Scrapie Prion Research Articles

Annual report of the Scientific Network on BSE‐TSE 2015

EFSA Supporting PublicationsVolume 12, Issue 12 915E Technical reportOpen Access Annual report of the Scientific Network on BSE-TSE 2015 European Food Safety Authority (EFSA), European Food Safety Authority (EFSA)Search for more papers by this author European Food Safety Authority (EFSA), European Food Safety Authority (EFSA)Search for more papers by this author First published: 11 December 2015 https://doi.org/10.2903/sp.efsa.2015.EN-915 Published date: 11 December 2015 Question number: EFSA-Q-2015-00738 AboutPDF ToolsExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat References EFSA (European Food Safety Authority), 2014. Annual report of the Scientific Network on BSE-TSE 2013. EFSA supporting publication 2014:EN-714, 9 pp. EFSA BIOHAZ Panel (EFSA Panel on Biological Hazards), 2015. Scientific Opinion on a request for a review of a scientific publication concerning the zoonotic potential of ovine scrapie prions. EFSA Journal, 2015; 13(8):4197, 58 pp. doi:10.2903/j.efsa.2015.4197 Volume12, Issue12December 2015915E ReferencesRelatedInformation

Mutated but Not Deleted Ovine PrP(C) N-Terminal Polybasic Region Strongly Interferes with Prion Propagation in Transgenic Mice.

Mammalian prions are proteinaceous infectious agents composed of misfolded assemblies of the host-encoded, cellular prion protein (PrP). Physiologically, the N-terminal polybasic region of residues 23 to 31 of PrP has been shown to be involved in its endocytic trafficking and interactions with glycosaminoglycans or putative ectodomains of membrane-associated proteins. Several recent reports also describe this PrP region as important for the toxicity of mutant prion proteins and the efficiency of prion propagation, both in vitro and in vivo. The question remains as to whether the latter observations made with mouse PrP and mouse prions would be relevant to other PrP species/prion strain combinations given the dramatic impact on prion susceptibility of minimal amino acid substitutions and structural variations in PrP. Here, we report that transgenic mouse lines expressing ovine PrP with a deletion of residues 23 to 26 (KKRP) or mutated in this N-terminal region (KQHPH instead of KKRPK) exhibited a variable, strain-dependent susceptibility to prion infection with regard to the proportion of affected mice and disease tempo relative to findings in their wild-type counterparts. Deletion has no major effect on 127S scrapie prion pathogenesis, whereas mutation increased by almost 3-fold the survival time of the mice. Deletion marginally affected the incubation time of scrapie LA19K and ovine bovine spongiform encephalopathy (BSE) prions, whereas mutation caused apparent resistance to disease. Recent reports suggested that the N-terminal polybasic region of the prion protein could be a therapeutic target to prevent prion propagation or toxic signaling associated with more common neurodegenerative diseases such as Alzheimer's disease. Mutating or deleting this region in ovine PrP completes the data previously obtained with the mouse protein by identifying the key amino acid residues involved.

Remarkable impairment of Wnt/β-catenin signaling in the brains of the mice infected with scrapie agents.

