Autophagy is an intracellular degradation pathway essential for cellular and energy homoeostasis. It functions in the clearance of misfolded proteins and damaged organelles, as well as recycling of cytosolic components during starvation to compensate for nutrient deprivation. This process is regulated by mTOR (mammalian target of rapamycin)-dependent and mTOR-independent pathways that are amenable to chemical perturbations. Several small molecules modulating autophagy have been identified that have potential therapeutic application in diverse human diseases, including neurodegeneration. Neurodegeneration-associated aggregation-prone proteins are predominantly degraded by autophagy and therefore stimulating this process with chemical inducers is beneficial in a wide range of transgenic disease models. Emerging evidence indicates that compromised autophagy contributes to the aetiology of various neurodegenerative diseases related to protein conformational disorders by causing the accumulation of mutant proteins and cellular toxicity. Combining the knowledge of autophagy dysfunction and the mechanism of drug action may thus be rational for designing targeted therapy. The present review describes the cellular signalling pathways regulating mammalian autophagy and highlights the potential therapeutic application of autophagy inducers in neurodegenerative disorders.

Autophagy as a cell survival process

The homoeostatic role of the proteolytic system is of fundamental importance for cellular functioning. Malfunction of these systems affects cellular vulnerability and longevity of organisms. Macroautophagy (hereinafter referred to as autophagy) is a major intracellular degradation pathway involved in the clearance of aggregation-prone proteins and damaged organelles [1]. Although this process was initially described as programmed cell death type II, its physiological role has evolved from a cell death phenomenon to a cell survival process. Autophagy plays a critical role in the removal of misfolded proteins and turnover of organelles for cellular homoeostasis. It also acts in sustaining energy homoeostasis during starvation by recycling of cytosolic components to compensate for nutrient deprivation. For example, tissue-specific abrogation of constitutive autophagy in mice causes dysfunction in the affected organs [26]. Moreover, autophagy-deficient newborn mice fail to survive after birth due to their inability to cope with starvation after detachment of the placental nutrient supply [7]. In addition, autophagy has a growing range of implications in a myriad of human physiological and pathological conditions, such as development, immunity, longevity, cancer, myopathies, liver diseases and neurodegeneration [1,8,9]. The concept of autophagy functioning predominantly as a cellular survival mechanism has been widely accepted to date, whereas autophagic cell death has been reported only in certain contexts [10,11].

The dynamic process of autophagy, defined as autophagic flux, encompasses the generation of autophagosomes and its fusion with late endosomes to form amphisomes, which subsequently fuse with lysosomes forming autolysosomes for degrading its cargo (Figure 1). This process is initiated by the formation of isolated membranes in the cytoplasm called phagophores. The membrane source of phagophores possibly arise from the ER (endoplasmic reticulum), plasma membrane, mitochondria and ER–mitochondria contact sites [1216]. The phagophores engulf cytoplasmic cargo during their elongation to form double-membraned vesicles called autophagosomes. Autophagosomes fuse with late endosomes to form single-membrane-bound hybrid organelles called amphisomes, which probably act as a sink for both the autophagic and endocytic pathways [17]. Amphisomes then fuse with the lysosomes to form acidic autolysosomes where the cargo is degraded. Whereas degradation of autophagic cargo is non-selective during nutrient deprivation, it can also be cargo-specific, as shown for p62 (sequestosome-1), mutant huntingtin, NBR1 (neighbour of BRCA1 gene 1) and certain pathogens [3,1821]. Furthermore, autophagy regulates metabolism of lipids (lipophagy) including cholesterol [22], which are essential structural components of membranes and govern intracellular trafficking [23]. Autophagy also acts in organelle quality control by regulating their turnover. For example, excess or damaged mitochondria are removed by autophagy, a process termed mitophagy [24].

The mammalian autophagy pathway and its regulatory machinery

Figure 1
The mammalian autophagy pathway and its regulatory machinery

Autophagy is initiated by the formation of phagophores in the cytoplasm, which engulf cytoplasmic cargo to form autophagosomes. Autophagosomes fuse with the late endosomes forming amphisomes that then fuse with the lysosomes to form autolysosomes where the autophagic cargo is degraded. Autophagy is controlled by mTOR-dependent and mTOR-independent pathways. The ULK1–Atg13–FIP200 complex regulates autophagosome synthesis downstream of the mTOR signalling pathways. The downstream components of mTOR-independent pathways regulating autophagosome biogenesis are not clear. The PI3KC3 complex, comprising hVps34, Beclin-1 and hVps15, also regulates autophagosome synthesis possibly downstream of the mTOR-independent pathways. Several Beclin-1 interactors, such as Atg14L, UVRAG, Bif-1 and Ambra1, or Rab5 interacting with hVps34, positively modulate this process. Two ubiquitin-like conjugation systems involving Atg proteins function in the elongation of phagophores. The Atg5–Atg12 conjugation involves Atg7 (E1-like) and Atg10 (E2-like), whereas the LC3–PE conjugation involves Atg7 (E1-like) and Atg3 (E2-like). The Atg5–Atg12 conjugate forms a complex with Atg16, Atg5–Atg12·Atg16, which has E3-like activity towards LC3–PE conjugation (LC3-II). LC3-II is a specific autophagy marker that is degraded in the autolysosomes as well as delipidated by Atg4 and recycled. Autophagosome maturation involving fusions with late endosomes and lysosomes are mediated by Rab7, ESCRT proteins, SNARE proteins and the UVRAG–Beclin-1–hVps34–hVps15 complex. Autophagic cargo consisting of non-specific or specific substrates such as p62, aggregation-prone proteins or mitochondria, are degraded in the autolysosomes.

Figure 1
The mammalian autophagy pathway and its regulatory machinery

Autophagy is initiated by the formation of phagophores in the cytoplasm, which engulf cytoplasmic cargo to form autophagosomes. Autophagosomes fuse with the late endosomes forming amphisomes that then fuse with the lysosomes to form autolysosomes where the autophagic cargo is degraded. Autophagy is controlled by mTOR-dependent and mTOR-independent pathways. The ULK1–Atg13–FIP200 complex regulates autophagosome synthesis downstream of the mTOR signalling pathways. The downstream components of mTOR-independent pathways regulating autophagosome biogenesis are not clear. The PI3KC3 complex, comprising hVps34, Beclin-1 and hVps15, also regulates autophagosome synthesis possibly downstream of the mTOR-independent pathways. Several Beclin-1 interactors, such as Atg14L, UVRAG, Bif-1 and Ambra1, or Rab5 interacting with hVps34, positively modulate this process. Two ubiquitin-like conjugation systems involving Atg proteins function in the elongation of phagophores. The Atg5–Atg12 conjugation involves Atg7 (E1-like) and Atg10 (E2-like), whereas the LC3–PE conjugation involves Atg7 (E1-like) and Atg3 (E2-like). The Atg5–Atg12 conjugate forms a complex with Atg16, Atg5–Atg12·Atg16, which has E3-like activity towards LC3–PE conjugation (LC3-II). LC3-II is a specific autophagy marker that is degraded in the autolysosomes as well as delipidated by Atg4 and recycled. Autophagosome maturation involving fusions with late endosomes and lysosomes are mediated by Rab7, ESCRT proteins, SNARE proteins and the UVRAG–Beclin-1–hVps34–hVps15 complex. Autophagic cargo consisting of non-specific or specific substrates such as p62, aggregation-prone proteins or mitochondria, are degraded in the autolysosomes.

Other types of autophagy include CMA (chaperone-mediated autophagy) and microautophagy. CMA utilizes the chaperone Hsc70 (heat-shock cognate protein 70) for specific substrate recognition that are delivered to the lysosomal membrane where they are unfolded and translocated into the lysosomal lumen by LAMP2a (lysosome-associated membrane protein 2a) [25]. Microautophagy, however, involves direct engulfment of cytoplasmic substrates by the lysosomes through invagination of the lysosomal membrane [26].

The autophagy machinery

Autophagy is evolutionarily conserved from yeast to mammals where several autophagy-related (Atg) proteins orchestrate the initiation, elongation, maturation and fusion stages of the pathway [27,28]. Two ubiquitin-like conjugation systems function in the initiation of autophagy (Figure 1). The first reaction involves Atg5–Atg12 conjugation, in which Atg7 (E1 ubiquitin-activating enzyme-like) activates the ubiquitin-like protein Atg12, which is then transferred to Atg10 (E2 ubiquitin-conjugating enzyme-like), and is ultimately linked covalently to Atg5 [29,30]. The Atg12–Atg5 conjugate forms a large complex with Atg16L1 (Atg12–Atg5·Atg16L1) that is essential for the elongation of phagophores, but dissociates after autophagosome formation [31]. The second system involves the conjugation of microtubule-associated protein 1 LC3 (light chain 3) (mammalian homologue of yeast Atg8) to PE (phosphatidylethanolamine). LC3 undergoes post-translational C-terminal cleavage by the protease Atg4B, resulting in the cytosolic LC3-I form, which is then conjugated to PE in a reaction involving Atg7 (E1-like) and Atg3 (E2-like) to generate the autophagosome-associated LC3-II form [32,33]. LC3-II mediates membrane tethering and hemifusion essential for the expansion and closure of phagophores to form autophagosomes where it remains associated throughout their lifespan [34]. The levels of LC3-II thus correlate with the steady-state number of autophagosomes [32]. LC3-II residing on the autophagosomal inner membrane is degraded within the autolysosomes, whereas those on the cytoplasmic surface of autolysosomes are delipidated by Atg4 and recycled [35]. Cross-talk occurs between the two conjugation systems, for example, the Atg12–Atg5·Atg16L1 complex acts like an E3 ubiquitin ligase to facilitate LC3-I—PE conjugation [36].

Distinct classes of PI3K (phosphoinositide 3-kinase) regulate autophagy in mammalian cells. The activity of the PI3KC3 (class III PI3K) member hVps34 (mammalian vacuolar protein sorting 34 homologue) is essential for autophagosome biogenesis, whereas inhibition of PI3KC1a (class Ia PI3K) activity stimulates autophagy by the mTOR (mammalian target of rapamycin) pathway [37] (Figure 1). The lipid kinase hVps34 phosphorylates phosphatidylinositol to form PtdIns3P, which possibly functions in autophagosome synthesis [38]. hVps34 is part of the autophagy-initiating macromolecular complex (PI3KC3) consisting of Beclin-1/Atg6, Atg14L and hVps15 [3942]. The activity of hVps34 is enhanced by its interaction with Beclin-1, which has several binding partners governing autophagosome formation [4345]. Beclin-1 interactors that positively regulate autophagy are Atg14L, Ambra1 (activating molecule in Beclin-1-regulated autophagy), UVRAG (UV irradiation resistance-associated gene) and Bif-1 (endophilin B1), whereas Rubicon, anti-apoptotic proteins Bcl-2 (B-cell lymphoma 2) and Bcl-XL, and the pro-apoptotic protein Bim bind to Beclin-1 to negatively regulate autophagy [42,4651]. The small GTPase Rab5 also binds to and activates hVps34 to induce autophagy [52] (Figure 1). Pharmacological inhibition of hVps34 activity by 3-methyladenine, or with PI3K inhibitors such as wortmannin and LY294002, inhibits autophagosome formation [37,53,54]. The crystal structure of Vps34 (vacuolar protein sorting 34) is important for designing new generations of Vps34 inhibitors with improved specificity [55].

Autophagosome biogenesis is also regulated by the ULK1 (UNC-51-like kinase 1)–Atg13–FIP200 (focal adhesion kinase family-interacting protein of 200 kDa) macromolecular complex [56] (described below). Membrane contribution possibly from other organelles for the elongation of phagophores is mediated by Atg9 [57]. A genome-wide screen under amino acid starvation-induced autophagy has identified several regulators, including the Golgi-resident SCOC (short coiled-coil protein) required for autophagosome formation [58]. Newly generated autophagosomes move bidirectionally along the microtubules with a bias towards the microtubule-organizing centre where the lysosomes are enriched [59,60]. Depolymerization of microtubules or inhibition of dynein-dependent transport impairs autophagosome maturation and blocks autophagic flux, suggesting a role for the dynein motor proteins [6163]. Autophagosomes first fuse with late endosomes to form amphisomes, which ultimately fuse with the lysosomes to yield autolysosomes. These fusion events are mediated by Rab7, ESCRTs (endosomal sorting complexes required for transport), SNAREs (N-ethylmaleimide-sensitive factor-attachment protein receptors) and the class C Vps proteins (Figure 1). During the formation of amphisomes, tethering between vesicular membranes is mediated by the small GTPase Rab7 and its effector/activator HOPS (homotypic fusion and vacuole protein sorting) complex [64,65]. Rab GTPase-activating proteins can also interact with LC3 to promote autophagy [66]. The Beclin-1-interacting protein UVRAG promotes autophagosome maturation by recruiting class C Vps proteins and activating Rab7 [48]. In contrast, another Beclin-1-binding protein Rubicon, which exists in a distinct Beclin-1 complex with hVps34, hVps15 and UVRAG, prevents autophagosome maturation [42,67]. Functional MVBs (multivesicular bodies) are also essential for the maturation of autophagosome since perturbations in MVB formation, such as with mutations in the ESCRT proteins, block autophagic flux [6870]. The formation of specific SNARE complexes mediating autophagosome maturation has been recently described, such as between autophagosomal syntaxin-17 and late endosomal/lysosomal VAMP8 (vesicle-associated membrane protein 8) mediated by SNAP-29 (synaptosome-associated protein 29) [71]. Other SNARE proteins implicated in the maturation of autophagosomes include VAMP3 (regulates amphisome formation) and VAMP7 (regulates fusion with lysosomes) [72,73]. The syntaxin-5 SNARE complex regulates ER–Golgi transport through the secretory pathway that is essential for the activity of lysosomal proteases such as cathepsins for autophagic degradation [74]. Impairing the lysosomal proteolytic function with the v-ATPase (vacuolar H+-ATPase) inhibitor bafilomycin A1, which prevents lysosomal acidification, retards autophagosome maturation and autophagic cargo clearance [75,76]. A proteomic analysis has uncovered the mammalian autophagy interaction landscape, revealing a network of 751 interactions under basal autophagy among 409 candidate proteins [77]. Regulation of autophagy by various signalling pathways is described below.

