Rapid cell-surface prion protein conversion revealed using a novel cell system

Abstract

Prion diseases are fatal neurodegenerative disorders with unique transmissible properties. The infectious and pathological agent is thought to be a misfolded conformer of the prion protein. Little is known about the initial events in prion infection because the infecting prion source has been immunologically indistinguishable from normal cellular prion protein (PrP(C)). Here we develop a unique cell system in which epitope-tagged PrP(C) is expressed in a PrP knockdown (KD) neuroblastoma cell line. The tagged PrP(C), when expressed in our PrP-KD cells, supports prion replication with the production of bona fide epitope-tagged infectious misfolded PrP (PrP(Sc)). Using this epitope-tagged PrP(Sc), we study the earliest events in cellular prion infection and PrP misfolding. We show that prion infection of cells is extremely rapid occurring within 1 min of prion exposure, and we demonstrate that the plasma membrane is the primary site of prion conversion.

Figure 1. Generation of prion-susceptible cell lines expressing epitope-tagged PrP^C and PrP^Sc.

(a) Western blots developed with anti-PrP and anti-MYC antibodies showing levels of PrP^C in PK1, PrP-KD, PrP-124GlyMYC and PrP-224AlaMYC cell extracts. Actin is used as a loading control. Quantitative densitometry of similar blots that showed PrP expression was reduced by 90–95% in our PrP-KD cells. (b) Merged confocal images of PK1, PrP-KD and PrP-224AlaMYC cells stained with anti-PrP antibodies (green) and counterstained with 6-diamidino-2-phenylindole (blue) are shown; scale bar, 20 μm. (c) Scrapie Cell Assay (SCA) data showing the relative prion susceptibility of cell lines expressing PrP-MYC constructs compared with PrP-KD cells. Cells exposed to RML prions (red bars) or control cells (blue bars) were processed for the SCA. PrP molecules with the MYC tag inserted near the C-terminus at Ala224 support prion propagation; 124GlyMYC cells, uninfected PrP-224AlaMYC cells and infected PrP-KD cells do not contain PrP^Sc. The mean±s.e.m. of six independent experiments are shown. (d) Tg20 mice inoculated intracerebrally with extracts of prion-infected PrP-224AlaMYC cells develop prion disease. Western blots developed with anti-PrP antibodies showing that tg20 mice inoculated with extracts of prion-infected PK1 and PrP-224AlaMYC cells generate PK-resistant PrP (PrP^Sc) with a triplet band pattern similar to that of RML prions. (e) Histological analysis of tg20 mice brains inoculated with extracts of RML prion-infected PK1 cells (iPK1) and PrP-224AlaMYC cells (iPrP-224AlaMYC) revealed classical prion neuropathology with PrP^Sc deposition (ICSM35 immunostaining), neuronal loss and spongiosis (hematoxylin and eosin, H&E staining) and gliosis (GFAP immunostaining). Brains from animals inoculated with extracts of RML prion-infected PrP-KD cells (iPrP-KD) or control uninfected PrP-224AlaMYC cells (PrP-224AlaMYC) did not contain PrP^Sc or show other diagnostic features. Scale bar is 80 μm on the H&E stained sections and 160 μm for all other panels.

Figure 2. Characterization of de novo MYC-tagged PrP^Sc distribution in RML prion-infected cells.

(a) Comparison of the methods used to visualize PrP^Sc in RML prion-infected cells. Uninfected PrP-224AlaMYC cells (top panels) or chronically RML prion-infected PrP-224AlaMYC cells (passage five following prion exposure—bottom panels) were fixed and treated with 98% formic acid, proteinase K (PK) or guanidinium hydrochloride (GdnHCl) before staining with anti-MYC antibodies (green) and counterstaining with 6-diamidino-2-phenylindole (DAPI, blue). Merged confocal images are shown; scale bar, 20 μm. A proportion of the RML prion-infected cells contain formic acid and PK-resistant PrP (PrP^Sc) with a characteristic plasma membrane/perinuclear compartment distribution (arrow). (b) Quantification of randomly chosen cell fields from uninfected PrP-224AlaMYC cells (blue bars) or chronically RML prion-infected PrP-224AlaMYC cells (red bars). Approximately 20% of the cells in cultures exposed to RML prions contain formic acid/PK-resistant PrP (PrP^Sc). The background staining observed in the uninfected PrP-224AlaMYC cells after formic acid and PK treatment is mostly attributable to clumped cells in which access to the PrP^C is restricted. The mean±s.e.m. from four independent experiments are shown. (c) Chronically RML prion-infected PrP-224AlaMYC cells were fixed and treated with 98% formic acid before staining with anti-MYC antibodies (green) and anti-PrP antibodies (red); the cells were counterstained with DAPI (blue). Single channels and merged confocal images are shown as indicated; scale bar, 20 μm.

