Accumulation Of Abnormal Prion Protein Research Articles

Unexpected decrease of full-length prion protein in macaques inoculated with prion-contaminated blood products.

The presence of prion infectivity in the blood of patients affected by variant Creutzfeldt-Jakob disease (v-CJD), the human prion disease linked to the bovine spongiform encephalopathy (BSE), poses the risk of inter-human transmission of this fatal prion disease through transfusion. In the frame of various experiments, we have previously described that several cynomolgus macaques experimentally exposed to prion-contaminated blood products developed c-BSE/v-CJD, but the vast majority of them developed an unexpected, fatal disease phenotype focused on spinal cord involvement, which does not fulfill the classical diagnostic criteria of v-CJD. Here, we show that extensive analyses with current conventional techniques failed to detect any accumulation of abnormal prion protein (PrPv-CJD) in the CNS of these myelopathic animals, i.e., the biomarker considered responsible for neuronal death and subsequent clinical signs in prion diseases. Conversely, in the spinal cord of these myelopathic primates, we observed an alteration of their physiological cellular PrP pattern: PrP was not detectable under its full-length classical expression but mainly under its physiological terminal-truncated C1 fragment. This observed disappearance of the N-terminal fragment of cellular PrP at the level of the lesions may provide the first experimental evidence of a link between loss of function of the cellular prion protein and disease onset. This original prion-induced myelopathic syndrome suggests an unexpected wide extension in the field of prion diseases that is so far limited to pathologies associated with abnormal changes of the cellular PrP to highly structured conformations.

Characterization of Classical Sheep Scrapie in White-tailed Deer after Experimental Oronasal Exposure.

Classic scrapie is a prion disease of sheep and goats that is associated with accumulation of abnormal prion protein (PrPSc) in the central nervous and lymphoid tissues. Chronic wasting disease (CWD) is the prion disease of cervids. This study was conducted to determine the susceptibility of white-tailed deer (WTD) to the classic scrapie agent. We inoculated WTD (n = 5) by means of a concurrent oral/intranasal exposure with the classic scrapie agent from sheep or oronasally with the classic scrapie agent from goats (n = 6). All deer exposed to the agent of classic scrapie from sheep accumulated PrPSc. PrPSc was detected in lymphoid tissues at preclinical time points, and necropsies in deer 28 months after inoculation showed clinical signs, spongiform lesions, and widespread PrPSc in neural and lymphoid tissues. Western blots on samples from the brainstem, cerebellum, and lymph nodes of scrapie-infected WTD have a molecular profile similar to CWD and distinct from samples from the cerebral cortex, retina, or the original classic scrapie inoculum. There was no evidence of PrPSc in any of the WTD inoculated with classic scrapie prions from goats. WTD are susceptible to the agent of classic scrapie from sheep, and differentiation from CWD may be difficult.

Calcineurin, Calcium-Dependent Serine-Threonine Phosphatase Activation by Prion Peptide 106-126 Enhances Nuclear Factor-κB-Linked Proinflammatory Response through Autophagy Pathway.

Prion diseases are mortal neurodegenerative pathologies that are caused by the accumulation of abnormal prion protein (PrPSc) in the brain. Recent advances reveal that calcineurin may play a critical role in regulating nuclear factor kappa B (NF-κB) in the calcium-calmodulin pathway. However, the exact mechanism by calcineurin remains unclear. In the present study, we observed that the prion peptide induces calcineurin and autophagy activation. Also, NF-κB and proinflammatory cytokines like interleukin (IL)-6 and tumor necrosis factor (TNF)-α are upregulated upon exposure to prion peptide in human neuroblastoma. The results show that the prion peptide induces calcineurin activation, leading to the activation of NF-κB transcription factor via autophagy signaling. Expression of TNF-α and IL-6 was increased by calcineurin activation and blocked by calcineurin inhibitor and autophagy inhibitor treatments. Collectively, these findings indicate that calcineurin activation mediated by prion protein induces NF-κB-driven neuroinflammation via autophagy pathway, suggesting that calcineurin and autophagy may be possible therapeutic targets for neuroinflammation in neurodegeneration diseases including prion disease.

Detection of cutaneous prion protein deposits could help diagnose GPI-anchorless prion disease with neuropathy.

