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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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Generation of prion-susceptible cell lines expressing epitope-tagged PrPC and PrPSc.(a) Western blots developed with anti-PrP and anti-MYC antibodies showing levels of PrPC 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 PrPSc. 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 (PrPSc) 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 PrPSc 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 PrPSc or show other diagnostic features. Scale bar is 80 μm on the H&E stained sections and 160 μm for all other panels.
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f1: Generation of prion-susceptible cell lines expressing epitope-tagged PrPC and PrPSc.(a) Western blots developed with anti-PrP and anti-MYC antibodies showing levels of PrPC 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 PrPSc. 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 (PrPSc) 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 PrPSc 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 PrPSc or show other diagnostic features. Scale bar is 80 μm on the H&E stained sections and 160 μm for all other panels.

Mentions: To study prion infection in cells expressing tagged PrPC constructs without any interference from the host wild-type PrPC, we produced PrP knockdown (KD) cells derived from the mouse PK1 prion-susceptible neuroblastoma cell line16, using RNA interference (RNAi) directed at the 3′ untranslated region (3′-UTR) of PrPC mRNA. This effectively silences PrP gene expression (Fig. 1a,b) and results in the complete inhibition of prion propagation (Fig. 1c). This strategy allows the expression of the mouse PrPC open reading frame from exogenous constructs lacking the 3′-UTR, and permits the expression of a panel of FLAG- and MYC-tagged PrPC chimerae. The PrP constructs were designed such that the epitope tags were inserted into regions of the murine PrPC sequence that are relatively unstructured, and therefore theoretically unlikely to interfere with prion conversion (Supplementary Fig. S1a)1217. Wild-type PK1 cells, PrP-KD cells and cells expressing tagged PrPC constructs were exposed to Rocky Mountain Laboratory (RML) mouse prions, and their ability to support prion propagation was assayed using the Scrapie Cell Assay16. PrP constructs 22CysFLAG and 30GlyFLAG were found to reconstitute prion propagation very efficiently and contained high levels of PrPSc. However, it became apparent during the course of our study that these amino (N)-terminal FLAG tags were readily cleaved during cellular processing such that a proportion of the tagged PrPSc in the cells was not detected by the anti-FLAG antibodies, making them unsuitable for this study. Cells expressing PrPC with epitope tags placed centrally within the PrP sequence (for example, PrP-124AlaMYC, Supplementary Fig. S1a) did not support prion propagation (Fig. 1c). However, cells expressing PrP-224AlaMYC with the epitope tag inserted near the carboxy (C)-terminus were found to propagate prions robustly and reproducibly (Fig. 1c). Immunofluorescence and immunoblot analyses demonstrate that the expression levels and the cellular distribution of PrP-224AlaMYC were similar to that of wild-type PrPC in PK1 cells (Fig. 1a,b). Immunoblotting of proteinase K (PK)-digested cell lysates from RML prion-infected PrP-224AlaMYC cells demonstrates that they contain protease-resistant PrP, a characteristic feature of PrPSc18 (Supplementary Fig. S1b).


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)

Generation of prion-susceptible cell lines expressing epitope-tagged PrPC and PrPSc.(a) Western blots developed with anti-PrP and anti-MYC antibodies showing levels of PrPC 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 PrPSc. 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 (PrPSc) 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 PrPSc 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 PrPSc or show other diagnostic features. Scale bar is 80 μm on the H&E stained sections and 160 μm for all other panels.
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f1: Generation of prion-susceptible cell lines expressing epitope-tagged PrPC and PrPSc.(a) Western blots developed with anti-PrP and anti-MYC antibodies showing levels of PrPC 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 PrPSc. 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 (PrPSc) 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 PrPSc 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 PrPSc or show other diagnostic features. Scale bar is 80 μm on the H&E stained sections and 160 μm for all other panels.
Mentions: To study prion infection in cells expressing tagged PrPC constructs without any interference from the host wild-type PrPC, we produced PrP knockdown (KD) cells derived from the mouse PK1 prion-susceptible neuroblastoma cell line16, using RNA interference (RNAi) directed at the 3′ untranslated region (3′-UTR) of PrPC mRNA. This effectively silences PrP gene expression (Fig. 1a,b) and results in the complete inhibition of prion propagation (Fig. 1c). This strategy allows the expression of the mouse PrPC open reading frame from exogenous constructs lacking the 3′-UTR, and permits the expression of a panel of FLAG- and MYC-tagged PrPC chimerae. The PrP constructs were designed such that the epitope tags were inserted into regions of the murine PrPC sequence that are relatively unstructured, and therefore theoretically unlikely to interfere with prion conversion (Supplementary Fig. S1a)1217. Wild-type PK1 cells, PrP-KD cells and cells expressing tagged PrPC constructs were exposed to Rocky Mountain Laboratory (RML) mouse prions, and their ability to support prion propagation was assayed using the Scrapie Cell Assay16. PrP constructs 22CysFLAG and 30GlyFLAG were found to reconstitute prion propagation very efficiently and contained high levels of PrPSc. However, it became apparent during the course of our study that these amino (N)-terminal FLAG tags were readily cleaved during cellular processing such that a proportion of the tagged PrPSc in the cells was not detected by the anti-FLAG antibodies, making them unsuitable for this study. Cells expressing PrPC with epitope tags placed centrally within the PrP sequence (for example, PrP-124AlaMYC, Supplementary Fig. S1a) did not support prion propagation (Fig. 1c). However, cells expressing PrP-224AlaMYC with the epitope tag inserted near the carboxy (C)-terminus were found to propagate prions robustly and reproducibly (Fig. 1c). Immunofluorescence and immunoblot analyses demonstrate that the expression levels and the cellular distribution of PrP-224AlaMYC were similar to that of wild-type PrPC in PK1 cells (Fig. 1a,b). Immunoblotting of proteinase K (PK)-digested cell lysates from RML prion-infected PrP-224AlaMYC cells demonstrates that they contain protease-resistant PrP, a characteristic feature of PrPSc18 (Supplementary Fig. S1b).

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