Central residues in prion protein PrPC are crucial for its conversion into the pathogenic isoform.

Conformational conversion of the cellular prion protein, PrPC, into the amyloidogenic isoform, PrPSc, is a key pathogenic event in prion diseases. However, the conversion mechanism remains to be elucidated. Here, we generated Tg(PrPΔ91-106)-8545/Prnp0/0 mice, which overexpress mouse PrP lacking residues 91-106. We showed that none of the mice became sick after intracerebral inoculation with RML, 22L, and FK-1 prion strains nor accumulated PrPScΔ91-106 in their brains except for a small amount of PrPScΔ91-106 detected in one 22L-inoculated mouse. However, they developed disease around 85 days after inoculation with bovine spongiform encephalopathy (BSE) prions with PrPScΔ91-106 in their brains. These results suggest that residues 91-106 are important for PrPC conversion into PrPSc in infection with RML, 22L, and FK-1 prions but not BSE prions. We then narrowed down the residues 91-106 by transducing various PrP deletional mutants into RML- and 22L-infected cells and identified that PrP mutants lacking residues 97-99 failed to convert into PrPSc in these cells. Our in vitro conversion assay also showed that RML, 22L, and FK-1 prions did not convert PrPΔ97-99 into PrPScΔ97-99, but BSE prions did. We further found that PrP mutants with proline residues at positions 97 to 99 or charged residues at positions 97 and 99 completely or almost completely lost their converting activity into PrPSc in RML- and 22L-infected cells. These results suggest that the structurally flexible and noncharged residues 97-99 could be important for PrPC conversion into PrPSc following infection with RML, 22L, and FK-1 prions but not BSE prions.

Spontaneous generation of distinct prion variants with recombinant prion protein from a baculovirus-insect cell expression system.

Prion diseases are transmissible and progressive neurodegenerative disorders characterized by abnormal prion protein (PrPSc) accumulation in the central nervous system. Generation of synthetic PrPSc in a cell-free conversion system and examination of its transmissibility to animals would facilitate testing of the protein-only hypothesis and the understanding of the molecular basis of sporadic prion diseases. In this study, we used recombinant prion protein from a baculovirus-insect cell expression system (Bac-rPrP) and insect cell-derived cofactors to determine whether Bac-rPrPSc is spontaneously produced in intermittent ultrasonic reactions. No spontaneous generation of Bac-rPrPSc was observed at 37 °C, but when the reaction temperature was increased to 45 °C, Bac-rPrPSc was generated in all trials. Some Bac-rPrPSc variants were transmissible to mice, but when the reaction was repeated for 40 rounds, the transmissibility was lost. Notably, a variety of Bac-rPrPSc variants, including non-transmissible ones, differing in resistance to proteinase K and cofactor dependence during amplification, was generated under the same experimental conditions, including the same sonication settings and cofactors. However, their characteristics also disappeared after 40 reaction rounds and the variety converged onto a single variant. These results indicate that various Bac-rPrPSc variants with different transmissibility to mice and structural properties are generated, which compete with each other and gradually converge onto a variant with a slightly faster amplification rate.

Extended application of the rapid post-mortem test kit for bovine spongiform encephalopathy to chronic wasting disease.

Chronic wasting disease (CWD) is a prion disease affecting cervid species primarily in the United States of America and Canada; however, it is now emerging in Scandinavian countries. Although CWD cases have not been reported in Japan, in case of a CWD outbreak occuring, it is critical to prepare for testing a large number of specimens. The present study showed that a rapid post-mortem test kit, which is used for bovine spongiform encephalopathy surveillance in Japan, is valid for the detection of CWD prion.

Ethanolamine Is a New Anti-Prion Compound.

