Protease-resistant Prion Protein Research Articles

Inhibition of Protease-Resistant Prion Protein Formation in a Transformed Deer Cell Line Infected with Chronic Wasting Disease

Chronic wasting disease (CWD) is an emerging transmissible spongiform encephalopathy (prion disease) of North American cervids, i.e., mule deer, white-tailed deer, and elk (wapiti). To facilitate in vitro studies of CWD, we have developed a transformed deer cell line that is persistently infected with CWD. Primary cultures derived from uninfected mule deer brain tissue were transformed by transfection with a plasmid containing the simian virus 40 genome. A transformed cell line (MDB) was exposed to microsomes prepared from the brainstem of a CWD-affected mule deer. CWD-associated, protease-resistant prion protein (PrP(CWD)) was used as an indicator of CWD infection. Although no PrP(CWD) was detected in any of these cultures after two passes, dilution cloning of cells yielded one PrP(CWD)-positive clone out of 51. This clone, designated MDB(CWD), has maintained stable PrP(CWD) production through 32 serial passes thus far. A second round of dilution cloning yielded 20 PrP(CWD)-positive subclones out of 30, one of which was designated MDB(CWD2). The MDB(CWD2) cell line was positive for fibronectin and negative for microtubule-associated protein 2 (a neuronal marker) and glial fibrillary acidic protein (an activated astrocyte marker), consistent with derivation from brain fibroblasts (e.g., meningeal fibroblasts). Two inhibitors of rodent scrapie protease-resistant PrP accumulation, pentosan polysulfate and a porphyrin compound, indium (III) meso-tetra(4-sulfonatophenyl)porphine chloride, potently blocked PrP(CWD) accumulation in MDB(CWD) cells. This demonstrates the utility of these cells in a rapid in vitro screening assay for PrP(CWD) inhibitors and suggests that these compounds have potential to be active against CWD in vivo.

Mefloquine, an Antimalaria Drug with Antiprion Activity In Vitro, Lacks Activity In Vivo

In view of the effectiveness of antimalaria drugs inhibiting abnormal protease-resistant prion protein (PrP-res) formation in scrapie agent-infected cells, we tested other antimalarial compounds for similar activity. Mefloquine (MF), a quinoline antimalaria drug, was the most active compound tested against RML and 22L mouse scrapie agent-infected cells, with 50% inhibitory concentrations of approximately 0.5 and approximately 1.2 microM, respectively. However, MF administered to mice did not delay the onset of intraperitoneally inoculated scrapie agent, the result previously observed with quinacrine. While most anti-scrapie agent compounds inhibit PrP-res formation in vitro, many PrP-res inhibitors have no activity in vivo. This underscores the importance of testing promising candidates in vivo.

Breaking an Absolute Species Barrier: Transgenic Mice Expressing the Mink PrP Gene Are Susceptible to Transmissible Mink Encephalopathy

Transmissible mink encephalopathy (TME) is a rare disease of the North American mink, which has never been successfully transmitted to laboratory mice. We generated transgenic mice expressing the mink prion protein (PrP) and inoculated them with TME or the mouse-adapted scrapie strain 79A. TME infected mink PrP-transgenic mice on a murine PrP knockout background. The absolute species barrier between the infectious agent of TME and mice was therefore broken. Following TME and 79A infection of mice carrying both mink and murine PrP(C), only proteinase-resistant PrP homologous to the incoming agent was detectable. The presence of the murine PrP(C) prolonged the incubation time of TME substantially.

Coexistence of multiple PrP Sc types in individuals with Creutzfeldt-Jakob disease

The molecular typing of sporadic Creutzfeldt-Jakob disease (CJD) is based on the size and glycoform ratio of protease-resistant prion protein (PrP(Sc)), and on PRNP haplotype. On digestion with proteinase K, type 1 and type 2 PrP(Sc) display unglycosylated core fragments of 21 kDa and 19 kDa, resulting from cleavage around amino acids 82 and 97, respectively. We generated anti-PrP monoclonal antibodies to epitopes immediately preceding the differential proteinase K cleavage sites. These antibodies, which were designated POM2 and POM12, recognise type 1, but not type 2, PrP(Sc). We studied 114 brain samples from 70 patients with sporadic CJD and three patients with variant CJD. Every patient classified as CJD type 2, and all variant CJD patients, showed POM2/POM12 reactivity in the cerebellum and other PrP(Sc)-rich brain areas, with a typical PrP(Sc) type 1 migration pattern. The regular coexistence of multiple PrP(Sc) types in patients with CJD casts doubts on the validity of electrophoretic PrP(Sc) mobilities as surrogates for prion strains, and questions the rational basis of current CJD classifications.

