Gregory J. Raymond

The most infectious prion protein particles

Neurodegenerative diseases such as Alzheimers, Parkinsons and the transmissible spongiform encephalopathies (TSEs) are characterized by abnormal protein deposits, often with large amyloid fibrils. However, questions have arisen as to whether such fibrils or smaller subfibrillar oligomers are the prime causes of disease. Abnormal deposits in TSEs are rich in PrPres, a protease-resistant form of the PrP protein with the ability to convert the normal, protease-sensitive form of the protein (PrPsen) into PrPres (ref. 3). TSEs can be transmitted between organisms by an enigmatic agent (prion) that contains PrPres (refs 4 and 5). To evaluate systematically the relationship between infectivity, converting activity and the size of various PrPres-containing aggregates, PrPres was partially disaggregated, fractionated by size and analysed by light scattering and non-denaturing gel electrophoresis. Our analyses revealed that with respect to PrP content, infectivity and converting activity peaked markedly in 17–27-nm (300–600 kDa) particles, whereas these activities were substantially lower in large fibrils and virtually absent in oligomers of ≤5 PrP molecules. These results suggest that non-fibrillar particles, with masses equivalent to 14–28 PrP molecules, are the most efficient initiators of TSE disease.

STRAIN-DEPENDENT DIFFERENCES IN BETA -SHEET CONFORMATIONS OF ABNORMAL PRION PROTEIN

Strain diversity in the transmissible spongiform encephalopathies (TSEs) has been proposed to be determined by variations in the conformation of the abnormal, protease-resistant form of prion protein (PrP-res). We have investigated whether infection of hamsters with three TSE strains resulted in the formation of PrP-res with different conformations using limited proteinase K (PK) digestion and infrared spectroscopy. PrP-res isolated from the brains of hamsters infected with the hyper (HY), drowsy (DY), and 263K TSE strains yielded similar SDS-polyacrylamide gel electrophoresis profiles prior to PK treatment. However, after limited digestion with PK, the PrP-res from the DY strain exhibited a fragmentation pattern that was distinct from that of the other two strains. Infrared spectra of HY and 263K PrP-res each had major absorption bands in the amide I region at 1626 and 1636 cm−1 both prior to and after digestion with PK. These bands were not evident in the DY PrP-res spectra, which had a unique band at 1629–1630 cm−1 and stronger band intensity at both 1616 and 1694–1695 cm−1. Because absorbances from 1616 to 1636 cm−1 of protein infrared spectra are attributed primarily to β-sheet structures, these findings indicate that the conformations of HY and 263K PrP-res differ from DY PrP-res at least in structural regions with β-sheet secondary structure. These results support the hypothesis that strain-specific PrP-res conformers can self-propagate by converting the normal prion protein to the abnormal conformers that induce phenotypically distinct TSE diseases.

Evidence of a molecular barrier limiting susceptibility of humans, cattle and sheep to chronic wasting disease.

Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy (TSE) of deer and elk, and little is known about its transmissibility to other species. An important factor controlling interspecies TSE susceptibility is prion protein (PrP) homology between the source and recipient species/genotypes. Furthermore, the efficiency with which the protease‐resistant PrP (PrP‐res) of one species induces the in vitro conversion of the normal PrP (PrP‐sen) of another species to the protease‐resistant state correlates with the cross‐species transmissibility of TSE agents. Here we show that the CWD‐associated PrP‐res (PrPCWD) of cervids readily induces the conversion of recombinant cervid PrP‐sen molecules to the protease‐resistant state in accordance with the known transmissibility of CWD between cervids. In contrast, PrPCWD‐induced conversions of human and bovine PrP‐sen were much less efficient, and conversion of ovine PrP‐sen was intermediate. These results demonstrate a barrier at the molecular level that should limit the susceptibility of these non‐cervid species to CWD.

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.

Mammalian Prions Generated from Bacterially Expressed Prion Protein in the Absence of Any Mammalian Cofactors

Transmissible spongiform encephalopathies (TSEs) are a group of neurodegenerative diseases that are associated with the conformational conversion of a normal prion protein, PrPC, to a misfolded aggregated form, PrPSc. The protein-only hypothesis asserts that PrPSc itself represents the infectious TSE agent. Although this model is supported by rapidly growing experimental data, unequivocal proof has been elusive. The protein misfolding cyclic amplification reactions have been recently shown to propagate prions using brain-derived or recombinant prion protein, but only in the presence of additional cofactors such as nucleic acids and lipids. Here, using a protein misfolding cyclic amplification variation, we show that prions causing transmissible spongiform encephalopathy in wild-type hamsters can be generated solely from highly purified, bacterially expressed recombinant hamster prion protein without any mammalian or synthetic cofactors (other than buffer salts and detergent). These findings provide strong support for the protein-only hypothesis of TSE diseases, as well as argue that cofactors such as nucleic acids, other polyanions, or lipids are non-obligatory for prion protein conversion to the infectious form.

Structural organization of brain-derived mammalian prions examined by hydrogen-deuterium exchange.

