Prion diseases are fatal neurodegenerative disorders in which early molecular responses to prion exposure remain incompletely understood, particularly in naturally susceptible hosts. In the present study, we performed RNA-sequencing analysis of ovine mesenchymal stem cell-derived neuron-like cells exposed to classical scrapie brain homogenate and compared their transcriptomic response with that of cells exposed to normal brain homogenate. Cells were analyzed at 2 and 4 days post-inoculation (dpi). Differential expression analysis identified 76 genes at 2 dpi and 124 genes at 4 dpi, with limited overlap between time points, indicating a dynamic response to scrapie exposure. At 2 dpi, enriched biological processes among upregulated genes were mainly related to signaling- and immune-associated transcriptional responses, whereas downregulated genes were enriched in developmental, morphogenetic and lipid-related processes. At 4 dpi, the transcriptional profile was dominated by broader gene repression and showed reduced functional specificity. RT-qPCR analysis of eight selected genes supported the direction of change detected by RNA-seq in most cases. Overall, these results indicate that scrapie exposure elicits a time-dependent transcriptional response in ovine MSC-derived neuron-like cells and provide an exploratory framework for studying early molecular responses associated with prion exposure in a naturally susceptible species.

The Kazakh coarse-wool fat-tailed sheep is an indigenous breed of Central Asia and represents a valuable genetic resource adapted to harsh continental environmental conditions. Despite its economic importance, its genomic architecture, population structure, and the genetic basis of disease resistance remain largely unexplored. Whole-genome sequencing was performed on 20 Kazakh coarse-wool fat-tailed sheep using the Illumina NovaSeq 6000 platform. Population structure was assessed by principal component analysis (PCA) using comparative genomic data from six domestic sheep breeds and the wild ancestor Ovis orientalis. Selection signatures were identified by integrating FST, nucleotide diversity (π), and Tajima's D statistics. Variants within the PRNP and TMEM154 loci were extracted from the filtered VCF files using bcftools. PCA revealed a distinct genetic cluster of the Kazakh coarse-wool fat-tailed sheep, showing close genetic affinity to Bashibai and Hu sheep while remaining clearly differentiated from European meat and short-tailed breeds. A total of 208 candidate selective sweep regions containing 297 overlapping genes were identified. Quantitative trait loci (QTL) annotation revealed enrichment for loci associated with resistance to gastrointestinal nematodes (14 QTLs), body weight (9 QTLs), susceptibility to pneumonia (2 QTLs), clinical mastitis (2 QTLs), and susceptibility to Mycobacterium avium subsp. paratuberculosis infection (2 QTLs). Five PRNP haplotypes were identified, with 60% of animals classified as high-risk (NSP3) for scrapie susceptibility. No animals carrying the fully resistant ARR/ARR genotype were detected. At the TMEM154 locus, 95% of individuals carried the E35E genotype, which is associated with increased genetic susceptibility to Maedi-Visna. This study provides the first comprehensive genomic characterization of the Kazakh coarse-wool fat-tailed sheep, revealing its unique population structure, genomic regions under selection associated with adaptation and disease resistance, as well as genetic predisposition susceptibility-associated genotypes for scrapie and Maedi-Visna. These findings provide a valuable genomic resource for marker-assisted breeding, genetic improvement, and conservation of indigenous sheep genetic resources in Kazakhstan.

Prion strains in infected animals may exist as heterogeneous populations composed of a major prion conformer responsible for the dominant disease phenotype and minor conformers that are phenotypically silent or difficult to detect. However, the presence and biological significance of such minor conformers in natural prion isolates remain poorly understood. Identifying and characterizing these hidden substrains is important for understanding prion strain diversity and for assessing potential transmission risks to humans and livestock. In this study, we utilized a modified protein misfolding cyclic amplification (PMCA) method to detect substrains within classical scrapie isolates. Our analysis provided evidence consistent with the coexistence of multiple prion conformers, including previously unrecognized conformers, within single prion isolates. Furthermore, we observed changes in the detectable substrain composition during passages in animal hosts. These findings support the idea that scrapie prion populations can contain diverse conformers and that changes in detectable conformer populations during passage may contribute to the overall diversity of scrapie prions.

