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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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The cell tropism of various prion strains changes after propagation in tgGPI− mice.Homogenates of GPI− or C57 brains infected with the strains indicated were subjected to the CPA. (A) The patterns elicited by 22L from both sources were very similar, however RML, 79A and 139A prions from wild-type brain were swa sensitive on PK1 cells and R332H11 incompetent, while those from tgGPI− brain were swa resistant and R332H1 competent. The RI600 (Response Index for 600 spots) on CAD, PK1, PK1+swa and R332H11 cells is given within the graphs (left upper corner) and the logarithm ± SD of the ratios RICAD/RIPK1 (blue) and RIPK1/RIPK1+swa (red) is plotted in the bar graph (B). The matrix (C) gives the p values for the pairwise comparison of two strains on the basis of their log[RICAD/RIPK1] (blue) and log[RIPK1/RIPK1+swa] (red) values. The framed “ns” indicates p values>0.1 for both log[ratios]. For example, C57[RML] and GPI−[RML] prions are significantly different (p = 0.0097 for log[RICAD/RIPK1] and p = 0.0001 for log[RIPK1/RIPK1+swa]), as are C57[79A] and GPI−[79A] prions, whereas C57[22L] and GPI−[22L] prions do not show a significant difference (framed “ns”; p>0.1) for both logRI ratios. By the same token C57[79A] and C57[RML], and GPI−[139A] and GPI−[RML] prions are not distinguishable, while C57[139A] and C57[RML] prions differ.
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ppat-1002746-g002: The cell tropism of various prion strains changes after propagation in tgGPI− mice.Homogenates of GPI− or C57 brains infected with the strains indicated were subjected to the CPA. (A) The patterns elicited by 22L from both sources were very similar, however RML, 79A and 139A prions from wild-type brain were swa sensitive on PK1 cells and R332H11 incompetent, while those from tgGPI− brain were swa resistant and R332H1 competent. The RI600 (Response Index for 600 spots) on CAD, PK1, PK1+swa and R332H11 cells is given within the graphs (left upper corner) and the logarithm ± SD of the ratios RICAD/RIPK1 (blue) and RIPK1/RIPK1+swa (red) is plotted in the bar graph (B). The matrix (C) gives the p values for the pairwise comparison of two strains on the basis of their log[RICAD/RIPK1] (blue) and log[RIPK1/RIPK1+swa] (red) values. The framed “ns” indicates p values>0.1 for both log[ratios]. For example, C57[RML] and GPI−[RML] prions are significantly different (p = 0.0097 for log[RICAD/RIPK1] and p = 0.0001 for log[RIPK1/RIPK1+swa]), as are C57[79A] and GPI−[79A] prions, whereas C57[22L] and GPI−[22L] prions do not show a significant difference (framed “ns”; p>0.1) for both logRI ratios. By the same token C57[79A] and C57[RML], and GPI−[139A] and GPI−[RML] prions are not distinguishable, while C57[139A] and C57[RML] prions differ.

Mentions: As shown by the CPA in Figure 2A, RML prions from C57 brain were swa sensitive on PK1 cells and R332H11 incompetent, i.e. unable to infect R332H11 cells efficiently, while RML-derived prions from GPI− brain were swa resistant and R332H11 competent; moreover, the RICAD/RIPK1 ratio was lower for the C57-derived than for the GPI−-derived samples. The bar diagram (Figure 2B) shows log[RICAD/RIPK1] (blue) and log[RIPK1/RIPK1+swa] (red) values plotted for 22L, RML, 79A, and 139A, propagated in C57 or GPI− brain. The quantified data clearly show that C57[RML] and GPI−[RML] give vastly different patterns, as do their 79A and 139A counterparts. The statistical significance of the “log[ratio]” differences between two strains is given in the matrix of Figure 2C. For example, the difference between C57[RML] and GPI−[RML] is highly significant for both ratios, whereas there is no significant difference between C57[22L] and GPI−[22L] by the criteria used here. Interestingly, the differences between C57[RML] and C57[139A] are significant for both ratios, but not those between C57[RML] and C57[79A], contradicting the common assumption that RML and 139A are the same strains [67] and confirming the cognate conclusions of Browning et al. [73] and Oelschlegel et al. (personal communication).


