Cells release prions in association with exosomes

Abstract

Prion diseases are infectious neurodegenerative disorders linked to the accumulation in the central nervous system of the abnormally folded prion protein (PrP) scrapie (PrPsc), which is thought to be the infectious agent. Once present, PrPsc catalyzes the conversion of naturally occurring cellular PrP (PrPc) to PrPsc. Prion infection is usually initiated in peripheral organs, but the mechanisms involved in infectious spread to the brain are unclear. We found that both PrPc and PrPsc were actively released into the extracellular environment by PrP-expressing cells before and after infection with sheep prions, respectively. Based on Western blot with specific markers, MS, and morphological analysis, our data revealed that PrPc and PrPsc in the medium are associated with exosomes, membranous vesicles that are secreted upon fusion of multivesicular endosomes with the plasma membrane. Furthermore, we found that exosomes bearing PrPsc are infectious. Our data suggest that exosomes may contribute to intercellular membrane exchange and the spread of prions throughout the organism.

Fig. 1.

The cell culture medium of infected cells contains infectious PrP. (A) The culture medium from 5 × 10⁶ infected Rov cells was submitted to differential centrifugation (11). The medium was submitted to a first centrifugation for 5 min at 4,500 × g to remove cells in suspension. The supernatant was ultracentrifuged at 10,000 × g for 30 min to remove cell debris, and, finally, the last supernatant was ultracentrifuged at 100,000 × g for 1 h. The resulting pellets of each centrifugation step were analyzed by Western blot for PrP. (B) The culture medium of 5 × 10⁶ infected Mov cells was analyzed for PrP as in A. (C) After 5 days of culture, the media of 2 × 10⁷ control Mov cells or scrapie-infected Mov cells were harvested and centrifuged for 5 min at 4,500 × g to remove potentially dissociated cells; the supernatant was then recentrifuged at 100,000 × g for 1 h. The pellets were digested with PK and analyzed for the presence of PK-resistant PrPsc by Western blotting with SAF 84. The lysate comprised Mov cells. (D) The medium of 3 × 10⁷ infected Rov cells was centrifuged and analyzed as in C. The lysate comprised Rov cells. (E) Cell culture medium from 3 × 10⁷ infected Rov cells was ultracentrifuged as above. The pellet was resuspended and incubated for 7 days with uninfected Rov cells. Cultures were grown for several weeks and monitored for the accumulation of cell-associated PK-resistant PrP. Total PrP was obtained by methanol precipitation of undigested cell lysates (lanes 1 and 3, 25 μg of proteins). PrP was isolated from PK-digested cell lysates (lanes 2 and 4, 250 μg of proteins). MW indicates molecular mass in all figures.

Fig. 2.

PrPsc released by infected cells is associated with exosome-like vesicles. (A)A continuous 0.25–2.5 M sucrose gradient was loaded on top of the 100,000 × g pellet isolated from Rov-infected cell culture medium (3 × 10⁷ cells) and ultracentrifuged to equilibrium. Fractions were PK-digested and analyzed by Western blotting for PrPsc. In the right lane, a lysate (non-PK-digested) from noninfected cells was probed for PrPc. (B–D and F–H) IEM analysis of membranes that were collected from the culture medium by centrifugation on a sucrose cushion. (B and C) ImmunoGold labeling for PrP before (B) and after (C) guanidium (3 M, 5 min) treatment. (D) Control (Cont.) with irrelevant antibody. (F and G) Single and double IEM for flotillin or flotillin and PrP, respectively. (H) Double IEM for Tsg101 and PrP on permeabilized exosomes. (E) Cell lysates and exosomes collected from the culture media of infected cells by centrifugation on a sucrose cushion were analyzed by Western blotting. IEM and Western blotting for PrP was performed with 8G8. TfR, transferrin receptor. (Scale bar, 100 nm.)

Fig. 4.

PrPc is associated with exosomes. (A) A continuous 0.25–2.5 M sucrose gradient was loaded on top of the 100,000 × g pellet obtained from Mov cell culture medium (4 × 10⁶ cells) and ultracentrifuged to equilibrium. Fractions were analyzed by Western blotting for PrP, Tsg101, Hsc70, and flotillin. (B) Equivalent protein loads of cell lysates (Cell). The 100,000 × g pellet of noninfected (NI) or infected (I) cell culture was fractionated by SDS/PAGE (12%) and stained with Coomassie blue. Some major proteins that were clearly stained and identified by MS (Table 2) are indicated.

Fig. 3.

Noninfected cells release PrPc in association with membrane vesicles. (A) Kinetics of PrP release by Mov cells. Culture media were collected after 1, 3, 5, or 7 days and centrifuged at 4,500 × g for 5 min and subsequently for 60 min at 100,000 × g. Pellets were analyzed by Western blotting for PrP. (B) The PrP content of uninfected Mov cell culture medium was analyzed by Western blot on pellets obtained after differential centrifugation. (C) GM1 content of the same fractions shown in B was analyzed by dot blot with cholera toxin. (D and E) IEM analysis of the 100,000 × g pellet for PrP and GM1. IEM and Western blotting for PrP was done with 8G8. (Scale bar, 200 nm.)

Fig. 5.

PrPc localizes to MVBs. (A and B). Ultrathin cryosections of Rov (A) and Mov (B) cells were ImmunoGold-labeled for PrP (PAG10). PrP localizes to both the limiting membrane and the internal vesicles of MVBs (stars). (C) Ultrathin cryosections of Mov cells were labeled with cholera toxin. Note the presence of GM1 in MVBs (stars), similar to PrP. (D) Before fixation, cells were allowed to internalize BSAG (see Materials and Methods). Exocytic fusion (arrows) is defined by the presence of externalized BSAG associated with the extracellular vesicles labeled for PrP (arrowheads). PM, plasma membrane. (Scale bar, 200 nm.)