Second Passage of Sheep Scrapie and Transmissible Mink Encephalopathy (TME) Agents in Raccoons (Procyon lotor)

To determine the transmissibility and pathogenicity of sheep scrapie and transmissible mink encephalopathy (TME) agents derived from raccoons (first passage), raccoon kits were inoculated intracerebrally with either TME (one source) or scrapie (two sources-each in separate groups of raccoons). Two uninoculated raccoon kits served as controls. All animals in the TME-inoculated group developed clinical signs of neurologic dysfunction and were euthanatized between postinoculation month (PIM) 6 and 8. Raccoons in the two scrapie-inoculated groups manifested similar clinical signs of disease, but such signs were observed much later and the animals were euthanized between PIM 12 and 18. Necropsy revealed no gross lesions in any of the raccoons. Spongiform encephalopathy was observed by use of light microscopy, and the presence of protease-resistant prion protein (PrPres) was detected by use of immunohistochemical (IHC) and Western blot analytic techniques. Results of IHC analysis indicated a distinct pattern of anatomic distribution of PrPres in the TME- and scrapie-inoculated raccoons. These findings confirm that TME and sheep scrapie are experimentally transmissible to raccoons and that the incubation periods and IHC distribution for both agents are distinct. Therefore, it may be possible to use raccoons for differentiating unknown transmissible spongiform encephalopathy (TSE) agents. Further studies, with regard to the incubation period and the pattern of PrPres deposition by use of IHC analysis in bovine spongiform encephalopathy and for other isolates of scrapie, chronic wasting disease, and TME in raccoons are needed before the model can be further characterized for differentiation of TSE agents.

A solid-phase immunoassay of protease-resistant prion protein with filtration blotting involving sodium dodecyl sulfate

The precise diagnosis for bovine spongiform encephalopathy (BSE) is crucial for preventing new transmission to humans. Several testing procedures are reported for determining protease-resistant prion protein in various tissues as a major hallmark of prion diseases such as BSE, scrapie, and Creutzfeldt–Jakob disease. However, contamination of materials from tissues or degradation of the specimens sometimes disturbs the accuracy of the assay. Here, we have developed a novel method for solid-phase immunoassay of the disease-specific conformational isoform, PrP Sc, using filtration blotting of protein in the presence of sodium dodecyl sulfate (SDS) followed by a filtration-based immunoassay with a single anti-prion protein antibody, together with the improved fractionation procedure involving high concentrations of surfactant/detergent. The SDS/heat treatment renders unfolded PrP Sc quantitative retention on a polyvinylidene difluoride filter and allows enhancement of the analyte signal with immunodetection; thus, all of the tested specimens are determined with 100% accuracy. In addition, the immunoassay is completed in approximately 1 h, indicating its usefulness not only for the screening of BSE specimens but probably also for the postmortem BSE diagnosis of fallen stock as the antibody recognizes the core part of PrP Sc. The solid-phase immunoassay method, including the filtration blotting with SDS, would be applicable to determining even more sensitively proteins other than PrP Sc, especially those having rigid conformations.

Redox metals and oxidative abnormalities in human prion diseases

Prion diseases are characterized by the accumulation of diffuse and aggregated plaques of protease-resistant prion protein (PrP) in the brains of affected individuals and animals. Whereas prion diseases in animals appear to be almost exclusively transmitted by infection, human prion diseases most often occur sporadically and, to a lesser extent, by inheritance or infection. In the sporadic cases (sporadic Creutzfeld-Jakob disease, sCJD), PrP-containing plaques are infrequent, whereas in transmitted (variant CJD) and inherited (Gerstmann-Straussler-Scheinker Syndrome) cases, plaques are a usual feature. In the current study, representative cases from each of the classes of human prion disease were analyzed for the presence of markers of oxidative damage that have been found in other neurodegenerative diseases. Interestingly, we found that the pattern of deposition of PrP, amyloid-beta, and redox active metals was distinct for the various prion diseases. Whereas 8-hydroxyguanosine has been shown to be increased in sCJD, and inducible NOS is increased in scrapie-infected mice, well-studied markers of oxidative damage that accumulate in the lesions of other neurodegenerative diseases (such as Alzheimer's disease, progressive supranuclear palsy, and Parkinson's disease), such as heme oxygenase-1 and lipid peroxidation, were not found around PrP deposits or in vulnerable neurons. These findings suggest an important distinction in prion-related oxidative stress, indicating that different neurodegenerative pathways are involved in different prion diseases.