Prion diseases are a group of fatal neurodegenerative disorders caused by accumulation of proteinaceous infectious particles, or prions, which mainly consist of the abnormally folded, amyloidogenic prion protein, designated PrPSc. PrPSc is produced through conformational conversion of the cellular isoform of prion protein, PrPC, in the brain. To date, no effective therapies for prion diseases have been developed. In this study, we incidentally noticed that mouse neuroblastoma N2a cells persistently infected with 22L scrapie prions, termed N2aC24L1-3 cells, reduced PrPSc levels when cultured in advanced Dulbecco's modified eagle medium (DMEM) but not in classic DMEM. PrPC levels remained unchanged in prion-uninfected parent N2aC24 cells cultured in advanced DMEM. These results suggest that advanced DMEM may contain an anti-prion compound(s). We then successfully identified ethanolamine in advanced DMEM has an anti-prion activity. Ethanolamine reduced PrPSc levels in N2aC24L1-3 cells, but not PrPC levels in N2aC24 cells. Also, oral administration of ethanolamine through drinking water delayed prion disease in mice intracerebrally inoculated with RML scrapie prions. These results suggest that ethanolamine could be a new anti-prion compound.

Strain-Dependent Prion Infection in Mice Expressing Prion Protein with Deletion of Central Residues 91-106.

Conformational conversion of the cellular prion protein, PrPC, into the abnormally folded isoform, PrPSc, is a key pathogenic event in prion diseases. However, the exact conversion mechanism remains largely unknown. Transgenic mice expressing PrP with a deletion of the central residues 91-106 were generated in the absence of endogenous PrPC, designated Tg(PrP∆91-106)/Prnp0/0 mice and intracerebrally inoculated with various prions. Tg(PrP∆91-106)/Prnp0/0 mice were resistant to RML, 22L and FK-1 prions, neither producing PrPSc∆91-106 or prions in the brain nor developing disease after inoculation. However, they remained marginally susceptible to bovine spongiform encephalopathy (BSE) prions, developing disease after elongated incubation times and accumulating PrPSc∆91-106 and prions in the brain after inoculation with BSE prions. Recombinant PrP∆91-104 converted into PrPSc∆91-104 after incubation with BSE-PrPSc-prions but not with RML- and 22L-PrPSc-prions, in a protein misfolding cyclic amplification assay. However, digitonin and heparin stimulated the conversion of PrP∆91-104 into PrPSc∆91-104 even after incubation with RML- and 22L-PrPSc-prions. These results suggest that residues 91-106 or 91-104 of PrPC are crucially involved in prion pathogenesis in a strain-dependent manner and may play a similar role to digitonin and heparin in the conversion of PrPC into PrPSc.

Self-propagating, protease-resistant, recombinant prion protein conformers with or without in vivo pathogenicity.

Prions, characterized by self-propagating protease-resistant prion protein (PrP) conformations, are agents causing prion disease. Recent studies generated several such self-propagating protease-resistant recombinant PrP (rPrP-res) conformers. While some cause prion disease, others fail to induce any pathology. Here we showed that although distinctly different, the pathogenic and non-pathogenic rPrP-res conformers were similarly recognized by a group of conformational antibodies against prions and shared a similar guanidine hydrochloride denaturation profile, suggesting a similar overall architecture. Interestingly, two independently generated non-pathogenic rPrP-res were almost identical, indicating that the particular rPrP-res resulted from cofactor-guided PrP misfolding, rather than stochastic PrP aggregation. Consistent with the notion that cofactors influence rPrP-res conformation, the propagation of all rPrP-res formed with phosphatidylglycerol/RNA was cofactor-dependent, which is different from rPrP-res generated with a single cofactor, phosphatidylethanolamine. Unexpectedly, despite the dramatic difference in disease-causing capability, RT-QuIC assays detected large increases in seeding activity in both pathogenic and non-pathogenic rPrP-res inoculated mice, indicating that the non-pathogenic rPrP-res is not completely inert in vivo. Together, our study supported a role of cofactors in guiding PrP misfolding, indicated that relatively small structural features determine rPrP-res' pathogenicity, and revealed that the in vivo seeding ability of rPrP-res does not necessarily result in pathogenicity.

