Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)
Daniel J. Klionsky1, Amal Kamal Abdel-Aziz2, Sara Abdelfatah3, Mahmoud Abdellatif4 +2980 more•Institutions (777)
TL;DR: In this article, the authors present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes.
Abstract: In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.
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University of Michigan1, Cornell University2, University of Pennsylvania3, University of Massachusetts Medical School4, University of Naples Federico II5, Baylor College of Medicine6, Spanish National Research Council7, Complutense University of Madrid8, New York University9, Boston Children's Hospital10, University of Rome Tor Vergata11, NewYork–Presbyterian Hospital12, University of Pittsburgh13, University of Paris14, French Institute of Health and Medical Research15, National University of Cuyo16, Albert Einstein College of Medicine17, University of New Mexico18, Goethe University Frankfurt19, Weizmann Institute of Science20, University of Turku21, Sapienza University of Rome22, Virginia Commonwealth University23, St. Jude Children's Research Hospital24, Discovery Institute25, University of Copenhagen26, University of Tromsø27, Eötvös Loránd University28, Merck & Co.29, University of Freiburg30, Babraham Institute31, University of South Australia32, University of Adelaide33, University of Oviedo34, University of Chicago35, University of Graz36, National Institutes of Health37, City University of New York38, Queens College39, University of Tokyo40, University of Zurich41, University of British Columbia42, Austrian Academy of Sciences43, University of California, San Francisco44, Russian Academy of Sciences45, University Medical Center Groningen46, University of Cambridge47, University of Glasgow48, Rutgers University49, University of Padua50, Kazan Federal University51, University of Bern52, University of Oxford53, Oslo University Hospital54, University of Oslo55, Foundation for Research & Technology – Hellas56, University of Crete57, Francis Crick Institute58, Osaka University59, Harvard University60, Chinese Academy of Sciences61, Icahn School of Medicine at Mount Sinai62, Shanghai Jiao Tong University63, Karolinska Institutet64
TL;DR: In this paper, preclinical data linking autophagy dysfunction to the pathogenesis of major human disorders including cancer as well as cardiovascular, neurodegenerative, metabolic, pulmonary, renal, infectious, musculoskeletal, and ocular disorders.
Abstract: Autophagy is a core molecular pathway for the preservation of cellular and organismal homeostasis. Pharmacological and genetic interventions impairing autophagy responses promote or aggravate disease in a plethora of experimental models. Consistently, mutations in autophagy-related processes cause severe human pathologies. Here, we review and discuss preclinical data linking autophagy dysfunction to the pathogenesis of major human disorders including cancer as well as cardiovascular, neurodegenerative, metabolic, pulmonary, renal, infectious, musculoskeletal, and ocular disorders.
365 citations
University of Bonn1, Charité2, Humboldt University of Berlin3, Max Planck Society4, Free University of Berlin5, Max Delbrück Center for Molecular Medicine6, University of Kiel7, German Center for Neurodegenerative Diseases8, German Cancer Research Center9, City University of Hong Kong10, Heidelberg University11
TL;DR: In this paper, the authors show that SARS-CoV-2 infection modulates cellular metabolism and limits autophagy, and identify druggable host pathways for virus inhibition.
Abstract: Viruses manipulate cellular metabolism and macromolecule recycling processes like autophagy. Dysregulated metabolism might lead to excessive inflammatory and autoimmune responses as observed in severe and long COVID-19 patients. Here we show that SARS-CoV-2 modulates cellular metabolism and reduces autophagy. Accordingly, compound-driven induction of autophagy limits SARS-CoV-2 propagation. In detail, SARS-CoV-2-infected cells show accumulation of key metabolites, activation of autophagy inhibitors (AKT1, SKP2) and reduction of proteins responsible for autophagy initiation (AMPK, TSC2, ULK1), membrane nucleation, and phagophore formation (BECN1, VPS34, ATG14), as well as autophagosome-lysosome fusion (BECN1, ATG14 oligomers). Consequently, phagophore-incorporated autophagy markers LC3B-II and P62 accumulate, which we confirm in a hamster model and lung samples of COVID-19 patients. Single-nucleus and single-cell sequencing of patient-derived lung and mucosal samples show differential transcriptional regulation of autophagy and immune genes depending on cell type, disease duration, and SARS-CoV-2 replication levels. Targeting of autophagic pathways by exogenous administration of the polyamines spermidine and spermine, the selective AKT1 inhibitor MK-2206, and the BECN1-stabilizing anthelmintic drug niclosamide inhibit SARS-CoV-2 propagation in vitro with IC50 values of 136.7, 7.67, 0.11, and 0.13 μM, respectively. Autophagy-inducing compounds reduce SARS-CoV-2 propagation in primary human lung cells and intestinal organoids emphasizing their potential as treatment options against COVID-19. Viruses manipulate host cell pathways to support infection. Here the authors show that SARS-CoV-2 infection modulates cellular metabolism and limits autophagy, and identify druggable host pathways for virus inhibition.
