Autophagy is the major intracellular degradation system by which cytoplasmic materials are delivered to and degraded in the lysosome. However, the purpose of autophagy is not the simple elimination of materials, but instead, autophagy serves as a dynamic recycling system that produces new building blocks and energy for cellular renovation and homeostasis. Here we provide a multidisciplinary review of our current understanding of autophagy’s role in metabolic adaptation, intracellular quality control, and renovation during development and differentiation. We also explore how recent mouse models in combination with advances in human genetics are providing key insights into how the impairment or activation of autophagy contributes to pathogenesis of diverse diseases, from neurodegenerative diseases such as Parkinson disease to inflammatory disorders such as Crohn disease.All living organisms undergo continuous renovation. In humans, cells and intracellular components are constantly remodeled and recycled. This is, in part, in order to replace old components with fresh, better-quality ones. However, when components are replaced with different types, a net change in character results. Such ‘‘cellular renovation’’ requires synthesis of new components but also degradation of pre-existing materials, which can serve as building blocks. Eukaryotic cells have two major degradation systems, the lysosome and the proteasome. Proteasomal degradation has high selectivity; the proteasome generally recognizes only ubiquitinated substrates, which are primarily short-lived proteins.

 By contrast, degradation in the lysosome does not follow such a simple pattern. Extracellular material and plasma membrane proteins can be delivered to lysosomes for degradation via the endocytic pathway. Furthermore, cytosolic components and organelles can also be delivered to the lysosome by autophagy (Figure 1). The lysosome is often described as a ‘‘cellular garbage can,’’ and its more positive roles in cellular renovation, particularly those involving autophagy, have not been well appreciated. In the 1990s, genetic studies in yeast identified a series of autophagy-related (ATG) genes (Klionsky et al., 2003; Nakatogawa et al., 2009). The results of these studies greatly increased our understanding of the mechanism and function of autophagy. In particular, analyses of autophagy-defective organisms have revealed numerous physiological and pathological roles of autophagy at both the cellular and whole-organism levels. In this Review, we summarize the current knowledge of autophagy and discuss the multidisciplinary function of autophagy in renovation of the cell and the organism. Mechanisms of Autophagy Autophagy is a generic term for all pathways by which cytoplasmic materials are delivered to the lysosome in animal cells or the vacuole in plant and yeast cells. There are roughly three classes of autophagy (Figure 1): macroautophagy, microautophagy, and chaperone-mediated autophagy. Macroautophagy uses the intermediate organelle ‘‘autophagosome.’’ An isolation membrane (also termed phagophore) sequesters a small portion of the cytoplasm, including soluble materials and organelles, to form the autophagosome.

 The autophagosome fuses with the lysosome to become an autolysosome and degrade the materials contained within it. Autophagosomes may fuse with endosomes before fusion with lysosomes. In microautophagy, the lysosome itself engulfs small components of the cytoplasm by inward invagination of the lysosomal membrane (Figure 1). Membrane dynamics during microautophagy may be quite similar or identical to that of endosomal sorting complex required for transport (ESCRT)-dependent multivesicular body (MVB) formation, which occurs in the late endosome. In fact, significant amounts of cytosolic proteins are incorporated into the endosomal lumen during MVB formation both in bulk and selectively (Sahu et al., 2011). The third type of autophagy is chaperone-mediated autophagy. This class does not involve membrane reorganization; instead, substrate proteins directly translocate across the lysosomal membrane during chaperone-mediated autophagy (Figure 1). The chaperone protein Hsc70 (heat shock cognate 70) and cochaperones specifically recognize cytosolic proteins that contain a KFERQ-like pentapeptide (Orenstein and Cuervo, 2010). The transmembrane protein Lamp-2A, which is an isoform of Lamp-2, acts as a receptor on the lysosome, and unfolded proteins are delivered into the lysosomal lumen through a multimeric translocation complex.

