Autophagy is a catabolic trafficking pathway for bulk destruction and turnover of long-lived proteins and organelles via regulated lysosomal degradation. In eukaryotic cells, autophagy occurs constitutively at low levels to perform housekeeping functions, such as the destruction of dysfunctional organelles. Up-regulation occurs in the presence of external stressors (e.g. starvation, hormonal imbalance and oxidative stress) and internal needs (e.g. removal of protein aggregates), suggesting that the process is an important survival mechanism. However, the occurrence of autophagic structures in dying cells of different organisms has led to the hypothesis that autophagy may also have a causative role in stress-induced cell death. The identification within the last decade of a full set of genes essential for autophagy in yeast, the discovery of human orthologues and the definition of signalling pathways regulating autophagy have accelerated our molecular understanding and interest in this fundamental process. A growing body of evidence indicates that autophagy is associated with heart disease, cancer and a number of neurodegenerative disorders, such as Alzheimer's, Parkinson's and Huntington's diseases. Furthermore, it has been demonstrated that autophagy plays a role in embryogenesis, aging and immunity. Recently, it has been shown that autophagy can be intensified by specific drugs. The pharmacological modulation of the autophagic pathway represents a major challenge for clinicians to treat human disease.

INTRODUCTION

A fine balance between protein synthesis, organelle biogenesis and degradation is essential for cell growth and development. Moreover, to maintain vital cellular functions, irreversibly damaged and potentially toxic proteins, which are prone to form aggregates, are rapidly degraded, thereby preventing further damage to cellular components. The two major protein degradation and recycling pathways in eukaryotic cells are the UPS (ubiquitin–proteasome system) and the autophagy–lysosome pathway. The UPS is responsible for the degradation of short-lived proteins, whereas autophagy regulates levels of long-lived proteins and organelles. In mammalian cells, three different mechanisms for the degradation of intracellular components within lysosomes have been identified: macroautophagy, microautophagy and CMA (chaperone-mediated autophagy) (Figure 1) [1,2]. Macroautophagy involves the sequestration and transport of complete regions of the cytoplasm, including both soluble proteins and entire organelles within double-membrane vacuoles, to the lysosomal system for degradation and recycling by lysosomal hydrolases. The bulk degradation of soluble proteins and cytoplasmic content generates free amino acids and non-esterified fatty acids (‘free fatty acids’), which can be metabolized further to respond to energy demand during periods of stress. This catabolic pathway is typically stimulated when nutrients are scarce. During microautophagy, the formation of intermediate vacuoles is not required and the cytoplasmic material is engulfed directly by invagination of the membrane of the lysosome, and degraded further by the luminal hydrolases. In contrast with the undiscriminated ‘in bulk’ removal and degradation of cytoplasmic materials by macro- or micro-autophagy, the third autophagy mechanism, CMA, involves the selective recognition of a cytosolic protein target by a dedicated chaperone complex, which allows its molecular recognition and transport into the lysosomal lumen via the lysosomal receptor LAMP2A (lysosomal-associated membrane protein 2A). These autophagic pathways are thought to act independently of each other, but studies have begun to reveal a complex interdependence among them and the occurrence of compensatory mechanisms when one of these degradation mechanisms is functionally disabled [3,4].

Three distinct forms of autophagy identified in mammalian cells

Macroautophagy, which is the main topic of the present review and referred further to as ‘autophagy’, is a dynamic and highly regulated process of self-digestion conserved in all eukaryotic organisms. The hallmark of autophagy is the de novo formation of a double-membrane vacuole, the autophagosome, originating from a largely undefined structure known as the phagophore or isolation membrane. The process proceeds in a step-wise fashion, with the elongation of the isolation membrane and maturation of the autophagosome, followed by its fusion with a lysosome, thereby generating an autophagolysosome or autolysosome. During this final step, the incorporation of the outer autophagosomal membrane with the lysosomal membrane eventually allows the degradation of the remaining inner single membrane and the cytoplasmic content of the autophagosome by lysosomal hydrolases. Generally, autophagy is a non-selective pathway of cytoplasmic degradation, but it may also function as a mechanism for the selective disposal of aberrant organelles. Indeed, autophagy pathways for the specific removal/degradation of mitochondria (mitophagy) [5], ER (endoplasmic reticulum; reticulophagy) [6] or peroxisomes (pexophagy) [7] have been described in several systems. At a basal rate, autophagy acts as a major housekeeping mechanism, crucially involved in the maintenance of normal cellular homoeostasis. When stimulated by cellular stress conditions, autophagy functions as a self-cannibalization pathway that promotes cell survival in an adverse environment; however, the occurrence of autophagic structures in dying cells has led to the hypothesis that autophagy may also have a causative role in stress-induced cell death. During development, for example, autophagy has been associated with a form of non-apoptotic cell death known as programmed cell death type II, which is distinct from apoptosis or programmed cell death type I. How can the pro-survival role of autophagy be switched into a cell death mechanism? One possibility is that the amplitude of autophagy is increased under certain circumstances above a threshold level incompatible with viability. Being a catabolic mechanism, unregulated autophagy may promote cell death through excessive self-digestion and degradation of essential cellular constituents, thus overwhelming the cellular synthetic capacity. Other reports suggest that autophagy may have a more direct cell killing role. This assumption is based on observations showing that, in cells with apoptosis defects exposed to different types of stress, the knockdown/knockout of essential autophagy genes (see below) is cytoprotective. To complicate this matter further, recent observations indicate that autophagy and apoptosis are often induced by the same stimuli, share similar effectors and regulators, and are subjected to a mutual control and complex cross-talk mechanisms [8].

