Macroautophagy is a major intracellular degradation process recognized as playing a central role in cell survival and longevity. This multistep process is extensively regulated at several levels, including post-translationally through the action of conserved longevity factors such as the nutrient sensor TOR. More recently, transcriptional regulation of autophagy genes has emerged as an important mechanism for ensuring the somatic maintenance and homeostasis necessary for a long life span. Autophagy is increased in many long-lived model organisms and contributes significantly to their longevity. In turn, conserved transcription factors, particularly the helixloop-helix transcription factor TFEB and the forkhead transcription factor FOXO, control the expression of many autophagy-related genes and are important for life-span extension. In this review, we discuss recent progress in understanding the contribution of these transcription factors to macroautophagy regulation in the context of aging. We also review current research on epigenetic changes, such as histone modification by the deacetylase SIRT1, that influence autophagy-related gene expression and additionally affect aging. Understanding the molecular regulation of macroautophagy in relation to aging may offer new avenues for the treatment of age-related diseases

 The membrane used to form the phagophore can originate from different locations, such as the endoplasmic reticulum, mitochondria, Golgi, endosomes, and plasma membrane (reviewed in refs. 2, 3). Integration of membrane lipids into the phagophore requires the synthesis of phosphatidylinositol 3-phosphate (PtdIns3P) by the phosphatidylinositol 3-kinase (PtdIns3K) nucleation complex. PtdIns3P is then recognized by PtdIns3P-binding proteins,4,5 which may possibly act as a shuttle by transferring membrane lipids between a membrane donor and the growing phagophore.6 Phagophore elongation is further dependent on 2 ubiquitin-like conjugation reactions. First, the ubiquitin-like protein ATG12 is covalently conjugated to ATG5 by ATG7 and ATG10 (E1- and E2-like enzymes, respectively). The ATG12–ATG5 conjugate, together with its interaction partner ATG16L1 is thought to act in part as a an E3-like ligase to promote conjugation of phosphatidylethanolamine (PE) to soluble LC3-I/Atg8, formed by cleavage of the ubiquitin-like protein LC3/Atg8 by the protease ATG4.7 The resulting PE-conjugated, membrane-bound LC3-II/Atg8–PE is important for phagophore elongation as well as cargo recognition.LC3-II/Atg8–PE can be bound by various cargo receptors; for example, SQSTM1/ p62, which recognizes ubiquitinated proteins or organelles targeted for degradation.8 The phagophore undergoes further maturation to fully encapsulate its cargo; the completed autophagosome releases outer membrane-associated autophagy-related proteins, after which it is ready to dock and fuse to the lysosome/vacuole to form an autolysosome. 

Upon fusion, the inner membrane of the autophagosome and the lumenal cargo are degraded, and the autolysosome reforms as a lysosome that is possibly again available for subsequent vesicular fusion events.9 Incomplete processing of autolysosomes can instead produce a residual body containing indigestible material.10 Notably, several age-related diseases, such as neurodegenerative disorders, are characterized by the accumulation of unprocessed autophagic vacuoles (reviewed in refs. 11, 12), suggesting that the ability of cells to efficiently coordinate and complete the autophagic process is gradually impaired with age.Upstream regulators of macroautophagy Autophagy is critical to cellular homeostasis under both normal and stressed conditions. Basal levels of autophagy ensure intracellular quality control, whereas autophagy induced by starvation and other stressors promotes cellular survival by maintaining adequate amino acid pools and cellular energy levels. Until recently, induction of autophagy was thought to be dependent primarily on post-translational regulation of nutrient-responsive pathways; however, it is now clear that alternative means of regulation exist. The emerging picture is that rapid induction of autophagy, e.g., in response to starvation, is mediated by posttranslational protein modifications and protein-protein interactions, whereas transcriptional mechanisms are necessary for a sustained response (reviewed in ref. 47). One key regulator of autophagy is the kinase MTOR/TOR (mammalian target of rapamycin), which is a component of 2 complexes, MTORC1 and MTORC2. 

