Infectivity of scrapie prion protein (PrPSc) following in vitro digestion with bovine gastrointestinal microbiota

The influence of a complex microflora residing in the gastrointestinal tract of cattle on the prion protein plays a crucial role with respect to early pathogenesis and the potential infectivity of faeces resulting in contamination of the environment. It is unknown whether infectious prion proteins, considered to be very stable, are inactivated by microbial processes in the gastrointestinal tract of animals during digestion. In our previous study it was shown that the scrapie-associated prion protein was degraded by ruminal and colonic microbiota of cattle, as indicated by a loss of anti-prion antibody 3F4 immunoreactivity in Western blot. Subsequently, in this study hamster bioassays with the pre-treated samples were performed. Although the PrP(Sc) signal was reduced up to immunochemically undetectable levels within 40 h of pre-treatment, significant residual prion infectivity was retained after degradation of infected hamster brain through the gastrointestinal microflora of cattle. The data presented here show that the loss of anti-prion antibody 3F4 immunoreactivity is obviously not correlated with a biological inactivation of PrP(Sc). These results highlight the deficiency of using Western blot in transmissible spongiform encephalopathies inactivation assessment studies and, additionally, point to the possibility of environmental contamination with faeces containing PrP(Sc) following an oral ingestion of prions.     

Generation of genuine prion infectivity by serial PMCA

Prions are the causative infectious agents of transmissible spongiform encephalopathies (TSEs). They are thought to arise from misfolding and aggregation of the prion protein (PrP). In serial transmission protein misfolding cyclic amplification (sPMCA) experiments, newly formed misfolded and proteinase K-resistant PrP (PrPres) catalysed the structural conversion of cellular prion protein (PrP(C)) as efficiently as PrP(Sc) from the brain of scrapie-infected (263K) hamsters confirming an autocatalytic misfolding cascade as postulated by the prion hypothesis. However, the fact that PrPres generated in vitro was associated with approximately 10 times less infectivity than an equivalent quantity of brain-derived PrP(Sc) casts doubt on the "protein-only" hypothesis of prion propagation and backs theories that suggest there are additional molecular species of infectious PrP or other agent-associated factors. By combining sPMCA with prion delivery on suitable carrier particles we were able to resolve the apparent discrepancy between the amount of PrPres and infectivity which we were then able to relate to differences in the size distribution of PrP aggregates and consecutive differences in regard to biological clearance. These findings demonstrate that we have designed an experimental set-up yielding in vitro generated prions that are indistinguishable from prions isolated from scrapie-infected hamster brain in terms of proteinase K resistance, autocatalytic conversion activity, and - most notably - specific biological infectivity.     

Alymphoplasia mice are resistant to prion infection via oral route

The major cause of infection in animal prion diseases is thought to be consumption of prion-contaminated stuff. There is evidence that the enteric nerve system (ENS) and gut-associated lymphoid tissues (GATL) are involved in the establishment of prion infection through alimentary tract. To elucidate the initial entry port for prion, we inoculated prion to alymphoplasia (aly) mice showing a deficiency in systemic lymph nodes and Peyer's patches. The aly/aly mice were susceptible to prion infection by intra-cranial inoculation and there were no differences in incubation periods between aly/aly mice and wild-type C57BL/6J mice. Incubation periods in aly/aly mice were about 20 days longer than those in C57BL/6J mice with the intra-peritoneal inoculation. The aly/aly mice were completely resistant to prion infection by per os administration, while C57BL/6J mice were sensitive as they entered the terminal stage of disease around 300 days post inoculation. PrP(Sc) were detected in the intestine and spleen of C57BL/6J mice inoculated with prion intraperitoneally or orally; however PrP(Sc) was not detected in the spleen and intestine of aly/aly mice. Prion infectivity was detected in the intestines and spleens of prion-inoculated C57BL/6J mice, even after the early stages of exposure, while no infectivity was detected in these tissues of prion-inoculated aly/aly mice. No apparent differences were observed in the organization of the enteric nerve system between wild-type and aly/aly mice. These results indicate that GALT rather than ENS acts as the primary entry port for prion after oral exposure.     

