Teaching the basics of autophagy and mitophagy to redox biologists--mechanisms and experimental approaches

Abstract

Autophagy is a lysosomal mediated degradation activity providing an essential mechanism for recycling cellular constituents, and clearance of excess or damaged lipids, proteins and organelles. Autophagy involves more than 30 proteins and is regulated by nutrient availability, and various stress sensing signaling pathways. This article provides an overview of the mechanisms and regulation of autophagy, its role in health and diseases, and methods for its measurement. Hopefully this teaching review together with the graphic illustrations will be helpful for instructors teaching graduate students who are interested in grasping the concepts and major research areas and introducing recent developments in the field.

Keywords: Aging; Beclin; LC3; Mitochondria; Neurodegenerative diseases; mTOR.

Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Figures

Graphical abstract

Fig. 1

The 3 types of autophagy. Lysosomal-mediated degradation of cellular contents, or autophagy, is divided into 3 general categories. 1. Microautophagy is defined by ultrastructural studies as performed by lysosomal/endosomal invagination of nearby cellular contents, or lysosomal extension to wrap around cellular contents, to engulf and degrade lipids, proteins and organelles. It has been shown to depend on ATP and microfilaments and be facilitated by HSC70-phospholipid interactions. 2. Chaperone-mediated autophagy is dependent on lysosomal LAMP-2A membrane receptor and chaperone HSC70 protein recognizing unfolded proteins that carry KFERQ consensus sequences. 3. Macroautophagy is characterized by formation of double membrane vesicles that recognize and encircle intracellular excessive or damaged lipids, proteins and organelles. The autophagic vesicles, or autophagosomes, then fuse with lysosomes, and their contents degraded by lysosomal enzymes. These 3 types of autophagy have shared as well as independent machineries, and their activities may compensate or regulate one another.

Fig. 2

Proteins involved in autophagosome formation. 1. Initiation of autophagosomal formation is regulated by mTOR inhibition. In response to insulin receptor withdrawal, amino acid starvation, low ATP, mTOR is inhibited, resulting in ULK1 activation and initiation of autophagy. 2. Beclin-1/VPS34/VPS15/Atg14 complex formation also plays an important role in nucleation of autophagic vesicles. 3. Extension of autophagosomal membranes are regulated by 2 ubiquitin-like conjugation pathways, with both LC3 and Atg12 resembling ubiquitin structure. One involves Atg7 and Atg10 acting as E1 and E2 enzymes, sequentially conjugating with Atg12 via glycine–cysteine thioester bonds, eventually conjugating Atg12 to Atg5 at a lysine residue through an isopeptide bond, resulting in Atg5/Atg12/Atg16 complex association with autophagosomal membranes. The other involves Atg4 cleaving pro-LC3 exposing a glycine residue at the C-terminal, conjugation of LC3-I with Atg7, then Atg3 via thioester bonds, resulting in conjugation of LC3-I with phosphotidylethanolamine (PE) through an amide bond, forming LC3-II and insertion into autophagosomal membranes. 4. Recognition of cargo can be mediated by adaptor proteins such as p62 that has an ubiquitin binding domain as well as an LC3-II interacting domain.

Fig. 3

mTOR in sensing autophagy signals : mTOR inhibition integrates amino acid starvation, growth factor deprivation, decreased ATP or oxygen levels, enhanced reactive oxygen species (ROS) to activate autophagy. Amino acid deprivation leads to mTOR dissociation with lysosomes. An association of AMPK with the late endosomes in response to glucose starvation also leads to dissociation and inactivation of mTOR. ATP depletion activates AMPK which can activate TSC1/2 and thereby inhibits mTOR, or activates ULK1 to activate autophagy. In addition, mitochondrial generated ROS may also induce autophagy via an AMPK mediated pathway. Growth factor deprivation leads to activation of TSC1/2, inactivation of Rheb, and inactivation of mTOR. Peroxisomal TSC1/2 localizes to peroxisomes through binding to PEX19 and PEX5, and can be activated by peroxisomal ROS to inhibit mTOR activities and induce autophagy. Oxygen deprivation may activate autophagy by HIF1α-mediated transcription activation of BNIP3 which inhibits Rheb. mTOR inhibition activates autophagy by activation of ULK1, VPS34 and TFEB, a master transcription activator of genes encoding autophagy and lysosomal proteins. Conformational inhibitors of mTOR such as rapamycin and catalytic inhibitors such as Torin1 have been shown to induce autophagy.

