Characteristics of prion protein (PrP(Sc)) in the brains of hamsters inoculated serially with a mouse-passaged scrapie strain

Syrian hamsters were inoculated intracerebrally with a mouse-passaged scrapie strain. After serial passage, the incubation period decreased, but the vacuolar lesion profiles remained unchanged. In immunoblot analysis, accumulated prion protein (PrP) showed hamster PrP characteristics from the first passage. However, immunohistochemical examination revealed a changing pattern of accumulated PrP with each passage. In particular, there were many PrP plaques in the subpial and subependymal region at the third passage, no such plaques having been observed in the same region at the first passage. These results suggest that the species barrier influences not only the incubation period but also the pattern of accumulation of PrP in affected brains.     

Infection specific prion protein (PrP) accumulates on neuronal plasmalemma in scrapie infected mice.

Prion protein (PrP) is an abundant membrane-associated host protein which accumulates in abnormal, relatively protease-resistant forms in the brains of animals with scrapie and related diseases. Using correlative light and electron microscopy we determined the sites of subcellular localisation of PrP in mice infected with the 87V strain of scrapie. Disease specific accumulation of PrP was observed at light microscopy as amyloid plaques or as diffuse or granular staining within the neuropil, often clearly associated with individual neurons. Serial electron microscopical preparations were immunostained for PrP by the immunogold method. Gold particles were located on amyloid fibrils and on the plasmalemma of neurites at the periphery of plaques and in the neuropil, irrespective of the morphological form of PrP accumulation when viewed by light microscopy. This suggests that amyloid fibrils are formed following the accumulation and aggregation of sub-unit proteins at the plasmalemma and, furthermore, that normal PrP may be converted to its pathological form at this site.     

Reduction of protein kinase MARK4 in the brains of experimental scrapie rodents and human prion disease correlates with deposits of PrP(Sc)

Microtubule affinity-regulating kinase 4 (MARK4) belongs to a family of kinases that are able to actively phosphorylate the neuronal microtubule-associate proteins (MAPs), such as tau, MAP2 and the ubiquitous MAP4. Abnormal changes in tubulin and the profiles of tau have been previously reported in the human brain and animal transmissible spongiform encephalopathies (TSEs), which may be associated with abnormal alterations of various cellular kinases. To elucidate the possible role of MARK4 in TSE pathogenesis, the MARK4 levels in the brain tissues of scrapie-infected rodents and human prion diseases were evaluated using western blotting and immunohistochemical assays. The results revealed that at terminal stages of the diseases, MARK4 levels in the brain tissues of the scrapie 263K-infected hamsters, 139A-infected mice and a case of Creutzfeldt-Jakob disease (CJD, G114V gCJD) correlated with amounts of PrP(Sc) deposits that were almost undetectable. On the other hand MARK4 signals were noticeable in the brain tissues of a fatal familial insomnia (FFI) patient without PrP(Sc). The reduction of MARK4 was closely related to the prolonged incubation times. These results could be reproduced in SK-N-SH and PC12 cell lines after being exposed to the synthetic peptide PrP106-126. Accordingly, the levels of phosphorylated tau at Ser262 (p-tau262) in cultured cells exposed to PrP106-126, or the ratios of p-tau262/total tau in the brain tissues of 263K-infected hamsters were also significantly decreased. According to our data there is a correlation between a TSE pathological-associated decline of MARK4 in the brain tissues with the deposits of PrP(Sc). Reduction of MARK4 will result in abnormalities of tau phosphorylation, and possibly induce further detachment of microtubules and hinder microtubule transportation.     

