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INVITED REVIEWJournal of PathologyJ Pathol 2012; 226: 255–273Published online in Wiley Online Library(wileyonlinelibrary.com) DOI: 10.1002/path.3025

Autophagy and disease: always two sides to a problemSunandini Sridhar,1 Yair Botbol,2 Fernando Macian2,3 and Ana Maria Cuervo1,3*

1 Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, New York, USA2 Department of Pathology, Albert Einstein College of Medicine, New York, USA3 Institute for Aging Studies, Albert Einstein College of Medicine, New York, USA

*Correspondence to: Ana Maria Cuervo, Department of Developmental and Molecular Biology, Institute for Aging Research, Albert EinsteinCollege of Medicine, Chanin Building, Room 504, 1300 Morris Park Avenue, Bronx, NY 10461, USA. e-mail: [email protected]

AbstractAutophagy is a process traditionally known to contribute to cellular cleaning through the removal of intracellularcomponents in lysosomes. In recent years, intensive scrutiny at the molecular level to which autophagy has beensubjected has also contributed to expanding our understanding of the physiological role of this pathway. Addedto the well-characterized role in quality control, autophagy has proved to be important in the maintenance ofcellular homeostasis and of the energetic balance, in cellular and tissue remodelling, and cellular defence againstextracellular insults and pathogens. It is not a surprise that, in light of this growing number of physiologicalfunctions, connections between autophagic malfunction and human pathologies have also been strengthened. Inthis review, we focus on several pathological conditions associated with primary or secondary defects in autophagyand comment on a recurring theme for many of them, ie the fact that autophagy can often exert both beneficialand aggravating effects on the progression of disease. Elucidating the factors that determine the switch betweenthese dual functions of autophagy in disease has become a priority when considering the potential therapeuticimplications of the pharmacological modulation of autophagy in many of these pathological conditions.Copyright 2011 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Keywords: cancer; cardiomyopathy; immunity; lysosomes; neurodegeneration; protein aggregation; proteolysis; proteotoxicity; T cellfunction

Received 6 August 2011; Revised 25 September 2011; Accepted 3 October 2011

No conflicts of interest were declared.

Introduction to autophagy

The Greek term ‘autophagy’ (‘self-eating’) was coinedalmost half a century ago by Christian DeDuve [1].Today, this intensely investigated pathway has cometo be recognized as an evolutionarily conserved intra-cellular process through which cytosolic components,ranging from proteins, lipids, sugars and nucleotides towhole organelles and invading pathogens, are targetedfor lysosomal degradation [2,3]. Autophagy contributesto both the removal of damaged long-lived proteins andorganelles and the normal turnover of these intracellu-lar components as part of its role in cellular qualitycontrol. In addition, autophagy also serves as a cellu-lar adaptive response to compromised conditions, suchas metabolic stress, when degradation of intracellularmaterials through this pathway becomes an alternativesource of energy [4].

In recent years, a growing number of functions havebeen added to these two basic cellular functions of theautophagic process—quality control and maintenanceof the cellular energetic balance—helping to shape thephysiological relevance of this pathway. Furthermore, abetter understanding of the molecular mechanisms thatmediate the autophagic process has helped to establish

connections between autophagy and the pathogenesisof different disorders and of ageing [3,5,6]. Findingsin recent years also support the existence of morethan one type of autophagy and the fact that differentautophagic pathways co-exist in most cells. In thisreview, we briefly describe the main characteristicsand molecular components of the different autophagicvariants and then provide a general view of the multiplephysiological functions of this cellular process. In thesecond part, we comment on specific autophagy-relatedpathologies, which either result from a primary defectin the autophagic process, or for which changes inautophagy have been described to exert a modulatoryeffect on the course of the disease.

The molecular dissection of the differentautophagic pathways

To date, three basic forms of autophagy have beendescribed, namely, macroautophagy, microautophagyand chaperone-mediated autophagy (CMA), which pri-marily differ in the way in which cytosolic components(cargo) are delivered to lysosomes [2,7,8] (Figure 1).Macroautophagy and microautophagy were both first

Copyright 2011 Pathological Society of Great Britain and Ireland. J Pathol 2012; 226: 255–273Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk www.thejournalofpathology.com

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Figure 1. Autophagic pathways. Three different general mechanisms of lysosomal delivery of cargo set the basis for the different typesof autophagy described in mammalian cells. (A) Macroautophagy : different extra- and intracellular signals activate the recruitment ofthe macroautophagy initiation complex to the sites of autophagosome formation. Shuttling of proteins and lipids to these regions andposttranslational modifications of the lipids initiate the formation of a limiting membrane, which grows through the assembly of proteinsconjugated to proteins or lipids while it sequesters components of the cytosol. Once the membrane seals to form the autophagosome,this double membrane vesicle is delivered to lysosomes where, upon membrane fusion, lysosomal hydrolases gain access to cargo.(B) Microautophagy : through stimuli that remain poorly identified, cytosolic soluble proteins and organelles are directly sequestered byinvaginations in the boundary membrane of lysosomes and late endosomes. Cargo internalized in the small luminal vesicles is degradedafter the vesicles pinch off from the limiting membrane. Although most microautophagy probably occurs in bulk, selective targeting byhsc70 of cytosolic proteins to microvesicles as they form in late endosomes has also been described. (C) Chaperone-mediated autophagy(CMA) is induced by stimuli such as prolonged starvation, oxidative stress and other conditions resulting in protein damage, but thesignalling mechanism activated by these stimuli remains unknown. When CMA is activated, selective cytosolic proteins bearing a targetingmotif are recruited by hsc70 and co-chaperones to the surface of lysosomes. Upon binding to the receptor protein LAMP-2A, substratescross the membrane through the LAMP-2A-dependent translocation complex and are then rapidly degraded in the lumen.

described as mechanisms for ‘in-bulk’ sequestra-tion of cytoplasmic components (including entireorganelles) into vesicular compartments. In macroau-tophagy, these vesicles form de novo from a limit-ing membrane of non-lysosomal origin that seals uponitself to form free-standing double membrane carriersknown as autophagosomes [9,10]. Fusion of lysosomeswith the limiting membrane of the autophagosomegrants lysosomal hydrolases access to the sequesteredcargo, assuring its complete degradation. In the caseof microautophagy, the engulfment of cargo occursthrough deformation of the lysosomal membrane itself,to form vesicles that invaginate towards the lysoso-mal lumen [11,12]. Pinch-off of these vesicles fromthe lysosomal membrane into the lumen, and the sub-sequent degradation of their limiting membrane by thelysosomal hydrolases, precede cargo degradation.