Fig. 4

Beclin1–VPS34 complex in regulation of autophagy. Beclin-1/VPS34/VPS15/ATG14 complex plays an important role in regulating autophagy and is subjected to transcriptional (e.g., by transcription factor NFκB and E2F) and post-translational controls (phosphorylation or ubiquitination). Beclin-1 can also complex with the anti-apoptotic Bcl-2 protein, attenuating its involvement in autophagic activities. Beclin-1–VPS34 complex is subject to regulation by UVRAG, Bif-1, Rubicon, Ambra1, NRBF2, and HMGB1. PI3P production by VPS34 can be sensed by WIPIs which control the localization of Atg9 and its involvement in autophagy. ALFY can also bind PI3P and associate with p62 and cytoplasmic protein aggregates and mediates their autophagic degradation. PI3K inhibitors wortmannin and 3-methyladenine can inhibit autophagy, although they may have additional impact on other PI3K activities.

Fig. 5

LC3 function and homologs. (A) LC3 is a microtubule-associated protein, MAP1 light chain 3. These microtubule associated proteins, including MAP2 (A, B, and C) that are restricted to the central nervous system, Tau that is enriched in the central nervous system but also expressed elsewhere, MAP4 that is widely expressed in mammalian tissues, and MAP1 (A, B, and C heavy chain, and light chain 1 and 2 that are enriched in the central nervous system, but also expressed in other tissues), are thought to be important for maintaining cell structure and shape. (B) LC3 has 6 mammalian homologs including LC3A, LC3B, LC3C, GATE16, GABARAP and GABARAPL1. All these homologs can be cleaved by Atg4 at the C-terminal which exposes the glycine residue generating LC3A-I, LC3B-I, LC3C-I, GATE16-I, GABARAP-I and GABARAPL1-I, allowing E1, E2, and E3-like conjugation by Atg7, Atg3 mediated reaction, ending with conjugation with phosphatidylethanolamine (PE), resulting in membrane associated form of LC3A-II, LC3B-II, LC3C-II, GATE16-II, GABARAP-II and GABARAPL1-II.

Fig. 6

Adaptor proteins that recognize autophagy substrates and/or bridge autophagy substrates with autophagy machineries. (A) P62/SQSTM1 is an important autophagy adaptor protein, with an LC3 interacting region (LIR) that can recognize autophagosomes through binding LC3/GABARAP family proteins, and an ubiquitin association domain (UBA) that can recognize ubiquitinated proteins that are autophagy substrates. Interestingly, p62 can also bind KEAP1 (via a KEAP1 interacting region, KIR), a redox sensor protein, to send KEAP1 for autophagy degradation. This is important for regulation of cellular redox status, since KEAP1 binding to NRF2 allows NRF2 degradation by the proteasomes, and KEAP1 modification or degradation frees NRF2 from the proteasomal degradation, therefore allowing NRF2 to activate transcription of antioxidants, as well as p62 itself. (B) P62/SQSTM1 mutations in the UBA have been found in Paget disease, in wide spread regions of the gene in amyloid lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). (C) NDP52 and optineurin also contain LIR and UBA and are important for recognition of ubiquitinated bacteria and degrade bacteria via xenophagy. (D) Other UBA containing proteins involved in autophagy include HDAC6 and ALFY that are important for aggrephagy. (E) Atg1, 3, 4, Nix, BNIP3, FUNDC1, FYCO1 and TRIM also contain LIR and regulate autophagy.

