Transmissible spongiform encephalopathy strain-associated diversity of N-terminal proteinase K cleavage sites of PrPSc from scrapie-infected and bovine spongiform encephalopathy-infected mice

Assessment of the different conformational states of the abnormal prion protein (PrP^Sc) in the CNS provides an established basis for distinguishing transmissible spongiform encephalopathy (TSE) strains. PrP^Sc conformers are variably resistant to N-terminal proteinase K (PK) digestion, and analysis of the consensus products (PrP^res) by immunoassay enables effective, but relatively low-resolution differentiation. Determination of the precise N-terminal amino acid profile (N-TAAP) of PrP^res presents a potential high-resolution means of TSE-strain typing, and thus of differential disease diagnosis. This approach was evaluated using individual mice affected by model scrapie (22A, ME7, 87V and 79A) and bovine spongiform encephalopathy (BSE) (301V) strains. Nano liquid chromatography–mass spectrometry (LC-MS) was used to determine PrP^res N-terminal tryptic digestion products. Four major N-terminal tryptic peptides were generated from all mouse TSE strains investigated, corresponding with predominant N-termination of PrP^res at G⁸¹, G⁸⁵, G⁸⁹ and G⁹¹. Both the mass spectrometric abundance of the individual peptides and the ratios of pairs of these peptides were evaluated as markers of conformation in relation to their potential for strain discrimination. The yield of peptides was significantly greater for BSE than scrapie strains and the relative quantities of particular peptide pairs differed between strains. Thus, whereas peptide G⁹¹–K¹⁰⁵ was a dominant peptide from 301V, this was not the case for other strains and, significantly, the ratio of peptides G⁹¹–K¹⁰⁵:G⁸⁹–K¹⁰⁵ was substantially higher for BSE-infected compared with scrapie-infected mice. These data support the potential of the N-TAAP approach for high-resolution TSE strain typing and differential diagnosis.     

Molecular analysis of the protease-resistant prion protein in scrapie and bovine spongiform encephalopathy transmitted to ovine transgenic and wild-type mice

The existence of different strains of infectious agents involved in scrapie, a transmissible spongiform encephalopathy (TSE) of sheep and goats, remains poorly explained. These strains can, however, be differentiated by characteristics of the disease in mice and also by the molecular features of the protease-resistant prion protein (PrP(res)) that accumulates into the infected tissues. For further analysis, we first transmitted the disease from brain samples of TSE-infected sheep to ovine transgenic [Tg(OvPrP4)] and to wild-type (C57BL/6) mice. We show that, as in sheep, molecular differences of PrP(res) detected by Western blotting can differentiate, in both ovine transgenic and wild-type mice, infection by the bovine spongiform encephalopathy (BSE) agent from most scrapie sources. Similarities of an experimental scrapie isolate (CH1641) with BSE were also likewise found following transmission in ovine transgenic mice. Secondly, we transmitted the disease to ovine transgenic mice by inoculation of brain samples of wild-type mice infected with different experimental scrapie strains (C506M3, 87V, 79A, and Chandler) or with BSE. Features of these strains in ovine transgenic mice were reminiscent of those previously described for wild-type mice, by both ratios and by molecular masses of the different PrP(res) glycoforms. Moreover, these studies revealed the diversity of scrapie strains and their differences with BSE according to labeling by a monoclonal antibody (P4). These data, in an experimental model expressing the prion protein of the host of natural scrapie, further suggest a genuine diversity of TSE infectious agents and emphasize its linkage to the molecular features of the abnormal prion protein.     

Molecular analysis of the abnormal prion protein during coinfection of mice by bovine spongiform encephalopathy and a scrapie agent