To investigate prion protein (PrP) deposits in cutaneous tissues of patients of glycosylphosphatidylinositol (GPI)-anchorless prion diseases with neuropathy. Cutaneous tissue samples from three patients with GPI-anchorless prion diseases were obtained, two cutaneous biopsy samples from the lower leg of Case 1 (Y162X) and Case 3 (D178fs25), and a cutaneous sample taken from the abdomen during an autopsy of Case 2 (D178fs25). We performed immunohistochemistry for PrP to look for abnormal PrP deposits. PrP deposits were observed in the dermal papilla, the sweat glands, the hair follicles, the arrector pili muscles, and peripheral nerves of all examined cases of GPI-anchorless prion disease with neuropathy. The abnormal PrP accumulation was frequently localized at the basement membrane, and colocalized with laminin. Immunohistochemical detection of PrP in cutaneous samples could be used to definitively diagnose GPI-anchorless PrP disease with neuropathy.

Novel Compounds Identified by Structure-Based Prion Disease Drug Discovery Using In Silico Screening Delay the Progression of an Illness in Prion-Infected Mice.

The accumulation of abnormal prion protein (PrPSc) produced by the structure conversion of PrP (PrPC) in the brain induces prion disease. Although the conversion process of the protein is still not fully elucidated, it has been known that the intramolecular chemical bridging in the most fragile pocket of PrP, known as the "hot spot," stabilizes the structure of PrPC and inhibits the conversion process. Using our original structure-based drug discovery algorithm, we identified the low molecular weight compounds that predicted binding to the hot spot. NPR-130 and NPR-162 strongly bound to recombinant PrP in vitro, and fragment molecular orbital (FMO) analysis indicated that the high affinity of those candidates to the PrP is largely dependent on nonpolar interactions, such as van der Waals interactions. Those NPRs showed not only significant reduction of the PrPSc levels but also remarkable decrease of the number of aggresomes in persistently prion-infected cells. Intriguingly, treatment with those candidate compounds significantly prolonged the survival period of prion-infected mice and suppressed prion disease-specific pathological damage, such as vacuole degeneration, PrPSc accumulation, microgliosis, and astrogliosis in the brain, suggesting their possible clinical use. Our results indicate that in silico drug discovery using NUDE/DEGIMA may be widely useful to identify candidate compounds that effectively stabilize the protein.

Administration of FK506 from Late Stage of Disease Prolongs Survival of Human Prion-Inoculated Mice

Human prion diseases are etiologically categorized into three forms: sporadic, genetic, and infectious. Sporadic Creutzfeldt-Jakob disease (sCJD) is the most common type of human prion disease that manifests as subacute progressive dementia. No effective therapy for sCJD is currently available. Potential therapeutic compounds are frequently tested in rodents infected with mouse-adapted prions that differ from human prions. However, therapeutic effect varies depending on the prion strain, which is one of the reasons why candidate compounds have shown little effect in sCJD patients. We previously reported that intraperitoneal administration of FK506 was able to prolong the survival of mice infected with a mouse-adapted prion by suppressing the accumulation of abnormal prion protein (PrP) and inhibiting the activation of microglia. In this study, we tested oral administration of FK506 in knock-in mice expressing chimeric human prion protein (KiChM) that were infected with sCJD to determine if this compound is also effective against a clinically relevant human prion, i.e., one that has not been adapted to mice. Treatment with FK506, started either just before or just after disease onset, suppressed typical sCJD pathology (gliosis) and slightly but significantly prolonged the survival of sCJD-inoculated mice. It would be worthwhile to conduct a clinical trial using FK506, which has been safety-approved and is widely used as a mild immunosuppressant.

Novel Compounds Identified by Structure-Based Prion Disease Drug Discovery Using <i>In Silico</i> Screening Delay the Progression of an Illness

