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

Abstract

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.

cPKCγ‐mediated down‐regulation of UCHL1 alleviates ischaemic neuronal injuries by decreasing autophagy viaERK‐mTOR pathway

“…The kinase mTOR is a considerable regulator of autophagy induction, when regulating mTOR positively can suppress autophagy and inhibiting mTOR can promote it. Rapamycin which directly inhibits mTOR is the most commonly used and specific inducer of autophagy 61. Our results showed that the mTOR phosphorylation level obviously decreased after 1‐hr OGD/24‐hr R, what's more, cPKCγ knockout resulted in more reduction in P‐mTOR.…”

Rotenone Inhibits Autophagic Flux Prior to Inducing Cell Death

“…To further delineate the involvement of macroautophagy in rotenoneinduced neuronal cell death, we assessed levels of autophagic vacuoles via Western blot analysis for MAP-light chain 3-II (LC3-II; Figure 2), an accepted and selective marker of autophagic vacuoles. 3 Treatment with rotenone induced a significant accumulation of autophagic vacuoles at both 6 h ( Figure 2B) and 24 h ( Figure 2C) after treatment. Since the accumulation of autophagic vacuoles may result from either Rotenone caused a significant increase in LC3-II immunoreactivity at both 6 and 24 h following treatment (A−C).…”

“…However, it is not entirely clear if rotenoneinduced accumulation of autophagic vacuoles results from either an increased induction of macroautophagy or from the inhibition of macroautophagy completion, which requires functional fusion of autophagic vacuoles with lysosomes. 3 Such information may be important for designing rational therapeutics that attenuate PD-associated neuronal dysfunction, in part through their maintenance of ALP function. Thus, the goal of this study was to delineate the mechanism by which autophagic vacuoles accumulate in cultured neuronal cells following treatment with an acute, death-inducing concentration of rotenone.…”

Updating the MISEV minimal requirements for extracellular vesicle studies: building bridges to reproducibility

“…We were interested to read a “guidelines to guidelines” recently published by Daniel J. Klionsky [7], noted autophagy researcher and the driving force behind the well-known autophagy guidelines. These guidelines, published in 2008 [8] and updated in 2012 [9] and 2016 [10], now include several thousand authors who reviewed the text and offered changes in a carefully guided manner. Klionsky’s staged writing process and a distributed author invitation plan ensure efficient progress but broad embrace of the global research body.…”