Change in the molecular properties of CH1641 prions after transmission to wild-type mice: Evidence for a single strain.

AIM: CH1641 was discovered in 1970 as a scrapie isolate that was unlike all other classical strains of scrapie isolated so far. We performed bio-assays of CH1641 in mice in order to further characterise this specific isolate. METHODS: We inoculated the original CH1641 isolate into ovine and bovine prion protein (PrP) transgenic mice as well as wild-type mice. In addition, we performed cross- and back passages between the various mouse lines to examine if one identical prion strain was isolated in all mouse lines or whether multiple prion strains exist in CH1641. RESULTS: We report the first successful transmission of CH1641 to wild-type RIII mice and via RIII mice to wild-type VM mice. Unexpectedly, analysis of the protease-resistant prion protein (PrPres ) in wild-type mice showed a classical scrapie banding pattern differing from the banding pattern of the original CH1641 isolate. Cross- and back passages of CH1641 between the various mouse lines confirmed that the same prion strain had been isolated in all mouse lines. CONCLUSIONS: The CH1641 isolate consists of a single prion strain but its molecular banding pattern of PrPres differs between wild-type mice and PrP transgenic mice. Consequently, molecular banding patterns of PrPres should be used with caution in strain typing since they do not solely depend on the properties of the prion strain but also on the host prion protein.

Disease phenotype of classical sheep scrapie is changed upon experimental passage through white-tailed deer.

Prion agents occur in strains that are encoded by the structure of the misfolded prion protein (PrPSc). Prion strains can influence disease phenotype and the potential for interspecies transmission. Little is known about the potential transmission of prions between sheep and deer. Previously, the classical US scrapie isolate (No.13-7) had a 100% attack rate in white-tailed deer after oronasal challenge. The purpose of this study was to test the susceptibility of sheep to challenge with the scrapie agent after passage through white-tailed deer (WTD scrapie). Lambs of various prion protein genotypes were oronasally challenged with WTD scrapie. Sheep were euthanized and necropsied upon development of clinical signs or at the end of the experiment (72 months post-inoculation). Enzyme immunoassay, western blot, and immunohistochemistry demonstrated PrPSc in 4 of 10 sheep with the fastest incubation occurring in VRQ/VRQ sheep, which contrasts the original No.13-7 inoculum with a faster incubation in ARQ/ARQ sheep. Shorter incubation periods in VRQ/VRQ sheep than ARQ/ARQ sheep after passage through deer was suggestive of a phenotype change, so comparisons were made in ovinized mice and with sheep with known strains of classical sheep scrapie: No. 13-7 and x-124 (that has a more rapid incubation in VRQ/VRQ sheep). After mouse bioassay, the WTD scrapie and x-124 isolates have similar incubation periods and PrPSc conformational stability that are markedly different than the original No. 13-7 inoculum. Furthermore, brain tissues of sheep with WTD scrapie and x-124 scrapie have similar patterns of immunoreactivity that are distinct from sheep with No. 13-7 scrapie. Multiple lines of evidence suggest a phenotype switch when No. 13-7 scrapie prions are passaged through deer. This represents one example of interspecies transmission of prions resulting in the emergence or selection of new strain properties that could confound disease eradication and control efforts.

In Vitro and In Vivo Evidence towards Fibronectin's Protective Effects against Prion Infection.

A distinctive signature of the prion diseases is the accumulation of the pathogenic isoform of the prion protein, PrPSc, in the central nervous system of prion-affected humans and animals. PrPSc 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 PrPSc. 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 PrPSc is tightly associated with proteins found in the systemic extracellular matrix, mostly fibronectin (FN). The interaction of PrPSc 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, PrPSc 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.

Experimental transmission of ovine atypical scrapie to cattle.

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.

Radotinib Decreases Prion Propagation and Prolongs Survival Times in Models of Prion Disease.

The conversion of cellular prion protein (PrPC) into pathogenic prion isoforms (PrPSc) 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, Tga20 and hamster cerebellar slice culture models, and 263K scrapie-infected hamster models. Radotinib inhibited PrPSc 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 PrPSc 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.

Conventional and State-of-the-Art Detection Methods of Bovine Spongiform Encephalopathy (BSE).

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 (PrPSc), which derives from a normal cellular protein (PrPC), 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 PrPSc 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.

