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. 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 vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased 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. 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. 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 autophagy assays, we hope to encourage technical innovation in the field.     

Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes

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.     

Methods for monitoring autophagy

Autophagy is an intracellular bulk degradation system that is found ubiquitously in eukaryotes. Autophagy is responsible for the degradation of most long-lived proteins and some organelles. Cytoplasmic constituents, including organelles, are sequestered into double-membraned autophagosomes, which subsequently fuse with lysosomes where their contents are degraded. This system has been implicated in various physiological processes including protein and organelle turnover, the starvation response, cellular differentiation, cell death, and pathogenesis. However, methods for monitoring autophagy have been very limited and unsatisfactory. The most standard method is conventional electron microscopy. In addition, some biochemical methods have been utilized to measure autophagic activity. Recently, the molecular basis of autophagosome formation has been extensively studied using yeast cells; these studies have provided useful marker proteins for autophagosomes. Importantly, most of these proteins are conserved in mammals. Using these probes, we can now specifically monitor autophagic activity.     

脑缺血后N-乙基马来酰亚胺敏感因子ATP酶失活致神经元自噬流障碍的病理机制

缺血性脑卒中是由脑血管梗塞引起的急性脑血管病,具有较高的发病率、致残率和致死率。研究发现,过度自噬或自噬不足均可导致细胞损伤。自噬包括自噬体的形成和成熟、自噬体与溶酶体融合、自噬底物在自噬溶酶体内的降解和清除,这些过程呈连续状态则称为自噬流。研究发现,脑缺血可导致自噬体与溶酶体间发生融合障碍,从而引发自噬流损伤。细胞内膜融合由3种核心组分介导,即N-乙基马来酰亚胺敏感因子（N-ethylmaleimide sensitive factor,NSF） ATP酶、可溶性NSF黏附蛋白（soluble NSF attachment protein,SNAP）及可溶性NSF黏附蛋白受体（soluble NSF attachment protein receptors,SNAREs）。当SNAREs介导自噬体与溶酶体融合后以非活性的复合体形式存留于自噬溶酶体膜,须被NSF再激活为单体后方可发挥新一轮的膜融合介导作用,而NSF是唯一可再激活SNAREs的ATP酶。新近研究表明,脑缺血可显著抑制NSF ATP酶活性,导致其对SNAREs再激活减少,这可能是自噬体与溶酶体间发生融合障碍并导致神经元自噬...     

SNAPIN is critical for lysosomal acidification and autophagosome maturation in macrophages

We previously observed that SNAPIN, which is an adaptor protein in the SNARE core complex, was highly expressed in rheumatoid arthritis synovial tissue macrophages, but its role in macrophages and autoimmunity is unknown. To identify SNAPIN's role in these cells, we employed siRNA to silence the expression of SNAPIN in primary human macrophages. Silencing SNAPIN resulted in swollen lysosomes with impaired CTSD (cathepsin D) activation, although total CTSD was not reduced. Neither endosome cargo delivery nor lysosomal fusion with endosomes or autophagosomes was inhibited following the forced silencing of SNAPIN. The acidification of lysosomes and accumulation of autolysosomes in SNAPIN-silenced cells was inhibited, resulting in incomplete lysosomal hydrolysis and impaired macroautophagy/autophagy flux. Mechanistic studies employing ratiometric color fluorescence on living cells demonstrated that the reduction of SNAPIN resulted in a modest reduction of H⁺ pump activity; however, the more critical mechanism was a lysosomal proton leak. Overall, our results demonstrate that SNAPIN is critical in the maintenance of healthy lysosomes and autophagy through its role in lysosome acidification and autophagosome maturation in macrophages largely through preventing proton leak. These observations suggest an important role for SNAPIN and autophagy in the homeostasis of macrophages, particularly long-lived tissue resident macrophages.     

