Dataset Information

Prion seeding activities of mouse scrapie strains with divergent PrPSc protease sensitivities and amyloid plaque content using RT-QuIC and eQuIC.

ABSTRACT: Different transmissible spongiform encephalopathy (TSE)-associated forms of prion protein (e.g. PrP(Sc)) can vary markedly in ultrastructure and biochemical characteristics, but each is propagated in the host. PrP(Sc) propagation involves conversion from its normal isoform, PrP(C), by a seeded or templated polymerization mechanism. Such a mechanism is also the basis of the RT-QuIC and eQuIC prion assays which use recombinant PrP (rPrP(Sen)) as a substrate. These ultrasensitive detection assays have been developed for TSE prions of several host species and sample tissues, but not for murine models which are central to TSE pathogenesis research. Here we have adapted RT-QuIC and eQuIC to various murine prions and evaluated how seeding activity depends on glycophosphatidylinositol (GPI) anchoring and the abundance of amyloid plaques and protease-resistant PrP(Sc) (PrP(Res)). Scrapie brain dilutions up to 10(-8) and 10(-13) were detected by RT-QuIC and eQuIC, respectively. Comparisons of scrapie-affected wild-type mice and transgenic mice expressing GPI anchorless PrP showed that, although similar concentrations of seeding activity accumulated in brain, the heavily amyloid-laden anchorless mouse tissue seeded more rapid reactions. Next we compared seeding activities in the brains of mice with similar infectivity titers, but widely divergent PrP(Res) levels. For this purpose we compared the 263K and 139A scrapie strains in transgenic mice expressing P101L PrP(C). Although the brains of 263K-affected mice had little immunoblot-detectable PrP(Res), RT-QuIC indicated that seeding activity was comparable to that associated with a high-PrP(Res) strain, 139A. Thus, in this comparison, RT-QuIC seeding activity correlated more closely with infectivity than with PrP(Res) levels. We also found that eQuIC, which incorporates a PrP(Sc) immunoprecipitation step, detected seeding activity in plasma from wild-type and anchorless PrP transgenic mice inoculated with 22L, 79A and/or RML scrapie strains. Overall, we conclude that these new mouse-adapted prion seeding assays detect diverse types of PrP(Sc).

SUBMITTER: Vascellari S

PROVIDER: S-EPMC3489776 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Prion seeding activities of mouse scrapie strains with divergent PrPSc protease sensitivities and amyloid plaque content using RT-QuIC and eQuIC.

Vascellari Sarah S Orrù Christina D CD Hughson Andrew G AG King Declan D Barron Rona R Wilham Jason M JM Baron Gerald S GS Race Brent B Pani Alessandra A Caughey Byron B

PloS one 20121105 11

Different transmissible spongiform encephalopathy (TSE)-associated forms of prion protein (e.g. PrP(Sc)) can vary markedly in ultrastructure and biochemical characteristics, but each is propagated in the host. PrP(Sc) propagation involves conversion from its normal isoform, PrP(C), by a seeded or templated polymerization mechanism. Such a mechanism is also the basis of the RT-QuIC and eQuIC prion assays which use recombinant PrP (rPrP(Sen)) as a substrate. These ultrasensitive detection assays h ...[more]

PMID: 23139828

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Project description:Although misfolding of normal prion protein (PrPC) into abnormal conformers (PrPSc) is critical for prion disease pathogenesis our current understanding of the underlying molecular pathophysiology is rudimentary. Exploiting an electrophysiology paradigm, herein we report that at least modestly proteinase K (PK)-resistant PrPSc (PrPres) species are acutely synaptotoxic. Brief exposure to ex vivo PrPSc from two mouse-adapted prion strains (M1000 and MU02) prepared as crude brain homogenates (cM1000 and cMU02) and cell lysates from chronically M1000-infected RK13 cells (MoRK13-Inf) caused significant impairment of hippocampal CA1 region long-term potentiation (LTP), with the LTP disruption approximating that reported during the evolution of murine prion disease. Proof of PrPSc (especially PrPres) species as the synaptotoxic agent was demonstrated by: significant rescue of LTP following selective immuno-depletion of total PrP from cM1000 (dM1000); modestly PK-treated cM1000 (PK+M1000) retaining full synaptotoxicity; and restoration of the LTP impairment when employing reconstituted, PK-eluted, immuno-precipitated M1000 preparations (PK+IP-M1000). Additional detailed electrophysiological analyses exemplified by impairment of post-tetanic potentiation (PTP) suggest possible heightened pre-synaptic vulnerability to the acute synaptotoxicity. This dysfunction correlated with cumulative insufficiency of replenishment of the readily releasable pool (RRP) of vesicles during repeated high-frequency stimulation utilised for induction of LTP. Broadly comparable results with LTP and PTP impairment were obtained utilizing hippocampal slices from PrPC knockout (PrPo/o) mice, with cM1000 serial dilution assessments revealing similar sensitivity of PrPo/o and wild type (WT) slices. Size fractionation chromatography demonstrated that synaptotoxic PrP correlated with PK-resistant species >100kDa, consistent with multimeric PrPSc, with levels of these species >6 ng/ml appearing sufficient to induce synaptic dysfunction. Biochemical analyses of hippocampal slices manifesting acute synaptotoxicity demonstrated reduced levels of multiple key synaptic proteins, albeit with noteworthy differences in PrPo/o slices, while such changes were absent in hippocampi demonstrating rescued LTP through treatment with dM1000. Our findings offer important new mechanistic insights into the synaptic impairment underlying prion disease, enhancing prospects for development of targeted effective therapies.

Dataset Information

Prion seeding activities of mouse scrapie strains with divergent PrPSc protease sensitivities and amyloid plaque content using RT-QuIC and eQuIC.

Publications

Prion seeding activities of mouse scrapie strains with divergent PrPSc protease sensitivities and amyloid plaque content using RT-QuIC and eQuIC.

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