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.

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.

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.

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.

Discrimination between L-type and C-type bovine spongiform encephalopathy by the strain-specific reactions of real-time quaking-induced conversion.

Real-time quaking-induced conversion (RT-QUIC) assays using Escherichia coli-derived purified recombinant prion protein (rPrP) enable us to amplify a trace amount of the abnormal form of PrP (PrPSc) from specimens. This technique can be useful for the early diagnosis of both human and animal prion diseases and the assessment of prion contamination. In the present study, we demonstrated that there are strain-specific differences in the RT-QUIC reactions between an atypical form of bovine spongiform encephalopathy (BSE), l-BSE, and classical BSE (C-BSE). Whereas mouse rPrP (rMoPrP) was efficiently converted to amyloid fibrils in the presence of PrPSc seed derived from either l-BSE or C-BSE, hamster rPrP (rHaPrP) was converted only in l-BSE, not C-BSE. These characteristics were preserved in the second round reaction, but gradually weakened in the subsequent rounds and were completely lost by the fifth round, most likely due to the selective growth advantage of nonspecific rPrP amyloid fibrils in the RT-QUIC. Our findings further enhance the discrimination of prion strains using RT-QUIC, and further our understanding of the molecular basis of prion strains.

[Elucidation of the Molecular Basis of Abnormal Prion Protein (PrP) Formation in a Cell-Free System Using Baculovirus and Insect Cell-derived Recombinant PrP].

The molecular basis underlying the conversion of normal prion protein (PrPC) into abnormal prion protein (PrPSc) has not been fully elucidated. The protein-misfolding cyclic amplification (PMCA) technique, which can amplify PrPSc in vitro with the use of intermittent sonication, mimics the process of in vivo PrPSc replication. Accumulating evidence suggests that co-factors other than PrP may play a crucial role in the faithful replication of PrPSc. In conventional PMCA, brain homogenates (BHs) from normal animals are used as the PrPC substrate. Since BHs contain many impurities, it is difficult to identify the co-factors using conventional PMCA. Thus, we developed a modified PMCA system using baculovirus and insect cell-derived recombinant PrP as a substrate (insect cell PMCA; iPMCA). We demonstrated that nucleic acids and glycosaminoglycans (GAGs) such as heparan sulfate (HS) or its analogue heparin (HP) are critical for PrPSc amplification in iPMCA. Of note, the addition of HS or HP restored the conversion efficiency in iPMCA under nucleic acid-depleted conditions. Moreover, the iPMCA products were infectious and preserved the strain properties of the input seed PrPSc. These data suggest that not only nucleic acids but also some GAGs play an important role in facilitating faithful replication of prions, at least in vitro.

Whole genome characterisation of G11P[25] and G9P[19] rotavirus A strains from adult patients with diarrhoea in Nepal.

Rotavirus A (RVA) causes acute diarrhoea in children and less frequently in adults. However, the knowledge about the genotype distribution of RVA strains circulating in adults is limited particularly in developing countries. This study aimed to characterise the RVA strains detected from adult patients with diarrhoea in Nepal. A total of 47 RVA positive stool samples from adult patients with diarrhoea in Kathmandu, Nepal during 2007-2008 were examined for the G and P genotypes by sequencing. Nearly half (49%) of the samples were genotyped as G9P[8] (n = 23), G1P[8], G2P[4] (n = 5 each), G12P[8] (n = 4), G12P[6] (n = 3), G1P[6] (n = 2), G3P[8] and G9P[6] (n = 1 each). Interestingly, two G11P[25] and one G9P[19] strains detected were further subjected to Illumina MiSeq next generation sequencing to determine their whole genome sequences. The genotype constellations of RVA/Human-wt/NPL/TK2615/2008/G11P[25] and RVA/Human-wt/NPL/TK2620/2008/G11P[25] were I12-R1-C1-M1-A1-N1-T1-E1-H1, whereas that of RVA/Human-wt/NPL/TK1797/2007/G9P[19] was I5-R1-C1-M1-A8-N1-T1-E1-H1. The 11 genes of TK2615 and TK2620 were virtually identical, and they were either porcine-like or unique except the VP2 and NSP1 genes which were of human RVA origin. The two G11P[25] strains were also very similar to KTM368, another G11P[25] isolated from a child in Nepal in 2004. On the other hand, no gene of TK1797 was likely to be of human RVA origin. The observation that porcine-like RVAs were detected from adult patients justifies further studies to explore the role of adults in the interspecies transmission of animal RVA to humans.

Identification of Alprenolol Hydrochloride as an Anti-prion Compound Using Surface Plasmon Resonance Imaging.

Prion diseases are transmissible neurodegenerative disorders of humans and animals, which are characterized by the aggregation of abnormal prion protein (PrPSc) in the central nervous system. Although several small compounds that bind to normal PrP (PrPC) have been shown to inhibit structural conversion of the protein, an effective therapy for human prion disease remains to be established. In this study, we screened 1200 existing drugs approved by the US Food and Drug Administration (FDA) for anti-prion activity using surface plasmon resonance imaging (SPRi). Of these drugs, 31 showed strong binding activity to recombinant human PrP, and three of these reduced the accumulation of PrPSc in prion-infected cells. One of the active compounds, alprenolol hydrochloride, which is used clinically as a ß-adrenergic blocker for hypertension, also reduced the accumulation of PrPSc in the brains of prion-infected mice at the middle stage of the disease when the drug was administered orally with their daily water from the day after infection. Docking simulation analysis suggested that alprenolol hydrochloride fitted into the hotspot within mouse PrPC, which is known as the most fragile structure within the protein. These findings provide evidence that SPRi is useful in identifying effective drug candidates for neurodegenerative diseases caused by abnormal protein aggregation, such as prion diseases.

