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Neurodegenerative diseases are characterised by neuronal loss and abnormal deposition of pathological proteins in the nervous system. Among the most common neurodegenerative diseases are Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease and transmissible spongiform encephalopathies (TSEs). Sleep and circadian rhythm disturbances are one of the most common symptoms in patients with neurodegenerative diseases. Currently, one of the main objectives in the study of TSEs is to try to establish an early diagnosis, as clinical signs do not appear until the damage to the central nervous system is very advanced, which prevents any therapeutic approach. In this paper, we provide the first description of sleep disturbance caused by classical scrapie in clinical and preclinical sheep using polysomnography compared to healthy controls. Fifteen sheep classified into three groups, clinical, preclinical and negative control, were analysed. The results show a decrease in total sleep time as the disease progresses, with significant changes between control, clinical and pre-clinical animals. The results also show an increase in sleep fragmentation in clinical animals compared to preclinical and control animals. In addition, sheep with clinical scrapie show a total loss of Rapid Eye Movement sleep (REM) and alterations in Non Rapid Eyes Movement sleep (NREM) compared to control sheep, demonstrating more shallow sleep. Although further research is needed, these results suggest that prion diseases also produce sleep disturbances in animals and that polysomnography could be a diagnostic tool of interest in clinical and preclinical cases of prion diseases.

Background: The most common natural prion disease that primarily affects sheep and goats is scrapie. It belongs to a group of disorders known as transmissible spongiform encephalopathies, which impact both humans and animals., Aim: The research is aimed to examine and confirm the presence of scrapie in Libya using immunohistochemistry (IHC) techniques., Methods: Brain samples were collected from thirty-three sheep older than two years of age showing clinical signs resembling to scrapie during the period between 2018 and 2023, regardless of race or gender. Three animals, six months old, healthy, and without any symptoms, were used as negative controls. Different parts of the brain, including the obex and cerebellum, were taken from each case. The IHC technique used in this study involved staining with monoclonal antibody L42 and DAB (3,3'-diaminobenzidine) as a chromogenic substrate., Results: The IHC examination showed the expression of prion proteins in brain tissue in twenty-three samples. The staining intensity was markedly observed in the neuronal cell bodies and around blood vessels., Conclusion: The findings of this study provide evidence that scrapie exists in Libya., Competing Interests: The authors declare no conflict of interest.

A distinctive signature of the prion diseases is the accumulation of the pathogenic isoform of the prion protein, PrP Sc , in the central nervous system of prion-affected humans and animals. PrP Sc is also found in peripheral tissues, raising concerns about the potential transmission of pathogenic prions through human food supplies and posing a significant risk to public health. Although muscle tissues are considered to contain levels of low prion infectivity, it has been shown that myotubes in culture efficiently propagate PrP Sc . Given the high consumption of muscle tissue, it is important to understand what factors could influence the establishment of a prion infection in muscle tissue. Here we used in vitro myotube cultures, differentiated from the C2C12 myoblast cell line (dC2C12), to identify factors affecting prion replication. A range of experimental conditions revealed that PrP Sc is tightly associated with proteins found in the systemic extracellular matrix, mostly fibronectin (FN). The interaction of PrP Sc with FN decreased prion infectivity, as determined by standard scrapie cell assay. Interestingly, the prion-resistant reserve cells in dC2C12 cultures displayed a FN-rich extracellular matrix while the prion-susceptible myotubes expressed FN at a low level. In agreement with the in vitro results, immunohistopathological analyses of tissues from sheep infected with natural scrapie demonstrated a prion susceptibility phenotype linked to an extracellular matrix with undetectable levels of FN. Conversely, PrP Sc deposits were not observed in tissues expressing FN. These data indicate that extracellular FN may act as a natural barrier against prion replication and that the extracellular matrix composition may be a crucial feature determining prion tropism in different tissues.

Eighteenth-century England witnessed the emergence of two neurological diseases in animals. Scrapie, a transmissible spongiform encephalopathy, is a fatal neurodegenerative disease of sheep and goats that appears in classical and atypical forms. Reports of classical scrapie in continental Europe with described symptoms date back to 1750 in what is now western Poland. However, two major outbreaks of scrapie appeared in England prior to the 1800s. References to a sheep disease with a resemblance to scrapie first appear in Southwestern England between 1693 and 1722 and in the East Midlands between 1693 and 1706. Concurrent with the descriptions of scrapie in sheep was a neurological disease of deer first appearing in the East of England. Two 18th-century writers remarked on the symptomatic similarities between the sheep and deer neurological diseases. Multiple outbreaks of the unknown deer disease existing as early as 1772 are examined and are identified as rabies.

Classical bovine spongiform encephalopathy (BSE) in cattle was caused by the recycling and feeding of meat and bone meal contaminated with a transmissible spongiform encephalopathy (TSE) agent but its origin remains unknown. This study aimed to determine whether atypical scrapie could cause disease in cattle and to compare it with other known TSEs in cattle. Two groups of calves (five and two) were intracerebrally inoculated with atypical scrapie brain homogenate from two sheep with atypical scrapie. Controls were five calves intracerebrally inoculated with saline solution and one non-inoculated animal. Cattle were clinically monitored until clinical end-stage or at least 96 months post-inoculation (mpi). After euthanasia, tissues were collected for TSE diagnosis and potential transgenic mouse bioassay. One animal was culled with BSE-like clinical signs at 48 mpi. The other cattle either developed intercurrent diseases leading to cull or remained clinical unremarkable at study endpoint, including control cattle. None of the animals tested positive for TSEs by Western immunoblot and immunohistochemistry. Bioassay of brain samples from the clinical suspect in Ov-Tg338 and Bov-Tg110 mice was also negative. By contrast, protein misfolding cyclic amplification detected prions in the examined brains from atypical scrapie-challenged cattle, which had a classical BSE-like phenotype. This study demonstrates for the first time that a TSE agent with BSE-like properties can be amplified in cattle inoculated with atypical scrapie brain homogenate., (© 2023. L’Institut National de Recherche en Agriculture, Alimentation et Environnement (INRAE).)

The emergence of bovine spongiform encephalopathy (BSE) prions from atypical scrapie has been recently observed upon experimental transmission to rodent and swine models. This study aimed to assess whether the inoculation of atypical scrapie could induce BSE-like disease in cattle. Four calves were intracerebrally challenged with atypical scrapie. Animals were euthanized without clinical signs of prion disease and tested negative for PrP Sc accumulation by immunohistochemistry and western blotting. However, an emergence of BSE-like prion seeding activity was detected during in vitro propagation of brain samples from the inoculated animals. These findings suggest that atypical scrapie may represent a potential source of BSE infection in cattle., (© 2023. L’Institut National de Recherche en Agriculture, Alimentation et Environnement (INRAE).)

Prion diseases, including chronic wasting disease (CWD) in cervids, are fatal neurodegenerative disorders caused by the misfolding of cellular prion proteins. CWD is known to spread among captive and free-ranging deer in North America. In 2016, an outbreak of contagious CWD was detected among wild reindeer in Norway, marking the first occurrence of the disease in Europe. Additionally, new sporadic forms of CWD have been discovered in red deer in Norway and moose in Fennoscandia. We used serial protein misfolding cyclic amplification to study the ability of Norwegian prion isolates from reindeer, red deer, and moose (two isolates), as well as experimental classical scrapie from sheep, to convert a panel of 16 brain homogenates (substrates) from six different species with various prion protein genotypes. The reindeer CWD isolate successfully converted substrates from all species except goats. The red deer isolate failed to convert sheep and goat substrates but exhibited amplification in all cervid substrates. The two moose isolates demonstrated lower conversion efficacies. The wild type isolate propagated in all moose substrates and in the wild type red deer substrate, while the other isolate only converted two of the moose substrates. The experimental classical scrapie isolate was successfully propagated in substrates from all species tested. Thus, reindeer CWD and classical sheep scrapie isolates were similarly propagated in substrates from different species, suggesting the potential for spillover of these contagious diseases. Furthermore, the roe deer substrate supported conversion of three isolates suggesting that this species may be vulnerable to prion disease., (© 2023. L’Institut National de Recherche en Agriculture, Alimentation et Environnement (INRAE).)