Comparison of protease-resistant prion protein inhibitors in cell cultures infected with two strains of mouse and sheep scrapie

The transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative diseases. A primary therapeutic target for TSE intervention has been a protease-resistant form of prion protein known as PrP Sc or PrP-res. In vitro testing of mouse scrapie-infected cell cultures has identified many PrP-res inhibitors that also have activity in vivo. Here we identify 32 new inhibitors of two strains of mouse scrapie PrP-res. Furthermore, to investigate the species-specificity of these and other PrP-res inhibitors, we have developed a high-throughput cell culture assay based on Rov9 cells chronically-infected with sheep scrapie. Of 32 inhibitors of murine PrP-res that were also tested in the Rov9 cells, only six showed inhibitory activity against sheep PrP-res. The three most potent inhibitors of both murine and ovine PrP-res formation (with 50% inhibition at ≤5 μM) were tannic acid, pentosan polysulfate and Fe(III) deuteroporphyrin 2,4-bisethyleneglycol. The latter two have anti-mouse scrapie activity in vivo. These results identify new inhibitors of murine and ovine PrP-res formation and reinforce the idea that compounds effective against PrP-res from one species or strain cannot be assumed to be active against others.

Australian sporadic CJD analysis supports endogenous determinants of molecular-clinical profiles

To define the protease-resistant prion protein (PrPres) types and associated clinical profiles in Australian patients with sporadic Creutzfeldt-Jakob disease (CJD) to allow comparison with those reported from other continents and concomitantly reaffirm absence of variant CJD (vCJD). Reassessment of available clinical and neuropathologic data on patients referred to the Australian National Creutzfeldt-Jakob Disease Registry (ANCJDR) who died between January 1, 1992, and June 30, 2003, was conducted. Molecular classification of PrPres was determined by immunoblot analysis of available frozen brain tissue. Brain homogenate pH and codon 129 genotype on the prion protein gene (PRNP) were established. PrPres patterns in 35 of 37 patients with sporadic CJD conformed to one of three common reported types. Of a range of clinical features assessed, illness duration was the only clinical feature significantly associated with PrPres type. Two patients displayed coexistence of more than one PrP type, with one displaying a novel pattern of three PrPres types in a single brain region. The absence of vCJD was reconfirmed, supported by the lack of the typical PrPres glycoform pattern. Given Australia's geographic isolation and environmental uniqueness, the general congruity of these results with those reported from other continents suggests that endogenous factors predominantly determine sporadic Creutzfeldt-Jakob disease (CJD) phenotypic subtypes or "strains." These results support a clinicopathologic classification system whereby both PrPres type and codon 129 genotype are utilized to most accurately depict phenotypic subtypes or strains of sporadic CJD.

Protease-resistant Prion Protein Amplification Reconstituted with Partially Purified Substrates and Synthetic Polyanions

Little is currently known about the biochemical mechanism by which induced prion protein (PrP) conformational change occurs during mammalian prion propagation. In this study, we describe the reconstitution of PrPres amplification in vitro using partially purified and synthetic components. Overnight incubation of purified PrP27-30 and PrPC molecules at a molar ratio of 1:250 yielded approximately 2-fold baseline PrPres amplification. Addition of various polyanionic molecules increased the level of PrPres amplification to approximately 10-fold overall. Polyanionic compounds that stimulated purified PrPres amplification to varying degrees included synthetic, homopolymeric nucleic acids such as poly(A) and poly(dT), as well as non-nucleic acid polyanions, such as heparan sulfate proteoglycan. Size fractionation experiments showed that synthetic poly(A) polymers must be >0.2 kb in length to stimulate purified PrPres amplification. Thus, one possible set of minimal components for efficient conversion of PrP molecules in vitro may be surprisingly simple, consisting of PrP27-30, PrPC, and a stimulatory polyanionic compound.