Regulation of autophagy by the mTOR pathway

The classical regulation of autophagy is governed by the mTOR pathway, which negatively regulates this process (Figure 2). The serine/threonine protein kinase mTOR also mediates vital cellular functions such as translation and cell growth. This pathway involves two distinct functional complexes: mTORC1 (mTOR complex 1) consisting of the mTOR catalytic subunit raptor (regulatory-associated protein of mTOR), PRAS40 (proline-rich Akt substrate of 40 kDa), mLST8 (mammalian lethal with SEC13 protein 8) and deptor (DEP-domain containing mTOR-interacting protein); and mTORC2 consisting of rictor (rapamycin-insensitive companion of mTOR), protor (protein observed with rictor), mSIN1 (mammalian stress-activated mitogen-activated protein kinase-interacting protein 1), mLST8 and deptor [78,79]. During starvation that inhibits mTORC1 activity, induction of autophagy recycles intracellular constituents to provide a source of energy. Inhibition of mTORC1 and induction of autophagy is associated with reduced phosphorylation of two of its downstream effectors, p70S6K (ribosomal protein S6 kinase-1) and 4E-BP1 (translation initiation factor 4E-binding protein-1) at Thr389/Thr421/Ser424 and Thr37/Thr46 respectively [80].

Regulation of autophagy by mTOR-dependent pathways

Figure 2
Regulation of autophagy by mTOR-dependent pathways

Diverse signals like amino acids, growth factors, energy status and stressors activate mTORC1, which negatively regulates autophagy. Insulin and growth factors act through the PI3KC1a/Akt/TSC/mTORC1 pathway by binding to their cell-surface receptors that activate PI3KC1a and Akt, leading to inhibition of TSC1/2, thereby allowing Rheb to activate mTORC1 and inhibit autophagy. Activation of p70S6K by mTORC1 also exerts a feedback loop by inhibiting IRS1 (insulin receptor substrate 1). In addition, activated Akt inhibits FoxO3-mediated transcription of autophagy genes. Influx of amino acids by their cell-surface transporters function through the Rag/mTORC1 pathway by activating Rag GTPases bound to the lysosome-resident Ragulator, thereby recruiting mTORC1 on the lysosomal surface where Rheb causes its activation, leading to suppression of autophagy. Lysosome-localized activated mTORC1 also sequesters TFEB to prevent its nuclear translocation and transcription of autophagy and lysosomal genes. Energy status and stress signals act through the AMPK/TSC/mTORC1 pathway to modulate autophagy. High ATP/AMP ratio, NO or cytoplasmic p53 inhibits AMPK, thereby preventing TSC1/2 activation that causes Rheb to activate mTORC1 and inhibit autophagy. The ULK1–Atg13–FIP200 complex regulates autophagosome synthesis downstream of mTORC1. Inhibition of mTORC1 during starvation or pharmacologically with rapamycin, CCI-779, Torin1 or PP242 stimulates autophagy. Dual inhibition of mTORC1 and PI3KK with Torin2, or PI3KC1a and mTORC1 with PI103, also activates autophagy. ATM, ataxia telangiectasia mutated; ATR, ataxia telangiectasia- and Rad3-related; IGF, insulin-like growth factor; PIP2, PtdIns(4,5)P2; PIP3, PtdIns(1,4,5)P3; PRAS40, proline-rich Akt substrate of 40 kDa.

Figure 2
Regulation of autophagy by mTOR-dependent pathways

Diverse signals like amino acids, growth factors, energy status and stressors activate mTORC1, which negatively regulates autophagy. Insulin and growth factors act through the PI3KC1a/Akt/TSC/mTORC1 pathway by binding to their cell-surface receptors that activate PI3KC1a and Akt, leading to inhibition of TSC1/2, thereby allowing Rheb to activate mTORC1 and inhibit autophagy. Activation of p70S6K by mTORC1 also exerts a feedback loop by inhibiting IRS1 (insulin receptor substrate 1). In addition, activated Akt inhibits FoxO3-mediated transcription of autophagy genes. Influx of amino acids by their cell-surface transporters function through the Rag/mTORC1 pathway by activating Rag GTPases bound to the lysosome-resident Ragulator, thereby recruiting mTORC1 on the lysosomal surface where Rheb causes its activation, leading to suppression of autophagy. Lysosome-localized activated mTORC1 also sequesters TFEB to prevent its nuclear translocation and transcription of autophagy and lysosomal genes. Energy status and stress signals act through the AMPK/TSC/mTORC1 pathway to modulate autophagy. High ATP/AMP ratio, NO or cytoplasmic p53 inhibits AMPK, thereby preventing TSC1/2 activation that causes Rheb to activate mTORC1 and inhibit autophagy. The ULK1–Atg13–FIP200 complex regulates autophagosome synthesis downstream of mTORC1. Inhibition of mTORC1 during starvation or pharmacologically with rapamycin, CCI-779, Torin1 or PP242 stimulates autophagy. Dual inhibition of mTORC1 and PI3KK with Torin2, or PI3KC1a and mTORC1 with PI103, also activates autophagy. ATM, ataxia telangiectasia mutated; ATR, ataxia telangiectasia- and Rad3-related; IGF, insulin-like growth factor; PIP2, PtdIns(4,5)P2; PIP3, PtdIns(1,4,5)P3; PRAS40, proline-rich Akt substrate of 40 kDa.

Pharmacological inhibitors of mTORC1 are used for research and possible anti-cancer drugs [81]. These include rapamycin (sirolimus) and its analogues such as CCI-779, which are potent inducers of autophagy in yeast to mammalian cells, including neurons and in vivo in mouse brain [18,8184] (Figure 2). In mammalian cells, rapamycin forms a complex with the immunophilin FKBP12 (FK506-binding protein of 12 kDa), thereby stabilizing the raptor–mTOR association and inhibiting mTORC1 kinase activity [85]. However, rapamycin does not completely suppress the activity of mTORC1, possibly due to rapamycin-resistant functions of mTORC1 such as cap-dependent translation and 4E-BP1 phosphorylation [86]. Selective ATP-competitive inhibitors of mTORC1 and mTORC2, such as Torin1 and PP242, induce autophagy with greater efficacy than rapamycin [8789]. Although the mechanism of PP242 in stimulating autophagy has not been assessed, the effect of Torin1 is mTORC2-independent, but mTORC1-dependent, due to the suppression of rapamycin-resistant functions of mTORC1 required for inhibiting autophagy [87]. Torin2 has been recently developed as a second-generation ATP-competitive inhibitor that is selective for both mTORC1 and mTORC2 with a superior pharmacokinetic profile to those of the previous inhibitors [90]. Unlike Torin1 and PP242, Torin2 also inhibits other PI3KKs (PI3K-related protein kinases) and may be relevant in contexts where mTOR inhibition alone is insufficient. Although Torin2 induces autophagy [90], its potency with respect to other mTOR inhibitors remains to be characterized.

A screen for compounds modulating autophagosome number has identified additional mTORC1 inhibitors that do not affect mTORC2. These include niclosamide, rottlerin, perhexilene and amiodarone, which possibly act upstream of mTORC1 to stimulate autophagy [91]. However, amiodarone has been also identified in two independent screens for autophagy modulators (described below), where it acts directly by inhibiting Ca2+ channels at a concentration considerably lower than that reported to inhibit mTORC1 [92,93]. Similarly, perhexiline is a Ca2+ channel blocker, and it is plausible that these compounds induce autophagy through mTOR-independent pathways. In addition, latrepirdine, a neuroactive compound, has been shown recently to induce autophagy by inhibiting the mTORC1 pathway [94].

Control of autophagy downstream of mTORC1 by the ULK1–Atg13–FIP200 complex

The molecular link between TOR (target of rapamycin) signalling and the autophagic machinery has been dissected in yeast where the Atg1 kinase and the Atg1–Atg13–Atg17 complex act downstream of TOR regulating autophagosome biogenesis [56]. In mammalian cells, Atg13 binds to the mammalian Atg1 homologues ULK1 or ULK2 and mediates the interaction of ULK1/2 with FIP200, leading to the formation of a ULK1–Atg13–FIP200 stable complex that signals to the autophagic machinery downstream of mTORC1 [95100] (Figure 2). Under nutrient-rich conditions, mTORC1 inhibits autophagy through direct interaction with this complex and mediates phosphorylation-dependent inactivation of the kinase activities of Atg13 and ULK1. Under starvation conditions or treatment with rapamycin, mTORC1 dissociates from this complex, resulting in the inhibition of mTORC1-mediated phosphorylation of Atg13 and ULK1. This leads to dephosphorylation-dependent activation of ULK1 and ULK1-mediated phosphorylation of Atg13, FIP200 and ULK1 itself, which triggers autophagy [9597]. The ULK1–Atg13–FIP200 complex thus acts as an integrator of the autophagy signals downstream of mTORC1 to regulate autophagosome synthesis. Diverse signals such as amino acids, growth factors, energy status (high ATP/AMP ratio) and stressors activate mTORC1 and suppress autophagy.

Control of autophagy by nutrient sensing through the Rag/mTORC1 pathway

Amino acids are the basic building blocks for protein synthesis. In addition, they serve as substrates in metabolic pathways and act as regulators in signal transduction. Although amino acids inhibit autophagosome synthesis by activating mTORC1 [101], their intracellular sensing mechanism has remained obscure until recently [102] (Figure 2). During the availability of extracellular amino acids, the influx of L-glutamine by its high-affinity transporter SLC1A5 (solute carrier 1A5) initially increases its intracellular concentration. The heterodimeric SLC7A5–SLC3A2 bidirectional transporter then uses the intracellular L-glutamine as an efflux substrate in exchange for the cellular uptake of essential amino acids, which subsequently activate mTORC1 [103]. The Rag (Ras-related GTP-binding protein) GTPases, which exist as heterodimers of either RagA or RagB bound to RagC or RagD, mediate amino acid signalling to mTORC1 activation. A multiprotein complex consisting of Rag GTPases, Ragulator and v-ATPase forms an amino acid-sensing machinery on the lysosomal surface [104,105]. When amino acids are absent, the Rag GTPases remain in an inactive conformation consisting of GDP-bound RagA/B and GTP-bound RagC/D. In the presence of amino acids, the Rag GTPases switch to an active conformation in which RagA/B is loaded with GTP and RagC/D is loaded with GDP. The active Rag heterodimer physically interacts with raptor, sequestering mTORC1 on to the surface of lysosomes where the Ragulator-bound Rag GTPases reside, thus enabling interaction with the mTORC1 activator Rheb (Ras homologue enriched in brain) bound to GTP. This causes activation of mTORC1 under nutrient-rich conditions to inhibit autophagy [106,107]. A recently described mechanism shows interaction of the lysosome-resident mTORC1 with TFEB (transcription factor EB) under nutrient-rich conditions, leading to phosphorylation of TFEB and its cytoplasmic retention (Figure 2). In contrast, starvation activates TFEB, which translocates to the nucleus causing expression of the lysosomal and autophagy genes associated with stimulation of autophagy [108110].

Starvation induces autophagy to restore cellular amino acid levels, which is essential for neonatal survival for energy homoeostasis since autophagy-deficient (deletion of Atg5) newborn mice exhibit reduced amino acid concentrations and fail to survive after birth [7]. The dynamics of intracellular lysosomal positioning co-ordinates mTORC1 signalling and autophagic flux. Activation of mTORC1 by nutrients correlates with its presence on peripheral lysosomes that are physically close to the upstream signalling modules, whereas starvation causes perinuclear clustering of lysosomes driven by changes in intracellular pH [111]. Starvation-induced autophagy has also been suggested to cause recruitment of mTORC1 on to the surface of autolysosomes from which mTORC1 facilitates the reformation of primary lysosomes, a process dependent on the mTORC1 kinase activity [112]. This reflects an intriguing paradigm that warrants further investigation. Expression of constitutively active Rag in mammalian cells under starvation conditions activates mTORC1 and inhibits autophagy, whereas dominant-negative Rag blocks the effects of amino acids on mTORC1 activation to stimulate autophagy [107]. Consistent with the findings in autophagy-deficient neonates, mice expressing a constitutively active form of RagA (GTP-bound) die considerably sooner after birth due to failure in inactivating mTORC1 to induce autophagy under starvation, compared with the wild-type littermates [113]. This study suggests that a glucose homoeostasis defect in fasted RagA(GTP/GTP) neonates correlates with their inability to trigger autophagy and to produce amino acids for de novo glucose production. The Rag GTPases thus integrate signals of both glucose and amino acid concentrations to regulate mTORC1 and autophagy.

Control of autophagy by growth factors through the PI3KC1a/Akt/TSC/mTORC1 pathway

mTORC1 is regulated by a multitude of growth factors, such as insulin, which relay information on the nutritional state of the organism, and consequently govern autophagy. A major signalling cascade regulating mTORC1 is the PI3KC1a pathway [37] (Figure 2). Binding of growth factors or insulin to their cell-surface receptors activates the PI3KC1a complex, which mediates conversion of the plasma membrane lipid PtdIns(4,5)P2 to generate PtdIns(3,4,5)P3. PtdIns(3,4,5)P3 recruits pleckstrin homology domain proteins, such as the serine/threonine kinases PDK1 (phosphoinositide-dependent kinase 1) and Akt, to the plasma membrane [114]. Stimulation of the PI3KC1a pathway increases PtdIns(3,4,5)P3, which inhibits autophagy by PDK1-mediated phosphorylation-dependent activation of Akt, whereas suppression of Akt or hyperactivity of the phosphoinositide phosphatase PTEN (phosphatase and tensin homologue deleted from chromosome 10), a tumour suppressor, stimulates autophagy by inhibiting this pathway [37,115,116]. Pharmacologically, a highly selective PI3KC1a inhibitor, PI103, which is also an ATP-competitive mTOR inhibitor, activates autophagy [117].

Phosphorylation of Akt by mTORC2 at Ser473 possibly facilitates PDK1-mediated phosphorylation at Thr308 [118]. Activated Akt mediates phosphorylation of the tumour-suppressor TSC (tuberous sclerosis complex), comprising the TSC1/2 heterodimer that affects cell growth and survival [118]. TSC1/2 is a GAP (GTPase-activating protein) for the Ras family GTP-binding protein Rheb, which remains inhibited (GDP-bound) when TSC1/2 is active. Akt-mediated phosphorylation of TSC2 inhibits its GAP activity for Rheb, thereby allowing the active GTP-bound form of Rheb to directly bind and activate mTORC1 [119,120]. Overexpression of Rheb sustains mTORC1 activity and inhibits autophagy, whereas loss of Rheb abrogates the effects of growth factors and nutrients on mTORC1 activation [18,121,122]. In addition to inhibiting TSC2 and activating mTORC1, Akt also causes phosphorylation-dependent inactivation of the transcription factor FoxO3 (forkhead box O3), leading to its cytoplasmic retention in response to insulin or growth factors [123]. A role for FoxO3 in regulating autophagy is shown in atrophying muscle from starved mice after denervation, where its activation mediates nuclear transcription of autophagy-related genes including LC3, Bnip3 (Bcl-2/adenovirus E1B 19 kDa-interacting protein 3), Vps34 and ULK1 [124,125] (Figure 2).