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)

The cell tropism of various prion strains changes after propagation in tgGPI− mice.Homogenates of GPI− or C57 brains infected with the strains indicated were subjected to the CPA. (A) The patterns elicited by 22L from both sources were very similar, however RML, 79A and 139A prions from wild-type brain were swa sensitive on PK1 cells and R332H11 incompetent, while those from tgGPI− brain were swa resistant and R332H1 competent. The RI600 (Response Index for 600 spots) on CAD, PK1, PK1+swa and R332H11 cells is given within the graphs (left upper corner) and the logarithm ± SD of the ratios RICAD/RIPK1 (blue) and RIPK1/RIPK1+swa (red) is plotted in the bar graph (B). The matrix (C) gives the p values for the pairwise comparison of two strains on the basis of their log[RICAD/RIPK1] (blue) and log[RIPK1/RIPK1+swa] (red) values. The framed “ns” indicates p values>0.1 for both log[ratios]. For example, C57[RML] and GPI−[RML] prions are significantly different (p = 0.0097 for log[RICAD/RIPK1] and p = 0.0001 for log[RIPK1/RIPK1+swa]), as are C57[79A] and GPI−[79A] prions, whereas C57[22L] and GPI−[22L] prions do not show a significant difference (framed “ns”; p>0.1) for both logRI ratios. By the same token C57[79A] and C57[RML], and GPI−[139A] and GPI−[RML] prions are not distinguishable, while C57[139A] and C57[RML] prions differ.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3369955&req=5

ppat-1002746-g002: The cell tropism of various prion strains changes after propagation in tgGPI− mice.Homogenates of GPI− or C57 brains infected with the strains indicated were subjected to the CPA. (A) The patterns elicited by 22L from both sources were very similar, however RML, 79A and 139A prions from wild-type brain were swa sensitive on PK1 cells and R332H11 incompetent, while those from tgGPI− brain were swa resistant and R332H1 competent. The RI600 (Response Index for 600 spots) on CAD, PK1, PK1+swa and R332H11 cells is given within the graphs (left upper corner) and the logarithm ± SD of the ratios RICAD/RIPK1 (blue) and RIPK1/RIPK1+swa (red) is plotted in the bar graph (B). The matrix (C) gives the p values for the pairwise comparison of two strains on the basis of their log[RICAD/RIPK1] (blue) and log[RIPK1/RIPK1+swa] (red) values. The framed “ns” indicates p values>0.1 for both log[ratios]. For example, C57[RML] and GPI−[RML] prions are significantly different (p = 0.0097 for log[RICAD/RIPK1] and p = 0.0001 for log[RIPK1/RIPK1+swa]), as are C57[79A] and GPI−[79A] prions, whereas C57[22L] and GPI−[22L] prions do not show a significant difference (framed “ns”; p>0.1) for both logRI ratios. By the same token C57[79A] and C57[RML], and GPI−[139A] and GPI−[RML] prions are not distinguishable, while C57[139A] and C57[RML] prions differ.
Mentions: As shown by the CPA in Figure 2A, RML prions from C57 brain were swa sensitive on PK1 cells and R332H11 incompetent, i.e. unable to infect R332H11 cells efficiently, while RML-derived prions from GPI− brain were swa resistant and R332H11 competent; moreover, the RICAD/RIPK1 ratio was lower for the C57-derived than for the GPI−-derived samples. The bar diagram (Figure 2B) shows log[RICAD/RIPK1] (blue) and log[RIPK1/RIPK1+swa] (red) values plotted for 22L, RML, 79A, and 139A, propagated in C57 or GPI− brain. The quantified data clearly show that C57[RML] and GPI−[RML] give vastly different patterns, as do their 79A and 139A counterparts. The statistical significance of the “log[ratio]” differences between two strains is given in the matrix of Figure 2C. For example, the difference between C57[RML] and GPI−[RML] is highly significant for both ratios, whereas there is no significant difference between C57[22L] and GPI−[22L] by the criteria used here. Interestingly, the differences between C57[RML] and C57[139A] are significant for both ratios, but not those between C57[RML] and C57[79A], contradicting the common assumption that RML and 139A are the same strains [67] and confirming the cognate conclusions of Browning et al. [73] and Oelschlegel et al. (personal communication).

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