Scrapie Strain Research Articles

RNA as a component of scrapie fibrils

Recently, electron cryo-microscopy (cryo-EM) maps of fibrils from the brains of mice and hamsters with five infectious scrapie strains have been published and deposited in the electron microscopy data bank (EMDB). As noted by the primary authors, the fibrils contain a second component other than protein. The aim of the present study was to identify the nature of this second component in the published maps using an in silico approach. Extra densities (EDs) containing this component were continuous, straight, axial, at right angles to protein rungs and within hydrogen-bonding distance of protein, consistent with a structural role. EDs co-located with strips of basic residues, notably lysines, and formed a conspicuous cladding over parts of the N-terminal lobe of the protein. A Y-shaped polymer consistent with RNA was found, in places forming a single chain and at one location forming a duplex, comprising two antiparallel chains, and raising the intriguing possibility of replicative behaviour. To reflect the monotonous nature of the protein interface, it is suggested that the RNA may be a short tandem repeat. Fibrils from brains of patients with Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis and other neurodegenerations also contain EDs and may be of a similar aetiology.

Temporal serum neurofilament light chain concentrations in sheep inoculated with the agent of classical scrapie.

Neurofilament light chain (Nf-L) has been used to detect neuroaxonal damage in the brain caused by physical injury or disease. The purpose of this study was to determine if serum Nf-L could be used as a biomarker for pre-symptomatic detection of scrapie in sheep. Four sheep with prion protein genotype AVQQ were intranasally inoculated with the classical scrapie strain x124. Blood was collected every 4 weeks until 44 weeks post-inoculation, at which point weekly collection commenced. Serum was analyzed using single molecule array (Quanterix SR-X) to evaluate Nf-L concentrations. Scrapie was confirmed in each sheep by testing homogenized brainstem at the level of the obex with a commercially available enzyme immunoassay. Increased serum Nf-L concentrations were identified above the determined cutoff during the last tenth of the respective incubation period for each sheep. Throughout the time course study, PrPSc accumulation was not detected antemortem by immunohistochemistry in rectal tissue at any timepoint for any sheep. RT-QuIC results were inconsistently positive throughout the timepoints tested for each sheep; however, each sheep had at least one timepoint detected positive. When assessing serum Nf-L utility using receiver operator characteristic curves against different clinical parameters, such as asymptomatic and symptomatic (pruritus or neurologic signs), results showed that Nf-L was most useful at being an indicator of disease only late in disease progression when neurologic signs were present. Serum Nf-L concentrations in the cohort of sheep increased as disease progressed; however, serum Nf-L did not increase during the presymptomatic window. The levels increased substantially throughout the final 10% of the animals' scrapie incubation period when other clinical signs were present. Serum Nf-L is not a reliable biomarker for pre-clinical detection of scrapie.

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

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. 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. 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. 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.

The APOE4 Allele Exacerbates Pathology in a Mouse Model of Prion Disease

AbstractBackgroundThe APOE4 allele is the strongest genetic risk factor in sporadic Alzheimer’s disease (AD). In allele‐dose dependent manner APOE4 increases AD risk, lowers age of onset, and accelerates the rate of dementia progression. Like AD, prionoses are conformational neurodegenerative disorders, which neuropathological hallmarks include accumulation of disease specific misfolded protein(s) and presence of reactive astrocytes and microglia. Accumulation of PrPSc, a toxic conformer of the cellular prion protein‐PrPC, is the initial culprit in prion pathogenesis. PrPSc buildup is associated with the presence of A1 neurotoxic astrocytes and neurodegenerative phenotype microglia (MGnD), which proinflammatory character contributes importantly to neurodegeneration in prionoses. Despite similarities between AD and prionoses, and established effects of APOE4 on misfolded protein accumulation and MGnD activation in AD, effects of the APOE genotype on prion pathology remain uninvestigated.MethodAPOE2, APOE3, and APOE4 targeted replacement mice were inoculated with 22L mouse adapted scrapie strain or normal brain homogenate (NBH) and subjected to repetitive behavioral testing. They were sacrificed at 15 or 23 weeks post inoculation (WPI), when they remained presymptomatic or showed overt neurological symptoms. Neuropathological and biochemical analyses were performed at either timepoint, while NanoStringTM analysis of microglia and astrocyte transcriptomic markers was done at 23WPI.ResultAPOE4 mice feature significantly shorter incubation period and more severe burden of neurological symptoms following prion inoculation than APOE2 and APOE3 mice. Neuropathological analysis reveals significantly higher burden of spongiforms changes in the brain cortex and greater load of reactive astrocytes and microglia in APOE4 mice compared to other APOE genotypes. APOE4 mice also show the highest level of total PrP and PrPSc and insoluble/soluble PrP index. Transcriptomic analysis reveals prion inoculation effects marked elevation of PAN and A1 reactive astrocyte markers, MGnD markers, and in pro‐inflammatory chemokines and cytokine receptors transcripts across all APOE genotypes but the NBH‐relative increase is the highest in APOE4 mice.ConclusionThe APOE4 allele promotes prion neurodegeneration through two separate mechanisms. It is associated with greater PrPSc accumulation and toxicity suggesting its role in the PrPSc metabolism and clearance. It also promotes acquisition of A1 and MGnD neurotoxic phenotypes by astrocytes and microglia, respectively.