Both macroautophagy and microautophagy were ini-tially described in mammalian cells, but their molec-ular characterization has been mostly carried out inyeast, taking advantage of the genetic analysis that canbe performed in this model organism. These geneticscreenings have identified that more than 30 differ-ent genes (known as ATG or autophagy-related genes)

participate in the execution and regulation of macroau-tophagy [2,13] (Figure 1). The protein products ofthese genes (Atg or autophagy-related proteins) orga-nize into functional complexes that mediate the fol-lowing steps in the autophagic process: nucleationof the limiting membrane, elongation of the mem-brane, sealing to form a vesicle and fusion of thedouble membrane vesicle with lysosomes. Nucleationis attained through post-translational modifications ofpre-existing lipids in the membranes of different intra-cellular organelles [endoplasmic reticulum (ER), mito-chondria, Golgi] as well as in the plasma membrane[14–16]. Coordinated recruitment of lipid-modifyingenzymes and proteins at the place of autophagosomeformation gives rise to the limiting membrane [17].Growth of this membrane occurs through two enzyme-regulated processes that mediate conjugation of (1) aprotein—light chain protein 3 or LC3—to one of theconstituent membrane lipids and (2) of two cytoso-lic proteins (Atg5 and Atg12) between them [9,18].SNARE-like proteins, molecular motors and additionallipid-modifying enzymes (phosphatases and kinases)participate in autophagosome-lysosome fusion. Mostof the regulatory components exert their action onthe nucleation complexes. For example, the Target of


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Rapamycin, a well-characterized negative regulator ofthis pathway, prevents the recruitment and interactionof specific Atgs of the nucleation complex to the siteof autophagosome formation [19–21]. Although lessis known about the regulation of later steps of theautophagic process, recent studies have identified thetranscription factor EB as a global regulator of Atgsthat can up-regulate initiation, elongation and fusion,as well as lysosomal biogenesis in response to nutri-tional stress [22]. Homologues for most yeast Atgshave been identified in many other species (Drosophila[23], Caenorhabditis elegans [24], Dictyostelium [25],Arabidopsis [26], Trypanosoma [27]) and in mammals,where these genes often have several variants withdiverse functions [28].

The number of yeast genes shown to participatein microautophagy does not exceed a dozen, butthis process also partially depends on Atgs sharedwith macroautophagy [29]. Microautophagy-specificproteins contribute to the formation and sealing ofthe vacuolar membrane (equivalent of the lysosome inyeast) [30]. Failure to identify mammalian homologuesof the yeast microautophagy genes initially led to theproposal that microautophagy was not evolutionarilyconserved. However, recent studies show otherwise,as a microautophagy-like process has been describedto occur in mammalian late endosomes. This process,known as endosomal microautophagy, has adopted themachinery involved in biogenesis of multivesicularbodies to form the invaginating vesicles that sequestercytosolic material for degradation [31].

In recent years, in addition to this ‘in bulk’ non-selective degradation of cytosolic components, specificsequestration of cargo has also been described forboth macro- and microautophagy. In the case of selec-tive macroautophagy, the distinctive characteristic isthe presence of a single type of cytosolic material inautophagosomes, in clear contrast to the ‘in-bulk’ pro-cess, where heterogeneous cargo occupies the lumenof the autophagosome. Selective forms of macroau-tophagy are named depending on the sequestered mate-rial, giving rise to terms such as mitophagy [32,33],pexophagy [34,35], reticulophagy [36,37], lipophagy[38], ribophagy [39] and aggrephagy [40]. Similar cri-teria apply to selective forms of microautophagy, suchas micropexophagy [34,35] micronucleophagy [41,42]and microglycophagy [43]. All these selective formsof autophagy utilize the same essential componentsof the autophagic machinery described for their ‘in-bulk’ variants, but they add an extra step related tocargo recognition. A specific subset of proteins, knownas cargo-recognition proteins, have been involved inthis step and include, among others, p62, neighbourof BRCA1 (NBR1), Nix and the PINK1/Parkin pair[44,45]. These adaptor proteins connect the degra-dation machinery with particular components—oftenubiquitin moieties—on the surface of the cytosolicorganelle to be sequestered [44,46,47]. Cytosolic chap-erones, in particular the heat shock cognate proteinof 70 kDa, or hsc70, and BAG-3, have also been

recently described to contribute to selective recogni-tion of aggregates by macroautophagy in chaperone-assisted selective autophagy (CASA) [48].

Selectivity is the distinctive feature of CMA, thethird type of mammalian autophagy, wherein particularsoluble proteins bearing in their sequence a pentapep-tide targeting motif biochemically related to KFERQ,are recognized by hsc70, which mediates their deliv-ery to the lysosomes for direct translocation acrossthe lysosomal membrane [8,49] (Figure 1). Althoughyeast genetic approaches could not be applied to themolecular dissection of CMA, since this pathway hasso far been described only in mammals, about eightdifferent proteins have been identified to participatein this pathway using biochemical procedures. Mostof the identified CMA components have promiscuousfunctions and are shared with other intracellular pro-cesses. Such is the case for the cytosolic chaperoneshsc70 and hsp90 [50,51] and the novel pair of reg-ulators, glial fibrillary acidic protein and elongationfactor 1α [52]. Interestingly, lysosome-specific vari-ants have been identified for each of these proteins,suggesting that post-translational modifications of thecytosolic proteins determine their association with thisdegradative compartment and their commitment toCMA. So far, the only CMA-exclusive component isthe lysosome-associated membrane protein type 2A(LAMP-2A), which acts as a receptor for substrates ofthis pathway [53]. Once the cytosolic proteins destinedfor CMA degradation bind to this single transmem-brane protein, they promote its assembly into a higher-order molecular complex required for translocation ofthe CMA substrates into the lysosomal lumen [51].

Interested readers are referred to more focusedreviews [2,3,8,29,54] for a comprehensive descriptionof the molecular apparatus executing these differentautophagic pathways. For the purposes of this review,the term ‘autophagy’ will represent macroautophagy,unless otherwise mentioned.

Physiological relevance of autophagy

As described in the Introduction, a growing number ofcellular functions have been attributed to autophagy.We highlight here some of the best-characterized func-tions that have contributed to expanding the physio-logical relevance of this catabolic process. The roleof autophagy in cellular defence, by participating ininnate and adaptive immunity, is discussed in detailin the section dedicated to autophagy and the immunefunction.

Autophagy in the cellular energetic balanceBoth macroautophagy and CMA are induced as anacute adaptive response to a variety of metabolicstressors, including, among others, nutrient starvation,growth factor withdrawal, high lipid content chal-lenges or hypoxia [4,55–57]. Under these conditions,


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free amino acids (especially branched-chain aminoacids), released by autophagic proteolysis of intracel-lular proteins and organelles, are recycled to main-tain protein synthesis even when nutrients are scarce.Autophagy in the liver converts this organ into amain source of amino acids that are then deliveredto other organs through the blood stream during star-vation [5,56,58,59]. In fact, the low plasma concen-trations of essential amino acids and decreased ATPlevels in most tissues observed in autophagy defectiveAtg5−/− and Atg7−/− neonates confirm the impor-tance of this pathway during nutritional deprivation,in this case as a result of the cessation in transpla-cental nutrient supply at birth [60,61]. Amino acidsresulting from autophagic breakdown could also beutilized for the production of cellular ATP throughdirect oxidation, or by fuelling the TCA cycle and glu-coneogenesis with intermediates such as oxaloacetate.This contribution of amino acids to energy homeostasishas been confirmed in specific conditions, such as inthe case of interleukin-3-deprived haematopoietic cells,where the addition of TCA cycle substrates, such asmethylpyruvate, is enough to preserve cellular viability[62]. However, amino acids are not an efficient sourceof energy. Instead, the array of energy stores mobi-lized by autophagy is now recognized to include moreenergetically efficient molecules, such as lipids, glyco-gen and nucleic acids. The hydrolytic products of thesemolecules, free fatty acids, glucose and nucleotides,can be funnelled into the TCA cycle, gluconeogenesisor glycolysis to produce ATP [4,43].