Molecular features of the proteinase K-resistant prion protein (PrP res) may discriminate among prion strains, and a specific signature could be found during infection by the infectious agent causing bovine spongiform encephalopathy (BSE). To investigate the molecular basis of BSE adaptation and selection, we established a model of coinfection of mice by both BSE and a sheep scrapie strain (C506M3). We now show that the PrP res features in these mice, characterized by glycoform ratios and electrophoretic mobilities, may be undistinguishable from those found in mice infected with scrapie only, including when mice were inoculated by both strains at the same time and by the same intracerebral inoculation route. Western blot analysis using different antibodies against sequences near the putative N-terminal end of PrP res also demonstrated differences in the main proteinase K cleavage sites between mice showing either the BSE or scrapie PrP res profile. These results, which may be linked to higher levels of PrP res associated with infection by scrapie, were similar following a challenge by a higher dose of the BSE agent during coinfection by both strains intracerebrally. Whereas PrP res extraction methods used allowed us to distinguish type 1 and type 2 PrP res, differing, like BSE and scrapie, by their electrophoretic mobilities, in the same brain region of some patients with Creutzfeldt-Jakob disease, analysis of in vitro mixtures of BSE and scrapie brain homogenates did not allow us to distinguish BSE and scrapie PrP res. These results suggest that the BSE agent, the origin of which remains unknown so far but which may have arisen from a sheep scrapie agent, may be hidden by a scrapie strain during attempts to identify it by molecular studies and following transmission of the disease in mice.     

Differences in proteinase K resistance and neuronal deposition of abnormal prion proteins characterize bovine spongiform encephalopathy (BSE) and scrapie strains.

Prion diseases are associated with the accumulation of an abnormal isoform of host-encoded prion protein (PrP(Sc)). A number of prion strains can be distinguished by "glycotyping" analysis of the respective deposited PrP(Sc) compound. In this study, the long-term proteinase K resistance, the molecular mass, and the localization of PrP(Sc) deposits derived from conventional and transgenic mice inoculated with 11 different BSE and scrapie strains or isolates were examined. Differences were found in the long-term proteinase K resistance (50 microg/ml at 37 degrees C) of PrP(Sc). For example, scrapie strain Chandler or PrP(Sc) derived from field BSE isolates were destroyed after 6 hr of exposure, whereas PrP(Sc) of strains 87V and ME7 and of the Hessen1 isolate were extremely resistant to proteolytic cleavage. Nonglycosylated, proteinase K-treated PrP(Sc) of BSE isolates and of scrapie strain 87V exhibited a 1-2 kD lower molecular mass than PrP(Sc) derived from all other scrapie strains and isolates. With the exception of strain 87V, PrP(Sc) was generally deposited in the cerebrum, cerebellum, and brain stem of different mouse lines at comparable levels. Long-term proteinase resistance, molecular mass, and the analysis of PrP(Sc) deposition therefore provide useful criteria in discriminating prion strains and isolates (e.g., BSE and 87V) that are otherwise indistinguishable by the PrP(Sc) "glycotyping" technique.     

Detection and localisation of PrP(Sc) in the liver of sheep infected with scrapie and bovine spongiform encephalopathy

Prions are largely contained within the nervous and lymphoid tissue of transmissible spongiform encephalopathy (TSE) infected animals. However, following advances in diagnostic sensitivity, PrP(Sc), a marker for prion disease, can now be located in a wide range of viscera and body fluids including muscle, saliva, blood, urine and milk, raising concerns that exposure to these materials could contribute to the spread of disease in humans and animals. Previously we demonstrated low levels of infectivity in the liver of sheep experimentally challenged with bovine spongiform encephalopathy. In this study we show that PrP(Sc) accumulated in the liver of 89% of sheep naturally infected with scrapie and 100% of sheep challenged with BSE, at both clinical and preclinical stages of the disease. PrP(Sc) was demonstrated in the absence of obvious inflammatory foci and was restricted to isolated resident cells, most likely Kupffer cells.     

Characterization of thermodynamic diversity between transmissible spongiform encephalopathy agent strains and its theoretical implications.

Some transmissible spongiform encephalopathy (TSE) (or "prion") strains, notably those derived from bovine spongiform encephalopathy, are highly resistant to total inactivation by heat. When three TSE strains derived from sheep with scrapie were heated, little inactivation took place at low temperatures, but at higher temperatures, considerable inactivation occurred. The temperature at which substantial inactivation first occurred varied according to TSE strain, and it was calculated to be 70 degrees C for the 22C strain, 84 degrees C for ME7, and 97 degrees C for 22A by fitting the data to a model based on competition between a destructive and a protective reaction. However, PrP(Sc) from mice infected with a range of TSE strains retained similar resistance to proteinase K digestion after heating to below or above these temperatures, showing that the properties of PrP(Sc) responsible for proteinase resistance do not correlate with those conferring thermostability on the TSE agent. The simplest explanation of these data is that the causal agent contains a macromolecular component that is structurally independent of the host, that it varies covalently between TSE strains, and that it is protected by other macromolecular components. The model is in accord with the virino hypothesis, which proposes a host-independent informational molecule protected by the host protein PrP.     