Prion diseases are a group of neurodegenerative diseases characterized by neuronal loss and spongiform degeneration, astrogliosis and aggregation of scrapie prion protein (PrPSc ) in the central nervous system (CNS). The Wnt signaling pathway is a highly evolutionarily conserved pathway in eukaryotes that regulates cell proliferation, differentiation and survival. Impairment of Wnt/β-catenin signaling has been reported in the CNS of various neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease. To investigate the functional state of Wnt/β-catenin signaling in the CNS tissues during the progression of prion disease, the components of Wnt/β-catenin signaling in the brains of the scrapie agents 139A- and ME7-infected mice were evaluated. Compared with the normal controls, the brain levels of phosphor-β-catenin (Ser33,37 and Thr41 ) in 139A- and ME7-infected mice were significantly increased, while those of cyclin D1, which is one of the target genes of Wnt signaling, were decreased. The levels of phosphor-glycogen synthase kinase-3β (GSK-3β) Ser9 were markedly reduced, representing an enhanced GSK-3β activity in scrapie-infected mice. Both western blot and immunohistochemical assays revealed a remarkable increase of Dickkopf-1, the antagonist of Wnt/β-catenin signaling, in the brains of scrapie-infected anim-als, which co-localized well with the remaining neurons in theimmunofluorescent tests. We also observed slightly decreased Wnt-3 and unchanged disheveled-3 (Dvl-3) in the brains of the infected mice. Our data, here, strongly indicate an impairment of Wnt/β-catenin pathway in the brains of prion disease, which shows a time-dependent progression along with the incubation period. Schematic for the impairment of canonical Wnt signaling during prion infection. The left and right parts represent the normal and prion-infected situations, respectively. Prion infection or PrPSc accumulation triggers the over-expression of Dickkopf WNT signaling pathway inhibitor 1 (DKK-1) and the enhancement of glycogen synthase kinase 3β (GSK-3β) activity, which subsequently promotes the phosphorylation and degradation of β-catenin. As a result, the impairment of β-catenin signaling leads to the down-regulation of Wnt target genes.

Objects in Contact with Classical Scrapie Sheep Act as a Reservoir for Scrapie Transmission.

Classical scrapie is an environmentally transmissible prion disease of sheep and goats. Prions can persist and remain potentially infectious in the environment for many years and thus pose a risk of infecting animals after re-stocking. In vitro studies using serial protein misfolding cyclic amplification (sPMCA) have suggested that objects on a scrapie-affected sheep farm could contribute to disease transmission. This in vivo study aimed to determine the role of field furniture (water troughs, feeding troughs, fencing, and other objects that sheep may rub against) used by a scrapie-infected sheep flock as a vector for disease transmission to scrapie-free lambs with the prion protein genotype VRQ/VRQ, which is associated with high susceptibility to classical scrapie. When the field furniture was placed in clean accommodation, sheep became infected when exposed to either a water trough (four out of five) or to objects used for rubbing (four out of seven). This field furniture had been used by the scrapie-infected flock 8 weeks earlier and had previously been shown to harbor scrapie prions by sPMCA. Sheep also became infected (20 out of 23) through exposure to contaminated field furniture placed within pasture not used by scrapie-infected sheep for 40 months, even though swabs from this furniture tested negative by PMCA. This infection rate decreased (1 out of 12) on the same paddock after replacement with clean field furniture. Twelve grazing sheep exposed to field furniture not in contact with scrapie-infected sheep for 18 months remained scrapie free. The findings of this study highlight the role of field furniture used by scrapie-infected sheep to act as a reservoir for disease re-introduction although infectivity declines considerably if the field furniture has not been in contact with scrapie-infected sheep for several months. PMCA may not be as sensitive as VRQ/VRQ sheep to test for environmental contamination.

Application of protein misfolding cyclic amplification to detection of prions in anaerobic digestate

The exceptional physio-chemical resistance of prions to established decontamination procedures poses a challenge to assessing the suitability of applied inactivation methods. Prion detection is limited by the sensitivity level of Western blotting or by the cost and time factors of bioassays. In addition, prion detection assays can be limited by either the unique or complex nature of matrices associated with environmental samples. To investigate anaerobic digestion (AD) as a practical and economical approach for potential conversion of specified risk materials (SRM) into value added products (i.e., renewable energy), challenges associated with detection of prions in a complex matrix need to be overcome to determine potential inactivation. Protein misfolding cyclic amplification (PMCA) assay, with subsequent Western blot visualization, was used to detect prions within the AD matrix. Anaerobic digestate initially inhibited the PMCA reaction and/or Western blot detection. However, at concentrations of ≤1% of anaerobic digestate, 263K scrapie prions could be amplified and semi-quantitatively detected. Infectious 263K prions were also proven to be bioavailable in the presence of high concentrations of digestate (10–90%). Development of the PMCA application to digestate provides extremely valuable insight into the potential degradation and/or fate of prions in complex biological matrices without requiring expensive and time-consuming bioassays.