Conformational changes and development of proteinase K resistance in surface-immobilized PrP

The prion protein, and an increasing number of other cellular proteins, can undergo conformational transitions leading to soluble oligomers and insoluble aggregates. We have previously shown that the transition of the prion protein from its native form to its infectious (PrP^Sc) conformation can be monitored with epitope specific antibodies while the protein is immobilized on the surface of a Biacore surface plasmon resonance sensor chip. (Leclerc et al EMBO J 20:1547–1554 2001). The folding pathways leading to insoluble aggregates (amyloids) and soluble oligomers are believed to be distinct. We report here the use of epitope-specific antibody Fab fragments and surface plasmon resonance measurements on immobilized PrP to investigate the conditions leading to either folding pathway. We found that full-length SHaPrP(29-231) and truncated SHaPrP(90-231) prion protein can be induced to undergo the transition to proteinase K-resistant PrP^Sc aggregates on a sensor chip. This transition is temperature and buffer dependent and can be blocked by the presence of antibody Fab fragments binding to epitopes important for the conformational change. We demonstrate that the use of monoclonal antibodies combined with surface plasmon resonance technology is suitable to monitor the environmental conditions leading to conformational changes in the prion protein. The methodology is applicable to other amyloid- and oligomer-forming proteins and should be useful for the evaluation of antibodies or small molecules preventing protein misfolding.     

Generation of monoclonal antibody that distinguishes PrP from PrP and neutralizes prion infectivity

To establish PrP^Sc-specific mAbs, we immunized Prnp^-/- mice with PrP^Sc purified from prion-infected mice. Using this approach, we obtained mAb 6H10, which reacted with PrP^Sc treated with proteinase K, but not with PrP^Sc pretreated with more than 3 M GdnHCl. In contrast, reactivity of pan-PrP mAbs increased with increasing concentrations of GdnHCl used for pretreatment of PrP^Sc. In histoblot analysis, mAb 6H10 showed a positive reaction on a non-denatured histoblot but reactivity was lower when the histoblot was pretreated by autoclaving. Epitope analysis suggested that the extreme C-terminus of PrP is likely to be part of the epitope for mAb 6H10. MAb 6H10 immunoprecipitated PrP^Sc from brains of mice, sheep, and cattle infected with prions. Furthermore, pretreatment of purified PrP^Sc with mAb 6H10 reduced the infectious titer more than 1 log. Taken together, these results suggest that mAb 6H10 recognizes a conformational epitope on PrP^Sc that is related to prion infectivity.     

Detection of PrP(Sc) in rectal biopsy and necropsy samples from sheep with experimental scrapie

Scrapie diagnosis is based on the demonstration of disease-associated prion protein (PrP(Sc)) in brain or, in the live animal, in readily accessible peripheral lymphoid tissue. Lymphatic tissues present at the rectoanal line were readily obtained from sheep without the need for anaesthesia. The presence of PrP(Sc) in such tissue was investigated in sheep infected orally with scrapie-infected brain material. The methods used consisted of immunohistochemistry and histoblotting on biopsy and post-mortem material. PrP(Sc) was detected in animals with PrP genotypes associated with high susceptibility to scrapie from 10 months after infection, i.e., from about the time of appearance of early clinical signs. In the rectal mucosa, PrP(Sc) was found in lymphoid follicles and in cells scattered in the lamina propria, often near and sometimes in the crypt epithelium. By Western blotting, PrP(Sc) was detected in rectal biopsy samples of sheep with the PrP genotype VRQ/VRQ, after electrophoresis of material equivalent to 8 mg of tissue. This study indicated that rectal biopsy samples should prove useful for the diagnosis of scrapie in sheep.     

Efficient in vitro amplification of a mouse-adapted scrapie prion protein

Protein misfolding cyclic amplification (PMCA) is a highly sensitive technique used to detect minute amounts of scrapie prion protein (PrP(Sc)), a major protein component of the infectious agents associated with prion diseases. Although exponential in vitro amplification of hamster scrapie PrP(Sc) has been established, the PMCA used was unsuccessful in achieving good amplification of PrP(Sc) from other animals. Here, we have investigated the cause of the insufficient PrP(Sc) amplification in mice and have developed an improved method suitable for amplification of the PrP(Sc) of the mouse-adapted scrapie prion strain Chandler. Mouse PrP(C), the cellular form of the prion protein, tends to become resistant to proteases during incubation independent of sonication. By adding digitonin to the reaction buffer as a lipid detergent, accumulation of the protease-resistant PrP(C) was inhibited; hence, mouse PrP(Sc) could be amplified to infinite levels. The present study is the first report describing effective amplification of PrP(Sc) of the mouse-adapted scrapie prion and this improved PMCA technique will contribute to prion research that uses mice as experimental animals.     