MOLECULAR PATHWAYS REGULATING AUTOPHAGY

Autophagy is regulated at the molecular level by a family of dedicated genes called Atg (AuTophaGy-related) genes [9]. Atgs were first discovered by genetic screens in yeast but, for many of these genes, an orthologue in higher eukaryotes has been found. Approx. 18 Atg gene products participate in protein complexes that are dynamically formed during assembly, docking and degradation of the autophagosome (Figure 2). Furthermore, lipid and protein kinases, phosphatases and GTPases contribute to the tight regulation of the autophagy process at different levels. Autophagic signalling from the cytoplasm toward the autophagy machinery, for example, is mainly relayed through the serine/threonine kinase mTOR (mammalian target of rapamycin). Although the mechanisms by which mTOR kinase inhibits autophagy are not fully elucidated yet, a major step involves the phosphorylation of Atg13 and its association with the mammalian Atg1 orthologue Ulk1 (Figure 2). Once phosphorylated, Atg13 associates less tightly with Ulk1 and the mammalian Atg17 orthologue FIP200 (focal adhesion kinase family interacting protein of 200 kDa), thereby disturbing the formation of the protein complex which promotes Ulk1 kinase activity. The exact molecular targets of Ulk1 remain largely unidentified, but the kinase activity of its counterpart in yeast, Atg1, has been shown to be required for autophagy [10].

Different structural phases of macroautophagy regulated by the autophagy-related genes and mTOR

The mTOR pathway is under the negative control of AMPK (AMP-dependent kinase), which acts as an energy and metabolic sensor. In a low-energy state, the AMP/ATP ratio increases and AMPK is activated. Active AMPK results in the repression of mTOR activity, thereby favouring catabolic pathways via stimulation of autophagy [11]. The best-characterized positive modifier of mTOR activity is the proto-oncogene Akt [also known as PKB (protein kinase B)], typically activated in response to growth factors, such as insulin and IGF-1 (insulin-like growth factor-1), resulting from the activation of class I PI3K (phosphoinositide 3-kinase). The class I PI3K/Akt pathway stimulates the import of nutrients in order to support the increased metabolic demand during growth and proliferation, inhibits apoptosis and concurrently activates mTOR. Once activated, mTOR promotes protein synthesis and blocks catabolism by inhibiting autophagy [11]. Thus the dynamic regulation of mTOR activity allows cells to sense nutrients and growth factor availability, and to couple this function with the inhibition of autophagy under nutrient-rich conditions, or to its stimulation when starvation occurs.

Nucleation of autophagic vesicles is mediated by the mammalian Vps34, a class III PI3K, whose activity generates PtdIns(3)P [9]. Activation of Vps34/class III PI3K requires a protein complex including Atg6, whose mammalian orthologue is beclin 1, the protein kinase Vps15 (or p150 in humans), essential for Vps34 membrane association, and Bif-1, also known as endophilin B1 (Figure 2). In mammals, two other beclin 1-interacting proteins, UVRAG (UV irradiation resistance-associated tumour suppressor gene) [12] and Ambra1 (activating molecule in beclin 1-regulated autophagy) [13], participate in this complex. Vesicle elongation is regulated by two ubiquitin-like conjugation systems (Figure 2) [9]. The first mechanism involves a protein-to-protein covalent conjugation of Atg5 to Atg12 [14]. In analogy with ubiquitination, this step is catalysed by an E1 ubiquitin-activating-like enzyme, Atg7, and an E2 ubiquitin-conjugating-like enzyme, Atg10. A small coiled-coil protein, Atg16, is recruited to the Atg5–Atg12 proteins forming an Atg12–Atg5–Atg16 multimeric complex mediated by Atg16 self-interaction [15]. The second conjugation process involves the protein-to-lipid conjugation of the ubiquitin-like protein Atg8 [or one of its mammalian orthologues, such as LC3-I (unconjugated microtubule-associated protein light-chain 3)] to PE (phosphatidylethanolamine) [16]. Similar to Atg5–Atg12, conjugation of LC3-I to PE, also known as LC3-II, is mediated by the E1-like Atg7 and the E2-like Atg3 proteins and requires Atg4, a cysteine protease that cleaves the C-terminus of proLC3 (LC3 precursor). Recently, the Atg5–Atg12 complex was shown to function as an E3-like ligase during the lipidation of LC3-I, at least In vitro [17]. LC3-II functions probably as a scaffold protein supporting membrane expansion, whereas recent findings support a role for the Atg5–Atg12 and Atg12–Atg5–Atg16 complexes in promoting LC3-I lipidation and its correct localization respectively [18,19]. Unlike the Atg12–Atg5–Atg16 complex, which is transiently associated with the autophagosome membrane and released in the cytosol when the mature autophagome is formed, a large amount of LC3-II remains associated within the mature autophagosome and is degraded, together with the cargo, by lysosomal hydrolases after the fusion of the autophagosome with a lysosome.

AUTOPHAGY IN EMBRYOGENESIS AND DEVELOPMENT

After fertilization, maternal proteins in oocytes are rapidly degraded and replaced by zygotic proteins. Although several proteins are degraded by the UPS [20], recent evidence indicates that autophagy is up-regulated within 4 h after fertilization and, thus, may be an essential protein degradation process during the oocyte-to-embryo transition [21]. Indeed, autophagy-defective oocytes derived from oocyte-specific Atg5-knockout mice fail to develop beyond the four- and eight-cell stages if they are fertilized by Atg5-null sperm. Because protein synthesis is reduced in autophagy-null embryos [21], it is tempting to speculate that autophagy is a crucial source of amino acids during preimplantation development. Oocyte-specific Atg5-deficient females ovulate normally in response to exogenous hormonal stimulation, and oocytes lacking Atg5 are fertilized normally in vivo, suggesting that autophagy is not important for folliculogenesis and oogenesis [21].

The importance of autophagy during further development has been highlighted by a number of approaches in both lower and higher eukaryotes [22]. One major finding is that Ambra1, an active component of the pro-autophagic multi-molecular complex involving beclin 1, Vps34 and possibly also other factors such as UVRAG, Vps15 and Bif-1 (Figure 2), is required for neurodevelopment [13]. Functional inactivation of Ambra1 in mice leads to lethality in utero and is characterized by severe neural tube defects associated with autophagy impairment, unbalanced cell proliferation, accumulation of ubiquitinated proteins and excessive apoptosis [13].

