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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TL;DR: It is shown that SUMO deletion in a humanized mouse HD model depletes mHTT and prevents brain shrinkage and behavioral abnormalities, and that blocking SUMO may ameliorate HD pathogenesis by regulating autophagy activities.
Abstract: Significance SUMO protein can decorate other proteins via a process called SUMOylation that can regulate toxicity of proteins linked to neurodegenerative diseases. The mutant huntingtin (mHTT) protein in Huntington disease (HD) degenerates nerve cells, and SUMOylation of mHTT makes it more soluble and more toxic to the nerve cells. Here, we show that SUMO deletion in a humanized mouse HD model depletes mHTT and prevents brain shrinkage and behavioral abnormalities. SUMO deletion blocked inflammation and enhanced autophagy, a beneficial cellular degradation pathway. Importantly, ginkgolic acid (GA), a widely used plant supplement that can inhibit SUMOylation, activates autophagy and promotes the degradation of mHTT in human HD cells. Thus, our study indicates GA and analogs might be therapeutically beneficial to HD patients. The CAG expansion of huntingtin (mHTT) associated with Huntington disease (HD) is a ubiquitously expressed gene, yet it prominently damages the striatum and cortex, followed by widespread peripheral defects as the disease progresses. However, the underlying mechanisms of neuronal vulnerability are unclear. Previous studies have shown that SUMO1 (small ubiquitin-like modifier-1) modification of mHtt promotes cellular toxicity, but the in vivo role and functions of SUMO1 in HD pathogenesis are unclear. Here, we report that SUMO1 deletion in Q175DN HD-het knockin mice (HD mice) prevented age-dependent HD-like motor and neurological impairments and suppressed the striatal atrophy and inflammatory response. SUMO1 deletion caused a drastic reduction in soluble mHtt levels and nuclear and extracellular mHtt inclusions while increasing cytoplasmic mHtt inclusions in the striatum of HD mice. SUMO1 deletion promoted autophagic activity, characterized by augmented interactions between mHtt inclusions and a lysosomal marker (LAMP1), increased LC3B- and LAMP1 interaction, and decreased interaction of sequestosome-1 (p62) and LAMP1 in DARPP-32–positive medium spiny neurons in HD mice. Depletion of SUMO1 in an HD cell model also diminished the mHtt levels and enhanced autophagy flux. In addition, the SUMOylation inhibitor ginkgolic acid strongly enhanced autophagy and diminished mHTT levels in human HD fibroblasts. These results indicate that SUMO is a critical therapeutic target in HD and that blocking SUMO may ameliorate HD pathogenesis by regulating autophagy activities.
13 citations
TL;DR: In this article, the authors discuss the importance of lipophagy in the most common central nervous system diseases and examine the latest evidence for the reported interplay between abnormalities in lipid accumulation and autophagy.
Abstract: Lipophagy is the process of selective degradation of lipid droplets (LDs) by autophagy. Several studies have highlighted the importance of lipophagy in regulating cellular lipid levels in various tissues and disease conditions. In recent years, disruption of autophagy and accumulation of LDs have been reported as pathological hallmarks in several neurodegenerative and neuroinflammatory diseases, raising the question whether lipophagy is a process that is important in the progression of these disorders. This supports the growing interest in lipid metabolism as a major player in neurodegeneration, and the emerging understanding of several neurological pathologies as not only proteinopathies but also lipidopathies. In this review we discuss the importance of lipophagy in the most common central nervous system diseases. We examine the latest evidence for the reported interplay between abnormalities in lipid accumulation and autophagy, and propose lipophagy as a potentially important mechanism in neurodegeneration.
13 citations
TL;DR: In this paper, the authors summarize the up-to-date knowledge regarding natural products that can modulate autophagy in various cancers, which will provide a new position to exploit more natural compounds as potential novel anticancer drugs and will lead to a better understanding of molecular pathways by targeting the various autoophagy stages of upcoming cancer therapeutics.