As part of the MTORC1 complex, TOR regulates cell growth, proliferation, survival, protein synthesis, and autophagy, whereas the MTORC2 complex primarily regulates different downstream signaling cascades modulating cell shape and metabolism, but can also regulate autophagy indirectly (reviewed in ref. 13). The MTORC1 complex (hereafter referred to as MTOR) directly inhibits autophagy through phosphorylation and inactivation of ULK/Atg1 and ATG13/Atg13, which are essential for the induction of autophagy (reviewed in ref. 14). TOR activity can be modulated by changes in amino acid abundance via interaction with RAG small GTPases.15 These enzymes are localized at the lysosomal surface through their interaction with the pentameric Ragulator complex.16 Elevated lysosomal amino acid levels, which may reflect the overall cellular abundance of amino acids, promote the guanine-nucleotide exchange factor function of the Ragulator complex17 and subsequent activation of RAG GTPases. These proteins recruit TOR to the lysosomal membrane where it is ultimately activated by the small GTPase RHEB.18 MTOR serves as a hub to integrate additional upstream signals from various sources, including INS/insulin, growth factors, and cellular energy levels (reviewed in refs. 13, 19). One notable pathway intersecting with MTOR is the INS-IGF1 pathway, which is involved in many functions necessary for metabolism, growth, and longevity.20 Formation of PtdIns(3,4,5)P3 by activated PtdIns3K, a downstream effector of the INS-IGF1 pathway, recruits the protein kinase AKT to the plasma membrane, where it is phosphorylated and activated by the PDPK1/2 (3-phosphoinositide dependent protein kinase 1/2).

 AKT modulates the function of TSC2, a component of the heterodimeric tuberous sclerosis complex (TSC). The TSC1/2 complex can inhibit MTOR signaling by preventing activation of RHEB.21 Upon growth factor or INS-IGF1 signaling, TSC2 is phosphorylated and inactivated by AKT, thereby allowing RHEB to activate MTOR.22 The TSC-RHEB-MTOR cascade therefore represents a major signaling axis for the control of growth and autophagy. Autophagy is also regulated by intracellular energy levels via the energy sensor AMPK (AMP-activated protein kinase), which directly activates the ULK/Atg1 initiation complex (reviewed in ref. 23). AMPK activity is sensitive to AMP levels and is further regulated by phosphorylation by upstream kinases such as STK11/LKB1 and CAMKK2/CamKKb (calcium/calmodulindependent protein kinase kinase 2, b).24  Although STK11/LKB1 is constitutively active, CAMKK2 has been implicated in AMPK regulation and autophagy induction only in response to changes in intracellular Ca2C levels.25 AMPK can also influence autophagy by inhibiting MTOR, either by phosphorylating the MTORC1 subunit RPTOR/raptor or by inhibiting phosphorylation of TSC1/2.26 Both AMPK and MTOR can themselves be phosphorylated by ULK/Atg1, providing an additional level of regulatory feedback to modulate and fine-tune autophagy (reviewed in ref. 27) In addition to regulating autophagy, AMPK and MTOR also modulate organismal aging in a conserved fashion. Specifically, inhibition of MTOR extends the life span of organisms ranging from yeast to mice,28 and overexpression of AMPK promotes longevity in worms and flies.24

 Collectively, these findings demonstrate that nutrient sensors are major regulators of life span in a variety of species and share autophagy as a downstream effector mechanism for the maintenance of health. Direct links between autophagy and aging Accumulating evidence over the past decade supports a direct role for autophagy in the aging process. Multiple genetic experiments have demonstrated a requirement for autophagy-related genes in many longevity paradigms, including inhibition of TOR activity in yeast (ATG1, ATG11, ATG7),29 worms (unc-51/Ulk/ ATG1, bec-1/Becn1/VPS30/ATG6, vps-34, atg-18/Wipi1/2) 30,31 and flies (Atg5).32 Similarly, life-span extension by dietary restriction is abrogated in autophagy-deficient yeast (ATG7, ATG5, ATG8, ATG15, and v-SNARE genes VAM3, and VAM7) 33,34 and in worms (unc-51/Ulk/ATG1, bec-1/Becn1/VPS30/ATG6, vps-34, atg-7).30,31,35 Similar links have been observed in other conserved longevity models (i.e., reduced INS-IGF1 signaling, germline removal; mitochondrial respiration, mRNA translation; as well as resveratrol and spermidine supplementation), which all cause induction of autophagy markers, and in each case, the resulting life-span extension is dependent on at least one autophagy gene (see Table 1 for a summary of direct links between autophagy-related genes and longevity). In support of such a link, overexpression of specific autophagy genes has been found to promote longevity in several different species. In mice, heterologous overexpression of ATG5 is sufficient to stimulate autophagy, promote a youthful appearance, and extend life span.36 In Drosophila, overexpression of Atg8a in the neurons and muscle of adult flies extends their life span.37,38