Bov-tA Short Interspersed Nucleotide Element Sequences in Circulating Nucleic Acids from Sera of Cattle with Bovine Spongiform Encephalopathy (BSE) and Sera of Cattle Exposed to BSE

Circulating nucleic acids (CNA) are known to be enriched in repetitive DNA sequences in humans. Here, bovine sera CNA were analyzed to determine if cell stress-related short interspersed nucleotide elements (SINEs) could be detected in sera from cattle associated with bovine spongiform encephalopathy (BSE). Nucleic acids were extracted, amplified, cloned, and sequenced from the sera of protease-resistant prion protein (PrP(res))-positive cattle (n = 2) and sera from BSE-cohort cows (n = 6); 150 out of 163 clones revealed the presence of, on average, an 80-bp sequence from the 3' region of Bov-tA SINE. A PCR protocol was developed that differentially identified SINE-associated CNA in BSE-exposed versus normal cattle. CNA were extracted from a serum vesicular fraction after controlled blood collection and processing procedures. Sera from four confirmed cases of BSE, 137 BSE-exposed cohort animals associated with eight confirmed BSE cases, and 845 healthy, PrP(res)-negative control cows were tested. All four sera from confirmed BSE cases were repeatedly reactive in the assay. BSE-exposed cohorts had a 100-fold higher occurrence of repeatedly reactive individuals per cohort (average = 63%; range = 33% to 91%), compared to healthy controls (average = 0.6%; P < 0.001). This study shows that BSE-confirmed and cohort animals possess a unique profile of SINE-associated serum CNA that can be utilized as a marker that highly correlates to BSE exposure.

Immunodetection of disease-associated mutant PrP, which accelerates disease in GSS transgenic mice

The absence of infectivity-associated, protease-resistant prion protein (PrP(Sc)) in the brains of spontaneously sick transgenic (Tg) mice overexpressing PrP linked to Gerstmann-Sträussler Scheinker syndrome, and the failure of gene-targeted mice expressing such PrP to develop disease spontaneously, challenged the concept that mutant PrP expression led to spontaneous prion production. Here, we demonstrate that disease in overexpressor Tg mice is associated with accumulation of protease-sensitive aggregates of mutant PrP that can be immunoprecipitated by the PrP(Sc)-specific monoclonal antibody designated 15B3. Whereas Tg mice expressing multiple transgenes exhibited accelerated disease when inoculated with disease-associated mutant PrP, Tg mice expressing mutant PrP at low levels failed to develop disease either spontaneously or following inoculation. These studies indicate that inoculated mutant PrP from diseased mice promotes the aggregation and accumulation of pre-existing pathological forms of mutant PrP produced as a result of transgene overexpression. Thus, while pathological mutant PrP possesses a subset of PrP(Sc) characteristics, we now show that the attribute of prion transmission suggested by previous studies is more accurately characterized as disease acceleration.

Uptake and Neuritic Transport of Scrapie Prion Protein Coincident with Infection of Neuronal Cells

Invasion of the nervous system and neuronal spread of infection are critical, but poorly understood, steps in the pathogenesis of transmissible spongiform encephalopathies or prion diseases. To characterize pathways for the uptake and intraneuronal trafficking of infectious, protease-resistant prion protein (PrP-res), fluorescent-labeled PrP-res was used to infect a neuronally derived murine cell line (SN56) and adult hamster cortical neurons in primary culture. Concurrent with the establishment of persistent scrapie infection, SN56 cells internalized PrP-res aggregates into vesicles positive for markers for late endosomes and/or lysosomes but not synaptic, early endocytic, or raft-derived vesicles. Internalized PrP-res was then transported along neurites to points of contact with other cells. Similar trafficking was observed with dextran, Alzheimer's Abeta1-42 fibrils and noninfectious recombinant PrP fibrils, suggesting that PrP-res is internalized by a relatively nonspecific pinocytosis or transcytosis mechanism. Hamster cortical neurons were also capable of internalizing and disseminating exogenous PrP-res. Similar trafficking of exogenous PrP-res by cortical neurons cultured from the brains of PrP knock-out mice showed that uptake and neuritic transport did not require the presence of endogenous cellular PrP. These experiments visualize and characterize the initial steps associated with prion infection and transport within neuronal cells.

