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Propagation of RML prions in mice expressing PrP devoid of GPI anchor leads to formation of a novel, stable prion strain.

Mahal SP, Jablonski J, Suponitsky-Kroyter I, Oelschlegel AM, Herva ME, Oldstone M, Weissmann C - PLoS Pathog. (2012)

Bottom Line: PrP(C), a host protein which in prion-infected animals is converted to PrP(Sc), is linked to the cell membrane by a GPI anchor.We found that RML and ME7, but not 22L prions propagated in tgGPI⁻ brain developed novel cell tropisms, as determined by the Cell Panel Assay (CPA).Surprisingly, the levels of proteinase K-resistant PrP(Sc) (PrP(res)) in RML- or ME7-infected tgGPI⁻ brain were 25-50 times higher than in wild-type brain.

View Article: PubMed Central - PubMed

Affiliation: Department of Infectology, Scripps Florida, Jupiter, Florida, United States of America.

ABSTRACT
PrP(C), a host protein which in prion-infected animals is converted to PrP(Sc), is linked to the cell membrane by a GPI anchor. Mice expressing PrP(C) without GPI anchor (tgGPI⁻ mice), are susceptible to prion infection but accumulate anchorless PrP(Sc) extra-, rather than intracellularly. We investigated whether tgGPI⁻ mice could faithfully propagate prion strains despite the deviant structure and location of anchorless PrP(Sc). We found that RML and ME7, but not 22L prions propagated in tgGPI⁻ brain developed novel cell tropisms, as determined by the Cell Panel Assay (CPA). Surprisingly, the levels of proteinase K-resistant PrP(Sc) (PrP(res)) in RML- or ME7-infected tgGPI⁻ brain were 25-50 times higher than in wild-type brain. When returned to wild-type brain, ME7 prions recovered their original properties, however RML prions had given rise to a novel prion strain, designated SFL, which remained unchanged even after three passages in wild-type mice. Because both RML PrP(Sc) and SFL PrP(Sc) are stably propagated in wild-type mice we propose that the two conformations are separated by a high activation energy barrier which is abrogated in tgGPI⁻ mice.

