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: A review of the current understanding of DOX-induced cardiomyopathy (DCM) with a focus on mitochondria, which were increasingly proven to be crucial determinants of DOx-induced cytotoxicity, and the current perspectives to contrast the development of DCM.
Abstract: Cardiotoxicity has emerged as a major side effect of doxorubicin (DOX) treatment, affecting nearly 30% of patients within 5 years after chemotherapy. Heart failure is the first non-cancer cause of death in DOX-treated patients. Although many different molecular mechanisms explaining the cardiac derangements induced by DOX were identified in past decades, the translation to clinical practice has remained elusive to date. This review examines the current understanding of DOX-induced cardiomyopathy (DCM) with a focus on mitochondria, which were increasingly proven to be crucial determinants of DOX-induced cytotoxicity. We discuss DCM pathophysiology and epidemiology and DOX-induced detrimental effects on mitochondrial function, dynamics, biogenesis, and autophagy. Lastly, we review the current perspectives to contrast the development of DCM, which is still a relatively diffused, invalidating, and life-threatening condition for cancer survivors.
16 citations
TL;DR: Wang et al. as mentioned in this paper identified an autophagy-related circular RNA prognostic factor of BCR for patients with Prostate Cancer (PCa) and developed a novel prognostic model with tenfold cross-validation and an L1 penalty.
Abstract: Prostate cancer (PCa) is a common high-incidence malignancy in men, some of whom develop biochemical recurrence (BCR) in the advanced stage. However, there are currently no accurate prognostic indicators of BCR in PCa. The aim of our study was to identify an autophagy-related circular RNA prognostic factor of BCR for patients with PCa. In this study, immunochemistry revealed that the classic autophagy marker MAP1LC3B was positively correlated with Gleason score. Least absolute shrinkage and selector operator regression were conducted to develop a novel prognostic model with tenfold cross-validation and an L1 penalty. Five autophagy-related circRNA signatures were included in the prognostic model. Patients with PCa were ultimately divided into high- and low-risk groups, based on the median risk score. Patients with PCa, who had a high risk score, were more likely to develop BCR in a shorter period of time. Univariate and multivariate Cox regression analyses demonstrated that the risk score was an independent variable for predicting BCR in PCa. In addition, a prognostic nomogram integrated with the risk score and numerous clinicopathological parameters was developed to accurately predict 3- and 5-year BCR of patients with PCa. Finally, the hsa_circ_0001747 signature was selected for further experimental verification in vitro and in vivo, which showed that downregulated hsa_circ_0001747 might facilitate PCa via augmenting autophagy. Our findings indicate that the autophagy-related circRNA signature hsa_circ_0001747 may serve as a promising indicator for BCR prediction in patients with PCa.
16 citations
TL;DR: This study evaluated the effectiveness and safety of XRK3F2 in LICs and demonstrated that mitophagy plays an indispensable role in the survival of LICs during AML development and progression, which can be impaired by blocking p62.
15 citations
TL;DR: In this paper, the mTOR-dependent mitophagy occlusion, along with suppression of mitochondrial fission was documented and quantified by using ultrastructural morphometry.
Abstract: Glioblastoma (GBM) cells feature mitochondrial alterations, which are documented and quantified in the present study, by using ultrastructural morphometry. Mitochondrial impairment, which roughly occurs in half of the organelles, is shown to be related to mTOR overexpression and autophagy suppression. The novelty of the present study consists of detailing an mTOR-dependent mitophagy occlusion, along with suppression of mitochondrial fission. These phenomena contribute to explain the increase in altered mitochondria reported here. Administration of the mTOR inhibitor rapamycin rescues mitochondrial alterations. In detail, rapamycin induces the expression of genes promoting mitophagy (PINK1, PARKIN, ULK1, AMBRA1) and mitochondrial fission (FIS1, DRP1). This occurs along with over-expression of VPS34, an early gene placed upstream in the autophagy pathway. The topographic stoichiometry of proteins coded by these genes within mitochondria indicates that, a remarkable polarization of proteins involved in fission and mitophagy within mitochondria including LC3 takes place. Co-localization of these proteins within mitochondria, persists for weeks following rapamycin, which produces long-lasting mitochondrial plasticity. Thus, rapamycin restores mitochondrial status in GBM cells. These findings add novel evidence about mitochondria and GBM, while fostering a novel therapeutic approach to restore healthy mitochondria through mTOR inhibition.
15 citations
TL;DR: In this paper , the role of the nociceptive nerve as a microenvironmental accomplice of cancer progression in nutrient-poor environments and upon nutrient-starvation therapies was revealed.
15 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