Fig. 7

Mitophagy. Mitophagy can be induced by mtDNA damage, mitochondrial electron transport chain inhibition, loss of mitochondrial membrane potential, mitochondrial fission or fragmentation, mitochondrial unfolded or damaged proteins. Mitophagy is important for mitochondrial quality control and thus essential for maintaining cellular energy, calcium homeostasis, redox signaling, mitochondrial–cytosol or mitochondrial–nuclear signaling, and sequestration of apoptotic factors. One major signaling pathway is through inactivation of mitochondrial protease PARL due to loss of membrane potential, thus stabilization of PINK1. PINK1 recruits Parkin which ubiquitinates mitochondrial associated proteins. P62 recognizes ubiquitinated proteins and brings mitochondria to autophagosomes by binding to LC3. Parkin and PINK1 can be regulated by a variety of cellular proteins in many recent studies, including those listed in this diagram. Deubiquitinase USP8 plays an important role in Parkin recruitment to the mitochondria and mitophagy. In contrast USP15 and 30 remove ubiquitin from Parkin substrates and thus attenuate mitophagy. Parkin/PINK1-independent mitophagy also exist, including those mediated or signaled by BNIP3, Nix, FUNDC1 or cardiolipin, which can bind LC3 and directly bring mitochondria to the autophagosomes for mitophagy. In addition, mitochondrial spheroids and mitochondria derived vesicles (MDV) may bring mitochondria to the lysosomes for degradation. In response to loss of mitochondrial membrane potential, choline dehydrogenase (CHDH) can also recruit p62 directly and target mitochondria to mitophagy.

Fig. 8

Plasma membrane contributes to autophagosomal biogenesis. Localization of autophagosomal associated protein Atg16L1 to plasma membrane and interaction of Atg16L1 with assembly polypeptide 2 (AP2) and clathrin heavy chain have been found to occur prior to the formation of subsequent endosomal-like structures. Knockdown of clathrin heavy chain of AP2 decreased both basal and starvation-induced autophagosomal biogenesis. Atg9 is also internalized by clathrin-mediated endocytosis in Atg16L1-free vesicles. Atg9- and Atg16L1-containing vesicles can undergo VAMP3-mediated heterotypic fusion, and this step also stimulates autophagosomal formation with plasma membrane as a contributing source. Atg16L1-containing vesicles can undergo VAMP7-mediated homotypic fusion which precedes the acquisition of LC3 by the plasma membrane-derived phagophore precursors.

Fig. 9

ER and Golgi contribute to autophagosomal biogenesis. (A) ER donates membranes to autophagosomes. In response to starvation, ULK complex and subsequently Atg14L moves to the ER at a site where vacuole membrane protein (VMP1) also transiently localizes, followed by DFCP1 moving to punctate structures called “omegasomes”, in a Beclin and VPS34 dependent manner, and colocalizes with LC3 and Atg5–Atg12–Atg16L1 complex, this begins the formation of autophagosomes. Recent studies have also found that VMP1-Beclin interaction plays an important role in autophagy induction. (B) ER–Golgi intermediate complex compartment (ERGIC) promotes LC3 lipidation. Autophagosomal formation from ERGIC depends on coat protein complex II (COPII) vesicles. (C) The mitochondria-associated ER membrane (MAM) donates membranes to autophagosomes. Glucose starvation also induces colocalization of LC3 and Atg5 with mitochondria, this localization has been shown to recruit mitochondrial lipids to autophagosomes. Formation of autophagosomes from MAM requires MFN2 or phosphofurin acidic cluster sorting protein 2 (PACS2). The SNARE protein syntaxin 17 (STX17) recruits Atg14L to the MAM sites and further recruits DFCP1.

Fig. 10

Autophagosome–lysosome fusion. Fusion of autophagosomes and lysosomes depends on lysosomal VAMP7/8 and insertion of SNARE protein syntaxin 17 (STX17) to the autophagosomal membrane and binding SNAP29. Additional factors required or inhibit fusion are listed.