 Macroautophagy is thought to be the major type of autophagy, and it has been studied most extensively compared to microautophagy and chaperone-mediated autophagy. Therefore, herein we refer to macroautophagy simply as ‘‘autophagy.’’ Autophagy is highly inducible, with starvation and other stresses rapidly increasing the number of autophagosomes. Autophagosomes are generated on or in close proximity to the endoplasmic reticulum (ER) (Figure 2) (Mizushima et al., 2011; Tooze and Yoshimori, 2010). However, it remains unclear whether the ER membrane is directly used for autophagosome formation. Recent studies suggest that additional membranes derived from the Golgi complex, the mitochondria, and the plasma membrane also contribute to autophagosome formation (Hailey et al., 2010; Mizushima et al., 2011; Ravikumar et al., 2010; Tooze and Yoshimori, 2010). Thus, autophagosome formation likely involves multiple, complex processes. Multiple Atg proteins govern autophagosome formation. Among the 35 Atg proteins thus far identified in yeast, Atg1–10, 12–14, 16, and 18 are the ‘‘core Atg proteins’’ (Nakatogawa et al., 2009). These proteins are required for autophagosome formation, in addition to Atg17, 29, and 31. The core Atg proteins are shared by other autophagy-related pathways, such as pexophagy (autophagic degradation of the peroxisome) and the cytoplasm-to-vacuole targeting pathway, which have been discussed in more detail in other reviews (Chen and Klionsky, 2011; Nakatogawa et al., 2009; Youle and Narendra, 2011). Figure 1. Different Types of Autophagy Macroautophagy: A portion of cytoplasm, including organelles, is enclosed by an isolation membrane (also called phagophore) to form an autophagosome. 

The outer membrane of the autophagosome fuses with the lysosome, and the internal material is degraded in the autolysosome. Microautophagy: Small pieces of the cytoplasm are directly engulfed by inward invagination of the lysosomal or late endosomal membrane. Chaperone-mediated autophagy: Substrate proteins containing a KFERQ-like pentapeptide sequence are first recognized by cytosolic Hsc70 and cochaperones. Then they are translocated into the lysosomal lumen after binding with lysosomal Lamp-2A. After all three types of autophagy, the resultant degradation products can be used for different purposes, such as new protein synthesis, energy production, and gluconeogenesis. The core Atg proteins are highly conserved in other eukaryotes, including mammals, and they act in a similar hierarchical manner in yeast and mammals (Itakura and Mizushima, 2010; Suzuki et al., 2007). Figure 2 summarizes their functional steps in mammalian cells, and more details for this process are described extensively elsewhere (Chen and Klionsky, 2011; Mizushima et al., 2011; Nakatogawa et al., 2009). Adaptive Metabolic Response The proteasome functions as a major generator of amino acids under normal nutrient-rich conditions, but autophagy’s contribution to amino acid production increases when cells are starved (Vabulas and Hartl, 2005).

 Limitation of various types of nutrients, such as amino acids, growth factors, oxygen, and energy, can induce autophagy (He and Klionsky, 2009; Kroemer et al., 2010). Among these nutrients, starvation of nitrogen or amino acids induces the highest levels of autophagy in yeast and cultured mammalian cells, respectively. This is quite reasonable because the main products of autophagy are amino acids derived from cellular proteins. Restoration of cellular (or local) levels of amino acids reactivates the serine/threonine protein kinase mTORC1 (mammalian target of rapamycin complex 1) and terminates autophagy (Yu et al., 2010). Autophagy, therefore, constitutes a negative feedback loop in response to nutrient starvation. When yeast cells are cultured in nitrogen-free medium, autophagy-deficient cells rapidly decrease intracellular amino acid levels (Onodera and Ohsumi, 2005) and lose their viability (Tsukada and Ohsumi, 1993). Similarly, mice with systemic deletion of Atg3 (Sou et al., 2008), Atg5 (Kuma et al., 2004), and Atg7 (Komatsu et al., 2005) die immediately after birth and show reduced amino acid levels in tissues and plasma during the neonatal starvation period. Thus, enhanced degradation of self-components by autophagy is a critical survival response against starvation conditions.An important question is, how do cells use these amino acids produced by autophagy (Figures 1 and 3)? Initial induction of autophagy is very rapid and occurs before energy fuels are completely exhausted. For instance, mice starved for 24 hr show increased autophagy in many tissues, but they still have sufficient lipids (glycogen may be consumed during the first day). Therefore, it is unlikely that autophagy simply supplies energy in these settings. 