The accumulation of abnormal prion protein (PrPSc) produced by the structure conversion of PrP (PrPC) in the brain induces prion disease. Although the conversion process of the protein is still not fully elucidated, the intramolecular chemical bridging in the most fragile pocket of PrP, known as the “Hot Spot”, can stabilize the structure of PrPC and inhibit the conversion process. Using our original structure-based drug discovery algorithm, we identified the low molecular weight compounds that can bind to the hot spot. Several candidates were identified as anti-prion drugs with strong binding to recombinant PrP in vitro that significantly reduced the levels of PrPSc. These compounds were able to decrease the number of aggresomes in persistently prion-infected cells. Remarkably, treatment with the predominant candidate compounds significantly prolonged the survival period of prion-infected mice and dominantly suppressed disease-specific pathological damage, such as vacuole degeneration, PrPSc accumulation, microgliosis and astrogliosis in the brain, suggesting their possible clinical use. Fragment molecular orbital (FMO) analysis indicated that the high affinity of the candidates to the protein is largely dependent on non-polar interactions, such as van der Waals interactions. Our results suggest that in silico drug discovery using NUDE/DEGIMA may be useful to identify candidate compounds that effectively stabilize the protein. Funding Statement: This work was supported in part by the a grant from a Grant-in-Aid of the Research Committee of Prion Disease and Slow Virus Infection from the Ministry of Health, Labour and Welfare of Japan (N.N.); a Grant-in-Aid of the Research Committee of Molecular Pathogenesis and Therapies for Prion Disease and Slow Virus Infection, the Practical Research Project for Rare and Intractable Disease from the Japan Agency for Medical Research and Development, AMED (D.I.); a grant from the Takeda Science Foundation (N.N. and D.I.); a grant from the Japan Intractable Disease Research Foundation (D.I.); a Grant-in-Aid from the Tokyo Biochemical Research Foundation (D.I.); a grant provided by the YOKOYAMA Foundation for Clinical Pharmacology (Grant No. YRY1502) (D.I.); the grant provided by the Ichiro Kanehara Foundation (D.I.); a grant provided by the Mochida Memorial Foundation for Medical and Pharmaceutical Research (D.I.); a grant provided by the Waksman Foundation of Japan Inc. (D.I.); and a grant provided by the Center for Clinical and Translational Research of Kyushu University Hospital (D.I.). Declaration of Interests: The authors state that they have no conflicts of interest. Ethics Approval Statement: Animal experiments were approved by the Animal Care and Use Committee of the Nagasaki University. All animals were treated and cared for in accordance with the Guidelines for Animal Experimentation of Nagasaki University.

The First Report of Genetic and Structural Diversities in the SPRN Gene in the Horse, an Animal Resistant to Prion Disease.

Prion diseases are fatal neurodegenerative diseases and are characterized by the accumulation of abnormal prion protein (PrPSc) in the brain. During the outbreak of the bovine spongiform encephalopathy (BSE) epidemic in the United Kingdom, prion diseases in several species were reported; however, horse prion disease has not been reported thus far. In previous studies, the shadow of prion protein (Sho) has contributed to an acceleration of conversion from normal prion protein (PrPC) to PrPSc, and the shadow of prion protein gene (SPRN) polymorphisms have been significantly associated with the susceptibility of prion diseases. We investigated the genotype, allele and haplotype frequencies of the SPRN gene using direct sequencing. In addition, we analyzed linkage disequilibrium (LD) and haplotypes among polymorphisms. We also investigated LD between PRNP and SPRN single nucleotide polymorphisms (SNPs). We compared the amino acid sequences of Sho protein between the horse and several prion disease-susceptible species using ClustalW2. To perform Sho protein modeling, we utilized SWISS-MODEL and Swiss-PdbViewer programs. We found a total of four polymorphisms in the equine SPRN gene; however, we did not observe an in/del polymorphism, which is correlated with the susceptibility of prion disease in prion disease-susceptible animals. The SPRN SNPs showed weak LD value with PRNP SNP. In addition, we found 12 horse-specific amino acids of Sho protein that can induce significantly distributional differences in the secondary structure and hydrogen bonds between the horse and several prion disease-susceptible species. To the best of our knowledge, this is the first report regarding the genetic and structural characteristics of the equine SPRN gene.

