Protease-Sensitive Synthetic Prions

Abstract

Prions arise when the cellular prion protein (PrPC) undergoes a self-propagating conformational change; the resulting infectious conformer is designated PrPSc. Frequently, PrPSc is protease-resistant but protease-sensitive (s) prions have been isolated in humans and other animals. We report here that protease-sensitive, synthetic prions were generated in vitro during polymerization of recombinant (rec) PrP into amyloid fibers. In 22 independent experiments, recPrP amyloid preparations, but not recPrP monomers or oligomers, transmitted disease to transgenic mice (n = 164), denoted Tg9949 mice, that overexpress N-terminally truncated PrP. Tg9949 control mice (n = 174) did not spontaneously generate prions although they were prone to late-onset spontaneous neurological dysfunction. When synthetic prion isolates from infected Tg9949 mice were serially transmitted in the same line of mice, they exhibited sPrPSc and caused neurodegeneration. Interestingly, these protease-sensitive prions did not shorten the life span of Tg9949 mice despite causing extensive neurodegeneration. We inoculated three synthetic prion isolates into Tg4053 mice that overexpress full-length PrP; Tg4053 mice are not prone to developing spontaneous neurological dysfunction. The synthetic prion isolates caused disease in 600–750 days in Tg4053 mice, which exhibited sPrPSc. These novel synthetic prions demonstrate that conformational changes in wild-type PrP can produce mouse prions composed exclusively of sPrPSc.

Progress towards structural understanding of infectious sheep PrP-amyloid

“…28 According to literature, the onset of neurological dysfunction in Syrian Gold hamsters or transgenic mice takes place between 380 and 750 days after inoculation of spontaneously generated recPrPamyloid. [29][30][31] Although our incubation periods are at the shorter end of this range, their high standard deviation also indicates the presence of low infectivity titers. Whether our relatively short incubation periods are a feature of the details of our preparation conditions, the ovine recPrP-sequence, or the associated conformational stability remains uncertain.…”

“…Whether our relatively short incubation periods are a feature of the details of our preparation conditions, the ovine recPrP-sequence, or the associated conformational stability remains uncertain. [29][30][31] After inoculation of the brain homogenates of these diseased mice into the same mouse line, 100 % of mice developed a prion disease with incubation periods characteristic for high prion infectivity. The incubation periods in transgenic ARQ mice even indicate prion infectivity as high as in brain-derived PrP Sc (see Table 1).…”

Prions

“…In other work, amyloid inoculation of Tg9949 mice overexpressing an amino-terminally truncated PrP resulted in novel, protease-sensitive, synthetic prions (Colby et al 2010). Although these strains lacked protease resistance, they caused severe neuropathology and were serially transmissible both in Tg9949 mice and in Tg mice moderately overexpressing full-length PrP.…”

“…Under the same conditions, PrP C and some forms of PrP Sc are completely hydrolyzed. Although resistance to limited proteolysis has proved to be a convenient tool for detecting PrP Sc , not all PrP Sc molecules are resistant to protease digestion Telling et al 1996;Safar et al 1998;Gambetti et al 2008;Colby et al 2010); these protease-sensitive PrP Sc forms are denoted sPrP Sc . Furthermore, PrP Sc from different species or prion strains may show different degrees of protease resistance.…”

Isolation of Novel Synthetic Prion Strains by Amplification in Transgenic Mice Coexpressing Wild-Type and Anchorless Prion Proteins

“…Inoculation of rPrP fibril preparations induced TSE disease after a prolonged incubation period in transgenic mice vastly overexpressing (16ϫ) N-terminally truncated PrP C , but many questions remained, in addition to concerns about the transgenic mouse model used (12,54). Subsequent studies have shown that a myriad of synthetic prion strains can be generated with properties modulated by the reaction conditions, albeit still via primary passage in transgenic mice overexpressing mouse PrP C (17,18). Potential issues associated with PrP C overexpression have been addressed in a hamster system, where inoculation of hamster rPrP fibrils subjected to a separate "annealing" procedure with heat, detergent, and normal brain homogenate allowed detection of infectivity after two serial passages in hamsters (35).…”