Heparan Sulfate and Heparin Promote Faithful Prion Replication in Vitro by Binding to Normal and Abnormal Prion Proteins in Protein Misfolding Cyclic Amplification.

The precise mechanism underlying the conversion of normal prion protein (PrPC) into abnormal prion protein (PrPSc) remains unclear. Protein misfolding cyclic amplification (PMCA), an in vitro technique used for amplifying PrPSc, results in PrPSc replication that preserves the strain-specific characteristics of the input PrPSc; thus, PMCA mimics the process of in vivo PrPSc replication. Previous work has demonstrated that in PMCA, nucleic acids are critical for PrPSc amplification, but little information has been reported on glycosaminoglycan (GAG) participation in PrPSc replication in vitro Here, we investigated whether GAGs play a role in the faithful replication of PrPSc by using a modified PMCA performed with baculovirus-derived recombinant PrP (Bac-PrP) as a substrate. The addition of heparan sulfate (HS) or its analog heparin (HP) restored the conversion efficiency in PMCA that was inhibited through nucleic acid depletion. Moreover, the PMCA products obtained under these conditions were infectious and preserved the properties of the input PrPSc These data suggest that HS and HP play the same role as nucleic acids in facilitating faithful replication of prions in PMCA. Furthermore, we showed that HP binds to both Bac-PrP and Bac-PrPSc through the sulfated groups present on HP and that the N-terminal domain of Bac-PrPSc might potentially not be involved in the binding to HP. These results suggest that the interaction of GAGs such as HS and HP with PrPC and/or PrPSc through their sulfate groups is critical for the faithful replication of prions.

Oral Transmission of L-Type Bovine Spongiform Encephalopathy Agent among Cattle.

To determine oral transmissibility of the L-type bovine spongiform encephalopathy (BSE) prion, we orally inoculated 16 calves with brain homogenates of the agent. Only 1 animal, given a high dose, showed signs and died at 88 months. These results suggest low risk for oral transmission of the L-BSE agent among cattle.

Experimental Infection of Cattle With a Novel Prion Derived From Atypical H-Type Bovine Spongiform Encephalopathy.

H-type bovine spongiform encephalopathy (H-BSE) is an atypical form of BSE in cattle. During passaging of H-BSE in transgenic bovinized (TgBoPrP) mice, a novel phenotype of BSE, termed BSE-SW emerged and was characterized by a short incubation time and host weight loss. To investigate the biological and biochemical properties of the BSE-SW prion, a transmission study was conducted in cattle, which were inoculated intracerebrally with brain homogenate from BSE-SW-infected TgBoPrP mice. The disease incubation period was approximately 15 months. The animals showed characteristic neurological signs of dullness, and severe spongiform changes and a widespread, uniform distribution of disease-associated prion protein (PrPSc) were observed throughout the brain of infected cattle. Immunohistochemical PrPSc staining of the brain revealed the presence of intraglial accumulations and plaque-like deposits. No remarkable differences were identified in vacuolar lesion scores, topographical distribution patterns, and staining types of PrPSc in the brains of BSE-SW- vs H-BSE-infected cattle. PrPSc deposition was detected in the ganglia, vagus nerve, spinal nerve, cauda equina, adrenal medulla, and ocular muscle. Western blot analysis revealed that the specific biochemical properties of the BSE-SW prion, with an additional 10- to 12-kDa fragment, were well maintained after transmission. These findings indicated that the BSE-SW prion has biochemical properties distinct from those of H-BSE in cattle, although clinical and pathologic features of BSW-SW in cattle are indistinguishable from those of H-BSE. The results suggest that the 2 infectious agents, BSE-SW and H-BSE, are closely related strains.

Multiple affinity purification of a baculovirus-derived recombinant prion protein with in vitro ability to convert to its pathogenic form.

