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: Higher- and lower-molecular-weight fractions of libraries are shown to contain distinct gene sets, and higher rates of gene discovery are associated with higher- molecular weight libraries.
Abstract: Gene and genome duplications are thought to be primary mechanisms of increasing the number of coding sequences subject to selection, leading to new proteins, morphogenic variations, and phenotypes (Ohno 1970; Holland et al. 1994; Sidow 1996). Members of the teleost family Salmonidae, including salmon, trout, char, grayling, and whitefish, all diverged from a common ancestor that is believed to have undergone a tetraploidization event 25 to 100 million years ago, after the teleost radiation (Allendorf and Thorgaard 1984). This relatively recent putative genome duplication in the salmonid lineage is supported by karyological and genome size data. Members of the family Clupeidae (e.g., herring, alewife), thought to maintain the ancestral diploid status, have 48 to 52 mostly acrocentric chromosomes per 2N cell and genome sizes of 0.8 to 1.4 pg/N, whereas salmonids have 52 to 102 chromosomes per 2N cell (over half metacentric or submetacentric) and genome sizes of 1.9 to 3.8 pg/N (Ohno et al. 1968; Phillips and Rab 2001; Gregory 2002). Because extant salmonids exhibit quadrivalents in meiosis (primarily in males; Ohno et al. 1965; Allendorf and Thorgaard 1984) and disomic and tetrasomic inheritance at different loci (Allendorf and Danzmann 1997), they appear to be in the process of re-establishing diploidy. Remarkably, ∼50% of examined salmonid loci persist as functional duplicates (Bailey et al. 1978). Research on salmonid genomes will shed light on poorly understood evolutionary phenomena such as genome duplication and duplicate gene silencing.
In addition to their scientific importance as recent tetraploids, salmonids also serve as prominent models for studies involving environmental toxicology (Katchamart et al. 2002), carcinogenesis (Bailey et al. 1996), comparative immunology (Shum et al. 2001), and the molecular genetics and physiology of the stress response (Basu et al. 2002), olfaction (Zhang et al. 2001), vision (Faillace et al. 2002), osmoregulation (Tipsmarck et al. 2002), growth (Devlin et al. 2001), and gametogenesis (Madigou et al. 2002). Furthermore, Atlantic salmon (AS; Salmo salar) are of particular importance to the global aquaculture industry. GRASP (Genomics Research on Atlantic Salmon Project), an initiative funded by Genome Canada, is intended to improve understanding of physiological and evolutionary processes influencing the survival and phenotype of salmonids and other fish in natural and aquaculture environments. GRASP has developed genomics resources to help achieve these goals. There is a rich literature in salmonid genetics, physiology, and ecology to support these genomics research tools.
A previously reported S. salar EST project surveyed 1152 ESTs from six cDNA libraries, with 510BLAST-identified sequences representing 178 salmon genes (Davey et al. 2001). There are currently (August 2003) ∼60,000 S. salar nucleotide sequences in GenBank, of which >51,000 were submitted by GRASP. In addition to forming an EST database containing >80,000 sequences from five salmonid species, GRASP has built a microarray from 3557 unique salmonid cDNAs. Initial cross-species testing of this microarray has shown it to be effective in hybridizations with salmon, trout, and whitefish targets.
300 citations
TL;DR: RalB signaling is a primary adaptive response to nutrient limitation that directly engages autophagocytosis through mobilization of the core vesicle nucleation machinery.
299 citations
TL;DR: PpAtg30 is a key player in the selection of peroxisomes as cargo and in their delivery to the autophagy machinery for pexophagy, and is required for formation of pexophile intermediates, such as the micropexophagy apparatus (MIPA) and the peXophagosome (Ppg).
299 citations
TL;DR: The cooperation between TRIM16 and Galectin-3 in targeting and activation of selective autophagy protects cells from lysosomal damage and Mycobacterium tuberculosis invasion.
299 citations
TL;DR: Increasing evidence obtained both in vitro and in vivo supports the hypothesis that a variety of cell death programs may be triggered in distinct circumstances, and the view that caspase-mediated apoptosis represents the standard programmed cell death is challenged.
Abstract: Programmed cell death is a major component of both normal development and disease. The roles of cell death during either embryogenesis or pathogenesis, the signals that modulate this event, and the mechanisms of cell demise are the major subjects that drive research in this field. Increasing evidence obtained both in vitro and in vivo supports the hypothesis that a variety of cell death programs may be triggered in distinct circumstances. Contrary to the view that caspase-mediated apoptosis represents the standard programmed cell death, recent studies indicate that an apoptotic morphology can be produced independent of caspases, that autophagic execution pathways of cell death may be engaged without either the involvement of caspases or morphological signs of apoptosis, and that even the necrotic morphology of cell death may be consistently produced in some cases, including certain plants. Alternative cell death programs may imply novel therapeutic targets, with important consequences for attempts to treat diseases associated with disregulated programmed cell death.
298 citations