Transgenic Rabbits Expressing Ovine PrP Are Susceptible to Scrapie.

Transmissible spongiform encephalopathies (TSEs) are a group of neurodegenerative diseases affecting a wide range of mammalian species. They are caused by prions, a proteinaceous pathogen essentially composed of PrPSc, an abnormal isoform of the host encoded cellular prion protein PrPC. Constrained steric interactions between PrPSc and PrPC are thought to provide prions with species specificity, and to control cross-species transmission into other host populations, including humans. Transgenetic expression of foreign PrP genes has been successfully and widely used to overcome the recognized resistance of mouse to foreign TSE sources. Rabbit is one of the species that exhibit a pronounced resistance to TSEs. Most attempts to infect experimentally rabbit have failed, except after inoculation with cell-free generated rabbit prions. To gain insights on the molecular determinants of the relative resistance of rabbits to prions, we generated transgenic rabbits expressing the susceptible V136R154Q171 allele of the ovine PRNP gene on a rabbit wild type PRNP New Zealand background and assessed their experimental susceptibility to scrapie prions. All transgenic animals developed a typical TSE 6–8 months after intracerebral inoculation, whereas wild type rabbits remained healthy more than 700 days after inoculation. Despite the endogenous presence of rabbit PrPC, only ovine PrPSc was detectable in the brains of diseased animals. Collectively these data indicate that the low susceptibility of rabbits to prion infection is not enciphered within their non-PrP genetic background.

Scientific Opinion on a request for a review of a scientific publication concerning the zoonotic potential of ovine scrapie prions

The factors that modulate the transmissibility of Transmissible Spongiform Encephalopathies (TSE) and the approaches for the study of their zoonotic potential are reviewed. The paper ‘ Evidence for zoonotic potential of ovine scrapie prions’ by Cassard et al. (2014) is scientifically appraised, focussing on the experimental design, the results and the conclusions. The paper provides evidence in a laboratory experiment that some Classical scrapie isolates can propagate in humanised transgenic mice and produce prions that on second passage are similar to those causing one form of sporadic Creutzfeldt-Jakob disease (sCJD). It is concluded that the results from the study raise the possibility that scrapie prions have the potential to be zoonotic, but do not provide evidence that transmission can or does take place under field conditions. The conclusions of the 2011 ECDC-EFSA ‘ Joint Scientific Opinion on any possible epidemiological or molecular association between TSEs in animals and humans’ are reviewed in the light of the new scientific evidence available since its publication. This supports and strengthens the conclusions of that opinion with regard to the potential for some animal TSE to be zoonotic, but does not provide evidence of a causal link between Classical or Atypical scrapie and human TSE. Current evidence does not establish this link, and no consistent risk factors have been identified for sCJD. The possibility of scrapie-related public health risks from the consumption of ovine products cannot be assessed. Recommendations are formulated on further studies and data that are needed to investigate the zoonotic potential of animal TSE and to estimate the amount of infectivity from TSE-infected products sourced from small ruminants and entering the food chain in the European Union.

Transgenic over-expression of mammalian heparanase delays prion disease onset and progression

Cellular heparan sulfate (HS) has a dual role in scrapie pathogenesis; it is required for PrPSc (scrapie prion protein) formation and facilitates infection of cells, mediating cellular uptake of prions. We examined the involvement of heparanase, a mammalian endoglycosidase degrading HS, in scrapie infection. In cultured cells, heparanase treatment or over-expression resulted in a profound decrease in PrPSc. Moreover, disease onset and progression were dramatically delayed in scrapie infected transgenic mice over-expressing heparanase. Together, our results provide direct in vivo evidence for the involvement of intact HS in the pathogenesis of prion disease and the protective role of heparanase both in terms of susceptibility to infection and disease progression.