Strategies for eliminating PrP(c) as substrate for prion conversion and for enhancing PrP(Sc) degradation

Prion diseases are fatal neurodegenerative infectious disorders for which no therapeutic or prophylactic regimens exist. Our work aims to eliminate PrP(c) as substrate for the conversion into PrP(Sc) and to increase the cellular clearance capacity of PrP(Sc). In order to achieve the first objective, we used chemical compounds which interfere with the subcellular trafficking of PrP(c), e.g. by intracellular re-routing. Recently, we found that PrP(c) requires cholesterol for cell surface localisation. Treatment with mevinolin significantly reduced the amount of cell surface PrP(c) and led to its accumulation in the Golgi compartment. These data show that cholesterol is essential for the cell surface localisation of PrP(c), which is in turn known to be necessary for the formation of PrP(Sc). Another anti-prion strategy uses RNA and peptide aptamers directed against PrP(c). We have selected peptide aptamers using a constrained peptide library presented on the active site loop of the Escherichia coli protein TrxA in a Y2H screen. Several peptides reproducibly binding to PrP(c) in several assays were identified. Preliminary data indicate that selected peptide aptamers are able to interfere with prion propagation in prion-infected cells. To obtain additive effects we have tried to clarify cellular mechanisms that enable cells to clear prion infectivity. This goal was achieved by selective interference in intracellular signalling pathways which apparently also increase the cellular autophagy machinery. Finally, we have tried to establish an active auto-vaccination approach directed against PrP, which gave some positive preliminary results in the mouse system. This might open the door to classical immunological interference techniques.     

Prion agent diversity and species barrier

Mammalian prions are the infectious agents responsible for transmissible spongiform encephalopathies (TSE), a group of fatal, neurodegenerative diseases, affecting both domestic animals and humans. The most widely accepted view to date is that these agents lack a nucleic acid genome and consist primarily of PrP(Sc), a misfolded, aggregated form of the host-encoded cellular prion protein (PrP(C)) that propagates by autocatalytic conversion and accumulates mainly in the brain. The BSE epizooty, allied with the emergence of its human counterpart, variant CJD, has focused much attention on two characteristics that prions share with conventional infectious agents. First, the existence of multiple prion strains that impose, after inoculation in the same host, specific and stable phenotypic traits such as incubation period, molecular pattern of PrP(Sc) and neuropathology. Prion strains are thought to be enciphered within distinct PrP(Sc) conformers. Second, a transmission barrier exists that restricts the propagation of prions between different species. Here we discuss the possible situations resulting from the confrontation between species barrier and prion strain diversity, the molecular mechanisms involved and the potential of interspecies transmission of animal prions, including recently discovered forms of TSE in ruminants.     

Detergents modify proteinase K resistance of PrP Sc in different transmissible spongiform encephalopathies (TSEs)

Prion diseases are diagnosed by the detection of their proteinase K-resistant prion protein fragment (PrP(Sc)). Various biochemical protocols use different detergents for the tissue preparation. We found that the resistance of PrP(Sc) against proteinase K may vary strongly with the detergent used. In our study, we investigated the influence of the most commonly used detergents on eight different TSE agents derived from different species and distinct prion disease forms. For a high throughput we used a membrane adsorption assay to detect small amounts of prion aggregates, as well as Western blotting. Tissue lysates were prepared using DOC, SLS, SDS or Triton X-100 in different concentrations and these were digested with various amounts of proteinase K. Detergents are able to enhance or diminish the detectability of PrP(Sc) after proteinase K digestion. Depending on the kind of detergent, its concentration - but also on the host species that developed the TSE and the disease form or prion type - the detectability of PrP(Sc) can be very different. The results obtained here may be helpful during the development or improvement of a PrP(Sc) detection method and they point towards a detergent effect that can be additionally used for decontamination purposes. A plausible explanation for the detergent effects described in this article could be an interaction with the lipids associated with PrP(Sc) that may stabilize the aggregates.     