Intriguingly, p70S6K is shown to be a positive regulator of autophagy in Drosophila, where loss of p70S6K activity prevents excessive autophagy in TOR-inactive states, probably by inhibiting the PI3KC1a pathway through a negative-feedback loop involving down-regulation of the insulin receptor substrates [126]. Several other kinases including AMPK (AMP-activated protein kinase) and ERK1/2 (extracellular-signal-regulated kinase 1/2) signal to mTORC1 by phosphorylating TSC2 and inhibiting TSC1/2 activity [79,118]. Thus TSC1/2 acts as an upstream integrator of various signals impinging on the mTORC1 pathway to regulate autophagy.

Control of autophagy by stress and energy sensing through the AMPK/TSC/mTORC1 pathway

Apart from amino acids, the activity of mTORC1 can be regulated by changes in the energy state through the AMPK/TSC pathway that influences autophagy [127] (Figure 2). Glycolysis and mitochondrial respiration convert nutrients into energy, which is stored in the form of ATP. AMPK is a cellular energy sensor of changes in the intracellular ATP/AMP concentrations [128]. During metabolic stress AMPK is phosphorylated and activated by the kinase LKB1, which is the Peutz–Jeghers syndrome gene product [129]. Under conditions of low energy (high AMP/ATP ratio) during nutrient deprivation, activated AMPK directly phosphorylates TSC2, providing the priming phosphorylation for subsequent phosphorylation by GSK3 (glycogen synthase kinase 3) [130,131]. Unlike the effects of Akt, these phosphorylation events activate the GAP activity of TSC1/2 towards Rheb to inhibit mTORC1 signalling. In addition, AMPK phosphorylates raptor that binds to 14-3-3 protein, leading mTORC1 inhibition [132]. This also prevents mTORC1-dependent protein synthesis under low energy, which is consistent with a role for AMPK to shut off ATP-dependent processes [128]. Activation of AMPK in yeast inhibits TOR and stimulates autophagy [133]. Likewise, inhibiting AMPK activity in mammalian cells with its dominant-negative form or pharmacologically with compound C suppresses autophagy [134]. However, the AMPK activator AICAR (5-amino-4-imidazolecarboxamide riboside) inhibits autophagy in mammalian cells [134], probably due to its effects unrelated to AMPK or it may act on additional targets in mammals.

Recent studies have shown that activation of AMPK increases autophagy not only indirectly through inactivation of mTORC1, but also directly through phosphorylation of ULK1 and Beclin-1. Activation of ULK1 by energy depletion occurs though a mechanism distinct to that mediated by amino acid starvation [135,136]. Although these studies differ in the AMPK-mediated ULK1 phosphorylation sites, nonetheless, the co-ordinated phosphorylation of ULK1 by mTORC1 and AMPK possibly integrate nutrient and energy signals to control autophagic flux in response to metabolic requirements. The LKB1/AMPK-dependent phosphorylation of p27 stabilizes this cyclin-dependent kinase inhibitor that allows cells to survive growth factor withdrawal and metabolic stress by activating autophagy [137]. In addition, AMPK differentially regulates different hVps34 complexes under energy depletion by inhibiting the non-autophagy hVps34 complexes through phosphorylation-dependent inactivation of hVps34, and activating the pro-autophagy hVps34 complexes by phosphorylating Beclin-1 [138]. These events are facilitated by Atg14L, thereby converting the AMPK-mediated inhibition of hVps34 into activation for stimulating autophagy.

Several stress signals regulate mTORC1 and autophagy presumably through the AMPK/TSC axis (Figure 2). For example, nitrosative stress mediated by NO (nitric oxide), which is generated by NOSs (nitric oxide synthases), inhibits autophagosome synthesis. NO S-nitrosylates and inhibits IKKβ (inhibitor of nuclear factor κB kinase β), leading to a decrease in AMPK activity and TSC1/2-dependent activation of mTORC1 [139]. Activation of the IKK complex phosphorylates AMPK and JNK1 (c-Jun N-terminal kinase 1) to stimulate autophagy independently of NF-κB (nuclear factor κB) [140]. Cytoplasmic p53 inhibits autophagy possibly through the AMPK/TSC/mTORC1 pathway, whereas pharmacological inhibition of p53 with pifithrin-α induces autophagy [141]. However, direct interaction of FIP200 with p53 is additionally shown to inhibit autophagy [142]. AMPK also mediates hypoxia-induced autophagy. During hypoxia, mitochondrial respiration is impaired, leading to a low ATP/AMP ratio that activates AMPK and inhibits mTORC1 [143].

Regulation of autophagy by mTOR-independent pathways

Apart from the regulation of autophagy by mTORC1 and diverse upstream signals impinging on it, various mTOR-independent autophagy pathways have been described that are amenable to chemical perturbations.

Control of autophagy by the inositol signalling pathway

The first mTOR-independent regulation of autophagy defines a role for the inositol signalling pathway that negatively regulates this process [144] (Figure 3). This pathway is stimulated by G-protein-coupled receptor-mediated activation of PLC (phospholipase C), which hydrolyses PtdIns(4,5)P2 to form Ins(1,4,5)P3 and DAG (diacylglycerol). Ins(1,4,5)P3 functions as a second messenger and binds to its receptors (IP3R) on the ER, thereby releasing ER Ca2+ into the cytoplasm that elicits a range of cellular responses [145]. Ins(1,4,5)P3 is degraded by a 5′-phosphatase and IPPase (inositol polyphosphate 1-phosphatase) to form InsP, which is further hydrolysed by IMPase (inositol monophosphatase) into free inositol that is required for the inositol signalling pathway [146].

Regulation of autophagy by mTOR-independent pathways

Figure 3
Regulation of autophagy by mTOR-independent pathways

Various signalling cascades involving cAMP/Epac/PLCϵ, Ca2+/calpain and inositol (Ins) signalling pathways regulate autophagy independently of mTORC1. Elevation of cAMP levels by adenylate cyclase (AC) activates Epac that in turn activates Rap2B, leading to PLCϵ-mediated hydrolysis of PtdIns(4,5)P2 (PIP2) to generate Ins(1,4,5)P3 (IP3), which inhibits autophagy. Ins(1,4,5)P3 binds to IP3Rs on the ER to release stored Ca2+ that impairs autophagosome maturation by blocking autophagosome–lysosome fusion. In addition, an increase in cytosolic Ca2+ levels due to Ca2+ efflux from ER stores as well as Ca2+ influx through L-type Ca2+ channels increases its cytosolic levels, resulting in activation of calpain which inhibits autophagy. Calpain activates G, increasing adenylate cyclase (AC) activity that generates cAMP to suppress autophagy, thereby creating an autophagy-inhibitory link between these pathways. Multiple drug targets acting at distinct stages of these mTOR-independent signalling pathways induce autophagy, such as IMPase inhibitors (lithium, L-690,330), inositol-lowering agents (carbamazepine, valproic acid), Ca2+ channel blockers (verapamil, loperamide, nitrendipine, niguldipine, nimodipine, amiodarone or pimozide), calpain inhibitors (calpastatin, calpeptin), G inhibitor (NF449), adenylate cyclase inhibitor (2′5′ddA), imidazoline receptor agonists (rilmenidine, clonidine) and IP3R antagonist (xestospongin B). Other mTOR-independent autophagy enhancers include trehalose, SMERs and Tat-Beclin-1. The NOS inhibitor L-NAME also stimulates autophagy, whereas NO generated by the NOS isoforms suppresses autophagy by inhibiting the JNK1/Beclin-1/PI3KC3 pathway. Binding of Bcl-2 to Beclin-1 inhibits autophagy, whereas BH3 mimetics like ABT737 induces autophagy by preventing this interaction. Starvation phosphorylates JNK1, which in turn causes Bcl-2 phosphorylation, thereby disrupting Beclin-1–Bcl-2 interaction to promote the Beclin-1–hVps34 autophagy-initiating complex. The PI3KC3 complex comprising Beclin-1–hVps34–hVps15 regulates autophagosome synthesis possibly downstream of the mTOR-independent pathways, although mechanistic details remain to be elucidated. Note that starvation and ABT737 also inhibit mTORC1 activity. DAG, diacylglycerol.

Figure 3
Regulation of autophagy by mTOR-independent pathways

Various signalling cascades involving cAMP/Epac/PLCϵ, Ca2+/calpain and inositol (Ins) signalling pathways regulate autophagy independently of mTORC1. Elevation of cAMP levels by adenylate cyclase (AC) activates Epac that in turn activates Rap2B, leading to PLCϵ-mediated hydrolysis of PtdIns(4,5)P2 (PIP2) to generate Ins(1,4,5)P3 (IP3), which inhibits autophagy. Ins(1,4,5)P3 binds to IP3Rs on the ER to release stored Ca2+ that impairs autophagosome maturation by blocking autophagosome–lysosome fusion. In addition, an increase in cytosolic Ca2+ levels due to Ca2+ efflux from ER stores as well as Ca2+ influx through L-type Ca2+ channels increases its cytosolic levels, resulting in activation of calpain which inhibits autophagy. Calpain activates G, increasing adenylate cyclase (AC) activity that generates cAMP to suppress autophagy, thereby creating an autophagy-inhibitory link between these pathways. Multiple drug targets acting at distinct stages of these mTOR-independent signalling pathways induce autophagy, such as IMPase inhibitors (lithium, L-690,330), inositol-lowering agents (carbamazepine, valproic acid), Ca2+ channel blockers (verapamil, loperamide, nitrendipine, niguldipine, nimodipine, amiodarone or pimozide), calpain inhibitors (calpastatin, calpeptin), G inhibitor (NF449), adenylate cyclase inhibitor (2′5′ddA), imidazoline receptor agonists (rilmenidine, clonidine) and IP3R antagonist (xestospongin B). Other mTOR-independent autophagy enhancers include trehalose, SMERs and Tat-Beclin-1. The NOS inhibitor L-NAME also stimulates autophagy, whereas NO generated by the NOS isoforms suppresses autophagy by inhibiting the JNK1/Beclin-1/PI3KC3 pathway. Binding of Bcl-2 to Beclin-1 inhibits autophagy, whereas BH3 mimetics like ABT737 induces autophagy by preventing this interaction. Starvation phosphorylates JNK1, which in turn causes Bcl-2 phosphorylation, thereby disrupting Beclin-1–Bcl-2 interaction to promote the Beclin-1–hVps34 autophagy-initiating complex. The PI3KC3 complex comprising Beclin-1–hVps34–hVps15 regulates autophagosome synthesis possibly downstream of the mTOR-independent pathways, although mechanistic details remain to be elucidated. Note that starvation and ABT737 also inhibit mTORC1 activity. DAG, diacylglycerol.

An elevation of intracellular inositol or Ins(1,4,5)P3 levels inhibits autophagosome synthesis [144]. This can be achieved pharmacologically by increasing inositol levels with myo-inositol or by increasing Ins(1,4,5)P3 levels through inhibition of prolyl oligopeptidase activity [147]. Inositol-lowering agents, such as mood-stabilizing drugs like lithium, carbamazepine or valproic acid [147], induce autophagy and facilitate the clearance of autophagy substrates without inhibiting mTORC1 activity [144]. Rapamycin does not affect inositol levels either, suggesting that the control of autophagy by Ins(1,4,5)P3 and mTOR is mediated by two independent pathways. Lithium primarily acts by inhibiting IMPase, thereby preventing inositol recycling and leading to depletion of the cellular inositol pool [145]. Likewise, valproic acid reduces inositol levels by inhibiting MIPS (myo-inositol-1-phosphate synthase) that catalyses the rate-limiting step of inositol biosynthesis [148]. The specificity of regulation by the inositol signalling pathway is shown using a competitive inhibitor of IMPase, L-690,330 [149], which prevents dephosphorylation of InsP, thereby depleting cellular inositol levels to stimulate autophagy [144]. Similarly, autophagy is induced by overexpression of cytosolic inositol-1,4,5-trisphosphate kinase A, which lowers Ins(1,4,5)P3 levels by phosphorylating it to Ins(1,3,4,5)P4 [93]. Consistent with a role for Ins(1,4,5)P3 in regulating autophagy, genetic knockdown or pharmacological inhibition of IP3R stimulates autophagy [150]. In addition, IP3R is a positive regulator of the autophagy-inhibitory Beclin-1–Bcl-2 complex. The IP3R antagonist xestospongin B induces autophagy by disrupting the interaction of Beclin-1 with the IP3R–Bcl-2 complex [151].

Control of autophagy by the Ca2+/calpain pathway

Autophagy is also regulated by changes in intracellular Ca2+ levels (Figure 3). An early study has shown impairment of autophagy by increased cytosolic Ca2+ in rat hepatocytes [152]. Subsequent studies have found that elevation in cytosolic Ca2+ has complex effects in impairing autophagy, which occurs at the level of both autophagosome formation and autophagosome–lysosome fusion. Pharmacological treatment with thapsigargin, an ER Ca2+/Mg2+-ATPase inhibitor that releases Ca2+ from ER stores, or ionomycin, a Ca2+ ionophore that releases Ca2+ from intracellular stores, blocks autophagic flux, thereby increasing the number of autophagosomes and retards autophagic cargo clearance [93,153]. In contrast, influx of extracellular Ca2+ causing elevation in its cytosolic concentration, such as with the L-type Ca2+ channel agonist (±)-Bay K8644, inhibits autophagy at the level of autophagosome synthesis [93]. An apparent conflict with this evidence has been reported in a study describing an induction of autophagy caused by treatment with Ca2+-mobilizing agents such as thapsigargin or ionomycin [154]. However, this study measured only the steady-state levels of autophagosomes without analysing autophagic flux, which can distinguish between autophagosome synthesis and autophagosome–lysosome fusion. It is therefore likely that the observations of an accumulation of autophagosomes by application of Ca2+ ionophores are consistent with that of a block in autophagic flux, as reported in the other studies [93,152,153]. Why an increase in cytosolic Ca2+ due to influx of extracellular Ca2+ or efflux from intracellular stores impairs autophagy at early or late stages of the pathway is not fully characterized.

