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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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PrPSc 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 (PrPSc) is formed on the cell surface of one of the cells in the field (arrow), no intracellular PrPSc 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 PrPSc 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).
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f5: PrPSc 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 (PrPSc) is formed on the cell surface of one of the cells in the field (arrow), no intracellular PrPSc 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 PrPSc 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).

Mentions: To determine whether PrP prion conversion can occur on the plasma membrane, we incubated pre-cooled PrP-224AlaMYC cells with RML prions at 4 °C. This temperature shift effectively abolished endocytosis in mammalian cells26. Significantly, PrPSc accumulated only on the cell surface under these conditions (Fig. 5a,b, 4 °C). Orthogonal projections of confocal sections taken through these infected cells revealed the plasma membrane distribution of MYC-tagged PrPSc (Fig. 5a—the z stack series is shown in Supplementary Fig. S6) and clearly demonstrated that endocytosis is not necessary to produce misfolded PrP.


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)

PrPSc 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 (PrPSc) is formed on the cell surface of one of the cells in the field (arrow), no intracellular PrPSc 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 PrPSc 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).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: PrPSc 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 (PrPSc) is formed on the cell surface of one of the cells in the field (arrow), no intracellular PrPSc 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 PrPSc 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).
Mentions: To determine whether PrP prion conversion can occur on the plasma membrane, we incubated pre-cooled PrP-224AlaMYC cells with RML prions at 4 °C. This temperature shift effectively abolished endocytosis in mammalian cells26. Significantly, PrPSc accumulated only on the cell surface under these conditions (Fig. 5a,b, 4 °C). Orthogonal projections of confocal sections taken through these infected cells revealed the plasma membrane distribution of MYC-tagged PrPSc (Fig. 5a—the z stack series is shown in Supplementary Fig. S6) and clearly demonstrated that endocytosis is not necessary to produce misfolded PrP.

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