A Novel Combination of Prion Strain Co-Occurrence in Patients with Sporadic Creutzfeldt-Jakob Disease

Six subgroups of sporadic Creutzfeldt-Jakob disease have been identified by distinctive clinicopathologic features, genotype at polymorphic codon 129 [methionine (M)/valine (V)] of the PRNP gene, and type of abnormal prion proteins (type 1 or 2). In addition to the pure subgroups, mixed neuropathologic features and the coexistence of two types of abnormal prion proteins in the same patient also have beenreported. Here, we found that a portion of the patients previously diagnosed as MM1 had neuropathologic characteristics of the MM2 thalamic form (ie, neuronal loss of the inferior olivary nucleus of the medulla). Furthermore, coexistence of biochemical features of the MM2 thalamic form also was confirmed in the identified cases. In addition, in transmission experiments using prion protein-humanized mice, the brain material from the identified case showed weak infectivity and generated characteristic abnormal prion proteins in the inoculated mice resembling those after inoculation with brain material of MM2 thalamic form. Taken together, these results show that the co-occurrence of MM1 and MM2 thalamic form is a novel entity of sporadic Creutzfeldt-Jakob disease prion strain co-occurrence. The present study raises the possibility that the co-occurrence of the MM2 thalamic form might have been overlooked so far because of the scarcity of abnormal prion protein accumulation and restricted neuropathology.

A domain responsible for spontaneous conversion of bank vole prion protein.

Bank vole is a small rodent that shows high susceptibility to infection with diverse prion strains. To determine whether the increased susceptibility of bank voles to prion diseases can be attributed to the intrinsic nature of bank vole prion protein (PrP) or to host factors other than PrP, we produced transgenic mice overexpressing bank vole PrP. These transgenic mice spontaneously developed neurological illness with spongiform changes and the accumulation of abnormal PrP in the brain. Then, we produced transgenic mice overexpressing chimeric mouse/bank vole PrP, which differs from mouse PrP only at two residues located at the C-terminus, to determine the minimum essential domain for the induction of spontaneous generation of abnormal PrP. These transgenic mice also developed spontaneous neurological illness with spongiform changes and the accumulation of abnormal PrP in the brain. In addition, knock-in mice expressing bank vole PrP at the same level as that of wild-type mice did not develop spontaneous disease but showed high susceptibility to infection with diverse prion strains, similarly to bank voles. Taken together, these findings show that bank vole PrP has a high propensity for the conformational conversion both in spontaneous disease and in prion infection, probably due to the characteristic structural properties of the C-terminal domain.

In Prion Disease Patients, Prion Seeding Activity in the Digestive System is Similar to Activity in CNS Tissue

Background: Human prion diseases (HPD) are neurodegenerative disorders caused by abnormal prion protein, and are associated with the accumulation of abnormal prion protein in the central nervous system. Infectivity had previously been detected only in the central nervous system and lymphoreticular tissues in sporadic Creutzfeldt-Jakob disease (CJD), and peripheral tissue involvement in prion disease, including abnormal prion accumulation in the tonsil, spleen, and lymph node, has been reported in variant CJD and several animal models of prion diseases. Furthermore, recent reports suggest that abnormal prion protein accumulates in various non-neuronal organs including liver, kidney and skin. The aim of this study was to examine abnormal prion accumulation in digestive organs of HPD patients. Methods and Methods: We reanalyzed autopsy samples collected from four sporadic (MM1) and two genetic HPD (E200K and P102L) cases. Analysis of prion seeding activities (PSA) based on real-time quacking-induced conversion assay indicated seeding activity exists in almost all digestive organs. PSA was 109.4-10.18 in brain of all HPD patients, and was 105-7 in digestive organs of all HPD patients. PSA in esophagus was 107.98-8.38 in two of the four sporadic HPD patients and one of the two genetic HPD patients, which was an infectious level similar to that in the central nervous system. Moreover, we identified that PSA was 107-8 in other digestive organs of patients with genetic prion disease. Conclusions: Clinicians should be careful to ensure the safety of endoscopic examinations and operation to prevent accidental transmission of prion protein. Funding: The study was financially supported by grants for scientific research from the Ministry of Health, Labour and Welfare of Japan (KSat: No. 14507303) and Japan Agency for Medical Research and Development (AMED) (grant number No.18ek0109362h0001). Declaration of Interest: All authors should state that none exist in the declarations of interests Ethical Approval: The protocol was approved by the Ethics Committee of Nagasaki University Hospital (ID: 100428423) and was also granted ethical approval for the use of specimens by the Japan CJD Surveillance Unit. The study was registered with the University Hospital Medical Information Network (ID: UMI03301).