Discriminating scrapie and bovine spongiform encephalopathy isolates by infrared spectroscopy of pathological prion protein

For the surveillance of transmissible spongiform encephalopathies (TSEs) in animals and humans, the discrimination of different TSE strains causing scrapie, BSE, or Creutzfeldt-Jakob disease constitutes a substantial challenge. We addressed this problem by Fourier transform-infrared (FT-IR) spectroscopy of pathological prion protein PrP27-30. Different isolates of hamster-adapted scrapie (263K, 22A-H, and ME7-H) and BSE (BSE-H) were passaged in Syrian hamsters. Two of these agents, 22A-H and ME7-H, caused TSEs with indistinguishable clinical symptoms, neuropathological changes, and electrophoretic mobilities and glycosylation patterns of PrP27-30. However, FT-IR spectroscopy revealed that PrP27-30 of all four isolates featured different characteristics in the secondary structure, allowing a clear distinction between the passaged TSE agents. FT-IR analysis showed that phenotypic information is mirrored in beta-sheet and other secondary structure elements of PrP27-30, also in cases where immunobiochemical typing failed to detect structural differences. If the findings of this study hold true for nonexperimental TSEs in animals and humans, FT-IR characterization of PrP27-30 may provide a versatile tool for molecular strain typing without antibodies and without restrictions to specific TSEs or mammalian species.     

A C-terminal protease-resistant prion fragment distinguishes ovine "CH1641-like" scrapie from bovine classical and L-Type BSE in ovine transgenic mice

The protease-resistant prion protein (PrP(res)) of a few natural scrapie isolates identified in sheep, reminiscent of the experimental isolate CH1641 derived from a British natural scrapie case, showed partial molecular similarities to ovine bovine spongiform encephalopathy (BSE). Recent discovery of an atypical form of BSE in cattle, L-type BSE or BASE, suggests that also this form of BSE might have been transmitted to sheep. We studied by Western blot the molecular features of PrP(res) in four "CH1641-like" natural scrapie isolates after transmission in an ovine transgenic model (TgOvPrP4), to see if "CH1641-like" isolates might be linked to L-type BSE. We found less diglycosylated PrP(res) than in classical BSE, but similar glycoform proportions and apparent molecular masses of the usual PrP(res) form (PrP(res) #1) to L-type BSE. However, the "CH1641-like" isolates differed from both L-type and classical BSE by an abundant, C-terminally cleaved PrP(res) product (PrP(res) #2) specifically recognised by a C-terminal antibody (SAF84). Differential immunoprecipitation of PrP(res) #1 and PrP(res) #2 resulted in enrichment in PrP(res) #2, and demonstrated the presence of mono- and diglycosylated PrP(res) products. PrP(res) #2 could not be obtained from several experimental scrapie sources (SSBP1, 79A, Chandler, C506M3) in TgOvPrP4 mice, but was identified in the 87V scrapie strain and, in lower and variable proportions, in 5 of 5 natural scrapie isolates with different molecular features to CH1641. PrP(res) #2 identification provides an additional method for the molecular discrimination of prion strains, and demonstrates differences between "CH1641-like" ovine scrapie and bovine L-type BSE transmitted in an ovine transgenic mouse model.     

Increased susceptibility of human-PrP transgenic mice to bovine spongiform encephalopathy infection following passage in sheep

