Paracrine diffusion of PrP(C) and propagation of prion infectivity by plasma membrane-derived microvesicles

Abstract

Cellular prion protein (PrP(C)) is a physiological constituent of eukaryotic cells. The cellular pathways underlying prions spread from the sites of prions infection/peripheral replication to the central nervous system are still not elucidated. Membrane-derived microvesicles (MVs) are submicron (0.1-1 microm) particles, that are released by cells during plasma membrane shedding processes. They are usually liberated from different cell types, mainly upon activation as well as apoptosis, in this case, one of their hallmarks is the exposure of phosphatidylserine in the outer leaflet of the membrane. MVs are also characterized by the presence of adhesion molecules, MHC I molecules, as well as of membrane antigens typical of their cell of origin. Evidence exists that MVs shedding provide vehicles to transfer molecules among cells, and that MVs are important modulators of cell-to-cell communication. In this study we therefore analyzed the potential role of membrane-derived MVs in the mechanism(s) of PrP(C) diffusion and prion infectivity transmission. We first identified PrP(C) in association with the lipid raft components Fyn, flotillin-2, GM1 and GM3 in MVs from plasma of healthy human donors. Similar findings were found in MVs from cell culture supernatants of murine neuronal cells. Furthermore we demonstrated that PrP(Sc) is released from infected murine neuronal cells in association with plasma membrane-derived MVs and that PrP(Sc)-bearing MVs are infectious both in vitro and in vivo. The data suggest that MVs may contribute both to the intercellular mechanism(s) of PrP(C) diffusion and signaling as well as to the process of prion spread and neuroinvasion.

Figure 1. PrP^C detection in MVs from human plasma.

MVs obtained from human plasma were analyzed for the presence of PrP^C. A: Western blot analysis with anti-PrP monoclonal 6H4 antibody of MVs compared with human peripheral blood (PB). B: Cytofluorimetric analysis with an anti-PrP (C-20 PE). MVs: MVs gated on the basis of forward and side scatter parameters using polystirene beads 0.8 µm as standard. Anti-PrP: MVs stained by anti-PrP PE (C-20 PE) vs IgG PE with irrelevant specificity. Anti-LBPA: MVs stained by anti-LBPA, followed by PE-conjugated anti-mouse IgG. Anti-MHC-I: MVs stained by anti-human MHC I vs IgG PE with irrelevant specificity.

Figure 2. Evidence for raft components in MVs from human plasma.

MVs obtained from human plasma were subjected to ganglioside extraction and analyzed by TLC immunostaining. A: reactivity of anti-GM3, followed by peroxidase-conjugated anti-mouse IgM MoAb; B: reactivity of HRP-CTxB. MVs obtained from human plasma were analyzed by Western blot using the anti-Fyn (FYN3) (C) and anti-flotillin-2 (D). Inset: electron microscopy negative staining of MVs obtained from human plasma.

Figure 3. Co-immunoprecipitation of PrP^C with gangliosides and p59 Fyn Kinase.

MVs from human plasma were immunoprecipitated with anti-PrP (C-20). The immunoprecipitates were subjected to ganglioside extraction and analyzed by TLC immunostaining or Western Blot with polyclonal anti-Fyn antibody (FYN3). A: reactivity of anti-GM3 (MoAb GMR6) with standard GM3, PrP^C immunoprecipitate and immunoprecipitate with IgG with irrelevant specificity. B: reactivity of CTxB perox with standard GM1, PrP^C immunoprecipitate and immunoprecipitate with IgG with irrelevant specificity. C: Western blot analysis with anti-Fyn (FYN3) of human peripheral blood, PrP^C immunoprecipitate and immunoprecipitate with IgG with irrelevant specificity.

Figure 4. Evidence for the presence of PrP^C in MVs from neuronal cells.

A MVs obtained from the Neuro-2a cells were analyzed by Western blot using anti-PrP SAF-32. Crude brain homogenate from wild type mice was included as positive control. B Left: electron microscopy negative staining of MVs obtained from Neuro-2a PK1 cells. Right: two representative examples of anti-PrP^C specific staining on the surface of MVs obtained from Neuro-2a PK1 cells. PrP^C was revealed by immunogold labelling (10 nm) and indicated on the images by the black arrows.

Figure 5. Evidence for raft components in MVs from neuronal cells.

A MVs from Neuro-2a cells were subjected to ganglioside extraction and analyzed by TLC immunostaining. a: reactivity of anti-GM2 (GMR6 MoAb) with standard GM2. MVs obtained from the Neuro-2a cells were analyzed by Western blot using different antibodies. b: reactivity with anti-Flotillin-2; c: reactivity with anti-GAP-43. Crude brain homogenate from wild type mice was included as positive control. B Co-immunoprecipitation of PrP^C and gangliosides. MVs from Neuro-2a cells were immunoprecipitated with anti-PrP (C-20). The immunoprecipitates were subjected to ganglioside extraction and analyzed by TLC immunostaining: reactivity of anti-GM2 (GMR6 MoAb) with standard GM2, PrP^C immunoprecipitate and immunoprecipitate with IgG with irrelevant specificity. The immunoprecipitate was revealed as PrP^C, as detected by Western blot, using the anti-PrP monoclonal 6H4 antibody.

Figure 6. Prion infected neuronal cells shed PrP^Sc bearing MVs.

Left panel: Detection of PrP^Sc in MVs isolated from culture supernatants of prion uninfected and infected Neuro-2a PK1 cells. Twenty µg total proteins of isolated MVs were PK digested. PrP^Sc was specifically detected in MVs released by the infected but not from the uninfected N2a PK1 cells. Right panel: Detection of PrP^Sc in infected Neuro-2a PK1 cells. Two hundred and fifty µg total proteins of cell lysates were PK digested. The presence of PK-resistant PrP^Sc was assessed with increasing amounts of proteinase K and detected by the use of the 6H4 antibody. The positions of the molecular weight standards (in kilodaltons) are indicated.

Figure 7. In vitro transmission of prion infectivity by PrP^Sc-bearing MVs.

Cell blot assay detecting de novo infected N2a PK1 cells at serial culture passages. N2a PK1 cells were incubated with MVs isolated from uninfected (Mvs^Uninf) or from infected (MVs^Inf) N2a PK1 cells. Mock and Chandler infections were also included as negative and positive control, respectively. A time-dependent increase of PrP^Sc levels was detected after proteinase K treatment (+PK).