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.

Differentiated cultures of an immortalized human neural progenitor cell line do not replicate prions despite PrPC overexpression

ABSTRACT Prions are misfolded proteins that accumulate within the brain in association with a rare group of fatal and infectious neurological disorders in humans and animals. A current challenge to research is a lack of in vitro model systems that are compatible with a wide range of prion strains, reproduce prion toxicity, and are amenable to genetic manipulations. In an attempt to address this need, here we produced stable cell lines that overexpress different versions of PrPC through lentiviral transduction of immortalized human neural progenitor cells (ReN VM). Differentiated cultures made from the neural progenitor cell lines overexpressed PrPC within 3D spheroid-like structures of TUBB3+ neurons and we observed evidence that PrPC modulates formation of these structures, consistent with PrPC’s role in neurogenesis. However, through repeated measurements of amyloid seeding activity in 6-week time course experiments, we failed to observe any evidence of prion replication within the differentiated ReN cultures following challenge with four prion isolates (human sCJD subtypes MM1 and VV2, and rodent adapted scrapie strains RML and 263K). We attributed amyloid seeding activity detected within the cultures to residual inoculum and concluded that PrPC overexpression was insufficient to confer permissiveness of ReN cultures to prion infection. While our ReN cell prion infection model was unsuccessful, additional efforts to develop cellular models of human prion disease are highly warranted.

PRNP Sequences of Tibetan Antelope, Blue Sheep, and Plateau Pika from the Qinghai-Tibet Plateau and Reactivity of PrP Proteins to Rodent-Adapted Scrapie Strains in RT-QuIC and PMCA

Background: Tibetan antelope (Rhinopithecus), blue sheep (Pseudois nayauris), and plateau pika (Ochotona curzoniae) are wild animals living on the Qinghai-Tibet Plateau. There have been no reports of naturally-occurring transmissible spongioform encephalopathies (TSEs) involving these animals. Furthermore, the PRNP genes have not been described in the literature. Methods: The PRNP genes from 21 Tibetan antelopes, 4 blue sheep, and 3 plateau pikas were obtained and sequenced. The recombinant proteins were then prepared. Using scrapie strains (263K, 139A, ME7, and S15) as the seeds, the reactivity of the PrP proteins from sheep (rSheepPrP25-234) and pika (rPikaPrP23-230) were tested using real-time quaking-induced conversion (RT-QuIC). Protein misfolding cyclic amplification (PMCA) tests of the brain homogenates from domestic sheep and rabbits were performed with the seeds of strains 263K and ME7. Results: The PRNP genes of bovids were 771 bp long and encoded 256 amino acids (aa), showing 100% homology with the wild-type sheep prion protein (PrP) aa sequence. The PRNP gene of pika was 759 bp long and encoded 252 amino acids, showing 92.1% homology with the aa sequence of domestic rabbits. The sheep and pika proteins revealed positive reactions in 10-5 diluted seeds. Only rPikaPrP23-230 produced positive curves in 10-7 diluted seeds. The PMCA tests failed to produce proteinase K (PK)-resistant PrP (PrPres). Conclusion: This is the first description of PRNP genes and PrP aa sequences of Tibetan antelope, blue sheep, and plateau pike from the Qinghai-Tibet Plateau. In the presence of rodent prions, the PrPs of sheep and pika efficiently induce fibrillation in RT-QuIC, but do not generate PrPres in PMCA. Our results indicate that pika, as one of the important links in the Qinghai-Tibet Plateau biological chain, may play an important role in the prion circulation. Pika PrP deserves further analysis for its potential application value in assays for human prion disease.