Development of a quick bioassay for the evaluation of transmission properties of acquired prion diseases

Evaluation of transmission properties is important for the differential diagnosis of a subgroup of acquired Creutzfeldt-Jakob disease (CJD) with methionine homozygosity at polymorphic codon 129 of the PRNP gene, an intermediate type abnormal prion protein (PrP), and kuru plaques, denoted as acquired CJD-MMiK. The present study aimed to develop a quick evaluation system of the transmission properties of acquired CJD-MMiK. In the PrP-humanized mice intraperitoneally inoculated with brain homogenates from an acquired CJD-MMiK patient, accumulation of abnormal PrP was observed in follicular dendritic cells of the spleen at 75 days post-inoculation. The transmission properties of acquired CJD-MMiK were quite different from those of sporadic CJD with the same PRNP codon 129 genotype. Moreover, even at 14 days post-inoculation, the characteristic transmission properties of acquired CJD-MMiK could be detected. These findings suggest that the bioassay using follicular dendritic cells of the spleen, named as a FDC assay, can be an easy, time-saving, and useful method to distinguish acquired CJD-MMiK from sporadic CJD.

Experimental transfusion of variant CJD-infected blood reveals previously uncharacterised prion disorder in mice and macaque

Exposure of human populations to bovine spongiform encephalopathy through contaminated food has resulted in <250 cases of variant Creutzfeldt–Jakob disease (vCJD). However, more than 99% of vCJD infections could have remained silent suggesting a long-term risk of secondary transmission particularly through blood. Here, we present experimental evidence that transfusion in mice and non-human primates of blood products from symptomatic and non-symptomatic infected donors induces not only vCJD, but also a different class of neurological impairments. These impairments can all be retransmitted to mice with a pathognomonic accumulation of abnormal prion protein, thus expanding the spectrum of known prion diseases. Our findings suggest that the intravenous route promotes propagation of masked prion variants according to different mechanisms involved in peripheral replication.

Prion pathogenesis is unaltered in the absence of SIRPα-mediated "don't-eat-me" signaling.

Prion diseases are neurodegenerative conditions caused by misfolding of the prion protein, leading to conspicuous neuronal loss and intense microgliosis. Recent experimental evidence point towards a protective role of microglia against prion-induced neurodegeneration, possibly through elimination of prion-containing apoptotic bodies. The molecular mechanisms by which microglia recognize and eliminate apoptotic cells in the context of prion diseases are poorly defined. Here we investigated the possible involvement of signal regulatory protein α (SIRPα), a key modulator of host cell phagocytosis; SIRPα is encoded by the Sirpa gene that is genetically linked to the prion gene Prnp. We found that Sirpa transcripts are highly enriched in microglia cells within the brain. However, Sirpa mRNA levels were essentially unaltered during the course of experimental prion disease despite upregulation of other microglia-enriched transcripts. To study the involvement of SIRPα in prion pathogenesis in vivo, mice expressing a truncated SIRPα protein unable to inhibit phagocytosis were inoculated with rodent-adapted scrapie prions of the 22L strain. Homozygous and heterozygous Sirpa mutants and wild-type mice experienced similar incubation times after inoculation with either of two doses of 22L prions. Moreover, the extent of neuronal loss, microgliosis and abnormal prion protein accumulation was not significantly affected by Sirpa genotypes. Collectively, these data indicate that SIRPα-mediated phagocytosis is not a major determinant in prion disease pathogenesis. It will be important to search for additional candidates mediating prion phagocytosis, as this mechanism may represent an important target of antiprion therapies.

Structure-Based Drug Discovery for Prion Disease Using a Novel Binding Simulation

The accumulation of abnormal prion protein (PrPSc) converted from the normal cellular isoform of PrP (PrPC) is assumed to induce pathogenesis in prion diseases. Therefore, drug discovery studies for these diseases have focused on the protein conversion process. We used a structure-based drug discovery algorithm (termed Nagasaki University Docking Engine: NUDE) that ran on an intensive supercomputer with a graphic-processing unit to identify several compounds with anti-prion effects. Among the candidates showing a high-binding score, the compounds exhibited direct interaction with recombinant PrP in vitro, and drastically reduced PrPSc and protein-aggresomes in the prion-infected cells. The fragment molecular orbital calculation showed that the van der Waals interaction played a key role in PrPC binding as the intermolecular interaction mode. Furthermore, PrPSc accumulation and microgliosis were significantly reduced in the brains of treated mice, suggesting that the drug candidates provided protection from prion disease, although further in vivo tests are needed to confirm these findings. This NUDE-based structure-based drug discovery for normal protein structures is likely useful for the development of drugs to treat other conformational disorders, such as Alzheimer's disease.