AUTOPHAGY IN HEART DISEASE

Induction of autophagy in the heart after I/R (ischaemia/reperfusion) or pressure overload

One of the first reports describing autophagy in the heart was published in the mid 1970s, approximately one decade after the initial description of autophagy in mammalian cells [23]. In that study, Sybers et al. [24] demonstrated that fetal mouse hearts in organ culture continue to beat for a period of weeks, but that degenerative changes occur. Electron microscopy revealed the formation of autophagic vacuoles containing damaged organelles in some cells after the first day, indicating focal cytoplasmic injury. This process was accelerated by transient deprivation of oxygen or glucose. A few years later, Decker and Wildenthal [25] and Decker et al. [26] observed the induction of autophagy in Langendorff-perfused rabbit hearts subjected to I/R. They found that 20 min of ischaemia did not induce autophagy, but that the number of autophagosomes increased when reperfusion was initiated. However, 40 min of ischaemia alone caused an increase in autophagy which was enhanced further during reperfusion. When ischaemia was extended to 60 min, the authors observed the presence of large and probable dysfunctional lysosomes during reperfusion, suggesting that prolonged ischaemia impaired the autophagic/lysosomal pathway. Recent evidence indicates that autophagy is triggered by cardiac ischaemia on a condition that AMPK is activated (Figure 3) [27,28]. Once active, AMPK probably induces autophagy through inhibition of mTOR [28,29]. Autophagy during the reperfusion phase is accompanied by the up-regulation of beclin 1, but not by the activation of AMPK (Figure 3) [27,28]. Indeed, AMPK is rapidly inactivated upon reperfusion, but overexpression of beclin 1 in cardiac myocytes following I/R enhances formation and downstream lysosomal degradation of autophagosomes (autophagic flux) and significantly reduces activation of the pro-apoptotic protein Bax [30]. Moreover, autophagosome formation during reperfusion is significantly inhibited in beclin 1+/− mice [27]. The mechanism that drives the up-regulation of beclin 1 in the heart is presently unknown. Recent findings suggest that NO is not involved in increased beclin 1 expression, although NO plays a crucial role in the process of I/R and heart failure by regulating several members of the caspase family [31]. In vascular smooth muscle cells, TNFα (tumour necrosis factor α) induces the up-regulation of beclin 1 expression and subsequent autophagy through the JNK (c-Jun N-terminal kinase) pathway [32]. As JNK is activated by reperfusion [33], beclin 1 may be up-regulated by JNK during the reperfusion phase. Besides I/R, profound and unremitting hypertension, a major risk factor for cardiac hypertrophy as seen in patients with poorly controlled high blood pressure, may trigger basal cardiomyocyte autophagy, particularly in the basal septum [34]. This effect is mediated by beclin 1, as heterozygous disruption of beclin 1 decreases pressure overload-induced autophagy and diminishes pathological remodelling. Conversely, beclin 1 overexpression heightens autophagic activity and accentuates hypertrophy upon pressure overload [34].

Induction of autophagy in the heart after I/R

Figure 3
Induction of autophagy in the heart after I/R

During ischaemia (I), oxygen and nutrient supplies are decreased, causing activation of AMPK and inactivation of mTOR, which in turn leads to autophagy for cell survival. Upon reperfusion (R), activation of AMPK and inactivation of mTOR are no longer observed. Instead, expression of beclin 1 is markedly up-regulated, which enhances the formation of autophagosomes and induction of autophagy. Furthermore, Bnip3 is involved in the up-regulation of autophagy and functions as a cytoprotective pathway to oppose I/R-related apoptosis.

Figure 3
Induction of autophagy in the heart after I/R

During ischaemia (I), oxygen and nutrient supplies are decreased, causing activation of AMPK and inactivation of mTOR, which in turn leads to autophagy for cell survival. Upon reperfusion (R), activation of AMPK and inactivation of mTOR are no longer observed. Instead, expression of beclin 1 is markedly up-regulated, which enhances the formation of autophagosomes and induction of autophagy. Furthermore, Bnip3 is involved in the up-regulation of autophagy and functions as a cytoprotective pathway to oppose I/R-related apoptosis.

Role of autophagy in the heart

Increasing evidence from In vitro and in vivo studies suggest that autophagy may have different functions in the heart. These roles are either beneficial or detrimental for normal cardiac function and morphology. First of all, autophagy under baseline conditions appears to have a special housekeeping role in the turnover of cytoplasmic constituents, as demonstrated by severe cardiac dysfunction in patients and mice showing defective autophagic degradation owing to a deficiency of the lysosomal protein LAMP2 [35,36], a disorder also known as Danon disease [37]. In line with these findings, cardiac-specific deficiency of Atg5 leads to cardiac hypertrophy, left ventricular dilation and contractile dysfunction in adult mice [38]. Moreover, Atg5-deficient hearts have increased levels of ubiquitination, a disorganized sarcomere structure and mitochondrial aggregation [38]. It should be noted, however, that embryonic Atg5-knockout mice are viable and live to adulthood without any detectable heart abnormalities [38], presumably due to compensatory mechanisms which also perform cellular maintenance. Given that the production of polyubiquitinated proteins is often increased during cardiomyopathy and chronic heart failure [39], it is possible that autophagy provides protection by degrading misfolded proteins and aberrant protein aggregates that may be toxic to cardiomyocytes. Tannous et al. [40] reported that aggregation of polyubiquitinated proteins after pressure overload is sufficient to induce cardiomyocyte autophagy. Attenuation of autophagic activity dramatically enhances aggresome size and abundance, consistent with a role for autophagic activity in protein aggregate clearance. Furthermore, autophagy can provide protection by removing damaged and dysfunctional mitochondria. Many studies have observed mitochondria sequestered inside autophagosomes in the myocardium after cellular distress [24,25,41]. The mitochondrial protein Bnip3 (Bcl-2/adenovirus E1B 19 kDa-interacting protein) is up-regulated in cardiomyocytes subjected to ischaemia and stimulates apoptotic cell death signalling during I/R injury of the heart via disruption of mitochondrial integrity, which in turn leads to enhanced superoxide production and the release of pro-apoptotic factors such as cytochrome c and AIF (apoptosis-inducing factor) [41]. Bnip3 activation is associated with the up-regulation of autophagy as determined by high levels of autophagosomes containing fragmented mitochondria. It has been proposed that the up-regulation of autophagy constitutes a protective response against Bnip3 death signalling by removing harmful and leaky mitochondria, thus preventing the activation of apoptosis (Figure 3) [41].

