neurodegeneration, aging, and heart settings, the self-cannibalistic or, paradoxically, be deleterious. This Review summarizes recent tions of autophagy and its possible roles in the Cell 132, January 11, 2008 ©2008 Elsevier Inc. 27 protein kinases (DAPk); the ER-membrane-associated protein, Ire-1; the stress-activated kinase, c-Jun-N-terminal kinase; the inositol-trisphosphate (IP3) receptor (IP3R); GTPases; Erk1/2; ceramide; and calcium (Criollo et al., 2007; Maiuri et al., 2007a; Meijer and Codogno, 2006; Rubinsztein et al., 2007). Administrator 高亮 Downstream of TOR kinase, there are more than 20 genes in yeast (known as the ATG genes) that encode proteins (many of which are evolutionarily conserved) that are essential for the execution of autophagy (Mizushima and Klionsky, 2007) (Figure 1). These include a protein serine/threonine kinase complex that responds to upstream signals such as TOR kinase (Atg1, Atg13, Atg17), a lipid kinase signaling complex that mediates vesicle nucleation (Atg6, Atg14, Vps34, and Vps15), two ubiquitin-like conjugation pathways that mediate vesicle expansion (the Atg8 and Atg12 systems), a recycling pathway that mediates the dis- assembly of Atg proteins from mature autophagosomes (Atg2, Atg9, Atg18), and vacuolar permeases that permit the efflux of amino acids from the degradative compartment (Atg22). In mam- mals, proteins that act more generally in lysosomal function are The identification of signals that regu- late autophagy and genes that execute autophagy has facilitated detection and manipulation of the autophagy pathway. Phosphatidylethanolamine (PE) conju- gation of yeast Atg8 or mammalian LC3 during autophagy results in a nonsoluble form of Atg8 (Atg8-PE) or LC3 (LC3-II) that stably associates with the autopha- gosomal membrane (Figure 1). Conse- quently, autophagy can be detected bio- chemically (by assessing the generation of Atg8-PE or LC3-II) or microscopically (by observing the localization pattern of fluorescently tagged Atg8 or LC3) (Mizushima and Klionsky, 2007). These approaches must be coupled with ancil- lary measures to discriminate between two physiologically distinct scenarios—increased autophagic flux without impair- ment in autophagic turnover (i.e., an increased “on-rate”) versus impaired clearance of autophagosomes (i.e., a “decreased off- rate”), which results in a functional defect in autophagic catabo- lism (Figure 2). Autophagy can be pharmacologically induced by inhibiting negative regulators such as TOR with rapamycin (Rubinsztein et al., 2007); the antiapoptotic proteins Bcl-2 and Bcl-XL that bind to the mammalian ortholog of yeast Atg6, Beclin 1, with ABT-737 (Maiuri et al., 2007b); IP3R with xestospongin B, an IP3R antagonist; or lithium, a molecule that lowers IP3 levels (Criollo et al., 2007). Autophagy can be pharmacologically inhibited by targeting the class III PI3K involved in autopha- Figure 1. The Cellular, Molecular, and Physiological Aspects of Autophagy The cellular events during autophagy follow dis- tinct stages: vesicle nucleation (formation of the isolation membrane/phagophore), vesicle elonga- tion and completion (growth and closure), fusion of the double-membraned autophagosome with the lysosome to form an autolysosome, and lysis of the autophagosome inner membrane and break- down of its contents inside the autolysosome. This process occurs at a basal level and is regulated by numerous different signaling pathways (see text for references). Shown here are only the regula- tory pathways that have been targeted pharma- cologically for experimental or clinical purposes. Inhibitors and activators of autophagy are shown in red and green, respectively. At the molecular level, Atg proteins form different complexes that function in distinct stages of autophagy. Shown here are the complexes that have been identified in mammalian cells, with the exception of Atg13 and Atg17 that have only been identified in yeast. The autophagy pathway has numerous proposed physiological functions; shown here are functions revealed by in vivo studies of mice that cannot un- dergo autophagy (see Table 1). 28 Cell 132, January 11, 2008 ©2008 Elsevier Inc. required for proper fusion with autophagosomes—such as the lysosomal transmembrane proteins, LAMP-2 and CLN3—and for the degradation of autophagosomal contents, such as the lysosomal cysteine proteases, cathepsins B, D, and L (Table 1). gosome formation with 3-methyladenine or by targeting the fusion of autophagosomes with lysosomes, using inhibitors of the lysosomal proton pump such as bafilomycin A1 or lysoso- motropic alkalines such as chloroquine and 3-hydroxychloro- quine (Rubinsztein et al., 2007) (Figure 1). It should be noted that all of these pharmacological agents lack specificity for the autophagy pathway. Therefore, although some of these agents such as rapamycin, lithium, and chloroquine are clinically available and may be helpful for