Abstract
Exosomes and other extracellular vesicles (EVs) participate in cell-cell communication. Herein, we isolated EVs from human plasma and demonstrated that these EVs activate cell signaling and promote neurite outgrowth in PC-12 cells. Analysis of human plasma EVs purified by sequential ultracentrifugation using tandem mass spectrometry indicated the presence of multiple plasma proteins, including α2-macroglobulin, which is reported to regulate PC-12 cell physiology. We therefore further purified EVs by molecular exclusion or phosphatidylserine affinity chromatography, which reduced plasma protein contamination. EVs subjected to these additional purification methods exhibited unchanged activity in PC-12 cells, even though α2-macroglobulin was reduced to undetectable levels. Nonpathogenic cellular prion protein (PrPC) was carried by human plasma EVs and essential for the effects of EVs on PC-12 cells, as EV-induced cell signaling and neurite outgrowth were blocked by the PrPC-specific antibody, POM2. In addition, inhibitors of the N-methyl-d-aspartate (NMDA) receptor (NMDA-R) and low-density lipoprotein receptor-related protein-1 (LRP1) blocked the effects of plasma EVs on PC-12 cells, as did silencing of Lrp1 or the gene encoding the GluN1 NMDA-R subunit (Grin1). These results implicate the NMDA-R-LRP1 complex as the receptor system responsible for mediating the effects of EV-associated PrPC. Finally, EVs harvested from rat astrocytes carried PrPC and replicated the effects of human plasma EVs on PC-12 cell signaling. We conclude that interaction of EV-associated PrPC with the NMDA-R-LRP1 complex in target cells represents a novel mechanism by which EVs may participate in intercellular communication in the nervous system.