基于自噬溶酶体形成机制调控缺血性脑卒中后神经元自噬流

脑卒中是由脑血管阻塞或出血引发的急性脑血管病，约84%的临床脑卒中患者由脑缺血引起。研究表明，自噬广泛参与并显著影响脑卒中病理生理进程。自噬是一个将陈旧蛋白质、损伤细胞器及多余胞质组分等呈递给溶酶体进行降解的代谢过程，其包括自噬的激活、自噬体的形成和成熟、自噬体与溶酶体融合、自噬产物在自噬溶酶体内消化和降解等过程。自噬流通常被定义为自噬/溶酶体信号机制。最近发现，自噬流障碍是导致缺血性脑卒中后神经元损伤的重要原因，而在自噬过程中任一步骤发生障碍均可导致自噬流损伤。本文重点对自噬体-溶酶体融合的机制，以及该机制在缺血性脑卒中后发生障碍的致病机理进行详细阐述，以期基于自噬体-溶酶体融合机制对神经元自噬流进行调节，进而诱导缺血性脑卒中后的神经保护。本文可为脑卒中病理机制研究指明方向，为脑卒中治疗探寻新的线索。     

CUP-5, the C. elegans ortholog of the mammalian lysosomal channel protein MLN1/TRPML1, is required for proteolytic degradation in autolysosomes

The process of macroautophagy (herein referred to as autophagy) involves the formation of a closed double-membrane structure, called the autophagosome, and its subsequent fusion with lysosomes to form an autolysosome. Lysosomes are regenerated from autolysosomes after degradation of the sequestrated materials. In this study, we showed that mutations in cup-5, encoding the C. elegans Mucolipin 1 homolog, cause defects in the autophagy pathway. In cup-5 mutants, a variety of autophagy substrates accumulate in enlarged vacuoles that display characteristics of late endosomes and lysosomes, indicating defective proteolytic degradation in autolysosomes. We further revealed that lysosomes in coelomocytes (scavenger cells located in the body cavity) are smaller in size and more numerous in mutants with loss of autophagy activity. Furthermore, the enlarged vacuole accumulation abnormality and embryonic lethality of cup-5 mutants are partially suppressed by reduced autophagy activity. Our results indicate that the basal constitutive level of autophagy activity regulates the size and number of lysosomes and provides insights into the molecular mechanisms underlying mucolipidosis type IV disease.     

The Anticancer Agent Di-2-pyridylketone 4,4-Dimethyl-3-thiosemicarbazone (Dp44mT) Overcomes Prosurvival Autophagy by Two Mechanisms: PERSISTENT INDUCTION OF AUTOPHAGOSOME SYNTHESIS AND IMPAIRMENT OF LYSOSOMAL INTEGRITY

Autophagy functions as a survival mechanism during cellular stress and contributes to resistance against anticancer agents. The selective antitumor and antimetastatic chelator di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT) causes lysosomal membrane permeabilization and cell death. Considering the integral role of lysosomes in autophagy and cell death, it was important to assess the effect of Dp44mT on autophagy to further understand its mechanism of action. Notably, Dp44mT affected autophagy by two mechanisms. First, concurrent with its antiproliferative activity, Dp44mT increased the expression of the classical autophagic marker LC3-II as a result of induced autophagosome synthesis. Second, this effect was supplemented by a reduction in autophagosome degradation as shown by the accumulation of the autophagic substrate and receptor p62. Conversely, the classical iron chelator desferrioxamine induced autophagosome accumulation only by inhibiting autophagosome degradation. The formation of redox-active iron or copper Dp44mT complexes was critical for its dual effect on autophagy. The cytoprotective antioxidant N-acetylcysteine inhibited Dp44mT-induced autophagosome synthesis and p62 accumulation. Importantly, Dp44mT inhibited autophagosome degradation via lysosomal disruption. This effect prevented the fusion of lysosomes with autophagosomes to form autolysosomes, which is crucial for the completion of the autophagic process. The antiproliferative activity of Dp44mT was suppressed by Beclin1 and ATG5 silencing, indicating the role of persistent autophagosome synthesis in Dp44mT-induced cell death. These studies demonstrate that Dp44mT can overcome the prosurvival activity of autophagy in cancer cells by utilizing this process to potentiate cell death.     

Monitoring autophagic flux by an improved tandem fluorescent-tagged LC3 (mTagRFP-mWasabi-LC3) reveals that high-dose rapamycin impairs autophagic flux in cancer cells

