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: The findings support the pathogenic significance of autophagic-lysosomal dysfunction in Alzheimer's disease and indicate the potential value of restoring normal autophagy as an innovative therapeutic strategy for Alzheimer’s disease.
Abstract: Autophagy, a major degradative pathway for proteins and organelles, is essential for survival of mature neurons. Extensive autophagic-lysosomal pathology in Alzheimer's disease brain contributes to Alzheimer's disease pathogenesis, although the underlying mechanisms are not well understood. Here, we identified and characterized marked intraneuronal amyloid-β peptide/amyloid and lysosomal system pathology in the Alzheimer's disease mouse model TgCRND8 similar to that previously described in Alzheimer's disease brains. We further establish that the basis for these pathologies involves defective proteolytic clearance of neuronal autophagic substrates including amyloid-β peptide. To establish the pathogenic significance of these abnormalities, we enhanced lysosomal cathepsin activities and rates of autophagic protein turnover in TgCRND8 mice by genetically deleting cystatin B, an endogenous inhibitor of lysosomal cysteine proteases. Cystatin B deletion rescued autophagic-lysosomal pathology, reduced abnormal accumulations of amyloid-β peptide, ubiquitinated proteins and other autophagic substrates within autolysosomes/lysosomes and reduced intraneuronal amyloid-β peptide. The amelioration of lysosomal function in TgCRND8 markedly decreased extracellular amyloid deposition and total brain amyloid-β peptide 40 and 42 levels, and prevented the development of deficits of learning and memory in fear conditioning and olfactory habituation tests. Our findings support the pathogenic significance of autophagic-lysosomal dysfunction in Alzheimer's disease and indicate the potential value of restoring normal autophagy as an innovative therapeutic strategy for Alzheimer's disease.
413 citations
TL;DR: Although the formation of autophagosomes is completely different from any other vesicular structures, their later itinerary appears to be very similar to those of other trafficking pathways, adding to the understanding of the mechanism and control of Autophagy.
Abstract: Macroautophagy, a constitutive process in higher eukaryotic cells, mediates degradation of many long-lived proteins and organelles. The actual events occurring during the process in the dynamic system of a living cell have never been thoroughly investigated. We aimed to develop a live-cell assay in which to follow the complete itinerary of an autophagosome. Our experiments show that autophagosomes are formed randomly in peripheral regions of the cell. They then move bidirectionally along microtubules, accumulating at the microtubule-organizing centre, in a similar way to lysosomes. Their centripetal movement is dependent on the motor protein dynein and is important for their fusion with lysosomes. Initially, autophagosomes dock on to lysosomes, independent of lysosomal acidification. Two kinds of fusion then occur: complete fusions, creating a hybrid organelle, or more often kiss-and-run fusions, i.e. transfer of some content while still maintaining two separate vesicles. Surprisingly, the autophagolysosomal compartment seems to be more long lived than expected. Our study documents many aspects of autophagosome behaviour, adding to our understanding of the mechanism and control of autophagy. Indeed, although the formation of autophagosomes is completely different from any other vesicular structures, their later itinerary appears to be very similar to those of other trafficking pathways.
413 citations
TL;DR: It is shown that the high-mobility group box 1 protein (HMGB1) is a redox-sensitive regulator of the balance between autophagy and apoptosis, representing a suitable target when coupled with conventional tumor treatments.
Abstract: The functional relationship and cross-regulation between autophagy and apoptosis is complex. In this study we show that the high-mobility group box 1 protein (HMGB1) is a redox-sensitive regulator of the balance between autophagy and apoptosis. In cancer cells, anticancer agents enhanced autophagy and apoptosis, as well as HMGB1 release. HMGB1 release may be a prosurvival signal for residual cells after various cytotoxic cancer treatments. Diminished HMGB1 by short hairpin RNA transfection or inhibition of HMGB1 release by ethyl pyruvate or other small molecules led predominantly to apoptosis and decreased autophagy in stressed cancer cells. In this setting, reducible HMGB1 binds to the receptor for advanced glycation end products (RAGEs), but not to Toll-like receptor 4, induces Beclin1-dependent autophagy and promotes tumor resistance to alkylators (melphalan), tubulin disrupting agents (paclitaxel), DNA crosslinkers (ultraviolet light) and DNA intercalators (oxaliplatin or adriamycin). On the contrary, oxidized HMGB1 increases the cytotoxicity of these agents and induces apoptosis mediated by the caspase-9/-3 intrinsic pathway. HMGB1 release, as well as its redox state, thus links autophagy and apoptosis, representing a suitable target when coupled with conventional tumor treatments.
413 citations
TL;DR: Stimulation of autophagosome formation by vinblastine was independent of nutrients and mTOR activity but was inhibited by depletion of the Autophagy proteins Atg5 and Atg6, known to be required for autophagy.
Abstract: Nutrient deprivation of eukaryotic cells provokes a variety of stress responses, including autophagy. Autophagy is carried out by autophagosomes which sequester cytosolic components and organelles for degradation after fusion with protease-containing endosomes. To determine the role of microtubules in autophagy, we used nocodazole and vinblastine to disrupt microtubules and independently measured formation and fusion of autophagsosomes in primary rat hepatocytes. By measuring the translocation of GFP-LC3, an autophagosomal marker, to autophagosomes and the lipidation of GFP-LC3, we quantified the rate and magnitude of autophagosome formation. Starvation increased both the rate of autophagosome formation over the basal level and the total number of autophagosomes per cell. Maximal autophagosome formation required an intact microtubule network. Fusion of autophagosomes with endosomes, assayed by acquisition of protease-inhibitor sensitivity as well as overlap with LysoTracker Red-positive endosomes, required intact microtubules. Live-cell imaging demonstrated that autophagosomes were motile structures, and their movement also required microtubules. Interestingly, vinblastine stimulated autophagosome formation more than twofold before any discernable change in the microtubule network was observed. Stimulation of autophagosome formation by vinblastine was independent of nutrients and mTOR activity but was inhibited by depletion of the Autophagy proteins Atg5 and Atg6, known to be required for autophagy.
412 citations
TL;DR: Pleckstrin homology domain containing protein family member 1 (PLEKHM1) directly interacts with HOPS complex and contains a LC3-interacting region (LIR) that mediates its binding to autophagosomal membranes.
412 citations