A Mass Spectrometry-Based Method of Quantifying the Contribution of the Lysine Polymorphism at Position 171 in Sheep PrP.

In sheep, the transmissibility and progression of scrapie, a sheep prion (PrPSc) disease, is strongly dependent upon specific amino acid polymorphisms in the natively expressed prion protein (PrPC). Sheep expressing PrPC 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 PrPSc. A tryptic peptide, 159R.YPNQVYYRPVDK.Y172, 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 15N-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.

5-Methylcytosine and 5-Hydroxymethylcytosine in Scrapie-Infected Sheep and Mouse Brain Tissues.

Scrapie is a neurodegenerative disorder belonging to the group of transmissible spongiform encephalopathies or prion diseases, which are caused by an infectious isoform of the innocuous cellular prion protein (PrPC) known as PrPSc. DNA methylation, one of the most studied epigenetic mechanisms, is essential for the proper functioning of the central nervous system. Recent findings point to possible involvement of DNA methylation in the pathogenesis of prion diseases, but there is still a lack of knowledge about the behavior of this epigenetic mechanism in such neurodegenerative disorders. Here, we evaluated by immunohistochemistry the 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) levels in sheep and mouse brain tissues infected with scrapie. Expression analysis of different gene coding for epigenetic regulatory enzymes (DNMT1, DNMT3A, DNMT3B, HDAC1, HDAC2, TET1, and TET2) was also carried out. A decrease in 5mC levels was observed in scrapie-affected sheep and mice compared to healthy animals, whereas 5hmC displayed opposite patterns between the two models, demonstrating a decrease in 5hmC in scrapie-infected sheep and an increase in preclinical mice. 5mC correlated with prion-related lesions in mice and sheep, but 5hmC was associated with prion lesions only in sheep. Differences in the expression changes of epigenetic regulatory genes were found between both disease models, being differentially expressed Dnmt3b, Hdac1, and Tet1 in mice and HDAC2 in sheep. Our results support the evidence that DNA methylation in both forms, 5mC and 5hmC, and its associated epigenetic enzymes, take part in the neurodegenerative course of prion diseases.

Kinetics of the reduction of Creutzfeldt-Jakob disease prion seeding activity by steam sterilization support the use of validated 134°C programmes.

BACKGROUND: Prions are renowned for their distinct resistance to chemical or physical inactivation, including steam sterilization. Impaired efficacy of inactivation poses a risk to patients for iatrogenic transmission of Creutzfeldt-Jakob disease (CJD) via contaminated surgical instruments. AIMS: Most established prion inactivation methods were validated against scrapie agents, although those were found to be generally less thermostable than human prions. Thus, knowledge gaps regarding steam-sterilization kinetics of CJD prions should be filled and current guidelines reviewed accordingly. METHODS: Prion inactivation through widely recommended steam sterilization at 134°C was assessed for several holding times by analysing the residual prion seeding activity using protein misfolding cyclic amplification (PMCA). FINDINGS: Scrapie 263K was found to be the least thermoresistant prion strain showing no seeding activity after 1.5 min at 134°C, while variant CJD was the most stable one demonstrating some seeding activity even after 18 min of steam sterilization. Sporadic CJD subtype VV2 exhibited residual seeding activity after 3 min, but no detectable activity after 5 min at 134°C. CONCLUSION: Validated steam sterilization for 5 min at 134°C as previously recommended for the routine reprocessing of surgical instruments in contact with high-risk tissues is able to substantially reduce the seeding activity of CJD agents, provided that no fixating chemical disinfection has been performed prior to sterilization and that thorough cleaning has reduced the protein load on the surface to less than 100 µg per instrument.

Cellular toxicity of scrapie prions in prion diseases; a biochemical and molecular overview.

Transmissible spongiform encephalopathies (TSEs) or prion diseases consist of a broad range of fatal neurological disorders affecting humans and animals. Contrary to Watson and Crick's 'central dogma', prion diseases are caused by a protein, devoid of DNA involvement. Herein, we briefly review various cellular and biological aspects of prions and prion pathogenesis focusing mainly on historical milestones, biosynthesis, degradation, structure-function of cellular and scrapie forms of prions .

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

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

Bona fide atypical scrapie faithfully reproduced for the first time in a rodent model.