A screen with FDA (U.S. Food and Drug Administration)-approved drugs to identify autophagy modulators has revealed L-type Ca2+ channel antagonists, such as verapamil, loperamide, amiodarone, nimodipine and nitrendipine, as enhancers of autophagy [93] (Figure 3). These Ca2+ channel blockers increase autophagosome synthesis and facilitate the clearance of autophagy substrates by decreasing the levels of cytosolic Ca2+. This screen has also identified K+ATP channel openers, such as minoxidil, which decreases whole-cell L-type Ca2+ channels to induce autophagy. In contrast, the K+ATP channel blockers, such as quinine sulfate and tolazamide, inhibit autophagy [93]. Some of the Ca2+ channel blockers have been also identified as autophagy activators in a high-throughput image-based screen, including niguldipine and pimozide [92]. These compounds do not inhibit mTORC1, and thus act as mTOR-independent autophagy modulators.

Increases in cytosolic Ca2+ activate calpains, which are Ca2+-dependent cysteine proteases, such as calpain 1 and calpain 2 [155]. Pharmacological inhibition of calpain with calpastatin and calpeptin or its genetic knockdown increases autophagic flux without perturbing mTORC1. In contrast, activation of calpain by Ca2+ channel openers or overexpression of constitutively active calpain 2 inhibits autophagosome synthesis [93]. Modulation of autophagy by L-type Ca2+ channel antagonists or agonists are abrogated by the activation or inhibition of calpain activity respectively, suggesting that calpain is a downstream mediator of the autophagy-regulatory effects of free cytosolic Ca2+ [93]. Although activation of calpains decreases autophagosome synthesis, some of the effects of raised cytosolic Ca2+ due to Ca2+ ionophores may be mediated by ER-related effects, resulting in impaired autophagsome–lysosome fusion (Figure 3). However, thapsigargin-mediated block in autophagy may not be absolute since calpastatin can partially alleviate this defect [93].

The autophagy-inhibitory effects of calpain are mediated by cleavage-dependent activation of the α-subunit of heterotrimeric G-proteins (G) [93], which increases the activity of adenylate cyclase, generating cAMP [156]. Inhibition of G activity, either chemically with NF449 or by genetic knockdown, increases autophagic flux, which rescues the impairment in autophagy arising due to calpain activation. Conversely, activation of G with its natural ligand, PACAP (pituitary adenylate cyclase-activating polypeptide), inhibits autophagy; an effect that is abolished by the adenylate cyclase inhibitor 2′5′ddA (2′,5′-dideoxyadenosine) that reduces cAMP levels, but not by the calpain inhibitor calpastatin [93]. These data designate G to be acting downstream of calpain and upstream of cAMP in regulating autophagy (Figure 3). In addition, the autophagy-inhibitory effects of activated calpain are rescued by inhibiting adenylate cyclase activity, suggesting that cAMP is a downstream target of calpain and G [93]. This creates a mechanistic link between the Ca2+/calpain and the cAMP pathways modulating autophagy.

Control of autophagy by cAMP/Epac/Ins(1,4,5)P3 pathway

Autophagy is regulated by the second messenger, cAMP. Elevation of intracellular cAMP levels inhibits autophagy [84] (Figure 3). Pharmacological inhibition of adenylate cyclase activity with 2′5′ddA induces autophagy and enhances the clearance of autophagic cargo independently of mTORC1, whereas agents increasing cAMP levels, such as forskolin or the cAMP analogue dibutyryl-cAMP, inhibit autophagy [93]. A chemical screen with FDA-approved drugs has identified the I1R (imidazoline-1 receptor) agonists clonidine and rilmenidine, which also induce mTOR-independent autophagy by reducing cAMP levels [93].

The two major cellular targets of cAMP are Epac (exchange protein directly activated by cAMP), a guanine-nucleotide-exchange factor for the small G-protein Rap, and PKA (protein kinase A) [157]. The regulation of autophagy by cAMP is mediated by Epac independently of PKA, since an Epac-specific cAMP analogue 8-CPT-2-Me-cAMP [8-(4-chlorophenylthio)-2′-O-methyladenosine 3′,5′-cyclic monophosphate], but not one that is PKA-specific, inhibits autophagy [93]. Activation of Epac by elevated cAMP levels activates Rap2B, a small G-protein of the Ras family [158]. Dominant-negative Rap2B induces autophagy and abrogates the inhibitory effects of the cAMP/Epac signal cascade, suggesting that Rap2B is acting downstream of this pathway. Subsequently, Rap2B activates PLCϵ, which inhibits autophagy [93]. Activation of PLCϵ hydrolyses PtdIns(4,5)P2 to generate Ins(1,4,5)P3, which inhibits autophagy as described previously [144]. An increase in intracellular Ins(1,4,5)P3 binds to its ER-resident IP3R to deplete the ER Ca2+ store, thereby increasing cytosolic Ca2+ that activates calpain [145,159]. The autophagy-inhibitory effects of activated PLCϵ, as well as that of raised cytosolic Ca2+, are rescued by calpain inhibition. Likewise, activation of calpain abrogates the autophagy-inducing effects of Ca2+ channel blockers and cAMP-reducing agents [93]. This evidence implies that the cAMP/Epac pathway converges into the inositol signalling and Ca2+/calpain pathways, thus creating an mTOR-independent cyclic link that negatively regulates autophagy.

Control of autophagy by the JNK1/Beclin-1/PI3KC3 pathway

The lipid kinase hVps34 is part of the autophagy-initiating PI3KC3 complex consisting of Beclin-1, along with other components described above [160] (Figure 1). Autophagosome synthesis requires PI3KC3 activity, which is enhanced by the interaction of hVps34 with Beclin-1 that has several binding partners modulating autophagy [37,44,46]. Autophagy is inhibited when Bcl-2 and Bcl-XL bind to Beclin-1 through a BH3 (Bcl-2 homology 3) domain, which mediates the docking of Beclin-1 to the BH3-binding groove of these anti-apoptotic proteins, thereby dissociating the Beclin-1–hVps34 pro-autophagy complex [46,51] (Figure 3). The BH3 mimetic ABT737 competitively disrupts the inhibitory interaction between the BH3 domain of Beclin-1 and Bcl-2, thus stimulating the Beclin-1-dependent allosteric activation of hVps34 to induce autophagy [51,161]. However, ABT737 has pleiotropic pro-autophagy effects by influencing multiple autophagy-regulatory components, including the inhibition of the mTORC1 pathway, possibly through off-target effects [161]. Interestingly, the autophagy-modulating strategy of microbial virulence factors through their interaction with Beclin-1 has been exploited to design an autophagy-inducing peptide, Tat-Beclin-1, consisting of the HIV-1 Tat (transactivator of transcription) protein transduction domain linked to a region of Beclin-1 that binds to the HIV-1 virulence factor Nef [162].

Starvation also regulates the JNK1/Beclin-1/PI3KC3 pathway. Starvation-induced activation of JNK1 phosphorylates Bcl-2, which dissociates from the autophagy-inhibitory Beclin-1–Bcl-2 complex, thus promoting the formation of the autophagy-stimulatory Beclin-1–hVps34 complex [46,163] (Figure 3). Although starvation also inhibits mTORC1, expression of constitutively active JNK1, however, does not perturb mTORC1 activity. Moreover, rapamycin has no effect on JNK1 and Bcl-2 phosphorylation, an effect consistent with no alterations of these phospho-proteins in TSC2-deficient cells where mTORC1 is active [139]. This evidence suggests that autophagy regulated by the JNK1/Beclin-1/PI3KC3 and mTORC1 pathways are possibly independent of each other, and any cross-talk requires further investigation. Generation of NO by various NOS isoforms inhibits autophagosome formation by S-nitrosylating and inactivating JNK1, thereby reducing Bcl-2 phosphorylation and disrupting the Beclin-1–hVps34 complex by allowing Bcl-2 to bind to Beclin-1. Inhibition of NOS by L-NAME (NG-nitro-L-arginine methyl ester) stimulates autophagy, but does not affect mTORC1 activity [139] (Figure 3). Nonetheless, amino acids have been suggested to stimulate mTORC1 by activating hVps34 by an unknown mechanism [164,165]. However, this reflects an intriguing paradigm that is difficult to reconcile since activation of hVps34 occurs during starvation-induced autophagy.

A genome-wide screen has identified various mTOR-independent signalling components under basal autophagy, such as calpain 1 and the MAPK (mitogen-activated protein kinase)/ERK1/2 pathway that impinge upon PI3KC3 to regulate autophagy [166]. It is possible that PI3KC3 also senses signals from the mTOR-independent pathways regulating autophagy described above, although a mechanistic link remains to be elucidated. It is plausible to speculate that the autophagy-initiating Beclin-1–hVps34 complex and the autophagy-inhibitory mTORC1 may be distinct and reflect different entry points that possibly act as a ‘speed and brake’ mechanism to steer this process.

Control of autophagy by other mTORindependent small molecules

Additional mTOR-independent autophagy enhancers have been reported, although their mechanism of action is not known (Figure 3). A potent autophagy activator is trehalose [167], a disaccharide found in various non-mammalian species where it acts as a chemical chaperone by preventing denaturation and aggregation of proteins through direct protein–trehalose interactions [168]. Although trehalose efficiently clears autophagic cargo, its ability to reduce neurodegeneration-associated protein aggregates was not observed in autophagy-deficient cells [167], suggesting that constitutive autophagy is necessary for the clearance of mutant proteins to decrease their aggregate load. Being a non-reducing stable sugar, its functions as an autophagy inducer and a chemical chaperone are of relevance in the context of protein conformational disorders where the mutant aggregation-prone proteins are autophagy substrates.

A screen with 50000 compounds for discovery of novel autophagy modulators has revealed various SMERs (small-molecule enhancers) and SMIRs (small-molecule inhibitors) of the cytostatic effects of rapamycin in yeast, which have been further screened in mammalian cells where several autophagy-inducing SMERs (SMER10, SMER18 and SMER28) and autophagy-inhibitory SMIRs were identified. Many structural analogues of these SMERs induce mTOR-independent autophagy [169]. Another image-based screen that identified Ca2+ channel blockers (described above) has found additional mTOR-independent autophagy inducers, such as fluspirilene, trifluoperazine (dopamine antagonist) and penitrem A (inhibitor of high conductance Ca2+-activated K+ channels) [92]. Many of the autophagy enhancers described above have potential therapeutic application in the neurodegenerative diseases described below.

Autophagy degrades neurodegenerationassociated aggregation-prone proteins

Autophagy is implicated in diverse human diseases, including neurodegeneration [1,8,170]. Neurodegenerative disorders, such as AD (Alzheimer's disease), PD (Parkinson's disease), ALS (amyotrophic lateral sclerosis), prion diseases and polyQ (polyglutamine) expansion disorders, including HD (Huntington's disease), SBMA (spinobulbar muscular atrophy), DRPLA (dentatorubral pallidoluysian atrophy) and a number of SCAs (spinocerebellar ataxias) (SCA1, SCA2, SCA3, SCA6, SCA7 and SCA17) belong to the class of protein conformational disorders associated with progressive neurodegeneration and cognitive decline [171,172]. The pathological hallmark includes the presence of intraneuronal ubiquitylated mutant protein aggregates (inclusions), although extracellular amyloid plaques occur in AD. The conformational change attributed to the mutant proteins is generally believed to promote the disease by a toxic gain-of-function. However, the nature of the toxic species (soluble, oligomeric or aggregated form) is not clear [171176].

The polyubiquitin-binding protein p62 is commonly found in inclusion bodies, such as Lewy bodies in PD, neurofibrillary tangles in AD and huntingtin aggregates in HD [18,177179]. It acts as a scaffold to facilitate aggregation of ubiquitylated mutant proteins since loss of p62 suppresses inclusion body formation in neurons [3]. Additionally, p62 is a specific autophagy substrate that targets the mutant proteins for autophagic degradation [3,21]. Several neurodegeneration-associated aggregation-prone proteins, such as A53T and A30P mutants of α-synuclein (familial PD), various polyQ-expanded proteins such as mutant huntingtin (HD), ataxin-1 (SCA1) and ataxin-3 (SCA3), mutant SOD1 (copper/zinc superoxide dismutase 1; familial ALS) and tau (AD) are predominantly degraded by autophagy, and are thus considered autophagy substrates [18,82,83,180184] (Figure 4). Sequestration of ubiquitylated mutant proteins into autophagosomes is mediated by p62 [3,21]. Phosphorylation of p62 in its UBA (ubiquitin-associated) domain by protein kinase CK2 increases the affinity for polyubiquitin chains, resulting in efficient targeting of the polyubiquitylated mutant proteins to the autophagosomes [185]. This adaptor protein also has an LC3-binding motif, thus enabling specific cargo recognition and degradation by autophagy [3,186]. Another autophagy adaptor, NBR1, also an autophagy substrate, contains both ubiquitin and LC3-binding domains that functions independently of p62 in selective autophagy-mediated clearance of ubiquitylated proteins [20].

Up-regulation of autophagy as a therapeutic strategy for neurodegenerative disease

Figure 4
Up-regulation of autophagy as a therapeutic strategy for neurodegenerative disease

Various cytosolic aggregation-prone proteins associated with neurodegenerative diseases are predominantly degraded by autophagy. Pharmacological induction of autophagy through mTOR-dependent or mTOR-independent pathways enhances the clearance of mutant proteins, leading to cytoprotection and rescue against neurodegeneration. Failure to degrade the mutant proteins leads to cytotoxicity and neurodegeneration.

Figure 4
Up-regulation of autophagy as a therapeutic strategy for neurodegenerative disease

Various cytosolic aggregation-prone proteins associated with neurodegenerative diseases are predominantly degraded by autophagy. Pharmacological induction of autophagy through mTOR-dependent or mTOR-independent pathways enhances the clearance of mutant proteins, leading to cytoprotection and rescue against neurodegeneration. Failure to degrade the mutant proteins leads to cytotoxicity and neurodegeneration.