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RML prions propagated in tgGPI− mice and returned to wild-type mice emerge as a novel, stable strain.Analysis of authentic RML prions propagated in C57BL/6 brain (C57[RML]), and of RML prions first propagated in GPI− brain (GPI−[RML]) and then once (C57/GPI−[RML]), twice (C57/C57/GPI−[RML]) or three times (C57/C57/C57/GPI−[RML]) in C57BL/6 brain. (A) shows the SSCA performed on PK1 cells in the presence or absence of kifunensine (kifu; 5 µg/ml) or swainsonine (swa; 2 µg/ml) for the samples indicated; C57[22L] was added as control. RI's were determined at 600 spots (RI600). Kifu strongly inhibited the propagation of C57[RML], but not of C57/GPI−[RML] and C57/C57/C57/GPI−[RML] prions on PK1 cells. (B) The bar graph shows the log[RIPK1/RIPK1+kifu] and log[RIPK1/RIPK1+swa] values for the samples listed in (A). The pairwise comparison in panel (C) shows that C57/GPI−[RML] and C57/C57/C57/GPI−[RML] prions do not differ significantly, but are vastly different from C57[RML] and GPI−[RML] prions. The framed “ns” indicates high p values (>0.1) for both log[ratios], indicating no significant difference between the samples. (D) Effect of castanospermine (csp; 50 µg/ml) or kifu (5 µg/ml) on propagation of the samples indicated on PK1 cells. As in (A), kifu strongly inhibits the propagation of C57[RML], but not C57/GPI−[RML] and C57/C57/GPI−[RML] prions. The same is true for csp, however to a lesser extent. (E) The bar graph depicts the log[RIPK1/RIPK1+kifu] (blue) and log[RIPK1/RIPK1+csp] (green) values. The RIPK1/RIPK1+kifu ratio was >500-fold lower for C57/GPI−[RML] and C57/C57/GPI−[RML] than for C57[RML] prions, again underscoring the difference between RML prions and the novel strain. The matrix (F) also shows that C57/GPI−[RML] and C57/C57/GPI−[RML] prions do not differ from each other, but that both differ significantly from C57[RML] prions. In summary, the figure sustains the conclusion that a new strain, designated SFL, emerged after RML prions were passaged through GPI− brain and returned C57 brain, and that they remained unchanged after two further passages.
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ppat-1002746-g003: RML prions propagated in tgGPI− mice and returned to wild-type mice emerge as a novel, stable strain.Analysis of authentic RML prions propagated in C57BL/6 brain (C57[RML]), and of RML prions first propagated in GPI− brain (GPI−[RML]) and then once (C57/GPI−[RML]), twice (C57/C57/GPI−[RML]) or three times (C57/C57/C57/GPI−[RML]) in C57BL/6 brain. (A) shows the SSCA performed on PK1 cells in the presence or absence of kifunensine (kifu; 5 µg/ml) or swainsonine (swa; 2 µg/ml) for the samples indicated; C57[22L] was added as control. RI's were determined at 600 spots (RI600). Kifu strongly inhibited the propagation of C57[RML], but not of C57/GPI−[RML] and C57/C57/C57/GPI−[RML] prions on PK1 cells. (B) The bar graph shows the log[RIPK1/RIPK1+kifu] and log[RIPK1/RIPK1+swa] values for the samples listed in (A). The pairwise comparison in panel (C) shows that C57/GPI−[RML] and C57/C57/C57/GPI−[RML] prions do not differ significantly, but are vastly different from C57[RML] and GPI−[RML] prions. The framed “ns” indicates high p values (>0.1) for both log[ratios], indicating no significant difference between the samples. (D) Effect of castanospermine (csp; 50 µg/ml) or kifu (5 µg/ml) on propagation of the samples indicated on PK1 cells. As in (A), kifu strongly inhibits the propagation of C57[RML], but not C57/GPI−[RML] and C57/C57/GPI−[RML] prions. The same is true for csp, however to a lesser extent. (E) The bar graph depicts the log[RIPK1/RIPK1+kifu] (blue) and log[RIPK1/RIPK1+csp] (green) values. The RIPK1/RIPK1+kifu ratio was >500-fold lower for C57/GPI−[RML] and C57/C57/GPI−[RML] than for C57[RML] prions, again underscoring the difference between RML prions and the novel strain. The matrix (F) also shows that C57/GPI−[RML] and C57/C57/GPI−[RML] prions do not differ from each other, but that both differ significantly from C57[RML] prions. In summary, the figure sustains the conclusion that a new strain, designated SFL, emerged after RML prions were passaged through GPI− brain and returned C57 brain, and that they remained unchanged after two further passages.

Mentions: C57BL/6 mice inoculated with prions from GPI−[RML] brain exhibited pronounced clinical signs of RML scrapie disease after 150±9 days, similar to those of mice inoculated with the original C57[RML] (Table S1, #4a,b; #1). However, as shown in Figure 3A, C57/GPI−[RML] prions neither retained the drug susceptibility pattern of GPI−[RML], nor did they regain the pattern characteristic for the original C57[RML] prions, even after two additional transfers through C57 mice. From the bar diagram (Figure 3B) it can be seen that the log[RIPK1/RIPK1+kifu] (blue) and log[RIPK1/RIPK1+swa] (red) values for C57/GPI−[RML] and C57/C57/C57/GPI−[RML] prions, 0.6, 0.7, and 0.7, 0.8, respectively, were indistinguishable, but far lower than those for C57[RML] prions, >2.8 and 1.4, respectively. The statistical evaluation shown in the matrix of Figure 3C confirms the significance of these conclusions.


