Guidelines for the use and interpretation of assays for monitoring autophagy

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure flux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation, it is imperative to target by gene knockout or RNA interference more than one autophagy-related protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular assays, we hope to encourage technical innovation in the field.

Research in autophagy continues to accelerate,(1) and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.(2,3) There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi). Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes. This set of guidelines is not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to verify an autophagic response.

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.

New horizon in autophagy research

Defective autophagy and Alzheimer's disease: is calcium the key?

In eukaryotic cells, excess or damaged cytoplasmic constituents are targeted into lytic compartments via autophagosomal membrane trafficking. Biogenesis of autophagosomes in fungi, metazoans, and plants relies on the conserved ATG (autophagy related) proteins. The machinery responsible for autophagosome turnover has been elucidated in yeast and metazoans, but not in plants. Here we examined 14 soluble N-ethylmaleimide-sensitive-factor attachment protein receptors (SNAREs) in Arabidopsis thaliana by autophagy marker and genetic analyses. We identified SYP22 (Syntaxin of Plants 22) as a SNARE that is necessary for the efficient fusion of autophagosomes with the vacuole. Genetic disruption of SYP22 led to a reduction in autophagic flux and the accumulation of autophagosomes. The vacuolar Qa-SNARE SYP22 interacted with autophagosomal proteins, such as ATG8 and the R-SNARE VAMP724. Overall, our molecular and genetic analyses of Arabidopsis SNAREs underscore the importance of autophagosome-vacuole fusion in autophagic flux, and provide an insight into how plant vacuolar SNARE proteins recognize the autophagosome and mediate its fusion. As a unique mutant defective in the turnover of autophagosomes, syp22 will be useful for overcoming bottlenecks in plant autophagy research. Abbreviations: AIM: Atg8-family interacting motif; ATG: autophagy related; BiFC: bimolecular fluorescence complementation; co-IP: co-immunoprecipitation; ConA: concanamycin A; DMSO: dimethyl sulfoxide; ER: endoplasmic reticulum; HOPS: homotypic fusion and protein sorting; LE: late endosome; PM: plasma membrane; PVC: prevacuolar compartment; SNARE: soluble N-ethylmaleimide-sensitive-factor attachment protein receptor; SYP: Syntaxin of Plants; TEM: transmission electron microscopy; TGN: trans-Golgi network; WT: wild type.

The power of forward genetics in yeast is the foundation on which the field of autophagy research firmly stands. Complementary work on autophagy in higher eukaryotes has revealed both the deep conservation of this process, as well as novel mechanisms by which autophagy is regulated in the context of development, immunity, and neuronal homeostasis. The recent emergence of new clustered regularly interspaced palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9)-based technologies has begun facilitating efforts to define novel autophagy factors and pathways by forward genetic screening in mammalian cells. Here, we set out to develop an expanded toolkit of autophagy reporters amenable to CRISPR/Cas9 screening. Genome-wide screening of our reporters in mammalian cells recovered virtually all known autophagy-related (ATG) factors as well as previously uncharacterized factors, including vacuolar protein sorting 37 homolog A (VPS37A), transmembrane protein 251 (TMEM251), amyotrophic lateral sclerosis 2 (ALS2), and TMEM41B. To validate this data set, we used quantitative microscopy and biochemical analyses to show that 1 novel hit, TMEM41B, is required for phagophore maturation. TMEM41B is an integral endoplasmic reticulum (ER) membrane protein distantly related to the established autophagy factor vacuole membrane protein 1 (VMP1), and our data show that these two factors play related, albeit not fully overlapping, roles in autophagosome biogenesis. In sum, our work uncovers new ATG factors, reveals a malleable network of autophagy receptor genetic interactions, and provides a valuable resource (http://crispr.deniclab.com) for further mining of novel autophagy mechanisms.

