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.     

Establishment of a chicken monoclonal antibody panel against mammalian prion protein

A panel of chicken monoclonal antibodies (mAbs) was developed against prion protein (PrP), the sequence of which is a highly conserved molecule among mammals. A portion of the splenocytes from chickens immunized with recombinant mouse PrP was fused with the chicken B cell line, MuH1. The remaining splenocytes were used to generate the recombinant mAbs by phage display. A total of 36 anti-PrP mAbs, 2 from cell fusion and 34 from phage display were established. The specificity of these mAbs was determined by Western blot and ELISA using various PrP antigens including recombinant PrPs, synthetic PrP peptides and PrPs from brains or scrapie-infected neuroblastoma cell line. These mAbs were classified into three main groups, protease K (PK)-sensitive (Group I), PK cleavage site proximal (Group II) and PK-resistant (Group III), based on their abilities to recognize PrP following PK-treatment. Some mAbs were found to selectively recognize different glycoforms of PrP as well as the metabolic fragments of PrP. Furthermore, we found that PrP recognition by chickens differed from that by PrP-knockout mouse. These results indicate that these newly generated PrP antibodies from chickens will help to research the PrP and to establish the diagnosis of prion disease.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Identification of a proteinase K resistant protein for use as an internal positive control marker in PrP Western blotting

The routine use of an internal positive control (IPC) marker could prove useful in the diagnosis of transmissible spongiform encephalopathy (TSE) diseases, particularly in surveillance programmes where large numbers of negative results are reported. Detection of an endogenous IPC protein in a negative sample adds confidence to the correct sample processing throughout the analytical procedure and could avoid the reporting of false negative diagnoses. Proteinase K (PK) resistance is one of the key diagnostic determinants of the disease-associated form of PrP (PrP(Sc)), the only disease-specific macromolecule currently associated with TSE disease. Additional PK resistant proteins, endogenous to TSE-suspect diagnostic tissue samples, were therefore assessed for use as IPC markers in the Western blot diagnosis of BSE and scrapie. Results indicated that, whilst essentially maintaining a standard PrP extraction and detection protocol, a ferritin heavy chain sub-unit of approximately 22kDa, was consistently detected in all PK treated TSE positive and negative tissue samples tested. Its presence in a range of sample types, any of which could be submitted under BSE and scrapie surveillance programmes, confirmed it as a suitable protein for an IPC marker in PrP(Sc) Western blotting.     

Progress and limits of TSE diagnostic tools

Following the two "mad cow" crises of 1996 and 2000, there was an urgent need for rapid and sensitive diagnostic methods to identify animals infected with the bovine spongiform encephalopathy (BSE) agent. This stimulated research in the field of prion diagnosis and led to the establishment of numerous so-called "rapid tests" which have been in use in Europe since 2001 for monitoring at-risk populations (rendering plants) and animals slaughtered for human consumption (slaughterhouse). These rapid tests have played a critical role in the management of the mad cow crisis by allowing the removal of prion infected carcasses from the human food chain, and by allowing a precise epidemiological monitoring of the BSE epizootic. They are all based on the detection of the abnormal form of the prion protein (PrP(Sc) or PrP(res)) in brain tissues and consequently are only suitable for post-mortem diagnosis. Since it is now very clear that variant Creutzfeldt-Jakob disease (vCJD) can be transmitted by blood transfusion, the development of a blood test for the diagnosis of vCJD is a top priority. Although significant progress has been made in this direction, including the development of the protein misfolding cyclic amplification (PMCA) technology, at the time this paper was written, this objective had not yet been achieved. This is the most important challenge for the years to come in this field of prion research.     

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.     

Stability of bovine spongiform encephalopathy prions: absence of prion protein degradation by bovine gut microbiota

Bovine spongiform encephalopathy (BSE) is transmitted by the oral route. However, the impacts of anaerobic fermentation processes in cattle on the stability of BSE-associated prion protein (PrP(Sc)) are still unresolved. In this study, experiments were designed to assess the ability of complex ruminal and colonic contents of bovines to degrade BSE-derived PrP(Sc). No significant decrease in PrP(Sc) levels in BSE brain homogenates was detected by Western blotting after up to 66 h of co-incubation with intestinal fluids. These results indicate that BSE-associated PrP(Sc) survive gastrointestinal digestion processes in cattle and might be excreted via faeces.     