Apart from its critical role in the removal of protein aggregates and damaged organelles, autophagy serves as a catabolic energy source in times of famine. Indeed, autophagy in cardiac myocytes has been suggested to provide a necessary source of energy between birth and suckling [42], and in a GFP–LC3 transgenic mouse model (where GFP is green fluorescent protein), cardiac myocytes from starved animals have high numbers of autophagosomes to survive the adverse conditions of nutrient deprivation [43]. Consistent with this idea, autophagy promotes survival during ischaemia by maintaining energy homoeostasis. Generation of ATP via oxidative phosphorylation is inhibited during ischaemia and reduced ATP levels have been shown to induce autophagy [44].

Overall, these results clearly indicate that autophagy has in the first place a pro-survival role in the heart. This statement is contradictory with the high levels of autophagy in the failing heart, which support the theory that excessive induction of autophagy underlies autophagic cell death and loss of cardiomyocytes. Indeed, dead and dying cardiomyocytes with characteristics of autophagy have been reported in heart failure caused by dilated cardiomyopathy [4547], valvular and hypertensive heart disease [48], chronic ischaemia [49] and stunned or hibernating myocardium [50,51], but not in the normal heart [46,50]. Importantly, the incidence of autophagic cardiomyocytes in failing hearts is greater than the incidence of apoptotic cells {0.03–0.3% compared with ≤0.002% based on staining for granular ubiquitin inclusions or TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling) respectively [45,46,50]}. As a consequence, autophagy is suggested to be an important mechanism underlying the cardiomyocyte drop-out responsible for the worsening of heart failure [52]. Direct proof for the causal role of autophagic cell death in the pathogenesis of heart failure was provided by Akazawa et al. [53]. By giving intramuscular injections of diphtheria toxin, these authors observed degeneration of cardiomyocytes within 7 days in transgenic mice that express the human diphtheria toxin receptor in the heart. Approx. 80% of the animals had pathophysiological features characteristic of heart failure and were dead within 14 days. Degenerated cardiomyocytes of the transgenic heart had several characteristics indicative of autophagic cell death, such as the up-regulation of lysomal markers and accumulation of autophagosomes [53]. We therefore assume that autophagy in the heart functions predominantly as a pro-survival pathway during nutrient deprivation and other forms of cellular stress; however, when autophagy is severely triggered, the autophagic machinery might also be used for self-destruction. In this way, autophagic cell death can occur in a detectable number of cardiac cells and finally leads to heart failure.

Therapeutic modulation of autophagy in the heart

Despite the discovery of many autophagy-specific genes and the dissection of signalling pathways involved in the regulation of autophagy, therapeutic approaches to modulate autophagy in cardiovascular disease are highly limited. Several possibilities can explain this discrepancy. First, the few autophagy inhibitors that are currently used in cell culture experiments, in particular the class III PI3K inhibitor 3-methyladenine, are unsuitable for in vivo applications because of their high toxicity [54]. Secondly, the most effective inducer of autophagy in mammalian cells is nutrient starvation, a strategy which is obviously not attractive in vivo and even dangerous from a cardiovascular point of view. Intermittent fasting in rats protects the heart from ischaemic injury and attenuates post-MI (myocardial infarction) cardiac remodelling [55], probably via anti-apoptotic/inflammatory mechanisms and possibly via induction of autophagy, but prolonged starvation triggers severe cardiovascular complications and cardiac death [56]. Several alternative strategies have recently been developed to regulate autophagy in cardiomyocytes. For example, treatment of UM-X7.1 hamsters, a model of cardiomyopathy and muscular dystrophy that is caused by lack of the δ-sarcoglycan gene [57], with G-CSF (granulocyte colony-stimulating factor) significantly improves survival, cardiac function and remodelling in these animals, and such beneficial effects were accompanied by a reduction in autophagy, an increase in cardiomyocyte size and a reduction in myocardial fibrosis [58]. Moreover, autophagy in cardiac myocytes after I/R is also reduced by the endogenous cardiac peptide urocortin [59] that inhibits beclin 1 expression. Other compounds that are able to regulate autophagy in the heart include the β-adrenoceptor antagonist propranolol, the calcium channel blocker verapamil (both have a stimulatory effect) and the β-adrenoceptor agonist isoproterenol that increases cAMP levels (and inhibits autophagy) [60,61]. These effects are in accordance with a recently identified mTOR-independent pathway involving cAMP/Ca2+/calpains/G, which leads to induction of autophagy after pharmacological inhibition [62]. Because verapamil, in contrast with propranolol, affects neither the β-adrenoreceptors nor the intracellular levels of the second messenger cAMP, it has been suggested that stimulation of autophagy is a regulatory step in the adaptation of the heart to a reduction in cardiac output [60]. The mTOR inhibitor and autophagy inducer rapamycin can also be useful in treating the heart, as it confers preconditioning-like protection against I/R injury in isolated mouse heart through opening of mitochondrial KATP channels [63]. In addition, rapamycin at low doses (25–100 nmol/l) reduces necrosis as well as apoptosis following simulated ischaemia/re-oxygenation in adult cardiomyocytes [63], although the direct impact of autophagy in these processes is unclear.