Prion-Seeding Activity Is widely Distributed in Tissues of Sporadic Creutzfeldt-Jakob Disease Patients.

Human prion diseases are neurodegenerative disorders caused by abnormally folded prion proteins in the central nervous system. These proteins can be detected using the quaking-induced conversion assay. Compared with other bioassays, this assay is extremely sensitive and was used in the present study to determine prion distribution in sporadic Creutzfeldt-Jakob disease patients at autopsy. Although infectivity of the sporadic form is thought to be restricted within the central nervous system, results showed that prion-seeding activities reach 106/g from a 50% seeding dose in non-neuronal tissues, suggesting that prion-seeding activity exists in non-neural organs, and we suggested that non-neural tissues of 106/g SD50 did not exist the infectivity.

A direct assessment of human prion adhered to steel wire using real-time quaking-induced conversion.

Accidental transmission of prions during neurosurgery has been reported as a consequence of re-using contaminated surgical instruments. Several decontamination methods have been studied using the 263K-hamster prion; however, no studies have directly evaluated human prions. A newly developed in vitro amplification system, designated real-time quaking-induced conversion (RT-QuIC), has allowed the activity of abnormal prion proteins to be assessed within a few days. RT-QuIC using human recombinant prion protein (PrP) showed high sensitivity for prions as the detection limit of our assay was estimated as 0.12 fg of active prions. We applied this method to detect human prion activity on stainless steel wire. When we put wires contaminated with human Creutzfeldt-Jakob disease brain tissue directly into the test tube, typical PrP-amyloid formation was observed within 48 hours, and we could detect the activity of prions at 50% seeding dose on the wire from 10(2.8) to 10(5.8) SD50. Using this method, we also confirmed that the seeding activities on the wire were removed following treatment with NaOH. As seeding activity closely correlated with the infectivity of prions using the bioassay, this wire-QuIC assay will be useful for the direct evaluation of decontamination methods for human prions.

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.

Rapid and Quantitative Assay of Amyloid-Seeding Activity in Human Brains Affected with Prion Diseases.

The infectious agents of the transmissible spongiform encephalopathies are composed of amyloidogenic prion protein, PrPSc. Real-time quaking-induced conversion can amplify very small amounts of PrPSc seeds in tissues/body fluids of patients or animals. Using this in vitro PrP-amyloid amplification assay, we quantitated the seeding activity of affected human brains. End-point assay using serially diluted brain homogenates of sporadic Creutzfeldt-Jakob disease patients demonstrated that 50% seeding dose (SD50) is reached approximately 10(10)/g brain (values varies 10(8.79-10.63)/g). A genetic case (GSS-P102L) yielded a similar level of seeding activity in an autopsy brain sample. The range of PrPSc concentrations in the samples, determined by dot-blot assay, was 0.6-5.4 µg/g brain; therefore, we estimated that 1 SD50 unit was equivalent to 0.06-0.27 fg of PrPSc. The SD50 values of the affected brains dropped more than three orders of magnitude after autoclaving at 121°C. This new method for quantitation of human prion activity provides a new way to reduce the risk of iatrogenic prion transmission.

Cytopathy of an infiltrating monocyte lineage during the early phase of infection with murinecoronavirus in the brain.

Viral spread during the early stages after infection was compared between a highly neurovirulent mouse hepatitis virus (MHV), JHMV cl-2 strain (cl-2), and its low-virulent mutant, soluble-receptor-resistant (srr)7. The infection of cells with srr7 (soluble-receptor-resistant mutant 7) is dependent on a known MHV receptor (MHVR), carcinoembryonic cell adhesion molecule 1a, whereas cl-2 shows MHVR-independent infection. Initial viral antigens were detected between 12 and 24 h post-inoculation (p.i) in the infiltrating cells that appeared in the subarachnoidal space of mouse brains infected with viruses. There were no significant differences in the intensity or spread of viral antigens in the inflammatory cells between the two viruses. However, 48 h after infection with cl-2, viral antigen-positive cells in the grey matter with the shape of neurons, which do not express MHVR, were detected, while srr7 infection was observed primarily in the white matter. Some of the viral antigen-positive inflammatory cells found in the subarachnoidal space during the early phase of infection reacted with anti-F4/80 or anti-CD11b monoclonal antibodies. Syncytial giant cells (SGCs) expressing viral and CD11b antigens were also detected among these inflammatory cells. These antigen-positive cells appeared in the subarachnoidal space prior to viral antigen spread into the brain parenchyma, indicating that viral encephalitis starts with the infection of infiltrating monocytes which express MHVR. Furthermore, the observation indicates that viral infection has cytopathic effects on the monocyte lineage, which plays a critical role in innate immunity, leading to the rapid spread of viruses during the early stage of infection.