In fact, several studies have suggested that autophagy-derived amino acids are used to synthesize proteins essential for starvation adaptation. Protein anabolism is generally downregulated in starved cells, but synthesis of certain types of proteins continues or is even upregulated during starvation. These include vacuolar/ lysosomal enzymes, respiratory chain proteins, antioxidant enzymes, and proteins involved in pathways of amino acid biosynthesis (Onodera and Ohsumi, 2005; Suzuki et al., 2011). Autophagy-deficient yeast cells fail to synthesize these proteins during starvation. As a result, these mutants loose respiratory function and accumulate higher levels of reactive oxygen species, which further decreases their mitochondrial DNA content (Suzuki et al., 2011). These appear to be the major mechanisms that rapidly kill autophagy-deficient yeast cells during nitrogen limitation. Nonetheless, autophagy seems to be an important energy generator in certain settings. Amino acids can be converted into intermediates of the tricarboxylic acid (TCA) cycle and thus contribute to ATP production. Metabolome analysis of Ras-expressing cancer cells shows that autophagy is important for maintenance of TCA cycle metabolites (Guo et al., 2011). It is interesting that citrate, aconitate, and isocitrate, which are solely produced in mitochondria, are specifically reduced, suggesting that autophagy could be important not only for providing TCA metabolites but also for quality control of mitochondria. In addition, breakdown of lipid droplets by autophagy (lipophagy) may also account for its energy-producing role, especially in the liver (Singh et al., 2009).

 Finally, amino acids are thought to be an important source for gluconeogenesis in the liver. In fact, when Atg7 is deleted specifically in the liver, the mutant mice show reduced levels of blood glucose and amino acids after 24 hr of starvation (Ezaki et al., 2011). This finding suggests that amino acids generated inside the liver are used for gluconeogenesis and maintenance of the plasma pool of amino acids. How much autophagy contributes to overall gluconeogenesis is still unknown. Intracellular Quality Control by Nonselective and Selective Autophagy Approximately 1%–1.5% of cellular proteins are catabolized per hour by autophagy, even under nutrient-rich conditions in the liver. It is unclear how much basal autophagy contributes to macromolecule synthesis and energy production in the steady state by supplying amino acids, glucose, and free fatty acids. Nevertheless, basal autophagy acts as the quality-control machinery for cytoplasmic components, and it is crucial for homeostasis of various postmitotic cells, such as neurons and hepatocytes. Although this quality control could be partially achieved by nonselective autophagy, increasing evidence indicates that ‘‘selective’’ autophagy degrades specific proteins, organelles, and invading bacteria (Figure 3). Selective autophagy occurs constitutively and can also be induced in response to cellular stresses. Selective Degradation of p62 One of the best characterized substrates of selective autophagy is p62, which is also known as sequestosome 1/SQSTM1. p62 is an ubiquitously expressed cellular protein, which is conserved in animals but not in plants and fungi. p62 directly interacts with LC3 (microtubule-associated protein light chain 3) on the isolation membrane through the LC3-interacting region (Figure 3).

 (LC3 is the mammalian homolog of Atg8 in yeast.) Subsequently, p62 is incorporated into the autophagosome and then degraded (Johansen and Lamark, 2011; Weidberg et al., 2011). Impairment of autophagy is accompanied by accumulation of p62. This leads to the formation of large aggregates, which include p62 and ubiquitin (Komatsu et al., 2007a). Similar inclusion bodies with p62 and ubiquitin have been identified in various neurodegenerative diseases, including Alzheimer disease, Parkinson disease, and amyotrophic lateral sclerosis; liver disorders, including alcoholic hepatitis and steatohepatitis; and cancers, including malignant glioma and hepatocellular carcinoma (Zatloukal et al., 2002). When Atg7 is disrupted in livers and brains of mice, p62-positive aggregates are observed in their hepatocytes and neurons, respectively. Interestingly, these aggregates, as well those in human hepatocellular carcinoma cells, are completely dispersed by the additional loss of p62 (Inami et al., 2011; Komatsu et al., 2007a). These findings implicate p62 in the formation of disease-related inclusion bodies (Figure 3). p62 functions as a signaling hub that may determine whether cells survive by activating the TNF receptor-associated factor 6 (TRAF6)–NF-kB pathway or die by facilitating the aggregation of caspase-8 and downstream effector caspases (Moscat and Diaz-Meco, 2009). On the other hand, p62 interacts with the Nrf2-binding site on Keap1, a component of Cullin3-type ubiquitin ligase for Nrf2. This interaction stabilizes Nrf2 and activates the transcription of Nrf2 target genes, including a battery of antioxidant proteins (Komatsu et al., 2010; Lau et al., 2010).