AUTOPHAGY IN NEURODEGENERATIVE DISEASES

In an early stage of many late-onset neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease and Huntington's disease, genetic mutations as well as post-translational modifications result in the intracellular presence of misfolded or conformationally altered proteins (usually a β-conformation) which aggregate [64] (Figure 4). It is not clear whether the aggregates are toxic or protective; however, studies have implicated pre-aggregate oligomers to be the most toxic species [64,65]. The soluble forms of the altered protein are removed by the UPS or macroautophagy. Because aggregates visible by light microscopy are not membrane-bound and larger than typical autophagosomes, it is likely that the monomeric and oligomeric species of the aggregate-prone proteins rather than the large aggregates are cleared by macroautophagy [66]. With aging or when aggravating conditions occur, such as oxidative stress, a failure in macroautophagy could be the main reason for the accumulation of toxic proteins, altered cell function and eventually cell death [67]. Indeed, different conditional knockout mice models for essential autophagic genes showed that the absence of autophagy in these animals leads to neurodegeneration [68,69]. Thus up-regulation of autophagy in neurodegenerative disorders confers an important defensive mechanism (Figure 4).

Role of autophagy during neurodegeneration

Figure 4
Role of autophagy during neurodegeneration

Autophagy is a homoeostatic process that prevents misfolded or conformationally altered proteins from accumulating to toxic levels. A failure in autophagy may lead to neurodegeneration.

Figure 4
Role of autophagy during neurodegeneration

Autophagy is a homoeostatic process that prevents misfolded or conformationally altered proteins from accumulating to toxic levels. A failure in autophagy may lead to neurodegeneration.

Autophagy in Huntington's disease

Huntington's disease is caused by a polyglutamine expansion mutation in the huntingtin protein that confers a toxic gain-of-function and causes the protein to become aggregate-prone, leading to neuronal loss in the striatum and cortex, and to the appearance of neuronal intranuclear inclusions of mutant huntingtin. Excitotoxicity, dopamine toxicity, metabolic impairment, mitochondrial dysfunction, oxidative stress, apoptosis and autophagy have been implicated in the progressive degeneration observed in Huntington's disease [70]. Evidence for a pure apoptotic process contributing to Huntington's disease cell death is controversial, although the activation of certain apoptotic pathways is likely to contribute to some degree to the pathology of the disease [71]. On the other hand, several studies provide evidence that autophagy plays an important role in the pathophysiology of Huntington's disease. (i) Brains of patients with Huntington's disease have endosomal and/or lysosomal organelles, multivesicular bodies and lipofuscin accumulation reminiscent of autophagy [72]. (ii) Expression of mutant huntingtin induces endosomal and/or lysosomal activity [73]. (iii) In response to ER stress, huntingtin releases from membranes and rapidly translocates into the nucleus. Huntingtin is then capable of nuclear export and re-association with the ER in the absence of stress. This release is inhibited when huntingtin contains the polyglutamine expansion seen in Huntington's disease. As a result, mutant huntingtin-expressing cells have a perturbed ER and an increase in autophagic vesicles [74]. In addition, molecular determinants of autophagic vacuole formation appear to be recruited more easily to cytoplasmic than to nuclear aggregates, which might help to explain why protein aggregates are more toxic when directed to the nucleus [75]. (iv) Aggregate-prone proteins with polyglutamine and polyalanine expansions are degraded by autophagy [76]. (v) In lymphoblasts from patients with Huntington's disease, the number of autophagic vacuoles correlates with the length of the polyglutamine expansion [77]. (vi) Blocking autophagy reduces cell viability and increases the number of cells bearing mutant huntingtin aggregates, whereas stimulating autophagy promotes huntingtin degradation [78]. (vii) mTOR is sequestrated into aggregates of mutant huntingtin with subsequent inhibition of its kinase activity [79] and induction of autophagy. This may promote huntingtin degradation and the clearance of aggregates [76,78,79]. (viii) The accumulation of mutant huntingtin can lead to mTOR-independent autophagy. Indeed, clearance of accumulated mutant huntingtin is also triggered by the activation of the insulin signalling pathway in a process that requires beclin 1 and Vps34 [80]. Activation of IRS2 (insulin receptor substrate 2), which mediates the signalling cascades of insulin and IGF-1, leads to an autophagy-mediated clearance of the accumulated proteins, despite the activation of Akt and mTOR [80]. Furthermore, sequestration of beclin 1 into neuronal intranuclear inclusions in patients with Huntington's disease might reduce the autophagic clearance of mutant huntingtin [81]. Expression of beclin 1 decreases in an age-dependent fashion in human brains. The age-dependent decrease in beclin 1 expression may lead to a reduction in autophagic activity during aging, which in turn promotes the accumulation of mutant huntingtin and the progression of the disease [81].

Taken together, autophagy may represent an initial attempt of the Huntington's disease cell to eliminate the mutant protein that over the course of the disease becomes overloaded, insufficient and dysfunctional, eventually resulting in cell degradation. Induction of autophagy enhances the clearance of both soluble and aggregated forms of mutant huntingtin, and protects against toxicity caused by these mutations in several models. Inhibition of autophagy has opposite effects. With aging or when aggravating conditions occur, such as oxidative stress provided by dopamine, a failure in autophagy could be the main factor responsible for the accumulation of toxic mutant huntingtin, altered cell function and eventually cell death [67,73].

