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

Goold R, Rabbanian S, Sutton L, Andre R, Arora P, Moonga J, Clarke AR, Schiavo G, Jat P, Collinge J, Tabrizi SJ - Nat Commun (2011)

Bottom Line: Prion diseases are fatal neurodegenerative disorders with unique transmissible properties.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)).

View Article: PubMed Central - PubMed

Affiliation: Department of Neurodegenerative Disease, Institute of Neurology, University College London, Queen Square, London WC1N 3BG, UK.

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.

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Plasma membrane PrPC 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 PrPSc 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 PrPSc as indicated by anti-MYC (green) and anti-PrP (red) immunostaining, which appears yellow in the merged image. No cells have generated MYC-tagged PrPSc 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).
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f6: Plasma membrane PrPC 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 PrPSc 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 PrPSc as indicated by anti-MYC (green) and anti-PrP (red) immunostaining, which appears yellow in the merged image. No cells have generated MYC-tagged PrPSc 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).

Mentions: An important role for plasma membrane PrPC expression in prion conversion was confirmed by experiments using phosphoinositide-specific phospholipase C (PIPLC) to deplete plasma membrane PrPC. This significantly reduced the generation of MYC-tagged PrPSc (Fig. 6a,b). It is interesting to note that many of the cells in the PrP-224AlaMYC cultures bind and/or internalize inocula-derived PrPSc following RML prion exposure, independent of surface PrPC expression (Fig. 6a). Further, striking examples of this phenomenon are included in Supplementary Figure S7. This suggests that other factors in addition to a simple physical association of cellular PrPC with PrPSc affect prion conversion, an observation in agreement with previously published data293031.


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

Goold R, Rabbanian S, Sutton L, Andre R, Arora P, Moonga J, Clarke AR, Schiavo G, Jat P, Collinge J, Tabrizi SJ - Nat Commun (2011)

Plasma membrane PrPC 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 PrPSc 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 PrPSc as indicated by anti-MYC (green) and anti-PrP (red) immunostaining, which appears yellow in the merged image. No cells have generated MYC-tagged PrPSc 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).
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f6: Plasma membrane PrPC 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 PrPSc 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 PrPSc as indicated by anti-MYC (green) and anti-PrP (red) immunostaining, which appears yellow in the merged image. No cells have generated MYC-tagged PrPSc 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).
Mentions: An important role for plasma membrane PrPC expression in prion conversion was confirmed by experiments using phosphoinositide-specific phospholipase C (PIPLC) to deplete plasma membrane PrPC. This significantly reduced the generation of MYC-tagged PrPSc (Fig. 6a,b). It is interesting to note that many of the cells in the PrP-224AlaMYC cultures bind and/or internalize inocula-derived PrPSc following RML prion exposure, independent of surface PrPC expression (Fig. 6a). Further, striking examples of this phenomenon are included in Supplementary Figure S7. This suggests that other factors in addition to a simple physical association of cellular PrPC with PrPSc affect prion conversion, an observation in agreement with previously published data293031.

Bottom Line: Prion diseases are fatal neurodegenerative disorders with unique transmissible properties.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)).

View Article: PubMed Central - PubMed

Affiliation: Department of Neurodegenerative Disease, Institute of Neurology, University College London, Queen Square, London WC1N 3BG, UK.

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.

Show MeSH
Related in: MedlinePlus