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Journal ArticleDOI

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

08 Feb 2021-Autophagy (Landes Bioscience)-Vol. 17, Iss: 1, pp 1-382
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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Journal ArticleDOI
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

Journal ArticleDOI
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

Journal ArticleDOI
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

Journal ArticleDOI
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

Journal ArticleDOI
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
Journal ArticleDOI
TL;DR: It is shown that trehalose also protects cells against subsequent pro-apoptotic insults via the mitochondrial pathway, which may be relevant to the treatment of HD and related diseases, where the mutant proteins are autophagy substrates.

1,024 citations

Journal ArticleDOI
11 Nov 2011-Cell
TL;DR: It is proposed that PINK1 phosphorylation of substrates triggers the subsequent action of Parkin and the proteasome, and the PINK/Parkin pathway may quarantine damaged mitochondria prior to their clearance.

1,022 citations

Journal ArticleDOI
TL;DR: TFEB is identified as a target of mTOR and a mechanism for matching the transcriptional regulation of genes encoding proteins of autophagosomes and lysosomes to cellular need is suggested.
Abstract: Lysosomes are the major cellular site for clearance of defective organelles and digestion of internalized material. Demand on lysosomal capacity can vary greatly, and lysosomal function must be adjusted to maintain cellular homeostasis. Here, we identified an interaction between the lysosome-localized mechanistic target of rapamycin complex 1 (mTORC1) and the transcription factor TFEB (transcription factor EB), which promotes lysosome biogenesis. When lysosomal activity was adequate, mTOR-dependent phosphorylation of TFEB on Ser(211) triggered the binding of 14-3-3 proteins to TFEB, resulting in retention of the transcription factor in the cytoplasm. Inhibition of lysosomal function reduced the mTOR-dependent phosphorylation of TFEB, resulting in diminished interactions between TFEB and 14-3-3 proteins and the translocation of TFEB into the nucleus, where it could stimulate genes involved in lysosomal biogenesis. These results identify TFEB as a target of mTOR and suggest a mechanism for matching the transcriptional regulation of genes encoding proteins of autophagosomes and lysosomes to cellular need. The closely related transcription factors MITF (microphthalmia transcription factor) and TFE3 (transcription factor E3) also localized to lysosomes and accumulated in the nucleus when lysosome function was inhibited, thus broadening the range of physiological contexts under which this regulatory mechanism may prove important.

1,020 citations

Journal ArticleDOI
TL;DR: Investigating ciliopathies has helped to understand the molecular mechanisms by which the cilium-associated basal body functions in early ciliogenesis, as well as how the transition zone functions in ciliary gating, and how intraflagellar transport enables cargo trafficking and signalling.
Abstract: Motile and non-motile (primary) cilia are nearly ubiquitous cellular organelles. The dysfunction of cilia causes diseases known as ciliopathies. The number of reported ciliopathies (currently 35) is increasing, as is the number of established (187) and candidate (241) ciliopathy-associated genes. The characterization of ciliopathy-associated proteins and phenotypes has improved our knowledge of ciliary functions. In particular, investigating ciliopathies has helped us to understand the molecular mechanisms by which the cilium-associated basal body functions in early ciliogenesis, as well as how the transition zone functions in ciliary gating, and how intraflagellar transport enables cargo trafficking and signalling. Both basic biological and clinical studies are uncovering novel ciliopathies and the ciliary proteins involved. The assignment of these proteins to different ciliary structures, processes and ciliopathy subclasses (first order and second order) provides insights into how this versatile organelle is built, compartmentalized and functions in diverse ways that are essential for human health.

1,017 citations

Journal ArticleDOI
25 Jun 2010-Cell
TL;DR: It is shown that macroautophagy requires the Alzheimer's disease-related protein presenilin-1 (PS1) for v-ATPase targeting to lysosomes, lysOSome acidification, and proteolysis during autophagy, which represents a basis for pathogenic protein accumulations and neuronal cell death in AD.

1,015 citations

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Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms.

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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.