Autophagy in Parkinson's disease

Parkinson's disease is a neurodegenerative disorder characterized by progressive cell loss confined mostly to dopaminergic neurons of the substantia nigra. Several factors, including mutants of the protein α-syn (α-synuclein), are involved in the degenerative process. Wild-type α-syn is normally translocated into lysosomes for degradation by CMA; however, the pathogenic A53T and A30P α-syn mutants bind to the receptor for this pathway on the lysosomal membrane and appear to act as uptake blockers, inhibiting both their own degradation and that of other substrates [82]. Pathogenic mutants of α-syn are therefore found in cytosolic inclusions, called Lewy bodies. Aggregates of α-syn can be degraded in lysosomes but only via macroautophagy [83]. Interestingly, a fraction of α-syn in Lewy bodies is monoubiquitinated [84], which promotes the aggregation of α-syn into amorphous aggregates and increases the formation of toxic inclusions within dopaminergic cells. Monoubiquitination as well as most other post-translational modifications of α-syn impair degradation of this protein by CMA further, but not degradation of other substrates. A special form of α-syn suggested to be responsible for neuron toxicity results from a non-covalent interaction of α-syn and oxidized dopamine. Dopamine-modified α-syn is not only poorly degraded by CMA, but also blocks degradation of other substrates, thereby increasing cellular vulnerability to stressors [85].

Autophagy in Alzheimer's disease

Alzheimer's disease is characterized by the extracellular accumulation of Aβ (β-amyloid) deposits, called senile plaques. These plaques are generated by the proteolytic cleavage of APP (amyloid precursor protein) via β- and γ-secretases that are localized in secretory and endocytic compartments of the affected cells. Autophagosomes and endosomes within neurons actively form in synapses and along neuritic processes. Efficient clearance of these compartments requires their retrograde transport towards the neuronal cell body, where lysosomes are most concentrated. In Alzheimer's disease, the maturation of autophagosomes and their retrograde transport towards the neuronal cell body are impeded, which leads to a massive accumulation of autophagic vacuoles within large swellings along dystrophic and degenerating neurites [86]. Given that APP and its processing enzymes have been detected in autophagic vacuoles, impaired clearance of these vesicles increases the enzyme–substrate interaction time. Therefore autophagy can be considered as a novel Aβ-generating pathway that is activated in Alzheimer's disease [87].

Recently, it has been shown that the expression of beclin 1 is reduced in the brains of patients with severe Alzheimer's disease [88]. In transgenic mouse models of Alzheimer's disease, heterozygous deletion of beclin 1 decreases neuronal autophagy and increases intraneuronal Aβ accumulation and extracellular Aβ deposition in aged (9-month-old) but not young (3.5-month-old) animals and promotes neurodegeneration [88,89]. Conversely, increased expression of beclin 1 in 6-month-old APP-transgenic mice did just the opposite. If reduced induction of autophagy is indeed the underlying mechanism for the observed higher levels of Aβ peptides and aggregates, then at first glance these results appear to be in contradiction with the above-mentioned findings that the maturation of autophagosomes may be impaired in Alzheimer's disease. However, the Aβ level is affected by both production and clearance [90]. Therefore defects in autophagosome maturation enhance Aβ production, whereas reduced autophagy through beclin 1 deficiency may inhibit Aβ clearance.

Therapeutic modulation of autophagy in neurodegenerative diseases

Induction of autophagy enhances the clearance of aggregate-prone proteins associated with neurodegenerative diseases, and protects against toxicity of these mutations in cell and animal models [9193]. However, the ability to maintain proper autophagic activity, rather than massive up-regulation of autophagy, should be the therapeutic goal in these protein conformational disorders [67].

Induction of autophagy by mTOR inhibition

Autophagy induction with the mTOR inhibitor rapamycin [79,94] accelerates the clearance of toxic substrates such as intracellular aggregate-prone proteins, albeit initial reports on the use of rapamycin were received with caution. Indeed, accelerated turnover of organelles could be a high price to pay for the removal of aggregate proteins. There is, however, a possible explanation why a rapamycin-induced autophagic response can be tolerated in neurodegenerative cells. The preferential accumulation of aggregate proteins in the perinuclear area and the delivery of autophagic vacuoles to this particular region may confer sufficient specificity to the degradation process [75]. Increasing the ratio of aggregate proteins to cytosolic components in these regions may guarantee that autophagosomes preferentially engulf the aggregates without disturbing cellular organelles. A second concern is the fact that rapamycin acts by inhibition of mTOR, a key regulator of translation, cell growth and proliferation [95]. The protective effects of rapamycin in vivo are independent of translation inhibition, as its protective effects against neurodegeneration are lost when key autophagy genes are knocked-down in various disease-associated fly models [94]. Furthermore, the side effects of rapamycin, especially immunosuppression, may preclude its use in certain patients.

Induction of autophagy by mTOR-independent pathways

Several drugs can induce autophagy in neurodegenerative diseases by mTOR-independent pathways. Lithium, often used for the treatment of patients with bipolar disorder, has been shown to activate autophagy in an mTOR-independent manner by inhibiting IMPase (inositol monophosphatase) and reducing inositol and Ins(1,4,5)P3 levels [96]. These effects of lithium reduce neurodegeneration in Drosophila models of Huntington's disease [93] and delay progression of ALS (amyotrophic lateral sclerosis) in ALS mouse models as well as in patients affected by ALS [97]. However, GSK-3β (glycogen synthase kinase-3β), another enzyme inhibited by lithium, has opposite effects. In contrast with IMPase inhibition that enhances autophagy, GSK-3β inhibition attenuates autophagy and mutant huntingtin clearance by activating mTOR. In order to counteract the autophagy-inhibitory effects of mTOR activation resulting from lithium treatment, Sarkar et al. [93] have used the mTOR inhibitor rapamycin in combination with lithium. This combination enhances macroautophagy by mTOR-independent (IMPase inhibition by lithium) and mTOR-dependent (mTOR inhibition by rapamycin) pathways and gives higher protection against neurodegeneration in a fly model of Huntington's disease compared with inhibition of either pathway alone [93].