Propagation of RML prions in mice expressing PrP devoid of GPI anchor leads to formation of a novel, stable prion strain.

Mahal SP, Jablonski J, Suponitsky-Kroyter I, Oelschlegel AM, Herva ME, Oldstone M, Weissmann C - PLoS Pathog. (2012)

RML prions propagated in tgGPI− mice and returned to wild-type mice emerge as a novel, stable strain.Analysis of authentic RML prions propagated in C57BL/6 brain (C57[RML]), and of RML prions first propagated in GPI− brain (GPI−[RML]) and then once (C57/GPI−[RML]), twice (C57/C57/GPI−[RML]) or three times (C57/C57/C57/GPI−[RML]) in C57BL/6 brain. (A) shows the SSCA performed on PK1 cells in the presence or absence of kifunensine (kifu; 5 µg/ml) or swainsonine (swa; 2 µg/ml) for the samples indicated; C57[22L] was added as control. RI's were determined at 600 spots (RI600). Kifu strongly inhibited the propagation of C57[RML], but not of C57/GPI−[RML] and C57/C57/C57/GPI−[RML] prions on PK1 cells. (B) The bar graph shows the log[RIPK1/RIPK1+kifu] and log[RIPK1/RIPK1+swa] values for the samples listed in (A). The pairwise comparison in panel (C) shows that C57/GPI−[RML] and C57/C57/C57/GPI−[RML] prions do not differ significantly, but are vastly different from C57[RML] and GPI−[RML] prions. The framed “ns” indicates high p values (>0.1) for both log[ratios], indicating no significant difference between the samples. (D) Effect of castanospermine (csp; 50 µg/ml) or kifu (5 µg/ml) on propagation of the samples indicated on PK1 cells. As in (A), kifu strongly inhibits the propagation of C57[RML], but not C57/GPI−[RML] and C57/C57/GPI−[RML] prions. The same is true for csp, however to a lesser extent. (E) The bar graph depicts the log[RIPK1/RIPK1+kifu] (blue) and log[RIPK1/RIPK1+csp] (green) values. The RIPK1/RIPK1+kifu ratio was >500-fold lower for C57/GPI−[RML] and C57/C57/GPI−[RML] than for C57[RML] prions, again underscoring the difference between RML prions and the novel strain. The matrix (F) also shows that C57/GPI−[RML] and C57/C57/GPI−[RML] prions do not differ from each other, but that both differ significantly from C57[RML] prions. In summary, the figure sustains the conclusion that a new strain, designated SFL, emerged after RML prions were passaged through GPI− brain and returned C57 brain, and that they remained unchanged after two further passages.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3369955&req=5

