Exosomes act as molecular vehicles contributing to cellular cholesterol efflux

Abstract

Exosomes are small vesicles of 50-100 nm diameter that derive from intraluminal vesicles (ILVs) and are secreted into the extracellular environment upon fusion of multiversicular bodies (MVBs) with the plasma membrane. Release of exosomes is reported for a variety of cell types and serves numerous functions including the shuttle of RNA, proteins and lipids for cell-to-cell communication, antigen presentation, and disposal of obsolete or superuous molecules. Exosomes display a cell type-specific lipid composition, for example those released by B lymphocytes are enriched in cholesterol. Since a tight regulation of cholesterol homeostasis is indispensable for the structural and functional integrity of each cell, we asked whether exosomes contribute to this process by facilitating cholesterol egress. This study shows that generation and release of exosomes is regulated by cellular cholesterol levels. While exosome release was decreased after methyl-beta-cyclodextrin-mediated cholesterol depletion of oligodendroglial cells, loading of cholesterol led to its increase. It is further demonstrated that flotillin-2, a membrane associated protein enriched in exosomes, is impaired in its exosomal secretion after mutating its putative cholesterol recognition/interaction amino acid consensus (CRAC) sequences. SiRNA-mediated down-regulation of flotillin-2 significantly reduced exosomal cholesterol release, highlighting flotillin's importance for cholesterol trafficking. Translocation of cholesterol from late endosomes/lysosomes to the ER and Golgi compartments has previously been shown to depend on NPC1 and NPC2 proteins. In Niemann- Pick type C disease (NPC), loss-of-function mutations of these proteins result in late endosomal/lysosomal accumulation of unesterified cholesterol and sphingolipids, clinically leading to progressive demyelination, neurodegeneration and eventually to death. Here, it is shown that oligodendroglial cells upregulate their exosomal cholesterol secretion upon induction of NPC-like late endosomal/lysosomal cholesterol trapping either by administration of U18666A or by siRNA-mediated down-regulation of NPC1. A comparable upregulation was seen in primary fibroblasts of NPC1 patients and in NPC1-mutant Chinese hamster ovary cells, where transient expression of wild-type NPC1 was able to reverse the phenotype. These findings indicate that exosomes may serve as molecular vehicles partially bypassing the cholesterol traffic block in NPC1 that causes pathological accumulation of cholesterol in late endosomal/lysosomal compartments. Moreover, they indicate a role for exosome secretion in contributing to the maintenance of cellular cholesterol homeostasis.