 It is thus possible that excess accumulation or aggregation of p62 leads to hyperactivation of these signaling pathways (Figure 3). Selective Degradation of Ubiquitinated Cargos Almost all tissues with defective autophagy display an accumulation of polyubiquitinated proteins (Mizushima and Levine, 2010). Loss of autophagy is thought to delay global turnover of cytoplasmic components (Hara et al., 2006) and impair the degradation of substrates destined for the proteasome (Korolchuk et al., 2009). These effects could partially explain the accumulation of misfolded and unfolded proteins followed by the formation of inclusion bodies.However, p62 has a ubiquitin-associated (UBA) domain. Thus, it has been proposed that p62 may be an autophagy receptor for degrading ubiquitinated cargos, including ubiquitinated aggregates, damaged mitochondria, ubiquitinated midbody rings, ubiquitin-tagged peroxisomes, ubiquitinated microbes, ribosomal proteins, and virus capsid proteins (Johansen and Lamark, 2011; Weidberg et al., 2011) (Figure 3). p62 and other adaptor proteins, such as NDP52 (Thurston et al., 2009) and optineurin (Wild et al., 2011), mediate the degradation of invading microbes via their interaction with ubiquitination (Figure 3). This selective autophagy could be regulated by posttranslational modification of the adaptors. For instance, TANK-binding kinase (TBK1) phosphorylates optineurin, which enhances its binding affinity to LC3 and thereby suppresses growth of invading microbes (Wild et al., 2011). Although a large number of studies suggest that p62 acts as an ubiquitin adaptor, it is still unknown whether soluble ubiquitinated proteins are also degraded through p62 binding. 

A mass spectrometric analysis clearly demonstrated that ubiquitinated proteins in autophagy-deficient livers and brains do not show any linkage specificity, indicating that specific polyubiquitin chain linkage is not the decisive signal for autophagic degradation (Riley et al., 2010). The simultaneous knockout of either p62 or Nrf2 completely suppresses the increase in ubiquitin conjugates in Atg7-deficient liver and brain (Riley et al., 2010). Therefore, the accumulation of ubiquitinated proteins in tissues defective in autophagy might be attributed to p62-mediated activation of Nrf2, resulting in global transcriptional changes to ubiquitin-associated genes. Further studies will be needed to elucidate more precisely the mechanism of degradation of ubiquitinated proteins by autophagy. Degradation of Damaged Mitochondria: Implications for the Pathogenesis of Parkinson Disease Recent studies have described the molecular mechanism by which damaged mitochondria are selectively targeted for autophagy, and these studies also suggest that the defects in this process underlie familial Parkinson disease (Youle and Narendra, 2011). PINK1, a mitochondrial kinase, and Parkin, an E3 ubiquitin ligase, have been genetically linked to both Parkinson disease and a pathway that prevents progressive mitochondrial damage and dysfunction. When mitochondria are damaged and depolarized, PINK1 becomes stabilized and recruits Parkin to the damaged mitochondria (Matsuda et al., 2010; Narendra et al., 2008; Narendra et al., 2010a; Vives-Bauza et al., 2010). Parkin ubiquitinates various mitochondrial outer membrane proteins, which could trigger mitophagy.