Monitoring autophagic flux is important for the analysis of autophagy. Tandem fluorescent-tagged LC3 (mRFP-EGFP-LC3) is a convenient assay for monitoring autophagic flux based on different pH stability of EGFP and mRFP fluorescent proteins. However, it has been reported that there is still weak fluorescence of EGFP in acidic environments (pH between 4 and 5) or acidic lysosomes. So it is possible that autolysosomes are labeled with yellow signals (GFP⁺RFP⁺ puncta), which results in misinterpreting autophagic flux results. Therefore, it is desirable to choose a monomeric green fluorescent protein that is more acid sensitive than EGFP in the assay of autophagic flux. Here, we report on an mTagRFP-mWasabi-LC3 reporter, in which mWasabi is more acid sensitive than EGFP and has no fluorescence in acidic lysosomes. Meanwhile, mTagRFP-mWasabi-LC3ΔG was constructed as the negative control for this assay. Compared with mRFP-EGFP-LC3, our results showed that this reporter is more sensitive and accurate in detecting the accumulation of autophagosomes and autolysosomes. Using this reporter, we find that high-dose rapamycin (30 μM) will impair autophagic flux, inducing many more autophagosomes than autolysosomes in HeLa cells, while low-dose rapamycin (500 nM) has an opposite effect. In addition, other chemical autophagy inducers (cisplatin, staurosporine and Z18) also elicit much more autophagosomes at high doses than those at low doses. Our results suggest that the dosage of chemical autophagy inducers would obviously influence autophagic flux in cells.     

A mammalian autophagosome maturation mechanism mediated by TECPR1 and the Atg12-Atg5 conjugate

Autophagy is a major catabolic pathway in eukaryotes associated with a broad spectrum of human diseases. In autophagy, autophagosomes carrying cellular cargoes fuse with lysosomes for degradation. However, the molecular mechanism underlying autophagosome maturation is largely unknown. Here we report that TECPR1 binds to the Atg12-Atg5 conjugate and phosphatidylinositol 3-phosphate (PtdIns[3]P) to promote autophagosome-lysosome fusion. TECPR1 and Atg16 form mutually exclusive complexes with the Atg12-Atg5 conjugate, and TECPR1 binds PtdIns(3)P upon association with the Atg12-Atg5 conjugate. Strikingly, TECPR1 localizes to and recruits Atg5 to autolysosome membrane. Consequently, elimination of TECPR1 leads to accumulation of autophagosomes and blocks autophagic degradation of LC3-II and p62. Finally, autophagosome maturation marked by GFP-mRFP-LC3 is defective in TECPR1-deficient cells. Thus, we propose that the concerted interactions among TECPR1, Atg12-Atg5, and PtdIns(3)P provide the fusion specificity between autophagosomes and lysosomes and that the assembly of this complex initiates the autophagosome maturation process.     

Decreased insulin-receptor signaling promotes the autophagic degradation of beta-amyloid peptide in C. elegans

Autophagy is a conserved membrane trafficking pathway that mediates the delivery of cytoplasmic substrates to the lysosome for degradation. Impaired autophagic function is implicated in the pathology of various neurodegenerative diseases. We have generated transgenic C. elegans that express human beta-amyloid peptide (Abeta) in order to examine the mechanism(s) of Abeta-toxicity. In this model, Abeta expression causes autophagosome accumulation, thereby mimicking a pathology found in brains of Alzheimer's disease patients. Furthermore, we demonstrate that decreased insulin-receptor signaling [using the daf-2(e1370) mutation] suppresses Abeta-induced paralysis by a mechanism that requires autophagy. Surprisingly, the daf-2 mutation also decreases Abeta-induced autophagosome accumulation. These observations can be explained by a model in which decreased insulin-receptor signaling promotes the maturation of autophagosomes into degradative autolysosomes, whereas Abeta impairs this process. Consistent with this model, we find that RNAi-mediated knock-down of lysosomal components results in enhanced Abeta-toxicity and autophagosome accumulation. Also, Abeta; daf-2(e1370) nematodes contain more lysosomes than either Abeta or control strains. Finally, we demonstrate that decreased insulin-receptor signaling promotes the autophagic degradation of Abeta.     

CUP-5, the C. elegans ortholog of the mammalian lysosomal channel protein MLN1/TRPML1, is required for proteolytic degradation in autolysosomes

The process of macroautophagy (herein referred to as autophagy) involves the formation of a closed double-membrane structure, called the autophagosome, and its subsequent fusion with lysosomes to form an autolysosome. Lysosomes are regenerated from autolysosomes after degradation of the sequestrated materials. In this study, we showed that mutations in cup-5, encoding the C. elegans Mucolipin 1 homolog, cause defects in the autophagy pathway. In cup-5 mutants, a variety of autophagy substrates accumulate in enlarged vacuoles that display characteristics of late endosomes and lysosomes, indicating defective proteolytic degradation in autolysosomes. We further revealed that lysosomes in coelomocytes (scavenger cells located in the body cavity) are smaller in size and more numerous in mutants with loss of autophagy activity. Furthermore, the enlarged vacuole accumulation abnormality and embryonic lethality of cup-5 mutants are partially suppressed by reduced autophagy activity. Our results indicate that the basal constitutive level of autophagy activity regulates the size and number of lysosomes and provides insights into the molecular mechanisms underlying mucolipidosis type IV disease.     