 Similarly, neuron-specific overexpression of Atg1 in adult Drosophila induces autophagy both cell autonomously and non-cell autonomously, and also results in life-span extension.39 Consistent with the physiological relevance of these observations, many autophagy genes (i.e., Atg1, Atg6, Atg7, Atg5, Atg8) show reduced expression with age in flies,37,40 and LC3 and ATG7 protein levels in muscle also decline with age in mice and humans.41 This culminates in the loss of autophagic capacity generally observed with normal aging (reviewed in ref. 42). Notably, genetic and age-related loss of adequate autophagic and lysosomal function has been linked to the development of several metabolic and neurodegenerative diseases (reviewed in ref. 11). For example, loss-of-function mutations in several genes with autophagy-related functions (e.g., Becn1/VPS30/ATG6, 43 Atg744, Atg545) result in decreased autophagy and increased accumulation of disordered and aggregated proteins in neurodegenerative disorders such as Huntington disease (HTT/huntingtin), Alzheimer disease (Ab and MAPT/tau), and Parkinson disease (SNCA/a-synuclein) (reviewed in ref. 46). Accumulating evidence thus supports a beneficial role for autophagy in aging, although the underlying mechanisms of autophagy regulation in long-lived organisms are not fully understood. In this review, we focus on the role of transcriptional and epigenetic regulation of autophagy in the context of aging by highlighting studies in longevity models, and noting relevance to age-related diseases where applicable.

 TFEB was first described as a key transcription factor in lysosomal biogenesis,58 and subsequent studies revealed additional roles in the regulation of genes involved in autophagosome formation (e.g., Atg9, Wipi1/2/Atg18, Lc3/Atg8), cargo recognition (e.g., Sqstm1), vacuolar fusion (e.g., Vps11, Vps18), vacuolar proton pumping (e.g., V-ATPase subunits), and lysosomal degradation (e.g., sulfatases and cathepsins).52 Thus, TFEB regulates autophagic flux by coordinating the expression of genes with functions at all stages of the autophagy process, from vesicle initiation to cargo degradation. Under nutrient-rich conditions, MTOR phosphorylates TFEB at the lysosomal surface, which results in the binding of YWHA/14–3–3 proteins and the retention of TFEB in the cytosol.56,57 The TFEB sequence contains several predicted MTOR and MAPK1/ERK2 phosphorylation sites, and mutation of serine 142 or serine 211 to nonphosphorylatable residues has been reported to induce nuclear localization of TFEB.55–57 Additionally, TFEB nuclear localization is induced by the TOR inhibitors rapamycin and Torin.55,56 Similarly, TFEB translocates to the nucleus in response to nutrient deprivation, causing increased lysosomal calcium release and activation of the phosphatase calcineurin (CaN) to directly dephosphorylate TFEB.169 Interestingly, another member of the MITF family, TFE3, can bind to similar promoter sequences found on autophagy-related target genes of TFEB (so-called CLEAR sites), displays conserved regulation by TOR, and has overlapping functions in lysosomal biogenesis and autophagy gene regulation under nutrient-scarce conditions. 

Moreover, overexpression of TFE3 promotes lysosomal biogenesis and autophagy. These findings suggest the potential nuclear coordination of MITF transcription factors in controlling autophagy.59 Nevertheless, TFEB and TFE3 must have distinct functions, as deletion of Tfeb in mice results in embryonic lethality, whereas deletion of Tfe3 has no apparent phenotype.60,61 Recent work has shown that TFEB is recruited to the lysosome for TOR phosphorylation by interaction with FLCN/folliculin,62,63 suggesting a complex regulatory network governs the intracellular localization of TFEB, and thus its activity (Fig. 1C). Taken together, recent studies demonstrate an important function for lysosomes, beyond their role in degradation, as sites for the integration of signaling by the nutrient sensor TOR. The TFEB homolog in C. elegans, HLH-30, plays a role similar to that of TFEB in the induction of orthologous target genes and activation of autophagy.64 Indeed, HLH-30 localization to the nucleus is induced by RNA interference (RNAi)-mediated inhibition of let-363/Tor, 64 nutrient deprivation,65 and by 4 additional conserved longevity paradigms (reduced insulin signaling, mRNA translation, and mitochondrial respiration, and removal of the germline) that induce autophagy markers and require autophagy genes for life-span extension.64 The induction of autophagy gene expression by either let-363/Tor inhibition or germline removal requires hlh-30, and hlh-30 is indispensable for the life-span extension observed in all of these 6 autophagydependent C. elegans longevity models.64,66 