Identification and characterization of two bovine spongiform encephalopathy cases diagnosed in the United States.

Bovine spongiform encephalopathy (BSE) is a transmissible spongiform encephalopathy of cattle, first detected in 1986 in the United Kingdom and subsequently in other countries. It is the most likely cause of variant Creutzfeldt-Jakob disease (vCJD) in humans, but the origin of BSE has not been elucidated so far. This report describes the identification and characterization of two cases of BSE diagnosed in the United States. Case 1 (December 2003) exhibited spongiform changes in the obex area of the brainstem and the presence of the abnormal form of the prion protein, PrP(Sc), in the same brain area, by immunohistochemistry (IHC) and Western blot analysis. Initial suspect diagnosis of BSE for case 2 (November 2004) was made by a rapid ELISA-based BSE test. Case 2 did not exhibit unambiguous spongiform changes in the obex area, but PrP(Sc) was detected by IHC and enrichment Western blot analysis in the obex. Using Western blot analysis, PrP(Sc) from case 1 showed molecular features similar to typical BSE isolates, whereas PrP(Sc) from case 2 revealed an unusual molecular PrP(Sc) pattern: molecular mass of the unglycosylated and monoglycosylated isoform was higher than that of typical BSE isolates and case 2 was strongly labeled with antibody P4, which is consistent with a higher molecular mass. Sequencing of the prion protein gene of both BSE-positive animals revealed that the sequences of both animals were within [corrected] the range of the prion protein gene sequence diversity previously reported for cattle.     

Degradation of scrapie associated prion protein (PrPSc) by the gastrointestinal microbiota of cattle

A food-borne origin of the transmission of bovine spongiform encephalopathy (BSE) to cattle is commonly assumed. However, the fate of infectious prion protein during polygastric digestion remains unclear. It is unknown at present, whether infectious prion proteins, considered to be very stable, are degraded or inactivated by microbial processes in the gastrointestinal tract of cattle. In this study, rumen and colon contents from healthy cattle, taken immediately after slaughter, were used to assess the ability of these microbial consortia to degrade PrP(Sc). Therefore, the consortia were incubated with brain homogenates of scrapie (strain 263K) infected hamsters under physiological anaerobic conditions at 37 degrees C. Within 20 h, PrP(Sc) was digested both with ruminal and colonic microbiota up to immunochemically undetectable levels. Especially polymyxin resistant (mainly gram-positive) bacteria expressed PrP(Sc) degrading activity. These data demonstrate the ability of bovine gastrointestinal microbiota to degrade PrP(Sc) during digestion.     

Neuroanatomical distribution of disease-associated prion protein in cases of bovine spongiform encephalopathy detected by fallen stock surveillance in Japan

Bovine spongiform encephalopathy (BSE) is a fatal neurodegenerative disorder of cattle characterized by accumulation of the disease-associated prion protein (PrP(Sc)) in the central nervous system (CNS). The immunohistochemical patterns and distribution of PrP(Sc) were investigated in the CNS, brains, and spinal cords of 7 naturally occurring BSE cases confirmed by the fallen stock surveillance program in Japan. No animals showed characteristic clinical signs of the disease. Coronal slices of 14 different brain areas in each case were immunohistochemically analyzed using an anti-prion protein antibody. Immunolabeled PrP(Sc) deposition was widely observed throughout each brain and spinal cord. Intense PrP(Sc) deposition was greater in the thalamus, brainstem, and spinal cord of the gray matter than in the neocortices. The topographical distribution pattern and severity of PrP(Sc) accumulation were mapped and plotted as immunohistochemical profiles of the different brain areas along the caudal-rostral axis of the brain. The distribution pattern and severity of the immunolabeled PrP(Sc) in the CNS were almost the same among the 7 cases analyzed, suggesting that the naturally occurring cases in this study were at the preclinical stage of the disease. Immunohistochemical mapping of the PrP(Sc) deposits will be used to clarify the different stages of BSE in cattle.     

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.