The conversion of cellular prion protein (PrP C ) into pathogenic prion isoforms (PrP Sc ) and the mutation of PRNP are definite causes of prion diseases. Unfortunately, without exception, prion diseases are untreatable and fatal neurodegenerative disorders; therefore, one area of research focuses on identifying medicines that can delay the progression of these diseases. According to the concept of drug repositioning, we investigated the efficacy of the c-Abl tyrosine kinase inhibitor radotinib, which is a drug that is approved for the treatment of chronic myeloid leukemia, in the treatment of disease progression in prion models, including prion-infected cell models, Tga 20 and hamster cerebellar slice culture models, and 263K scrapie-infected hamster models. Radotinib inhibited PrP Sc deposition in neuronal ZW13-2 cells that were infected with the 22L or 139A scrapie strains and in cerebellar slice cultures that were infected with the 22L or 263K scrapie strains. Interestingly, hamsters that were intraperitoneally injected with the 263K scrapie strain and intragastrically treated with radotinib (100 mg/kg) exhibited prolonged survival times (159 ± 28.6 days) compared to nontreated hamsters (135 ± 9.9 days) as well as reduced PrP Sc deposition and ameliorated pathology. However, intraperitoneal injection of radotinib exerted a smaller effect on the survival rate of the hamsters. Additionally, we found that different concentrations of radotinib (60, 100, and 200 mg/kg) had similar effects on survival time, but this effect was not observed after treatment with a low dose (30 mg/kg) of radotinib. Interestingly, when radotinib was administered 4 or 8 weeks after prion inoculation, the treated hamsters survived longer than the vehicle-treated hamsters. Additionally, a pharmacokinetic assay revealed that radotinib effectively crossed the blood-brain barrier. Based on our findings, we suggest that radotinib is a new candidate anti-prion drug that could possibly be used to treat prion diseases and promote the remission of symptoms.

Bovine spongiform encephalopathy (BSE) is a fatal neurodegenerative disease that belongs to a group of diseases known as transmissible spongiform encephalopathies (TSEs). It is believed that the infectious agent responsible for prion diseases is abnormally folded prion protein (PrP Sc ), which derives from a normal cellular protein (PrP C ), which is a cell surface glycoprotein predominantly expressed in neurons. There are three different types of BSE, the classical BSE (C-type) strain and two atypical strains (H-type and L-type). BSE is primarily a disease of cattle; however, sheep and goats also can be infected with BSE strains and develop a disease clinically and pathogenically indistinguishable from scrapie. Therefore, TSE cases in cattle and small ruminants require discriminatory testing to determine whether the TSE is BSE or scrapie and to discriminate classical BSE from the atypical H- or L-type strains. Many methods have been developed for the detection of BSE and have been reported in numerous studies. Detection of BSE is mainly based on the identification of characteristic lesions or detection of the PrP Sc in the brain, often by use of their partial proteinase K resistance properties. The objective of this paper was to summarize the currently available methods, highlight their diagnostic performance, and emphasize the advantages and drawbacks of the application of individual tests.

In sheep, the transmissibility and progression of scrapie, a sheep prion (PrP Sc ) disease, is strongly dependent upon specific amino acid polymorphisms in the natively expressed prion protein (PrP C ). Sheep expressing PrP C with lysine (K) polymorphism at position 171 (K171) are partially resistant to oronasal dosing of classical sheep scrapie. In addition, scrapie infected sheep expressing the K171 polymorphism show a longer incubation period compared to sheep homozygous (glutamine (Q)) at position 171. Quantitating the amount of the K171 polymorphism in a sheep scrapie sample can provide important information on the composition of PrP Sc . A tryptic peptide, 159 R.YPNQVYYRPVDK.Y 172 , derived from the digestion of 171K recombinant PrP, was identified as an analyte peptide suitable for a multiple reaction monitoring-based analysis. This method, using 15 N-labeled analogs and another internal peptide from the proteinase K-resistant core, permits the simultaneous quantitation of the total amount of PrP and the proportion of K171 polymorphism in the sample. Background molecules with similar retention times and transitions were present in samples from scrapie-infected sheep. Proteinase K digestion followed by ultracentrifugation-based isolation or phosphotungstic acid-based isolation were employed to minimize the contribution of those background molecules, making this approach suitable for quantitating the amount of the K171 polymorphism in heterozygous scrapie infected sheep.

This study examines the effect of various infectious prion titers within the dynamic range as measured by ELISA on incubation period. We inoculated ovinized transgenic mice with seven decreasing dilutions of a fast-incubating scrapie strain. The highest inoculum group was a 20% w/v brain homogenate from a sheep with scrapie. The subsequent six inoculum dilutions ranged from the highest ELISA optical density reading of 4.000 to a dilution where scrapie prions were not detectable by ELISA. Multiple comparison analysis demonstrated variation in the incubation periods between some inoculum groups. Incubation periods were similar between inoculum groups unless their optical density differed by more than ≈2 units of absorbance. These data will inform the interpretation of future studies that compare incubation periods in experimentally inoculated animals for TSE research., (© 2022. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Prions, which cause transmissible spongiform encephalopathies (TSEs), are a notorious group of infectious agents with possibly the highest resistance to complete inactivation. Although various gas plasma instruments have been developed, studies on prion inactivation using gas plasma instruments are limited. Among them, the hydrogen peroxide gas plasma instrument, STERRAD ® (Advanced Sterilization Products; ASP, Johnson & Johnson, Irvine, CA, USA), is recommended for prion inactivation of heat-sensitive medical devices. However, STERRAD ® is not a plasma sterilizer but a hydrogen peroxide gas sterilizer. In STERRAD ® , plasma generated by radio frequency (RF) discharge removes excess hydrogen peroxide gas and does not contribute to sterilization. This is also supported by evidence that the instrument was not affected by the presence or absence of RF gas plasma. However, recent studies have shown that other gas plasma instruments derived from air, nitrogen, oxygen, Ar, and a mixture of gases using corona, dielectric barrier, microwave, and pulse discharges can inactivate scrapie prions. As inactivation studies on prions other than scrapie are limited, further accumulation of evidence on the effectiveness of gas plasma using human-derived prion samples is warranted for practical purposes.

Objective: To describe the global profiles of acetylated proteins in the brains of scrapie agents 139A- and ME7-infected mice collected at mid-early, mid-late, and terminal stages., Methods: The acetylated proteins from the cortex regions of scrapie agent (139A- and ME7)-infected mice collected at mid-early (80 days postinfection, dpi), mid-late (120 dpi), and terminal (180 dpi) stages were extracted, and the global profiles of brain acetylated proteins were assayed with proteomic mass spectrometry. The proteins in the infected mice showing 1.5-fold higher or lower levels than that of age-matched normal controls were considered as differentially expressed acetylated peptides (DEAPs)., Results: A total of 118, 42, and 51 DEAPs were found in the brains of 139A-80, 139A-120, and 139A-180 dpi mice, respectively. Meanwhile, 390, 227, and 75 DEAPs were detected in the brains of ME7-80, ME7-120, and ME7-180 dpi mice, respectively. The overwhelming majority of DEAPs in the mid-early stage were down-regulated, and more portions of DEAPs in the mid-late and late stages were up-regulated. Approximately 22.1% (328/1,485) of acetylated peptides mapped to 74 different proteins were mitochondrial associated. Kyoto Encyclopedia of Genes and Genomes pathway analysis identified 39 (80 dpi), 13 (120 dpi), and 10 (180 dpi) significantly changed pathways in 139A-infected mice. Meanwhile, 55, 25, and 18 significantly changed pathways were observed in the 80, 120, and 180 dpi samples of 139A- and ME7-infected mice ( P < 0.05), respectively. Six pathways were commonly involved in all tested samples. Moreover, many steps in the citrate cycle (tricarboxylic acid cycle) were affected, represented by down-regulated acetylation for relevant enzymes in the mid-early stage and up-regulated acetylation in the mid-late and late stages., Conclusion: Our data here illustrated the changes in the global profiles for brain acetylated proteins during prion infection, showing remarkably inhibited acetylation in the early stage and relatively enhanced acetylation in the late stage., (Copyright © 2022 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.)