Fig. 11

Autophagy assessment by LC3-II levels. (A) During normal autophagic flux, LC3-I is conjugated with phosphatidylethanolamine to become LC3-II and inserts into autophagosomal membranes. LC3-II level has been found to be proportional to the amount of autophagosomes and thus has been used for autophagy assessment. (B) Inhibition of autophagy prior to LC3-II generation, either by 3-methyladenine (3-MA) or by knockout/knockdown of Atg7 decreases LC3-II. (C) Activation of autophagy either by starvation or by inhibitors of mTOR such as rapamycin increases LC3-II. (D) Inhibition of lysosomal clearance of autophagosomes, such as chloroquine, bafilomycin, E64+pepstatin A (PepA) also increases LC3-II accumulation. Therefore, to assess if a chemical or a genetic manipulation activates autophagy prior to LC3-I to LC3-II conversion, one approach is to compare LC3-II in response to manipulation alone, lysosomal blockade alone (chloroquine, bafilomycin or E64+pepstatin A), and manipulation in the presence of lysosomal blockade.

Fig. 12

Autophagic flux assessment using RFP–GFP-LC3. To assess autophagic flux from the autophagosomes to the lysosomes, RFP–GFP-LC3 plasmids can be transfected into cells. Under conditions with minimum autophagosomal accumulation, RFP–GFP-LC3-I is diffused in the cytosol. When RFP–GFP-LC3-I is converted into RFP–GFP-LC3-II and inserted into autophagosomal membrane, RFP and GFP signals colocalize to punctate structures. As autophagosomes fuse with lysosomes, the outer membrane RFP–GFP-LC3-II is delipidated to RFP–GFP-LC3-I and diffused to the cytosolic space, the inner membrane RFP–GFP-LC3-II is quenched for the GFP signal but not the RFP signal. This is due to the lysosomal acidic environment that protonates the fluorophore of the GFP but not RFP because of differences in their pKa values. Therefore, the red only puncta indicate the flux of RFP–GFP-LC3-II protein into the acidic environment of the lysosomes.

Fig. 13

Mitophagy assessment. Mitophagy is characterized by colocalization of autophagy proteins (such as p62, LC3, Parkin or PINK1) with the mitochondria; colocalization of mitochondria with lysosomal markers (either due to autophagosomal–lysosomal fusion or due to mitochondrial derived vesicles (MDV) fusing with the lysosomes). These characteristics can be used to assess mitophagy activities. In addition, the consequences of mitophagy can be assessed by either a decrease of mitochondrial proteins or DNA, or changes of mitochondrial activities such as reactive species production, oxygen consumption or changes of mitochondrial membrane potential.

Fig. 14

Role of autophagy and mitophagy in health and diseases. (A) Deficient autophagy due to inhibition of autophagic proteins or mutations of autophagy genes can lead to accumulation of gene mutations, dysfunctional mitochondria and protein aggregates, as well as inability to clear viral particles, as occurs in tumorigenesis, neurodegeneration, aging and infections. Autophagy induction, for example, by rapamycin, trehalose, caloric restriction, Atg5 or Beclin overexpression, has been shown to provide benefits to attenuate disease pathogenesis and extend lifespan. (B) Autophagy has also been shown to provide a detrimental role in established cancer by enhancing survival of cancer cells in nutrient deprived conditions or in response to chemotherapeutics. Inhibiting autophagy, for example by chloroquine treatment, has been investigated as a cancer therapeutic strategy.

Fig. 15

Future directions. Although we now know that many different conditions (including growth factor, glucose, amino acids or ATP deprivation, deficient or excessive oxygen, reactive species, damage to DNA, lipid, proteins or organelles) may stimulate autophagy, the exactly mechanisms are unclear. Understanding the genetic, epigenetic, transcriptional, and post-translational regulation of autophagy, understanding the regulation of membrane formation, fusion and movement, and understanding the specificity, timing, duration, location of these regulations, understanding the cross regulation of autophagy with proteasomal and NRF2 transcription activities (in regulating expression of antioxidant proteins and p62), and understanding how to apply pharmacological or genetic interventions to achieve maximal beneficial impact without negative outcomes will be important in autophagy biology and its application to medicine.