Activation of autophagy as a therapeutic strategy in neurodegenerative diseases

Up-regulation of autophagy is considered a therapeutic strategy for several neurodegenerative diseases [187189]. Cytoplasmic aggregation-prone proteins causing neurodegeneration are good autophagy substrates in general, and therefore enhancing autophagy facilitates their clearance, whereas inhibiting this process leads to accumulation of these toxic species (Figure 4). Although stimulation of autophagy reduces both the soluble and aggregated forms, the oligomeric species are likely to be sequestered in the autophagosomes [170]. This preferential proteolytic route over the ubiquitin–proteasome pathway is possibly due to the inability of oligomeric and aggregated species to be unfolded for entering the narrow proteasome barrel [190192]. Although activation of autophagy decreases mutant protein aggregates, it is unclear whether large aggregates are removed directly by autophagy or as a consequence of degrading the soluble oligomeric species. Notably, some of their wild-type counterparts such as huntingtin and α-synuclein that are not aggregation-prone show a substantially lower dependency on autophagy-mediated clearance [83,167,180,193,194], although autophagy degrades both wild-type and mutant tau [82]. Since the physiological functions of these wild-type proteins are not completely understood, preferential clearance of the mutant proteins may provide a more rational therapeutic approach.

The beneficial effects of autophagy up-regulation in neurodegenerative diseases first came with the mTORC1 inhibitor rapamycin/CCI-779, which ameliorates the disease phenotype in transgenic HD mice [18]. Subsequently, rapamycin has been shown to be protective in various cell and animal models of neurodegenerative diseases. The protective effects of rapamycin appear to be autophagy-dependent in vivo, since its ability to rescue against toxicity is abrogated in transgenic Drosophila heterozygous for an essential autophagy gene, Atg1 [82]. These findings open a promising new therapeutic intervention. However, the use of rapamycin has been impeded by the fact that rapamycin not only exhibits immune-suppressive effects, but also may have undesirable side effects pertaining to mTOR inhibition that governs critical cellular processes, such as translation and cell growth, and whole body metabolism [78,195]. Therefore such treatment may not be ideal for patients with neurodegenerative diseases requiring long-term drug administration, possibly for years. The discovery of mTOR-independent autophagy enhancers may thus provide an alternative to stimulating this process. A number of studies and chemical screens have identified small molecules regulating mTOR-independent autophagy, including some that are FDA-approved drugs [187189,196198] (Table 1). Nonetheless, it is critical to characterize any underlying autophagy defects in disease-specific contexts to determine treatment strategy.

Table 1
List of selected small-molecule autophagy enhancers and their mechanism of autophagy activation

GAPR-1, Golgi-associated plant pathogenesis-related protein 1.

Chemical inducers of autophagy Mode of drug action Mechanism of autophagy activation Reference(s) 
mTOR-dependent autophagy inducers    
 Rapamycin, CCI-779, RAD001 mTORC1 inhibitor Inhibition of mTORC1 activity [18,81,83,84
 Torin1, PP242 ATP-competitive mTOR (mTORC1 and mTORC2) inhibitor Direct inhibition of mTORC1 [87,88
 Torin2 Dual ATP-competitive mTOR (mTORC1 and mTORC2) and multiple PI3KK inhibitor Direct inhibition of mTORC1 [90
 PI103 Dual ATP-competitive mTOR (mTORC1 and mTORC2) and selective PI3KC1a inhibitor Direct inhibition of mTORC1 and selective inhibition of class 1a PI3K [117
 Rottlerin Inhibitor of various protein kinases Inhibition of mTORC1 pathway [91
 Niclosamide Anti-parasitic agent Inhibition of mTORC1 pathway [91
 Latrepirdine Neuroactive agent Inhibition of mTORC1 pathway [94
 ABT737 BH3 mimetic Inhibition of Beclin-1–Bcl-2 interaction [161
 Pifithrin-α p53 inhibitor Unknown; possibly by activation of AMPK and inhibition of mTORC1 pathway [141
mTOR-independent autophagy inducers    
 Lithium, L-690,330 IMPase inhibitor Reduction in inositol and Ins(1,4,5)P3 levels [144,275
 Valproic acid MIPS inhibitor Reduction in inositol and Ins(1,4,5)P3 levels [144
 Carbamazepine Anti-epileptic agent Reduction in inositol and Ins(1,4,5)P3 levels [144
 Verapamil, loperamide, nitrendipine, niguldipine, nimodipine, nicardipine, pimozide, amiodarone Ca2+ channel antagonist Reduction in cytosolic Ca2+ levels [92,93
 Minoxidil KATP channel agonist Unknown [93
 Penitrem A High conductance Ca2+-activated K+ channel inhibitor Unknown [92
 Fluspirilene, trifluoperazine Dopamine antagonist Unknown [92
 Trehalose Chemical chaperone Unknown [167
 SMER10, SMER18, SMER28 andanalogues Unknown Unknown [169
 Calpastatin, calpeptin Calpain inhibitor Inhibition of calpain [93
 Rilmenidine, clonidine I1R agonist Reduction in cAMP levels [93
 2′5′ddA Adenylate cyclase inhibitor Reduction in cAMP levels [93
 NF449 G inhibitor Reduction in cAMP levels [93
 Xestospongin B IP3R antagonist Inhibition of Beclin-1 interaction with IP3R–Bcl-2 complex [150
 l-NAME NOS inhibitor Inhibition of NOS activity [139
 Resveratrol Sirtuin-1 activator Unknown; suggested to be by deacetylation of essential Atg proteins [323
 Spermidine Histone acetyltransferase inhibitor Unknown; suggested to be by Atg gene expression [322
 Tat-Beclin-1 Autophagy-inducing peptide Unknown; suggested to be by interaction with the negative autophagy regulator GAPR-1 [162
Chemical inducers of autophagy Mode of drug action Mechanism of autophagy activation Reference(s) 
mTOR-dependent autophagy inducers    
 Rapamycin, CCI-779, RAD001 mTORC1 inhibitor Inhibition of mTORC1 activity [18,81,83,84
 Torin1, PP242 ATP-competitive mTOR (mTORC1 and mTORC2) inhibitor Direct inhibition of mTORC1 [87,88
 Torin2 Dual ATP-competitive mTOR (mTORC1 and mTORC2) and multiple PI3KK inhibitor Direct inhibition of mTORC1 [90
 PI103 Dual ATP-competitive mTOR (mTORC1 and mTORC2) and selective PI3KC1a inhibitor Direct inhibition of mTORC1 and selective inhibition of class 1a PI3K [117
 Rottlerin Inhibitor of various protein kinases Inhibition of mTORC1 pathway [91
 Niclosamide Anti-parasitic agent Inhibition of mTORC1 pathway [91
 Latrepirdine Neuroactive agent Inhibition of mTORC1 pathway [94
 ABT737 BH3 mimetic Inhibition of Beclin-1–Bcl-2 interaction [161
 Pifithrin-α p53 inhibitor Unknown; possibly by activation of AMPK and inhibition of mTORC1 pathway [141
mTOR-independent autophagy inducers    
 Lithium, L-690,330 IMPase inhibitor Reduction in inositol and Ins(1,4,5)P3 levels [144,275
 Valproic acid MIPS inhibitor Reduction in inositol and Ins(1,4,5)P3 levels [144
 Carbamazepine Anti-epileptic agent Reduction in inositol and Ins(1,4,5)P3 levels [144
 Verapamil, loperamide, nitrendipine, niguldipine, nimodipine, nicardipine, pimozide, amiodarone Ca2+ channel antagonist Reduction in cytosolic Ca2+ levels [92,93
 Minoxidil KATP channel agonist Unknown [93
 Penitrem A High conductance Ca2+-activated K+ channel inhibitor Unknown [92
 Fluspirilene, trifluoperazine Dopamine antagonist Unknown [92
 Trehalose Chemical chaperone Unknown [167
 SMER10, SMER18, SMER28 andanalogues Unknown Unknown [169
 Calpastatin, calpeptin Calpain inhibitor Inhibition of calpain [93
 Rilmenidine, clonidine I1R agonist Reduction in cAMP levels [93
 2′5′ddA Adenylate cyclase inhibitor Reduction in cAMP levels [93
 NF449 G inhibitor Reduction in cAMP levels [93
 Xestospongin B IP3R antagonist Inhibition of Beclin-1 interaction with IP3R–Bcl-2 complex [150
 l-NAME NOS inhibitor Inhibition of NOS activity [139
 Resveratrol Sirtuin-1 activator Unknown; suggested to be by deacetylation of essential Atg proteins [323
 Spermidine Histone acetyltransferase inhibitor Unknown; suggested to be by Atg gene expression [322
 Tat-Beclin-1 Autophagy-inducing peptide Unknown; suggested to be by interaction with the negative autophagy regulator GAPR-1 [162

Impairment of autophagy in neurodegenerative diseases and therapeutic application of small-molecule autophagy enhancers

Emerging evidence suggests that certain neurodegeneration-associated mutant proteins can cause perturbations in the autophagy pathway (Table 2). Autophagy dysfunction has been implicated in several neurodegenerative diseases in the accumulation of misfolded protein and in cellular toxicity [199,200]. In addition, these diseases are commonly associated with nitrosative stress and Ca2+ excititoxicity [201,202], which can inhibit autophagic flux [93,139]. Brain-specific abrogation of basal autophagy in normal mice results in neurodegeneration in the absence of any disease-causing mutations, suggesting that impaired autophagy contributes to the aetiology of neurodegenerative diseases. Deletion of essential autophagy genes, such as Atg5 or Atg7, yielded a neurodegenerative phenotype in mouse brain characterized by accumulation of intraneuronal, ubiquitylated protein aggregates [2,4]. These genetic data suggest that autophagy plays a crucial role in neuronal homoeostasis by removing naturally occurring misfolded proteins. Compromised autophagy contributes to the accumulation of aggregation-prone proteins and other toxic substrates, including damaged mitochondria by impeding mitophagy [24,199,203]. Inappropriate accumulation of autophagy substrates may contribute to cellular toxicity during autophagy dysfunction by increasing the susceptibility to pro-apoptotic insults [203205] (Figure 4). Defective autophagy in neurodegenerative diseases (Table 2) and the beneficial effects of autophagy enhancers (Table 3) are described in disease-specific contexts.

Table 2
Perturbations in autophagy pathways caused by mutant proteins associated with neurodegenerative diseases
Disease Mutant protein/species Perturbations in autophagy Mechanism of autophagy modulation Reference(s) 
AD PS1 Inhibition of autophagosome maturation Impairment in lysosomal acidification due to defective v-ATPase targeting [214
AD Aβ Induction of autophagosome synthesis Generation of ROS (reactive oxygen species) acting through the PI3KC3 pathway [209
AD APP Inhibition of autophagosome synthesis Reduction in Beclin-1 [210
AD Tau Unknown Unknown  
PD α-Synuclein Inhibition of autophagosome synthesis Inhibition of Rab1a and mislocalization of Atg9; sequestration of TFEB [237,240
  (Inhibition of CMA) (Inhibition of uptake by CMA) ([235]) 
PD Parkin Inhibition of mitophagy Targeting depolarized mitochondria to autophagosomes [249251
PD PINK1 Inhibition of mitophagy Recruitment of parkin to depolarized mitochondria [249,252
PD DJ-1 Not clear; elevation in autophagosomes Unknown [248
PD LRRK2 Not clear; elevation in autophagosomes Activation of CaMKKβ/AMPK pathway [260
  (Inhibition of CMA) (Inhibition of CMA translocation complex) ([262]) 
HD Huntingtin Induction of autophagosome synthesis; inhibition of cargo clearance Sequestration of mTOR into aggregates and inactivation of mTORC1 activity; failure in autophagic cargo recognition [18,270
SCA1 Ataxin-1 Unknown Unknown  
SCA3 Ataxin-3 Not clear; elevation in autophagosomes Unknown [277
SCA7 Ataxin-7 Inhibition of autophagosome synthesis Disruption of ULK1–FIP200–Atg13 complex [283
SBMA AR Unknown Unknown  
DRPLA Atrophin-1 Inhibition of cargo clearance Inhibition of lysosomal proteolysis [290
ALS Dynein Inhibition of autophagosome maturation Inhibition of microtubule-based autophagosome trafficking and autophagosome–lysosome fusion [61,295
ALS Dynactin Unknown Unknown  
ALS ESCRT-III Inhibition of autophagosome maturation Inhibition of autophagosome fusion with late endosomes and lysosomes [69,300
ALS SOD1 Not clear; elevation in autophagosomes Inhibition of mTORC1 activity [302
LD Laforin Inhibition of autophagosome synthesis Inhibition of mTORC1 activity [314
LD Malin Inhibition of autophagosome synthesis Unknown [315
Disease Mutant protein/species Perturbations in autophagy Mechanism of autophagy modulation Reference(s) 
AD PS1 Inhibition of autophagosome maturation Impairment in lysosomal acidification due to defective v-ATPase targeting [214
AD Aβ Induction of autophagosome synthesis Generation of ROS (reactive oxygen species) acting through the PI3KC3 pathway [209
AD APP Inhibition of autophagosome synthesis Reduction in Beclin-1 [210
AD Tau Unknown Unknown  
PD α-Synuclein Inhibition of autophagosome synthesis Inhibition of Rab1a and mislocalization of Atg9; sequestration of TFEB [237,240
  (Inhibition of CMA) (Inhibition of uptake by CMA) ([235]) 
PD Parkin Inhibition of mitophagy Targeting depolarized mitochondria to autophagosomes [249251
PD PINK1 Inhibition of mitophagy Recruitment of parkin to depolarized mitochondria [249,252
PD DJ-1 Not clear; elevation in autophagosomes Unknown [248
PD LRRK2 Not clear; elevation in autophagosomes Activation of CaMKKβ/AMPK pathway [260
  (Inhibition of CMA) (Inhibition of CMA translocation complex) ([262]) 
HD Huntingtin Induction of autophagosome synthesis; inhibition of cargo clearance Sequestration of mTOR into aggregates and inactivation of mTORC1 activity; failure in autophagic cargo recognition [18,270
SCA1 Ataxin-1 Unknown Unknown  
SCA3 Ataxin-3 Not clear; elevation in autophagosomes Unknown [277
SCA7 Ataxin-7 Inhibition of autophagosome synthesis Disruption of ULK1–FIP200–Atg13 complex [283
SBMA AR Unknown Unknown  
DRPLA Atrophin-1 Inhibition of cargo clearance Inhibition of lysosomal proteolysis [290
ALS Dynein Inhibition of autophagosome maturation Inhibition of microtubule-based autophagosome trafficking and autophagosome–lysosome fusion [61,295
ALS Dynactin Unknown Unknown  
ALS ESCRT-III Inhibition of autophagosome maturation Inhibition of autophagosome fusion with late endosomes and lysosomes [69,300
ALS SOD1 Not clear; elevation in autophagosomes Inhibition of mTORC1 activity [302
LD Laforin Inhibition of autophagosome synthesis Inhibition of mTORC1 activity [314
LD Malin Inhibition of autophagosome synthesis Unknown [315
Table 3
List of selected autophagy enhancers and their beneficial effects in various models of neurodegenerative diseases

GAPR-1, Golgi-associated plant pathogenesis-related protein 1; Htt, Huntingtin; α-Syn, α-synuclein.