The structures of prion protein (PrP)-based mammalian prions have long been elusive. However, cryo-EM has begun to reveal the near-atomic resolution structures of fully infectious ex vivo mammalian prion fibrils as well as relatively innocuous synthetic PrP amyloids. Comparisons of these various types of PrP fibrils are now providing initial clues to structural features that correlate with pathogenicity. As first indicated by electron paramagnetic resonance and solid-state NMR studies of synthetic amyloids, all sufficiently resolved PrP fibrils of any sort (n > 10) have parallel in-register intermolecular β-stack architectures. Cryo-EM has shown that infectious brain-derived prion fibrils of the rodent-adapted 263K and RML scrapie strains have much larger ordered cores than the synthetic fibrils. These bona fide prion strains share major structural motifs, but the conformational details and the overall shape of the fibril cross sections differ markedly. Such motif variations, as well as differences in sequence within the ordered polypeptide cores, likely contribute to strain-dependent templating. When present, N-linked glycans and glycophosphatidylinositol (GPI) anchors project outward from the fibril surface. For the mouse RML strain, these posttranslational modifications have little effect on the core structure. In the GPI-anchored prion structures, a linear array of GPI anchors along the twisting fibril axis appears likely to bind membranes in vivo, and as such, may account for pathognomonic membrane distortions seen in prion diseases. In this review, we focus on these infectious prion structures and their implications regarding prion replication mechanisms, strains, transmission barriers, and molecular pathogenesis., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Published by Elsevier Inc.)

Prion diseases are diagnosed in the symptomatic stage, when the neuronal damage is spread throughout the central nervous system (CNS). The assessment of biological features that allow the detection of asymptomatic cases is needed, and, in this context, scrapie, where pre-symptomatic infected animals can be detected through rectal biopsy, becomes a good study model. Neurogranin (Ng) and neurofilament light chain (NfL) are proteins that reflect synaptic and axonal damage and have been studied as cerebrospinal fluid (CSF) biomarkers in different neurodegenerative disorders. In this study, we evaluated Ng and NfL both at the protein and transcript levels in the CNS of preclinical and clinical scrapie-affected sheep compared with healthy controls and assessed their levels in ovine CSF. The correlation between these proteins and the main neuropathological events in prion diseases, PrP Sc deposition and spongiosis, was also assessed. The results show a decrease in Ng and NfL at the protein and gene expression levels as the disease progresses, and significant changes between the control and preclinical animals. On the contrary, the CSF levels of NfL increased throughout the progression of the disease. Negative correlations between neuropathological markers of prion disease and the concentration of the studied proteins were also found. Although further research is needed, these results suggest that Ng and NfL could act as biomarkers for neurodegeneration onset and intensity in preclinical cases of scrapie.

Prion diseases are chronic and fatal neurodegenerative diseases characterized by the accumulation of disease-specific prion protein (PrP Sc ), spongiform changes, neuronal loss, and gliosis. Growing evidence shows that the neuroinflammatory response is a key component of prion diseases and contributes to neurodegeneration. Toll-like receptors (TLRs) have been proposed as important mediators of innate immune responses triggered in the central nervous system in other human neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. However, little is known about the role of TLRs in prion diseases, and their involvement in the neuropathology of natural scrapie has not been studied. We assessed the gene expression of ovine TLRs in four anatomically distinct brain regions in natural scrapie-infected sheep and evaluated the possible correlations between gene expression and the pathological hallmarks of prion disease. We observed significant changes in TLR expression in scrapie-infected sheep that correlate with the degree of spongiosis, PrP Sc deposition, and gliosis in each of the regions studied. Remarkably, TLR4 was the only gene upregulated in all regions, regardless of the severity of neuropathology. In the hippocampus, we observed milder neuropathology associated with a distinct TLR gene expression profile and the presence of a peculiar microglial morphology, called rod microglia, described here for the first time in the brain of scrapie-infected sheep. The concurrence of these features suggests partial neuroprotection of the hippocampus. Finally, a comparison of the findings in naturallyinfected sheep versus an ovinized mouse model (tg338 mice) revealed distinct patterns of TLRgene expression.

Prion diseases are fatal and infectious neurodegenerative diseases in humans and other mammals caused by templated misfolding of the endogenous prion protein (PrP). Although there is currently no vaccine or therapy against prion disease, several classes of small-molecule compounds have been shown to increase disease-free incubation time in prion-infected mice. An apparent obstacle to effective anti-prion therapy is the emergence of drug-resistant strains during static therapy with either single compounds or multi-drug combination regimens. Here, we treated scrapie-infected mice with dynamic regimens that alternate between different classes of anti-prion drugs. The results show that alternating regimens containing various combinations of Anle138b, IND24 and IND116135 reduce the incidence of combination drug resistance, but do not significantly increase long-term disease-free survival compared to monotherapy. Furthermore, the alternating regimens induced regional vacuolation profiles resembling those generated by a single component of the alternating regimen, suggesting the emergence of strain dominance.

Portugal was among the first European countries to report cases of Atypical Scrapie (ASc), the dominant form of Transmissible Spongiform Encephalopathy (TSE) in Portuguese small ruminants. Although the diagnostic phenotypes observed in Portuguese ASc cases seem identical to those described for Nor98, unequivocal identification requires TSE strain-typing using murine bioassays. In this regard, we initiated characterization of ASc isolates from sheep either homozygous for the ARQ genotype or the classical scrapie-resistant ARR genotype. Isolates from such genotypes were transmitted to TgshpXI mice expressing ovine PrPARQ. Mean incubation periods were 414 ± 58 and 483 ± 107 days in mice inoculated with AL 141 RQ/AF 141 RQ and AL 141 RR/AL 141 RR sheep isolates, respectively. Both isolates produced lesion profiles similar to French ASc Nor98 'discordant cases', where vacuolation was observed in the hippocampus (G6), cerebral cortex at the thalamus (G8) level, cerebellar white matter (W1) and cerebral peduncles (W3). Immunohistochemical PrP Sc deposition was observed in the hippocampus, cerebellar cortex, cerebellar white matter and cerebral peduncles in the form of aggregates and fine granules. These findings were consistent with previously reported cases of ASc Nor98 transmitted to transgenic TgshpXI mice, confirming that the ASc strain present in Portuguese sheep corresponds to ASc Nor98.

Classical scrapie is a prion disease of small ruminants, the infectious agent of which has been shown to be extremely persistent in the environment. Cleaning and disinfection (C&D) after a scrapie outbreak is currently recommended by many governments' veterinary advisors and implemented in most farms affected. Yet, the effectiveness of these procedures remains unclear. The aim of this study was to review existing literature and guidelines regarding farm C&D protocols following classical scrapie outbreaks and assess their effectiveness and the challenges that translation of policy and legislative requirements present at a practical level. A review of the literature was conducted to identify the on-farm C&D protocols used following outbreaks of scrapie, assess those materials with high risk for persistence of the scrapie agent on farms, and review the existing evidence of the effectiveness of recommended C&D protocols. An expert workshop was also organised in Great Britain (GB) to assess: the decision-making process used when implementing C&D protocols on GB farms, the experts' perceptions on the effectiveness of these protocols and changes needed, and their views on potential recommendations for policy and research. Outputs of the literature review revealed that the current recommended protocol for C&D [1 h treatment with sodium hypochlorite containing 20,000 ppm free chlorine or 2 M sodium hydroxide (NaOH)] is based on laboratory experiments. Only four field farm experiments have been conducted, indicating a lack of data on effectiveness of C&D protocols on farms by the re-occurrence of scrapie infection post re-stocking. Recommendations related to the control of outdoor environment, which are difficult and expensive to implement, vary between countries. The expert workshop concluded that there are no practical, cost-effective C&D alternatives to be considered at this time, with control therefore based on C&D only in combination with additional time restrictions on re-stocking and replacement with non-susceptible livestock or more genetically resistant types, where available. Participants agreed that C&D should still be completed on scrapie affected farms, as it is considered to be "good disease practice" and likely to reduce the levels of the prion protein. Participants felt that any additional protocols developed should not be "too prescriptive" (should not be written down in specific policies) because of significant variation in farm types, farm equipment and installations. Under this scenario, control of classical scrapie on farms should be designed with a level of C&D in combination with re-stocking temporal ban and replacement with livestock of limited susceptibility., (Crown Copyright © 2021. Published by Elsevier B.V. All rights reserved.)

