Cholesterol regulates mitochondrial raft-like domains during TNF/Fas-mediated hepatocellular apoptosis

Trabajo presentado a la 51st International Conference on the Bioscience of Lipids (ICBL) celebrada en Bilbao del 7 al 11 de septiembre de 2010.Lipid rafts constitute distinctive domains of biological membranes characterized by a specific lipid composition enriched in cholesterol and sphingolipids, which determine their detergent insolubility. These domains are thought to play a critical role in signal transduction and apoptosis, particularly in the plasma membrane by providing a platform for specific interactions of membrane receptors with their corresponding ligands and adaptor molecules. However, during death receptor (Fas/TNF)-mediated apoptosis of lymphocytes mitochondrial raft-like domains have been described and characterized by a multimolecular signaling complex that recruits proapoptotic Bcl-2 family proteins. Since cholesterol is a key component of raft-like domains and its trafficking to mitochondria has been shown to regulate hepatocellular susceptibility to TNF/Fas, the purpose of this study was to examine the role of cholesterol in the regulation of mitochondrial raft-like domains. [Methods]: Hepatic mitochondrial fractions were prepared from models of hepatic cholesterol loading, including cholesterol or alcohol feeding, transgenic Tg-SREBP-2 mice and NPC1 knockout mice. Mitochondrial cholesterol was determined by HPLC, while ganglioside GD3 was analyzed by immunoelectron microscopy and immunoTLC. Detergent soluble and insoluble mitochondrial fractions were processed for Western blotting for caveolin-1, human Fis 1 proteins (hFis1) and VDAC-1. [Results]: hFis1 and VDAC-1 levels in detergent-insoluble fractions were higher in mitochondria from mice fed the hypercholesterolemic diet (MitH) compared to fractions of control mice (MitC). Furthermore, cholesterol enrichment was associated with enhanced GD3 levels as examined by immunostaining and immunoelectron microscopy in MitH fractions. Interestingly, caveolin-1 was present in detergent-insoluble mitochondrial fractions only in MitH but not in MitC samples. Similar findings regarding the association of cholesterol and GD3, and the presence of hFis1, VDAC-1 and caveolin-1 in detergent-insoluble fractions were observed in mitochondria from Tg-SREBP-2 and NPC1 KO mice. In all these models, hepatocytes were highly susceptible to TNF/Fas-mediated apoptosis that was accompanied by increased GD3 levels and recruitment of Bax. [Conclusions]: These findings indicate the existence of raft-like domains in hepatic mitochondria in cholesterol loading models, which may be of significance in steatohepatitis due to the hepatocellular sensitization to TNF/Fas. The findings showing the correlation of mitochondrial caveolin-1 and cholesterol levels, suggest a potential role caveolin-1 in mitochondrial cholesterol downregulationPeer Reviewe

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

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