Suzette A. Priola

Ultrasensitive detection of scrapie prion protein using seeded conversion of recombinant prion protein.

The scrapie prion protein isoform, PrPSc, is a prion-associated marker that seeds the conformational conversion and polymerization of normal protease-sensitive prion protein (PrP-sen). This seeding activity allows ultrasensitive detection of PrPSc using cyclical sonicated amplification (PMCA) reactions and brain homogenate as a source of PrP-sen. Here we describe a much faster seeded polymerization method (rPrP-PMCA) which detects ≥50 ag of hamster PrPSc (≈0.003 lethal dose) within 2–3 d. This technique uses recombinant hamster PrP-sen, which, unlike brain-derived PrP-sen, can be easily concentrated, mutated and synthetically tagged. We generated protease-resistant recombinant PrP fibrils that differed from spontaneously initiated fibrils in their proteolytic susceptibility and by their infrared spectra. This assay could discriminate between scrapie-infected and uninfected hamsters using 2-μl aliquots of cerebral spinal fluid. This method should facilitate the development of rapid, ultrasensitive prion assays and diagnostic tests, in addition to aiding fundamental studies of structure and mechanism of PrPSc formation.

Molecular assessment of the potential transmissibilities of BSE and scrapie to humans

More than a million cattle infected with bovine spongiform encephalopathy (BSE) may have entered the human food chain. Fears that BSE might transmit to man were raised when atypical cases of Creutzfeldt–Jakob disease (CJD), a human transmissible spongiform encephalopathy (TSE), emerged in the UK,. In BSE and other TSE diseases, the conversion of the protease-sensitive host prion protein (PrP-sen) to a protease-resistant isoform (PrP-res) is an important event in pathogenesis. Biological aspects of TSE diseases are reflected in the specificities of in vitro PrP conversion reactions. Here we show that there is a correlation between in vitro conversion efficiencies and known transmissibilities of BSE, sheep scrapie and CJD. On this basis, we used an in vitro system to gauge the potential transmissibility of scrapie and BSE to humans. We found limited conversion of human PrP-sen to PrP-res driven by PrP-res associated with both scrapie (PrPSc) and BSE (PrPBSE). The efficiencies of these heterologous conversion reactions were similar but much lower than those of relevant homologous conversions. Thus the inherent ability of these infectious agents of BSE and scrapie to affect humans following equivalent exposure may be finite but similarly low.

Simplified ultrasensitive prion detection by recombinant PrP conversion with shaking