The risk of the transmission of ruminant transmissible spongiform encephalopathy (TSE) to humans was thought to be low due to the lack of association between sheep scrapie and the incidence of human TSE. However, a single TSE agent strain has been shown to cause both bovine spongiform encephalopathy (BSE) and human vCJD, indicating that some ruminant TSEs are transmissible to humans. While the transmission of cattle BSE to humans in transgenic mouse models has been inefficient, indicating the presence of a significant transmission barrier between cattle and humans, BSE has been transmitted to a number of other species. Here, we aimed to further investigate the human transmission barrier following the passage of BSE in a sheep. Following inoculation with cattle BSE, gene-targeted transgenic mice expressing human PrP showed no clinical or pathological signs of TSE disease. However, following inoculation with an isolate of BSE that had been passaged through a sheep, TSE-associated vacuolation and proteinase K-resistant PrP deposition were observed in mice homozygous for the codon 129-methionine PRNP gene. This observation may be due to higher titers of the BSE agent in sheep or an increased susceptibility of humans to BSE prions following passage through a sheep. However, these data confirm that, contrary to previous predictions, it is possible that a sheep prion is transmissible to humans and that BSE from other species is a public health risk.     

Isolation from cattle of a prion strain distinct from that causing bovine spongiform encephalopathy

To date, bovine spongiform encephalopathy (BSE) and its human counterpart, variant Creutzfeldt-Jakob disease, have been associated with a single prion strain. This strain is characterised by a unique and remarkably stable biochemical profile of abnormal protease-resistant prion protein (PrP(res)) isolated from brains of affected animals or humans. However, alternate PrP(res) signatures in cattle have recently been discovered through large-scale screening. To test whether these also represent separate prion strains, we inoculated French cattle isolates characterised by a PrP(res) of higher apparent molecular mass--called H-type--into transgenic mice expressing bovine or ovine PrP. All mice developed neurological symptoms and succumbed to these isolates, showing that these represent a novel strain of infectious prions. Importantly, this agent exhibited strain-specific features clearly distinct from that of BSE agent inoculated to the same mice, which were retained on further passage. Moreover, it also differed from all sheep scrapie isolates passaged so far in ovine PrP-expressing mice. Our findings therefore raise the possibility that either various prion strains may exist in cattle, or that the BSE agent has undergone divergent evolution in some animals.     

Strain‐specific viral properties of variant Creutzfeldt–Jakob disease (vCJD) are encoded by the agent and not by host prion protein

Human CJD, endemic sheep scrapie, epidemic bovine spongiform encephalopathy (BSE), and other transmissible spongiform encephalopathies (TSEs), are caused by a group of related but molecularly uncharacterized infectious agents. The UK‐BSE agent infected many species, including humans where it causes variant CJD (vCJD). As in most viral infections, different TSE disease phenotypes are determined by both the agent strain and the host species. TSE strains are most reliably classified by incubation time and regional neuropathology in mice expressing wild‐type (wt) prion protein (PrP). We compared vCJD to other human and animal derived TSE strains in both mice and neuronal cultures expressing wt murine PrP. Primary and serial passages of the human vCJD agent, as well as the highly selected mutant 263K sheep scrapie agent, revealed profound strain‐specific characteristics were encoded by the agent, not by host PrP. Prion theory posits that PrP converts itself into the infectious agent, and thus short incubations require identical PrP sequences in the donor and recipient host. However, wt PrP mice injected with human vCJD brain homogenates showed dramatically shorter primary incubation times than mice expressing only human PrP, a finding not in accord with a PrP species barrier. All mouse passage brains showed the vCJD agent derived from a stable BSE strain. Additionally, both vCJD brain and monotypic neuronal cultures produced a diagnostic 19 kDa PrP fragment previously observed only in BSE and vCJD primate brains. Monotypic cultures can be used to identify the intrinsic, strain‐determining molecules of TSE infectious particles. J. Cell. Biochem. 106: 220–231, 2009. © 2008 Wiley‐Liss, Inc.     

Prion protein alleles showing a protective effect on the susceptibility of sheep to scrapie and bovine spongiform encephalopathy

The susceptibility of sheep to classical scrapie and bovine spongiform encephalopathy (BSE) is mainly influenced by prion protein (PrP) polymorphisms A136V, R154H, and Q171R, with the ARR allele associated with significantly decreased susceptibility. Here we report the protective effect of the amino acid substitution M137T, I142K, or N176K on the ARQ allele in sheep experimentally challenged with either scrapie or BSE. Such observations suggest the existence of additional PrP alleles that significantly decrease the susceptibility of sheep to transmissible spongiform encephalopathies, which may have important implications for disease eradication strategies.     