This capability of autophagy to maintain ATP pro-duction and support macromolecular synthesis makesit a pro-survival pathway of particular importance inorgans with high energetic requirements, such as theheart or skeletal muscles. Alterations of this specificautophagic function also constitute the basis of somecommon metabolic disorders.

Autophagy and cellular quality control

The proteome and cellular organelles are susceptible todifferent toxic insults that can lead to the generation ofmisfolded or modified soluble proteins, protein cross-linking and oligomerization into high-order irreversiblestructures or aggregates, and accumulation of defec-tive, no longer functional, organelles that could becomeharmful for the cells [63–65]. Basal autophagy, oftenreferred to as ‘quality control autophagy’, is integralto the cellular surveillance machinery responsible forrecognition and removal of these aberrant structures[66,67]. Basal autophagy is particularly important innon-dividing post-mitotic cells that cannot dilute thecellular damage through division. For example, condi-tional knock-out of the essential autophagy genes atg7or atg5 in hepatocytes, neurons and cardiomyocytesleads to marked accumulation of ubiquitin-positive pro-tein aggregates and damaged organelles, even in theabsence of an added toxic challenge [60,68,69].

Exposure to stressors, such as oxidative stress, ERstress or other conditions resulting in massive amountsof unfolded proteins and organelle damage, elicits acti-vation of inducible forms of autophagy [70–73]. In thiscontext, activation of CMA contributes to the selec-tive removal of soluble (aggregate-prone) proteins,whereas macroautophagy facilitates the clearance ofprotein aggregates [40,63,74,75] and whole organelles[33,36,37]. However, it is possible that macroau-tophagy ameliorates proteotoxicity not only throughaggregate removal but also by the in-bulk sequestra-tion of ‘still-soluble’ forms of the pathogenic proteinsbefore they aggregate [68,76,77]. Autophagy is alsoimportant to restore organelle homeostasis [36,72,78],to eliminate damaged organelles after stress [37,79]and to assist cells to adapt their organelle content tothe changing environmental conditions (eg it controlsperoxisome number during nutrient auxotrophy or totalmitochondria mass for energetic balance) [80].

Autophagy-mediated control of protein and organellequality, as well as organelle number, is essential for themaintenance of cellular homeostasis and to guaranteecellular survival during stress.

Cellular remodelling and autophagyCellular differentiation often requires the eliminationof large subsets of proteins, nucleic acids or organellesin order to switch into the next developmental stage orfinal differentiated state. Furthermore, cellular remod-elling is an energy-demanding process often associ-ated with nutrient-deprived developmental phases. Itis no surprise, therefore, that autophagy has emergedthrough evolution as one of the favoured cellulartools to accomplish these developmental remodellingtasks. The capability to eliminate whole regions of thecytosol, unique to some of the autophagy variants, andits recycling properties underlie the important role thatthis process plays during development. Autophagy hasbeen shown to be necessary in yeast during sporula-tion [81], in C. elegans during dauer formation [24],in Drosophila during the transition from the larval tothe pupal stage [82] and in Dictyostelium for switch-ing from amoeba to fruiting body [25]. Examplesof autophagy-mediated differentiation events in mam-malian cells include, among others, the removal ofmaternal macromolecules during early embryogenesis[83] and the clearance of mitochondria during ery-throcyte [84–86], lymphocyte [87,88] and adipocyte[89,90] differentiation.

Autophagy in cellular death and cell survivalAs indicated in the previous sections, the ability ofautophagy to assist cells to adapt to the changing envi-ronment, to protect them against the damage caused bytoxic insults and to defend them from pathogens has ledto the classification of this pathway as a cell-survivalmechanism. However, interplay between autophagyand mechanisms related to cell death have also beendescribed. The best-accepted example of autophagy as



a cell death effector is the degradation of the salivaryglands during pupal transition in Drosophila [82,91]. Incontrast, direct evidence of a similar role for autophagyin mammals is sparse [92]. In fact, many of the orig-inal claims of ‘cell death by autophagy’ have cur-rently been revised and reformulated as ‘cell deathwith autophagy’, since the main support for autophagicinvolvement in many of these cases was the obser-vation of autophagic vacuoles in the dying cell [93].Although an increase in the number of autophagic vac-uoles could result from their increased formation, thelarge capability of the lysosomal system often preventstheir accumulation by facilitating their rapid clearance.It is more frequent that the increase in autophagosomecontent results from a compromise in their clearanceby lysosomes and, consequently, from impaired ratherthan enhanced macroautophagy. Systematic analysis ofautophagic flux, rather than autophagic markers, shouldhelp to clarify these issues. However, independent ofthis initial controversy, it has become clear that, asdescribed in the following sections, once in a diseasecontext interactions between autophagy and apoptosis(or type I cell death) are complex and need to be anal-ysed in the specific disorder or cell type [94–96]. Infact, autophagy and apoptosis share some molecularcomponents and appear to be in a delicate balance witheach other in most systems examined [97,98].

Pathology of autophagy

The broad array of physiological functions attributedto autophagy justifies why alterations in this catabolicprocess lead to cellular malfunctioning and often celldeath, and has set the basis for its contribution to thepathogenesis of severe human disorders. Rather thanproviding an exhaustive list of disease conditions inwhich autophagy has been shown to be altered, inthe following sections we have selected four commonpathological conditions, cancer, neurodegeneration,heart dysfunction and immune and infectious diseases,to comment on their interplay with the autophagic sys-tem. The reason behind our selections is the fact thatin all of these pathologies growing evidence supportsa dual role of autophagy in their pathogenesis and dis-ease progression, which leads to the question, ‘Shouldwe inhibit or activate autophagy in these pathologies?’.

Autophagy and cancerCancer was the first human pathology connectedto autophagy through the discovery that Beclin1(Atg6/VPS30 homologue), a core component of theautophagosome nucleation complex, was monoalleli-cally deleted in 40–75% of sporadic human breast,ovarian and prostate cancers [99]. Independent stud-ies verified that heterozygous Beclin1+/− mice developspontaneous tumours, including lymphomas, lung car-cinomas, hepatocellular carcinomas and mammary pre-cancerous lesions [100,101]. Analogous generation

of spontaneous hyperproliferating tumours was alsoreported later upon deletion of other autophagy-relatedgenes, such as the ultraviolet resistance-associatedgene (UVRAG), the Bax interacting factor-1 (Bif-1 )and the LC3 (Atg8 homologue) processing proteaseAtg4c [102–104]. Furthermore, the products of com-mon oncogenes, such as class I PI3K, PKB, TOR andBcl-2, have been shown to act as autophagy repres-sors, whereas tumour suppressor gene products suchas p53, PTEN, DAPk and TSC1/TSC2 exert a stim-ulatory effect on autophagy (reviewed in [3,98,105]).Although, in light of this opposite effect of oncogenesand tumour suppressors on autophagy, this process wasinitially classified as an anti-oncogenic mechanism,this conclusion has been challenged by experimen-tal evidence supporting that under certain conditionsautophagy can also be pro-oncogenic (Figure 2).