Absence of Apolipoprotein E Exacerbates Prion Pathology and Promote Microglial Mediated Neurodegeneration

AbstractBackgroundBoth Alzheimer’s disease and prionoses are neurogenerative disorders, which involve misfolded protein deposition and transition of homeostatic microglia to their neurodegenerative phenotype. In prionoses, accumulation of toxic PrPSc protein constitutes the culprit of pathogenesis. Neurodegenerative phenotype microglia (MGnD) appear early in the course of disease and play opposing roles in PrPSc mediated neurodegeneration. Whilst clearance of PrPSc has a disease‐limiting effect, MGnD‐driven neuroinflammation is deleterious to neurons. Apolipoprotein (apo) E is a pleiotropic protein, which controls MGnD phagocytic and inflammatory characteristics. Despite prominent role of microglia in prionoses, involvement of apoE in their pathogenesis has not been established.MethodWild type (WT) and Apoe‐/‐ mice, both on the B6 background, were inoculated with 22L mouse adapted scrapie strain or normal brain homogenate. Neuropathological and biochemical analyses were performed 15 and 23 weeks post inoculation (WPI) on asymptomatic and overly symptomatic animals, respectively. NanoStringTM analysis of microglia and astrocytic transcriptomic markers was performed at 23WPI.ResultPrion infection of WT mice upregulates total brain apoE level and effects cell‐type shift in the apoE expression, downregulating astrocytic apoE while increasing microglial apoE 24‐folds. 22L Apoe‐/‐ mice have significantly shorter disease incubation period, higher load of spongiform changes, and increased total brain PrP and PrPSc levels compared to 22LWT mice. In 22L Apoe ‐/‐ mice astro and micro gliosis appears earlier and is more robust compared to 22LWT mice. Transcriptomic analysis shows significantly higher level of PAN and A1, but not A2 astrocytic markers and MGnD markers in 22L Apoe ‐/‐ mice compared to 22LWT animals. Microglia in 22L Apoe ‐/‐ mice feature impaired neuronal phagocytosis and produce increased level of pro‐inflammatory interleukins and cytokines. 22L Apoe ‐/‐ mice show higher neuronal loss and greater tempo of neuronal degeneration than 22LWT mice.ConclusionOur results indicate the net effect of apoE in prion pathogenesis is protective. ApoE facilitates clearance of PrPSc and dying neurons by microglia, which becomes ineffective in the absence of apoE. Remnants of disintegrating neurons promote MGnD phenotype transition and inflammatory cascade propagating the vicious cycle of neuronal death and neuroinflammation. Our data also indicate homeostatic microglia transit to the MGnD even if they do not express apoE.

Effect of inoculation with prion dilutions within the dynamic range of ELISA absorbance on prion incubation period

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.

Combinatorial treatment of brain samples from sheep with scrapie using sodium percarbonate, sodium dodecyl sulfate, and proteinase K increases survival time in inoculated susceptible sheep

