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.
Highlights
Introduction exhibited unchanged activity in PC12 cells, evenExtracellular vesicles (EVs) are produced by dithough α2M was reduced to undetectable levels
Non-pathogenic cellular prion protein (PrPC) was verse cells and include exosomes, which form by inward budding of multivesicular bodies in the carried by human plasma extracellular vesicles (EVs) and was essential endosomal transport pathway, microvesicles that for the effects of EVs on PC-12 cells, as EV-inshed from the cell surface, and membrane blebs duced cell-signaling and neurite outgrowth were blocked by the PrPC-specific antibody, POM2
We previously showed that a recombinant protein (S-PrP), corresponding closely in sequence to a form of PrPC released from cell surfaces by ADAM10 [20], activates cell-signaling and promotes neurite outgrowth in PC-12 and N2a cells by engaging a cell-signaling receptor assembly that includes low-density lipoprotein receptorrelated protein-1 (LRP1) and the NMDA Receptor (NMDA-R) [21]
Summary
NTA and bicinchoninic acid (BCA) protein assays were performed to compare UC, SEC, and P-AC EVs from human plasma. In cells in which Lrp or Grin was silenced, the response to UC EVs was blocked, as was the response to S-PrP and α2M (Fig. 4C, D), confirming that the NMDA-R/LRP1 system mediates ERK1/2 activation in PC-12 cells treated with UC EVs. Mattei et al [48] reported that tPA-initiated cell-signaling requires target cell PrPC. The activity of α2M was not inhibited by POM1 or POM2, despite the apparent requirement for PrPC as an NMDAR/LRP1 co-receptor for α2M, identified in Prnp gene-silencing studies (see Fig. 4E) These results suggest that, in experiments with plasma EVs, POM2 targets EV-associated PrPC and not PC-12 cell PrPC.
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