From the perspective of cellular energetics, auto-phagy is likely a pro-survival mechanism in both earlyand late stages of cancer development, as it suppliescells with the energy and essential macromolecules tosustain normal cellular functions. However, it has beenproposed that in the early stages of cancer develop-ment, quality control by autophagy, particularly overgenome maintenance, inhibits tumourigenesis, confer-ring anti-oncogenic functions upon this pathway. Inaddition, autophagy could also play a role in the main-tenance or entry of cells into the G0 phase and con-sequently, prevent spontaneous hyperproliferation ofcells [106,107]. In contrast, in the late stages of onco-genesis, autophagy is necessary for cancer survival,as it contributes both energy for the rapidly dividingcancer cells and quality control functions to eliminateintracellular damage caused by the aggressive tumourmicroenvironment and by anti-oncogenic interventions(Figure 2). In fact, consistent with the pro-survivalfunction of autophagy, compromise of this pathway(ie Beclin1 interference) in apoptosis-deficient tumourcells impairs their survival in metabolic stress condi-tions in vitro and in vivo [108]. In general, blockageof autophagy sensitizes cells to metabolic stress, oftenleading to necrotic cell death accompanied by inflam-mation, a condition shown to promote tumourigene-sis [109–111]. Metabolic stress is intrinsic to rapidlygrowing tumours, in which poor vascularization resultsin lack of nutrients and oxygen for long periods of time[112–116]. Activation of macroautophagy under theseconditions has the potential to accommodate the acuteenergy demands by providing cancer cells with aminoacids, free fatty acids and glucose, required to gener-ate energy through the TCA cycle and via β-oxidation(Figure 2). Macroautophagy has also been recentlyimplicated in facilitating the unique utilization of glu-cose by cancer cells, known as the ‘Warburg effect’[117,118], by which cells favour glycolysis to accel-erate ATP production and generate glycolysis inter-mediates required for the transformed cells [119,120].Blockage of autophagy has been shown to prevent Ras-mediated oncogenic transformation by reducing glu-cose uptake and its utilization toward glycolysis [121].


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Figure 2. Autophagy and cancer. Autophagy may play opposite roles in the oncogenic process. Anti-tumoural effect (left): activemaintenance of cellular quality control for cytosolic pro-oncogenic proteins such as p62 prevents malignant transformation of non-tumoural cells. In addition, the supply of energy provided through macroautophagy activation reduces the dependence on glycolysis,while assuring the energy required for maintenance of a stable genome, further preventing oncogenesis. Pro-oncogenic effect (right): thereduction in macroautophagic activity in early stages of the oncogenic process favours malignant transformation, as the accumulationof molecules such as p62 activates signalling mechanisms that promote necrosis and inflammation. Poor quality control as a result ofdiminished macroautophagy can also result in accumulation of defective mitochondria, with the subsequent release of harmful molecules(cytochrome c and reactive oxygen species) that contribute to further altering genome maintenance. However, as the tumour progresses,activation of macroautophagy is observed in many oncogenic processes, in part to compensate for the poor nutritional supply associatedwith rapidly growing tumours and to defend cancer cells against damage induced by anti-oncogenic therapies. In addition, enhancedmitochondrial degradation in this stage may contribute to the up-regulation of glycolysis to maintain the energetic balance (Warburgeffect) characteristic of malignant cells.

Although it is clear that interrupting glycolysis elimi-nates the energetic advantage of the cancer cells andcompromises proliferation, the mechanism by whichautophagy up-regulates glycolysis is not known. It hasbeen proposed that this effect could in part be medi-ated by activation of the removal of mitochondria viaautophagy (mitophagy) to force energy dependence onglycolysis [119,120,122] (Figure 2). Autophagy couldimpact oncogenic transformation in multiple ways; inaddition to the proposed effect on glycolysis, a bettermaintenance of highly efficient mitochondria throughmitophagy has been recently proposed to also con-tribute to meeting the energetic requirements of thetransforming cells [123]. Furthermore, an additionalfactor in this already complex scenario is the possi-ble impact that metabolic changes in the surroundingstroma cells could have on the cancer cells. In fact,an autophagy-dependent metabolic switch in the can-cer stroma fibroblasts has been recently proposed. Thisswitch in the stroma could provide cancer cells withglycolytic intermediates necessary for efficient ATPproduction by oxidative phosphorylation [122].

Reduction in cellular energy upon autophagic com-promise also decreases the fidelity of cellular pro-cesses such as DNA replication and mitosis, result-ing in genetic aberrations [124]. The occurrence of

these aberrations in tumour cells with defective qual-ity control of organelles and toxic proteins due to theautophagic compromise can precipitate death of thecancer cells. In this respect, accumulation of the pro-tein p62 (normally degraded by macroautophagy) whenthis pathway is compromised leads to the formationof p62 aggregates capable of stimulating inflammationby inhibition of nuclear factor-κB (NF-κB) and activa-tion of NF-E2 related factor 2 (Nrf-2) [109,125,126](Figure 2).

Little is known to date about the contributions ofother forms of autophagy (CMA and microautophagy)to cancer biology. Recent studies have revealed thatCMA contributes to the turnover of inactive forms ofthe M2 isoform of pyruvate kinase, a key enzymein the maintenance of glycolysis in cancer cells[127]. A number of additional glycolytic enzymeshave been recognized as CMA substrates, includ-ing glyderaldehyde-3-phosphate dehydrogenase, phos-phoglycerate kinase, aspartate aminotransferase andaldolase B, strengthening the possible link betweenCMA and the energetic balance of cancer cells[49,128].

Collectively, the relationship between autophagy andcancer has proved to be a very complex one. This dualcapability of autophagy to, on the one hand facilitate



tumour progression and, on the other hand diminishmalignant transformation, has reinforced the need tocritically evaluate autophagy in a disease-context andstage-specific manner, especially during the designof targeted therapy, as autophagy function is tightlyrelated to these factors [78,94].

Effectors and modulators of macroautophagy havebecome attractive targets in the treatment of can-cer. Autophagy up-regulation has been described inmany different types of cancers in response to anti-oncogenic drugs, when it often exerts a protectiveeffect [129,130]. Based on these findings, down-regulation of autophagy should be an effective way ofenhancing the cytotoxic effect of different chemother-apy protocols. In fact, inhibition of macroautophagyhas proved to be effective in enhancing the toxicity ofdrugs commonly used in cancer therapy, such as oxalo-platin [129] and the kinase inhibitors sorafenib [131],epirubicin [132] or 5-fluorouracil [133].