Abstract: Macro-autophagy (autophagy) is a highly conserved eukaryotic intracellular process of self-digestion caused by lysosomes on demand, which is upregulated as a survival strategy upon exposure to various stressors, such as metabolic insults, cytotoxic drugs, and alcohol abuse. Paradoxically, autophagy dysfunction also contributes to cancer and aging. It is well known that regulating autophagy by targeting specific regulatory molecules in its machinery can modulate multiple disease processes. Therefore, autophagy represents a significant pharmacological target for drug development and therapeutic interventions in various diseases, including cancers. According to the framework of autophagy, the suppression or induction of autophagy can exert therapeutic properties through the promotion of cell death or cell survival, which are the two main events targeted by cancer therapies. Remarkably, natural products have attracted attention in the anticancer drug discovery field, because they are biologically friendly and have potential therapeutic effects. In this review, we summarize the up-to-date knowledge regarding natural products that can modulate autophagy in various cancers. These findings will provide a new position to exploit more natural compounds as potential novel anticancer drugs and will lead to a better understanding of molecular pathways by targeting the various autophagy stages of upcoming cancer therapeutics.
13 citations
TL;DR: In this article , the authors show that 1,2-dioleoyl-sn-glycero-3-phospho-(1'-myo-inositol) [PI(18:1/18):1] is a SCD1-derived signaling lipid, which inhibits p38 mitogen-activated protein kinase activation, counteracts UPR, endoplasmic reticulum-associated protein degradation, and apoptosis, regulates autophagy, and maintains cell morphology and proliferation.
Abstract: Cytotoxic stress activates stress-activated kinases, initiates adaptive mechanisms, including the unfolded protein response (UPR) and autophagy, and induces programmed cell death. Fatty acid unsaturation, controlled by stearoyl-CoA desaturase (SCD)1, prevents cytotoxic stress but the mechanisms are diffuse. Here, we show that 1,2-dioleoyl-sn-glycero-3-phospho-(1'-myo-inositol) [PI(18:1/18:1)] is a SCD1-derived signaling lipid, which inhibits p38 mitogen-activated protein kinase activation, counteracts UPR, endoplasmic reticulum-associated protein degradation, and apoptosis, regulates autophagy, and maintains cell morphology and proliferation. SCD1 expression and the cellular PI(18:1/18:1) proportion decrease during the onset of cell death, thereby repressing protein phosphatase 2 A and enhancing stress signaling. This counter-regulation applies to mechanistically diverse death-inducing conditions and is found in multiple human and mouse cell lines and tissues of Scd1-defective mice. PI(18:1/18:1) ratios reflect stress tolerance in tumorigenesis, chemoresistance, infection, high-fat diet, and immune aging. Together, PI(18:1/18:1) is a lipokine that links fatty acid unsaturation with stress responses, and its depletion evokes stress signaling.
13 citations
TL;DR: It is found that LINC01207 was overexpressed in OSCC cells and tissues and promotes OSCC progression via the miR-1301-3p/LDHA axis.
Abstract: Long noncoding RNAs (lncRNAs) have been reported to participate in the progression of various cancers, including oral squamous cell carcinoma (OSCC). This study aims to find out whether lncRNA LINC01207 regulates the progression of OSCC. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was conducted to evaluate gene expression in OSCC cells and tissues. Cell viability, proliferation, migration, apoptosis, and autophagy were detected using Cell Counting Kit-8 (CCK-8), colony formation, Transwell assays, flow cytometry, and western blot analysis. Luciferase reporter and RNA immunoprecipitation (RIP) assays were conducted to assess the interactions among genes. We found that LINC01207 was overexpressed in OSCC cells and tissues. LINC01207 silencing inhibited OSCC cell proliferation and migration but promoted apoptosis and autophagy, and LINC01207 overexpression had an opposite result. LINC01207 interacted with microRNA-1301-3p (miR-1301-3p) while lactate dehydrogenase isoform A (LHDA) was targeted by miR1301-3p. Effects caused by LINC01207 downregulation on OSCC cells were reversed by overexpression of LDHA. Overall, LINC01207 promotes OSCC progression via the miR-1301-3p/LDHA axis.
13 citations
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TL;DR: A new criterion for triggering the extension of word hits, combined with a new heuristic for generating gapped alignments, yields a gapped BLAST program that runs at approximately three times the speed of the original.