treating diseases associated with autophagy deregulation, genetic approaches to inhibiting autophagy—for example, knockout of ATG genes by homolo- gous recombination or knockdown by small-interfering RNA (siRNA)—have yielded more conclusive information about the biologic roles of autophagy in health and disease. Physiological Functions of Autophagy bulk form of degradation generates free amino and fatty acids that can be recycled in a cell-autonomous fashion or delivered systemically to distant sites within the organism. Presumably, the amino acids generated are used for the de novo synthesis of proteins that are essential for stress adaptation. The molec- ular basis for the recycling function of autophagy has only recently begun to be defined with the identification of yeast Atg22 as a vacuolar permease required for the efflux of amino acids resulting from autophagic degradation (Mizushima and Klionsky, 2007). It is presumed that the recycling function of autophagy is conserved in mammals and other higher organ- isms, although direct data proving this concept are lacking. atg5F/F:MerCreMer (car- diomyocytes, tamoxifen- inducible) Ventricular dilatation, contractile dysfunction, disorganized sarcomeres, and misaligned/ aggregated mitochondria after tamoxifen injection. Nakai et al., 2007 atg5−/− (dendritic cells) Normal dendritic cell development but impaired autophagic delivery to endosomal Toll- like receptors and interferon production during virus infection. Lee et al., 2007 atg5−/− (T cells) Increased spontaneous apoptosis in vivo (CD8+ T cells) and defective activation-induced proliferation in vitro (CD4+ and CD8+ T cells). Pua et al., 2007 atg6/beclin 1−/− (all tissues) Abnormal ectodermal layer with reduced cavitation and early embryonic lethality. Yue et al., 2003; Qu et al., 2007 atg6/beclin 1+/− (all tissues) Increased frequency of spontaneous malignancies (especially lymphomas) and mam- mary neoplasia. Decreased pressure overload-induced heart failure. Decreased cardiac injury during ischemia/reperfusion. Qu et al., 2003; Yue et al., 2003; Zhu et al., 2007; Matsui et al., 2007 atg7−/− (all tissues) Death within 24 hr after birth presumably due to nutrient and energy depletion. Komatsu et al., 2005 atg7F/F:nestin-Cre (neurons) Progressive neurodegeneration associated with ubiquitinated protein aggregates and inclusion bodies. Increased frequency of TUNEL+ neurons. Komatsu et al., 2006 atg7F/F:Mx1-Cre (liver) Ubiquitinated protein aggregates, deformed mitochondria, and aberrant membranous structures in hepatocytes. Reduced removal of peroxisomes after chemical treatment. Komatsu et al., 2005; Iwata et al., 2006 ambra1−/− (all tissues) Decreased autophagy, increased apoptosis, and increased cell proliferation in fetal brain. Neural tube defects and embryonic death. Fimia et al., 2007 bif-1−/− Increased frequency of spontaneous lymphomas and solid tumors. Takahashi et al., 2007 Mutations Affecting Lysosomal Clearance of Autophagosomes lamp-2−/− (all tissues) Autophagosome accumulation in multiple tissues. Impaired hepatocyte long-lived pro- tein degradation. Vacuolar cardiomyopathy and skeletal myopathy. Tanaka et al., 2000 cln3 (all tissues) Juvenile neuronal ceroid lipofuscinosis, with autophagic vacuolization and LC3-I to LC3- II conversion. Cao et al., 2006 cathepsin D−/− (all tissues) Neuronal ceroid lipofuscinosis with autophagic vacuolization and LC3-I to LC3-II conver- sion. Bax knockout reduces enhanced apoptosis but not autophagic degeneration and neuronal loss. Koike et al., 2005; Shacka et al., 2007 cathepsin B−/−L−/− (all tissues) Severe brain atrophy with enhanced apoptosis, autophagic vacuolization, and LC3-I to LC3-II conversion. Felbor et al., 2002; Koike et al., 2005 Table 1. Phenotypes of Mice with Mutations in Autophagy Genes Genotype (Organ) Phenotype Mutations Affecting Formation of Autophagosomes atg4C−/− (all tissues) Diaphragm-specific autophagy defect duri fibrosarcomas. atg5−/− (all tissues) Death within 24 hr after birth presumably d defect. Increased numbers of apoptotic ce atg5F/F:nestin-Cre (neurons) Progressive neurodegeneration associated inclusion bodies. atg5F/F:MLC2v-Cre (cardio- myocytes) Normal hearts under basal conditions but dilatation and heart failure. Autophagy Defends against Metabolic Stress Autophagy is activated as an adaptive catabolic process in response to different forms of metabolic stress, including nutrient deprivation, growth factor depletion, and hypoxia. This Reference ng starvation. Increased chemically induced Marino et al., 2007 ue to nutrient and energy depletion. Suckling lls in embryos. Kuma et al., 2004; Qu et al., 2007 with ubiquitinated protein aggregates and Hara et al., 2006 increased pressure load-induced ventricular Nakai et al., 2007 Cell 132, January 11, 2008 ©2008 Elsevier Inc. 29 The amino acids liberated from autophagic degradation can be further processed and, together with the