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.

Both dauer formation (a stage of developmental arrest) and adult life-span in Caenorhabditis elegans are negatively regulated by insulin-like signaling, but little is known about cellular pathways that mediate these processes. Autophagy, through the sequestration and delivery of cargo to the lysosomes, is the major route for degrading long-lived proteins and cytoplasmic organelles in eukaryotic cells. Using nematodes with a loss-of-function mutation in the insulin-like signaling pathway, we show that bec-1, the C. elegans ortholog of the yeast and mammalian autophagy gene APG6/VPS30/beclin1, is essential for normal dauer morphogenesis and life-span extension. Dauer formation is associated with increased autophagy and also requires C. elegans orthologs of the yeast autophagy genes APG1, APG7, APG8, and AUT10. Thus, autophagy is a cellular pathway essential for dauer development and life-span extension in C. elegans.

Autophagy is postulated to play a role in antiviral innate immunity. However, it is unknown whether viral evasion of autophagy is important in disease pathogenesis. Here we show that the herpes simplex virus type 1 (HSV-1)-encoded neurovirulence protein ICP34.5 binds to the mammalian autophagy protein Beclin 1 and inhibits its autophagy function. A mutant HSV-1 virus lacking the Beclin 1-binding domain of ICP34.5 fails to inhibit autophagy in neurons and demonstrates impaired ability to cause lethal encephalitis in mice. The neurovirulence of this Beclin 1-binding mutant virus is restored in pkr(-/-) mice. Thus, ICP34.5-mediated antagonism of the autophagy function of Beclin 1 is essential for viral neurovirulence, and the antiviral signaling molecule PKR lies genetically upstream of Beclin 1 in host defense against HSV-1. Our findings suggest that autophagy inhibition is a novel molecular mechanism by which viruses evade innate immunity and cause fatal disease.

The plant innate immune response includes the hypersensitive response (HR), a form of programmed cell death (PCD). PCD must be restricted to infection sites to prevent the HR from playing a pathologic rather than protective role. Here we show that plant BECLIN 1, an ortholog of the yeast and mammalian autophagy gene ATG6/VPS30/beclin 1, functions to restrict HR PCD to infection sites. Initiation of HR PCD is normal in BECLIN 1-deficient plants, but remarkably, healthy uninfected tissue adjacent to HR lesions and leaves distal to the inoculated leaf undergo unrestricted PCD. In the HR PCD response, autophagy is induced in both pathogen-infected cells and distal uninfected cells; this is reduced in BECLIN 1-deficient plants. The restriction of HR PCD also requires orthologs of other autophagy-related genes including PI3K/VPS34, ATG3, and ATG7. Thus, the evolutionarily conserved autophagy pathway plays an essential role in plant innate immunity and negatively regulates PCD.

The eIF2␣ kinases are a family of evolutionarily conserved serine͞ threonine kinases that regulate stress-induced translational arrest. Here, we demonstrate that the yeast eIF2␣ kinase, GCN2, the target phosphorylation site of Gcn2p, Ser-51 of eIF2␣, and the eIF2␣-regulated transcriptional transactivator, GCN4, are essential for another fundamental stress response, starvation-induced autophagy. The mammalian IFN-inducible eIF2␣ kinase, PKR, rescues starvationinduced autophagy in GCN2-disrupted yeast, and pkr null and Ser-51 nonphosphorylatable mutant eIF2␣ murine embryonic fibroblasts are defective in autophagy triggered by herpes simplex virus infection. Furthermore, PKR and eIF2␣ Ser-51-dependent autophagy is antagonized by the herpes simplex virus neurovirulence protein, ICP34.5. Thus, autophagy is a novel evolutionarily conserved function of the eIF2␣ kinase pathway that is targeted by viral virulence gene products. During nutrient starvation and viral infection, cells need a strategy to synthesize essential proteins in the face of a limited supply caused either by environmental depletion or intracellular parasitism. This strategy must involve not only a mechanism for strict translational regulation, but also for generating new pools of amino acids from existing proteins. Accordingly, in nutrientlimiting conditions, eukaryotic cells simultaneously decrease overall protein synthesis and increase rates of protein degradation by an autophagic pathway (1), a process involving the bulk degradation of cellular contents by autophagolysosomes (2, 3). It is not known whether stress-induced translational repression and stress-induced autophagy are regulated by a common or by genetically distinct pathways.The phosphorylation of eukaryotic initiation factor-2-␣ (eIF2␣) at Ser-51 by a conserved family of eIF2␣ protein kinases is a central mechanism in stress-induced translation regulation (4-6), but the mechanisms of stress-induced regulation of autophagy are not well understood (7). Autophagy is required for survival during amino acid starvation of eukaryotic cells (2, 3), and several yeast and mammalian APG and AUT genes have been identified that are essential for autophagy (8-13). At least in yeast, these genes act downstream of the autophagy-inhibitory target of rapamycin (TOR) signaling pathway (14). However, little is known about upstream cellular genes that are essential for initiating the process of autophagy. Furthermore, it is not known whether stimuli other than amino acid starvation such as viral infection, endoplasmic reticulum stress, or heme depletion, which trigger stress-induced translational arrest, also trigger stress-induced autophagy.We developed the hypotheses that eIF2␣ kinases, well characterized regulators of stress-induced translational control programs, are also involved in the regulation of stress-induced autophagy, and that specific stress stimuli that activate eIF2␣ kinase-dependent translational arrest also activate eIF2␣ kinase-dependent autophagy. In yeast, there is only one known eIF2␣ k...