To the editor: A key problem in managing prion diseases is the lack of a rapid, practical assay for prions (infectivity) at low-level infectious, or sub-infectious, amounts. Prion diseases involve the accumulation of a pathological, typically protease-resistant form of prion protein, termed PrPSc, which appears to propagate itself in infected hosts by inducing the conversion of its normal hostencoded precursor, PrP-sen, into additional PrPSc (refs. 1–4). In crude brain homogenates, PrPSc and infectivity can be amplified from endogenous PrP-sen during multiple rounds of intermittent sonication and serial dilution into fresh normal brain homogenate2,4. This ultrasensitive assay, termed PMCA, allows detection of ~1 ag of PrPSc in ~3 weeks5. To improve the speed and practicality of prion detection assays, we recently developed a cell-free conversion reaction that supports sustained PrPSc-seeded conversion of recombinant PrP-sen (rPrP-sen) to specific protease-resistant (rPrP-res) forms. This method (which we previously reported in Nature Methods), called rPrP-PMCA, uses periodic sonication and serial reaction rounds of the PMCA method, but is faster6. To circumvent problems associated with sonication in the PMCA and rPrP-PMCA methods (see Supplementary Results online), we have now developed a new prion assay, abbreviated QUIC for quaking-induced conversion, which uses rPrP-sen as a substrate and automated tube shaking rather than sonication. This assay can detect about one lethal prion dose within a day, and is faster and simpler than previous described PMCA6 and rPrP-PMCA5 assays. Initial testing of QUIC reaction conditions revealed that periodic shaking enhanced PrPSc-seeded conversion of hamster rPrP-sen (residues 23–231) into PK-resistant conversion products (rPrP-res(Sc), where (Sc) refers to seeding by PrPSc; Supplementary Fig. 1 and Supplementary Methods online). Consistent with our previous observations with rPrP-PMCA reactions6, the rPrP-res(Sc) reaction products had 17-, 13-, 12and 11-kDa fragments, which represented different C-terminal PrP fragments (Supplementary Fig. 2 online). These results showed that periodic shaking could substitute for sonication in promoting rPrP-res(Sc) formation. Additional experiments revealed that rPrP-res(Sc) generation was also sensitive to rPrP-sen concentration, reaction volume (Supplementary Fig. 1), reaction time (Supplementary Fig. 2), number of serial reactions (Supplementary Fig. 3 online), temperature (Supplementary Fig. 4 online) and shaking cycle (Supplementary Results). In QUIC reactions performed at 45 °C, we observed rPrP-res(Sc) formation in single 46-h QUIC reactions seeded with ≥100 ag of PrPSc (Fig. 1a). In contrast, 21 negative control reactions seeded with comparable dilutions of normal brain homogenate or buffer alone produced no rPrP-res (Fig. 1b). We obtained results similar to those shown in Figure 1a,b in an independent repeat experiment done in triplicate (data not shown). When we diluted products of PrPSc-seeded reactions 1,000-fold into fresh rPrP-sen to seed the subsequent reaction rounds, we observed strong propagation of rPrP-res(Sc) through at least 4 serial reactions (Supplementary Fig. 5 online). Elevation of QUIC reaction temperatures accelerated rPrP-res(Sc) formation. At 55 °C, we detected rPrP-res(Sc) in single 8-h reactions seeded with as little as 10 fg PrPSc (~2 lethal intracerebral doses; Supplementary Fig. 4). We detected 1 fg in 18-h reactions (Supplementary Fig. 6 online). At 65 °C, we detected 100 fg PrPSc seed with a 4-h reaction (Supplementary Fig. 4). However, at 65 °C, there was also 25 20

Astrocyte-specific expression of hamster prion protein (PrP) renders PrP knockout mice susceptible to hamster scrapie

Transmissible spongiform encephalopathies are characterized by spongiosis, astrocytosis and accumulation of PrPSc, an isoform of the normal host protein PrPC. The exact cell types responsible for agent propagation and pathogenesis are still uncertain. To determine the possible role of astrocytes, we generated mice devoid of murine PrP but expressing hamster PrP transgenes driven by the astrocyte‐specific GFAP promoter. After inoculation with hamster scrapie, these mice accumulated infectivity and PrPSc to high levels, developed severe disease after 227 ± 5 days and died 7 ± 4 days later. Therefore, astrocytes could play an important role in scrapie pathogenesis, possibly by an indirect toxic effect on neurons. Interestingly, mice expressing the same transgenes but also endogenous murine PrP genes propagated infectivity without developing disease.

Neuron-specific expression of a hamster prion protein minigene in transgenic mice induces susceptibility to hamster scrapie agent

Abstract To study the effect of cell type-restricted hamster PrP expression on susceptibility to the hamster scrapie agent, we generated transgenic mice using a 1 kb hamster cDNA clone containing the 0.76 kb HPrP open reading frame under control of the neuron-specific enolase promoter. In these mice, expression of HPrP was detected only in brain tissue, with highest levels found in neurons of the cerebellu, hippocampus, thalamus, and cerebral cortex. These transgenic mice were susceptible to infection by the 263K strain of hamster scrapie with an average incubation period of 93 days, compared to 72 days in normal hamsters. In contrast, nontransgenic mice were not susceptible to this agent. These results indicate that neuron-specific expression of the 1 kb HPrP minigene including the HPrP open-reading frame is sufficient to mediate susceptibility to hamster scraple, and that HPrP expression in nonneuronal brain cells is not necessary to overcome the TSE species barrier.

Susceptibility of Common Fibroblast Cell Lines to Transmissible Spongiform Encephalopathy Agents

The risk of contamination of tissue culture cells with transmissible spongiform encephalopathy (TSE) agents as a result of the use of animal products as medium components has been considered to be low, in part, because only a few brain-derived cell lines have been reported to be susceptible to TSE infection. In the present study, we demonstrate that the common laboratory fibroblast cell lines NIH/3T3 and L929, which express low levels of cellular mouse prion protein, are susceptible to infection with mouse-adapted scrapie. Our results show that the susceptibility of a cell line to TSE infection cannot be predicted on the basis of its tissue origin or its level of expression of the cellular prion protein, and they suggest that any cell line expressing normal host prion protein could have the potential to support propagation of TSE agents. Thus, testing of cells for TSE susceptibility might be necessary for all cell lines that are routinely used in vaccine production and in other medical applications.