 However, the precise substrate of Parkin, which is essential for mitophagy, is still unknown (Figure 3). Of note, mutations in PINK1 and Parkin, which are associated with Parkinson disease, are known to impair mitophagy (Matsuda et al., 2010; Narendra et al., 2008, 2010a; Vives-Bauza et al., 2010), suggesting that there is a link between defective mitophagy and Parkinson disease. Accumulation of damaged mitochondria would cause oxidative stress and loss of neuronal cells. Nevertheless, many questions remain about mitophagy and Parkin. First, it is unknown how the autophagosome recognizes these ubiquitinated mitochondria. Although p62 has been implicated in this recognition process, elimination of mitochondria occurs normally in p62-deficient cells (Narendra et al., 2010b; Okatsu et al., 2010). p62 seems to be required for the clustering of depolarized mitochondria in the perinuclear region. The role of other mitochondrial adaptor proteins such as Nix remains unknown (Novak et al., 2010). Second, Parkin appears to function in other degradative processes besides mitophagy. Parkin can induce degradation of a broad range of mitochondrial outer membrane proteins, such as mitofusin 1/2 and Tom20 (Chan et al., 2011; Tanaka et al., 2010; Yoshii et al., 2011), which may also be important for mitochondrial quality control. Third, in contrast to in vitro studies, a recent in vivo study showed that depolarized mitochondria do not recruit Parkin in dopamine neurons (Sterky et al., 2011). Thus, Parkin seems to have multiple functions in mitochondrial quality control and neuronal cell survival. More studies are needed to fully understand the physiological and pathological role of this protein.

 Renovation during Differentiation and Development Development and differentiation processes are often accompanied by drastic cellular and tissue remodeling, which requires enhanced degradation. Autophagy can ‘‘kill two birds with one stone’’ during this remodeling; it eliminates pre-existing materials and provides support for the subsequent creation of new components (Mizushima and Levine, 2010). For example, autophagy has been shown to be required for formation of spores in yeast (Tsukada and Ohsumi, 1993) and dauer larvae in Caenorhabditis elegans (Mele´ ndez et al., 2003), both of which are triggered by starvation to sustain the organism during adverse conditions. Autophagy also plays a crucial role in insect metamorphosis (Ryoo and Baehrecke, 2010). It is possible that autophagy-derived amino acids can be used as essential building blocks in these processes. The most dramatic cellular renovation may occur shortly after fertilization. Maternal proteins and messenger RNAs (mRNAs) are extensively degraded while new proteins encoded by the zygotic genome are synthesized. In mammals, fertilization induces massive autophagy, which plays an essential role in early embryogenesis (Tsukamoto et al., 2008) (Figure 4). Nutrient availability may be limited in embryos until implantation, and autophagy functions as a major nutrient-providing system during this period. In addition, autophagy in early embryos is essential for selective elimination of paternal mitochondria in C. elegans. This could be a key mechanism underlying maternal inheritance of mitochondrial DNA and may be conserved in mammals (Sato and Sato, 2011; Al Rawi et al., 2011).

 Atg3
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(Sou et al., 2008), Atg5
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(Kuma et al., 2004), Atg7
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(Komatsu et al., 2005), Atg9
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(Saitoh et al., 2009), and Atg16L1
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(Saitoh et al., 2008) mice die shortly after birth without apparent anatomical abnormalities. However, tissuespecific gene-targeting studies have later revealed that autophagy functions in several specific lineages of differentiation, such as adipocytes, erythrocytes, T cells, and B-1a cells (Figure 4). Autophagy may play an important role in complete or partial elimination of mitochondria during these processes (Mizushima and Levine, 2010). In a separate mechanism, autophagy in thymic epithelial cells could be involved in the establishment of self-tolerance of T cells; specifically, autophagy fine tunes epithelial cells’ presentation of self-antigens to thymocytes, which leads to the elimination of some populations of self-reactive T cells (Nedjic et al., 2008) (Figure 4). Tissue Homeostasis and Renovation in Health and Disease Liver Regardless of the nutritional situation, basal autophagy metabolizes cytoplasmic components to prevent accumulation of degenerated proteins and organelles (Figure 4). Loss of Atg7 in mouse hepatocytes causes marked accumulation of swollen and deformed mitochondria and the appearance of concentric membranous structures consisting of ER. In addition, loss of Atg7 leads to an increased number of peroxisomes and lipid droplets, as well as the formation of protein aggregates positive for p62 and ubiquitin (Komatsu et al., 2005). These mutant mice exhibit severe hepatomegaly (i.e., an enlarged liver) and hepatocytic hypertrophy, followed by hepatitis (Komatsu et al., 2005).