Strain-Dependent Effect of Macroautophagy on Abnormally Folded Prion Protein Degradation in Infected Neuronal Cells

Prion diseases are neurodegenerative disorders caused by the accumulation of abnormal prion protein (PrPSc) in the central nervous system. With the aim of elucidating the mechanism underlying the accumulation and degradation of PrPSc, we investigated the role of autophagy in its degradation, using cultured cells stably infected with distinct prion strains. The effects of pharmacological compounds that inhibit or stimulate the cellular signal transduction pathways that mediate autophagy during PrPSc degradation were evaluated. The accumulation of PrPSc in cells persistently infected with the prion strain Fukuoka-1 (FK), derived from a patient with Gerstmann–Sträussler–Scheinker syndrome, was significantly increased in cultures treated with the macroautophagy inhibitor 3-methyladenine (3MA) but substantially reduced in those treated with the macroautophagy inducer rapamycin. The decrease in FK-derived PrPSc levels was mediated, at least in part, by the phosphatidylinositol 3-kinase/MEK signalling pathway. By contrast, neither rapamycin nor 3MA had any apparently effect on PrPSc from either the 22L or the Chandler strain, indicating that the degradation of PrPSc in host cells might be strain-dependent.

Glycosylphosphatidylinositol Anchoring Directs the Assembly of Sup35NM Protein into Non-fibrillar, Membrane-bound Aggregates

In prion-infected hosts, PrPSc usually accumulates as non-fibrillar, membrane-bound aggregates. Glycosylphosphatidylinositol (GPI) anchor-directed membrane association appears to be an important factor controlling the biophysical properties of PrPSc aggregates. To determine whether GPI anchoring can similarly modulate the assembly of other amyloid-forming proteins, neuronal cell lines were generated that expressed a GPI-anchored form of a model amyloidogenic protein, the NM domain of the yeast prion protein Sup35 (Sup35(GPI)). We recently reported that GPI anchoring facilitated the induction of Sup35(GPI) prions in this system. Here, we report the ultrastructural characterization of self-propagating Sup35(GPI) aggregates of either spontaneous or induced origin. Like membrane-bound PrPSc, Sup35(GPI) aggregates resisted release from cells treated with phosphatidylinositol-specific phospholipase C. Sup35(GPI) aggregates of spontaneous origin were detergent-insoluble, protease-resistant, and self-propagating, in a manner similar to that reported for recombinant Sup35NM amyloid fibrils and induced Sup35(GPI) aggregates. However, GPI-anchored Sup35 aggregates were not stained with amyloid-binding dyes, such as Thioflavin T. This was consistent with ultrastructural analyses, which showed that the aggregates corresponded to dense cell surface accumulations of membrane vesicle-like structures and were not fibrillar. Together, these results showed that GPI anchoring directs the assembly of Sup35NM into non-fibrillar, membrane-bound aggregates that resemble PrPSc, raising the possibility that GPI anchor-dependent modulation of protein aggregation might occur with other amyloidogenic proteins. This may contribute to differences in pathogenesis and pathology between prion diseases, which uniquely involve aggregation of a GPI-anchored protein, versus other protein misfolding diseases.

Efficacy and Mechanism of a Glycoside Compound Inhibiting Abnormal Prion Protein Formation in Prion-Infected Cells: Implications of Interferon and Phosphodiesterase 4D-Interacting Protein