Transmission of scrapie prions to primate after an extended silent incubation period.

Classical bovine spongiform encephalopathy (c-BSE) is the only animal prion disease reputed to be zoonotic, causing variant Creutzfeldt-Jakob disease (vCJD) in humans and having guided protective measures for animal and human health against animal prion diseases. Recently, partial transmissions to humanized mice showed that the zoonotic potential of scrapie might be similar to c-BSE. We here report the direct transmission of a natural classical scrapie isolate to cynomolgus macaque, a highly relevant model for human prion diseases, after a 10-year silent incubation period, with features similar to those reported for human cases of sporadic CJD. Scrapie is thus actually transmissible to primates with incubation periods compatible with their life expectancy, although fourfold longer than BSE. Long-term experimental transmission studies are necessary to better assess the zoonotic potential of other prion diseases with high prevalence, notably Chronic Wasting Disease of deer and elk and atypical/Nor98 scrapie.

Enhanced Formation of Scrapie Prion Protein in Cultured Cells by Treatment with Mycosporine-like Amino Acids(MAAs)

Enhanced Formation of Scrapie Prion Protein in Cultured Cells by Treatment with Mycosporine-like Amino Acids(MAAs)

Use of a 2-aminothiazole to Treat Chronic Wasting Disease in Transgenic Mice.

Treatment with the 2-aminothiazole IND24 extended the survival of mice infected with mouse-adapted scrapie but also resulted in the emergence of a drug-resistant prion strain. Here, we determined whether IND24 extended the survival of transgenic mice infected with prions that caused scrapie in sheep or prions that caused chronic wasting disease (CWD; hereafter "CWD prions") in deer, using 2 isolates for each disease. IND24 doubled the incubation times for mice infected with CWD prions but had no effect on the survival of those infected with scrapie prions. Biochemical, neuropathologic, and cell culture analyses were used to characterize prion strain properties following treatment, and results indicated that the CWD prions were not altered by IND24, regardless of survival extension. These results suggest that IND24 may be a viable candidate for treating CWD in infected captive cervid populations and raise questions about why some prion strains develop drug resistance whereas others do not.

Distinct pathological phenotypes of Creutzfeldt-Jakob disease in recipients of prion-contaminated growth hormone

IntroductionThe present study compares the clinical, pathological and molecular features of a United States (US) case of growth hormone (GH)-associated Creutzfeldt-Jakob disease (GH-CJD) (index case) to those of two earlier referred US cases of GH-CJD and one case of dura mater (d)-associated CJD (dCJD). All iatrogenic CJD (iCJD) subjects were methionine (M) homozygous at codon 129 (129MM) of the prion protein (PrP) gene and had scrapie prion protein (PrPSc) type 1 (iCJDMM1).ResultsThe index subject presented with ataxia, weight loss and changes in the sleep pattern about 38 years after the midpoint of GH treatment. Autopsy examination revealed a neuropathological phenotype reminiscent of both sCJDMV2-K (a sporadic CJD subtype in subjects methionine/valine heterozygous at codon 129 with PrPSc type 2 and the presence of kuru plaques) and variant CJD (vCJD). The two earlier cases of GH-CJDMM1 and the one of dCJDMM1 were associated with neuropathological phenotypes that differed from that of the index case mainly because they lacked PrP plaques. The phenotype of the earlier GH-CJDMM1 cases shared several, but not all, characteristics with sCJDMM1, whereas dCJDMM1 was phenotypically indistinguishable from sCJDMM1. Two distinct groups of dCJDMM1 have also been described in Japan based on clinical features, the presence or absence of PrP plaques and distinct PK-resistant PrPSc (resPrPSc) electrophoretic mobilities. The resPrPSc electrophoretic mobility was, however, identical in our GH-CJDMM1 and dCJDMM1 cases, and matched that of sCJDMM1.ConclusionsOur study shows that receipt of prion-contaminated GH can lead to a prion disease with molecular features (129MM and PrPSc type 2) and phenotypic characteristics that differ from those of sporadic prion disease (sCJDMM1), a difference that may reflect adaptation of “heterologous” prion strains to the 129MM background.