  Models of neurodegenerative diseases expressing mutant proteins as indicated, where protective effects have been reported with up-regulation of autophagy (with references) 
Chemical inducers of autophagy Mechanism of autophagy activation Cell Drosophila Zebrafish Mouse 
mTOR-dependent autophagy inducers      
 Rapamycin, CCI-779 Inhibition of mTORC1 activity Htt [83], α-Syn [180], tau [82], ataxin-1 [82], ataxin-3 [82], ataxin-7 [282], polyQ [82Htt [18,275], tau [82], polyQ [82Htt [93,139Htt [18], tau [228], ataxin-3 [280], APP [222], PS1/APP/tau [223
 Latrepirdine Inhibition of mTORC1 pathway α-Syn [94Not tested Not tested APP [225
mTOR-independent autophagy inducers      
 Lithium Inhibition of IMPase and reduction in inositol and Ins(1,4,5)P3 levels Htt [144], α-Syn [144Htt [275Not tested Tau [230], APP/PS1 [224
 Valproic acid Inhibition of MIPS and reduction in inositol and Ins(1,4,5)P3 levels Htt [144], α-Syn [144Htt [93Not tested Not tested 
 Carbamazepine Reduction in inositol and Ins(1,4,5)P3 levels Htt [144], α-Syn [144Not tested Not tested APP/PS1 [220
 Verapamil Inhibition of L-type Ca2+ channels and reduction in cytosolic Ca2+ levels Htt [93], α-Syn [93Htt [93Htt [93Not tested 
 Loperamide, nitrendipine, nimodipine, amiodarone Inhibition of L-type Ca2+ channels and reduction in cytosolic Ca2+ levels Htt [93], α-Syn [93Not tested Not tested Not tested 
 Niguldipine, nicardipine, pimozide Inhibition of Ca2+ channels and reduction in cytosolic Ca2+ levels PolyQ [92Not tested Not tested Not tested 
 Minoxidil Unknown Htt [93], α-Syn [93Not tested Not tested Not tested 
 Penitrem A, fluspirilene, trifluoperazine Unknown PolyQ [92Not tested Not tested Not tested 
 Trehalose Unknown Htt [167], α-Syn [167], tau [226], SOD1 [311Not tested Not tested APP [221], tau [227], tau/parkin [229], SOD1 [311
 SMER28 Unknown Htt [169], α-Syn [169], APP [219Htt [169Not tested Not tested 
 Calpastatin Inhibition of calpain Htt [93], α-Syn [93Not tested Htt [93Not tested 
 Rilmenidine I1R agonist and reduction in cAMP levels Htt [93], α-Syn [93Not tested Not tested Htt [274
 Clonidine I1R agonist and reduction in cAMP levels Htt [93], α-Syn [93Htt [93Htt [93Not tested 
 2′5′ddA Inhibition of adenylate cyclase and reduction in cAMP levels Htt [93], α-Syn [93Not tested Htt [93Not tested 
 l-NAME Inhibition of NOS activity Htt [139], α-Syn [139Htt [139Htt [139Not tested 
 Tat-Beclin-1 Unknown; suggested to be by interaction with GAPR-1 Htt [162Not tested Not tested Not tested 
  Models of neurodegenerative diseases expressing mutant proteins as indicated, where protective effects have been reported with up-regulation of autophagy (with references) 
Chemical inducers of autophagy Mechanism of autophagy activation Cell Drosophila Zebrafish Mouse 
mTOR-dependent autophagy inducers      
 Rapamycin, CCI-779 Inhibition of mTORC1 activity Htt [83], α-Syn [180], tau [82], ataxin-1 [82], ataxin-3 [82], ataxin-7 [282], polyQ [82Htt [18,275], tau [82], polyQ [82Htt [93,139Htt [18], tau [228], ataxin-3 [280], APP [222], PS1/APP/tau [223
 Latrepirdine Inhibition of mTORC1 pathway α-Syn [94Not tested Not tested APP [225
mTOR-independent autophagy inducers      
 Lithium Inhibition of IMPase and reduction in inositol and Ins(1,4,5)P3 levels Htt [144], α-Syn [144Htt [275Not tested Tau [230], APP/PS1 [224
 Valproic acid Inhibition of MIPS and reduction in inositol and Ins(1,4,5)P3 levels Htt [144], α-Syn [144Htt [93Not tested Not tested 
 Carbamazepine Reduction in inositol and Ins(1,4,5)P3 levels Htt [144], α-Syn [144Not tested Not tested APP/PS1 [220
 Verapamil Inhibition of L-type Ca2+ channels and reduction in cytosolic Ca2+ levels Htt [93], α-Syn [93Htt [93Htt [93Not tested 
 Loperamide, nitrendipine, nimodipine, amiodarone Inhibition of L-type Ca2+ channels and reduction in cytosolic Ca2+ levels Htt [93], α-Syn [93Not tested Not tested Not tested 
 Niguldipine, nicardipine, pimozide Inhibition of Ca2+ channels and reduction in cytosolic Ca2+ levels PolyQ [92Not tested Not tested Not tested 
 Minoxidil Unknown Htt [93], α-Syn [93Not tested Not tested Not tested 
 Penitrem A, fluspirilene, trifluoperazine Unknown PolyQ [92Not tested Not tested Not tested 
 Trehalose Unknown Htt [167], α-Syn [167], tau [226], SOD1 [311Not tested Not tested APP [221], tau [227], tau/parkin [229], SOD1 [311
 SMER28 Unknown Htt [169], α-Syn [169], APP [219Htt [169Not tested Not tested 
 Calpastatin Inhibition of calpain Htt [93], α-Syn [93Not tested Htt [93Not tested 
 Rilmenidine I1R agonist and reduction in cAMP levels Htt [93], α-Syn [93Not tested Not tested Htt [274
 Clonidine I1R agonist and reduction in cAMP levels Htt [93], α-Syn [93Htt [93Htt [93Not tested 
 2′5′ddA Inhibition of adenylate cyclase and reduction in cAMP levels Htt [93], α-Syn [93Not tested Htt [93Not tested 
 l-NAME Inhibition of NOS activity Htt [139], α-Syn [139Htt [139Htt [139Not tested 
 Tat-Beclin-1 Unknown; suggested to be by interaction with GAPR-1 Htt [162Not tested Not tested Not tested 

Regulation of autophagy in Alzheimer's disease

AD is a late-onset dementia with degeneration of neurons in the basal forebrain and hippocampus. Both intracellular (neurofibrillary tangles) and extracellular (neuritic plaques) protein aggregates occur in AD. The neurofibrillary tangles arise due to mutations in the microtubule-associated protein tau, associated with tauopathies such as FTDs (frontotemporal dementias) with parkinsonism [206]. The neuritic plaques consist primarily of self-aggregating Aβ (amyloid β-peptide) (Aβ42), derived by cleavage of the APP (amyloid precursor protein) either due to mutations in APP itself or in PS (presenilin) 1 or 2 that are linked to autosomal dominant forms of FAD (familial AD) [207,208]. Multiple studies reporting autophagy alterations in AD have described contrasting effects. Stimulation of autophagy is supported by Aβ-induced generation of ROS (reactive oxygen species) increasing autophagosome synthesis through the PI3KC3 pathway [209]. In contrast, inhibition of autophagosome biogenesis is suggested due to decrease in Beclin-1 in AD patient brain [210]. Furthermore, heterozygous deletion of Beclin-1 in mice decreases neuronal autophagy, resulting in neurodegeneration. Similarly, reducing Beclin-1 in APP(K670M/N671L/V717I) mutant mice increases Aβ deposition associated with neuronal loss, whereas overexpression of Beclin-1 induces autophagy and suppresses amyloid pathology in these mice [210].

Accumulation of autophagosomes and late AVs (autophagic vacuoles), however, is observed in the dystrophic dendrites in AD patients and transgenic mouse brain. This is suggested to be caused by their defective clearance, although neuronal autophagy is induced early before extracellular Aβ deposition in APP(K670M/N671L)/PS1(M146V) mutant mice [211213]. AVs purified from AD mouse brain contain APP and its β-cleaved forms, along with PS1. In addition, modulation of autophagy in neuronal cells affects Aβ production, suggesting that autophagy influences Aβ levels and designating AVs as Aβ-generating compartments [213]. Subsequently, PS1 is shown to be essential for targeting v-ATPase to the lysosomes that is required for lysosomal acidification and proteolysis during autophagy [214,215]. Depletion of PS1 in mice impairs autophagic degradation arising from insufficient autolysosome acidification and cathepsin activation, suggesting that PS1 mutations causing FAD may block autophagic flux [214]. However, a direct conflict with the role of presenilins in lysosomal proteolysis has been reported, where no alterations in lysosomal acidification and its proteolytic function have been found in mice lacking PS1 and PS2 [216]. Nonetheless, a recent study addresses this issue using a multitude of lysosomal probes to measure lysosomal pH. Defective lysosomal acidification is found in PS1/PS2-deficient cells and patient fibroblasts, including neurons from APP(K670M/N671L)/PS1(M146V) mutant mice [217]. Accordingly, enhancing lysosomal cathepsin activity by deletion of cystatin B, an endogenous inhibitor of lysosomal cysteine proteases, rescues this pathology and reduces the accumulation of Aβ42 peptide and other autophagic substrates in APP(K670M/N671L/V717F) mutant mice [218].

Activating autophagy by starvation or mTOR-independently with SMER28 facilitates the clearance of Aβ peptide and APP-derived C-terminal fragment in cell lines and primary neurons in an Atg5-dependent manner [219]. Treatment with autophagy enhancers in various AD mouse models have yielded beneficial effects associated with a reduction in Aβ42 levels. Stimulation of autophagy in APP(K670M/N671L)/PS1(ΔE9) double-transgenic mice with the mTOR-independent autophagy inducer carbamazepine alleviates memory deficits by increasing autophagic flux and reducing amyloid plaques and Aβ42 levels [220]. Likewise, trehalose reduces anaesthesia-induced amyloid pathology and tau plaques in APP(K670M/N671L) transgenic mice [221], whereas rapamycin is shown to lower Aβ42 levels and extended lifespan in APP(K670M/N671L/V717F) mice [222]. Notably, inducing autophagy with rapamycin before, but not after, the formation of amyloid plaques decreases plaques and tangles and ameliorates cognitive defects in triple-transgenic AD [PS1(M146V)/APP(K670M/N671L)/Tau(P130L)] mice [223]. Treatment with lithium also suppresses the disease pathology in aged APP(K670M/N671L)/PS1(A246E) mutant mice, although this effect may be due to GSK3β inhibition affecting tau phosphorylation [224]. Latrepirdine (Dimebon), a neuroactive compound shown recently to induce autophagy and decrease Aβ42 levels by inhibiting the mTORC1 pathway, also improves cognition in APP(K670M/N671L/V717F) mutant mice and thus is of clinical interest [225]. This evidence suggests that stimulating autophagy is protective in AD mouse models possibly by enhancing Aβ42 clearance, despite the hypothesis of abnormality in lysosomal proteolytic function.

Induction of autophagy is also beneficial in models of tauopathies. Rapamycin enhances the clearance of both wild-type and insoluble mutant tau in cell models and alleviates toxicity in Drosophila expressing mutant tau(R406W) [82]. Similarly, trehalose reduces tau aggregates and suppresses cytotoxicity in a neuronal model of tauopathy [226]. Up-regulation of autophagy with rapamycin or trehalose in mutant tau(P301S) mice attenuates neurodegeneration [227,228]. Trehalose reduces insoluble mutant tau and its aggregates, and increases neuronal survival in the cerebral cortex and brainstem, effects that are attributable to increased autophagy in the brain. However, trehalose fails to activate autophagy in the spinal cord, where it has no impact on the levels of insoluble tau [227]. Likewise, rapamycin induces autophagy and reduces p62 aggregates and cortical tau tangles, as well as insoluble and hyperphosphorylated tau in transgenic mice [228]. Trehalose also ameliorates dopaminergic and tau pathology in a mouse model expressing human mutant tau(G272V/P301L/R406W) on a null background for parkin [229]. Additionally, lithium increases autophagy and reduces p62 aggregates and neurofibrillary tangles in the neurons of mutant tau(P301S) mice where the motor dysfunction is attenuated [230]. Thus stimulating autophagy is a promising therapeutic strategy for tauopathies.

Regulation of autophagy in Parkinson's disease

PD is a neurodegenerative disease characterized by movement dysfunction and tremor, and is caused by degeneration of dopaminergic neurons in the substantia nigra of the midbrain [231]. Early-onset autosomal dominant forms of PD are associated with α-synuclein point mutations (A53T and A30P) or gene multiplication, or mutations in LRRK2 (leucine-rich repeat kinase 2). Recessive early-onset PD is caused by mutations in parkin, PINK1 (PTEN-induced kinase 1) or DJ-1 [232]. The pathological hallmark of adult-onset PD is the intraneuronal Lewy body, a major constituent of which is aggregated α-synuclein [171,233]. Increased severity of PD correlates with high α-synuclein dosage, and thus deregulated expression of wild-type α-synuclein is possibly a toxic mediator in sporadic PD [234].