Prion protein self-interaction in prion disease therapy approaches

Transmissible spongiform encephalopathies (TSEs) or prion diseases are unique disorders that are not caused by infectious micro-organisms (bacteria or fungi), viruses or parasites, but rather seem to be the result of an infectious protein. TSEs are comprised of fatal neurodegenerative disorders affecting both human and animals. Prion diseases cause sponge-like degeneration of neuronal tissue and include (among others) Creutzfeldt-Jacob disease in humans, bovine spongiform encephalopathy (BSE) in cattle and scrapie in sheep. TSEs are characterized by the formation and accumulation of transmissible (infectious) disease-associated protease-resistant prion protein (PrP(Sc)), mainly in tissues of the central nervous system. The exact molecular processes behind the conversion of PrP(C) into PrP(Sc) are not clearly understood. Correlations between prion protein polymorphisms and disease have been found, however in what way these polymorphisms influence the conversion processes remains an enigma; is stabilization or destabilization of the prion protein the basis for a higher conversion propensity? Apart from the disease-associated polymorphisms of the prion protein, the molecular processes underlying conversion are not understood. There are some notions as to which regions of the prion protein are involved in refolding of PrP(C) into PrP(Sc) and where the most drastic structural changes take place. Direct interactions between PrP(C) molecules and/or PrP(Sc) are likely at the basis of conversion, however which specific amino acid domains are involved and to what extent these domains contribute to conversion resistance/sensitivity of the prion protein or the species barrier is still unknown.     

Enrichment of prion protein in exosomes derived from ovine cerebral spinal fluid

Prion diseases are transmissible neurodegenerative disorders affecting humans and a wide variety of animal species including sheep and cattle. The transmissible agent, the prion, is an abnormally folded form (PrP(Sc)) of the host encoded cellular prion protein (PrP(C)). Distribution of the prion protein in the fluids of species susceptible to these diseases is of importance to human health and the iatrogenic spread of prion disease. Aside from blood which is confirmed to be a source of prion infectivity, it is currently unclear which other body fluids harbor a significant transmission risk. In the current study we examined two ovine fluids; pseudo-afferent lymph and cerebral spinal fluid (CSF), for the presence of exosomes and concurrent enrichment of the normal, cellular form of the prion protein (PrP(C)). Here we demonstrate the existence of exosomes in both pseudo-afferent lymph and CSF isolated from sheep. In the CSF derived exosomes we were able to show an enrichment of PrP(C) over unfractionated CSF. This experimental approach suggests that CSF derived exosomes could be used as a novel means of detecting abnormal forms of the prion protein and provide an in vivo link between these vesicles and prion disease pathogenesis.     

Recent developments in prion disease research: diagnostic tools and in vitro cell culture models

After prion infection, an abnormal isoform of prion protein (PrP(Sc)) converts the cellular isoform of prion protein (PrP(C)) into PrP(Sc). PrP(C)-to-PrP(Sc) conversion leads to PrP(Sc) accumulation and PrP(C) deficiency, contributing etiologically to induction of prion diseases. Presently, most of the diagnostic methods for prion diseases are dependent on PrP(Sc) detection. Highly sensitive/accurate specific detection of PrP(Sc) in many different samples is a prerequisite for attempts to develop reliable detection methods. Towards this goal, several methods have recently been developed to facilitate sensitive and precise detection of PrP(Sc), namely, protein misfolding cyclic amplification, conformation-dependent immunoassay, dissociation-enhanced lanthanide fluorescent immunoassay, capillary gel electrophoresis, fluorescence correlation spectroscopy, flow microbead immunoassay, etc. Additionally, functionally relevant prion-susceptible cell culture models that recognize the complexity of the mechanisms of prion infection have also been pursued, not only in relation to diagnosis, but also in relation to prion biology. Prion protein (PrP) gene-deficient neuronal cell lines that can clearly elucidate PrP(C) functions would contribute to understanding of the prion infection mechanism. In this review, we describe the trend in recent development of diagnostic methods and cell culture models for prion diseases and their potential applications in prion biology.     