Trehalose, a disaccharide present in many non-mammalian species, protects cells against various environmental stresses, partly by its chemical chaperone properties. In addition, trehalose may function as an mTOR-independent autophagy activator and enhances the clearance of the autophagy substrates mutant α-syn and huntingtin, associated with Parkinson's disease and Huntington's disease respectively [98]. Recently, an mTOR-independent autophagy pathway involving cAMP/Ca2+/calpains/G has been described [62]. Pharmacological inhibition of this mTOR-independent pathway induces autophagy and provides protection against neurodegeneration in fly and zebra fish models of Huntington's disease [62]. This can be achieved by agents that reduce cAMP levels [e.g. clonidine, rilmenidine and the adenylate cyclase inhibitor 2′5′ddA (2′5′-dideoxyadenosine)], agents that lower inositol and Ins(3)P levels (e.g. carbamazepine, valproic acid and lithium), L-type Ca2+ channel antagonists (e.g. verapamil), calpain inhibitors (e.g. calpastatin and calpeptin) and G inhibitors (such as NF449). Finally, three SMERs (small-molecule enhancers) of rapamycin have been identified using a small-molecule screen in the yeast Saccharomyces cerevisiae. These SMERs induce autophagy independently of rapamycin and reduce toxicity in models of Huntington's disease [92].

It should be noted that combination therapy with a low dose of each compound may be safer for long-term treatment compared with therapy using a high dose of only one pharmacological agent. Moreover, overexpression of beclin 1 (through administration of a viral vector) reduces both intracellular and extracellular deposition of Aβ in APP-transgenic mice [88]. These findings in animal models of Alzheimer's disease suggest that modulation of beclin 1 activity, possibly in combination with other interventions, may be an attractive therapeutic approach for patients with Alzheimer's disease, although the molecular and cellular mechanisms for the observed effects remain to be determined [89].

AUTOPHAGY IN CANCER

Role of autophagy in cancer

Autophagic degradation is less pronounced in cancer cells [99] or in cells treated with carcinogens [100] than in their normal or untreated counterparts. Therefore autophagy was initially believed to function as a bona fide mechanism of tumour suppression [101]. The first indication for a direct link between defective autophagy and tumour development was the mono-allelic deletion of beclin 1 in ovarian, breast, brain and prostate carcinomas [102,103]. More recently, somatic mutations in genes that are required for the regulation of autophagy and for autophagosomal sequestration have been identified [104,105]. In this light, it is worth mentioning that many signalling molecules involved in the PI3K/Akt/mTOR pathway are altered in cancer cells. This may result in the constitutive activation of mTOR signalling and consequently in inhibition of autophagy. Furthermore, autophagy serves to limit genomic instability (Figure 5), especially in a context of inactivated p53 and retinoblastoma protein checkpoints and/or defects in apoptosis frequently observed in cancer cells [106]. Under metabolic stress, defects in autophagy divert cells to chronic necrosis when apoptosis is compromised. This in turn can result in chronic inflammation, macrophage infiltration and tumour progression. Alternatively, defects in autophagy result in the reduced removal of potential sources of genotoxic stress such as ROS (reactive oxygen species) from leaky damaged mitochondria or other organelles [107]. Accumulation of ROS may be an important source of DNA damage and tumour growth. Notwithstanding these findings, increasing evidence suggests that autophagy might play a different role in cancer depending on the stage of the disease. Indeed, in advanced stages of tumour progression, autophagy is frequently up-regulated [108] and localized in regions of the tumour with increased metabolic stress [106,109,110]. Therefore autophagy should also be considered as a survival pathway of cancer cells in stressful conditions. Several observations support this theory. First, autophagy is a quick way for cancer cells to cope with situations where nutrient and oxygen supply is limited [106,109,110]. This starvation-induced autophagy serves to protect cancer cells from cell death by maintaining metabolic activity. It should be noted that this process is self-limiting, since persistent growth factor deprivation leads to cell death, probably due to degradation of essential organelles and macromolecules. Secondly, metastatic cancer cells may escape from anoikis, an apoptotic cell death programme induced by anchorage-dependent cell detachment from the surrounding extracellular matrix, via the induction of autophagy [111]. The mechanism is still unclear, but studies indicate that cells detach from the extracellular matrix and lose survival signalling through integrins and that disruption of β1 integrin functions as an activator of cellular dormancy [112].

Role of autophagy in cancer

Figure 5
Role of autophagy in cancer

Autophagy acts in tumour suppression by removing damaged organelles and reducing DNA damage. Defective autophagy (e.g. due to mono-allelic loss of beclin 1) results in genomic instability, thereby facilitating tumour progression. However, autophagy may also act as a cytoprotective mechanism that helps cancer cells to resist anticancer treatments and to survive in conditions of low nutrient supply. Autophagy augmentation may therefore be effective in preventing tumour formation and progression, whereas autophagy inhibition may be helpful in promoting tumour regression.

Figure 5
Role of autophagy in cancer

Autophagy acts in tumour suppression by removing damaged organelles and reducing DNA damage. Defective autophagy (e.g. due to mono-allelic loss of beclin 1) results in genomic instability, thereby facilitating tumour progression. However, autophagy may also act as a cytoprotective mechanism that helps cancer cells to resist anticancer treatments and to survive in conditions of low nutrient supply. Autophagy augmentation may therefore be effective in preventing tumour formation and progression, whereas autophagy inhibition may be helpful in promoting tumour regression.