Abstract: The BLAST programs are widely used tools for searching protein and DNA databases for sequence similarities. For protein comparisons, a variety of definitional, algorithmic and statistical refinements described here permits the execution time of the BLAST programs to be decreased substantially while enhancing their sensitivity to weak similarities. A new criterion for triggering the extension of word hits, combined with a new heuristic for generating gapped alignments, yields a gapped BLAST program that runs at approximately three times the speed of the original. In addition, a method is introduced for automatically combining statistically significant alignments produced by BLAST into a position-specific score matrix, and searching the database using this matrix. The resulting Position-Specific Iterated BLAST (PSIBLAST) program runs at approximately the same speed per iteration as gapped BLAST, but in many cases is much more sensitive to weak but biologically relevant sequence similarities. PSI-BLAST is used to uncover several new and interesting members of the BRCT superfamily.
70,111 citations
TL;DR: This paper identified the small molecule ferrostatin-1 as a potent inhibitor of ferroptosis in cancer cells and glutamate-induced cell death in organotypic rat brain slices, suggesting similarities between these two processes.
7,192 citations
TL;DR: It is demonstrated that the rat microtubule‐associated protein 1 light chain 3 (LC3), a homologue of Apg8p essential for autophagy in yeast, is associated to the autophagosome membranes after processing.
Abstract: Little is known about the protein constituents of autophagosome membranes in mammalian cells. Here we demonstrate that the rat microtubule-associated protein 1 light chain 3 (LC3), a homologue of Apg8p essential for autophagy in yeast, is associated to the autophagosome membranes after processing. Two forms of LC3, called LC3-I and -II, were produced post-translationally in various cells. LC3-I is cytosolic, whereas LC3-II is membrane bound. The autophagic vacuole fraction prepared from starved rat liver was enriched with LC3-II. Immunoelectron microscopy on LC3 revealed specific labelling of autophagosome membranes in addition to the cytoplasmic labelling. LC3-II was present both inside and outside of autophagosomes. Mutational analyses suggest that LC3-I is formed by the removal of the C-terminal 22 amino acids from newly synthesized LC3, followed by the conversion of a fraction of LC3-I into LC3-II. The amount of LC3-II is correlated with the extent of autophagosome formation. LC3-II is the first mammalian protein identified that specifically associates with autophagosome membranes.
6,244 citations
Clotilde Théry1, Kenneth W. Witwer2, Elena Aikawa3, María José Alcaraz4 +414 more•Institutions (209)
TL;DR: The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities, and a checklist is provided with summaries of key points.
Abstract: The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines for the field in 2014. We now update these “MISEV2014” guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points.
5,988 citations
TL;DR: A molecular mechanism for regulation of the mammalian autophagy-initiating kinase Ulk1, a homologue of yeast ATG1, is demonstrated and a signalling mechanism for UlK1 regulation and autophagic induction in response to nutrient signalling is revealed.
Abstract: Autophagy is a process by which components of the cell are degraded to maintain essential activity and viability in response to nutrient limitation. Extensive genetic studies have shown that the yeast ATG1 kinase has an essential role in autophagy induction. Furthermore, autophagy is promoted by AMP activated protein kinase (AMPK), which is a key energy sensor and regulates cellular metabolism to maintain energy homeostasis. Conversely, autophagy is inhibited by the mammalian target of rapamycin (mTOR), a central cell-growth regulator that integrates growth factor and nutrient signals. Here we demonstrate a molecular mechanism for regulation of the mammalian autophagy-initiating kinase Ulk1, a homologue of yeast ATG1. Under glucose starvation, AMPK promotes autophagy by directly activating Ulk1 through phosphorylation of Ser 317 and Ser 777. Under nutrient sufficiency, high mTOR activity prevents Ulk1 activation by phosphorylating Ulk1 Ser 757 and disrupting the interaction between Ulk1 and AMPK. This coordinated phosphorylation is important for Ulk1 in autophagy induction. Our study has revealed a signalling mechanism for Ulk1 regulation and autophagy induction in response to nutrient signalling.
5,314 citations