The agent of scrapie is resistant to most chemical and physical methods of inactivation. Prions bind to soils, metals, and various materials and persist in the environment confounding the control of prion diseases. Most methods of prion inactivation require severe conditions such as prolong exposure to sodium hypochlorite or autoclaving, which may not be suitable for field conditions. We evaluated the efficacy of a combinatorial approach to inactivation of US scrapie strain x124 under the mild conditions of treating scrapie-affected brain homogenate with sodium percarbonate (SPC), sodium dodecyl sulfate (SDS), or in combination followed by proteinase K (PK) digestion at room temperature. Western blot analysis of treated brain homogenate demonstrates partial reduction in PrPSc immunoreactivity. Genetically susceptible VRQ/ARQ Suffolk sheep were oronasally inoculated with 1 g of SPC (n = 1), SDS (n = 2), SDS + PK (n = 2), and SPC + SDS + PK (n = 4) treated brain homogenate. Sheep were assessed daily for clinical signs, euthanized at the development of clinical disease, and tissues were assessed for accumulation of PrPSc. Scrapie status in all sheep was determined by western blot, enzyme immunoassay, and immunohistochemistry. Mean incubation periods (IPs) for SPC (11.9 months, 0% survival), SDS (12.6 months, 0% survival), SDS + PK (14.0 months, 0% survival), and SPC + SDS + PK (12.5 months, 25% survival) were increased compared to positive control sheep (n = 2, 10.7 months, 0% survival) by 1.2, 1.9, 3.3, and 1.8 months, respectively. Treatment did not influence PrPSc accumulation and distribution at the clinical stage of disease. Differences in mean IPs and survival indicates partial but not complete reduction in scrapie infectivity.

Cryo-EM structure of anchorless RML prion reveals variations in shared motifs between distinct strains

Little is known about the structural basis of prion strains. Here we provide a high (3.0 Å) resolution cryo-electron microscopy-based structure of infectious brain-derived fibrils of the mouse anchorless RML scrapie strain which, like the recently determined hamster 263K strain, has a parallel in-register β-sheet-based core. Several structural motifs are shared between these ex vivo prion strains, including an amino-proximal steric zipper and three β-arches. However, detailed comparisons reveal variations in these shared structural topologies and other features. Unlike 263K and wildtype RML prions, the anchorless RML prions lack glycophosphatidylinositol anchors and are severely deficient in N-linked glycans. Nonetheless, the similarity of our anchorless RML structure to one reported for wildtype RML prion fibrils in an accompanying paper indicates that these post-translational modifications do not substantially alter the amyloid core conformation. This work demonstrates both common and divergent structural features of prion strains at the near-atomic level.

Primary glia cells from bank vole propagate multiple rodent-adapted scrapie prions

Since the beginning prion research has been largely dependent on animal models for deciphering the disease, drug development or prion detection and quantification. Thereby, ethical as well as cost and labour-saving aspects call for alternatives in vitro. Cell models can replace or at least complement animal studies, but their number is still limited and the application usually restricted to certain strains and host species due to often strong transmission barriers. Bank voles promise to be an exception as they or materials prepared from them are uniquely susceptible to prions from various species in vivo, in vitro and in cell-free applications. Here we present a mainly astrocyte-based primary glia cell assay from bank vole, which is infectible with scrapie strains from bank vole, mouse and hamster. Stable propagation of bank vole-adapted RML, murine 22L and RML, and hamster 263K scrapie is detectable from 20 or 30 days post exposure onwards. Thereby, the infected bank vole glia cells show similar or even faster prion propagation than likewise infected glia cells of the corresponding murine or hamster hosts. We propose that our bank vole glia cell assay could be a versatile tool for studying and comparing multiple prion strains with different species backgrounds combined in one cell assay.

Detecting Differences in Prion Protein Conformation by Quantifying Methionine Oxidation.

A prion’s pathogenic character is enciphered in its conformation, which also defines the chemical environments of its amino acids. Differences in chemical environments influence the reactivity of amino acid side chains, in a conformation-dependent manner. Chemical oxidation of susceptible methionines would identify those methionines on the surface of a prion, which would reveal conformation-dependent information. We identified a set of methionine-containing peptides derived from the tryptic, chymotryptic, or tryptic/chymotryptic digestion of recombinant prion protein and the Sc237 strain of hamster-adapted scrapie. We developed a multiple reaction monitoring-based method of quantifying the extent of the methionine oxidation in those peptides. This approach can be used to define a prion’s conformation and to distinguish among prion strains, which is an important component of food safety.