The association between PrP and infectivity in scrapie and BSE infected mouse brain

Summary The structure of the scrapie agent remains unknown. However, scrapie infectivity tends to co-sediment with an infection specific fraction of the glycoprotein PrP (PrP^Sc) under conditions which solubilise the normal form of this protein (PrP^c); accordingly, PrP has been proposed as a candidate component of the agent. To investigate this further we have been examining a new scrapie-related murine model in conjunction with established scrapie models. A bovine spongiform encephalopathy (BSE) derived murine model has short incubation periods, high infectivity titre and low amounts of PrP deposited in the brain. A membrane fraction from scrapie/BSE infected brain is solubilised with Sarkosyl at pH9.0. Most PrP is also solubilised. In models of the disease with little deposition of PrP in the brain, this solubilisation step is particularly effective in reducing the amounts of PrP sedimented from brain extracts. Gradient centrifugation of the sedimented fraction shows further separation of infectivity and the residual PrP. It is concluded that at least some PrP^Sc in the brain need not be associated directly with infectious agent but is deposited in brain solely as a pathological product of infection. However, a residual sedimentable fraction contains PrP which may be a component of the agent.     

ST1859 reduces prion infectivity and increase survival in experimental scrapie

On the basis of the structural homologies between ST1859 (1[(2-hydroxy-1-naphtyl)methyl]-2-naphthol) and the anti-prion agents and its anti-amyloidogenic activity, we tested whether this molecule altered the biochemical properties of aggregates formed in vitro by synthetic prion peptides and affected prion infectivity in experimental scrapie. Co-incubation of ST1859 with the peptides PrP 106–126 and PrP 82–146 reduced their fibrillogenic capacity and their resistance to digestion with protease K. Hamsters inoculated with the ST1859-treated homogenate showed a significant delay in the onset of clinical signs of disease and longer survival. Survival was also significantly longer in infected hamsters treated peripherally with ST1859 for the whole post-inoculation period until the onset of clinical symptoms. Similar results were found with the analogue ST1745. Our data indicate that ST1859 reduces prion infectivity and can exert a therapeutic effect in experimental scrapie.     

The paraffin-embedded tissue blot detects PrP(Sc) early in the incubation time in prion diseases

With the appearance of bovine spongiform encephalopathy (BSE) and a new variant of Creutzfeldt-Jakob disease (nvCJD) that seems to be caused by BSE, there is an increased need for improvement of diagnostic techniques and recognition of all variants of prion diseases in humans and animals. Publications on the immunohistochemical identification of PrP(Sc) in the tonsils and appendix in the incubation period of nvCJD indicate that new and more sensitive techniques for the detection of PrP(Sc) in various tissues may be a valuable tool for early diagnosis in prion diseases. We developed a new and sensitive technique to detect PrP(Sc) in formalin-fixed and paraffin-embedded tissue, the paraffin-embedded tissue blot (PET blot), and reinvestigated archival brain material from CJD as well as BSE and scrapie. In addition, C57/Bl6 mice experimentally infected with the ME7 strain were investigated sequentially during the incubation time to compare this new technique with conventional methodologies. The PET blot detects PrP(Sc) in idiopathic (sporadic) and acquired prion diseases, even in cases with equivocal or negative immunohistochemistry, and is more sensitive than the conventional Western blot and histoblot techniques. The PET blot makes possible the detection of PrP(Sc) during the incubation period long before the onset of clinical disease and in prion disease variants with very low levels of PrP(Sc). In mice experimentally infected with the ME7 strain, the PET blot detects PrP(Sc) in the brain 30 days after intracerebral inoculation-145 days before the onset of clinical signs. Its anatomical resolution is superior to that of the histoblot technique. It may therefore be of particular interest in biopsy diagnosis. Thus it complements other tissue-based techniques for the diagnosis of prion diseases in humans and animals.     