Toxic gain-of-function mutants of α-synuclein (A53T or A30P) have been described where these mutant proteins inhibit CMA by inhibiting their uptake and that of other CMA substrates [235]. Although wild-type α-synuclein is degraded by CMA [235], its post-translational dopamine-modified form blocks CMA [236]. Nonetheless, these α-synuclein point mutants, but not the wild-type protein, are autophagy substrates [180] and their overexpression does not perturb autophagy [237]. Accordingly, stimulating autophagy with rapamycin or several mTOR-independent chemical inducers such as lithium, carbamazepine, trehalose, SMERs, calpastatin, rilmenidine and Ca2+ channel blockers facilitates the clearance of mutant α-synuclein in cell models [93,144,167,169,180]. In contrast, overexpression of wild-type α-synuclein, which reflects PD-associated increased gene dosage by triplication of the α-synuclein locus [234], inhibits autophagosome synthesis in mammalian cells and transgenic mice through inhibition of Rab1a, causing mislocalization of Atg9 that plays a crucial role in the expansion of phagophores [237]. This increases aggregation of mutant huntingtin [238], probably by retarding its clearance. Consistently, overexpression of wild-type α-synuclein in two mouse models of HD (expressing exon 1 huntingtin-Q115 or N-terminal huntingtin-Q82) enhances the onset of tremors and weight loss, whereas deletion of α-synuclein in both of these transgenic mice increases autophagy, thereby significantly delaying the onset of disease phenotypes [239]. Moreover, α-synuclein overexpression in rat midbrain has been suggested to inhibit autophagy by sequestering TFEB into aggregates and causing its cytoplasmic retention, whereas overexpression of TFEB induces autophagy and protects against α-synuclein-induced neurotoxicity [240]. Lentiviral overexpression of the autophagy activator Beclin-1 in the brain of α-synuclein transgenic mice also ameliorates neuronal pathology with a reduction in α-synuclein accumulation [241]. Recently, pharmacological activation of CMA by retinoic acid derivatives, such as AR7 (atypical retinoid 7), GR1 (guanidine retinoid 1) and GR2, is shown to increase cellular viability and reduce oligomeric α-synuclein species in cells expressing α-synuclein treated with the pro-oxidant paraquat [242]. Further in vivo trials with chemical enhancers of autophagy remain to be tested.

Mitochondrial dysfunction is a major pathological feature in PD [243,244], thus requiring their elimination through mitophagy. A functional correlation between a defect in mitophagy with recessive PD-associated proteins (parkin, PINK1 and DJ-1) has been established, suggesting that their wild-type counterparts regulate this process in metazoan cells. Parkin, an E3 ubiquitin ligase, and PINK1 act in the same molecular pathway to influence mitochondrial dynamics [245247], whereas DJ-1 is likely to function in a parallel pathway [248]. Parkin is recruited to depolarized mitochondria by PINK1 for mediating mitophagy [249252], which may require PINK1-mediated phosphorylation of parkin in its ubiquitin-like domain [253]. Subsequently, parkin mediates the formation of Lys27-linked polyubiquitin chains on VDAC1 (voltage-dependent anion channel 1), an outer mitochondrial membrane protein [250]. This causes sequestration of the polyubiquitin-binding autophagy adaptor p62, which possibly directs damaged mitochondria for autophagy-mediated clearance through its interaction with LC3 on the expanding phagophores. Various pathogenic mutations in parkin and PINK1 associated with familial PD abrogate mitophagy at distinct steps, leading to accumulation of dysfunctional mitochondria and augmenting cellular toxicity [24,249252]. On the other hand, DJ-1 also localizes to depolarized mitochondria upon oxidative stress [254], whereas loss of DJ-1 causes mitochondrial fragmentation and accumulation of autophagosomes [248], although the mechanism of autophagy perturbation is not known.

Familial PD is also linked to mutations in LRRK2, which possesses both kinase and GTPase functions and is implicated in autophagy [255], although mechanistic detail remains to be elucidated. Transgenic mice expressing mutant LRRK2(G2019S) show increased AVs in the striatum and cortex [256], whereas loss of LRRK2 has been suggested to impair autophagy [257]. Overexpression of mutant LRRK2(G2019S) or LRRK2(R1441C) increases autophagosome number in cell models [258260]; however, changes in autophagic flux remain to be characterized. LRRK2-mediated alteration in autophagy has been suggested through CaMKKβ (Ca2+-dependent protein kinase kinase-β) and AMPK pathways [260]. Brain-specific abrogation of autophagy (by deletion of Atg7) in mice causes accumulation of LRRK2 [261], although its specificity for autophagy-mediated clearance has not been assessed. In fact, wild-type LRRK2 is a CMA substrate, whereas LRRK2(G2019S) is poorly degraded through this pathway [262], raising the possibility of LRRK2(G2019S) being degraded by autophagy. However, mutations in LRRK2 impair CMA, as evident in the brain of LRRK2 patients and transgenic mice, as well as in hiPSC (human induced pluripotent stem cell)-derived dopaminergic neurons [262]. Since α-synuclein is also degraded by CMA [235], the CMA-inhibitory effects of LRRK2 may reflect cross-talk in the clearance of these proteins. Indeed, a possible pathophysiological interplay is suggested from a study in α-synuclein(A53T) transgenic mice, in which overexpression of LRRK2 accelerates the progression of neuropathological changes, whereas deletion of LRRK2 alleviates these alterations [263].

The deleterious effects of failure in autophagy and mitophagy resemble PD-like phenotypes, such as increase in protein aggregation, mitochondrial dysfunction and susceptibility to pro-apoptotic insults. Although efficacy of autophagy activation in vivo warrants investigation, lack of robust neurodegenerative phenotypes including dopaminergic neuronal vulnerability in various PD transgenic mice [264] has hindered treatment trials.

Regulation of autophagy in Huntington's disease

HD is an autosomal dominant neurodegenerative disorder characterized by degeneration of the striatum along with motor dysfunction and cognitive decline. It is a polyQ disorder caused by CAG repeat expansion encoding an abnormally large polyQ tract in huntingtin, thus rendering the protein aggregation-prone and pathogenic [265]. PolyQ-expanded N-terminal fragments comprising the first 100–150 residues generated from the cleavage of mutant huntingtin form aggregates, and thus HD pathogenesis is frequently modelled with huntingtin exon 1 or N-terminal fragments containing expanded polyQ repeats [266,267]. Early observations in striatal neurons either transiently expressing mutant huntingtin or from HD mice has revealed accumulation of AVs [268,269], suggesting alterations in autophagy either due to increased autophagosome synthesis or impaired clearance. Subsequently, mutant huntingtin aggregates have been shown to sequester and inactivate mTOR in HD patient brain tissues and cellular models. Since mTORC1 negatively regulates autophagy, accumulation of autophagosomes in HD is linked to an activation of autophagy [18]. In contrast, mutant huntingtin aggregates also recruit Beclin-1 that regulates autophagy initiation, thereby impairing its function [193]. However, another study has identified a failure in autophagic cargo recognition in HD transgenic models without any substantial changes in autophagosome number, thereby implying a decline in the functional activity of the pathway, even though mutant huntingtin could be sequestered into the autophagosomes and degraded [270].

Mutant huntingtin (full-length or toxic N-terminal fragments) is a well-established autophagy substrate [18,83,193]. The PtdIns3P-binding protein Alfy scaffolds a complex between p62-bound mutant huntingtin aggregates and the autophagic effectors, such as Atg5 and LC3, thus enabling selective elimination of this aggregation-prone protein through autophagy [21,271]. Stimulation of autophagy with rapamycin facilitates the clearance of mutant huntingtin in cell models of HD and attenuates neurodegeneration in transgenic Drosophila expressing N-terminal huntingtin-Q120 [18,82,83]. Consistently, the rapamycin ester CCI-779 ameliorates HD pathology in HD mice (expressing N-terminal huntingtin-Q82) by reducing mutant huntingtin aggregates through activation of autophagy, leading to improved motor function [18]. In contrast, thiol antioxidants such as NAC (N-acetylcysteine) and glutathione inhibit autophagy in a dose-dependent manner. NAC exacerbates neurodegeneration in Drosophila HD, and abrogates the beneficial effects of autophagy inducers in HD zebrafish model (expressing exon 1 huntingtin-Q71) [272]. Since supplements with putative antioxidant properties are commonly used by patients with neurodegenerative disease, this evidence emphasizes the need for a tight regulation of drug administration.

Interestingly, deleting the polyQ stretch of full-length mutant huntingtin in a knockin HD mouse model activates neuronal autophagy and longevity [273]. Several mTOR-independent autophagy inducers, such as inositol-lowering agents (lithium, carbamazepine or sodium valproate), Ca2+ channel blockers (verapamil, loperamide, amiodarone, nimodipine or nitrendipine), calpain inhibitor (calpastatin), cAMP-reducing agents (rilmenidine or clonidine), NOS inhibitor (L-NAME), anti-psychotic drugs (fluspirilene or pimozide), trehalose, SMERs and Tat-Beclin-1 peptide enhance the clearance of mutant huntingtin and rescue its toxicity in HD cell models. Many of these compounds, some of which are FDA-approved drugs, exhibit protective effects in transgenic fruitflies, as well as in a zebrafish HD model expressing exon 1 huntingtin-Q71 [92,93,139,144,162,167,169]. Notably, the mTOR-independent autophagy inducer rilmenidine, which is an FDA-approved centrally acting drug, increases autophagy in the brain and attenuates the disease pathology in HD mice expressing N-terminal huntingtin-Q82 [274]. A combination treatment strategy for inducing autophagy with greater efficacy (described below) by simultaneously stimulating through the mTOR-dependent (rapamycin) and mTOR-independent (lithium) pathways cause a significant rescue against neurodegeneration in HD fruitflies compared with the protective effects of individual compounds [275]. Thus up-regulation of autophagy in HD is a promising treatment strategy in patients.

Regulation of autophagy in spinocerebellar ataxias

PolyQ disorders comprise at least nine late-onset progressive neurodegenerative disorders, including SCAs. The unstable CAG repeat expansion in disease-specific proteins is believed to cause dominant gain-of-function neurotoxicity, although this issue is still debatable [172,174176]. SCA3 is the most common form of dominantly inherited ataxias, characterized by aggregation of mutant ataxin-3 and neurodegeneration [172]. A genome-wide screen for identifying modifiers of pathogenic ataxin-3-mediated neurodegeneration in transgenic Drosophila expressing truncated ataxin-3-Q78 has found a role of autophagy in the accumulation of this misfolded protein [276]. Although accumulation of LC3 and p62 aggregates is seen in SCA3 patient brain tissues and transgenic mice, Beclin-1 levels are also decreased, suggesting intriguing alterations in the autophagy pathway [277]. However, it is unclear whether any defect occurs at the formation or fusion stages of autophagosomes and the mechanistic basis for it. Mutant ataxin-3 directly binds to parkin and facilitates its autophagic degradation [278]; an effect consistent with reduced parkin levels in SCA3 mice that may imply some of the parkinsonism features in the disease [278,279]. In addition, full-length mutant ataxin-3 itself is an autophagy substrate [82]. Inducing autophagy by overexpressing Beclin-1 enhances the clearance of mutant ataxin-3 and is neuroprotective in neuronal cultures and rat model of SCA3 expressing full-length ataxin-3-Q71 [277]. Chemical activation of autophagy with rapamycin or its analogue CCI-779 also reduces the levels of mutant ataxin-3, and ameliorates its toxicity in cell and mouse (expressing full-length ataxin-3-Q70) models of SCA3 [82,280]. However, a comprehensive rescue of the disease pathology could not be assessed due to the mild phenotype of SCA3 transgenic mice [280]. Further trials are required in mouse models exhibiting a more robust phenotype and examining the efficacy of inducing autophagy with mTOR-independent compounds.

A role for the proteolytic systems is also identified in a genome-wide screen for modifiers of mutant ataxin-1-induced neurodegeneration in a Drosophila model of SCA1 expressing full-length ataxin-1-Q82 [281]. Full-length mutant ataxin-1, as well as mutant ataxin-7, can be degraded by autophagy, and therefore inducing autophagy with rapamycin facilitates their clearance and reduces toxicity in cell models of SCA1 and SCA7 respectively [82,181,282]. On the other hand, mutant ataxin-7 inhibits autophagosome synthesis by increasing the interaction between p53 and FIP200 and sequestrating them into its aggregates, thereby disrupting the ULK1–FIP200–Atg13 complex that regulates autophagy initiation. This defect in autophagy is restored in the SCA7 cell model by compounds inhibiting p53 or ataxin-7 aggregation [283]. Furthermore, a reduction in cathepsin B in a SCA6 transgenic mice associated with Purkinje cell degeneration may affect lysosomal proteolysis and the function of the autophagy pathway [284].

Regulation of autophagy in other polyQ diseases

SBMA is an inherited MND (motor neuron disease) caused by polyQ-expanded AR (androgen receptor) [175]. Autophagy is implicated in clearing mutant AR, since depletion of p62 increases accumulation of this mutant protein by retarding its recognition and clearance by the autophagy pathway, leading to exacerbation of motor phenotypes in a mouse model of SBMA [285]. Indeed, cytoplasmic retention of mutant AR facilitates its degradation through autophagy [286]. Stimulating this pathway with 17-AAG [17-(allylamino)-17-demethoxygeldanamycin], an Hsp90 (heat-shock protein 90) inhibitor, reduces the levels of the mutant protein, leading to amelioration of motor neuron degeneration in SBMA transgenic mice expressing AR-Q97 [287,288]. Further studies using well-characterized autophagy inducers are required to assess the benefits of autophagy up-regulation.

DRPLA is another neurodegeneration-associated polyQ disorder caused by CAG expansion in atrophin-1 [175,176]. Intraneuronal inclusions in DRPLA patient brains, although largely co-stained with p62, are negative for LC3 [289]. However, mutant atrophin-1 has been suggested to perturb autophagy indirectly in a Drosophila model expressing atrophin-1 with Gln79 in the polyproline domain and Gln66 in the C-terminal end. Expression of this aggregation-prone protein causes neurodegeneration, along with an increase in autophagosome number [290]. Although autophagosome–lysosome fusion is not affected, mutant atrophin-1 impairs lysosomal proteolysis and retards autophagic degradation. Consequently, up-regulation of autophagy by rapamycin or expressing dominant-negative TOR fail to rescue mutant atrophin-1-mediated neurodegeneration in transgenic fruitflies [290]. In this context, increasing the efficacy of lysosomal proteolysis may be a more rational approach than enhancing autophagy. The regulation of autophagy in many of the neurodegeneration-associated polyQ diseases is not fully characterized, and requires further work towards the development of transgenic animal models recapitulating the disease phenotypes and treatment trials with autophagy inducers.

Regulation of autophagy in amyotrophic lateral sclerosis

ALS is a neurodegenerative disorder belonging to the group of MND, characterized by degeneration of the motor neurons [291]. Mutations affecting microtubule transport, such as in the dynein motor machinery, have been implicated in MNDs [292294]. Perturbing dynein function that is essential for microtubule-based autophagosome trafficking and their delivery to the lysosomes blocks autophagy, thus causing accumulation of autophagosomes and aggregation-prone proteins in transgenic mice [61,295]. Consequently, mutations in dynein machinery augment neurodegeneration in HD transgenic mice by impairing autophagy [61].