We previously showed that baculovirus-derived recombinant prion protein (Bac-PrP) can be converted to the misfolded infectious form (PrPSc) by protein misfolding cyclic amplification, an in vitro conversion technique. Bac-PrP, with post-translational modifications, would be useful for various applications such as using PrP as an immunogen for generating anti-PrP antibody, developing anti-prion drugs or diagnostic assays using in vitro conversion systems, and establishing an in vitro prion propagation model. For this purpose, highly purified Bac-PrP with in vitro conversion activity is necessary for use as a PrPC source, to minimize contamination. Furthermore, an exogenous affinity tag-free form is desirable to avoid potential steric interference by the affinity tags during the conversion process. In this study, we established purification methods for the untagged Bac-PrP under native conditions by combining exogenous double-affinity tags, namely, a polyhistidine-tag and a profinity eXact tag, with an octarepeat sequence of the N-terminal region of PrP, which has metal ion-binding affinity. The untagged Bac-PrP with near-homogeneity was obtained by three-step affinity purification, and it was shown that the final, purified Bac-PrP could convert to its pathogenic form. The presented purification procedure could be applied not only to PrP but also to other eukaryotic, recombinant proteins that require high purity and intact physiological activity.

Proximity of SCG10 and prion protein in membrane rafts.

The conversion of normal cellular prion protein (PrPC) into its pathogenic isoform (PrPSc) is an essential event in prion pathogenesis. In culture models, membrane rafts are suggested to play a critical role in PrPSc formation. To identify the candidate molecules capable of interacting with PrPC and facilitating PrPSc formation in membrane rafts, we applied a novel biochemical labeling method termed enzyme-mediated activation of radical sources. Enzyme-mediated activation of radical sources was applied to the Lubrol WX insoluble detergent-resistant membrane fractions from mouse neuroblastoma (N2a) cells in which the surface PrPC was labeled with HRP-conjugated anti-PrP antibody. Two-dimensional western blots of these preparations revealed biotinylated spots of approximately 20 kDa with an isoelectric point of 8.0-9.0. Liquid chromatography-tandem mass spectrometry analysis resulted in the identification of peptides containing SCG10, the neuron-specific microtubule regulator. Proximity of SCG10 and PrPC was confirmed using proximity ligation assay and co-immunoprecipitation assay. Transfection of persistently 22L prion-infected N2a cells with SCG10 small interfering RNA reduced SCG10 expression, but did not prevent PrPSc accumulation, indicating that SCG10 appears to be unrelated to PrPSc formation of 22L prion. Immunofluorescence and western blot analyses showed reduced levels of SCG10 in the hippocampus of prion-infected mice, suggesting a possible association between SCG10 levels and the prion neuropathogenesis. By applying a novel biochemical labeling method against detergent-resistant membrane fractions from mouse neuroblastoma cells, the neuron-specific microtubule-destabilization protein, SCG10 was identified as a novel candidate that is proximate to normal prion protein (PrP) in membrane rafts. SCG10 seemed unrelated to disease-related PrP formation under certain conditions, while there is a possible association between SCG10 levels and prion neuropathogenesis. Cover Image for this issue: doi: 10.1111/jnc.13310.

Ultrasensitive detection of PrP(Sc) in the cerebrospinal fluid and blood of macaques infected with bovine spongiform encephalopathy prion.