Different reports have also documented that the cyto-toxic effect of some common anti-cancer agents isin part due to their ability to up-regulate autophagy[134–138]. These findings now raise a note of cautionin the universal applicability of autophagy inhibitorsas anti-cancer drugs. However, additional studies arerequired to clarify the contribution of the observedincrease in autophagy in cancer cell death under theseconditions. Often, the proposed increase in autophagyis based on the fact that levels of autophagy-relatedproteins are higher during the treatment. Up-regulationof autophagy markers does not necessarily meanincreased autophagic flux. In fact, if autophagy is func-tioning properly, accumulation of autophagic compart-ments rarely occurs. It is thus possible that the observedincrease in autophagic markers in some of these studiesis actually a reflection of a compromise in the clear-ance of autophagic compartments, rather than in theirformation. Accumulation of autophagolysosomes cancontribute to cell toxicity and be detrimental for theaffected cells. Interestingly, this toxic effect of the‘congestion’ of the autophagic pathway is currentlybeing explored for therapeutic purposes. Althoughsome success has been obtained by directly targetingthe final steps of autophagy, the lysosomal degrada-tion, the characteristic plasticity of the lysosomal com-partment makes a very prolonged inhibition necessarybefore this intervention becomes detrimental. One wayaround this problem, which is currently utilized in dif-ferent clinical trials, is to combine the lysosomal block-age with enhanced formation of autophagosomes (egby inhibition of the mTOR complex with rapamycin)[139–141]. The higher content of autophagic vacuoles,as a result of both increased formation and reducedclearance, can compromise cell viability by differentmechanisms. Accumulation of these compartments inthe cytoplasm interferes with intracellular traffickingand prevents recycling of essential cellular compo-nents (amino acids, free fatty acids, etc.). In addition,as autophagolysosomes persist inside cells longer than

usual, their membranes often become leaky, facilitat-ing the release of potent lysosomal enzymes in thecytosol, further enhancing cellular damage and toxi-city.

Autophagy and neurodegenerationMaintenance of cellular homeostasis is essential inneurons, a typical example of non-dividing differen-tiated cells. The importance of autophagy in neuronalhomeostasis has now been well supported, despite orig-inal disagreement on whether autophagy was activein neurons. The reason for this contention was thefact that autophagosomes, the morphological featureof macroautophagy, were rarely observed in neurons.However, recent studies support that this pathway isconstitutively active and that it can be further up-regulated in neurons in response to very different stres-sors [142–144]. Perhaps the most conclusive supportfor the existence of basal autophagy in neurons and forits contribution to the maintenance of neuronal survivalcame from studies in transgenic mouse models con-ditionally knocked out for essential autophagy genesin the central nervous system [68,69]. These animals,even in the absence of any added stressor, displayedmarked neurodegeneration associated with the accumu-lation of intracellular protein inclusions and neuronalloss. The efficient capability of the lysosomal systemto remove newly formed autophagosomes could bebehind the low occurrence of detectable autophago-somes in neurons in a given moment.

As with many other cells, neurons up-regulateautophagy in response to common stressors but alsoin defence against neuron-specific injuries, such asaxotomy, neuronal ischaemia or excitatory toxicity[145–147] (Figure 3). Often, failure to up-regulateautophagy under these conditions or primary com-promise of autophagic activity during the stress pre-cipitates neuronal death post-injury [148]. However,activation of autophagy in neurons may have a rela-tively narrow window of opportunity, because recentreports have revealed that genetic or chemical inhibi-tion of autophagy becomes beneficial at some specificstates post-injury [105]. These findings suggest thateither excessive up-regulation of autophagy, or up-regulation of this pathway under conditions in whichautophagosome formation or clearance cannot be guar-anteed, may be harmful to neurons [149]. In furthersupport of the possible detrimental effect of massiveup-regulation of autophagy, recent studies have shownthat, under specific conditions, autophagosomes canbecome a source of reactive oxygen species and thusaggravate neurotoxicity [150].

Changes in autophagic activity have been describedin many protein conformational disorders, and amongthem neurodegenerative diseases have received particu-lar attention. The feature shared by all these conditionsis the presence of pathogenic proteins that accumulateinside neurons, organized in the form of oligomericor multimeric structures. The disease originates from


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Figure 3. Autophagy and Neurodegeneration. (A) Protective autophagy : macroautophagy and CMA both contribute to the maintenance ofneuronal homeostasis and are necessary in the defence of neurons against injury and stressors. (B) Defective macroautophagy : defects inmacroautophagy have been described to occur at very different levels in neurodegenerative diseases. Some of the possible steps affectedin this process are highlighted in the model, and the conditions in which they have been observed are described in the text. (C) DefectiveCMA: primary defects in CMA have been described both in Parkinson’s disease and in certain tauopathies. While in the former condition,pathogenic proteins such as α-synuclein can block access of other cytosolic proteins to lysosomes via CMA by abnormally binding to thetranslocation machinery, in tauopathies the accumulation at the surface of lysosomes of oligomeric forms of pathogenic tau targetedvia CMA destabilizes the lysosomal membrane and results in leakage of lysosomal enzymes into the cytosol, which often triggers cellulardeath.

both the loss of function of the pathogenic protein andthe toxicity associated with the presence of the abnor-mal protein structures, which often leads to neuronaldeath. The first connection between neurodegenera-tion and autophagy originated from the observationthat protein aggregates can be eliminated by autophagy[40,74] (Figure 3). In fact, pharmacological activationof this catabolic process in experimental animal mod-els of Huntington’s disease reduced cellular toxicityand slowed down progression of the disease [151].After these initial reports, the degradation of manyother pathogenic proteins by autophagy and the ben-eficial effect of up-regulation of this pathway haveproved to be true in experimental models of manyother neurodegenerative conditions [152]. In fact, notonly the acute pharmacological activation of autophagybut even chronic conditions that lead to a progressivemaintained up-regulation of this process, such as underconditions when ER stress is maintained, have beenshown to have a positive effect [153]. These findingshave now opened the possibility of utilizing modulatorsof autophagy as the basis for therapeutic approaches for

these types of pathology and, in fact, ongoing chemi-cal screenings aim at identifying chemical modulatorsof this pathway with higher selectivity and potency[154].

However, there are certain limitations to the uni-versal use of up-regulation of macroautophagy foranti-neurodegenerative purposes. In fact, it has beenreported that not all protein aggregates are recog-nized by the macroautophagic machinery. Despitethe current dissection of molecules that participatein autophagic recognition of aggregates (p62, NBR1and several cytosolic chaperones, including hsc70 andBAG-3), the presence of these proteins in an aggre-gate, although necessary, does not seem to be suffi-cient for autophagic recognition. Thus, some proteininclusions positive for these cargo-recognition proteinsremain unnoticed by the autophagic machinery, mak-ing superfluous any attempt to enhance autophago-some formation as a way to eliminate these toxicprotein products [155]. Further studies are needed toaddress whether expression of other cellular compo-nents or specific post-translational modifications in the



aggregated proteins could enhance their recognition bythe autophagic systems.