One of the mysteries in prion research is the structure of the infectious form of mammalian prion protein PrPSc. Here we used mass spectrometry analysis of hydrogen-deuterium exchange to examine brain-derived PrPSc. Our data indicate that, contrary to popular models, prion-protein conversion involves refolding of the entire region from residue ~80–90 to the C-terminus, which in PrPSc consists of β-strands and relatively short turns and/or loops, with no native α-helices present.

Aggregates of scrapie-associated prion protein induce the cell-free conversion of protease-sensitive prion protein to the protease-resistant state

INTRODUCTION Scrapie infection instigates the in vivo conversion of normal, protease-sensitive prion protein (PrPC) into a protease-resistant form (PrPSc) by an unknown mechanism. In vitro studies have indicated that PrPSc can induce this conversion, consistent with proposals that PrPSc itself might be the infectious scrapie agent. Using this cell-free model of the PrPC to PrPSc conversion, we have studied the dependence of conversion on reactant concentration, and the properties of the PrPSc-derived species that has converting activity. RESULTS The cell-free conversion of 35S PrPC to the proteinase K-resistant form was dependent on the reaction time and initial concentrations of PrPSc (above an apparent minimum threshold concentration) and 35S PrPC. Analysis of the physical size of the converting activity indicated that detectable converting activity was associated only with aggregates. Under mildly chaotropic conditions, which partially disaggregated PrPSc and enhanced the converting activity, the active species were heterogeneous in size, but larger than either effectively solubilized PrP or molecular weight standards of approximately 2000 kDa. CONCLUSIONS The entity responsible for the converting activity was many times larger than a soluble PrP monomer and required a threshold concentration of PrPSc. These results are consistent with a nucleated polymerization mechanism of PrPSc formation and inconsistent with a heterodimer mechanism.

Long-Term Subclinical Carrier State Precedes Scrapie Replication and Adaptation in a Resistant Species: Analogies to Bovine Spongiform Encephalopathy and Variant Creutzfeldt-Jakob Disease in Humans

ABSTRACT Cattle infected with bovine spongiform encephalopathy (BSE) appear to be a reservoir for transmission of variant Creutzfeldt-Jakob disease (vCJD) to humans. Although just over 100 people have developed clinical vCJD, millions have probably been exposed to the infectivity by consumption of BSE-infected beef. It is currently not known whether some of these individuals will develop disease themselves or act as asymptomatic carriers of infectivity which might infect others in the future. We have studied agent persistence and adaptation after cross-species infection using a model of mice inoculated with hamster scrapie strain 263K. Although mice inoculated with hamster scrapie do not develop clinical disease after inoculation with 10 million hamster infectious doses, hamster scrapie infectivity persists in brain and spleen for the life span of the mice. In the present study, we were surprised to find a 1-year period postinfection with hamster scrapie where there was no evidence for replication of infectivity in mouse brain. In contrast, this period of inactive persistence was followed by a period of active replication of infectivity as well as adaptation of new strains of agent capable of causing disease in mice. In most mice, neither the early persistent phase nor the later replicative phase could be detected by immunoblot assay for protease-resistant prion protein (PrP). If similar asymptomatic carriers of infection arise after exposure of humans or animals to BSE, this could markedly increase the danger of additional spread of BSE or vCJD infection by contaminated blood, surgical instruments, or meat. If such subclinical carriers were negative for protease-resistant PrP, similar to our mice, then the recently proposed screening of brain, tonsils, or other tissues of animals and humans by present methods such as immunoblotting or immunohistochemistry might be too insensitive to identify these individuals.

Inhibition of Protease-Resistant Prion Protein Accumulation In Vitro by Curcumin

ABSTRACT Inhibition of the accumulation of protease-resistant prion protein (PrP-res) is a prime strategy in the development of potential transmissible spongiform encephalopathy (TSE) therapeutics. Here we show that curcumin (diferoylmethane), a major component of the spice turmeric, potently inhibits PrP-res accumulation in scrapie agent-infected neuroblastoma cells (50% inhibitory concentration, ∼10 nM) and partially inhibits the cell-free conversion of PrP to PrP-res. In vivo studies showed that dietary administration of curcumin had no significant effect on the onset of scrapie in hamsters. Nonetheless, other studies have shown that curcumin is nontoxic and can penetrate the brain, properties that give curcumin advantages over inhibitors previously identified as potential prophylactic and/or therapeutic anti-TSE compounds.

Subclinical Scrapie Infection in a Resistant Species: Persistence, Replication, and Adaptation of Infectivity during Four Passages

Cross-species infection with transmissible spongiform encephalopathy agents may lead to subclinical infection and to adaptation of the infection to new species. This is of particular concern for the millions of people possibly exposed to bovine spongiform encephalopathy (BSE) by consumption of BSE-infected beef. Subclinical infection was studied by making 4 serial passages of hamster scrapie agent (263K) in mice. At each step, infectivity was followed by inoculation of hamsters and mice. Subclinical infection was demonstrated either by detection of abnormal protease-resistant prion protein (PrP-res) or in the absence of PrP-res by detection of infectivity. Replication and adaptation of hamster infectivity in mice was shown in year 2 after initial mouse passage. In third and fourth passages, dual-tropic, mouse-tropic, and hamster-tropic infectivity was found in different animals. In some cases infectivity similar to the original 263K hamster scrapie strain was found after 2 or 3 serial mouse passages totaling 1200-1550 days.