An antibody to the aggregated synthetic prion protein peptide (PrP106-126) selectively recognizes disease-associated prion protein (PrP) from human brain specimens

Human prion diseases are characterized by the conversion of the normal host cellular prion protein (PrP^C) into an abnormal misfolded form [disease-associated prion protein (PrP^Sc)]. Antibodies that are capable of distinguishing between PrP^C and PrP^Sc may prove to be useful, not only for the diagnosis of these diseases, but also for a better understanding of the molecular mechanisms involved in disease pathogenesis. In an attempt to produce such antibodies, we immunized mice with an aggregated peptide spanning amino acid residues 106 to 126 of human PrP (PrP106–126). We were able to isolate and single cell clone a hybridoma cell line (P1:1) which secreted an IgM isotype antibody [monoclonal antibody (mAb P1:1)] that recognized the aggregated, but not the monomeric form of the immunogen. When used in immunoprecipitation assays, the antibody did not recognize normal PrP^C from non-prion disease brain specimens, but did selectively immunoprecipitate full-length PrP^Sc from cases of variant and sporadic Creutzfeldt–Jakob disease and Gerstmann–Straussler–Scheinker disease. These results suggest that P1:1 recognizes an epitope formed during the structural rearrangement or aggregation of the PrP that is common to the major PrP^Sc types found in the most common forms of human prion disease.     

Cellular uptake of the prion protein fragment PrP106-126 in vitro

The aetiological agent of prion disease is proposed to be an aberrant isoform of the cell surface glycoprotein known as the prion protein (PrP^c). This pathological isoform (PrP^Sc) is abnormally deposited in the extracellular space of diseased CNS. Neurodegeneration in these disease has been shown to be associated with accumulation of PrP^Sc in affected tissue. To investigate the possible uptake mechanisms that may be required for PrP^Sc-induced neurodegeneration we studied the cellular trafficking of the neurotoxic fragment, PrP106-126. We were able to detect, by fluorescence microscopy, PrP106-126 inclusions in murine neurones, astrocytes and microglia in vitro. These inclusions were abundant after 24 hour exposure and still present 48h post-exposure. Shorter exposure times yielded only occasional cells with inclusions. Large extracellular aggregates of PrP106-126 could also be detected, which appeared in a time dependent manner. The appearance of inclusions or aggregates was not dependent on PrP^c expression as determined by exposure of peptides from PrP-null mice. Using transmission electron microscopy and gold particle detection, positively labelled osmiophilic inclusions of peptide could be detected in the cytoplasm of exposed cells. These results demonstrate that cultured cells are capable of sequestering PrP106-126 and may indicate uptake pathways for PrP^Sc in various cell types. Toxicity of PrP106-126 may thus be mediated via a sequestration pathway that is not effective for this peptide in PrP-null cells.     

Influence of the prion protein gene,Prnp, on scrapie susceptibility in sheep

Natural scrapie in sheep occurs through a complex interplay between host genetic elements and various strains of the infectious scrapie agent. Scrapie-related polymorphisms in the coding region of the prion protein (PrP) gene, Prnp, have been studied in a number of breeds. The disease-promoting V136 allele, and the susceptibility-reducing R171 allele, have proved to be most important. However, variation in the coding region of Prnp cannot alone explain the diverse patterns of scrapie susceptibility in various breeds. For instance, in many breeds plagued with scrapie, the V136 allele appears to be a rarity. The R171 allele greatly reduces scrapie susceptibility This lays the molecular foundation for marker-assisted breeding for reduced scrapie susceptibility now underway in many countries. Although potentially important, and still under investigation, variable expression level and pattern of the ovine Prnp appears to be of little importance for the occurrence of natural scrapie. Studies of scrapie in mice also indicate that genetic elements other than Prnp may have a strong influence on scrapie incubation time, and hence susceptibility. Narrowing down the search to focus on these elements and identification of candidate genes are important tasks for future research in sheep scrapie.     