fatty acids, used by the tricarboxylic acid cycle (TCA) to maintain cellular ATP production. The importance of autophagy in fueling the TCA cycle is supported by studies showing that certain phenotypes of autophagy-deficient cells can be reversed by supplying them with a TCA substrate such as pyruvate (or its membrane- permeable derivative methylpyruvate). For example, meth- ylpyruvate can maintain ATP production and survival in growth factor-deprived autophagy-deficient cells that would otherwise quickly die (Lum et al., 2005). It can also restore ATP produc- tion, the generation of engulfment signals, and effective corpse removal in autophagy-deficient cells during embryonic devel- opment (Qu et al., 2007). This role of autophagy in maintaining macromolecular in organismal survival during nutrient stress (Maiuri et al., 2007a). Yeast cells lacking ATG genes display reduced tol- erance to nitrogen or carbon deprivation and are defective in starvation-induced sporulation. Similarly, null mutations in ATG genes in slime molds limit viability and differentiation during nutrient depri- vation. Loss-of-function mutations in ATG genes in plants reduce tolerance to nitrogen or carbon depletion, resulting in enhanced chlorosis, reduced seed set, and accelerated leaf senescence (Bassham et al., 2006). Furthermore, siRNA-mediated knockdown of atg genes in nematodes decreases survival during starvation (Kang et al., 2007). Autophagy also enables mammals to withstand nutrient depletion (Table 1). Mice lacking either atg5−/− or atg7−/− are born at normal Mendelian ratios yet die within hours after birth, presumably due to their inability to adapt to the neonatal starvation period. Thus, a critical physiological role of autophagy appears to be the mobilization of intracellular energy resources to meet cellular and organismal demands for metabolic sub- strates. The requirement for this function of autophagy is not limited to settings of nutrient starvation. Because growth factors are often required for nutrient uptake, loss of growth factor signaling can result in reduced intracellular metabo- lite concentrations and activation of autophagy-dependent survival mechanisms (Lum et al., 2005). It is also possible Figure 2. Alterations in Different Stages of Autophagy Have Different Consequences An increased on-rate of autophagy occurs in re- sponse to stress signals, resulting in increased autophagosomal and autolysosomal accumula- tion and successful execution of the adaptive physiological functions of autophagy. In certain disease states or upon treatment with lysosomal inhibitors, there is a reduced off-rate resulting in impaired lysosomal degradation of autophago- somes. This results in increased autophagosomal accumulation and adverse pathophysiological consequences related to unsuccessful comple- tion of the autophagy pathway. A decreased on-rate is observed if signaling activation of au- tophagy is defective or mutations are present in ATG genes. This results in decreased autopha- gosomal accumulation, the accumulation of pro- tein aggregates and damaged organelles, and pathophysiological consequences related to de- ficient protein and organelle turnover. The physi- ological and pathophysiological consequences listed for “increased on-rate,” “reduced off-rate,” and “decreased on-rate” are based on knockout studies of the ATG genes in model organisms. 30 Cell 132, January 11, 2008 ©2008 Elsevier Inc. synthesis and ATP production is likely a critical mechanism underlying its evolutionarily conserved prosurvival function. Gene knockout or knockdown studies in diverse phyla provide strong evidence that autophagy plays an essential function that in certain settings, especially when cells suddenly have high metabolic needs, autophagy may be needed in a cell-autonomous fashion to generate sufficient intracellular metabolic substrates to maintain cellular energy homeosta- sis. This hypothesis may explain why there are high levels of autophagy in the mouse heart and diaphragm immediately following birth (Kuma et al., 2004). Autophagy Works as a Cellular Housekeeper The repertoire of routine housekeeping functions performed by autophagy includes the elimination of defective proteins and organelles, the prevention of abnormal protein aggregate accumulation, and the removal of intracellular pathogens. Figure 3. Autophagy, Protein Quality Control, and Neurodegeneration Normal proteins are routinely turned over by different protein degradation sys- tems, including the ubiquitin-proteasome system (UPS), chaperone-mediated autophagy (CMA), and macroautophagy (referred to herein as “autophagy”). In autophagy-deficient neurons, there is an accumulation of ubiquitinated pro- tein aggregates that is associated with neurodegeneration. Similar effects of autophagy deficiency are observed in other postmitotic cells (hepatocytes, cardiomyocytes) under basal conditions. Proteins altered by mu