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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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Characterization of de novo MYC-tagged PrPSc distribution in RML prion-infected cells.(a) Comparison of the methods used to visualize PrPSc 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 (PrPSc) 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 (PrPSc). 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 PrPC 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.
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f2: Characterization of de novo MYC-tagged PrPSc distribution in RML prion-infected cells.(a) Comparison of the methods used to visualize PrPSc 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 (PrPSc) 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 (PrPSc). 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 PrPC 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.

Mentions: To perform a detailed analysis of prion infection in cultured cells, it is necessary to distinguish newly synthesized PrPSc from the host cellular PrPC. Three established methods achieve this by utilizing the biochemical differences between native PrPC and misfolded PrPSc: These are PK digestion, guanidinium hydrochloride (GdnHCl) denaturation and formic acid extraction2021. We compared each method in detail by immunostaining our PrP-224AlaMYC-expressing cells with anti-MYC antibodies. Both PK and formic acid treatment revealed a population of PK and formic acid-resistant MYC-tagged PrPSc in prion-infected cells (Fig. 2a,b), with a strong signal at the plasma membrane and in the perinuclear compartment (PNC), consistent with previous observations of the sub-cellular distribution of PrPSc (refs 20,22,23). In contrast, only very low levels of background immunostaining were detected in uninfected cells, indicating that PrPC had been effectively removed by these treatments. GdnHCl denaturation was less effective at removing PrPC staining (Fig. 2a,b). Similar results were obtained using ScPK1 cells that express wild-type PrPSc (ref. 20) (Supplementary Fig. S2a,b), indicating that our MYC-tagged PrPSc displays a physiologically relevant cellular localization.


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)

Characterization of de novo MYC-tagged PrPSc distribution in RML prion-infected cells.(a) Comparison of the methods used to visualize PrPSc 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 (PrPSc) 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 (PrPSc). 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 PrPC 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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3104518&req=5

f2: Characterization of de novo MYC-tagged PrPSc distribution in RML prion-infected cells.(a) Comparison of the methods used to visualize PrPSc 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 (PrPSc) 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 (PrPSc). 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 PrPC 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.
Mentions: To perform a detailed analysis of prion infection in cultured cells, it is necessary to distinguish newly synthesized PrPSc from the host cellular PrPC. Three established methods achieve this by utilizing the biochemical differences between native PrPC and misfolded PrPSc: These are PK digestion, guanidinium hydrochloride (GdnHCl) denaturation and formic acid extraction2021. We compared each method in detail by immunostaining our PrP-224AlaMYC-expressing cells with anti-MYC antibodies. Both PK and formic acid treatment revealed a population of PK and formic acid-resistant MYC-tagged PrPSc in prion-infected cells (Fig. 2a,b), with a strong signal at the plasma membrane and in the perinuclear compartment (PNC), consistent with previous observations of the sub-cellular distribution of PrPSc (refs 20,22,23). In contrast, only very low levels of background immunostaining were detected in uninfected cells, indicating that PrPC had been effectively removed by these treatments. GdnHCl denaturation was less effective at removing PrPC staining (Fig. 2a,b). Similar results were obtained using ScPK1 cells that express wild-type PrPSc (ref. 20) (Supplementary Fig. S2a,b), indicating that our MYC-tagged PrPSc displays a physiologically relevant cellular localization.

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