Preparation and characterization of antibodies against mouse prion protein (PrP) peptides.

Antisera were raised in rabbits against three peptides, representing amino acid sequences 150 to 159, 165 to 174, and 213 to 226 of mouse prion (PrP), which were synthesized by using a multiple antigenic peptide (MAP) system. The reactivities of these sera to PrP were examined by an enzyme-linked immunosorbent assay (ELISA), Western immunoblotting (WB), and immunohistochemical procedures. The results of both ELISA and WB showed that antisera to peptide sequence 150 to 159 (Ab150-159) did not react with purified mouse PrP. On the other hand, sera to the sequence 165 to 174 (Ab165-174) reacted weakly with purified mouse PrP, as detected by WB but not by ELISA. However, antiserum to peptide sequence 213 to 226 (Ab213-226) reacted strongly with mouse, Syrian hamster, and sheep PrP by WB and with mouse PrP as shown by the results of ELISA. Moreover, Ab213-226 clearly detected PrP immunohistochemically in mouse, Syrian hamster, and sheep brains affected with scrapie as well as in the brain of a cow with bovine spongiform encephalopathy. From these data, we conclude that rabbit antiserum against the MAP representing amino acid sequence 213 to 226 of mouse PrP is useful as a diagnostic tool for prion disease of animals.     

Cellular prion protein (PrP) and superoxide dismutase (SOD) in vascular cells under oxidative stress

The PrP^C is expressed in several cell types but its physiological function is unknown. Some studies associate the PrP^C with copper metabolism and the antioxidant activity of SOD. Our hypothesis was that changes in PrP^C expression lead to abnormal copper regulation and induce SOD downregulation in the vascular wall. Objectives: to study whether the PrP^C expression undergoes induction by agents that trigger endoplasmic reticulum stress (ERS) and, in this context, to evaluate the SOD activity. Methods: To trigger ERS, in vitro, rabbit aortic smooth muscle cells were challenged for 4, 8 and 18 hours, with angiotensin-II, tunicamycin and 7-ketocholesterol. For in vivo studies rabbit aortic arteries were subjected to injury by balloon catheter. Results: In vitro baseline SOD activity, determined through inhibition of cytochrome-c reduction, was 13.9±1.2 U/mg protein, angiotensin-II exposed for 8 hours produced an increase in SOD activity, and cellular copper concentration was about 9 times greater only under these conditions. Western blotting analysis for SOD isoenzymes showed an expression profile that was not correlated with the enzymatic activity. PrP^C expression decreased after exposure to all agents after different incubation periods. RT-PCR assay showed increased mRNA expression for PrP^C only in cells stimulated for 8 hours with the different stressors. The PrP^C mRNA expression in rabbit aortic artery fragments, subjected to balloon catheter injury, showed a pronounced increase immediately after overdistension. The results obtained indicated a PrP^C protection factor during the early part of the ERS exposure period, but did not demonstrate a SOD-like profile for the PrP^C.     

Short-term study of the uptake of PrP(Sc) by the Peyer's patches in hamsters after oral exposure to scrapie

The disease-associated prion protein (PrP(Sc)) has been detected in the ileal Peyer's patches of lambs as early as one week after oral exposure to scrapie. In hamsters, the earliest reported time of PrP(Sc) detection in the Peyer's patches after oral exposure to scrapie is 69 days post-infection. To evaluate the acute uptake of inoculum and to investigate whether the Peyer's patches constitute the primary site of entry for scrapie after oral exposure, hamsters were each exposed orally to 1 ml of a 10% brain homogenate from hamsters in the terminal stage of infection with the 263 K strain of the scrapie agent. PrP(Sc) was demonstrated in the Peyer's patches only a few days after exposure, i.e., much earlier than previously reported. This study supports the view that the Peyer's patches constitute at least one of the primary entry sites of PrP(Sc) after oral exposure to scrapie.     