Prion disease is a fatal neurodegenerative disease with a broad host range in humans and animals. It is caused by proteinase K-resistant prion protein (PrPres). In previous studies, a heterogeneous infection in Cervidae and Caprinae was reported. Chronic wasting disease (CWD) has been frequently reported as the only prion disease in Korea that occurs in livestock. Thus, there is a possibility of transmission of CWD to Korean native black goats. However, PrPres has not been investigated thus far in Korean native black goats. We found strong linkage disequilibrium between c.126G>A and c.414T>C (r2 = 1) and between c.718C>T and c.126G>A (r2 = 0.638). In addition, the haplotype GTGTAAAC (representing codons 42, 102, 127, 138, 143, 146, 218 and 240) showed the highest frequency with 45.1%. Among 41 Korean native black goats, 20 animals (48.78%) were homozygous for the susceptible haplotypes (histidine at codon 143, asparagine at codon 146 and arginine at codon 154). Interestingly, we did not detect PrPres bands in any of the tested animals, including the 20 animals carrying potential scrapie susceptible haplotypes.

Transmissible spongiform encephalopathies, or prion diseases, are fatal neurodegenerative diseases affecting humans and animals. Although many host tissues express PrP C (essential for prion replication), relatively few cell types accumulate significant levels of infectivity, including neurons and other cell types in the nervous system, and follicular dendritic cells in secondary lymphoid organs. This suggests that tissue or cell-specific receptors or cofactors could play a role in controlling differential susceptibility to infection. Endogenous retroviruses (ERV), the remnants of ancient retroviral integration into the host germline, may represent one such cofactor. We examined the effect of scrapie infection on expression of three ovine ERV families (enJSRV/β1-OERV, γ1-OERV, γ2-OERV) in secondary lymphoid tissues of sheep at different time points following subcutaneous inoculation, using RT-qPCR. These OERVs were constitutively expressed in the prescapular lymph node and spleen of uninfected sheep. However, we were unable to find convincing evidence of specific differential expression of OERV in the same tissues following scrapie infection, in contrast to previous studies of ERV expression in brains of prion-infected mice and macaques. This study is the first to quantify the expression of potentially functional OERV transcripts in sheep lymphoid tissues, opening up interesting questions about the consequences for host immune function., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Activation of inflammatory cells and upregulations of a number of cytokines in the central nervous system (CNS) of patients with prion diseases are frequently observed. To evaluate the potential changes of some brain cytokines that were rarely addressed during prion infection, the levels of 17 different cytokines in the brain homogenates of mice infected with different scrapie mouse-adapted agents were firstly screened with Luminex assay. Significant upregulations of interferon gamma-induced protein 10 (IP10), keratinocyte chemoattractant (KC) and macrophage colony stimulating factor (M-CSF) were frequently detected in the brain lysates of many strains of scrapie infected mice. The upregulations of those three cytokines in the brains of scrapie infected mice were further validated by the individual specific ELISA and immunohistochemical assay. Increased specific mRNAs of IP10, M-CSF and KC in the brains of scrapie infected mice were also detected by the individual specific qRT-PCRs and IP10-specific digital PCR. Dynamic analyses of the brain samples collected at different time points post infection revealed the time-dependent increases of those three cytokines, particularly IP10 during the incubation period of scrapie infection. In addition, we also found that the levels of IP10 in cerebral spinal fluid (CSF) of 45 sporadic Creutzfeldt-Jakob disease (sCJD) patients were slightly but significantly higher than those of the cases who were excluded the diagnosis of prion diseases. These data give us a better understanding of inflammatory reaction during prion infection and progression of prion disease.

Resveratrol shows the ability to block prion replication in a scrapie-infected cell line, SMB-S15, and remove the infectivity of the treated cell lysates in an experimental bioassay. In this study, we compared the effectiveness of three stilbene compounds, resveratrol (Res), pterostilbene (Pte), and piceatannol (Pic), on inhibiting prion propagations in the levels of cell culture, PMCA, and RT-QuIC. All three chemicals showed active suppressions on PrP Sc replication in SMB-S15 cells, in which Res seemed to be the most active one, followed by Pic and Pte. Mouse PrP-based PMCA tests using the lysates of SMB-S15 cells and brain homogenates of scrapie agents S15-, 139A-, or ME7-infected mice verified that Res, Pte, and Pic inhibited the amplifications of PK-resistant signals. Res was also the most effective one. Mouse PrP-based RT-QuIC using the above seeds demonstrated that three stilbenes efficiently inhibited the fibril formation. However, Pic was the most effective one, followed by Res and Pte. Furthermore, the inhibition activities of the three stilbenes on the brain-derived prion from a 263K-infected hamster were tested with hamster PrP-based PMCA and RT-QuIC. The results indicated that Pic was the most effective one apparently, followed by Res and Pte. According to the results of Biacore, Res showed binding affinities much stronger than those of Pte, whereas both revealed markedly stronger binding affinities with mouse PrP. Our data here indicate that different stilbenes have the ability to block PrP Sc replication in vitro with different prion species. The suppressive effects of stilbene compounds are likely associated with their molecular binding activities with PrPs.

Prion diseases are fatal infectious neurodegenerative disorders in human and animals caused by misfolding of the cellular prion protein (PrP C ) into the infectious isoform PrP Sc . These diseases have the potential to transmit within or between species, and no cure is available to date. Targeting the unfolded protein response (UPR) as an anti-prion therapeutic approach has been widely reported for prion diseases. Here, we describe the anti-prion effect of the chemical compound Sephin1 which has been shown to protect in mouse models of protein misfolding diseases including amyotrophic lateral sclerosis (ALS) and multiple sclerosis (MS) by selectively inhibiting the stress-induced regulatory subunit of protein phosphatase 1, thus prolonging eIF2α phosphorylation. We show here that Sephin1 dose and time dependently reduced PrP Sc in different neuronal cell lines which were persistently infected with various prion strains. In addition, prion seeding activity was reduced in Sephin1-treated cells. Importantly, we found that Sephin1 significantly overcame the endoplasmic reticulum (ER) stress induced in treated cells, as measured by lower expression of stress-induced aberrant prion protein. In a mouse model of prion infection, intraperitoneal treatment with Sephin1 significantly prolonged survival of prion-infected mice. When combining Sephin1 with the neuroprotective drug metformin, the survival of prion-infected mice was also prolonged. These results suggest that Sephin1 could be a potential anti-prion drug selectively targeting one component of the UPR pathway.

Mitophagy is an important process for removing damaged mitochondria in cells, the dysfunction of which has been directly linked to an increasing number of neurodegenerative disorders. However, the details of mitophagy in prion diseases still need to be deeply explored. In this study, we identified more autophagosomes and large swelling mitochondria structures in the prion-infected cultured cell line SMB-S15 by transmission electron microscopy, accompanying the molecular evidence of activated autophagic flux. Western blots illustrated that the levels of Pink1 and Parkin, particularly in the mitochondrial fraction, were increased in SMB-S15 cells, whereas the levels of mitochondrial membrane proteins TIMM44, TOMM20, and TIMM23 were decreased. The amount of whole polyubiquitinated proteins decreased, but that of phosphor-polyubiquitinated proteins increased in SMB-S15 cells. The level of MFN2 in SMB-S15 cells were down-regulated, but its polyubiquitinated form was up-regulated. Knockdown of the expressions of Pink1 and Parkin by the individual SiRNAs in SMB-S15 cells reduced autophagic activity but did not seem to influence the expressions of TOMM20 and TIMM23. Moreover, we also demonstrated that the brain levels of Pink1 and Parkin in the mice infected with scrapie strains 139A and ME7 were remarkably increased at the terminal stage of the disease by Western blot and immunohistochemical (IHC) assays. Immunofluorescent assays revealed that Pink1 signals widely colocalized with GAFP-, Iba1-, and NeuN-positive cells in the brains of scrapie-infected mice. IHC assays with serial sections of the brain tissues infected with agents 139A and ME7 showed more Pink1- and Parkin-positive cells located at the areas with more PrP Sc deposit. These results suggest an activated mitophagy in prion-infected cells and prion-infected experimental mice, probably via an enhanced Pink-Parkin pathway.