Incubation of ovine scrapie with environmental matrix results in biological and biochemical changes of PrPSc over time

Ovine scrapie can be transmitted via environmental reservoirs. A pool of ovine scrapie isolates were incubated on soil for one day or thirteen months and eluted prion was used to challenge tg338 mice transgenic for ovine PrP. After one-day incubation on soil, two PrPSc phenotypes were present: G338 or Apl338ii. Thirteen months later some divergent PrPSc phenotypes were seen: a mixture of Apl338ii with either G338 or P338, and a completely novel PrPSc deposition, designated Cag338. The data show that prolonged ageing of scrapie prions within an environmental matrix may result in changes in the dominant PrPSc biological/biochemical properties.

Central Nervous System Diseases Due to Slow Viruses and Prions

Several central nervous system diseases whose common elements include a long incubation period and a progressive clinical course were once called slow virus infections, because most of them are in fact caused by viruses. However, one group of these CNS diseases is now believed to be caused by abnormally configured proteins known as prions; rather than an etiologic designation, therefore, on the whole these diseases are better characterized by their chronicity, their transmissibility, and at this point, their inexorably deteriorating natural history. This chapter reviews the more common of these: HIV-associated dementia (HAD or HIVD), human T cell lymphotropic virus type 1 (HTLV-1)-associated myelopathy, Creutzfeldt-Jakob disease (CJD), progressive multifocal leukoencephalopathy (PML), and subacute sclerosing encephalitis (SSPE), which is associated with a variant of measles virus. Figures illustrate the pathogenesis and the pathology of HIV dementia, propagation of scrapie prion protein (PrP) in brain neurons, and spongiform brain changes of CJD. Tables list the stages of HAD and the clinical and pathologic characteristics distinguishing classic CJD and varient CJD. This module contains ­5 highly rendered figures, 2 tables, 57 references, and 5 MCQs.

Stability and Reproducibility Underscore Utility of RT-QuIC for Diagnosis of Creutzfeldt-Jakob Disease

Real-time quaking-induced conversion (RT-QuIC) allows the amplification of miniscule amounts of scrapie prion protein (PrPSc). Recent studies applied the RT-QuIC methodology to cerebrospinal fluid (CSF) for diagnosing human prion diseases. However, to date, there has not been a formal multi-centre assessment of the reproducibility, validity and stability of RT-QuIC in this context, an indispensable step for establishment as a diagnostic test in clinical practice. In the present study, we analysed CSF from 110 prion disease patients and 400 control patients using the RT-QuIC method under various conditions. In addition, “blinded” ring trials between different participating sites were performed to estimate reproducibility. Using the previously established cut-off of 10,000 relative fluorescence units (rfu), we obtained a sensitivity of 85 % and a specificity of 99 %. The multi-centre inter-laboratory reproducibility of RT-QuIC revealed a Fleiss’ kappa value of 0.83 (95 % CI: 0.40–1.00) indicating an almost perfect agreement. Moreover, we investigated the impact of short-term CSF storage at different temperatures, long-term storage, repeated freezing and thawing cycles and the contamination of CSF with blood on the RT-QuIC seeding response. Our data indicated that the PrPSc seed in CSF is stable to any type of storage condition but sensitive to contaminations with blood (>1250 erythrocytes/μL), which results in a false negative RT-QuIC response. Fresh blood-contaminated samples (3 days) can be rescued by removal of erythrocytes. The present study underlines the reproducibility and high stability of RT-QuIC across various CSF storage conditions with a remarkable sensitivity and specificity, suggesting RT-QuIC as an innovative and robust diagnostic method.Electronic supplementary materialThe online version of this article (doi:10.1007/s12035-015-9133-2) contains supplementary material, which is available to authorized users.