Competing intrachain interactions regulate the formation of beta-sheet fibrils in bovine PrP peptides

At the heart of the pathogenesis of transmissible spongiform encephalopathies (TSEs), such as BSE, scrapie, and Creutzfeldt-Jakob disease, lies a poorly understood structural rearrangement of PrP, an abundant glycoprotein of the nervous and lymphoid systems. The normal form (PrP(C)), rich in alpha-helix, converts into an aberrant beta-sheet-dominated form (PrP(Sc)), which seems to be at the center of the pathotoxic symptoms observed in TSEs. To understand this process better at a molecular level, we have studied the interactions between different peptides derived from bovine PrP and their structural significance. We show that two unstructured peptides derived from the central region of bovine PrP, residues 115-133 and 140-152, respectively, interact stoichiometrically under physiological conditions to generate beta-sheet-dominated fibrils. However, when both peptides are incubated in the presence of a third peptide derived from an adjoining alpha-helical region (residues 153-169), the formation of beta-sheet-rich fibrils is abolished. These data indicate that native PrP(C) helix 1 might inhibit the strong intrinsic beta-sheet-forming propensity of sequences immediately N-terminal to the globular core of PrP(C), by keeping in place intrachain interactions that would prevent these amyloidogenic regions from triggering aggregation. Moreover, these results indicate new ways in which PrP(Sc) formation could be prevented.     

PET-blot analysis contributes to BSE strain recognition in C57Bl/6 mice.

Identification of the strain of agent responsible for bovine spongiform encephalopathy (BSE) can be made histologically through the analysis of both distribution and intensity of brain vacuolar lesions after BSE transmission to mouse. Another useful way to distinguish the BSE agent from other prion strains is the study of the distribution of the abnormal prion protein (PrP(res)). For that purpose, paraffin-embedded tissue blot (PET-blot) method was applied on brains from C57Bl/6 mice infected with cattle BSE, experimental sheep BSE, or feline spongiform encephalopathy (FSE) from a cheetah. PrP(res) distribution was comparable, whichever of the three BSE agent sources was considered and was distinct from the PrP(res) distribution in C57Bl/6 mice inoculated with a French scrapie isolate or with a mouse-adapted scrapie strain (C506M3). These data confirm a common origin of infectious agent responsible for the British and French cattle BSE. They also indicate that PET-blot method appears as a precise complementary tool in prion strain studies because it offers easy and quick assessment of the PrP(res) mapping. Advantages and limits of the PET-blot method are discussed and compared with other established and validated methods of strain typing.     

Molecular behaviors of "CH1641-like" sheep scrapie isolates in ovine transgenic mice (TgOvPrP4)

Molecular analyses of the protease-resistant prion protein (PrP(res)) from a few natural scrapie isolates showed by Western blotting some partial similarities with those observed in experimental ovine bovine spongiform encephalopathy (BSE). They showed a low apparent molecular mass of unglycosylated PrP(res), although diglycosylated PrP(res) was less abundant than in ovine BSE. The prototype of such cases is the CH1641 experimental scrapie isolate. We analyzed PrP(res) molecular features from three French natural "CH1641-like" isolates, in comparison with CH1641 and BSE, after transmission of the disease in ovine transgenic mice (TgOvPrP4). One of these isolates (TR316211) behaved like the CH1641 isolate, with PrP(res) features in mice similar to those in the sheep brain. From two other isolates (O100 and O104), two distinct PrP(res) phenotypes were identified in mouse brains, with either high (h-type) or low (l-type) apparent molecular masses of unglycosylated PrP(res), the latter being similar to that observed with CH1641, TR316211, or BSE. Both phenotypes could be found in variable proportions in the brains of the individual mice. In contrast with BSE, l-type PrP(res) from "CH1641-like" isolates showed lower levels of diglycosylated PrP(res). From one of these cases (O104), a second passage in mice was performed for two mice with distinct PrP(res) profiles. This showed a partial selection of the l-type phenotype in mice infected with a mouse brain with predominant l-type PrP(res), and it was accompanied by a significant increase in the proportions of the diglycosylated band. These results are discussed in relation to the diversity of scrapie and BSE strains.     