An overview of transmissible spongiform encephalopathies

Transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative disorders of humans and animals associated with an accumulation of abnormal isoforms of prion protein (PrP) in nerve cells. The pathogenesis of TSEs involves conformational conversions of normal cellular PrP (PrP(c)) to abnormal isoforms of PrP (PrP(Sc)). While the protein-only hypothesis has been widely accepted as a causal mechanism of prion diseases, evidence from more recent research suggests a possible involvement of other cellular component(s) or as yet undefined infectious agent(s) in PrP pathogenesis. Although the underlying mechanisms of PrP strain variation and the determinants of interspecies transmissibility have not been fully elucidated, biochemical and molecular findings indicate that bovine spongiform encephalopathy in cattle and new-variant Creutzfeldt-Jakob disease in humans are caused by indistinguishable etiological agent(s). Cumulative evidence suggests that there may be risks of humans acquiring TSEs via a variety of exposures to infected material. The development of highly precise ligands is warranted to detect and differentiate strains, allelic variants and infectious isoforms of these PrPs. This article describes the general features of TSEs and PrP, the current understanding of their pathogenesis, recent advances in prion disease diagnostics, and PrP inactivation.     

Conformational change in hamster scrapie prion protein (PrP27-30) associated with proteinase K resistance and prion infectivity

The scrapie prion protein (PrP27-30) is a crucial component of the prion and is responsible for its transmissibility. Structural information on this protein is limited because it is insoluble and shows aggregated properties. In this study, PrP27-30 was effectively dispersed using sonication under the weak alkaline condition. Subsequently, the small PrP27-30 aggregates were subjected to different pH, heat, and denaturing conditions. The loss of proteinase K (PK) resistance of PrP27-30 and prion infectivity were monitored along with spectroscopic changes. Prion inactivation could not be achieved by the loss of PK resistance alone; a significant loss of the PrP27-30 amyloid structure, which was represented by a decrease in thioflavin T fluorescence, was required for the loss of transmissibility.     

Susceptibility of transgenic mice expressing chimeric sheep,bovine and human PrP genes to sheep scrapie

The use of Transgenic (Tg) mice expressing chimeric sheep/mouse (Sh/Mo) prion protein (PrP) and chimeric bovine/mouse (Bo/Mo) PrP genes was evaluated as a sheep scrapie model. We also investigated the potential for the transmission of sheep scrapie to a human/mouse (Hu/Mo) PrP Tg mouse line. The Sh/Mo PrP and Bo/Mo PrP Tg Prnp(+/+) or Prnp(0/0) mouse lines were inoculated intracerebrally with brain homogenates from three sheep with natural scrapie (KU, Y5 or S2). Incubation periods were slightly shorter in Sh/Mo PrP Tg Prnp(+/+), than in non-Tg mice inoculated with KU brain homogenate. In contrast, the incubation period was significantly prolonged (p<0.05) in Bo/Mo PrP Tg Prnp(+/+) mice inoculated with KU brain homogenate. The incubation period was significantly longer in all Tg Prnp(+/+) and Prnp(0/0), than in non-Tg mice (p<0.01) inoculated withY5 brain homogenate. None of the Tg Prnp(0/0) mice inoculated with S2 brain homogenate developed clinical signs and PrP(Sc) was undetectable in their brains. These results suggested that expression of the Sh/Mo PrP or Bo/Mo PrP transgenes does not confer susceptibility to sheep prions upon mice, and thus none of the Tg mouse lines could be a suitable model of sheep scrapie. Hu/Mo PrP Tg Prnp(0/0) mice inoculated with natural and experimental scrapie or mouse prions did not develop clinical signs of scrapie and PrP(Sc) was undetectable. These results suggested that neither sheep nor mouse strains of scrapie are highly transmissible to humans.     

In situ detection of cellular and abnormal isoforms of prion protein in brains of cattle with bovine spongiform encephalopathy and sheep with scrapie by use of a histoblot technique.

To detect prion protein, brains from 5 cattle naturally affected with bovine spongiform encephalopathy (BSE) and 3 sheep naturally affected with scrapie were examined and compared with brains of normal cattle and sheep using a histoblot technique. The technique enabled the in situ distinctive detection of the cellular (PrP(C)) and abnormal (PrP(Sc)) isoforms of the prion protein. In BSE- or scrapie-affected brains, the Prp(C) signal decreased, especially in those areas where the PrP(Sc) signal was detected.     