Activation of complement system in central nervous system (CNS) of the patients suffering from prion diseases or animal models infected with prion agents experimentally is reported repeatedly, but which pathways are involved in the complement system during prion infection is not well documented. Here, we evaluated the level of complement factor B (CFB), which is the key factor that triggers alterative pathway (AP) of complement in the brain tissues of scrapie-infected mice with various methodologies. We found that the levels of mRNA and protein of CFB significantly increased in the brain tissues of scrapie-infected mice. Morphologically, the increased CFB-specific signal overlapped with the elevated C3 signal in brain sections of scrapie-infected mice, meanwhile overlapped with damaged neurons and activated microglia, but not with the proliferative astrocytes. Additionally, the level of complement factor P (CFP), the key positive regulator of AP, also increased remarkably in the brain tissues of infected mice. The transcriptional levels of CD55 and CD46, two negative regulators of AP, decreased without significance in brain tissues of scrapie-infected mice at the terminal stage. However, the mRNA and protein levels of CFH, another negative regulator of AP, increased. Through the dynamic analyses of the expressions of CFB, CFP, and CFH in brain sections of 139A-infected mice, which were collected at different time-points during incubation period, illustrated time-dependent increase levels of each factor during the incubation period of scrapie infection. Taken together, our data here demonstrate that the AP of complement cascade is activated in the CNS microenvironment during prion infection.

Mammalian transmissible spongiform encephalopathies (TSEs) display marked activation of astrocytes and microglia that precedes neuronal loss. Investigation of clinical parallels between TSEs and other neurodegenerative protein misfolding diseases, such as Alzheimer's disease, has revealed similar patterns of neuroinflammatory responses to the accumulation of self-propagating amyloids. The contribution of glial activation to the progression of protein misfolding diseases is incompletely understood, with evidence for mediation of both protective and deleterious effects. Glial populations are heterogeneously distributed throughout the brain and capable of dynamic transitions along a spectrum of functional activation states between pro- and antiinflammatory polarization extremes. Using a murine model of Rocky Mountain Laboratory scrapie, the neuroinflammatory response to prion infection was characterized by evaluating glial activation across 15 brain regions over time and correlating it to traditional markers of prion neuropathology, including vacuolation and PrP Sc deposition. Quantitative immunohistochemistry was used to evaluate glial expression of iNOS and Arg1, markers of classical and alternative glial activation, respectively. The results indicate progressive upregulation of iNOS in microglia and a mixed astrocytic profile featuring iNOS expression in white matter tracts and detection of Arg1-positive populations throughout the brain. These data establish a temporospatial lesion profile for this prion infection model and demonstrate evidence of multiple glial activation states.

Prion diseases in mammals are caused by a conformational transition of the cellular prion protein from its native conformation (PrP C ) to a pathological isoform called "prion protein scrapie" (PrP Sc ). A molecular level of understanding of this conformational transition will be helpful in unveiling the disease etiology. Experimental structural biological techniques (NMR and X-ray crystallography) have been used to unravel the atomic level structural information for the prion and its binding partners. More than one hundred three-dimensional structures of the mammalian prions have been deposited in the protein databank. Structural studies on the prion protein and its structural transitions will deepen our understanding of the molecular basis of prion pathogenesis and will provide valuable guidance for future structure-based drug discovery endeavors., (© 2019 The Protein Society.)

Activation of microglia and increased expression of TNF-α are frequently observed in the brains of human and animal prion diseases. As an important cytokine, TNF-α participates in not only pro-inflammatory responses but also in cellular communication, cell differentiation, and cell death. However, the role of TNF-α in the pathogenesis of prion disease remains ambiguous. In this study, the activities of a scrapie-infected cell line SMB-S15 and its normal partner SMB-PS exposed to the supernatant of a LPS-activated microglia cell line BV2 were evaluated. After it was exposed to the LPS-stimulated supernatant of BV2 cells, the cell viability of SMB-S15 cells was markedly decreased, whereas that of the SMB-PS cells remained unchanged. The level of TNF-α was significantly increased in the LPS-stimulated supernatant of BV2 cells. Further, we found that the recombinant TNF-α alone induced the decreased cell viability of SMB-S15 and the neutralizing antibody for TNF-α completely antagonized the decreased cell viability caused by the LPS-stimulated supernatant of BV2 cells. Stimulation with TNF-α induced the remarkable increases of apoptosis-associated proteins in SMB-PS cells, such as cleaved caspase-3 and RIP1, whereas an obvious increase of necroptosis-associated protein in SMB-S15 cells, such as p-MLKL. Meanwhile, the upregulation of caspase-8 activity in SMB-PS cells was more significant than that of SMB-S15 cells. The decreased cell viability of SMB-S15 and the increased expression of p-MLKL induced by TNF-α were completely rescued by Necrostatin-1. Moreover, we verified that removal of PrP Sc propagation in SMB-S15 cells by resveratrol partially rescues the cell tolerance to the stimulation of TNF-α. These data indicate that the prion-infected cell line SMB-S15 is more vulnerable to the stimulations of activated microglia and TNF-α, which is likely due to the outcome of necroptosis rather than apoptosis. Furthermore, significant upregulation of p-MLKL, MLKL, and RIP3 was detected in the post-mortem cortical brains of the patients of various types of human prion diseases, including sporadic Creutzfeldt-Jakob disease (sCJD), G114 V-genetic CJD (gCJD), and fatal familial insomnia (FFI).

Scrapie is a mammalian transmissible spongiform encephalopathy or prion disease that predominantly affects sheep and goats. Scrapie has been shown to overcome the species barrier via experimental infection of other rodents. To confirm the re-transmissibility of the mouse-adapted ME7 scrapie strain to ovine prion protein (PrP) transgenic mice, mice of an ovinized transgenic mouse line carrying the Suffolk sheep PrP gene that contained the A 136 R 154 Q 171 /ARQ allele were intracerebrally inoculated with brain homogenates obtained from terminally ill ME7-infected C57BL/6J mice. Herein, we report that the mouse-adapted ME7 scrapie strain was successfully re-transmitted to the transgenic mice expressing ovine PrP. In addition, we observed changes in the incubation period, glycoform profile, and pattern of scrapie PrP (PrP Sc ) deposition in the affected brains. PrP Sc deposition in the hippocampal region of the brain of 2nd-passaged ovine PrP transgenic mice was accompanied by plaque formation. These results reveal that the mouse-adapted ME7 scrapie strain has the capacity to act as a template for the conversion of ovine normal monomeric precursors into a pathogenic form in ovine PrP transgenic mice. The change in glycoform pattern and the deposition of plaques in the hippocampal region of the brain of the 2nd-passaged PrP transgenic mice are most likely cellular PrP species dependent rather than being ME7 scrapie strain encoded., Competing Interests: Conflicts of Interest: The authors declare no conflicts of interest.