Prion diseases are characterized by the prominent accumulation of the misfolded form of a normal cellular protein (PrP(Sc)) in the central nervous system. The pathological features and biochemical properties of PrP(Sc) in macaque monkeys infected with the bovine spongiform encephalopathy (BSE) prion have been found to be similar to those of human subjects with variant Creutzfeldt-Jakob disease (vCJD). Non-human primate models are thus ideally suited for performing valid diagnostic tests and determining the efficacy of potential therapeutic agents. In the current study, we developed a highly efficient method for in vitro amplification of cynomolgus macaque BSE PrP(Sc). This method involves amplifying PrP(Sc) by protein misfolding cyclic amplification (PMCA) using mouse brain homogenate as a PrP(C) substrate in the presence of sulfated dextran compounds. This method is capable of amplifying very small amounts of PrP(Sc) contained in the cerebrospinal fluid (CSF) and white blood cells (WBCs), as well as in the peripheral tissues of macaques that have been intracerebrally inoculated with the BSE prion. After clinical signs of the disease appeared in three macaques, we detected PrP(Sc) in the CSF by serial PMCA, and the CSF levels of PrP(Sc) tended to increase with disease progression. In addition, PrP(Sc) was detectable in WBCs at the clinical phases of the disease in two of the three macaques. Thus, our highly sensitive, novel method may be useful for furthering the understanding of the tissue distribution of PrP(Sc) in non-human primate models of CJD.

Prion replication elicits cytopathic changes in differentiated neurosphere cultures.

The molecular mechanisms of prion-induced cytotoxicity remain largely obscure. Currently, only a few cell culture models have exhibited the cytopathic changes associated with prion infection. In this study, we introduced a cell culture model based on differentiated neurosphere cultures isolated from the brains of neonatal prion protein (PrP)-null mice and transgenic mice expressing murine PrP (dNP0 and dNP20 cultures). Upon exposure to mouse Chandler prions, dNP20 cultures supported the de novo formation of abnormal PrP and the resulting infectivity, as assessed by bioassays. Furthermore, this culture was susceptible to various prion strains, including mouse-adapted scrapie, bovine spongiform encephalopathy, and Gerstmann-Sträussler-Scheinker syndrome prions. Importantly, a subset of the cells in the infected culture that was mainly composed of astrocyte lineage cells consistently displayed late-occurring, progressive signs of cytotoxicity as evidenced by morphological alterations, decreased cell viability, and increased lactate dehydrogenase release. These signs of cytotoxicity were not observed in infected dNP0 cultures, suggesting the requirement of endogenous PrP expression for prion-induced cytotoxicity. Degenerated cells positive for glial fibrillary acidic protein accumulated abnormal PrP and exhibited features of apoptotic death as assessed by active caspase-3 and terminal deoxynucleotidyltransferase nick-end staining. Furthermore, caspase inhibition provided partial protection from prion-mediated cell death. These results suggest that differentiated neurosphere cultures can provide an in vitro bioassay for mouse prions and permit the study of the molecular basis for prion-induced cytotoxicity at the cellular level.

Different antigenicities of the N-terminal region of cellular and scrapie prion proteins.

Limited information is available about conformational differences between the abnormal isoform of prion protein (PrP(Sc) ) and cellular prion protein (PrP(C) ) under native conditions. To clarify conformational differences between these two isoforms, PrP-deficient mice were immunized with brain homogenates of normal and scrapie-infected animals. All mice generated anti-PrP antibodies. Peptide array analysis of these serum samples revealed a distinctive epitope of PrP(Sc) consisting of QGSPGGN (PrP41-47) at the N-terminus. This study demonstrated a conformational dissimilarity at the N-terminus between PrP(Sc) and PrP(C) , a finding that may provide novel information about conformational features of PrP(Sc) .

Glycosylphosphatidylinositol anchor-dependent stimulation pathway required for generation of baculovirus-derived recombinant scrapie prion protein.