Extended period of asymptomatic prion disease after low dose inoculation: Assessment of detection methods and implications for infection control

We used quantal dose-titration of a mouse-adapted human transmissible spongiform encephalopathy strain (M470) to compare different analytical methods for their ability to detect asymptomatic brain prion infection after low dose inoculation. At a time point approximately 2.5-fold beyond the mean incubation period of high dose inocula, asymptomatic brain infection was commonly observed using histologic examination, Western blot, and "blind" bioassay following intracerebral inoculation with low titer inocula. At this time point, when a clinical end-point titration would usually be determined, evidence of infection was seen in all healthy animals inoculated with up to 100-fold lower inoculation doses than the lowest causing consistent clinical disease. For the assessment of the presence of asymptomatic infection, we compared different Western immunoblot and histopathological methods in relation to "blind" bioassay using transgenic Tga/20 mice overexpressing mouse prion protein (PrP). Sodium phosphotungstic acid (NaPTA) precipitation of protease-resistant PrP isoforms (PrP(res)) prior to Western blotting was found to approach the sensitivity of the Tga/20 bioassay and was superior to conventional Western blot and histopathological methods, wherein infectivity was commonly found when both of the latter were negative. Re-scaling the original titer by incorporating "blind" transmission data from surviving asymptomatic mice revises the estimate two orders of magnitude higher than the value derived using the conventional clinical disease outcome approach. We also found that the sensitivity of the NaPTA Western blot technique, if used with a diluent such as PBS compared with 10% normal brain homogenate, is adversely affected by up to around 20-fold. We postulate that infectious titer estimates based on more sensitive detection systems such as we report provide a more accurate indication of ultimate transmission risk.

In Vitro Generation of Infectious Scrapie Prions

Prions are unconventional infectious agents responsible for transmissible spongiform encephalopathy (TSE) diseases. They are thought to be composed exclusively of the protease-resistant prion protein (PrPres) that replicates in the body by inducing the misfolding of the cellular prion protein (PrPC). Although compelling evidence supports this hypothesis, generation of infectious prion particles in vitro has not been convincingly demonstrated. Here we show that PrPC --> PrPres conversion can be mimicked in vitro by cyclic amplification of protein misfolding, resulting in indefinite amplification of PrPres. The in vitro-generated forms of PrPres share similar biochemical and structural properties with PrPres derived from sick brains. Inoculation of wild-type hamsters with in vitro-produced PrPres led to a scrapie disease identical to the illness produced by brain infectious material. These findings demonstrate that prions can be generated in vitro and provide strong evidence in support of the protein-only hypothesis of prion transmission.

Creutzfeldt-Jakob disease in a patient with an R208H mutation of the prion protein gene (PRNP) and a 17-kDa prion protein fragment

A case of Creutzfeldt-Jakob disease (CJD) with a rare mutation of the prion protein (PrP) gene (PRNP) at codon 208 (R208H) is described. By comparison with two preceding reports, the case described here displayed two distinct biochemical and neuropathological features. Western blot analysis of brain homogenates showed, in addition to the commonly observed three bands of abnormal protease-resistant PrP isoform (PrP(Sc)), an additional band of about 17 kDa. Neuropathological examination of the post mortem brain revealed tau pathology in the hippocampus and entorhinal cortex, as well as ballooned neurons in the cortex, hippocampus and subcortical gray matter.

Neuroinvasion by Scrapie following Inoculation via the Skin Is Independent of Migratory Langerhans Cells

Many natural transmissible spongiform encephalopathy (TSE) infections are likely to be acquired peripherally, and studies in mice show that skin scarification is an effective means of scrapie transmission. After peripheral exposure, TSE agents usually accumulate in lymphoid tissues before spreading to the brain. The mechanisms of TSE transport to lymphoid tissues are not known. Langerhans cells (LCs) reside in the epidermis and migrate to the draining lymph node after encountering antigen. To investigate the potential role of LCs in scrapie transportation from the skin, we utilized mouse models in which their migration was blocked either due to CD40 ligand deficiency (CD40L-/- mice) or after caspase-1 inhibition. We show that the early accumulation of scrapie infectivity in the draining lymph node and subsequent neuroinvasion was not impaired in mice with blocked LC migration. Thus, LCs are not involved in TSE transport from the skin. After intracerebral inoculation with scrapie, wild-type mice and CD40L-/- mice develop clinical disease with similar incubation periods. However, after inoculation via skin scarification CD40L-/- mice develop disease significantly earlier than do wild-type mice. The shorter incubation period in CD40L-/- mice is unexpected and suggests that a CD40L-dependent mechanism is involved in impeding scrapie pathogenesis. In vitro studies demonstrated that LCs have the potential to acquire and degrade protease-resistant prion protein, which is thought to be a component of the infectious agent. Taken together, these data suggest that LCs are not involved in scrapie transport to draining lymphoid tissues but might have the potential to degrade scrapie in the skin.

