Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

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

PGC-1α4 GENE EXPRESSION IS SUPPRESSED BY THE IL-6-ERK1/2 MAPK SIGNALING AXIS

Jacob L. Brown1, Megan E. Rosa1, David E. Lee1, Lemuel A. Brown1, Richard A. Perry1, James A. Carson2, Sami Dridi1, Tyrone A. Washington1,2, Nicholas P. Greene1,2 1University of Arkansas, Fayetteville, AR; 2University of South Carolina, Columbia, SC; e-mail: [email protected] Maintenance and promotion of skeletal muscle mass and function are critical for both daily living and exercise performance. Lost muscle mass and function are associated with reduced quality of life in many disease conditions. Recently, the peroxisome proliferator-activated receptor γ coactivator-1α4 (PGC-1α4) isoform has been shown to play a key role in the promotion of skeletal muscle mass; however its specific regulation under normal and stress (inflammatory) conditions is still unknown. PURPOSE: To elicit signaling mechanisms regulating the gene expression of Pgc-1α4 during inflammation and particularly by IL-6. METHODS: Female C57BL/6J (WT) and C57BL/6J × IL-6−/− (IL-6 KO) mice were euthanized and tibialis anterior muscles were excised, snap-frozen in liquid nitrogen and processed for gene expression analysis. To further determine the underlying mechanisms by which IL-6 governs PGC-1α4 expression, C2C12 myotubes were treated with recombinant IL-6 (50 ng/ml), recombinant TNF-α (20 ng/ml), Pyrrolidine dithiocarbamate (PDTC, 50 µM) and PD098059 (20 µM; MEK1/2 inhibitor preventing ERK1/2 activity). RNA was isolated from WT and IL-6 KO mice as well as C2C12 myotubes. RNA was analyzed by real time RT PCR for mRNA content analysis of 18S and Pgc-1α4. Protein analysis was performed by immunoblot for P-STAT3, STAT3, P-ERK 1/2 and ERK 1/2 and normalized to Ponceau S. A student t-test or one way ANOVA was employed as appropriate with α set at 0.05. RESULTS: Pgc-1α4 mRNA content was 10-fold greater in IL-6 KO mice compared to WT. In C2C12 myotubes, IL-6 significantly repressed Pgc-1α4 mRNA content by ~70% while TNF-α and PDTC treated cells were not different from control. In the following experiment, similar IL-6 repression of Pgc-1α4 mRNA was abrogated when IL-6 was combined with ERK1/2 inhibition via MEK (PD098059 + IL6). There was a significant 40% increase in the P-STAT3/STAT3 ratio and the P-ERK/ ERK ratio with IL-6 treatment while inhibitors of STAT3 (PDTC) and ERK 1/2 (PD098059) significantly decreased these ratios by 30% and 50 %, respectively. CONCLUSION: PGC-1α4 expression is repressed by IL-6 through the ERK1/2 signaling axis. These findings may have significant impact in treatment to prevent and ameliorate muscle wasting conditions