Absence of Apolipoprotein E is associated with exacerbation of prion pathology and promotes microglial neurodegenerative phenotype

Prion diseases or prionoses are a group of rapidly progressing and invariably fatal neurodegenerative diseases. The pathogenesis of prionoses is associated with self-replication and connectomal spread of PrPSc, a disease specific conformer of the prion protein. Microglia undergo activation early in the course of prion pathogenesis and exert opposing roles in PrPSc mediated neurodegeneration. While clearance of PrPSc and apoptotic neurons have disease-limiting effect, microglia-driven neuroinflammation bears deleterious consequences to neuronal networks. Apolipoprotein (apo) E is a lipid transporting protein with pleiotropic functions, which include controlling of the phagocytic and inflammatory characteristics of activated microglia in neurodegenerative diseases. Despite the significance of microglia in prion pathogenesis, the role of apoE in prionoses has not been established. We showed here that infection of wild type mice with 22L mouse adapted scrapie strain is associated with significant increase in the total brain apoE protein and mRNA levels and also with a conspicuous cell-type shift in the apoE expression. There is reduced expression of apoE in activated astrocytes and marked upregulation of apoE expression by activated microglia. We also showed apoE ablation exaggerates PrPSc mediated neurodegeneration. Apoe−/− mice have shorter disease incubation period, increased load of spongiform lesion, pronounced neuronal loss, and exaggerated astro and microgliosis. Astrocytes of Apoe−/− mice display salient upregulation of transcriptomic markers defining A1 neurotoxic astrocytes while microglia show upregulation of transcriptomic markers characteristic for microglial neurodegenerative phenotype. There is impaired clearance of PrPSc and dying neurons by microglia in Apoe−/− mice along with increased level of proinflammatory cytokines. Our work indicates that apoE absence renders clearance of PrPSc and dying neurons by microglia inefficient, while the excess of neuronal debris promotes microglial neurodegenerative phenotype aggravating the vicious cycle of neuronal death and neuroinflammation.

Mice Treated Subcutaneously with Mouse LPS-Converted PrPres or LPS Alone Showed Brain Gene Expression Profiles Characteristic of Prion Disease

Previously, we showed that bacterial lipopolysaccharide (LPS) converts mouse PrPC protein to a beta-rich isoform (moPrPres) resistant to proteinase K. In this study, we aimed to test if the LPS-converted PrPres is infectious and alters the expression of genes related to prion pathology in brains of terminally sick mice. Ninety female FVB/N mice at 5 weeks of age were randomly assigned to 6 groups treated subcutaneously (sc) for 6 weeks either with: (1) Saline (CTR); (2) LPS from Escherichia coli 0111:B4 (LPS), (3) one-time sc administration of de novo generated mouse recombinant prion protein (moPrP; 29-232) rich in beta-sheet by incubation with LPS (moPrPres), (4) LPS plus one-time sc injection of moPrPres, (5) one-time sc injection of brain homogenate from Rocky Mountain Lab (RLM) scrapie strain, and (6) LPS plus one-time sc injection of RML. Results showed that all treatments altered the expression of various genes related to prion disease and neuroinflammation starting at 11 weeks post-infection and more profoundly at the terminal stage. In conclusion, sc administration of de novo generated moPrPres, LPS, and a combination of moPrPres with LPS were able to alter the expression of multiple genes typical of prion pathology and inflammation.

Deletion of Kif5c Does Not Alter Prion Disease Tempo or Spread in Mouse Brain

In prion diseases, the spread of infectious prions (PrPSc) is thought to occur within nerves and across synapses of the central nervous system (CNS). However, the mechanisms by which PrPSc moves within axons and across nerve synapses remain undetermined. Molecular motors, including kinesins and dyneins, transport many types of intracellular cargo. Kinesin-1C (KIF5C) has been shown to transport vesicles carrying the normal prion protein (PrPC) within axons, but whether KIF5C is involved in PrPSc axonal transport is unknown. The current study tested whether stereotactic inoculation in the striatum of KIF5C knock-out mice (Kif5c−/−) with 0.5 µL volumes of mouse-adapted scrapie strains 22 L or ME7 would result in an altered rate of prion spreading and/or disease timing. Groups of mice injected with each strain were euthanized at either pre-clinical time points or following the development of prion disease. Immunohistochemistry for PrP was performed on brain sections and PrPSc distribution and tempo of spread were compared between mouse strains. In these experiments, no differences in PrPSc spread, distribution or survival times were observed between C57BL/6 and Kif5c−/− mice.