Autophagy is a critical mechanism by which methamphetamine (METH) induces neuronal damage and neurotoxicity. Prolonged METH exposure can result in the accumulation of autophagosomes within cells. The autophagy process encompasses several essential vesicle-related biological steps, collectively referred to as the autophagic flux. However, the precise mechanisms by which METH modulates the autophagic flux and the underlying pathways remain to be elucidated. In this study, we utilized a chronic METH exposure mouse model and cell model to demonstrate that METH treatment leads to an increase in p62 and LC3B-II and the accumulation of autophagosomes in striatal neurons and SH-SY5Y cells. To assess autophagic flux, this study utilized autophagy inhibitors and inducers. The results demonstrated that the lysosomal inhibitor chloroquine exacerbated autophagosome accumulation; however, blocking autophagosome formation with 3-methyladenine did not prevent METH-induced autophagosome accumulation. Compared to the autophagy activator rapamycin, METH significantly reduced autophagosome-lysosome fusion, leading to autophagosome accumulation. Rab7a is a critical regulator of autophagosome-lysosome fusion. Although Rab7a expression was upregulated in SH-SY5Y cells and brain tissues after METH treatment, immunoprecipitation experiments revealed weakened interactions between Rab7a and the lysosomal protein RILP. Overexpression of active Rab7a (Rab7a Q67L) significantly alleviated the METH-induced upregulation of LC3-II and p62. PTEN, a key regulator of Rab7a dephosphorylation, was downregulated following METH treatment, resulting in decreased Rab7a dephosphorylation and reduced Rab7a activity, thereby contributing to autophagosome accumulation. We further investigated the role of neuronal exosomes in the autophagy process. Our results demonstrated that the miRNA expression profiles in exosomes released by METH-induced SH-SY5Y cells were significantly altered, with 122 miRNAs upregulated and 151 miRNAs downregulated. KEGG and GO enrichment analyses of these differentially expressed miRNAs and their target genes revealed significant associations with the autophagy pathway and potential regulation of PTEN expression. Our experiments confirmed that METH-induced exosomes reduced PTEN expression levels and decreased Rab7a dephosphorylation, thereby exacerbating autophagic flux impairment and autophagosome accumulation. In conclusion, our study indicated that METH and its induced neuronal exosomes downregulate PTEN expression, leading to reduced Rab7a dephosphorylation. This, in turn, hinders the fusion of autophagosomes and lysosomes, ultimately resulting in autophagic flux impairment and neuronal damage.

In the conserved autophagy pathway, autophagosomes (APs) engulf cellular components and deliver them to the lysosome for degradation. Before fusing with the lysosome, APs have to close via an unknown mechanism. We have previously shown that the endocytic Rab5-GTPase regulates AP closure. Therefore, we asked whether ESCRT, which catalyzes scission of vesicles into late endosomes, mediates the topologically similar process of AP sealing. Here, we show that depletion of representative subunits from all ESCRT complexes causes late autophagy defects and accumulation of APs. Focusing on two subunits, we show that Snf7 and the Vps4 ATPase localize to APs and their depletion results in accumulation of open APs. Moreover, Snf7 and Vps4 proteins complement their corresponding mutant defects in vivo and in vitro. Finally, a Rab5-controlled Atg17-Snf7 interaction is important for Snf7 localization to APs. Thus, we unravel a mechanism in which a Rab5-dependent Atg17-Snf7 interaction leads to recruitment of ESCRT to open APs where ESCRT catalyzes AP closure.

During the last decade, autophagy has been pointed out as a central process in cellular homeostasis with the consequent implication in most cellular settings and human diseases pathology. At present, there is significant data available about molecular mechanisms that regulate autophagy. Nevertheless, autophagy pathway itself and its importance in different cellular aspects are still not completely clear. In this article, we are focused in four main aspects: (a) Induction of Autophagy: Autophagy is an evolutionarily conserved mechanism induced by nutrient starvation or lack of growth factors. In higher eukaryotes, autophagy is a cell response to stress which starts as a consequence of organelle damage, such as oxidative species and other stress conditions. (b) Initiation of Autophagy; The two major actors in this signaling process are mTOR and AMPK. These multitasking protein complexes are capable to summarize the whole environmental, nutritional, and energetic status of the cell and promote the autophagy induction by means of the ULK1-Complex, that is the first member in the autophagy initiation. (c) ULK1-Complex: This is a highly regulated complex responsible for the initiation of autophagosome formation. We review the post-transductional modifications of this complex, considering the targets of ULK1. (d)The mechanisms involved in autophagosome formation. In this section we discuss the main events that lead to the initial structures in autophagy. The BECN1-Complex with PI3K activity and the proper recognition of PI3P are one of these. Also, the transmembrane proteins, such as VMP1 and ATG9, are critically involved. The membrane origin and the cellular localization of autophagosome biogenesis will be also considered. Hence, in this article we present an overview of the current knowledge of the molecular mechanisms involved in the initial steps of mammalian cell autophagosome biogenesis.

Autophagy is the chief machinery for bulk degradation of superfluous or aberrant cytoplasmic components. This study used the rat moderate fluid percussion injury model to investigate whether the autophagy pathway plays a key role after traumatic brain injury (TBI). Induction of autophagy is manifested by accumulation of autophagosomes (APs), observable under transmission electron microscopy (EM). Two hallmarks of autophagy, i.e., the microtubule-associated protein light chain 3 (LC3)-II and the autophagy-related gene (ATG)12-ATG5 conjugates, were explored by biochemical and confocal microscopic analyses of brain tissues. Under EM, both APs and autolysosomes were markedly accumulated in neurons from 4 h onward after TBI. Western blot analysis showed that ATG12-ATG5 conjugate was markedly redistributed during 5 to 15 days in brain tissues after TBI. LC3-II conjugate was initially unchanged but was drastically upregulated from 24 h onward in the pre-AP-containing fraction after TBI. LC-3 immunostaining was mainly located in living neurons under confocal microscopy. These results clearly show that the autophagy pathway is persistently activated after TBI. Because the autophagy pathway is the chief machinery for bulk elimination of aberrant cell components, we propose that activation of this pathway serves as a protective mechanism for maintaining cellular homeostasis after TBI.

