The Role of Cardiolipin as a Scaffold Mitochondrial Phospholipid in Autophagosome Formation: In Vitro Evidence.

Valeria Manganelli,Tina Garofalo,Vincenzo Mattei,Gloria Riitano,Maurizio Sorice,Antonella Capozzi,Roberta Misasi,Serena Recalchi,Agostina Longo

doi:10.3390/biom11020222

Abstract

Cardiolipin (CL) is a hallmark phospholipid localized within the inner mitochondrial membrane. Upon several mitochondrial stress conditions, CL is translocated to specialized platforms, where it may play a role in signaling events to promote mitophagy and apoptosis. Recent studies characterized the molecular composition of MAM-associated lipid microdomains and their implications in regulating the autophagic process. In this study we analyzed the presence of CL within MAMs following autophagic stimulus and the possible implication of raft-like microdomains enriched in CL as a signaling platform in autophagosome formation. Human 2FTGH fibroblasts and SKNB-E-2 cells were stimulated under nutrient deprivation with HBSS. MAM fraction was obtained by an ultracentrifugation procedure and analyzed by HPTLC immunostaining. CL interactions with mitofusin2 (MFN2), calnexin (CANX) and AMBRA1 were analyzed by scanning confocal microscopy and coimmunoprecipitation. The analysis revealed that CL accumulates in MAMs fractions following autophagic stimulus, where it interacts with MFN2 and CANX. It associates with AMBRA1, which in turn interacts with BECN1 and WIPI1. This study demonstrates that CL is present in MAM fractions following autophagy triggering and interacts with the multimolecular complex (AMBRA1/BECN1/WIPI1) involved in autophagosome formation. It may have both structural and functional implications in the pathophysiology of neurodegenerative disease(s).

Highlights

IntroductionThe distribution of CL is mostly confined to the inner mitochondrial membrane (IMM), it distributes in the outer mitochondrial membrane (OMM), in the contact sites formed between the inner and outer membranes, where its content is even higher (about 27% of total phospholipids) [6]
We previously reported that the presence of lipid rafts in the mitochondria-associated membranes (MAMs) could be pivotal in the mitochondria–endoplasmic reticulum (ER) crosstalk leading to autophagosome formation [37]
Since CL may be present in raft-like microdomains at contact sites between inner and outer mitochondrial membranes, we first tested the presence of CL in MAM fractions

Summary

Introduction

The distribution of CL is mostly confined to the IMM, it distributes in the outer mitochondrial membrane (OMM), in the contact sites formed between the inner and outer membranes, where its content is even higher (about 27% of total phospholipids) [6]. These contact sites represent a relevant portion of mitochondrial membrane, since about 115 such sites were calculated in one isolated mitochondrion (ranging from 78 to 270 depending on cell type) [7].

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Conclusion