Stimulating the Release of Exosomes Increases the Intercellular Transfer of Prions

Abstract

Exosomes are small extracellular vesicles released by cells and play important roles in intercellular communication and pathogen transfer. Exosomes have been implicated in several neurodegenerative diseases, including prion disease and Alzheimer disease. Prion disease arises upon misfolding of the normal cellular prion protein, PrP(C), into the disease-associated isoform, PrP(Sc). The disease has a unique transmissible etiology, and exosomes represent a novel and efficient method for prion transmission. The precise mechanism by which prions are transmitted from cell to cell remains to be fully elucidated, although three hypotheses have been proposed: direct cell-cell contact, tunneling nanotubes, and exosomes. Given the reported presence of exosomes in biological fluids and in the lipid and nucleic acid contents of exosomes, these vesicles represent an ideal mechanism for encapsulating prions and potential cofactors to facilitate prion transmission. This study investigates the relationship between exosome release and intercellular prion dissemination. Stimulation of exosome release through treatment with an ionophore, monensin, revealed a corresponding increase in intercellular transfer of prion infectivity. Conversely, inhibition of exosome release using GW4869 to target the neutral sphingomyelinase pathway induced a decrease in intercellular prion transmission. Further examination of the effect of monensin on PrP conversion revealed that monensin also alters the conformational stability of PrP(C), leading to increased generation of proteinase K-resistant prion protein. The findings presented here provide support for a positive relationship between exosome release and intercellular transfer of prion infectivity, highlighting an integral role for exosomes in facilitating the unique transmissible nature of prions.

Keywords: cell biology; exosome (vesicle); exosomes; extracellular vesicles; monensin; neutral sphingomy; neutral sphingomyelinase; prion; prion disease; transwell.

FIGURE 1.

Successful intercellular transmission of prions across transwell membranes. A, schematic depicting the setup for a transwell assay with a non-infected population of cells (recipient, blue) separated from a prion-infected population (donor, red) by a membrane with 0.4-μm pores. B, cell blot assay confirming the presence of PrP^Sc and transmission of prion infection to recipient cells via the transwell assay.

FIGURE 2.

Monensin stimulates exosome release, which is associated with a corresponding increase in intercellular prion transmission. A, AChE quantitation of isolated exosomes, indicating increasing exosome release with MON treatment. B, cell blot analysis revealing increasing levels of PrP^Sc and increasing transfer of prion infectivity in recipient cells after exposure to donor cells treated with increasing amounts of MON in a transwell assay. Data are presented as mean ± S.E. (n = 3). *, p < 0.05; **, p < 0.01.

FIGURE 3.

Monensin similarly stimulates exosome release and prion transmission in neuronal cells. A, increasing concentrations of MON were tested for toxicity to GT1-7 cells, and no toxicity was observed at all concentrations tested. B, Western blotting analysis for the presence of PrP^Tot and PrP^Sc in control- and monensin-treated, prion-infected GT1-7 cell lysates and exosomes (Exo) released from prion-infected GT1-7 cells. The blot confirms that exosomes released from monensin-treated cells contain PrP^Sc. C, AChE assay of isolated exosomes indicating that MON also stimulates exosome release from GT1-7 cells. Cell blot analysis of recipient cells reveals increased levels of PrP^Sc and prion transmission following exposure to prion-infected GT1-7 donor cells in the presence of MON. D, AChE assay of isolated exosomes indicating that treatment with GW4869 decreases exosome release from GT1-7 cells. Cell blot analysis of recipient cells exposed to prion-infected GT1-7 cells in the presence of GW4869 reveals a corresponding decrease in PrP^Sc and prion transmission. Data are presented as mean ± S.E. (n = 3). **, p < 0.01; ***, p < 0.001. DMSO, dimethyl sulfoxide.

FIGURE 4.

Monensin does not induce de novo generation of PrP^Sc but impairs Golgi trafficking of PrP. A, immunoblot analysis of PMCA and −80 °C controls indicating no change in de novo generation of PrP^Sc in the presence of MON. Infected brain homogenates (IBH) are included as a reference point. B, confocal microscopy analysis of PrP^C (green) in non-infected vehicle- and MON-treated GT1-7 cells. Treatment with MON abolishes colocalization of PrP^C with GM130-positive compartments (red), indicating impaired Golgi trafficking. Visualization of cell surface PrP^C revealed unhindered trafficking of PrP^C to the plasma membrane. Scale bars = 20 μm.

FIGURE 5.

Monensin induces long-term accumulation of PrP^Sc. A, immunoblot analysis of PrP in prion-infected GT1-7 cells immediately after vehicle or MON treatment. B, densitometric analysis showed no significant differences in PrP^Tot and PrP^Sc levels. C, immunoblot analysis of PrP in prion-infected GT1-7 cells three passages after vehicle or MON treatment. D, densitometric analysis revealed a significant increase in PrP^Tot and PrP^Sc levels. E, the levels of prion infectivity in vehicle- and MON-treated GT1-7 cells were quantitated using PICA. A dose-response curve of the relative fluorescence units (RFU) of PrP^Sc, as a percentage of the background levels in controls cells, was plotted against the log of inoculum used. No significant differences were observed between the infectious responses associated with vehicle- or MON-treated lysates. Data are presented as mean ± S.E. (n = 3). *, p < 0.05; ns, not significant.