These observations are consistent with HLH-30 transactivation causing an increase in autophagic flux, which is necessary for life-span extension in these longevity paradigms. In support of this, overexpression of HLH-30/TFEB is sufficient to activate autophagy and to moderately extend C. elegans life span.64 HLH-30/TFEB is negatively regulated by another helix-loop-helix transcription factor, MXL3, which provides precise temporal control of HLH-30/TFEB activity to maintain homeostasis.67 In mammals, the transcription factor ZNF24/ZSCAN3 works in opposition to TFEB by acting as a repressor of TFEB target genes.68 Nuclear TFEB levels are elevated in liver cells of long-lived, dietary-restricted mice,64 indicating that TFEB is a component of a conserved longevity mechanism. Other targets of HLH-30/TFEB suggested to play a role in life-span extension (Table 1) include the lysosomal acid lipases lipl-1 and lipl-3, 67 supporting previous observations suggesting that increased lipolysis69 via lipophagy by the homologous lysosomal acid lipase lipl-4, 70 may represent a central life-spanextending mechanism. Of note, TFEB has been linked to changes in lipid metabolism induced by starvation.65,67 Lipophagy-mediated remodeling and utilization of lipids for energy generation and signaling via lipophagic products may represent possible mechanisms by which cellular homeostasis, organelle biogenesis, and survival are ensured during nutrient deprivation. Indeed, elevation of TFEB activity by overexpression or by starvation leads to an increase in PPARA/PPARa and PPARGC1A/PGC1a expression,65,71 suggesting an enhanced cellular ability to respond to lipid signals. 

Thus, TFEB-mediated transcriptional induction of autophagy may be central for increasing autophagic flux in order to provide a dynamic pool of metabolites, particularly lipids, for synthetic and signaling pathways conducive to longevity. The conserved regulation of TFEB activity by MTOR supports the notion that nutrient signaling is pivotal for the control of autophagic flux. Thus, TFEB stimulation of autophagy-related and lysosomal gene expression coordinates each step of the process to provide a vital source of metabolites during periods of nutrient deprivation. Recent work suggests an emerging role for TFEB, beyond aging and metabolism, in the pathology of diseases. For example, overexpression of TFEB has beneficial effects on cellular clearance in models of lysosomal storage diseases, Parkinson disease, and a1-antitrypsin deficiency.52,72,73 In addition, heterologous overexpression of TFEB in mouse brain increases the clearance of aberrant MAPT/tau protein, a key player in Alzheimer disease, in part by improving lysosomal function.74,74 Benefits of TFEB induction in the turnover of disease-related proteins such as HTT/huntingtin also involves interactions with PPARGC1A,75 a transcriptional cofactor involved in mitochondrial biogenesis and recently linked to longevity in Drosophila. 76 Conversely, in X-linked spinal and bulbar muscle atrophy, TFEB transactivation is inhibited by interaction with polyglutamine-expanded ARs/androgen receptors, with a subsequent impairment of autophagy.77

 This recent finding raises the possibility that TFEB activity may be impaired by the age-related accumulation and aggregation of disordered proteins in a variety of neurodegenerative diseases. Interestingly, TFEB is also required for the response to infection in both worms and mice, suggesting a broad and conserved role for this transcription factor in survival associated with a variety of stressors.78 Taken together, these studies highlight a critical role of TFEB in promoting autophagy under physiological and pathological conditions, including the increasingly prevalent age-related diseases.Forkhead transcription factors Another major family of transcriptional regulators of autophagy with a conserved role in aging is the forkhead transcription factors (FOXO), which play well-recognized and central roles in cellular homeostasis through the regulation of genes involved in lipid and glucose metabolism and in mitochondrial function.79 A role for the FOXO family in autophagy was first described in murine models of muscle atrophy, an age-related condition80,81 to which several degradative pathways contribute, especially autophagy.82 In the muscle, FOXO1 and FOXO3 elevate the autophagic flux by increasing the expression of autophagy genes mainly working as part of the core machinery (Ulk2/ATG1, Pik3c3/VPS34, Becn1/VPS30/ATG6, Atg4B/ATG4, Lc3b/ATG8, GabarapL1/ATG8, Atg12/ATG12, Bnip3) 80,81,83,84 and additionally increase protein degradation via the proteasomal pathway.80,85,171 In particular, FOXO3 increases the capacity of the lysosome to degrade incoming cargo, indicating a role for lysosomal function in muscle atrophy.