A new type of antiprion compound, Gly-9, was found to inhibit abnormal prion protein formation in prion-infected neuroblastoma cells, in a prion strain-independent manner, when the cells were treated for more than 1 day. It reduced the intracellular prion protein level and significantly modified mRNA expression levels of genes of two types: interferon-stimulated genes were downregulated after more than 2 days of treatment, and the phosphodiesterase 4D-interacting protein gene, a gene involved in microtubule growth, was upregulated after more than 1 day of treatment. A supplement of interferon given to the cells partly restored the abnormal prion protein level but did not alter the normal prion protein level. This interferon action was independent of the Janus activated kinase-signal transducer and activator of transcription signaling pathway. Therefore, the changes in interferon-stimulated genes might be a secondary effect of Gly-9 treatment. However, gene knockdown of phosphodiesterase 4D-interacting protein restored or increased both the abnormal prion protein level and the normal prion protein level, without transcriptional alteration of the prion protein gene. It also altered the localization of abnormal prion protein accumulation in the cells, indicating that phosphodiesterase 4D-interacting protein might affect prion protein levels by altering the trafficking of prion protein-containing structures. Interferon and phosphodiesterase 4D-interacting protein had no direct mutual link, demonstrating that they regulate abnormal prion protein levels independently. Although the in vivo efficacy of Gly-9 was limited, the findings for Gly-9 provide insights into the regulation of abnormal prion protein in cells and suggest new targets for antiprion compounds. This report describes our study of the efficacy and potential mechanism underlying the antiprion action of a new antiprion compound with a glycoside structure in prion-infected cells, as well as the efficacy of the compound in prion-infected animals. The study revealed involvements of two factors in the compound's mechanism of action: interferon and a microtubule nucleation activator, phosphodiesterase 4D-interacting protein. In particular, phosphodiesterase 4D-interacting protein was suggested to be important in regulating the trafficking or fusion of prion protein-containing vesicles or structures in cells. The findings of the study are expected to be useful not only for the elucidation of cellular regulatory mechanisms of prion protein but also for the implication of new targets for therapeutic development.

Amyloid-Binding Compounds and their Anti-Prion Potency

Prion diseases, or transmissible spongiform encephalopathies, are characterized by abnormal prion protein accumulation in the brain. Abnormal prion proteins, having properties of amyloids when extracted from the brain, are observed as amyloid plaque deposits in the brain in some prion diseases such as variant Creutzfeldt-Jakob disease and Gerstmann-Sträussler-Scheinker syndrome. This article reviews amyloid-binding compounds from the perspective of their usefulness for diagnosis and therapy of prion diseases. Styrylbenzoazole derivatives and phenylhydrazine derivatives are recently developed amyloid binding compounds that present benefits for prion-disease-related medicinal applications. For instance, styrylbenzoazole derivative BF-227, currently used as an amyloid imaging probe of positron emission tomography in Alzheimer disease, is useful also for the diagnosis of Gerstmann-Sträussler-Scheinker syndrome. A phenylhydrazine derivative, compB, has remarkable prophylactic effects on intracerebrally infected animals with certain prion strains, even when administered orally. These amyloid-binding compounds, however, are not applicable to prion strains or prion diseases of all types. For example, amyloid-binding compounds are ineffective for inhibiting prion strains such as 263K. They are not feasible for detecting abnormal prion protein accumulation in the brain for prion diseases having no amyloid plaques. To elucidate the limitations of amyloid-binding compounds, further investigation is necessary to clarify the binding mode of the compounds to abnormal prion protein structures at an atomic level.

Susceptibility of cattle to the agent of chronic wasting disease from elk after intracranial inoculation

Cattle could be exposed to the agent of chronic wasting disease (CWD) through contact with infected farmed or free-ranging cervids or exposure to contaminated premises. The purpose of the current study was to assess the potential for CWD derived from elk to transmit to cattle after intracranial inoculation. Calves (n = 14) were inoculated with brain homogenate derived from elk with CWD to determine the potential for transmission and to define the clinicopathologic features of disease. Cattle were necropsied if clinical signs occurred or at the end of the study (49 months postinoculation; MPI). Clinical signs of poor appetite, weight loss, circling, and bruxism occurred in 2 cattle (14%) at 16 and 17 MPI, respectively. Accumulation of abnormal prion protein (PrP(Sc)) occurred in only the 2 clinically affected cattle and was confined to the central nervous system, with the most prominent immunoreactivity in midbrain, brainstem, and hippocampus with lesser immunoreactivity in the cervical spinal cord. The rate of transmission was lower than in cattle inoculated with CWD derived from mule deer (38%) or white-tailed deer (86%). Additional studies are required to fully assess the potential for cattle to develop CWD through a more natural route of exposure, but a low rate of transmission after intracranial inoculation suggests that risk of transmission through other routes is low. A critical finding is that if CWD did transmit to exposed cattle, currently used diagnostic techniques would detect and differentiate it from other prion diseases in cattle based on absence of spongiform change, distinct pattern of PrP(Sc) deposition, and unique molecular profile.