ppat-1002746-g003: RML prions propagated in tgGPI− mice and returned to wild-type mice emerge as a novel, stable strain.Analysis of authentic RML prions propagated in C57BL/6 brain (C57[RML]), and of RML prions first propagated in GPI− brain (GPI−[RML]) and then once (C57/GPI−[RML]), twice (C57/C57/GPI−[RML]) or three times (C57/C57/C57/GPI−[RML]) in C57BL/6 brain. (A) shows the SSCA performed on PK1 cells in the presence or absence of kifunensine (kifu; 5 µg/ml) or swainsonine (swa; 2 µg/ml) for the samples indicated; C57[22L] was added as control. RI's were determined at 600 spots (RI600). Kifu strongly inhibited the propagation of C57[RML], but not of C57/GPI−[RML] and C57/C57/C57/GPI−[RML] prions on PK1 cells. (B) The bar graph shows the log[RIPK1/RIPK1+kifu] and log[RIPK1/RIPK1+swa] values for the samples listed in (A). The pairwise comparison in panel (C) shows that C57/GPI−[RML] and C57/C57/C57/GPI−[RML] prions do not differ significantly, but are vastly different from C57[RML] and GPI−[RML] prions. The framed “ns” indicates high p values (>0.1) for both log[ratios], indicating no significant difference between the samples. (D) Effect of castanospermine (csp; 50 µg/ml) or kifu (5 µg/ml) on propagation of the samples indicated on PK1 cells. As in (A), kifu strongly inhibits the propagation of C57[RML], but not C57/GPI−[RML] and C57/C57/GPI−[RML] prions. The same is true for csp, however to a lesser extent. (E) The bar graph depicts the log[RIPK1/RIPK1+kifu] (blue) and log[RIPK1/RIPK1+csp] (green) values. The RIPK1/RIPK1+kifu ratio was >500-fold lower for C57/GPI−[RML] and C57/C57/GPI−[RML] than for C57[RML] prions, again underscoring the difference between RML prions and the novel strain. The matrix (F) also shows that C57/GPI−[RML] and C57/C57/GPI−[RML] prions do not differ from each other, but that both differ significantly from C57[RML] prions. In summary, the figure sustains the conclusion that a new strain, designated SFL, emerged after RML prions were passaged through GPI− brain and returned C57 brain, and that they remained unchanged after two further passages.
Mentions: C57BL/6 mice inoculated with prions from GPI−[RML] brain exhibited pronounced clinical signs of RML scrapie disease after 150±9 days, similar to those of mice inoculated with the original C57[RML] (Table S1, #4a,b; #1). However, as shown in Figure 3A, C57/GPI−[RML] prions neither retained the drug susceptibility pattern of GPI−[RML], nor did they regain the pattern characteristic for the original C57[RML] prions, even after two additional transfers through C57 mice. From the bar diagram (Figure 3B) it can be seen that the log[RIPK1/RIPK1+kifu] (blue) and log[RIPK1/RIPK1+swa] (red) values for C57/GPI−[RML] and C57/C57/C57/GPI−[RML] prions, 0.6, 0.7, and 0.7, 0.8, respectively, were indistinguishable, but far lower than those for C57[RML] prions, >2.8 and 1.4, respectively. The statistical evaluation shown in the matrix of Figure 3C confirms the significance of these conclusions.

Bottom Line: PrP(C), a host protein which in prion-infected animals is converted to PrP(Sc), is linked to the cell membrane by a GPI anchor.We found that RML and ME7, but not 22L prions propagated in tgGPI⁻ brain developed novel cell tropisms, as determined by the Cell Panel Assay (CPA).Surprisingly, the levels of proteinase K-resistant PrP(Sc) (PrP(res)) in RML- or ME7-infected tgGPI⁻ brain were 25-50 times higher than in wild-type brain.

View Article: PubMed Central - PubMed

Affiliation: Department of Infectology, Scripps Florida, Jupiter, Florida, United States of America.

ABSTRACT
PrP(C), a host protein which in prion-infected animals is converted to PrP(Sc), is linked to the cell membrane by a GPI anchor. Mice expressing PrP(C) without GPI anchor (tgGPI⁻ mice), are susceptible to prion infection but accumulate anchorless PrP(Sc) extra-, rather than intracellularly. We investigated whether tgGPI⁻ mice could faithfully propagate prion strains despite the deviant structure and location of anchorless PrP(Sc). We found that RML and ME7, but not 22L prions propagated in tgGPI⁻ brain developed novel cell tropisms, as determined by the Cell Panel Assay (CPA). Surprisingly, the levels of proteinase K-resistant PrP(Sc) (PrP(res)) in RML- or ME7-infected tgGPI⁻ brain were 25-50 times higher than in wild-type brain. When returned to wild-type brain, ME7 prions recovered their original properties, however RML prions had given rise to a novel prion strain, designated SFL, which remained unchanged even after three passages in wild-type mice. Because both RML PrP(Sc) and SFL PrP(Sc) are stably propagated in wild-type mice we propose that the two conformations are separated by a high activation energy barrier which is abrogated in tgGPI⁻ mice.

Show MeSH
Related in: MedlinePlus