140 citations
TL;DR: The role of autophagy in the pathogenesis of metabolic diseases associated with or occurring in the context of ageing, including insulin resistance, T2DM and sarcopenic obesity, was discussed in this article.
Abstract: Autophagy is an evolutionarily conserved, lysosome-dependent catabolic process whereby cytoplasmic components, including damaged organelles, protein aggregates and lipid droplets, are degraded and their components recycled. Autophagy has an essential role in maintaining cellular homeostasis in response to intracellular stress; however, the efficiency of autophagy declines with age and overnutrition can interfere with the autophagic process. Therefore, conditions such as sarcopenic obesity, insulin resistance and type 2 diabetes mellitus (T2DM) that are characterized by metabolic derangement and intracellular stresses (including oxidative stress, inflammation and endoplasmic reticulum stress) also involve the accumulation of damaged cellular components. These conditions are prevalent in ageing populations. For example, sarcopenia is an age-related loss of skeletal muscle mass and strength that is involved in the pathogenesis of both insulin resistance and T2DM, particularly in elderly people. Impairment of autophagy results in further aggravation of diabetes-related metabolic derangements in insulin target tissues, including the liver, skeletal muscle and adipose tissue, as well as in pancreatic β-cells. This Review summarizes the role of autophagy in the pathogenesis of metabolic diseases associated with or occurring in the context of ageing, including insulin resistance, T2DM and sarcopenic obesity, and describes its potential as a therapeutic target. The cellular consequences of dysfunctional autophagy contribute to numerous diseases. In this Review, Kitada and Koya consider the relationship between impaired autophagy and age-related metabolic derangements, including insulin resistance, type 2 diabetes mellitus and sarcopenic obesity, and discuss candidate autophagy-based therapies.
109 citations
TL;DR: In this article, the authors systematically screened 28 viral proteins of SARS-CoV-2 and identified that ORF3a strongly inhibited autophagic flux by blocking the fusion of autophagosomes with lysosomes.
Abstract: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the ongoing coronavirus disease 2019 pandemic. How SARS-CoV-2 regulates cellular responses to escape clearance by host cells is unknown. Autophagy is an intracellular lysosomal degradation pathway for the clearance of various cargoes, including viruses. Here, we systematically screened 28 viral proteins of SARS-CoV-2 and identified that ORF3a strongly inhibited autophagic flux by blocking the fusion of autophagosomes with lysosomes. ORF3a colocalized with lysosomes and interacted with VPS39, a component of the homotypic fusion and protein sorting (HOPS) complex. The ORF3a-VPS39 interaction prohibited the binding of HOPS with RAB7, which prevented the assembly of fusion machinery, leading to the accumulation of unfused autophagosomes. These results indicated the potential mechanism by which SARS-CoV-2 escapes degradation; that is, the virus interferes with autophagosome-lysosome fusion. Furthermore, our findings will facilitate strategies targeting autophagy for conferring potential protection against the spread of SARS-CoV-2.
106 citations
TL;DR: The latest advances in the understanding of the regulating mechanisms and signaling pathways of STING1 in autophagy and cell death are outlined, which may shed light on new targets for therapeutic interventions.
Abstract: Cell death and immune response are at the core of life. In past decades, the endoplasmic reticulum (ER) protein STING1 (also known as STING or TMEM173) was found to play a fundamental role in the production of type I interferons (IFNs) and pro-inflammatory cytokines in response to DNA derived from invading microbial pathogens or damaged hosts by activating multiple transcription factors. In addition to this well-known function in infection, inflammation, and immunity, emerging evidence suggests that the STING1-dependent signaling network is implicated in health and disease by regulating autophagic degradation or various cell death modalities (e.g., apoptosis, necroptosis, pyroptosis, ferroptosis, mitotic cell death, and immunogenic cell death [ICD]). Here, we outline the latest advances in our understanding of the regulating mechanisms and signaling pathways of STING1 in autophagy and cell death, which may shed light on new targets for therapeutic interventions.
78 citations
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TL;DR: Results suggest that NBR1 is the specific autophagy receptor for pexophagy, and that substrate selectivity is partly achieved by N BR1 itself by coincident binding of the J and UBA domains to peroxisomes.
Abstract: Selective macro-autophagy is an intracellular process by which large cytoplasmic materials are selectively sequestered and degraded in the lysosomes. Substrate selection is mediated by ubiquitylation and recruitment of ubiquitin-binding autophagic receptors such as p62, NBR1, NDP52 and Optineurin. Although it has been shown that these receptors act cooperatively to target some types of substrates to nascent autophagosomes, their precise roles are not well understood. We examined selective autophagic degradation of peroxisomes (pexophagy), and found that NBR1 is necessary and sufficient for pexophagy. Mutagenesis studies of NBR1 showed that the amphipathic α-helical J domain, the ubiquitin-associated (UBA) domain, the LC3-interacting region and the coiled-coil domain are necessary to mediate pexophagy. Strikingly, substrate selectivity is partly achieved by NBR1 itself by coincident binding of the J and UBA domains to peroxisomes. Although p62 is not required when NBR1 is in excess, its binding to NBR1 increases the efficiency of NBR1-mediated pexophagy. Together, these results suggest that NBR1 is the specific autophagy receptor for pexophagy.