A second scenario, in which chemical up-regulationof macroautophagy may not be effective againstneurodegeneration, is in those conditions in whichmacroautophagy itself is compromised. Understandingthe macroautophagy step or steps defective in specificneurodegenerative disorders is a priority if autophagyenhancers are to be moved forward in the treatment ofthese conditions. Examples of defects in almost all ofthe steps of macroautophagy have been described indifferent neurodegenerative disorders [66] (Figure 3).For example, initiation of autophagy may be com-promised because of altered signalling through theinsulin or mTOR pathways, which is tightly bound toactivation of autophagy. Also, conditions resulting inlower content of available Atgs will reduce neuronalautophagic capability, such as the recently describeddepletion of LC3-II through abnormal interaction andaggregation with p62 in the presence of dopaminergicneurotoxin [156]. Along the same lines, abnormal inter-action between mutant α-synuclein (the protein thataccumulates in protein inclusions in Parkinson’s dis-ease) and Rab1 has been shown to misplace Atg9, aprotein required for the formation of autophagosomes,out of the ER membrane [157]. Under other conditions,problems arise at the level of cargo recognition, eitherbecause of alterations in the organelle-specific mark-ers for degradation or in the autophagic machinery. Aprimary defect at the level of the limiting membranethat seems to prevent selective recognition of cargo hasbeen recently described in cellular and animal modelsof Huntington’s disease [158]. In these cases, mutanthuntingtin binds tightly to the inner surface of the form-ing autophagosome, resulting in an abnormally highinteraction with p62 in this compartment that affects itsability to bind cargo. In other conditions, autophago-somes form and sequester the pertinent cargo but theyfail to be cleared from the cytosol. Problems withclearance could result from alterations at very differ-ent levels. For example, problems with vesicular traf-ficking could indirectly interfere with the mobilizationof autophagosomes toward the lysosomal compartment[159]. Pathogenic proteins can also interfere with thefusion step, which, although still not completely elu-cidated at the molecular level, is known to dependon different SNARE proteins, the actin cytoskeletonand the histone deacetylase 6 (HDAC6) [160]. Con-sequently, changes in any of these components couldlead to intracellular accumulation of autophagosomes.Lastly, primary defects in the lysosomal compartmentalso have a negative impact on clearance of autophago-somes in different neurological disorders. The rea-sons for lysosomal failure could be multiple. In prin-ciple, most lysosomal storage disorders could com-promise autophagosome clearance because often theaccumulation of the undegraded product inside lyso-somes limits their degradative capacity [161]. In addi-tion, other conditions that alter lysosomal membrane

stability, decrease lysosomal biogenesis or change lyso-somal pH could also alter autophagosome clearance.In fact, defective acidification of lysosomes, becauseof compromised targeting of a component of the pro-ton pump from the ER to the lysosomal membrane,has been already identified as causative of the reducedrates of autophagy in some models of Alzheimer’sdisease [162]. As functional analysis of autophagybecomes more broadly utilized in the study of neurode-generative diseases, it is likely that new connectionsbetween these pathologies and autophagy will becomeevident.

Alterations of CMA have also been described insome neurodegenerative disorders (Figure 3). Althoughin many of them, this pathway becomes up-regulatedas a consequence of the failure in macroautophagy, dif-ferent pathogenic proteins have been shown to directlyinterfere with CMA activity [163]. Mutant forms of α-synuclein can be targeted to lysosomes via CMA but,in contrast to the wild-type protein that binds to thelysosomal receptor and rapidly reaches the lumen fordegradation, the mutant variants fail to translocate. Per-sistence of mutant α-synuclein tightly bound to theCMA receptor at the lysosomal membrane inhibitslysosomal degradation of other cytosolic proteinsvia this pathway [164]. A similar effect was laterdescribed for wild-type α-synuclein upon modificationby dopamine, which makes CMA blockage relevant forsporadic forms of Parkinson’s disease, more commonthan the familial forms [165]. Later studies have alsoproposed interference of CMA by other Parkinson’sdisease-related proteins, such as mutant forms of UCH-L1 [166]. A higher degree of lysosomal compromise asa result of lysosomal targeting of pathogenic proteinsvia CMA has been described in certain tauopathies,where the mutant forms of the protein not only interactabnormally with CMA components blocking this path-way, but also disrupt the lysosomal membrane as theyorganize into toxic oligomeric species on their surface[167].

To date, systematic studies analysing the contribu-tion of alterations in microautophagy to neurodegen-eration have not been performed. However, the factthat protein unfolding is not a prerequisite for microau-tophagy makes attractive the possibility that micro-aggregates can be delivered to this pathway for degra-dation.

Growing evidence supports that neurons utilizediverse ways to eliminate pathogenic proteins and thatmultiple steps of the autophagic process are oftenaffected in a given neurodegenerative disorder. Thus,for example, in the case of Alzheimer’s disease (AD),macroautophagy contributes to the elimination of thetoxic product of the amyloid precursor protein (APP)A-β. However, clearance of A-β is compromised as thedisease progresses, due to the failure in this autophagicpathway [168]. In most forms of AD, the autophagicdefect occurs in the late steps of macroautophagy.Thus, autophagosomes form properly sequestering theusual cargo but they are not eliminated through the


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lysosomal system [168]. As described above, deficientacidification of the lysosomal lumen, due to the inabil-ity to target to lysosomes one of the subunits of theproton pump that usually acidifies this compartment,has been recently described in some types of AD [169].Persistence of autophagosomes in AD neurons is par-ticularly toxic because the enzymes responsible forprocessing of APP into A-β as well as APP accumu-late in these compartments. Autophagosomes becomein this case a new site of intracellular production oftoxic A-β [168]. Failure of the autophagic system com-promises the elimination of aggregate forms of tau, aprotein that also accumulates in AD neurons. In fact,for certain types of tau mutations, this pathogenic pro-tein could contribute to the failure of macroautophagy,due to the toxic effect that the still soluble forms ofthe protein exert in the membrane of lysosomes whenthey are delivered to this compartment by CMA [167].

Similarly, in the case of Parkinson’s disease (PD)the most common pathogenic protein, α-synuclein, isusually degraded by different autophagic and non-autophagic pathways. Soluble forms of the proteinare substrates of both the ubiquitin proteasome sys-tem and of CMA [164,170]. However, macroautophagyis the only plausible way for the elimination of thepathogenic variants of this protein once they aggregate[171]. As in the other disorders, pathogenic forms of α-synuclein interfere with the normal functioning of theubiquitin proteasome pathway [170], CMA [164,165]and even macroautophagy at the level of autophago-some formation [157]. The recently identified physio-logical role for Parkin, a protein also related to PD,in mitophagy, suggests that alterations of this selec-tive form of autophagy could also contribute to thepathogenesis of this disease [172].

Macroautophagy has proved to be an effective mech-anism for the degradation of aggregates of huntingtin,the protein mutated in Huntington’s disease (HD)and main component of the inclusions observed inthe affected neurons [151,173]. Huntingtin is a verydynamic protein that undergoes physiological cleavageinto different functional fragments that likely are turnedover by different proteolytic systems. Mutant hunt-ingtin, even before aggregation, seems to be primarily atarget for macroautophagic degradation [174], althoughspecific posttranslational modifications can also favourits elimination by CMA [175]. As in the other neu-rodegenerative disorders, growing evidence supportsa direct toxic effect of the mutant protein on theautophagic system. For example, mutant huntingtin hasbeen described to accumulate on the luminal side of thelimiting membrane as the autophagosome forms andthus interfere with the recognition of cytosolic cargo,including huntingtin protein aggregates [158]. Alter-ations at the level of autophagosome trafficking andclearance have also been described in some experimen-tal models of HD [176]. Although huntingtin bears sev-eral CMA-targeting motifs in its amino acid sequence,the contribution of this pathway to the removal of thepathogenic protein is, overall, modest. However, recent

studies support a beneficial effect of the artificial tar-geting of mutant huntingtin for CMA degradation thatcould be explored for therapeutic purposes [177].

It is anticipated that a similar dual interaction ofdifferent pathogenic proteins with the autophagic sys-tems—by which they can be eliminated through thesepathways but also interfere with their normal function-ing—could also contribute to pathogenesis in otherneurodegenerative disorders.

