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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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ME7 prions propagated in tgGPI− mice for one or two passages acquire novel characteristics.(A) Serially diluted brain homogenates were analyzed on LD9, CAD and PK1 cells by the CPA. After the first passage of ME7 prions in GPI− mice (GPI−[ME7]), the RI on CAD cells dropped 20-fold, most likely reflecting low titers, but increased about 30-fold after the second passage (GPI−/GPI−[ME7]), indicating adaptation to the GPI− environment. Returning prions from the first passage in GPI− brain to wild-type brain (C57/GPI−[ME7]) restored the original CPA pattern. (B) Log[RILD9/RICAD] (red) and log[RILD9/RIPK1] (blue) are plotted as a bar graph. (C) The matrix shows that C57[ME7] and C57/GPI−[ME7] prions do not differ, while both differ in at least one log[ratio] from GPI−[ME7] and GPI−/GPI−[ME7] prions.
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ppat-1002746-g004: ME7 prions propagated in tgGPI− mice for one or two passages acquire novel characteristics.(A) Serially diluted brain homogenates were analyzed on LD9, CAD and PK1 cells by the CPA. After the first passage of ME7 prions in GPI− mice (GPI−[ME7]), the RI on CAD cells dropped 20-fold, most likely reflecting low titers, but increased about 30-fold after the second passage (GPI−/GPI−[ME7]), indicating adaptation to the GPI− environment. Returning prions from the first passage in GPI− brain to wild-type brain (C57/GPI−[ME7]) restored the original CPA pattern. (B) Log[RILD9/RICAD] (red) and log[RILD9/RIPK1] (blue) are plotted as a bar graph. (C) The matrix shows that C57[ME7] and C57/GPI−[ME7] prions do not differ, while both differ in at least one log[ratio] from GPI−[ME7] and GPI−/GPI−[ME7] prions.

Mentions: Typical scrapie signs were not observed in ME7-inoculated GPI− mice by 300–305 days, at which time mice showing mild clinical signs were euthanized and homogenates of their brains were subjected to the CPA. The level of infectivity of GPI−[ME7] brain homogenates as measured on CAD and PK1 cells was very low (RI<103), however LD9 cells showed a higher response (RI = 1.1×104), albeit at a level about 25 times lower than that found for C57[ME7] brains 2.8×105 (Figure 4). The GPI−[ME7] brain homogenates were infectious to wild-type mice, leading to disease at 170±0 dpi (Table S1, #20), as compared to 138±4 days for “normal” C57[ME7] (Table S1, #18b), and yielded prions indistinguishable from the original ME7, as judged by the CPA. A second transmission of GPI−[ME7] prions to GPI− mice resulted in disease at 447±64 dpi (Table S1, #21) and, interestingly, to high levels of PrPres, as determined by sandwich ELISA (Figure 1C, 1D), and infectivity, as measured on CAD cells (RI = 1.5×105; Figure 4A). GPI−/GPI−[ME7] prions differed from C57[ME7] prions by their log[RILD9/RICAD] value, -0.03 versus 0.7; whether or not they revert to the original ME7 after being returned to C57 is still under investigation (Table S1, 22.) (Figure 4B, C). In summary, repeated propagation of ME7 prions through GPI− brain resulted in a distinct alteration of their properties, reflecting progressive adaptation to the modified environment. However, prions returned from GPI−[ME7] mice to C57 mice resulted in reversion to prions indistinguishable from the original C57[ME7] prions.


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)

ME7 prions propagated in tgGPI− mice for one or two passages acquire novel characteristics.(A) Serially diluted brain homogenates were analyzed on LD9, CAD and PK1 cells by the CPA. After the first passage of ME7 prions in GPI− mice (GPI−[ME7]), the RI on CAD cells dropped 20-fold, most likely reflecting low titers, but increased about 30-fold after the second passage (GPI−/GPI−[ME7]), indicating adaptation to the GPI− environment. Returning prions from the first passage in GPI− brain to wild-type brain (C57/GPI−[ME7]) restored the original CPA pattern. (B) Log[RILD9/RICAD] (red) and log[RILD9/RIPK1] (blue) are plotted as a bar graph. (C) The matrix shows that C57[ME7] and C57/GPI−[ME7] prions do not differ, while both differ in at least one log[ratio] from GPI−[ME7] and GPI−/GPI−[ME7] prions.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3369955&req=5

ppat-1002746-g004: ME7 prions propagated in tgGPI− mice for one or two passages acquire novel characteristics.(A) Serially diluted brain homogenates were analyzed on LD9, CAD and PK1 cells by the CPA. After the first passage of ME7 prions in GPI− mice (GPI−[ME7]), the RI on CAD cells dropped 20-fold, most likely reflecting low titers, but increased about 30-fold after the second passage (GPI−/GPI−[ME7]), indicating adaptation to the GPI− environment. Returning prions from the first passage in GPI− brain to wild-type brain (C57/GPI−[ME7]) restored the original CPA pattern. (B) Log[RILD9/RICAD] (red) and log[RILD9/RIPK1] (blue) are plotted as a bar graph. (C) The matrix shows that C57[ME7] and C57/GPI−[ME7] prions do not differ, while both differ in at least one log[ratio] from GPI−[ME7] and GPI−/GPI−[ME7] prions.
Mentions: Typical scrapie signs were not observed in ME7-inoculated GPI− mice by 300–305 days, at which time mice showing mild clinical signs were euthanized and homogenates of their brains were subjected to the CPA. The level of infectivity of GPI−[ME7] brain homogenates as measured on CAD and PK1 cells was very low (RI<103), however LD9 cells showed a higher response (RI = 1.1×104), albeit at a level about 25 times lower than that found for C57[ME7] brains 2.8×105 (Figure 4). The GPI−[ME7] brain homogenates were infectious to wild-type mice, leading to disease at 170±0 dpi (Table S1, #20), as compared to 138±4 days for “normal” C57[ME7] (Table S1, #18b), and yielded prions indistinguishable from the original ME7, as judged by the CPA. A second transmission of GPI−[ME7] prions to GPI− mice resulted in disease at 447±64 dpi (Table S1, #21) and, interestingly, to high levels of PrPres, as determined by sandwich ELISA (Figure 1C, 1D), and infectivity, as measured on CAD cells (RI = 1.5×105; Figure 4A). GPI−/GPI−[ME7] prions differed from C57[ME7] prions by their log[RILD9/RICAD] value, -0.03 versus 0.7; whether or not they revert to the original ME7 after being returned to C57 is still under investigation (Table S1, 22.) (Figure 4B, C). In summary, repeated propagation of ME7 prions through GPI− brain resulted in a distinct alteration of their properties, reflecting progressive adaptation to the modified environment. However, prions returned from GPI−[ME7] mice to C57 mice resulted in reversion to prions indistinguishable from the original C57[ME7] prions.

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