Increased infectivity of anchorless mouse scrapie prions in transgenic mice overexpressing human prion protein.

Prion protein (PrP) is found in all mammals, mostly as a glycoprotein anchored to the plasma membrane by a C-terminal glycosylphosphatidylinositol (GPI) linkage. Following prion infection, host protease-sensitive prion protein (PrPsen or PrPC) is converted into an abnormal, disease-associated, protease-resistant form (PrPres). Biochemical characteristics, such as the PrP amino acid sequence, and posttranslational modifications, such as glycosylation and GPI anchoring, can affect the transmissibility of prions as well as the biochemical properties of the PrPres generated. Previous in vivo studies on the effects of GPI anchoring on prion infectivity have not examined cross-species transmission. In this study, we tested the effect of lack of GPI anchoring on a species barrier model using mice expressing human PrP. In this model, anchorless 22L prions derived from tg44 mice were more infectious than 22L prions derived from C57BL/10 mice when tested in tg66 transgenic mice, which expressed wild-type anchored human PrP at 8- to 16-fold above normal. Thus, the lack of the GPI anchor on the PrPres from tg44 mice appeared to reduce the effect of the mouse-human PrP species barrier. In contrast, neither source of prions induced disease in tgRM transgenic mice, which expressed human PrP at 2- to 4-fold above normal. Prion protein (PrP) is found in all mammals, usually attached to cells by an anchor molecule called GPI. Following prion infection, PrP is converted into a disease-associated form (PrPres). While most prion diseases are species specific, this finding is not consistent, and species barriers differ in strength. The amino acid sequence of PrP varies among species, and this variability affects prion species barriers. However, other PrP modifications, including glycosylation and GPI anchoring, may also influence cross-species infectivity. We studied the effect of PrP GPI anchoring using a mouse-to-human species barrier model. Experiments showed that prions produced by mice expressing only anchorless PrP were more infectious than prions produced in mice expressing anchored PrP. Thus, the lack of the GPI anchor on prions reduced the effect of the mouse-human species barrier. Our results suggest that prion diseases that produce higher levels of anchorless PrP may pose an increased risk for cross-species infection.

Mechanism of Scrapie Prion Precipitation with Phosphotungstate Anions

The phosphotungstate anion (PTA) is widely used to facilitate the precipitation of disease-causing prion protein (PrPSc) from infected tissue for applications in structural studies and diagnostic approaches. However, the mechanism of this precipitation is not understood. In order to elucidate the nature of the PTA interaction with PrPSc under physiological conditions, solutions of PTA were characterized by NMR spectroscopy at varying pH. At neutral pH, the parent [PW12O40]3– ion decomposes to give a lacunary [PW11O39]7– (PW11) complex and a single orthotungstate anion [WO4]2– (WO4). To measure the efficacy of each component of PTA, increasing concentrations of PW11, WO4, and mixtures thereof were used to precipitate PrPSc from brain homogenates of scrapie prion-infected mice. The amount of PrPSc isolated, quantified by ELISA and immunoblotting, revealed that both PW11 and WO4 contribute to PrPSc precipitation. Incubation with sarkosyl, PTA, or individual components of PTA resulted in separation of higher-density PrP aggregates from the neuronal lipid monosialotetrahexosylganglioside (GM1), as observed by sucrose gradient centrifugation. These experiments revealed that yield and purity of PrPSc were greater with polyoxometalates (POMs), which substantially supported the separation of lipids from PrPSc in the samples. Interaction of POMs and sarkosyl with brain homogenates promoted the formation of fibrillar PrPSc aggregates prior to centrifugation, likely through the separation of lipids like GM1 from PrPSc. We propose that this separation of lipids from PrP is a major factor governing the facile precipitation of PrPSc by PTA from tissue and might be optimized further for the detection of prions.