Figure 3. MYC-tagged PrP^Sc is synthesized rapidly, following RML prion exposure.

(a) PrP-224AlaMYC cells were exposed to RML prions for 72 h, then fixed immediately or washed and cultured in fresh media (without RML prions) for the indicated durations before fixation and formic acid extraction. The proportion of anti-MYC-positive (RML prion infected) cells fixed at the indicated durations was quantified (red). As a control, PrP-KD cells were exposed to RML prions and processed in parallel (blue). The mean±s.e.m. from four independent experiments are shown. (b) PrP-224AlaMYC cells were exposed to RML prions for 0–72 h, then fixed and extracted with formic acid. The proportion of anti-MYC-positive (RML prion infected) cells fixed at the indicated durations was quantified. The mean±s.e.m. from four independent experiments are shown. Before RML prion exposure (0 h), few PrP-positive cells are detected, whereas RML prion exposure causes the rapid build-up of formic acid-resistant PrP (PrP^Sc) detectable as early as 2 h or less. (c) PrP-224AlaMYC cells were exposed to RML prions for 0–32 min, then fixed and extracted with formic acid. The proportion of anti-MYC-positive (prion infected) cells fixed at the indicated durations was quantified. The mean±s.e.m. from four independent experiments are shown; at the 0-, 1- and 2-min time points, the mean±s.e.m. from eight experiments is shown (*P<0.05, **P<0.001, two-tailed t-test). (d) PrP-224AlaMYC cells were exposed to RML prions for 2 min, then fixed and digested with PK. A confocal image stained with anti-MYC antibodies (green) and counterstained with 6-diamidino-2-phenylindole (blue) is shown. PK-resistant PrP (that is, PrP^Sc) was generated after just 2 min of RML prion exposure; scale bar, 20 μm.

Figure 4. Prion conversion first occurs at the cell surface within 1 min of prion exposure.

(a) PrP224-AlaMYC cells were exposed to RML prions for the indicated durations, and then fixed and extracted with formic acid before staining with anti-MYC antibodies (green) and counterstaining with 6-diamidino-2-phenylindole (DAPI, blue). Orthogonal projections (red and green squares) of serial confocal sections are shown alongside one z-section taken from the middle of the cell (as indicated by the guidelines). A cell fixed after a 1-min exposure to RML prions shows PrP^Sc immunostaining only at the cell surface (1 min, white arrow). An adjacent cell in the field that has high levels of plasma membrane PrP^Sc also shows low intracellular levels of PrP^Sc (1 min, yellow arrow). Cells exposed to RML prions for 180 min show the typical steady-state distribution of PrP^Sc, with strong immunostaining at the plasma membrane (white arrows) and in the perinuclear region (yellow arrow −180 min); scale bar, 10 μm. (b) Quantification of cell phenotypes observed at different time points following prion exposure (the mean±s.e.m. from four independent experiments are shown; *P<0.05, **P<0.01, two-tailed t-test. The exact P-values are: membrane 1–2 min, 0.022; membrane 1–4 min, 0.049; membrane/PNC 1–2 min, 0.0049; membrane PNC 1–4 min, 0.0057). (c) PrP-224AlaMYC cells were exposed to RML prions and Texas red-labelled transferrin (red) for the indicated durations, then fixed and extracted with formic acid. Merged confocal images of cells stained with anti-MYC antibodies (green) and counterstained with DAPI (blue) are shown; scale bar, 20 μm. Initially, PrP^Sc shows a plasma membrane distribution (1 min, arrows) and/or diffuse intracellular distribution (2 min, arrows) and then rapidly attains its steady-state distribution concentrated at the plasma membrane and PNC (4 min, white arrow indicates strong PNC stain). Initially, transferrin could be observed in small puncta at the cell periphery, reaching its equilibrium distribution in recycling endosomes at the PNC (yellow arrows) between 8 and 16 min.