Prions are composed solely of the pathological isoform (PrP Sc ) of the normal cellular prion protein (PrP C ). Identification of different PrP Sc structures is crucially important for understanding prion biology because the pathogenic properties of prions are hypothesized to be encoded in the structures of PrP Sc However, these structures remain yet to be identified, because of the incompatibility of PrP Sc with conventional high-resolution structural analysis methods. Previously, we reported that the region between the first and the second α-helix (H1∼H2) of PrP C might cooperate with the more C-terminal side region for efficient interactions with PrP Sc From this starting point, we created a series of PrP variants with two cysteine substitutions (C;C-PrP) forming a disulfide-crosslink between H1∼H2 and the distal region of the third helix (Ctrm). We then assessed the conversion capabilities of the C;C-PrP variants in N2a cells infected with mouse-adapted scrapie prions (22L-ScN2a). Specifically, Cys substitutions at residues 165, 166, or 168 in H1∼H2 were combined with cysteine scanning along Ctrm residues 220-229. We found that C;C-PrPs are expressed normally with glycosylation patterns and subcellular localization similar to WT PrP, albeit differing in expression levels. Interestingly, some C;C-PrPs converted to protease-resistant isoforms in the 22L-ScN2a cells, but not in Fukuoka1 prion-infected cells. Crosslink patterns of convertible C;C-PrPs indicated a positional change of H1∼H2 toward Ctrm in PrP Sc -induced conformational conversion. Given the properties of the C;C-PrPs reported here, we propose that these PrP variants may be useful tools for investigating prion strain-specific structures and structure-phenotype relationships of PrP Sc ., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Microglial cells in the central nervous system play important roles in neurodevelopment and resistance to infection, yet microglia can become neurotoxic under some conditions. An early event during prion infection is the activation of microglia and astrocytes in the brain prior to damage or death of neurons. Previous prion disease studies using two different strategies to manipulate signaling through the microglial receptor CSF-1R reported contrary effects on survival from prion disease. However, in these studies, reductions of microglial numbers and function were variable, thus confounding interpretation of the results. In the present work, we used oral treatment with a potent inhibitor of CSF-1R, PLX5622, to eliminate 78 to 90% of microglia from cortex early during the course of prion infection. Oral drug treatment early after infection with the RML scrapie strain significantly accelerated vacuolation, astrogliosis, and deposition of disease-associated prion protein. Furthermore, drug-treated mice had advanced clinical disease requiring euthanasia 31 days earlier than untreated control mice. Similarly, PLX5622 treatment during the preclinical phase at 80 days postinfection with RML scrapie also accelerated disease and resulted in euthanasia of mice 33 days earlier than infected controls. PLX5622 also accelerated clinical disease after infection with scrapie strains ME7 and 22L. Thus, microglia are critical in host defense during prion disease. The early accumulation of PrPSc in the absence of microglia suggested that microglia may function by clearing PrPSc, resulting in longer survival. IMPORTANCE Microglia contribute to many aspects of health and disease. When activated, microglia can be beneficial by repairing damage in the central nervous system (CNS) or they can turn harmful by becoming neurotoxic. In prion and prionlike diseases, the involvement of microglia in disease is unclear. Previous studies suggest that microglia can either speed up or slow down disease. In this study, we infected mice with prions and depleted microglia from the brains of mice using PLX5622, an effective CSF-1R tyrosine kinase inhibitor. Microglia were markedly reduced in brains, and prion disease was accelerated, so that mice needed to be euthanized 20 to 33 days earlier than infected control mice due to advanced clinical disease. Similar results occurred when mice were treated with PLX5622 at 80 days after infection, which was just prior to the start of clinical signs. Thus, microglia are important for removing prions, and the disease is faster when microglia are depleted., (Copyright © 2018 American Society for Microbiology.)

We previously reported that mice intracerebrally inoculated with the mouse-adapted scrapie strain ME7 have markedly diminished T zones in the spleen due to the decreased expression of CCL19 and CCL21. In addition, follicular dendritic cell networks in germinal centers were larger in ME7-infected spleens compared to uninfected spleens. As an extension of that study, we set out to determine how ME7 infection affects spleen structure and follicular helper T (Tfh) cell responses in mice. For this study, mice were intraperitoneally inoculated with brain homogenate of the ME7 inoculum and spleens were analyzed 50, 130, and 200 days after inoculation and compared with those from uninfected mice. The result showed that ME7- infected mice had increased Tfh cell responses which were maintained until end-stage prion disease. Although CD4 T cells decreased in white pulps, they increased in germinal centers, and expressed higher levels of the Tfh-related genes, such as Bcl6, Il21, Cxcr5, Icos, and Pdcd1. In addition, ME7-infected spleens had increased numbers of CD4 memory T cells. These data indicate that although ME7 infection led to impaired splenic white pulp structure, CD4 memory T cells were increased and Tfh cell responses were required and prolonged to provide help for the replication and accumulation of pathogenic prion protein in germinal centers.

Scrapie is a transmissible spongiform encephalopathy of sheep and goats, and scrapie eradication programs in many parts of the world rely on strong genetic resistance to classical scrapie in sheep. However, the utility of putative resistance alleles in goats has been a focus of research because goats can transmit scrapie to sheep and may serve as a scrapie reservoir. Prior work showed that disease-free survival time was significantly extended in orally inoculated goats singly heterozygous for prion amino acid substitutions S146 or K222, but average durations were only around 3 years post-inoculation. The aim of this study was to investigate whether extended survival would exceed 6 years, which represents the productive lifetimes of most commercial goats. While all control homozygotes were clinically affected by an average of <2 years, none of the NS146 or QK222 goats developed clinical scrapie or had PrP Sc -positive rectal biopsies. Several NS146 and QK222 goats developed other conditions unrelated to scrapie, but tissue accumulation of PrP Sc was not detected in any of these animals. The NS146 heterozygotes have remained disease-free for an average of 2734days (approximately 7.5 years), the longest duration of any classical scrapie challenge experiment with any genotype to date. The QK222 heterozygotes have remained disease-free for an average of 2450days (approximately 6.7 years), the longest reported average duration for QK222 goats challenged with classical scrapie. This research is ongoing, but the current results demonstrate S146 and K222 confer strong resistance to classical scrapie in goats., (Published by Elsevier Ltd.)

Scrapie was the first prion disease to be recognised and the study of this disease in sheep and goats has provided a wealth of information not only for scrapie but also for the other prion diseases. All prion diseases are under strong genetic control of the prion gene PRNP, independent of whether they are typical or atypical scrapie and which of the different prion strains is causing infection. Decades of studies using experimental disease challenges and field surveys have established disease association models, in which species-specific amino acid variations in the prion or PrP protein, encoded by the PRNP gene, can predict disease susceptibility or resistance. PRNP genetics represents an important and successful basis for implementing scrapie eradication strategies in sheep and goats. In general terms these studies have revealed that there appear to be many more amino acid changes in PrP leading to increased resistance than to higher susceptibility. Most changes are in the globular part of PrP protein and three regions appear to have major influence. This knowledge can be transferred into prion diseases of other species to facilitate genetic control strategies. However, an obstacle remains with the lack of fully understanding the underlying molecular mechanism, impeding our ability to deal with the difference in the genetic control between typical and atypical forms of scrapie or to predict association in newly infected species. This chapter will discuss the advances in both typical and atypical scrapie from a genetic perspective., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Transcription factor NF-κB functions as a pleiotropic regulator of target genes controlling physiological function as well as pathological processes of many different diseases, including some neurodegenerative diseases. However, the role of NF-κB in the pathogenesis of prion disease remains ambiguous. In this study, the status of NF-κB (p65) in a prion-infected cell line SMB-S15 was first evaluated. Significantly lower levels of p65 and the phosphorylated form of p65 (p-p65) were detected in SMB-S15 cells, compared with its normal partner cell line SMB-PS. Markedly slower responses of the NF-κB system to the stimulation of TNF-α were observed in SMB-S15 cells. Removal of PrP Sc replication in SMB-S15 cells rescued the expression and activity of NF-κB. However, overexpression of p65 in SMB-S15 cells did not influence the propagation of PrP Sc . Moreover, significant decline of p65 level was also observed in the brain tissues of mice infected with the lysates of SMB-S15 cells and hamsters infected with scrapie agent 263K at terminal stage. Immunofluorescence assays (IFAs) on brain sections from either normal or scrapie-infected rodents revealed colocalization of p65 with neuronal nuclear (NeuN) protein positive cells but not with glial fibrillary acidic protein (GFAP) positive cells. Assays of the agents involving in the regulation of NF-κB showed down-regulated phosphoinositide 3-kinase (PI3K) and protein kinase B (PKB/Akt) both in SMB-S15 cells and in the brains of scrapie-infected rodents. Those data indicate a remarkable repression of the classical NF-κB pathway during prion infection both in vitro and in vivo. The alteration of NF-κB (p65) shows close association with the replication and accumulation of PrP Sc in the cells.

