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.
Citations
More filters
••
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
More filters
••
TL;DR: The findings indicate that the pathological process associated with p62 accumulation results in hyperactivation of Nrf2 and delineates unexpected roles of selective autophagy in controlling the transcription of cellular defence enzyme genes.
Abstract: Impaired selective turnover of p62 by autophagy causes severe liver injury accompanied by the formation of p62-positive inclusions and upregulation of detoxifying enzymes. These phenotypes correspond closely to the pathological conditions seen in human liver diseases, including alcoholic hepatitis and hepatocellular carcinoma. However, the molecular mechanisms and pathophysiological processes in these events are still unknown. Here we report the identification of a novel regulatory mechanism by p62 of the transcription factor Nrf2, whose target genes include antioxidant proteins and detoxification enzymes. p62 interacts with the Nrf2-binding site on Keap1, a component of Cullin-3-type ubiquitin ligase for Nrf2. Thus, an overproduction of p62 or a deficiency in autophagy competes with the interaction between Nrf2 and Keap1, resulting in stabilization of Nrf2 and transcriptional activation of Nrf2 target genes. Our findings indicate that the pathological process associated with p62 accumulation results in hyperactivation of Nrf2 and delineates unexpected roles of selective autophagy in controlling the transcription of cellular defence enzyme genes.
1,918 citations
••
TL;DR: In this article, the authors present guidelines for the reporting of tumor marker studies, which encourage transparent and complete reporting so that the relevant information will be available to others to help them to judge the usefulness of the data and understand the context in which the conclusions apply.
Abstract: Despite years of research and hundreds of reports on tumor markers in oncology, the number of markers that have emerged as clinically useful is pitifully small. Often, initially reported studies of a marker show great promise, but subsequent studies on the same or related markers yield inconsistent conclusions or stand in direct contradiction to the promising results. It is imperative that we attempt to understand the reasons that multiple studies of the same marker lead to differing conclusions. A variety of methodologic problems have been cited to explain these discrepancies. Unfortunately, many tumor marker studies have not been reported in a rigorous fashion, and published articles often lack sufficient information to allow adequate assessment of the quality of the study or the generalizability of study results. The development of guidelines for the reporting of tumor marker studies was a major recommendation of the National Cancer Institute-European Organisation for Research and Treatment of Cancer (NCI-EORTC) First International Meeting on Cancer Diagnostics in 2000. As for the successful CONSORT initiative for randomized trials and for the STARD statement for diagnostic studies, we suggest guidelines to provide relevant information about the study design, preplanned hypotheses, patient and specimen characteristics, assay methods, and statistical analysis methods. In addition, the guidelines suggest helpful presentations of data and important elements to include in discussions. The goal of these guidelines is to encourage transparent and complete reporting so that the relevant information will be available to others to help them to judge the usefulness of the data and understand the context in which the conclusions apply.
1,892 citations
••
TL;DR: Misregulated ferroptosis has been implicated in multiple physiological and pathological processes, including cancer cell death, neurotoxicity, neurodegenerative diseases, acute renal failure, drug-induced hepatotoxicity, hepatic and heart ischemia/reperfusion injury, and T-cell immunity.
Abstract: Ferroptosis is a recently recognized form of regulated cell death. It is characterized morphologically by the presence of smaller than normal mitochondria with condensed mitochondrial membrane densities, reduction or vanishing of mitochondria crista, and outer mitochondrial membrane rupture. It can be induced by experimental compounds (e.g., erastin, Ras-selective lethal small molecule 3, and buthionine sulfoximine) or clinical drugs (e.g., sulfasalazine, sorafenib, and artesunate) in cancer cells and certain normal cells (e.g., kidney tubule cells, neurons, fibroblasts, and T cells). Activation of mitochondrial voltage-dependent anion channels and mitogen-activated protein kinases, upregulation of endoplasmic reticulum stress, and inhibition of cystine/glutamate antiporter is involved in the induction of ferroptosis. This process is characterized by the accumulation of lipid peroxidation products and lethal reactive oxygen species (ROS) derived from iron metabolism and can be pharmacologically inhibited by iron chelators (e.g., deferoxamine and desferrioxamine mesylate) and lipid peroxidation inhibitors (e.g., ferrostatin, liproxstatin, and zileuton). Glutathione peroxidase 4, heat shock protein beta-1, and nuclear factor erythroid 2-related factor 2 function as negative regulators of ferroptosis by limiting ROS production and reducing cellular iron uptake, respectively. In contrast, NADPH oxidase and p53 (especially acetylation-defective mutant p53) act as positive regulators of ferroptosis by promotion of ROS production and inhibition of expression of SLC7A11 (a specific light-chain subunit of the cystine/glutamate antiporter), respectively. Misregulated ferroptosis has been implicated in multiple physiological and pathological processes, including cancer cell death, neurotoxicity, neurodegenerative diseases, acute renal failure, drug-induced hepatotoxicity, hepatic and heart ischemia/reperfusion injury, and T-cell immunity. In this review, we summarize the regulation mechanisms and signaling pathways of ferroptosis and discuss the role of ferroptosis in disease.
1,871 citations
••
TL;DR: The maturation programme entails a dramatic transformation of these dynamic organelles disconnecting them functionally and spatially from early endosomes and preparing them for their unidirectional role as a feeder pathway to lysosomes.
Abstract: Being deeply connected to signalling, cell dynamics, growth, regulation, and defence, endocytic processes are linked to almost all aspects of cell life and disease. In this review, we focus on endosomes in the classical endocytic pathway, and on the programme of changes that lead to the formation and maturation of late endosomes/multivesicular bodies. The maturation programme entails a dramatic transformation of these dynamic organelles disconnecting them functionally and spatially from early endosomes and preparing them for their unidirectional role as a feeder pathway to lysosomes.
1,859 citations
••
TL;DR: A new mode of protein lipidation is reported, in which Apg8 is covalently conjugated to phosphatidylethanolamine through an amide bond between the C-terminal glycine and the amino group of phosph atidyleanolamine, mediated by a ubiquitination-like system.
Abstract: Autophagy is a dynamic membrane phenomenon for bulk protein degradation in the lysosome/vacuole1,2. Apg8/Aut7 is an essential factor for autophagy in yeast3,4,5. We previously found that the carboxy-terminal arginine of nascent Apg8 is removed by Apg4/Aut2 protease, leaving a glycine residue at the C terminus6. Apg8 is then converted to a form (Apg8-X) that is tightly bound to the membrane6. Here we report a new mode of protein lipidation. Apg8 is covalently conjugated to phosphatidylethanolamine through an amide bond between the C-terminal glycine and the amino group of phosphatidylethanolamine. This lipidation is mediated by a ubiquitination-like system. Apg8 is a ubiquitin-like protein that is activated by an E1 protein, Apg7 (refs 7, 8), and is transferred subsequently to the E2 enzymes Apg3/Aut1 (ref. 9). Apg7 activates two different ubiquitin-like proteins, Apg12 (ref. 10) and Apg8, and assigns them to specific E2 enzymes, Apg10 (ref. 11) and Apg3, respectively. These reactions are necessary for the formation of Apg8-phosphatidylethanolamine. This lipidation has an essential role in membrane dynamics during autophagy6.
1,849 citations