The pathogenic isoform (PrP(Sc)) of the host-encoded cellular prion protein (PrP(C)) is considered to be an infectious agent of transmissible spongiform encephalopathy (TSE). The detailed mechanism by which the PrP(Sc) seed catalyzes the structural conversion of endogenous PrP(C) into nascent PrP(Sc) in vivo still remains unclear. Recent studies reveal that bacterially derived recombinant PrP (recPrP) can be used as a substrate for the in vitro generation of protease-resistant recPrP (recPrP(res)) by protein-misfolding cyclic amplification (PMCA). These findings imply that PrP modifications with a glycosylphosphatidylinositol (GPI) anchor and asparagine (N)-linked glycosylation are not necessary for the amplification and generation of recPrP(Sc) by PMCA. However, the biological properties of PrP(Sc) obtained by in vivo transmission of recPrP(res) are unique or different from those of PrP(Sc) used as the seed, indicating that the mechanisms mediated by these posttranslational modifications possibly participate in reproductive propagation of PrP(Sc). In the present study, using baculovirus-derived recombinant PrP (Bac-PrP), we demonstrated that Bac-PrP is useful as a PrP(C) substrate for amplification of the mouse scrapie prion strain Chandler, and PrP(Sc) that accumulated in mice inoculated with Bac-PrP(res) had biochemical and pathological properties very similar to those of the PrP(Sc) seed. Since Bac-PrP modified with a GPI anchor and brain homogenate of Prnp knockout mice were both required to generate Bac-PrP(res), the interaction of GPI-anchored PrP with factors in brain homogenates is essential for reproductive propagation of PrP(Sc). Therefore, the Bac-PMCA technique appears to be extremely beneficial for the comprehensive understanding of the GPI anchor-mediated stimulation pathway.

Cytochalasin D enhances the accumulation of a protease-resistant form of prion protein in ScN2a cells: involvement of PI3 kinase/Akt signalling pathway.

The conversion of a host-encoded PrPsen (protease-sensitive cellular prion protein) into a PrPres (protease-resistant pathogenic form) is a key process in the pathogenesis of prion diseases, but the intracellular mechanisms underlying PrPres amplification in prion-infected cells remain elusive. To assess the role of cytoskeletal proteins in the regulation of PrPres amplification, the effects of cytoskeletal disruptors on PrPres accumulation in ScN2a cells that were persistently infected with the scrapie Chandler strain have been examined. Actin microfilament disruption with cytochalasin D enhanced PrPres accumulation in ScN2a cells. In contrast, the microtubule-disrupting agents, colchicine, nocodazole and paclitaxel, had no effect on PrPres accumulation. In addition, a PI3K (phosphoinositide 3-kinase) inhibitor, wortmannin and an Akt kinase inhibitor prevented the cytochalasin D-induced enhancement of PrPres accumulation. Cytochalasin D-induced extension of neurite-like processes might correlate with enhanced accumulation of PrPres. The results suggest that the actin cytoskeleton and PI3K/Akt pathway are involved in the regulation of PrPres accumulation in prion-infected cells.

Ultrasensitive detection of scrapie prion protein derived from ARQ and AHQ homozygote sheep by interspecies in vitro amplification.

Prions, infectious agents causing TSEs, are composed primarily of the pathogenic form (PrP(Sc)) of the PrP(C). The susceptibility of sheep to scrapie is determined by polymorphisms in the coding region of the PRNP, mainly at codons 136, 154, and 171. The efficiency of in vitro amplification of sheep PrP(Sc) seems to be linked also to the PrP genotype. PrP(Sc) derived from sheep with V(136)R(154)Q(171)-associated genotypes can be amplified efficiently by PMCA in the presence of additional polyanion such as poly A, but there are no reports that cite ultrasensitive detection of PrP(Sc) derived from sheep of other PrP genotypes. We report here that sheep PrP(Sc) derived from ARQ and AHQ homozygotes was amplified efficiently by serial PMCA using mouse brain homogenate as PrP(C) substrate. ARQ/ARQ PrP(Sc) was detected in infected brain homogenates diluted up to 10(-10) after five rounds of amplification, and AHQ/AHQ PrP(Sc) was detected in samples diluted up to 10(-8) after four rounds of amplification. On the other hand, amplification of PrP(Sc) from VRQ/ARQ sheep seemed to be less efficient under the experimental conditions used. The interspecies PMCA developed in this study may be useful in the detailed analysis of PrP(Sc) distribution in classical scrapie-infected ARQ and AHQ homozygote sheep.

