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

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.

(1999) J. Biol. Chem. 274, 27529 -27535). In the present investigation, we define a second pathway contributing to CREB-dependent up-regulation of Bcl-2 expression as a novel anti-apoptotic function of Akt signaling. To examine the role of Akt on Bcl-2 expression, a series of transient transfections using a luciferase reporter gene driven by the promoter region of Bcl-2 containing a CRE were carried out. Pharmacological inhibition of phosphatidylinositol (PI) 3-kinase, the upstream kinase of Akt, with LY294002 led to a 45% decrease in Bcl-2 promoter activity. The reporter activity was enhanced 2.3-fold by overexpression of active p110 subunit of PI 3-kinase and inhibited 44% by the dominant negative p85 subunit of PI 3-kinase. Cotransfection with 3-phosphoinositide-dependent kinase (PDK1), which is required for the full activation of Akt, resulted in enhanced luciferase activity. Insulin-like growth factor-I-mediated induction of Bcl-2 promoter activity was decreased significantly (p < 0.01) by the dominant negative forms of p85 subunit of PI 3-kinase, PDK1, and Akt. These data indicate that regulation of Bcl-2 expression by IGF-I involves a signaling cascade mediated by PI 3-kinase/PDK1/Akt/CREB. Furthermore, we measured the Bcl-2 mRNA in PC12 cells overexpressing Akt by real-time quantitative reverse transcriptionpolymerase chain reaction using the TaqMan TM fluorogenic probe system. We observed a 2.1-fold increase in Bcl-2 mRNA levels in the Akt cell line compared with control PC12 cells, supporting the observation that enhanced CREB activity by Akt signaling leads to increased Bcl-2 promoter activity and cell survival.The serine threonine kinase Akt/protein kinase B is an important mediator of metabolic as well as survival responses to insulin and growth factors (1). Akt is activated by translocation to plasma membrane when the PI 3-kinase-generated 3-phosphoinositides bind to its pleckstrin homology domain (2). For its full activation it needs to be further phosphorylated by 3-phosphoinositide-dependent kinase1 (PDK1) 1 at Thr-308 and by PDK2 at Ser-473. The metabolic actions of insulin mediated by Akt include stimulation of GLUT4 translocation and activation of glycogen synthase and the glycolytic enzyme 6-phosphofructose-

Agents that elevate intracellular cyclic AMP (cAMP) levels promote neuronal survival in a manner independent of neurotrophic factors. Inhibitors of phosphatidylinositol 3 kinase and dominant-inactive mutants of the protein kinase Akt do not block the survival effects of cAMP, suggesting that another signaling pathway is involved. In this report, we demonstrate that elevation of intracellular cAMP levels in rat cerebellar granule neurons leads to phosphorylation and inhibition of glycogen synthase kinase 3␤ (GSK-3␤). The increased phosphorylation of GSK-3␤ by protein kinase A (PKA) occurs at serine 9, the same site phosphorylated by Akt. Purified PKA is able to phosphorylate recombinant GSK-3␤ in vitro. Inhibitors of GSK-3 block apoptosis in these neurons, and transfection of neurons with a GSK-3␤ mutant that cannot be phosphorylated interferes with the prosurvival effects of cAMP. These data suggest that activated PKA directly phosphorylates GSK-3␤ and inhibits its apoptotic activity in neurons.Neurons require continuous exposure to extracellular trophic factors for survival, and those that fail to receive sufficient trophic factor support undergo apoptotic cell death (34). Among the extracellular factors shown to influence neuronal survival are the neurotrophins, which include nerve growth factor, brain-derived neurotrophic factor, neurotrophin 3 and neurotrophin 4, the fibroblast growth factors, ciliary growth factor, insulin, and insulin-like growth factors (2, 31). Agents that elevate intracellular cyclic AMP (cAMP) also promote neuronal survival in a manner independent of neurotrophic factors (24, 38). Substantial progress has been made over the last several years in delineating signal transduction pathways that mediate trophic factor-induced cell survival. Less is known about the survival pathways activated by cAMP in neurons.Recent reports have established a key role for phosphatidylinositol (PI)-3 kinase in regulating trophic factor-dependent survival of neurons (18,22,23). The Akt protein kinase (also termed proteins kinase B [PKB] and Rac) has been implicated as the transducer of PI-3 kinase-dependent survival signals generated by serum and certain growth factors (6,22,28). In response to PI-3 kinase activation, Akt binds to phosphorylated membrane lipids via its pleckstrin homology domain and is phosphorylated at threonine 308 and serine 473 (17). Phosphorylation of Akt at these two sites leads to its activation and the propagation of an antiapoptotic signal. Several downstream targets of Akt implicated in cell survival include the Bcl-2 family member BAD (13), caspase 9 (8), and FKHRL1, a member of the Forkhead family of transcription factors (5). Another Akt substrate recently implicated in cell fate decisions is glycogen synthase kinase 3 (GSK-3). Mammalian GSK-3 exists as two isoforms termed ␣ (51 kDa) and ␤ (47 kDa), each encoded by a distinct gene (45-47). The GSK-3 isoforms share 85% homology at the amino acid level and are ubiquitously expressed (45-47). Although GSK-3 was originally identifie...