Subcellular Localization of Disease-Associated Prion Protein in the Human Brain

Disease-associated prion protein (PrP(TSE)) deposits in distinct immunostaining patterns in the brain in Creutzfeldt-Jakob disease, including synaptic, extracellular, and cell-associated localizations. After having developed an appropriate pretreatment protocol to enhance immunostaining for PrP(TSE) without damaging epitopes of other antigens, we systematically evaluated co-localization patterns of distinct PrP(TSE) immunodeposits by confocal laser microscopy, including optical serial sectioning. As shown by quantification, the most prominent co-localization of PrP(TSE) is with synaptophysin, but PrP(TSE) may also co-deposit with connexin-32, a gap junction-related protein. Furthermore, neuronal cell bodies, dendrites, axons, astrocytes, and microglia harbor granular PrP(TSE) deposits. Highly aggregated deposits are focally ubiquitinated. We conclude that PrP(TSE) is not exclusively associated with chemical but also with electric synapses, axonal transport may be a relevant route of PrP(TSE) spread in the brain, and activated microglia and astrocytes may play a role in PrP(TSE) processing, degradation, or removal.

Effects on the serotoninergic system in sub-acute transmissible spongiform encephalopathies: current data, hypotheses, suggestions for experimentation

Sub-acute transmissible spongiform encephalopathies (TSEs), or prion diseases, are affections in which little is known of their etiology. The predominant theory stipulates that an abnormal protease-resistant prion protein (PrPres) would be infectious by directly inducing its defective conformation to the normal native protein (PrPC). The function of PrPC remains unknown. The preferred localization of PrPC at the level of the synapses supposes a function in neuronal transmission. Several neurotransmitter systems (acetylcholine, GABA, dopamine, etc.) are damaged in TSEs, mainly the serotonin (5-HT) system. At a hypothetical level, PrPC would play a trophic and functional role by regulating the capture of amino acid precursors of neurotransmitters and the functions of neuroreceptors, in particular regarding tryptophan and 5-HT receptors. By comparison with the modes of action of Ras proteins and of the envelope glycoprotein of jaagsiekte sheep retrovirus, the adaptation of an oncogenic model is suggested for the mode of action of PrPres. The sequence of events could be the following: capture of PrPres and forming of an abnormal receptor, chronic disturbance of transduction pathways, more particularly of the phosphatidylinositol-3 kinase (PI-3K)/protein kinase B (Akt)/glycogen synthetase kinase 3 (GSK 3)/Wnt-beta catenin pathway, deregulation of the PrP gene and infrequent and transitory forming of abnormal RNA messengers and, finally, the forming of abnormal proteins and the deterioration of the serotoninergic system. The involvement of endogenous nucleic acids is supposed. The infectious agent of TSEs could be an ancestral form of retrovirus, such as a retrotransposon using the prion protein as an envelope glycoprotein. Pharmacological tests, by comparison with a rare disease of unknown etiology in cattle, bovine spastic paresis, are suggested with the amino acid precursors of neuromediators (tryptophan, tyrosine, glutamic acid, etc.) and with lithium, neuroprotector and regulator of the serotonergic system.

Effective antigen-retrieval method for immunohistochemical detection of abnormal isoform of prion proteins in animals

For immunohistochemistry of the prion diseases, several pretreatment methods to enhance the immunoreactivity of human and animal abnormal proteinase-resistant prion protein (PrP(Sc)) on the tissue sections have been employed. The method of 121 degree C hydrated autoclaving pretreatment or the combination method of 121 degree C hydrated autoclaving with a certain chemical reagent (formic acid or proteinase K, etc) are now widely used. We found that an improved hydrated autoclaving method at 135 degrees C, more effectively enhanced PrP(Sc) immunoreactivity for the antibodies recognizing the linear epitope. In addition, this method was more effective for the long-term fixation samples as compared with other previous methods. However, this modified method could not retrieve PrP(Sc) antigenic epitopes composed of conformational structures or several discontinuous epitopes. We describe the comparative studies between our improved method and other antigen-retrieval procedures reported previously. Based on the differences of reaction among the antibodies, we also discuss the mechanisms of the hydrated autoclaving methods to retrieve PrP(Sc) immunoreactivity.