Second passage of chronic wasting disease of mule deer to sheep by intracranial inoculation compared to classical scrapie.

The origin of chronic wasting disease (CWD) in cervids is unclear. One hypothesis suggests that CWD originated from scrapie in sheep. We compared the disease phenotype of sheep-adapted CWD to classical scrapie in sheep. We inoculated sheep intracranially with brain homogenate from first-passage mule deer CWD in sheep (sCWDmd). The attack rate in second-passage sheep was 100% (12 of 12). Sheep had prominent lymphoid accumulations of PrPSc reminiscent of classical scrapie. The pattern and distribution of PrPSc in the brains of sheep with CWDmd was similar to scrapie strain 13-7 but different from scrapie strain x124. The western blot glycoprofiles of sCWDmd were indistinguishable from scrapie strain 13-7; however, independent of sheep genotype, glycoprofiles of sCWDmd were different than x124. When sheep genotypes were evaluated individually, there was considerable overlap in the glycoprofiles that precluded significant discrimination between sheep CWD and scrapie strains. Our data suggest that the phenotype of CWD in sheep is indistinguishable from some strains of scrapie in sheep. Given our results, current detection techniques would be unlikely to distinguish CWD in sheep from scrapie in sheep if cross-species transmission occurred naturally. It is unknown if sheep are naturally vulnerable to CWD; however, the susceptibility of sheep after intracranial inoculation and lymphoid accumulation indicates that the species barrier is not absolute.

Classical scrapie in small ruminants is caused by at least four different prion strains

The diversity of goat scrapie strains in Europe has recently been studied using bioassays in a wide collection of rodent models, resulting in the classification of classical scrapie into four different categories. However, the sole use of the first passage does not lead to isolate adaptation and identification of the strains involved and might therefore lead to misclassification of some scrapie isolates. Therefore, this work reports the complete transmission study of a wide collection of goat transmissible spongiform encephalopathy (TSE) isolates by intracranial inoculation in two transgenic mouse lines overexpressing either small ruminant (TgGoat-ARQ) or bovine (TgBov) PrPC. To compare scrapie strains in sheep and goats, sheep scrapie isolates from different European countries were also included in the study. Once the species barrier phenomenon was overcome, an accurate classification of the isolates was attained. Thus, the use of just two rodent models allowed us to fully differentiate at least four different classical scrapie strains in small ruminants and to identify isolates containing mixtures of strains. This work reinforces the idea that classical scrapie in small ruminants is a prion disease caused by multiple different prion strains and not by a single strain, as is the case for epidemic classical bovine spongiform encephalopathy (BSE-C). In addition, the clear dissimilarity between the different scrapie strains and BSE-C does not support the idea that classical scrapie is the origin of epidemic BSE-C.

Involvement of Cellular Prion Protein in Invasion and Metastasis of Lung Cancer by Inducing Treg Cell Development.

The cellular prion protein (PrPC) is a cell surface glycoprotein expressed in many cell types that plays an important role in normal cellular processes. However, an increase in PrPC expression has been associated with a variety of human cancers, where it may be involved in resistance to the proliferation and metastasis of cancer cells. PrP-deficient (Prnp0/0) and PrP-overexpressing (Tga20) mice were studied to evaluate the role of PrPC in the invasion and metastasis of cancer. Tga20 mice, with increased PrPC, died more quickly from lung cancer than did the Prnp0/0 mice, and this effect was associated with increased transforming growth factor-beta (TGF-β) and programmed death ligand-1 (PD-L1), which are important for the development and function of regulatory T (Treg) cells. The number of FoxP3+CD25+ Treg cells was increased in Tga20 mice compared to Prnp0/0 mice, but there was no significant difference in either natural killer or cytotoxic T cell numbers. In addition, mice infected with the ME7 scrapie strain had decreased numbers of Treg cells and decreased expression of TGF-β and PD-L1. These results suggest that PrPC plays an important role in invasion and metastasis of cancer cells by inducing Treg cells through upregulation of TGF-β and PD-L1 expression.