Defects in autophagy due to dysfunctional ESCRT machinery has also been implicated in contributing to neurodegeneration in ALS and FTD3 (FTD linked to chromosome 3). The ESCRT machinery consists of four complexes: ESCRT-I, ESCRT-II, ESCRT-III and ESCRT-IV, which function in the biogenesis of intraluminal vesicles in MVBs and mediates fusion of autophagosomes with the endosomal system [296]. Mutations in CHMP2B (charged multivesicular body protein 2B), an ESCRT-III subunit, are associated with certain forms of ALS and FTD3 [297299]. CHMP2B is required for autophagosome maturation, since expression of its deletion mutant in cell and fruitfly models causes accumulation of autophagosomes by impairing autophagic flux [69,300].

Familial ALS is caused due to mutations in SOD1 [301]. Increased autophagosomes with a reduction in mTORC1 activity has been reported in the motor neurons of mutant SOD1(G93A) mice [302]; however, mechanistic details remain to be elucidated. Mutant SOD1 is degraded by autophagy mediated by its Lys63-linked ubiquitylation that promotes its aggregation [183,303]. Additionally, p62 can bind to mutant SOD1 in an ubiquitin-independent manner to target its degradation through autophagy [304]. Mutations in p62 have been found in patients with ALS and FTD3 [305], which open the possibility of a failure in cargo sequestration that requires further investigation. Stimulating autophagy independently of mTOR with lithium facilitates the clearance of mutant SOD1(G93A), delays the disease onset and is neuroprotective in a mouse model of ALS. This study has also reported a marked attenuation of disease progression in ALS patients with lithium treatment [306]. However, subsequent studies using lithium found no obvious benefits in clinical trials with patients or in transgenic mice [307310]. Although lithium has various autophagy-independent side effects, up-regulation of mTOR-independent autophagy by trehalose has been reported recently to be beneficial in mutant SOD1(G86R) mice [311]. Trehalose prolongs lifespan and attenuates disease pathology in these ALS transgenic mice, which is associated with increased autophagy and reduction in mutant SOD1 aggregates in motor neurons [311]. Further trials using FDA-approved autophagy inducers in mouse models of ALS will be important to ascertain the efficacy of autophagy modulation for treatment.

Regulation of autophagy in Lafora disease

LD (Lafora disease) is a lethal autosomal recessive neurodegenerative disorder that manifests with myoclonus epilepsy. It is characterized by the accumulation of intracytosolic ubiquitylated polyglucosan inclusion bodies called Lafora bodies, comprising insoluble poorly-branched glycogen polymers that are present in brain, spinal cord and other tissues [312]. The majority of LD-causing mutations occur in EPM2A, which encodes laforin, a member of the dual-specificity protein phosphatase family, and EPM2B, which codes for malin, a protein with an N-terminal RING finger domain characteristic of an important group of E3 ubiquitin ligases [313]. Laforin and malin functions in the biogenesis, degradation and branching of glycogen. Mice deficient for either laforin or malin exhibit a defect in autophagosome synthesis [314,315]. Loss of laforin due to disease-causing mutations inhibits autophagy by activating mTORC1. In contrast, laforin positively regulates autophagy by inhibiting mTORC1, although the exact mechanism is unclear since specific laforin substrates governing autophagy remains to be identified [314]. However, loss of malin inhibits autophagy without affecting mTORC1 activity, the mechanism of which needs to be elucidated [315]. Since defects in autophagy lead to accumulation of glycogen and ubiquitylated protein aggregates, impaired autophagy in LD is likely to contribute to the disease pathology by accumulating Lafora bodies. Thus it may be possible to rescue the function of autophagy in LD by stimulating this process with mTOR-independent autophagy enhancers, which can facilitate autophagic cargo clearance under conditions when the mTORC1 is active [93].

Combination strategy for enhancing autophagy by mTOR-dependent and mTOR-independent pathways

Additive effects in enhancing autophagy can be achieved through simultaneous stimulation by mTOR inhibition and mTOR-independent routes, which suggests that these regulatory pathways are independent of each other. Apart from the ability of lithium to induce mTOR-independent autophagy by inhibiting IMPase [144], GSK3β, another intracellular target of lithium, has opposing effects on autophagy by activating mTORC1 in a TSC2-dependent fashion [130,275]. The autophagy-inhibitory effects of mTORC1 activation resulting from lithium treatment due to GSK3β inhibition can be abrogated with rapamycin in combination with lithium, thereby allowing autophagy induction to occur through inhibition of the mTOR pathway and by reduction of Ins(1,4,5)P3 levels [275]. Combination of IMPase inhibitors, such as lithium or L-690,330, along with rapamycin causes greater induction of autophagy compared with the effects of the individual compounds [144,275]. Consequently, substantial clearance of autophagy substrates, such as mutant huntingtin and α-synuclein, is attained with the combined treatment, compared with that mediated by either treatment alone. Similar combination effects for activating autophagy have been shown in cell culture using rapamycin with mTOR-independent autophagy enhancers, such as trehalose [167], calpastatin [93] or SMERs [169]. The mTOR-independent autophagy modulators can stimulate autophagy even in Rheb-expressing cells where mTORC1 is activated. Likewise, rapamycin induces autophagy even when cellular levels of inositol and Ins(1,4,5)P3 have been elevated by pharmacological means that inhibit autophagy independently of mTORC1 [144]. These data suggest that stimulation of autophagy by either pathway can overcome the inhibition caused by the other route. Such paradigm may be relevant in neurodegenerative diseases where autophagy is perturbed in certain contexts, and thus determining the route of autophagy stimulation may be rational to designing the treatment strategy with mTOR-independent autophagy modulators.

Combination therapy for enhancing autophagy with lower doses of each chemical inducer may be safer for long-term drug administration in neurodegenerative diseases and may lessen the drug-specific side effects, compared with using higher doses of either compound alone that result in more severe perturbations of a single pathway. The rational combination treatment approach has been confirmed in vivo in a Drosophila model of HD, in which lithium or rapamycin exhibited protective effects against neurodegeneration, whereas a combination treatment exerted a significantly greater rescue compared with either compounds acting alone [275]. This application has been validated further by showing greater protection against neurodegeneration with lithium treatment in HD fruitflies heterozygous for a null allele of Drosophila TOR, compared with the effects of inhibiting either TOR or inositol signalling pathways in HD fruitflies alone [275]. Whereas the combination treatment strategy opens a new possibility for achieving greater efficacy with therapeutic stimulation of autophagy, demonstration of this approach in mouse models of neurodegenerative diseases remains to be investigated.

Concluding remarks and future perspectives

Regulation of mammalian autophagy by mTOR-dependent and mTOR-independent pathways is amenable to chemical perturbations. A number of small-molecule inducers of autophagy has been identified, some of which are FDA-approved drugs [187189,196198]. Chemical activation of autophagy rescues the disease phenotype in various transgenic models of neurodegenerative diseases. Autophagy also acts as a protective pathway in other human diseases. For example, stimulation of autophagy by carbamazepine reduces hepatic fibrosis and α1-antitrypsin Z load in a mouse model of α1-antitrypsin deficiency-associated liver disease [316]. Inducing autophagy with rapamycin also restores myofibre survival and ameliorates dystrophic phenotype in mouse model of muscular dystrophy linked to collagen VI deficiency [317]. Similarly, SMERs or rapamycin enhance the elimination of mycobacteria in primary macrophages by up-regulating autophagy, implying possible application in infectious diseases [318,319]. Likewise, lithium or trehalose increases autophagic clearance of pathological prion proteins [320,321]. In addition, stimulating autophagy with spermidine or resveratrol promotes organismal longevity [322,323]. In contrast, inhibitors of autophagy are being considered as anti-cancer drugs, although the situation is complex [324]. Thus modulation of autophagy has a growing range of treatment prospects in diverse pathological conditions, raising the possibility for creating a common therapeutic paradigm in certain contexts.

However, not all chemical regulators of autophagy may be optimal for clinical use. Although there are over 21000 drug products, it is estimated after discarding duplications of active ingredients and salt forms that the availability of small-molecule drugs is only approximately 1200 and that of biological drugs is less than 200 [325]. Technological advancements in designing efficient chemical screening platforms and high-throughput drug discovery may reveal novel pharmacological probes for accurately manipulating autophagy. One challenging aspect is the identification of small molecules targeting the autophagy machinery involving the Atg conjugation systems without affecting the upstream signalling pathways, which may provide greater specificity in modulating this process. Nonetheless, side effects related to current pharmacological induction of autophagy are not known, and, if any, are possibly masked by the therapeutic benefits achieved through up-regulation of autophagy. Recent advances in hiPSC technology and establishment of hiPSC patient-specific disease models may shed light on the perturbations of autophagy in disease-specific clinically relevant cellular contexts, such as in neuronal cells generated from the differentiation of neurodegenerative disease hiPSCs [326]. Combining the knowledge of autophagy dysfunction and the mechanism of drug action may be rational for designing targeted therapy.

Early Career Research Award Delivered at Royal Holloway, University of London on 28 March 2012, as part of the LRRK2: Function and Dysfunction Focused Meeting Sovan Sarkar

Abbreviations

     
  • amyloid β-peptide

  •  
  • AD

    Alzheimer’s disease

  •  
  • ALS

    amyotrophic lateral sclerosis

  •  
  • Ambra1

    activating molecule in Beclin-1-regulated autophagy

  •  
  • AMPK

    AMP-activated protein kinase

  •  
  • APP

    amyloid precursor protein

  •  
  • AR

    androgen receptor

  •  
  • Atg

    autophagy-related

  •  
  • AV

    autophagic vacuole

  •  
  • Bcl

    B-cell lymphoma

  •  
  • BH3

    Bcl-2 homology 3

  •  
  • CaMKKβ

    Ca2+-dependent protein kinase kinase β

  •  
  • CHMP2B

    charged multivesicular body protein 2B

  •  
  • CMA

    chaperone-mediated autophagy

  •  
  • 2′5′ddA

    2′,5′-dideoxyadenosine

  •  
  • deptor

    DEP-domain containing mTOR-interacting protein

  •  
  • DRPLA

    dentatorubral pallidoluysian atrophy

  •  
  • 4E-BP1

    translation initiation factor 4E-binding protein-1

  •  
  • Epac

    exchange protein directly activated by cAMP

  •  
  • ER

    endoplasmic reticulum

  •  
  • ERK1/2

    extracellular-signal-regulated kinase 1/2

  •  
  • ESCRT

    endosomal sorting complex required for transport

  •  
  • FAD

    familial AD

  •  
  • FDA

    U.S. Food and Drug Administration

  •  
  • FIP200

    focal adhesion kinase family-interacting protein of 200 kDa

  •  
  • FoxO3

    forkhead box O3

  •  
  • FTD

    frontotemporal dementia

  •  
  • FTD3

    FTD linked to chromosome 3

  •  
  • GAP

    GTPase-activating protein

  •  
  • GR

    guanidine retinoid

  •  
  • GSK3

    glycogen synthase kinase 3

  •  
  • HD

    Huntington’s disease

  •  
  • hiPSC

    human induced pluripotent stem cell

  •  
  • hVps

    mammalian vacuolar protein sorting homologue

  •  
  • IKK

    inhibitor of nuclear factor κB kinase

  •  
  • IMPase

    inositol monophosphatase

  •  
  • IP3R

    Ins(1,4,5)P3 receptor

  •  
  • I1R

    imidazoline-1 receptor

  •  
  • JNK1

    c-Jun N-terminal kinase 1

  •  
  • LC3

    light chain 3

  •  
  • LD

    Lafora disease

  •  
  • L-NAME

    NG-nitro-L-arginine methyl ester

  •  
  • LRRK2

    leucine-rich repeat kinase 2

  •  
  • MIPS

    myo-inositol-1-phosphate synthase

  •  
  • mLST8

    mammalian lethal with SEC13 protein 8

  •  
  • MND

    motor neuron disease

  •  
  • mTOR

    mammalian target of rapamycin

  •  
  • mTORC

    mTOR complex

  •  
  • MVB

    multivesicular body

  •  
  • NAC

    N-acetylcysteine

  •  
  • NBR1

    neighbour of BRCA1 gene 1

  •  
  • NOS

    nitric oxide synthase

  •  
  • p70S6K

    ribosomal protein S6 kinase-1

  •  
  • PD

    Parkinson’s disease

  •  
  • PDK1

    phosphoinositide-dependent kinase 1

  •  
  • PE

    phosphatidylethanolamine

  •  
  • PI3K

    phosphoinositide 3-kinase

  •  
  • PI3KC1a

    class Ia PI3K

  •  
  • PI3KC3

    class III PI3K

  •  
  • PI3KK

    PI3K-related protein kinase

  •  
  • PINK1

    PTEN-induced kinase 1

  •  
  • PKA

    protein kinase A

  •  
  • PLC

    phospholipase C

  •  
  • polyQ

    polyglutamine

  •  
  • PS

    presenilin

  •  
  • PTEN

    phosphatase and tensin homologue deleted from chromosome 10

  •  
  • Rag

    Ras-related GTP-binding protein

  •  
  • raptor

    regulatory-associated protein of mTOR

  •  
  • Rheb

    Ras homologue enriched in brain

  •  
  • rictor

    rapamycin-insensitive companion of mTOR

  •  
  • SBMA

    spinobulbar muscular atrophy

  •  
  • SCA

    spinocerebellar ataxia

  •  
  • SLC

    solute carrier

  •  
  • SMER

    small-molecule enhancer of rapamycin

  •  
  • SMIR

    small-molecule inhibitor of rapamycin

  •  
  • SNARE

    N-ethylmaleimide-sensitive factor-attachment protein receptor

  •  
  • SOD1

    copper/zinc superoxide dismutase 1

  •  
  • TFEB

    transcription factor EB

  •  
  • TOR

    target of rapamycin

  •  
  • TSC

    tuberous sclerosis complex

  •  
  • ULK1

    UNC-51-like kinase 1

  •  
  • UVRAG

    UV irradiation resistance-associated gene

  •  
  • VAMP

    vesicle-associated membrane protein

  •  
  • v-ATPase

    vacuolar H+-ATPase

  •  
  • Vps

    vacuolar protein sorting

I am thankful to the Biochemical Society for the Early Career Research Award in Cell Biology, and Tom DiCesare (Whitehead Institute) for assistance with illustrations.

Funding

I am grateful for a Gates Cambridge Scholarship, a Hughes Hall Research Fellowship and to the laboratory of D.C. Rubinsztein for funding in the past, and the laboratory of R. Jaenisch and the Whitehead Institute for current funding. S.S. is also a Former Fellow at Hughes Hall, University of Cambridge, Cambridge, U.K.

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