ATG14 controls SNARE-mediated autophagosome fusion with a lysosome

Autophagosome fusion with a lysosome constitutes the last barrier for autophagic degradation. It is speculated that this fusion process is precisely and tightly regulated. Recent genetic evidence suggests that a set of SNARE proteins, including STX17, SNAP29, and VAMP8, are essential for the fusion between autophagosomes and lysosomes. However, it remains unclear whether these SNAREs are fusion competent and how their fusogenic activity is specifically regulated during autophagy. Using a combination of biochemical, cell biology, and genetic approaches, we demonstrated that fusogenic activity of the autophagic SNARE complex is temporally and spatially controlled by ATG14/Barkor/Atg14L, an essential autophagy-specific regulator of the class III phosphatidylinositol 3-kinase complex (PtdIns3K). ATG14 directly binds to the STX17-SNAP29 binary complex on autophagosomes and promotes STX17-SNAP29-VAMP8-mediated autophagosome fusion with lysosomes. ATG14 homo-oligomerization is required for SNARE binding and fusion promotion, but is dispensable for PtdIns3K stimulation and autophagosome biogenesis. Consequently, ATG14 homo-oligomerization is required for autophagosome fusion with a lysosome, but is dispensable for autophagosome biogenesis. These data support a key role of ATG14 in controlling autophagosome fusion with a lysosome.     

Accumulation of autophagosomes in Semliki Forest virus-infected cells is dependent on expression of the viral glycoproteins

Autophagy is a cellular process that sequesters cargo in double-membraned vesicles termed autophagosomes and delivers this cargo to lysosomes to be degraded. It is enhanced during nutrient starvation to increase the rate of amino acid turnover. Diverse roles for autophagy have been reported for viral infections, including the assembly of viral replication complexes on autophagic membranes and protection of host cells from cell death. Here, we show that autophagosomes accumulate in Semliki Forest virus (SFV)-infected cells. Despite this, disruption of autophagy had no effect on the viral replication rate or formation of viral replication complexes. Also, viral proteins rarely colocalized with autophagosome markers, suggesting that SFV did not utilize autophagic membranes for its replication. Further, we found that SFV infection, unlike nutrient starvation, did not inactivate the constitutive negative regulator of autophagosome formation, mammalian target of rapamycin, suggesting that SFV-dependent accumulation of autophagosomes was not a result of enhanced autophagosome formation. In starved cells, addition of NH(4)Cl, an inhibitor of lysosomal acidification, caused a dramatic accumulation of starvation-induced autophagosomes, while in SFV-infected cells, NH(4)Cl did not further increase levels of autophagosomes. These results suggest that accumulation of autophagosomes in SFV-infected cells is due to an inhibition of autophagosome degradation rather than enhanced rates of autophagosome formation. Finally, we show that the accumulation of autophagosomes in SFV-infected cells is dependent on the expression of the viral glycoprotein spike complex.     

Dissection of autophagy in tobacco BY-2 cells under sucrose starvation conditions using the vacuolar H+-ATPase inhibitor concanamycin A and the autophagy-related protein Atg8

Tobacco BY-2 cells undergo autophagy in sucrose-free culture medium, which is the process mostly responsible for intracellular protein degradation under these conditions. Autophagy was inhibited by the vacuolar H⁺-ATPase inhibitors concanamycin A and bafilomycin A₁, which caused the accumulation of autophagic bodies in the central vacuoles. Such accumulation did not occur in the presence of the autophagy inhibitor 3-methyladenine, and concanamycin in turn inhibited the accumulation of autolysosomes in the presence of the cysteine protease inhibitor E-64c. Electron microscopy revealed not only that the autophagic bodies were accumulated in the central vacuole, but also that autophagosome-like structures were more frequently observed in the cytoplasm in treatments with concanamycin, suggesting that concanamycin affects the morphology of autophagosomes in addition to raising the pH of the central vacuole. Using BY-2 cells that constitutively express a fusion protein of autophagosome marker protein Atg8 and green fluorescent protein (GFP), we observed the appearance of autophagosomes by fluorescence microscopy, which is a reliable morphological marker of autophagy, and the processing of the fusion protein to GFP, which is a biochemical marker of autophagy. Together, these results suggest the involvement of vacuole type H⁺-ATPase in the maturation step of autophagosomes to autolysosomes in the autophagic process of BY-2 cells. The accumulation of autophagic bodies in the central vacuole by concanamycin is a marker of the occurrence of autophagy; however, it does not necessarily mean that the central vacuole is the site of cytoplasm degradation.     