Prion pathogenesis in the absence of NLRP3/ASC inflammasomes.

The accumulation of the scrapie prion protein PrPSc, a misfolded conformer of the cellular prion protein PrPC, is a crucial feature of prion diseases. In the central nervous system, this process is accompanied by conspicuous microglia activation. The NLRP3 inflammasome is a multi-molecular complex which can sense heterogeneous pathogen-associated molecular patterns and culminates in the activation of caspase 1 and release of IL 1β. The NLRP3 inflammasome was reported to be essential for IL 1β release after in vitro exposure to the amyloidogenic peptide PrP106-126 and to recombinant PrP fibrils. We therefore studied the role of the NLRP3 inflammasome in a mouse model of prion infection. Upon intracerebral inoculation with scrapie prions (strain RML), mice lacking NLRP3 (Nlrp3-/-) or the inflammasome adaptor protein ASC (Pycard-/-) succumbed to scrapie with attack rates and incubation times similar to wild-type mice, and developed the classic histologic and biochemical features of prion diseases. Genetic ablation of NLRP3 or ASC did not significantly impact on brain levels of IL 1β at the terminal stage of disease. Our results exclude a significant role for NLRP3 and ASC in prion pathogenesis and invalidate their claimed potential as therapeutic target against prion diseases.

Efficient uptake and dissemination of scrapie prion protein by astrocytes and fibroblasts from adult hamster brain.

Prion infections target neurons and lead to neuronal loss. However, the role of non-neuronal cells in the initiation and spread of infection throughout the brain remains unclear despite the fact these cells can also propagate prion infectivity. To evaluate how different brain cells process scrapie prion protein (PrPres) during acute infection, we exposed neuron-enriched and non-neuronal cell cultures from adult hamster brain to fluorescently-labeled purified PrPres and followed the cultures by live cell confocal imaging over time. Non-neuronal cells present in both types of cultures, specifically astrocytes and fibroblasts, internalized PrPres more efficiently than neurons. PrPres was trafficked to late endosomal/lysosomal compartments and rapidly transported throughout the cell bodies and processes of all cell types, including contacts between astrocytes and neurons. These observations suggest that astrocytes and meningeal fibroblasts play an as yet unappreciated role in prion infections via efficient uptake and dissemination of PrPres.

HTERT-immortalized ovine microglia propagate natural scrapie isolates

Ex vivo propagation of natural prion isolates (i.e., propagated solely in the natural host) is crucial for the characterization and study of transmissible spongiform encephalopathies (TSEs). Several well-established, prion-permissive cell culture systems are available; however, only a few cell lines are permissive to natural prion isolates and these cells are not pathophysiologically relevant (e.g., renal epithelium and fibroblast-like cells). Therefore, a pathophysiologically relevant cell line derived from a natural TSE host could be used for propagation of natural prion isolates. In this study, ovine brain macrophages (microglia) were immortalized by transfection with the human telomerase reverse transcriptase (hTERT) gene to identify cell lines (hTERT-microglia) permissive to natural scrapie prion isolates. Following transfection, hTERT-microglia were passaged up to 100 times and their lifespan was significantly longer compared to parental cells (Fisher's exact test, P<0.001). Multiple sublines were permissive to cell culture-adapted prions; two sublines were also permissive to natural scrapie isolates (i.e., derived from brain homogenates of sheep infected with scrapie). Prion infectivity and partial protease resistance of the prion protein were maintained in hTERT-microglia. Comparisons between scrapie-permissive and non-permissive hTERT-microglia sublines revealed that overall quantity of the normal cellular prion protein was not associated with prion permissiveness. The use of hTERT-microglia in future TSE studies may be more germane to the characterization of the cellular and subcellular pathophysiology of natural scrapie prion isolates and to investigate host-specific factors involved in prion replication.