Cellular pathogenesis in prion diseases

Prion diseases are characterised by neuronal loss, vacuolation (spongiosis), reactive astrocytosis, microgliosis and in most cases by the accumulation in the central nervous system of the abnormal prion protein, named PrP(Sc). In this review on the "cellular pathogenesis in prion diseases", we have chosen to highlight the main mechanisms underlying the impact of PrP(C)/PrP(Sc) on neurons: the neuronal dysfunction, the neuronal cell death and its relation with PrP(Sc) accumulation, as well as the role of PrP(Sc) in the microglial and astrocytic reaction.     

Counting of single prion particles bound to a capture-antibody surface (surface-FIDA)

Hitherto accredited prion tests use the PK resistance of PrP(Sc), the pathogenic isoform of the prion protein, as a marker for the disease. Because of variations in the amount of disease-related aggregated PrP, which is not PK-resistant, these prion tests offer only limited sensitivity. Therefore, a prion detection method that does not rely on PK digestion would allow for the detection of both PK-resistant as well as PK-sensitive PrP(Sc). Furthermore, single particle counting is more sensitive than methods measuring an integrated signal. Our new test system is based on dual-colour fluorescence correlation spectroscopy (FCS). This method quantifies the number of protein aggregates that have been simultaneously labelled with two different antibodies using dual-colour fluorescence intensity distribution analysis (2D-FIDA). This only counts PrP aggregates, and not PrP monomers. To increase the sensitivity, PrP(Sc) was concentrated in a two-dimensional space by immobilizing it so that the antibodies could be captured on the surface of the slide (surface-FIDA). When the surface was systematically scanned, even single prion particles were detected. Using this new technique, the sensitivity to identify samples from scrapie-infected hamster as well as BSE-infected cattle can be dramatically increased in comparison with identification using FIDA in solution.     

Effect of tissue deterioration on postmortem BSE diagnosis by immunobiochemical detection of an abnormal isoform of prion protein

Surveillance for bovine spongiform encephalopathy (BSE) in fallen stock in Japan is conducted with a commercial enzyme-linked immunosorbent assay (ELISA) for mass screening, with Western blotting (WB) and immunohistochemistry performed for confirmation of the ELISA. All tests are based on immunological detection of an abnormal isoform of the prion protein (PrP(Sc)) in brain tissues, which have sometimes deteriorated by the time samples from fallen stock reach a diagnostic laboratory. To evaluate BSE surveillance procedures for fallen stock, we examined PrP(Sc) detection from artificially deteriorated BSE-affected bovine brain tissues with a commercial ELISA kit and compared the results with those of WB. The optical density (OD) values of the ELISA decreased with advancing deterioration of the tissues, whereas no reduction in the signal for PrP(Sc) was observed in WB, even when performed after 4 days of incubation at 37 degrees C. The progressive decrease in the OD values in the ELISA appear to be caused by a partial loss of the N-terminal moiety of PrP(Sc) due to digestion by endogeneous and/or contaminated microbial enzymes, and by the presence of ELISA inhibitors that are generated in deteriorated tissues. These results suggest that WB is the most reliable test for fallen stock, especially for cattle brains within decaying carcasses.     

Experimental transmission of US scrapie agent by nasal, peritoneal, and conjunctival routes to genetically susceptible sheep

Scrapie is a naturally occurring fatal neurodegenerative disease of sheep and goats. This study documents incubation periods, pathologic findings, and distribution of abnormal prion proteins (PrP(Sc)) by immunohistochemistry in tissues of genetically susceptible sheep inoculated with US sheep scrapie agent. Four-month-old Suffolk lambs (QQ at codon 171) were inoculated by 1 of 3 different routes (nasal, peritoneal, and conjunctival) with an inoculum (No. 13-7) consisting of a pool of scrapie-affected sheep brains. Except for 3 sheep, all inoculated animals were euthanized when advanced clinical signs of scrapie were observed between 19 and 46 months postinoculation (MPI). Spongiform lesions in the brains and labeling of PrP(Sc) in central nervous system and lymphoid tissues were present in these sheep. One intranasally inoculated sheep euthanized at 12 MPI had presence of PrP(Sc) that was confined to the pharyngeal tonsil. These results indicate that the upper respiratory tract, specifically the pharyngeal tonsil, may serve as a portal of entry for prion protein in scrapie-infected environments.     