Galectin-1 (Gal-1) shows neuroprotective activity in brain ischemia, spinal cord injury, and autoimmune neuroinflammation. To evaluate the Gal-1 situation in the brains of prion disease, the brain levels of Gal-1 in several scrapie-infected experimental rodent models were tested by Western blot, including agents 263K-infected hamsters, 139A-, ME7-, and S15-infected mice. Remarkable increases of brain Gal-1 were observed in all tested scrapie-infected rodents at the terminal stage. The brain levels of Gal-1 showed time-dependent increases along with the prolonging of incubation times. Immunohistochemical assays illustrated much stronger stainings in the brain sections of scrapie-infected rodents. Quantitative RT-PCR of Gal-1 gene demonstrated increased transcription in the brains of scrapie-infected mice. Gal-1 was colocalized with GFAP- and NeuN-positive cells, but not with Iba-1-positive cells in immunofluorescent test. Increases of Gal-1 were also detected in the several postmortem cortex regions of human prion diseases. Moreover, the S-nitrosylated forms of Gal-1 in the brains of scrapie-infected rodents were significantly higher than those of normal ones. Our finding here demonstrates markedly increased brain Gal-1 and S-nitrosylated Gal-1 both in scrapie-infected rodents and human prion diseases.

Transmission of prions between mammalian species is limited by a "species barrier," a biological effect involving an increase in incubation period to decrease the percentage of animals succumbing to disease. In this study, we used protein misfolding cyclic amplification (PMCA) technique, which accelerates the conversion of prion proteins in vitro. Direct interspecies PMCA involving 144 cycles confirmed that both mouse-adapted scrapie strain 139A and hamster-adapted 263K could use brain homogenates of opposite species to form proteinase K (PK)-resistant PrP proteins (PrP res ). Newly formed interspecies prions could stably propagate themselves in subsequent serial PMCA passages. The two types of PMCA-generated cross-species PrP res changed their glycosylation profiles, which was similar to that observed during interspecies infection by the mouse agent 139A in vivo. These profiles were distinct from individual seeded PrP Sc and possessed properties of new hosts. Comparative analysis with respect to PK resistance showed no significant diversity between PMCA-PrP res and native PrP Sc or between brain and muscle PrP res . However, PrP res from the relatively early cycles of serial PMCA showed lower PK resistance than those from later cycles. Inoculation of these PMCA products amplified with homogeneous or heterogeneous brain tissues (cross-species products) induced experimental transmissible spongiform encephalopathies. These results suggested that PMCA can help prion strains to overcome species barrier and to propagate efficiently both in vitro and in vivo.

Ovine scrapie is a worldwide spread prion disease that is transmitted horizontally under field conditions. Placenta from scrapie-infected ewes is an important source of infection, since this tissue can accumulate high amounts of PrP Sc depending on the foetal genotype. Therefore, placentas carrying susceptible foetuses can accumulate PrP Sc but there is not PrP Sc accumulation in presence of foetuses with at least one ARR haplotype. In scrapie eradication programs, ARR/ARR males are used for breeding to increase the resistant progeny and reduce the horizontal transmission of the disease through the placenta. The development of highly sensitive techniques, that allow the detection of minimal amounts of PrP Sc , has caused many secretions/excretions and tissues that had previously been deemed negative to be relabeled as positive for PrP Sc . This has raised concerns about the possible presence of minimal amounts of PrP Sc in placentas from ARR foetuses that conventional techniques had indicated were negative. In the present study we examined 30 placentas from a total of 23 gestations; 15 gestations resulted from naturally ARQ/ARQ scrapie-infected ewes mated with ARR/ARR rams. The absence of PrP Sc in placentas carrying the foetal ARR haplotype (n=19) was determined by IDEXX HerdChek scrapie/BSE Antigen EIA Test, Prionics ® -Check WESTERN and corroborated by the highly sensitive Protein Misfolding Cyclic Amplification technique (PMCA). By immunohistochemistry, several unspecific stainings that might mislead a diagnosis were observed. The results of the present study support that using ARR/ARR males in scrapie eradication programs efficiently decreases the spreading of the agent in the environment via shed placentas., (Copyright © 2017. Published by Elsevier B.V.)

The infections of prion agents may cause progressive and fatal neurodegenerative diseases in humans and a serial of animal species. Previous studies have proposed that the levels of nitric oxide (NO) and nitric oxide synthase (NOS) in the brains of some neurodegeneration diseases changed, while S-nitrosylation (SNO) of many brain proteins altered in prion diseases. To elucidate the potential changes of brain NO levels during prion infection, the NO levels and NOS activities in the brain tissues of three scrapie experimental rodents were measured, including scrapie agent 263 K-infected hamsters and 139A- and ME7-infected mice. Both NO levels and NOS activities, including total NOS (TNOS) and inducible NOS (iNOS), were increased at the terminal stages of scrapie-infected animals. Assays of the brain samples collected at different time points during scrapie infection showed that the NO levels and NOS activities started to increase at early stage, reached to the peak in the middle stage, and dropped down at late stage. Western blots for brain iNOS revealed increased firstly and decreased late, especially in the brains of 139A- and ME7-infected mice. In line with those alterations, the levels of the SNO forms of several selected brain proteins such as aquaporin-1 (AQP1), calcium/calmodulin-dependent protein kinase II (CaMKII), neurogranin, and opalin, underwent similar changing trends, while their total protein levels did not change obviously during scrapie infection. Our data here for the first time illustrate the changing profile of brain NO and NOS during prion infection. Time-dependent alterations of brain NO level and the associated protein S-nitrosylation process may contribute greatly to the neuropathological damage in prion diseases.

In pursuit of a tractable bioassay to assess blood prion infectivity, we have generated prion protein (PrP) transgenic Drosophila, which show a neurotoxic phenotype in adulthood after exposure to exogenous prions at the larval stage. Here, we determined the sensitivity of ovine PrP transgenic Drosophila to ovine prion infectivity by exposure of these flies to a dilution series of scrapie-infected sheep brain homogenate. Ovine PrP transgenic Drosophila showed a significant neurotoxic response to dilutions of 10 -2 to 10 -10 of the original scrapie-infected sheep brain homogenate. Significantly, we determined that this prion-induced neurotoxic response in ovine PrP transgenic Drosophila was transmissible to ovine PrP transgenic mice, which is indicative of authentic mammalian prion detection by these flies. As a consequence, we considered that PrP transgenic Drosophila were sufficiently sensitive to exogenous mammalian prions to be capable of detecting prion infectivity in the blood of scrapie-infected sheep. To test this hypothesis, we exposed ovine PrP transgenic Drosophila to scrapie-infected plasma, a blood fraction notoriously difficult to assess by conventional prion bioassays. Notably, pre-clinical plasma from scrapie-infected sheep induced neurotoxicity in PrP transgenic Drosophila and this effect was more pronounced after exposure to samples collected at the clinical phase of disease. The neurotoxic phenotype in ovine PrP transgenic Drosophila induced by plasma from scrapie-infected sheep was transmissible since head homogenate from these flies caused neurotoxicity in recipient flies during fly-to-fly transmission. Our data show that PrP transgenic Drosophila can be used successfully to bioassay prion infectivity in blood from a prion-diseased mammalian host., (© 2016 The Author(s).)

The environment plays a key role in horizontal transmission of prion diseases, since prions are extremely resistant to classical inactivation procedures. In prior work, we observed the high stability of bovine spongiform encephalopathy (BSE) infectivity when these prions were incubated in aqueous media such as phosphate-buffered saline (PBS) or wastewater for nearly nine months. As a continuation of this experiment, the same samples were maintained in PBS or wastewater for five additional years and residual BSE infectivity was assessed in bovine PrP C transgenic mice. Over this long time period (more than six years), BSE infectivity was reduced by three and one orders of magnitude in wastewater and PBS respectively. To rule out a possible agent specific effect, sheep scrapie prions were subjected to the same experimental protocol, using eight years as the experimental end-point. No significant reduction in scrapie infectivity was observed over the first nine months of wastewater incubation while PBS incubation for eight years only produced a two logarithmic unit reduction in infectivity. By contrast, the dynamics of PrP Res persistence was different, disappearing progressively over the first year. The long persistence of prion infectivity observed in this study for two different agents provides supporting evidence of the assumed high stability of these agents in aquatic environments and that environmental processes or conventional wastewater treatments with low retention times would have little impact on prion infectivity. These results could have great repercussions in terms of risk assessment and safety for animals and human populations., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Prions or PrP(Sc) are proteinaceous infectious agents that consist of misfolded, self-replicating states of the prion protein or PrP(C) PrP(C) is posttranslationally modified with N-linked glycans and a sialylated glycosylphosphatidylinositol (GPI) anchor. Conformational conversion of PrP(C) gives rise to glycosylated and GPI-anchored PrP(Sc) The question of the sialylation status of GPIs within PrP(Sc) has been controversial. Previous studies that examined scrapie brains reported that both sialo- and asialo-GPIs were present in PrP(Sc), with the majority being asialo-GPIs. In contrast, recent work that employed cultured cells claimed that only PrP(C) with sialylo-GPIs could be recruited into PrP(Sc), whereas PrP(C) with asialo-GPIs inhibited conversion. To resolve this controversy, we analyzed the sialylation status of GPIs within PrP(Sc) generated in the brain, spleen, or cultured N2a or C2C12 myotube cells. We found that recruiting PrP(C) with both sialo- and asialo-GPIs is a common feature of PrP(Sc) The mixtures of sialo- and asialo-GPIs were observed in PrP(Sc) universally regardless of prion strain as well as host, tissue, or type of cells that produced PrP(Sc) Remarkably, the proportion of sialo- versus asialo-GPIs was found to be controlled by host, tissue, and cell type but not prion strain. In summary, this study found no strain-specific preferences for selecting PrP(C) with sialo- versus asialo-GPIs. Instead, this work suggests that the sialylation status of GPIs within PrP(Sc) is regulated in a cell-, tissue-, or host-specific manner and is likely to be determined by the specifics of GPI biosynthesis., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

