Abstract
Different transmissible spongiform encephalopathy (TSE)-associated forms of prion protein (e.g. PrPSc) can vary markedly in ultrastructure and biochemical characteristics, but each is propagated in the host. PrPSc propagation involves conversion from its normal isoform, PrPC, 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 (rPrPSen) 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 PrPSc (PrPRes). 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 PrPRes levels. For this purpose we compared the 263K and 139A scrapie strains in transgenic mice expressing P101L PrPC. Although the brains of 263K-affected mice had little immunoblot-detectable PrPRes, RT-QuIC indicated that seeding activity was comparable to that associated with a high-PrPRes strain, 139A. Thus, in this comparison, RT-QuIC seeding activity correlated more closely with infectivity than with PrPRes levels. We also found that eQuIC, which incorporates a PrPSc 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 PrPSc.
Highlights
Misfolding of cellular prion protein (PrPC) into the scrapie prion protein (PrPSc) isoform is a key event in the pathogenesis of prion disorders [1,2]
PrPSc amyloid is a prominent feature of some genetic human prion diseases such as Gerstmann-Straussler-Scheinker syndrome (GSS) [27] and prion protein cerebral amyloid angiopathy (PrP-CAA) [28]
normal brain homogenate (NBH) controls gave rPrPspon when moPrPSen23–231 was used with higher NaCl concentrations ($200 mM)
Summary
Misfolding of cellular prion protein (PrPC) into the scrapie prion protein (PrPSc) isoform is a key event in the pathogenesis of prion disorders [1,2]. In the brain PrPSc can accumulate in deposits ranging from large fibrillar amyloid plaques [21,22,23,24] to smaller diffuse nonamyloid oligomers [25,26]. In scrapie-infected transgenic mice expressing prion protein lacking the glycosylphosphatidylinositol anchor (GPI), PrPSc appears to be exclusively contained in amyloid plaques [29,30]. Both large amyloid fibrils and nonamyloid aggregates of PrPSc are associated with high levels of infectivity [13,29], but smaller non-fibrillar oligomers have been found to have the highest specific infectivity per unit protein with several scrapie strains [13,31]. Infectivity can sometimes be associated with forms of PrPSc that are largely proteinase K (PK)-sensitive (sPrPSc)
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