Multiple Amino Acid Residues within the Rabbit Prion Protein Inhibit Formation of Its Abnormal Isoform

ABSTRACT Transmissible spongiform encephalopathies (TSEs) are neurological diseases that are associated with the conversion of the normal host-encoded prion protein (PrP-sen) to an abnormal protease-resistant form, PrP-res. Transmission of the TSE agent from one species to another is usually inefficient and accompanied by a prolonged incubation time. Species barriers to infection by the TSE agent are of particular importance given the apparent transmission of bovine spongiform encephalopathy to humans. Among the few animal species that appear to be resistant to infection by the TSE agent are rabbits. They survive challenge with the human kuru and Creutzfeldt-Jakob agents as well as with scrapie agent isolated from sheep or mice. Species barriers to the TSE agent are strongly influenced by the PrP amino acid sequence of both the donor and recipient animals. Here we show that rabbit PrP-sen does not form PrP-res in murine tissue culture cells persistently infected with the mouse-adapted scrapie agent. Unlike other TSE species barriers that have been studied, critical amino acid residues that inhibit PrP-res formation are located throughout the rabbit PrP sequence. Our results suggest that the resistance of rabbits to infection by the TSE agent is due to multiple rabbit PrP-specific amino acid residues that result in a PrP structure that is unable to refold to the abnormal isoform associated with disease.

Glycosylation influences cross-species formation of protease-resistant prion protein

A key event in the transmissible spongiform encephalopathies (TSEs) is the formation of aggregated and protease‐resistant prion protein, PrP‐res, from a normally soluble, protease‐sensitive and glycosylated precursor, PrP‐sen. While amino acid sequence similarity between PrP‐sen and PrP‐res influences both PrP‐res formation and cross‐species transmission of infectivity, the influence of co‐ or post‐translational modifications to PrP‐sen is unknown. Here we report that, if PrP‐sen and PrP‐res are derived from different species, PrP‐sen glycosylation can significantly affect PrP‐res formation. Glycosylation affected PrP‐res formation by influencing the amount of PrP‐sen bound to PrP‐res, while the amino acid sequence of PrP‐sen influenced the amount of PrP‐res generated in the post‐binding conversion step. Our results show that in addition to amino acid sequence, co‐ or post‐translational modifications to PrP‐sen influence PrP‐res formation in vitro. In vivo, these modifications might contribute to the resistance to infection associated with transmission of TSE infectivity across species barriers.

Susceptibilities of nonhuman primates to chronic wasting disease.

A species barrier may protect humans from this disease.

N-terminal Truncation of Prion Protein Affects Both Formation and Conformation of Abnormal Protease-resistant Prion Protein Generatedin Vitro

Transmissible spongiform encephalopathy diseases are characterized by conversion of the normal protease-sensitive host prion protein, PrP-sen, to an abnormal protease-resistant form, PrP-res. In the current study, deletions were introduced into the flexible tail of PrP-sen (23) to determine if this region was required for formation of PrP-res in a cell-free assay. PrP-res formation was significantly reduced by deletion of residues 34–94 relative to full-length hamster PrP. Deletion of another nineteen amino acids to residue 113 further reduced the amount of PrP-res formed. Furthermore, the presence of additional proteinase K cleavage sites indicated that deletion to residue 113 generated a protease-resistant product with an altered conformation. Conversion of PrP deletion mutants was also affected by post-translational modifications to PrP-sen. Conversion of unglycosylated PrP-sen appeared to alter both the amount and the conformation of protease-resistant PrP-res produced from N-terminally truncated PrP-sen. The N-terminal region also affected the ability of hamster PrP to block mouse PrP-res formation in scrapie-infected mouse neuroblastoma cells. Thus, regions within the flexible N-terminal tail of PrP influenced interactions required for both generating and disrupting PrP-res formation.