We have previously demonstrated that prion protein-deficient (Prnp(0/0)) Zürich I mice display impaired T zone structure resulting from decreased splenic expression of the T cell homing chemokines, CCL19 and CCL21. Prions are transported to, and colonise in, the secondary lymphoid tissues. Therefore, in order to investigate how scrapie infection affects the splenic white pulp structure, we infected C57BL/6 mice with the mouse-adapted scrapie strain ME7 and analysed end-stage prion disease. We found that the white pulp regions of ME7-infected spleens were smaller, and contained markedly diminished T zones, as compared to control spleens. Although lymphoid tissue inducer cells were not affected, the expression of both CCL19 and CCL21 was decreased. In addition, the networks of follicular dendritic cells, which are known to express high levels of the cellular prion protein (PrP(C)) and to accumulate PrP(Sc) following scrapie infection, were larger in ME7-infected spleens. Further, they were associated with increased numbers of B cells expressing high levels of IgM. These data indicate that ME7-infected spleens display phenotype characteristics different from those reported for Prnp(0/0) spleens mainly due to the gain of PrP(Sc) function and suggest that the PrP(C) is required, not only to form the splenic white pulp structure, but also to maintain the intact T zone structure., (Copyright © 2016. Published by Elsevier GmbH.)

Tauopathies are a family of neurodegenerative diseases in which fibrils of human hyperphosphorylated tau (P-tau) are believed to cause neuropathology. In Alzheimer disease, P-tau associates with A-beta amyloid and contributes to disease pathogenesis. In familial human prion diseases and variant CJD, P-tau often co-associates with prion protein amyloid, and might also accelerate disease progression. To test this latter possibility, here we compared progression of amyloid prion disease in vivo after scrapie infection of mice with and without expression of human tau. The mice used expressed both anchorless prion protein (PrP) and membrane-anchored PrP, that generate disease associated amyloid and non-amyloid PrP (PrPSc) after scrapie infection. Human P-tau induced by scrapie infection was only rarely associated with non-amyloid PrPSc, but abundant human P-tau was detected at extracellular, perivascular and axonal deposits associated with amyloid PrPSc. This pathology was quite similar to that seen in familial prion diseases. However, association of human and mouse P-tau with amyloid PrPSc did not diminish survival time following prion infection in these mice. By analogy, human P-tau may not affect prion disease progression in humans. Alternatively, these results might be due to other factors, including rapidity of disease, blocking effects by mouse tau, or low toxicity of human P-tau in this model.

Rapid and sensitive detection of prions is important in managing prion diseases. The real-time quaking-induced conversion (RT-QuIC) assay for prion seeding activity has been applied to many prion diseases and provides for specific antemortem diagnostic testing. We evaluated RT-QuIC's long-term consistency and varied multiple reaction parameters. Repeated assays of a single scrapie sample using multiple plate readers and recombinant prion protein (rPrP(Sen)) substrates gave comparable results. N-terminal truncated hamster rPrP(Sen) (residues 90-231) hastened both prion-seeded and prion-independent reactions but maintained a clear kinetic distinction between the two. Raising temperatures or shaking speeds accelerated RT-QuIC reactions without compromising specificity. When applied to nasal brushings from Creutzfeldt-Jakob disease patients, higher temperatures accelerated RT-QuIC kinetics, and the use of hamster rPrP(Sen) (90-231) strengthened RT-QuIC responses. Elongation of shaking periods reduced scrapie-seeded reaction times, but continuous shaking promoted false-positive reactions. Furthermore, pH 7.4 provided for more rapid RT-QuIC reactions than more acidic pHs. Additionally, we show that small variations in the amount of sodium dodecyl sulfate (SDS) significantly impacted the assay. Finally, RT-QuIC performed in multiplate thermoshakers followed by fluorescence readings in separate plate readers enhanced assay throughput economically. Collectively, these results demonstrate improved speed, efficacy and practicality of RT-QuIC assays and highlight variables to be optimized for future applications.

The mouse-adapted scrapie prion strain RML is one of the most widely used in prion research. The introduction of a cell culture-based assay of RML prions, the scrapie cell assay (SCA) allows more rapid and precise prion titration. A semi-automated version of this assay (ASCA) was applied to explore a range of conditions that might influence the infectivity and properties of RML prions. These include resistance to freeze-thaw procedures; stability to endogenous proteases in brain homogenate despite prolonged exposure to varying temperatures; distribution of infective material between pellet and supernatant after centrifugation, the effect of reducing agents and the influence of detergent additives on the efficiency of infection. Apparent infectivity is increased significantly by interaction with cationic detergents. Importantly, we have also elucidated the relationship between the duration of exposure of cells to RML prions and the transmission of infection. We established that the infection process following contact of cells with RML prions is rapid and followed an exponential time course, implying a single rate-limiting process.

Background: A clear association of amino acid variation in the prion protein gene (PRNP) with susceptibility and resistance to classical scrapie exists in sheep, but not in goats. In this study we examined DNA sequence variation in the PRNP of 149 animals from two scrapie-infected herds of Saanen dairy goats, and identified 6 non-synonymous variants in the coding region., Results: In the larger herd, all of the 54 scrapie-affected goats tested had at least one allele with the arginine (R) codon at position 211, with 52 being homozygous for that variant. No animal homozygous for the glutamine (Q) codon at 211 were affected and only two heterozygotes (R/Q) were affected. A weak association was found at position 146 and no significant associations were found with amino acid variation at the remaining four variant positions (142, 143, 222 and 240), however, the allelic variation was low. Similar patterns were observed in the second scrapie-affected herd., Conclusion: We also evaluated previous studies on goat herds affected with scrapie and this relationship of R susceptibility and Q resistance at 211 was present independent of the genotypes at the other positions including 222. The fact that glutamine at 211 provides a significant protective property to scrapie irrespective of the other positions could be important for breeding strategies aimed at improving herd resistance to scrapie, while maintaining important productivity traits.

Prion diseases, or transmissible spongiform encephalopathies (TSEs) are progressive, fatal neurodegenerative diseases with no effective treatment. The pathology of these diseases involves the conversion of a protease sensitive form of the cellular prion protein (PrP(C)) into a protease resistant infectious form (PrP(res)). The efficiency of this conversion is predicated upon a number of factors, most notably a strong homology between cellular PrP(C) and PrP(res). In our recently published study, we infected mice with the RML-Chandler strain of scrapie and treated them with heterologous hamster prion proteins. This treatment was seen to reduce clinical signs of prion disease, to delay the onset of clinical symptoms and to prolong survival. In this current article we discuss potential mechanisms of action of treatment with heterologous prion proteins. We also discuss potential extensions of these studies using a heterologous rabbit PrP-based treatment strategy or a peptide based strategy, and improvement of treatment delivery including a lentiviral-based system.

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., (Copyright © 2015 Elsevier B.V. All rights reserved.)