302 citations
TL;DR: Trehalose treatment led to a significant upregulation in the expression of key autophagy-related genes at the mRNA level including Lc3, Becn1, Sqstm1 and Atg5, and trehalose administration enhanced the nuclear translocation of FOXO1, an important transcription factor involved in the activation of autophagic in neurons.
Abstract: Amyotrophic lateral sclerosis (ALS) is a fatal motoneuron disease with no current effective treatment. Accumulation of abnormal protein inclusions containing SOD1, TARDBP, FUS, among other proteins, is a pathological hallmark of ALS. Autophagy is the major degradation pathway involved in the clearance of damaged organelles and protein aggregates. Although autophagy has been shown to efficiently degrade ALS-linked mutant protein in cell culture models, several studies suggest that autophagy impairment may also contribute to disease pathogenesis. In this report, we tested the potential use of trehalose, a disaccharide that induces MTOR-independent autophagy, in the development of experimental ALS. Administration of trehalose to mutant SOD1 transgenic mice significantly prolonged life span and attenuated the progression of disease signs. These effects were associated with decreased accumulation of SOD1 aggregates and enhanced motoneuron survival. The protective effects of trehalose were associated with incre...
302 citations
TL;DR: This study characterized the putative compartment from which both autophagosomes and the analogous Cvt vesicles may originate and identified a perivacuolar membrane as the resident compartment for both the Apg-Cvt9 signaling complex, which mediates the switching between autophagic and Cvt transport, and the autophagy/Cvt-specific phosphatidylinositol 3-kinase complex.
301 citations
TL;DR: The most commonly used pH probes for sensitivity and localisation are evaluated, and LysoSensor yellow/blue‐dextran, among currently used probes, are identified as having the optimal profile of properties for measuring lysosomal pH.
Abstract: Autophagy is a lysosomal degradative process which recycles cellular waste and eliminates potentially toxic damaged organelles and protein aggregates. The important cytoprotective functions of autophagy are demonstrated by the diverse pathogenic consequences that may stem from autophagy dysregulation in a growing number of neurodegenerative disorders. In many of the diseases associated with autophagy anomalies, it is the final stage of autophagy-lysosomal degradation that is disrupted. In several disorders, including Alzheimer's disease (AD), defective lysosomal acidification contributes to this proteolytic failure. The complex regulation of lysosomal pH makes this process vulnerable to disruption by many factors, and reliable lysosomal pH measurements have become increasingly important in investigations of disease mechanisms. Although various reagents for pH quantification have been developed over several decades, they are not all equally well suited for measuring the pH of lysosomes. Here, we evaluate the most commonly used pH probes for sensitivity and localisation, and identify LysoSensor yellow/blue-dextran, among currently used probes, as having the optimal profile of properties for measuring lysosomal pH. In addition, we review evidence that lysosomal acidification is defective in AD and extend our original findings, of elevated lysosomal pH in presenilin 1 (PS1)-deficient blastocysts and neurons, to additional cell models of PS1 and PS1/2 deficiency, to fibroblasts from AD patients with PS1 mutations, and to neurons in the PS/APP mouse model of AD.
300 citations
TL;DR: N2,N4-Dibenzylquinazoline-2,4-diamine (DBeQ) was identified as a selective, potent, reversible, and ATP-competitive p97 inhibitor that blocks multiple processes that have been shown by RNAi to depend on p97, including degradation of ubiquitin fusion degradation and endoplasmic reticulum-associated degradation pathway reporters.
Abstract: A specific small-molecule inhibitor of p97 would provide an important tool to investigate diverse functions of this essential ATPase associated with diverse cellular activities (AAA) ATPase and to evaluate its potential to be a therapeutic target in human disease. We carried out a high-throughput screen to identify inhibitors of p97 ATPase activity. Dual-reporter cell lines that simultaneously express p97-dependent and p97-independent proteasome substrates were used to stratify inhibitors that emerged from the screen. N2,N4-dibenzylquinazoline-2,4-diamine (DBeQ) was identified as a selective, potent, reversible, and ATP-competitive p97 inhibitor. DBeQ blocks multiple processes that have been shown by RNAi to depend on p97, including degradation of ubiquitin fusion degradation and endoplasmic reticulum-associated degradation pathway reporters, as well as autophagosome maturation. DBeQ also potently inhibits cancer cell growth and is more rapid than a proteasome inhibitor at mobilizing the executioner caspases-3 and -7. Our results provide a rationale for targeting p97 in cancer therapy.
300 citations