Atypical Scrapie, which is not linked to epidemics, is assumed to be an idiopathic spontaneous prion disease in small ruminants. Therefore, its occurrence is unlikely to be controlled through selective breeding or other strategies as it is done for classical scrapie outbreaks. Its spontaneous nature and its sporadic incidence worldwide is reminiscent of the incidence of idiopathic spontaneous prion diseases in humans, which account for more than 85% of the cases in humans. Hence, developing animal models that consistently reproduce this phenomenon of spontaneous PrP misfolding, is of importance to study the pathobiology of idiopathic spontaneous prion disorders. Transgenic mice overexpressing sheep PrPC with I112 polymorphism (TgShI112, 1-2 × PrP levels compared to sheep brain) manifest clinical signs of a spongiform encephalopathy spontaneously as early as 380 days of age. The brains of these animals show the neuropathological hallmarks of prion disease and biochemical analyses of the misfolded prion protein show a ladder-like PrPres pattern with a predominant 7-10 kDa band. Brain homogenates from spontaneously diseased transgenic mice were inoculated in several models to assess their transmissibility and characterize the prion strain generated: TgShI112 (ovine I112 ARQ PrPC), Tg338 (ovine VRQ PrPC), Tg501 (ovine ARQ PrPC), Tg340 (human M129 PrPC), Tg361 (human V129 PrPC), TgVole (bank vole I109 PrPC), bank vole (I109I PrPC), and sheep (AHQ/ARR and AHQ/AHQ churra-tensina breeds). Our analysis of the results of these bioassays concludes that the strain generated in this model is indistinguishable to that causing atypical scrapie (Nor98). Thus, we present the first faithful model for a bona fide, transmissible, ovine, atypical scrapie prion disease.

Global Profiles of Acetylated Proteins in Brains of Scrapie Agents 139A- and ME7-Infected Mice Collected at Mid-Early, Mid-Late, and Terminal Stages.

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.

Impairment of Neuronal Mitochondrial Quality Control in Prion-Induced Neurodegeneration.

Mitochondrial dynamics continually maintain cell survival and bioenergetics through mitochondrial quality control processes (fission, fusion, and mitophagy). Aberrant mitochondrial quality control has been implicated in the pathogenic mechanism of various human diseases, including cancer, cardiac dysfunction, and neurological disorders, such as Alzheimer's disease, Parkinson's disease, and prion disease. However, the mitochondrial dysfunction-mediated neuropathological mechanisms in prion disease are still uncertain. Here, we used both in vitro and in vivo scrapie-infected models to investigate the involvement of mitochondrial quality control in prion pathogenesis. We found that scrapie infection led to the induction of mitochondrial reactive oxygen species (mtROS) and the loss of mitochondrial membrane potential (ΔΨm), resulting in enhanced phosphorylation of dynamin-related protein 1 (Drp1) at Ser616 and its subsequent translocation to the mitochondria, which was followed by excessive mitophagy. We also confirmed decreased expression levels of mitochondrial oxidative phosphorylation (OXPHOS) complexes and reduced ATP production by scrapie infection. In addition, scrapie-infection-induced aberrant mitochondrial fission and mitophagy led to increased apoptotic signaling, as evidenced by caspase 3 activation and poly (ADP-ribose) polymerase cleavage. These results suggest that scrapie infection induced mitochondrial dysfunction via impaired mitochondrial quality control processes followed by neuronal cell death, which may have an important role in the neuropathogenesis of prion diseases.

Enhanced M-CSF/CSF1R Signaling Closely Associates with PrPSc Accumulation in the Scrapie-Infected Cell Line and the Brains of Scrapie-Infected Experimental Rodents.