Properties of L-type bovine spongiform encephalopathy in intraspecies passages

The origin and transmission routes of atypical bovine spongiform encephalopathy (BSE) remain unclear. To assess whether the biological and biochemical characteristics of atypical L-type BSE detected in Japanese cattle (BSE/JP24) are conserved during serial passages within a single host, 3 calves were inoculated intracerebrally with a brain homogenate prepared from first-passaged BSE/JP24-affected cattle. Detailed immunohistochemical and neuropathologic analysis of the brains of second-passaged animals, which had developed the disease and survived for an average of 16 months after inoculation, revealed distribution of spongiform changes and disease-associated prion protein (PrP(Sc)) throughout the brain. Although immunolabeled PrP(Sc) obtained from brain tissue was characterized by the presence of PrP plaques and diffuse synaptic granular accumulations, no stellate-type deposits were detected. Western blot analysis suggested no obvious differences in PrP(Sc) molecular mass or glycoform pattern in the brains of first- and second-passaged cattle. These findings suggest failures to identify differences in mean incubation period and biochemical and neuropathologic properties of the BSE/JP24 prion between the first and second passages in cattle.     

Sensitive detection of PrPSc by Western blot assay based on streptomycin sulphate precipitation

Transmissible spongiform encephalopathies, also termed prion diseases, are fatal neurodegenerative disorders that affect both humans and animals, which are characterized by presences of protease-resistance disease-associated prion protein (PrP(Sc)) in brains. In the present study, we optimized the Western blot assay for PrP(Sc) with a precipitation procedure of streptomycin sulphate. After incubated with suitable amount of streptomycin sulphate, the detective sensitivity for PrP(Sc) was remarkably improved. The precipitation of PrP(Sc) was obviously influenced by pH value in the solution. Employs of PrP(Sc) stock sample into various mimic specimens, including normal hamster brain homogenate, human cerebrospinal fluid and urine, demonstrated that streptomycin precipitation markedly increased the detective sensitivity of PrP(Sc), regardless in low concentration or in large volume. In addition, the PrP(Sc) from a human brain tissue of familiar Creutzfeldt-Jakob disease (fCJD) was efficiently precipitated with streptomycin sulphate. As a sensitive, specific, rapid and flexible protocol for PrP(Sc), the protocol in this study has the potential, alone or combined with other techniques, to detect low levels of PrP(Sc) in the specimens not only from central nerve system, but also from peripheral organs or fluids.     

Abnormal prion accumulation associated with retinal pathology in experimentally inoculated scrapie-affected sheep

The purpose of this study was to characterize the patterns of PrP(Sc) immunoreactivity in the retinae of scrapie-affected sheep and to determine the extent of retinal pathology as indicated by glial fibrillary acidic protein immunoreactivity (GFAP-IR) of Müller glia. Sections from the retina of 13 experimentally inoculated scrapie-affected and 2 negative control sheep were examined with immunohistochemical staining for PrP(Sc), GFAP, and PrP(Sc)/GFAP double staining. GFAP-IR of Müller glia is suggestive of retinal pathology in the absence of morphologic abnormality detected by light microscopy. Sheep with the least amount of PrP(Sc) in the retina have multifocal punctate aggregates of prion staining in the outer half of the inner plexiform layer and rarely in the outer plexiform layer. In these retinae, GFAP-IR is not localized with prion accumulation, but rather is present in moderate numbers of Müller glia throughout the sections of retina examined. The majority of sheep with retinal accumulation of PrP(Sc) have intense, diffuse PrP(Sc) staining in both plexiform layers, with immunoreactivity in the cytoplasm of multiple ganglion cells and lesser amounts in the optic fiber layer and between nuclei in nuclear layers. This intense PrP(Sc) immunoreactivity is associated with diffuse, intense GFAP-IR that extends from the inner limiting membrane to the outer limiting membrane. This is the first report of a prion disease in a natural host that describes the accumulation of PrP(Sc) in retina associated with retinal pathology in the absence of overt morphologic changes indicative of retinal degeneration.