Pentosan polysulfate induces low-level persistent prion infection keeping measurable seeding activity without PrP-res detection in Fukuoka-1 infected cell cultures.

Each prion strain has its own characteristics and the efficacy of anti-prion drugs varies. Screening of prion disease therapeutics is typically evaluated by measuring amounts of protease-resistant prion protein (PrP-res). However, it remains unclear whether such measurements correlate with seeding activity, which is evaluated by real-time quaking-induced conversion (RT-QuIC). In this study, the effects of anti-prion compounds pentosan polysulfate (PPS), Congo red, and alprenolol were measured in N2a58 cells infected with Fukuoka-1 (FK1) or 22L strain. The compounds abolished PrP-res and seeding activity, except for N2a58/FK1 treated with PPS. Interestingly, the seeding activity of N2a58/FK1, which was reduced in the presence of PPS, was not lost and remained at low levels. However, upon removal of PPS, both were gradually restored to their original levels. These results indicate that low-level persistent prion infection keeping measurable seeding activity is induced by PPS in a strain-dependent manner. Furthermore, for protein misfolding cyclic amplification (PMCA), the anti-prion effect of PPS decreased in FK1 compared to 22L, suggesting that the differences occur at the level of the direct conversion. Our findings demonstrate that the advantages of RT-QuIC and PMCA can be exploited for more accurate assessment of therapeutic drug screening, reflecting strain differences.

Tracing conformational transition of abnormal prion proteins during interspecies transmission by using novel antibodies.

Conformational differences in abnormal prion proteins (PrP(Sc)) have been postulated to produce different prion phenotypes. During the interspecies transmission of prions, the conformation of PrP(Sc) may change with passage; however, little is known about the mechanism of PrP(Sc) transition. In this study, novel PrP(Sc)-specific monoclonal antibodies (mAbs) were developed that could detect the PrP(Sc) of mouse but not that of sheep. By using these mAbs, we attempted to examine PrP(Sc) accumulated in mice inoculated with sheep scrapie serially up to five passages. The presence of PrP(Sc) in the mice was confirmed at all passages; however, mAb-bound PrP(Sc) conformer was detected only from the third passage onward. The generated mAb enabled tracing of a particular conformer during adaptation in sheep-to-mice transmission of prion, suggesting that the conformational transition of PrP(Sc) was caused by propagation of this conformer. Such mAbs capable of discriminating conformational differences may allow us to address questions concerning PrP(Sc) conformation and strain diversity.

Experimental H-type bovine spongiform encephalopathy characterized by plaques and glial- and stellate-type prion protein deposits.

Atypical bovine spongiform encephalopathy (BSE) has recently been identified in Europe, North America, and Japan. It is classified as H-type and L-type BSE according to the molecular mass of the disease-associated prion protein (Pr(PSc)). To investigate the topographical distribution and deposition patterns of immunolabeled Pr(PSc), H-type BSE isolate was inoculated intracerebrally into cattle. H-type BSE was successfully transmitted to 3 calves, with incubation periods between 500 and 600 days. Moderate to severe spongiform changes were detected in the cerebral and cerebellar cortices, basal ganglia, thalamus, and brainstem. H-type BSE was characterized by the presence of PrP-immunopositive amyloid plaques in the white matter of the cerebrum, basal ganglia, and thalamus. Moreover, intraglial-type immunolabeled Pr(PSc) was prominent throughout the brain. Stellate-type immunolabeled Pr(PSc) was conspicuous in the gray matter of the cerebral cortex, basal ganglia, and thalamus, but not in the brainstem. In addition, Pr(PSc) accumulation was detected in the peripheral nervous tissues, such as trigeminal ganglia, dorsal root ganglia, optic nerve, retina, and neurohypophysis. Cattle are susceptible to H-type BSE with a shorter incubation period, showing distinct and distinguishable phenotypes of Pr(PSc) accumulation.