Prion strain discrimination using luminescent conjugated polymers

The occurrence of multiple strains of prions may reflect conformational variability of PrP(Sc), a disease-associated, aggregated variant of the cellular prion protein, PrP(C). Here we used luminescent conjugated polymers (LCPs), which emit conformation-dependent fluorescence spectra, for characterizing prion strains. LCP reactivity and emission spectra of brain sections discriminated among four immunohistochemically indistinguishable, serially mouse-passaged prion strains derived from sheep scrapie, chronic wasting disease (CWD), bovine spongiform encephalopathy (BSE), and mouse-adapted Rocky Mountain Laboratory scrapie prions. Furthermore, using LCPs we differentiated between field isolates of BSE and bovine amyloidotic spongiform encephalopathy, and identified noncongophilic deposits in prion-infected deer and sheep. We found that fibrils with distinct morphologies generated from chemically identical recombinant PrP yielded unique LCP spectra, suggesting that spectral characteristic differences resulted from distinct supramolecular PrP structures. LCPs may help to detect structural differences among discrete protein aggregates and to link protein conformational features with disease phenotypes.     

Prion protein and the transmissible spongiform encephalopathies

Transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative diseases that occur in a wide variety of mammals. In humans, TSE diseases include kuru, sporadic and iatrogenic Creutzfeldt-Jakob disease (CJD), Gerstmann-Str?ussler-Scheinker syndrome (GSS), and fatal familial insomnia (FFI). So far, TSE diseases occur only rarely in humans; however, scrapie is a widespread problem in sheep, and the recent epidemic of bovine spongiform encephalopathy (BSE or mad cow disease) has seriously affected the British cattle industry. Of special concern is the recent appearance of a new variant of CJD in humans that is suspected of being caused by infections from BSE-infected cattle products. In all these diseases, an abnormal form of a host protein, prion protein (PrP), is essential for the pathogenic process. The relationship of this protein to the transmissible agent is currently the subject of great interest and controversy and is the subject of this review.     

PrP(c) mRNA, but not PrP(Sc) is found in the salivary glands of scrapie-infected sheep

Transmission studies in transmissible spongiform encephalopathies (TSEs) have become increasingly important due to the possible transmission of bovine spongiform encephalopathy to humans resulting in new variant Creutzfeldt-Jacob disease. The horizontal transmission of scrapie, a TSE of sheep, is poorly understood. Possible sources of horizontal transmission are the submandibular and parotid salivary glands. TSEs like natural sheep scrapie are characterized by the conversion of a normal protease sensitive prion protein, PrP(c), to an abnormal protease resistant prion protein, PrP(Sc). Since the presence of PrP(Sc) is an indicator of disease, the salivary glands of scrapie-infected sheep were examined for the presence of PrP(Sc). Although PrP(c) mRNA was detected in the salivary glands, PrP(Sc) was not found in the salivary glands of scrapie-infected sheep. These data suggest that the salivary glands are unlikely sources of horizontal transmission of natural sheep scrapie.     

Atypical/Nor98 scrapie infectivity in sheep peripheral tissues

Atypical/Nor98 scrapie was first identified in 1998 in Norway. It is now considered as a worldwide disease of small ruminants and currently represents a significant part of the detected transmissible spongiform encephalopathies (TSE) cases in Europe. Atypical/Nor98 scrapie cases were reported in ARR/ARR sheep, which are highly resistant to BSE and other small ruminants TSE agents. The biology and pathogenesis of the Atypical/Nor98 scrapie agent in its natural host is still poorly understood. However, based on the absence of detectable abnormal PrP in peripheral tissues of affected individuals, human and animal exposure risk to this specific TSE agent has been considered low. In this study we demonstrate that infectivity can accumulate, even if no abnormal PrP is detectable, in lymphoid tissues, nerves, and muscles from natural and/or experimental Atypical/Nor98 scrapie cases. Evidence is provided that, in comparison to other TSE agents, samples containing Atypical/Nor98 scrapie infectivity could remain PrP(Sc) negative. This feature will impact detection of Atypical/Nor98 scrapie cases in the field, and highlights the need to review current evaluations of the disease prevalence and potential transmissibility. Finally, an estimate is made of the infectivity loads accumulating in peripheral tissues in both Atypical/Nor98 and classical scrapie cases that currently enter the food chain. The results obtained indicate that dietary exposure risk to small ruminants TSE agents may be higher than commonly believed.