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
References
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TL;DR: In vivo evidence is provided that growth arrest, autophagy, and atg genes are required for physiological autophagic cell death and that multiple degradation pathways cooperate in the efficient clearance of cells during development.
588 citations
TL;DR: The results suggest that Bnip3 contributes to I/R injury which triggers a protective stress response with upregulation of autophagy and removal of damaged mitochondria.
Abstract: Ischemia and reperfusion (I/R) injury is associated with extensive loss of cardiac myocytes. Bnip3 is a mitochondrial pro-apoptotic Bcl-2 protein which is expressed in the adult myocardium. To investigate if Bnip3 plays a role in I/R injury, we generated a TAT-fusion protein encoding the carboxyl terminal transmembrane deletion mutant of Bnip3 (TAT-Bnip3ΔTM) which has been shown to act as a dominant negative to block Bnip3-induced cell death. Perfusion with TAT-Bnip3ΔTM conferred protection against I/R injury, improved cardiac function, and protected mitochondrial integrity. Moreover, Bnip3 induced extensive fragmentation of the mitochondrial network and increased autophagy in HL-1 myocytes. 3D rendering of confocal images revealed fragmented mitochondria inside autophagosomes. Enhancement of autophagy by ATG5 protected against Bnip3-mediated cell death, whereas inhibition of autophagy by ATG5K130R enhanced cell death. These results suggest that Bnip3 contributes to I/R injury which triggers a protective stress response with upregulation of autophagy and removal of damaged mitochondria.
586 citations
TL;DR: The consequence of phagocytosis of dead cells is strongly affected by those components of the autophagy pathway involved in LAP, and the engagement of LAP upon uptake of apoptotic, necrotic, and RIPK3-dependent nec rotic cells by macrophages is described.
Abstract: The recognition and clearance of dead cells is a process that must occur efficiently to prevent an autoimmune or inflammatory response. Recently, a process was identified wherein the autophagy machinery is recruited to pathogen-containing phagosomes, termed MAPLC3A (LC3)-associated phagocytosis (LAP), which results in optimal degradation of the phagocytosed cargo. Here, we describe the engagement of LAP upon uptake of apoptotic, necrotic, and RIPK3-dependent necrotic cells by macrophages. This process is dependent on some members of the classical autophagy pathway, including Beclin1, ATG5, and ATG7. In contrast, ULK1, despite being required for autophagy, is dispensable for LAP induced by uptake of microbes or dead cells. LAP is required for efficient degradation of the engulfed corpse, and in the absence of LAP, engulfment of dead cells results in increased production of proinflammatory cytokines and decreased production of anti-inflammatory cytokines. LAP is triggered by engagement of the TIM4 receptor by either phosphatidylserine (PtdSer)-displaying dead cells or PtdSer-containing liposomes. Therefore, the consequence of phagocytosis of dead cells is strongly affected by those components of the autophagy pathway involved in LAP.
585 citations
TL;DR: Comparison of reporter accumulation and cleavage of fluorogenic substrates demonstrates that the rate-limiting chymotrypsin-like activity of the proteasome can be substantially curtailed without significant effect on ubiquitin-dependent proteolysis.
Abstract: The ubiquitin/proteasome-dependent proteolytic pathway is an attractive target for therapeutics because of its critical involvement in cell cycle progression and antigen presentation. However, dissection of the pathway and development of modulators are hampered by the complexity of the system and the lack of easily detectable authentic substrates. We have developed a convenient reporter system by producing N-end rule and ubiquitin fusion degradation (UFD)-targeted green fluorescent proteins that allow quantification of ubiquitin/proteasome-dependent proteolysis in living cells. Accumulation of these reporters serves as an early predictor of G2/M arrest and apoptosis in cells treated with proteasome inhibitors. Comparison of reporter accumulation and cleavage of fluorogenic substrates demonstrates that the rate-limiting chymotrypsin-like activity of the proteasome can be substantially curtailed without significant effect on ubiquitin-dependent proteolysis. These reporters provide a new powerful tool for elucidation of the ubiquitin/proteasome pathway and for high throughput screening of compounds that selectively modify proteolysis in vivo.
585 citations
Wellcome Trust Sanger Institute1, National Autonomous University of Mexico2, University of Buenos Aires3, Iowa State University4, University of Zurich5, University of the Republic6, University of Oxford7, Beijing Institute of Genomics8, University of Toronto9, University of Würzburg10, Natural History Museum11, National University of Ireland, Galway12, University of Calgary13, McGill University14
TL;DR: An analysis of tapeworm genome sequences using the human-infective species Echinococcus multilocularis, E. granulosus, Taenia solium and the laboratory model Hymenolepis microstoma offers insights into the evolution of parasitism and identifies new potential drug targets.
Abstract: Tapeworms (Cestoda) cause neglected diseases that can be fatal and are difficult to treat, owing to inefficient drugs. Here we present an analysis of tapeworm genome sequences using the human-infective species Echinococcus multilocularis, E. granulosus, Taenia solium and the laboratory model Hymenolepis microstoma as examples. The 115- to 141-megabase genomes offer insights into the evolution of parasitism. Synteny is maintained with distantly related blood flukes but we find extreme losses of genes and pathways that are ubiquitous in other animals, including 34 homeobox families and several determinants of stem cell fate. Tapeworms have specialized detoxification pathways, metabolism that is finely tuned to rely on nutrients scavenged from their hosts, and species-specific expansions of non-canonical heat shock proteins and families of known antigens. We identify new potential drug targets, including some on which existing pharmaceuticals may act. The genomes provide a rich resource to underpin the development of urgently needed treatments and control.
581 citations