Activation and proliferation of microglia are one of the hallmarks of prion disease and is usually accompanied by increased levels of various cytokines and chemokines. Our previous study demonstrated that the level of brain macrophage colony-stimulating factor (M-CSF) was abnormally elevated during prion infection, but its association with PrPSc is not completely clear. In this study, colocalization of the increased M-CSF with accumulated PrPSc was observed by IHC with serial brain sections. Reliable molecular interaction between total PrP and M-CSF was observed in the brain of 263 K-infected hamsters and in cultured prion-infected cell line. Immunofluorescent assays showed that morphological colocalization of M-CSF with neurons and microglia, but not with astrocytes in brains of scrapie-infected animals. The transcriptional and expressing levels of CSF1R were also significantly increased in prion-infected cell line and mice, and colocalization of CSF1R with neurons and microglia was observed in the brains of prion-infected mouse models. Removal of PrPSc replication by resveratrol in SMB-S15 cells induced limited reductions of cellular levels of M-CSF and CSF1R. In addition, we found that the level of IL-34, another ligand of CSF1R, did not change significantly after prion infection, but its distribution on the cell types in the brains shifted from neurons in healthy mice to the proliferated astrocytes and microglia in scrapie-infected mice. Our data demonstrate activation of M-CSF/IL-34/CSF1R signaling in the microenvironment of prion infection, strongly indicating its vital role in the pathophysiology of prions. It provides solid scientific evidence for the therapeutic potential of inhibiting M-CSF/CSF1R signaling in prion diseases.

Structural biology of ex vivo mammalian prions.

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 ß-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.

Strain Typing of Classical Scrapie and Bovine Spongiform Encephalopathy (BSE) by Using Ovine PrP (ARQ/ARQ) Overexpressing Transgenic Mice.

Transmissible spongiform encephalopathies (TSE), caused by abnormal prion protein (PrPSc), affect many species. The most classical scrapie isolates harbor mixtures of strains in different proportions. While the characterization of isolates has evolved from using wild-type mice to transgenic mice, no standardization is established yet. Here, we investigated the incubation period, lesion profile and PrPSc profile induced by well-defined sheep scrapie isolates, bovine spongiform encephalopathy (BSE) and ovine BSE after intracerebral inoculation into two lines of ovine PrP (both ARQ/ARQ) overexpressing transgenic mice (Tgshp IX and Tgshp XI). All isolates were transmitted to both mouse models with an attack rate of almost 100%, but genotype-dependent differences became obvious between the ARQ and VRQ isolates. Surprisingly, BSE induced a much longer incubation period in Tgshp XI compared to Tgshp IX. In contrast to the histopathological lesion profiles, the immunohistochemical PrPSc profiles revealed discriminating patterns in certain brain regions in both models with clear differentiation of both BSE isolates from scrapie. These data provide the basis for the use of Tgshp IX and XI mice in the characterization of TSE isolates. Furthermore, the results enable a deeper appreciation of TSE strain diversity using ovine PrP overexpressing transgenic mice as a biological prion strain typing approach.

Distinctive Toll-like Receptors Gene Expression and Glial Response in Different Brain Regions of Natural Scrapie.

Prion diseases are chronic and fatal neurodegenerative diseases characterized by the accumulation of disease-specific prion protein (PrPSc), 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, PrPSc 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.

The Effect of Curcuma phaeocaulis Valeton (Zingiberaceae) Extract on Prion Propagation in Cell-Based and Animal Models.

Prion diseases are neurodegenerative disorders in humans and animals for which no therapies are currently available. Here, we report that Curcuma phaeocaulis Valeton (Zingiberaceae) (CpV) extract was partly effective in decreasing prion aggregation and propagation in both in vitro and in vivo models. CpV extract inhibited self-aggregation of recombinant prion protein (PrP) in a test tube assay and decreased the accumulation of scrapie PrP (PrPSc) in ScN2a cells, a cultured neuroblastoma cell line with chronic prion infection, in a concentration-dependent manner. CpV extract also modified the course of the disease in mice inoculated with mouse-adapted scrapie prions, completely preventing the onset of prion disease in three of eight mice. Biochemical and neuropathological analyses revealed a statistically significant reduction in PrPSc accumulation, spongiosis, astrogliosis, and microglia activation in the brains of mice that avoided disease onset. Furthermore, PrPSc accumulation in the spleen of mice was also reduced. CpV extract precluded prion infection in cultured cells as demonstrated by the modified standard scrapie cell assay. This study suggests that CpV extract could contribute to investigating the modulation of prion propagation.

Circulation of Nor98 Atypical Scrapie in Portuguese Sheep Confirmed by Transmission of Isolates into Transgenic Ovine ARQ-PrP Mice.

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 AL141RQ/AF141RQ and AL141RR/AL141RR 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 PrPSc 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.