Autophagy and the failing heartThe heart is comprised of long-lived, post-mitotic cellswith little regenerative capacity, which are continu-ally subjected to stress, such as ischaemia, pressureoverload and ischaemia–reperfusion injury. Adapta-tion to these conditions is attained through remodelling(myocytes elongate and undergo hypertrophy) and fail-ure to do so frequently constitutes the basis of coronaryartery disease, hypertension and congestive heart fail-ure [178,179]. Evidence for the role of basal autophagyin the quality control and housekeeping of cardiomy-ocytes was first obtained through genetic models ofLAMP-2 knock-out mice (defective for autophago-some–lysosome fusion) that demonstrated cardiomy-opathy and abnormal accumulation of autophagicvacuoles, similar to that observed in Danon’s diseasepatients [180,181]. In fact, mutations in the LAMP-2 gene were subsequently described in these patients[182]. Studies in cardiomyocyte-specific Atg5 and Atg7knock-out models have reiterated the need for func-tional autophagy to preserve normal cardiac function,both under basal conditions and in response to stress[183]. In fact, pronounced loss of autophagy in car-diomyocytes with age seems to be behind differentforms of age-related cardiomyopathy [184].

Added to the key role of basal macroautophagyin cardiomyocyte homeostasis, growing evidence alsosupports an important contribution of this pathwayin the heart in response to stressors. As describedin the brain, macroautophagy has also proved tobe essential for the prevention of cardiomyocyteproteinopathies, such as those arising from desminand α-β-crystallin accumulation [185,186]. Autophagyinduced in response to cardiac stress (especially duringischaemia–reperfusion injury) also plays a cytopro-tective role in the heart. In fact, in support of thisprosurvival function, different studies have demon-strated that pharmacological inhibition of autophagyduring mild ischaemic stress enhances cardiomyocytedeath [187,188], whereas macroautophagy activationis cardioprotective [189,190]. The beneficial effect ofmacroautophagy up-regulation under these conditionscould result, in part, from an efficient removal ofthe mitochondrial population compromised under theseconditions. Thus, mitochondrial membrane depolar-ization and increased production of reactive oxygenspecies by mitochondria are common features asso-ciated to ischaemia–reperfusion. Degradation of thealtered mitochondria by macroautophagy under these



conditions has been shown to efficiently amelioratecellular damage by reducing the activation of pro-apoptotic cascades [191–193].

However, induction of macroautophagy in thestressed heart can also be detrimental and contributeto heart failure, judging by the fact that pharmacolog-ical or genetic blockage of macroautophagy enhancescell survival post-ischaemia–reperfusion injury in spe-cific settings [194]. The timing and the nature of thecondition that induced cardiac stress may be essentialfor the switch in the effect of macroautophagy. Thus,some studies suggest that macroautophagy is cardio-protective during ischaemic recovery but maladaptiveduring reperfusion recovery [195]. It is possible that,during reperfusion, the need for removal of dysfunc-tional mitochondria and oxidatively damaged cellularstructures that accumulated during the ischaemic periodcould drive the autophagic response to be more aggres-sive than desired, resulting in cell death. In fact, geneticup-regulation of autophagy in response to the haemo-dynamic stress-induced hypertrophic growth responsehas also proved to be maladaptive and to result inpathological cardiac hypertrophy [196].

In summary, as in the case of tumour biologyand neurodegeneration, the role of autophagy in heartpathology is also context-dependent and excessive orinsufficient macroautophagy is often associated withdisease. However, when maintained under strict con-trol, up-regulation of macroautophagy by compoundssuch as resveratrol has proved to be a useful cardiopro-tective strategy during ischaemia–reperfusion injury[197–199].

Although the heart is one of the organs in whichCMA is up-regulated most rapidly in response to star-vation [200], the contribution of CMA to cardiomy-ocyte homeostasis and to heart pathology has not beenexplored in depth. As mentioned above, mutations inLAMP-2 occur in patients with Danon’s cardiomyopa-thy, but the phenotype seems, for the most part, to berelated to the presence of large autophagic vacuolesin muscle, reflective of compromised macroautophagy.This could be explained by the fact that the lamp2 geneundergoes splicing, giving rise to three protein variants,A, B and C [201] and that mutations in only the B vari-ant, tightly related to macroautophagy, are enough toreproduce the full vacuolar phenotype [182], whereaschanges in LAMP-2A, the variant required for CMA,do not affect macroautophagy [202].

Autophagy, infectious disorders and autoimmunityThe autophagy machinery can act as a cell-autonomousdefence against invading pathogens through a processknown as xenophagy [203]. Activation of this processhas been extensively reported, for example, in responseto Group A Streptococcus infection in epithelial cells(Figure 4). The fact that Atg5 deficiency allows bacte-ria to survive and multiply robustly supports the normalcontribution of autophagy in the elimination of thispathogen [204]. Autophagic surveillance is not limited

to bacteria as, in fact, a protective role for autophagyhas been also demonstrated for the vesicular stomatitisvirus, herpes simplex virus 1 or HIV-1, among others[205–207].

Cytosolic autophagy often becomes a second surveil-lance point for pathogens such as Listeria monocy-togenes that can escape phagosomes by puncturingtheir membrane to replicate in the cytoplasm. How-ever, once in the cytoplasm, the autophagic surveil-lance mechanism is activated to entrap the escapedbacteria and deliver them to endocytic and lysoso-mal compartments for degradation [208]. Pathogensthat survive inside vesicular compartments can also becontrolled by macroautophagy. For example, Mycobac-terium tuberculosis resides in phagosomes by avoidingtheir fusion to lysosomes. Interestingly, this blockagecan be overcome by induction of autophagy, leading tothe degradation of this pathogen [209].

Autophagy proteins may also help to acceleratephagosome maturation through a process that has beentermed ‘LC3-associated phagocytosis’. Engagement oftoll-like receptors has been shown to induce recruit-ment of LC3 to the phagosomal membrane and favourfusion with the lysosome [210].

Despite this active involvement of autophagy inpathogen elimination, some pathogens have evolvedto take advantage of the different compartments ofthe autophagy pathway to establish replicative niches[211,212] (Figure 4). Evidence that the autophagosomecompartment is required for the replication of cer-tain pathogens, such as Porphyromonas gingivalis orCoxiella burnetti was provided by demonstrating thattreatment with 3-methyladenine, a well-characterizedinhibitor of autophagosome formation, was efficient indecreasing the survival of these pathogens [213,214].In fact, some of these microorganisms that utilize com-partments of the autophagic system have developedmechanisms to expand the size and number of thesecompartments. For example, Staphylococcus aureus iscapable of secreting a factor that activates autophagy[215]. Seclusion inside the autophagosomes may bebeneficial for pathogens as a way to escape the cytoso-lic surveillance mechanisms, but these compartmentsmay also contain cytosolic materials that can be uti-lized as an energy source by the microorganisms.

This bivalent role of autophagy in pathogen defencesuggests that, although it should be possible to phar-macologically manipulate the autophagic system toeliminate different pathogens, it is essential to firstunderstand the characteristics of the pathogen–autophagy interaction in each of the individualinstances [216,217].

