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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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MYC-tagged PrPSc 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 (PrPSc) 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, PrPSc) was generated after just 2 min of RML prion exposure; scale bar, 20 μm.
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f3: MYC-tagged PrPSc 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 (PrPSc) 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, PrPSc) was generated after just 2 min of RML prion exposure; scale bar, 20 μm.

Mentions: Our novel cell system expressing MYC-tagged PrPSc combined with effective PrPC removal by formic acid or PK treatment allows de novo synthesized PrPSc to be clearly distinguished from the host RML prion inocula. This makes a detailed temporal and spatial study of the early stages of cellular prion infection feasible. Previous work in the field suggests that a 72-h exposure to prion-infected material is necessary to fully infect susceptible cells16. Therefore, we first infected PrP-224AlaMYC cells with RML prions for 3 days, and either fixed immediately or passaged in fresh media before fixation, formic acid extraction and anti-MYC staining. Maximum numbers of RML prion-infected MYC-tagged PrPSc cells were seen 6 days post infection (DPI; Fig. 3a) and stable prion propagation in ∼20% of the cells was observed up to 14 DPI (Fig. 3a). PrP-KD cells treated in the same way showed no anti-MYC immunostaining up to 6 DPI (Fig. 3a), indicating that the observed staining was dependent on PrP-224AlaMYC expression. Interestingly, a significant proportion of the PrP-224AlaMYC cells already contained MYC-tagged PrPSc after 3 days of RML prion exposure (Fig. 3a). This prompted us to examine the first 72 h of prion exposure in more detail. Figure 3b shows the proportion of infected cells at different time points of RML prion exposure and reveals that PrP-224AlaMYC cells produce PrPSc rapidly, with MYC-tagged PrPSc-positive prion-infected cells clearly detectable after 2 h of RML prion exposure. These observations suggest that cellular prion infection is faster than previously thought. Therefore, we examined the first few minutes of prion exposure in more detail. PrP-224AlaMYC cells were exposed to RML prions for very short periods before rapid washing and fixation. A significant proportion of the cells showed clearly detectable levels of formic acid-resistant MYC-tagged PrPSc after as little as 1 min of exposure to RML prions (Fig. 3c), the shortest time period technically possible to reproduce accurately within the limitations of our system. PK digestion of similar cells confirmed that protease-resistant MYC-tagged PrPSc was generated after very short periods of prion exposure (Fig. 3d).


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)

MYC-tagged PrPSc 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 (PrPSc) 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, PrPSc) was generated after just 2 min of RML prion exposure; scale bar, 20 μm.
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Related In: Results  -  Collection

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f3: MYC-tagged PrPSc 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 (PrPSc) 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, PrPSc) was generated after just 2 min of RML prion exposure; scale bar, 20 μm.
Mentions: Our novel cell system expressing MYC-tagged PrPSc combined with effective PrPC removal by formic acid or PK treatment allows de novo synthesized PrPSc to be clearly distinguished from the host RML prion inocula. This makes a detailed temporal and spatial study of the early stages of cellular prion infection feasible. Previous work in the field suggests that a 72-h exposure to prion-infected material is necessary to fully infect susceptible cells16. Therefore, we first infected PrP-224AlaMYC cells with RML prions for 3 days, and either fixed immediately or passaged in fresh media before fixation, formic acid extraction and anti-MYC staining. Maximum numbers of RML prion-infected MYC-tagged PrPSc cells were seen 6 days post infection (DPI; Fig. 3a) and stable prion propagation in ∼20% of the cells was observed up to 14 DPI (Fig. 3a). PrP-KD cells treated in the same way showed no anti-MYC immunostaining up to 6 DPI (Fig. 3a), indicating that the observed staining was dependent on PrP-224AlaMYC expression. Interestingly, a significant proportion of the PrP-224AlaMYC cells already contained MYC-tagged PrPSc after 3 days of RML prion exposure (Fig. 3a). This prompted us to examine the first 72 h of prion exposure in more detail. Figure 3b shows the proportion of infected cells at different time points of RML prion exposure and reveals that PrP-224AlaMYC cells produce PrPSc rapidly, with MYC-tagged PrPSc-positive prion-infected cells clearly detectable after 2 h of RML prion exposure. These observations suggest that cellular prion infection is faster than previously thought. Therefore, we examined the first few minutes of prion exposure in more detail. PrP-224AlaMYC cells were exposed to RML prions for very short periods before rapid washing and fixation. A significant proportion of the cells showed clearly detectable levels of formic acid-resistant MYC-tagged PrPSc after as little as 1 min of exposure to RML prions (Fig. 3c), the shortest time period technically possible to reproduce accurately within the limitations of our system. PK digestion of similar cells confirmed that protease-resistant MYC-tagged PrPSc was generated after very short periods of prion exposure (Fig. 3d).

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