Distinct proteinase K-resistant prion protein fragment in goats with no signs of disease in a classical scrapie outbreak

Considerable efforts have been directed toward the identification of small-ruminant prion diseases, i.e., classical and atypical scrapie as well as bovine spongiform encephalopathy (BSE). Here we report the in-depth molecular analysis of the proteinase K-resistant prion protein core fragment (PrP(res)) in a highly scrapie-affected goat flock in Greece. The PrP(res) profile by Western immunoblotting in most animals was that of classical scrapie in sheep. However, in a series of clinically healthy goats we identified a unique C- and N-terminally truncated PrP(res) fragment, which is akin but not identical to that observed for atypical scrapie. These findings reveal novel aspects of the nature and diversity of the molecular PrP(res) phenotypes in goats and suggest that these animals display a previously unrecognized prion protein disorder.     

Creutzfeldt-Jakob disease (CJD) of a short duration with prion protein (PrP) plaques.

We report here PrP-immunohistochemistry performed on brains from CJD cases from Poland. Only one out of five definitive CJD cases exhibited typical PrP-immunoreactive kuru-like plaques and this was case of a short duration. Thus, we confirmed the low percentage of PrP plaques in CJD of Eastern and Central European origin.     

Epitope mapping of a PrP(Sc)-specific monoclonal antibody: identification of a novel C-terminally truncated prion fragment

Monoclonal antibodies (mAbs) against prion proteins (PrPs) are indispensable in research and diagnosis of prion diseases, however the majority of these bind both the cellular (PrP^C) and the disease-associated (PrP^Sc) isoforms. According to the widely accepted protein-only hypothesis the two isoforms share the same sequence, but differ in their conformation. In the present study we set to determine the critical binding residues of our PrP^Sc-specific mAbs with the view of discerning which residues play a key role in the conformational transition between PrP^C and PrP^Sc. Focussing on the V5B2 mAb that provided differential labelling of prion-affected tissue from individuals positive for transmissible spongiform encephalopathies, we performed alanine scanning and phage-display epitope mapping to elucidate the antigenic determinants of this mAb and gain insight into its specificity on a molecular level. We observed that instead of discriminating between the two prion protein isoforms based on conformational differences, V5B2 binds a previously uncharacterized C-terminally truncated form of PrP^Sc that ends with the residue Y226, which we named PrP226*. The addition of a single C-terminal amino-acid residue completely abolished V5B2 binding, while Western blots using recombinant full-length PrPs and PrPs terminating at Y226 confirmed that the V5B2 mAb discriminates between the two based on their difference in length.     

Immunolocalization of the prion protein in scrapie affected rodent retinas

The effects of intrathecal administration of NMDA (N-methyl-D-aspartic acid) receptor antagonist AP-5 (2-Amino-5-phosphonopentanoic acid), a competitive and specific NMDA antagonist, and glycine on the neuronal expression of c-fos protein (Fos) in the dorsal neurons lumbar segments four and five were studied after noxious heat stimulation. Heat (52 degrees C, 3 s per application, repeated 10 times) was applied to the hindpaws of rats. NMDA receptor antagonist AP-5 (0.1 mmol/10 ml, i.t.) suppressed the noxious heat-induced Fos immunoreactivity by 65% as compared to animals pre-treated with saline. In contrast, glycine (0.1 micromol/10 microl, it.) did not influence Fos expression induced by the noxious heat stimulation. This study suggests that excitatory amino acids, e.g. glutamate but not the inhibitory aminos acid, glycine, plays a role in thermal nociception which in turn is mediated, in part, by c-fos activity.