Figure 5. PrP^Sc forms at the cell surface.

(a) Pre-cooled PrP-224AlaMYC cells were exposed to RML prions for 2 min on ice, then fixed and extracted with formic acid. Orthogonal reconstructions of serial confocal slices are shown (red and green boxes) alongside one z-slice taken from near the middle of the cells (as indicated by the guide lines). The cells were fixed after 2 min and stained with anti-MYC antibodies (green) and counterstained with 6-diamidino-2-phenylindole (DAPI, blue); scale bar, 20 μm. Formic acid-resistant PrP (PrP^Sc) is formed on the cell surface of one of the cells in the field (arrow), no intracellular PrP^Sc was detected. (b) PrP-224AlaMYC cells were mock-transfected or transfected with RNAi directed at CHC, pretreated with dynasore (80 μM), EIPA (100 μM) or pre-cooled to 4 °C. RML prions were added in the continued presence of the treatments for 180 min and Texas red-labelled transferrin (red) was added for the final 10 min of incubation. The cells were then fixed and extracted with formic acid. Merged confocal images of cells stained with anti-MYC antibodies (green) and counterstained with DAPI (blue) are shown; scale bar, 20 μm. Following prion exposure at 4 °C, MYC-tagged PrP^Sc was detected only at the plasma membrane (arrow). (c) Lysates from PrP-224AlaMYC cells mock-transfected or transfected with RNAi directed at CHC were collected and subjected to SDS–polyacrylamide gel electrophoresis. Immunoblots developed with anti-CHC and anti-actin antibodies are shown. Quantitative densitometry of similar blots showed that CHC expression was reduced by ∼70% in the RNAi-treated cells. (d) PrP-224AlaMYC cells were treated as in b. The percentage of anti-MYC-positive (RML prion infected) cells observed was quantified for each condition. The mean±s.e.m. from three independent experiments are shown (background levels found in uninfected cells for each condition have been subtracted from the mean).

Figure 6. Plasma membrane PrP^C is required for efficient prion infection.

(a) Upper panel: PrP-224AlaMYC cells were treated with PIPLC (0.5 U ml⁻¹) for 2 h before fixation and immunostaining with anti-PrP antibodies (red) and counter staining with 6-diamidino-2-phenylindole (DAPI, blue). Merged confocal images are shown; scale bar, 20 μm. Control cells (−PIPLC) show strong plasma membrane (white arrow) and intracellular (yellow arrow) PrP immunostaining. PIPLC effectively removed surface plasma membrane-bound PrP (+PIPLC), but residual intracellular PrP immunostaining was seen (yellow arrow). Lower panel: Untreated and PIPLC-treated PrP224AlaMYC cells were exposed to RML prions for 180 min, then fixed and extracted with formic acid. Merged confocal images of cells stained with anti-MYC (green) and anti-PrP antibodies (red), then counterstained with DAPI (blue) are shown; scale bar, 20 μm. Formic acid-resistant PrP^Sc derived from the inocula and immunostained with anti-PrP antibodies only (red) can be seen closely associated with all the cells independent of PIPLC treatment (examples are indicated by white arrows). One untreated cell (−PIPLC, yellow arrow) in the field shown has generated de novo MYC-tagged PrP^Sc as indicated by anti-MYC (green) and anti-PrP (red) immunostaining, which appears yellow in the merged image. No cells have generated MYC-tagged PrP^Sc following PIPLC treatment (+PIPLC). (b) PrP-224AlaMYC cells were treated as in Figure 6a lower panel. The percentage of anti-MYC-positive (RML prion infected) cells was quantified for each condition. The mean±s.e.m. from three independent experiments are shown (background levels found in uninfected cells for each condition have been subtracted from the mean). (c) PrP-224AlaMYC cells were pretreated with vehicle (DMSO) or filipin (5 μg ml⁻¹) for 30 min, or U18666A (1 μg ml⁻¹) for 16 h, then exposed to RML prions for 180 min in the continued presence of the vehicle or inhibitor. Cells were then fixed and extracted with formic acid before immunostaining with anti-MYC antibodies. The percentage of anti-MYC-positive (RML prion-infected) cells was quantified. The mean±s.e.m. from three independent experiments are shown (background levels found in uninfected cells for each condition for have been subtracted from the mean).