Mutations in LRRK2 cause a dominantly inherited form of Parkinson’s disease (PD) and are the most common known genetic determinant of PD. Inhibitor-based therapies targeting LRRK2 have emerged as a key therapeutic strategy in PD; thus, understanding the consequences of inhibiting the normal cellular functions of this protein is vital. Despite much interest, the physiological functions of LRRK2 remain unclear. Several recent studies have linked the toxicity caused by overexpression of pathogenic mutant forms of LRRK2 to defects in the endolysosomal and autophagy pathways, raising the question of whether endogenous LRRK2 might play a role in these processes. Here, we report the characterization of multiple novel ethyl methanesulfonate (EMS)-induced nonsense alleles in the Drosophila LRRK2 homolog, lrrk. Using these alleles, we show that lrrk loss-of-function causes striking defects in the endolysosomal and autophagy pathways, including the accumulation of markedly enlarged lysosomes that are laden with undigested contents, consistent with a defect in lysosomal degradation. lrrk loss-of-function also results in the accumulation of autophagosomes, as well as the presence of enlarged early endosomes laden with mono-ubiquitylated cargo proteins, suggesting an additional defect in lysosomal substrate delivery. Interestingly, the lysosomal abnormalities in these lrrk mutants can be suppressed by a constitutively active form of the small GTPase rab9, which promotes retromer-dependent recycling from late endosomes to the Golgi. Collectively, our data provides compelling evidence of a vital role for lrrk in lysosomal function and endolysosomal membrane transport in vivo, and suggests a link between lrrk and retromer-mediated endosomal recycling.

Improving autophagy-lysosome fusion has been considered a key method in the treatment of Alzheimer's disease (AD). Cornel iridoid glycoside (CIG) is extracted from Cornus officinalis and has been shown to promote the clearance of tau oligomers via the autophagy pathway. However, the mechanisms of CIG on autophagy deficits are not understood. Here, we found autophagy deficit and tau aggregation in the brains of P301S tau transgenic mice and MAPT cells edited using CRISPR-Cas9 technology. CIG decreased tau aggregation and alleviated autophagic markers involving the JNK/Beclin-1 signaling pathway which demonstrated CIG that might enhance lysosome formation by upregulating ATPase Vps4A expression. Knocking down VPS4A increased autophagosome accumulation and attenuated the effect of CIG on p62. In addition, CIG had no effect on tau oligomers but still inhibited the level of tau monomer in VPS4A knockout cells. The effective component (Sweroside, SWE) of CIG attenuated tau oligomers accumulation and increased Vps4A level but not CHMP2B. SWE could not change the level of tau oligomers in VPS4A knockout cells. In conclusion, CIG suppressed autophagosome accumulation by regulating the ATPase Vps4A/JNK. SWE is a core of active factors of CIG in Vps4A regulation. These findings suggest CIG may be a potential drug in AD treatment.

Autophagy is a regulated pathway for bulk degradation of cytoplasmic contents and organelles, an important process involved in many physiological and pathological conditions in multiple organs, including the nervous system. It has been proposed that developing autophagosomes fuse with late endosomal compartments before their fusion with lysosomes; however, little is known about the functional relationship between the autophagy and endocytic pathways. In the endosomal-lysosomal pathway, a key step in sorting transmembrane cargo proteins is regulated by multimeric complexes called ESCRT (endosomal sorting complex required for transport). We recently reported that dysfunction of ESCRT-III, either by depletion of its essential subunit mSnf7-2 or by expression of a mutant CHMP2B protein associated with frontotemporal dementia linked to chromosome 3 (FTD3), caused autophagosome accumulation and dendritic retraction before neurodegeneration in cultured mature cortical neurons. This defect is likely a result of an abnormal fusion process between autophagosomes and endosomal compartments or lysosomes. This study suggests that defects in the late steps of the autophagy pathway may contribute to the pathogenesis of FTD and potentially other neurodegenerative diseases.Addendum to: Lee J-A, Beigneux A, Ahmad ST, Young SG, Gao F-B. ESCRT-III dysfunction causes autophagosome accumulation and neurodegeneration. Curr Biol 2007; 17:1561-67.

Gas6 or Mer deficiency ameliorates silica-induced autophagosomes accumulation in mice lung