Autophagy modulation as a potential therapeutic target for diverse diseases

Abstract

Autophagy is an essential, conserved lysosomal degradation pathway that controls the quality of the cytoplasm by eliminating protein aggregates and damaged organelles. It begins when double-membraned autophagosomes engulf portions of the cytoplasm, which is followed by fusion of these vesicles with lysosomes and degradation of the autophagic contents. In addition to its vital homeostatic role, this degradation pathway is involved in various human disorders, including metabolic conditions, neurodegenerative diseases, cancers and infectious diseases. This article provides an overview of the mechanisms and regulation of autophagy, the role of this pathway in disease and strategies for therapeutic modulation.

Figure 1. Overview of the regulation of macroautophagy and potential drug targets

Two major signalling pathways are depicted here: the pathway involving class I phosphoinositide 3-kinase (PI3K), protein kinase B (PKB) and mammalian target of rapamycin complex 1 (mTORC1), and a cyclical mTOR-independent pathway; the basic helix–loop–helix leucine zipper transcription factor EB (TFEB)-mediated pathway is also depicted. TFEB regulates the expression of the genes involved in the different stages of autophagy between autophagosome formation and cargo degradation (see FIG. 2). In nutrient-rich medium, TFEB is phosphorylated by mTORC1 and is retained in the cytoplasm. In starved cells, TFEB is dephosphorylated and is translocated into the nucleus. TFEB is a potential target for drugs. Retaining TFEB in the cytoplasm would inhibit autophagy, as illustrated in the figure. By contrast, promoting the nuclear translocation of TFEB would stimulate autophagy. Activating the class I PI3K–PKB–mTORC1 pathway by growth factors and amino acids blocks autophagy by inhibiting the initiation of autophagosome formation by the UNC51-like kinase 1 (ULK1) complex. Glucose starvation increases the AMP/ATP ratio, which activates AMP-activated protein kinase (AMPK). This enzyme induces autophagy by inhibiting mTORC1 (via directly targeting elements of the complex) or by phosphorylating and activating tuberous sclerosis 2 (TSC2) in the TSC1–TSC2 complex. Recent data suggest that mTOR interacts with TFEB, as the two proteins are colocalized on lysosomal membranes. Phosphorylation of mTOR inhibits TFEB acivity, whereas ATP-competitive mTOR inhibitors enable TFEB dephosphorylation, thus allowing its nuclear translocation and activation. AMPK also phosphorylates and thus activates the ULK1 complex. Activators of AMPK (such as metformin) stimulate autophagy, as do inhibitors of mTORC1 (for example, rapamycin or rapalogues) and inhibitors of class I PI3Ks (for example, PI-103). Amino acids activate mTORC1 via RAG GTPases, and also via the vacuolar V-ATPase located in the lysosomal membrane. Inhibitors of V-ATPases, such as bafilomycin A1, block the maturation of autophagosomes. However, blocking V-ATPase also inhibits the amino-acid-dependent activation of mTORC1, which would have a stimulatory effect on autophagosome formation. Elevation of the intracellular levels of cyclic AMP (cAMP) by adenylyl cyclase downstream of G protein-coupled receptors blocks autophagy by activating the exchange protein directly activated by cAMP (EPAC), the small G protein RAP2B and phospholipase Cε (PLCε). Activation of PLCε results in the production of inositol-1,4,5-trisphosphate (Ins(1,4,5)P₃) and, consequently, the release of Ca²⁺ from the endoplasmic reticulum via the Ins(1,4,5)P₃ receptor (Ins(1,4,5)P₃R). Influx of Ca²⁺ into the cytoplasm is also triggered by l-type Ca²⁺ channel agonists. Drugs acting at the various different steps of the cAMP–EPAC–PLCε– Ins(1,4,5)P₃ pathway regulate autophagy. Calpains activated by Ca²⁺ block autophagy by cleaving and constitutively activating G_sα proteins (also called G_αs), which increases cAMP levels. Thus, inhibitors of calpains would stimulate autophagy in this setting. The binding of B cell lymphoma 2 (BCL-2) to beclin 1 inhibits autophagy, and this effect can be counteracted by BCL-2 homology 3 (BH3) mimetics. DAG, diacylglycerol; IMPase, inositol monophosphatase; InsP, inositol monophosphate; Ins(1,4)P₂, inositol-1,4-bisphosphate; PtdIns(4,5)P₂, phosphatidylinositol-4,5-bisphosphate; RHEB, RAS homologue enriched in brain (GTP binding protein).

Figure 2. Effects of drugs on the different steps of the autophagic pathway

The formation of the autophagosome depends on the initiation and/or nucleation of a specific membrane structure known as the phagophore, and on the elongation and closure of the phagophore to form a double-membrane-bound vacuole. The biogenesis of autophagosomes requires the ordered intervention of autophagy-regulated (ATG) proteins that act on different modules. Some of these modules are shown on the figure, including the UNC51-like kinase 1 (ULK1) and phosphoinositide 3-kinase (PI3K) complexes, ATG12 (the ATG12–ATG5 complex) and microtubule-associated protein 1 light-chain 3 (LC3; for example, the LC3–phosphatidylethanolamine conjugates); ATG4B is a protein that is involved in the regulation of LC3 lipidation and delipidation. The ULK1 complex is composed of ULK1 (the mammalian orthologue of yeast Atg1) and RB1-inducible coiled-coil 1 (RB1CC1; also known as FIP200; the mammalian orthologue of yeast Atg13, Atg17 and Atg101). The PI3K complex is composed of beclin 1 (the mammalian orthologue of yeast Atg6 and Atg14), class III PI3K (PIK3C3), PIK3R4 (PI3K regulatory subunit 4) and AMBRA1 (activating molecule in BECN1-regulated autophagy protein 1). Both complexes congregate at the phagophore assembly site to initiate autophagy in response to nutrient starvation. The kinase activity of ULK1 is controlled by the kinase mammalian target of rapamycin (mTOR) in mTOR complex 1 (mTORC1), which is sensitive to rapamycin. The protein ATG14 appears to have a key role in the targeting of the PI3K complex to the endoplasmic reticulum (ER). How the ULK1 and PI3K complexes are coordinately regulated remains to be elucidated. The production of phosphatidylinositol-3-phosphate (PtdIns3P) by human PIK3C3 recruits the PtdIns3P-binding proteins ATG18 (also known as WIPI1 and WIPI2) and double FYVE domain-containing protein 1 (DFPC1). Cargos can be incorporated into autophagosomes in a non-selective manner (known as bulk autophagy) or in a selective manner (known as selective autophagy). Maturation occurs when the autophagosome fuses with the endolysosomal compartment. The final degradation of cargos occurs in autolysosomes. The activity of the class III PI3K complex can be manipulated by both activators (such as BH3 (BCL-2 homology 3) mimetics) and inhibitors (such as spautin-1; specific and potent autophagy inhibitor 1). PIK3C3, which is a component of the class III PI3K complex, is a potential target for drugs. Some other potential targets are also indicated on the figure. Autophagosome closure can be inhibited by reducing ATG4B activity. Compounds that interfere with LC3-interacting region (LIR) motifs could potentially block the selective recruitment of cargos such as mitochondria, viruses and bacteria. Inhibitors of lysosomal enzymes (such as cystatin B), and lysosomotropic agents that increase the lysosomal pH (such as chloroquine and hydrochloroquine) block the degradative activity of autolysosomes. NDP52, nuclear dot protein 52; p62, ubiquitin-binding protein p62 (also known as sequestosome 1).