Altered degradation of α-synuclein (α-syn) has been implicated in the pathogenesis of Parkinson disease (PD).We have shown that α-syn can be degraded via chaperone-mediated autophagy (CMA), a selective lysosomal mechanism for degradation of cytosolic proteins. Pathogenic mutants of α-syn block lysosomal translocation, impairing their own degradation along with that of other CMA substrates. While pathogenic α-syn mutations are rare, α-syn undergoes posttranslational modifications, which may underlie its accumulation in cytosolic aggregates in most forms of PD. Using mouse ventral medial neuron cultures, SH-SY5Y cells in culture, and isolated mouse lysosomes, we have found that most of these posttranslational modifications of α-syn impair degradation of this protein by CMA but do not affect degradation of other substrates. Dopamine-modified α-syn, however, is not only poorly degraded by CMA but also blocks degradation of other substrates by this pathway. As blockage of CMA increases cellular vulnerability to stressors, we propose that dopamine-induced autophagic inhibition could explain the selective degeneration of PD dopaminergic neurons.

Continuous turnover of intracellular components by autophagy is necessary to preserve cellular homeostasis in all tissues. Alterations in macroautophagy, main responsible for bulk autophagic degradation, have been proposed to contribute to pathogenesis in Huntington’s disease (HD), a genetic neurodegenerative disorder caused by an expanded polyglutamine tract in huntingtin protein. However, the precise mechanism behind macroautophagy malfunctioning in HD is poorly understood. In this work, using cellular and mouse models of HD and cells from HD patients, we have identified a primary defect in the ability of autophagic vacuoles to recognize cytosolic cargo in HD cells. Autophagic vacuoles form at normal or even enhanced rates in HD cells and are adequately eliminated by lysosomes, but they fail to efficiently trap cytosolic cargo in their lumen. We propose that inefficient engulfment of cytosolic components by autophagosomes is responsible for their slower turnover, functional decay and accumulation inside HD cells.

Autophagy functions in antiviral immunity. However, it is not yet known whether endogenous autophagy genes protect against viral disease in vertebrates. Using three different approaches to inactivate the autophagy gene Atg5 in virally-infected neurons, we found that loss of Atg5 function increases mouse susceptibility to lethal Sindbis virus CNS infection. This phenotype is associated with delayed clearance of viral proteins, increased accumulation of the cellular p62 adaptor protein, and increased cell death in neurons, but not with altered levels of CNS viral replication. In vitro, p62 interacts with Sindbis virus capsid protein and genetic knockdown of p62 blocks the targeting of viral capsid to autophagosomes. Moreover, p62 or autophagy gene knockdown increases viral capsid accumulation and accelerates virus-induced cell death without affecting virus replication. These results suggest a novel function for autophagy in mammalian antiviral defense: a cell-autonomous mechanism in which p62 adaptor-mediated autophagic viral protein clearance promotes cell survival.