Valosin-containing protein (VCP) is required for autophagy and is disrupted in VCP disease

Mutations in valosin-containing protein (VCP) cause inclusion body myopathy (IBM), Paget''s disease of the bone, and frontotemporal dementia (IBMPFD). Patient muscle has degenerating fibers, rimmed vacuoles (RVs), and sarcoplasmic inclusions containing ubiquitin and TDP-43 (TARDNA-binding protein 43). In this study, we find that IBMPFD muscle also accumulates autophagosome-associated proteins, Map1-LC3 (LC3), and p62/sequestosome, which localize to RVs. To test whether VCP participates in autophagy, we silenced VCP or expressed adenosine triphosphatase–inactive VCP. Under basal conditions, loss of VCP activity results in autophagosome accumulation. After autophagic induction, these autophagosomes fail to mature into autolysosomes and degrade LC3. Similarly, IBMPFD mutant VCP expression in cells and animals leads to the accumulation of nondegradative autophagosomes that coalesce at RVs and fail to degrade aggregated proteins. Interestingly, TDP-43 accumulates in the cytosol upon autophagic inhibition, similar to that seen after IBMPFD mutant expression. These data implicate VCP in autophagy and suggest that impaired autophagy explains the pathology seen in IBMPFD muscle, including TDP-43 accumulation.     

Decreased Insulin-Receptor Signaling Promotes the Autophagic Degradation of β-Amyloid Peptide in C. elegans

Autophagy is a conserved membrane trafficking pathway that mediates the delivery of cytoplasmic substrates to the lysosome for degradation. Impaired autophagic function is implicated in the pathology of various neurodegenerative diseases. We have generated transgenic C. elegans that express human β-amyloid peptide (Aβ) in order to examine the mechanism(s) of Aβ-toxicity. In this model, Aβ expression causes autophagosome accumulation, thereby mimicking a pathology found in brains of Alzheimer’s disease patients. Furthermore, we demonstrate that decreased insulin-receptor signaling [using the daf-2(e1370) mutation] suppresses Aβ-induced paralysis by a mechanism that requires autophagy. Surprisingly, the daf-2 mutation also decreases Aβ-induced autophagosome accumulation. These observations can be explained by a model in which decreased insulin-receptor signaling promotes the maturation of autophagosomes into degradative autolysosomes, whereas Aβ impairs this process. Consistent with this model, we find that RNAi-mediated knock-down of lysosomal components results in enhanced Aβ-toxicity and autophagosome accumulation. Also, Aβ; daf-2(e1370) nematodes contain more lysosomes than either Aβ or control strains. Finally, we demonstrate that decreased insulin-receptor signaling promotes the autophagic degradation of Aβ.     

Constitutive activation of chaperone-mediated autophagy in cells with impaired macroautophagy

Three different types of autophagy-macroautophagy, microautophagy, and chaperone-mediated autophagy (CMA)-contribute to degradation of intracellular components in lysosomes in mammalian cells. Although some level of basal macroautophagy and CMA activities has been described in different cell types and tissues, these two pathways are maximally activated under stress conditions. Activation of these two pathways is often sequential, suggesting the existence of some level of cross-talk between both stress-related autophagic pathways. In this work, we analyze the consequences of blockage of macroautophagy on CMA activity. Using mouse embryonic fibroblasts deficient in Atg5, an autophagy-related protein required for autophagosome formation, we have found that blockage of macroautophagy leads to up-regulation of CMA, even under basal conditions. Interestingly, different mechanisms contribute to the observed changes in CMA-related proteins and the consequent activation of CMA during basal and stress conditions in these macroautophagy-deficient cells. This work supports a direct cross-talk between these two forms of autophagy, and it identifies changes in the lysosomal compartment that underlie the basis for the communication between both autophagic pathways.