Apart from its role in cell-autonomous immunity,autophagy plays a role in activation of adaptiveimmunity through its recently described involvementin antigen processing and presentation to lympho-cytes [218–220] (Figure 4). In professional APCs,phagosome-processed extracellular antigens are pre-sented through MHC class II molecules to CD4 T


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Figure 4. Autophagy and the immune system. (A) Macroautophagy against pathogens: macroautophagy contributes to the elimination ofdifferent types of pathogens—bacteria and viruses—when they escape to the cytosol after internalization in the phagosome. Under certainconditions, fusion of autophagosomes with phagosomes is required before degradation can occur. (B) Pathogens using macroautophagy :growing evidence supports that certain pathogens have evolved to utilize autophagosomes as a site of replication and can activelyprevent the fusion of this compartment, with lysosomes to guarantee their survival. (C) Antigen presentation: all three forms of autophagy,macroautophagy, CMA and microautophagy have been shown to contribute to antigen loading of MHC class II molecules for presentationof antigens to activate T cells.

cells. However, several cytosolic and nuclear pro-teins have been found associated with MHC IImolecules [221,222]. In fact, pharmacological inhibi-tion of macroautophagy has been shown to be efficientin reducing MHC class II intracellular antigen pre-sentation [223–225], whereas activation of autophagypromotes this type of presentation [226]. Later stud-ies have confirmed the presence of intracellular anti-gens in autophagosomes, and marked reduction ofMHC II presentation in lymphoblastoid cell lines uponknock-down of essential Atgs, as well as in vivo inAtg5-deficient dendritic cells [227,228]. Interestingly,presentation of self-antigens by class II molecules isnot limited to macroautophagy but may also involveother types of autophagy. In fact, over-expression ofLAMP-2A, the receptor for CMA, has been shown toenhance MHC II presentation of intracellular antigens[229], and a new microautophagy-dependent processof antigen delivery into late endosomes in dendriticcells has also been recently characterized [31]. Fur-thermore, participation of autophagy in antigen pre-sentation may not be limited to presentation throughMHC II. Although it is traditionally accepted that MHCclass I presents intracellular antigens processed throughthe proteasomal pathway, growing evidence supportsthat macroautophagy could also assist in MHC I

presentation by collaborating with the proteasome inthe processing of intracellular proteins [230,231].

Also of interest is the recently proposed contributionof autophagy in the establishment of tolerance to selfantigens. Self-reactive T cells are usually actively elim-inated through MHC class II presentation in epithelialcells of the thymus to allow T cell tolerance to self-antigens [232]. Proof that autophagy regulates centralT cell tolerance has been provided by a recent studyshowing that transplantation into athymic nude hostsof Atg5-deficient thymus results in defects in posi-tive and negative selection and the development ofautoimmunity [233]. Interestingly, the antigen-loadingcompartments for MHC class II in immature dendriticcells, which are involved in peripheral tolerance, con-tinuously receive input from autophagosomes [234].Furthermore, genetic polymorphisms in two differentautophagy genes (ATG16L1 and IRGM ) have beenlinked to Crohn’s disease [235,236]. Compromisedautophagic function may result in insufficient induc-tion of tolerance against commensals or self-antigensin the gut, [235,236], although it may also affect thesecretion of antimicrobial proteins by Paneth cells [7].

This novel role of autophagy in the establishmentand maintenance of tolerance also suggests a possi-ble role for autophagy in autoimmunity [237]. In this



respect, autophagy is known to contribute to apoptotic-cell clearance [238], and defective clearance of apop-totic cells has been proposed to lead to autoimmunediseases, such as systemic lupus erythematosus [239].

As any other cell type, cells of the immune systemalso depend on autophagy for the maintenance of theirhomeostasis [240], but changes in autophagy in T cellsin particular have also been described to contribute tomodulation of specific cell functions. Thus, the activa-tion of macroautophagy observed upon T cell activa-tion is required to promote T cell differentiation andsurvival and allows cells to accommodate the bioener-getic requirements of these conditions [241–243].

Overall, recent studies support that autophagydefends the cell in a bimodal fashion; first, it directlyeliminates invading pathogens [244]; and second, itsimultaneously assists the immune system of the hostorganism to mount a specialized immune responseagainst the invader by processing pathogenic anti-gens for presentation to T cells [6,203,245]. Thereforeautophagy can be envisaged as an immediate as wellas a persistent mechanism of cellular defence.

Conclusions and future perspectives

The better molecular characterizations of the differ-ent autophagic pathways, as well as the possibilityof genetically manipulating these cellular processes,have helped to establish tight connections betweenautophagic malfunctioning and disease. The initialexcitement about the possibility of modulating auto-phagy with therapeutic purposes in different disordershas been followed by some degree of confusion asto whether autophagy should be up- or down-regulatein these conditions. As described in this review, bothup- and down-regulation of macroautophagy have beenshown to be protective against cancer, neurodegener-ation, infectious diseases and ischaemic insult in theheart. How to decide what to do? Further analysis inmost of these conditions is required to fully under-stand the contribution of autophagic malfunctioning tothe disease. Questions, such as when in the course ofthe disease autophagic function becomes compromised,what changes in each autophagic pathway and whetherthese changes are primary or compensatory to failure inother systems, need to be answered before autophagymodulators can be systematically used in the treatmentof these conditions.

A considerable advance in this respect has beenthe introduction of functional read-outs of autophagyto complement the initial morphological analysis.For example, the presence of a higher number ofautophagosomes in many disease conditions, initiallyinterpreted as an increase in macroautophagy, is nowmore cautiously analysed, because blockage in the latesteps of this pathway can also have a similar mor-phological signature. Thus, conditions initially labelledas having ‘too much autophagy’ are being currently

revised as having ‘a blockage in autophagic clearance’.Blocking autophagosome formation may only be ben-eficial then, when excessive autophagosome contentand autophagic clogging contribute to the pathology,but not when autophagy is up-regulated in the diseaseto compensate for failure in other systems. Along thesame lines, the ultimate goal may not be to repressautophagosome formation in some of these cases, butinstead to facilitate their clearance by directly repair-ing possible defects in the late steps of the autophagicprocess.

In addition to determining the right timing for mod-ulating autophagy in many of these diseases, andwhether this process should be up- or down-regulated,further expansion of the chemical options to manip-ulate autophagy is needed for it to become a routinetherapeutic target. To date, most of the macroautophagyinhibitors used in clinical trials act during the late stepsof this process, on the lysosomal compartment, whichis also shared by other autophagic pathways. In con-trast, for activators, most of the available drugs havean effect on initiation and autophagosome formation,but few compounds have been shown to be effective atincreasing the overall autophagic flux. Ongoing chem-ical screenings are currently addressing the need forthese types of modulators and should render usablemolecules in a timely fashion [154].

AcknowledgmentWe thank Dr Susmita Kaushik for critically review-ing the manuscript. Work in our laboratories is sup-ported by the National Institutes of Health/NationalInstitute on Aging (Grant Nos. AG021904 to AMCand AG031782 to AMC and FM) and the NationalInstitute of Allergy and Infectious Diseases (GrantNo. AI059738 to FM). AMC is also supported by aHirschl/Weill–Caulier Career Scientist Award.

Author’s contributions

All authors contributed separate sections to this reviewand all read and edited the final submitted version.

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