Inactivation of p53 is one of the most frequent alterations that characterize cancer cells. It has recently been found that knockout, knockdown or chemical inhibition of p53 stimulates autophagy in most mammalian cell lines or tissues [113]. This effect is a cell-cycle-dependent phenomenon that occurs mostly in the G1-phase, less so in the S-phase but not in the G2-and M-phases [114]. p53 is best known as a transcription factor that operates in the nucleus, but it also acts in the cytoplasm as an inducer of mitochondrial membrane permeabilization and apoptosis. Although p53 mutants have generally been visualized in the nucleus of tumour cells, In vitro large-scale analyses have demonstrated that many p53 variants can be found in either the nucleus and cytoplasm [115]. Nuclear and cytoplasmic p53 exert an apparently contradictory role in the control of autophagy. Although nuclear p53 can act as an autophagy-promoting transcription factor, cytoplasmic p53 inhibits autophagy [113,115]. Thus p53 turns out to be a central player in the regulation of autophagy, particularly in cancer cells.

Therapeutic modulation of autophagy in cancer

Induction of autophagic cell death is a useful approach to eliminate cancer cells independently or in cross-talk with apoptosis. For example, autophagic cell death has been reported in tamoxifen-treated MCF-7 cells [116] and soya bean saponin-treated colon cancer cells [117]. Moreover, stimulation of autophagy inhibits angiogenesis. Because many highly vascularized tumours form as a result of elevated mTOR signalling, inhibition of mTOR with rapamycin can diminish the process of angiogenesis and thus also tumour growth [118]. TRAIL (TNF-related apoptosis-inducing ligand) induces apoptotic cell death in a variety of cancer cells, but many cancer cells with apoptotic defects are resistant against TRAIL treatment, limiting its potential as an anticancer therapeutic. The single-chain fragment variable HW1 that specifically binds to TRAIL receptor 2 has been shown to induce autophagic cell death in a variety of both TRAIL-sensitive and TRAIL-resistant cancer cells [119]. On the other hand, genetic or pharmacological inhibition of autophagy enhances cytotoxicity of cancer chemotherapeutic agents [120,121]. Consequently, clinical trials are in progress to disrupt autophagic degradation to maximize the effects of cancer cytotoxic agents. Thus it may be necessary to differentially target autophagy in a context- and disease-stage-specific manner. Autophagy augmentation may be effective in preventing tumour formation and progression, whereas autophagy inhibition may be helpful in promoting tumour regression (Figure 5) [1].

AUTOPHAGY IN OTHER DISEASES

Growing evidence reveals that autophagy is involved in the progression or prevention of many other human diseases, such as atherosclerosis [122], diabetes [123] and muscular disease [124,125], as well as liver [126] and gastrointestinal disorders, including pancreatitis and Crohn's disease [127,128]. Furthermore, the activation of autophagy could be an effective way of eliminating infectious agents that access the cytosol either directly through the plasma membrane or after being internalized in phagosomes [129,130]. In this light, it is important to note that autophagy plays a crucial role in the delivery of microbial antigenic material to the innate and adaptive immune system, as well as in lymphocyte homoeostasis [131]. Finally, it is worth mentioning that autophagic activity decreases with age [132] and that this effect could mediate the functional deterioration of aging persons. Caloric restriction, the only intervention known to slow down aging, appears to improve autophagy induction, possibly owing to lower levels of insulin, an autophagy inhibitor.

Conclusions

A large body of evidence has demonstrated that autophagy in disease functions as a survival mechanism by maintaining viability during periods of stress, and by removing damaged organelles and toxic metabolites, such as protein aggregates or intracellular pathogens. Moreover, autophagy may act as a tumour suppressor mechanism by restraining necrosis and inflammation and by maintaining genetic stability under conditions of metabolic stress, which are hallmarks of the tumour microenvironment. Several drugs that regulate autophagy have been reported recently, suggesting that the autophagic machinery can be manipulated to treat human diseases. However, considering its dual capacity in cytoprotection and cell death, we need to improve our understanding of the various regulatory pathways before autophagy can be manipulated for therapeutic purposes. In particular, CMA and microautophagy need to be studied in more detail. Given the rate at which investigations into the mechanisms regulating autophagy accumulate and our understanding of this process progresses, it can be expected that many of these conundrums will be clarified soon.

FUNDING

The authors' work was supported by the Fund for Scientific Research (FWO)-Flanders (Belgium) [grant numbers G.0308.04, G.0113.06, G.0112.08]; the Bekales Foundation, the University of Antwerp (NOI-BOF and TOP-BOF); the Concerted Research Initiative of Ghent University [GOA project number 01G013A7]; and the Catholic University of Leuven [project number OT/06/49]. W. M. is a postdoctoral fellow of the FWO-Flanders.

Abbreviations

     
  • ALS

    amyotrophic lateral sclerosis

  •  
  • Ambra1

    activating molecule in beclin 1-regulated autophagy 1

  •  
  • AMPK

    AMP-dependent kinase

  •  
  • APP

    amyloid precursor protein

  •  
  • Atg

    AuTophaGy-related

  •  
  • β-amyloid

  •  
  • Bnip3

    Bcl-2/adenovirus E1B 19 kDa-interacting protein

  •  
  • CMA

    chaperone-mediated autophagy

  •  
  • ER

    endoplasmic reticulum

  •  
  • GSK-3β

    glycogen synthase kinase-3β

  •  
  • I/R

    ischaemia/reperfusion

  •  
  • IGF-1

    insulin-like growth factor-1

  •  
  • IMPase

    inositol monophosphatase

  •  
  • JNK

    c-Jun N-terminal kinase

  •  
  • LAMP

    lysosomal-associated membrane protein

  •  
  • LC3

    microtubule-associated protein light-chain 3

  •  
  • mTOR

    mammalian target of rapamycin

  •  
  • PE

    phosphatidylethanolamine

  •  
  • PI3K

    phosphoinositide 3-kinase

  •  
  • ROS

    reactive oxygen species

  •  
  • SMER

    small-molecule enhancer

  •  
  • α-syn

    α-synuclein

  •  
  • TNF

    tumour necrosis factor

  •  
  • TRAIL

    TNF-related apoptosis-inducing ligand

  •  
  • UPS

    ubiquitin–proteasome system

  •  
  • UVRAG

    UV irradiation resistance-associated tumour suppressor gene

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