Scrapie is a fatal transmissible spongiform encephalopathy affecting small ruminants, with susceptibility strongly influenced by polymorphisms in the prion protein gene (PRNP). The aim of this study was to evaluate trends in PRNP haplotype and genotype frequencies in the Valle del Belìce sheep breed before and after the implementation of the Sicilian scrapie Resistance Plan. Furthermore, a simulation analysis was performed to assess the expected genetic response under a revised selection strategy. A total of 4341 animals were genotyped for the PRNP gene. In the overall population, ARR (0.543) and ARQ (0.406) were the most frequent haplotypes. In animals born before the implementation of the selection plan (pre-2016), the frequency of the ARR haplotype was 0.462 (females = 0.459; males = 0.537). In animals born after the introduction of the plan, ARR frequency increased to 0.589 (females = 0.566; males = 0.774), reflecting the greater selection intensity applied to breeding rams. These results support the effectiveness of targeted genetic selection in increasing the frequency of the resistant ARR haplotype, while emphasizing the often-overlooked role of ewes as a reservoir of susceptible genotypes. Simulation results suggest that extending selection to ewes and implementing controlled mating schemes could accelerate genetic progress, leading to near-fixation of the ARR haplotype within approximately seven to eight years. This study demonstrates that the inclusion of the entire breeding population is essential to optimize scrapie resistance programs. The findings provide a quantitative basis for revising current regional guidelines and offer broader insights for the design of sustainable genetic control strategies.

The conversion of native prion protein (PrP) into its misfolded isoform, scrapie (PrP[Sc]) and its intracellular accumulation represent central events in the pathogenesis of prion diseases. Reduction of native PrP in the central nervous system (CNS) has emerged as a promising strategy for treatment and prevention of prion diseases in humans. To facilitate translation into clinical practice, it is essential to identify at-risk individuals through biomarker development and to elucidate PrP behaviour across prion disease subtypes and biological fluids. Measurements of PrP in accessible biofluids, such as plasma and cerebrospinal fluid (CSF), may provide a pharmacodynamic readout and enable monitoring for PrP-targeted therapies. This study systematically quantifies PrP in plasma and CSF of individuals with sporadic and genetic prion diseases, healthy controls (HC), patients with non-neurodegenerative neurological conditions (ND) and Alzheimer's disease (AD). We analysed 136 plasma and 84 CSF samples, including HC, AD, sporadic Creutzfeldt-Jakob disease (sCJD), as well as symptomatic patients and asymptomatic carriers of the mutations D178N, E200K and P102L. Quantification of PrP was performed using a BetaPrion Human ELISA. Statistical analyses assessed differences between diagnostic groups, associations with demographic factors and PRNP codon 129 polymorphism, and diagnostic accuracy via ROC curves. Plasma PrP was significantly reduced in patients with sCJD (P = 0.043), in symptomatic patients with the E200K (P = 0.0078) and in both symptomatic and asymptomatic D178N carriers (P = 0.0002) compared to HC. Furthermore, symptomatic and asymptomatic D178N carriers had significantly lower plasma PrP levels than patients with AD (P = 0.0025 and P = 0.0041, respectively). In CSF, PrP concentrations were notably lower in D178N symptomatic patients (P = 0.0026) versus non-neurodegenerative (ND) controls. Plasma PrP levels showed no association with age, sex or disease onset and were lower in genetic prion disease patients with the methionine/valine (MV) genotype at PRNP codon 129. The diagnostic accuracy for PrP quantification in plasma as a biomarker discriminated D178N asymptomatic carriers [area under the curve (AUC) = 0.96] and D178N symptomatic patients (AUC = 0.90) from HC with excellent accuracy. In CSF, PrP quantification discriminated D178N symptomatic patients from ND with good accuracy (AUC = 0.64). Taken together, this study defines a characteristic profile of persistently low plasma and CSF PrP in D178N symptomatic and asymptomatic mutation carriers. Low plasma levels in sCJD and E200K, in contrast to P102L, are puzzling. Mutation-specific patterns of PrP need to be considered for monitoring purposes in clinical trials.

We report 6 cases of camel prion disease in dromedaries in Tunisia, confirming widespread occurrence of the disease in North Africa. Affected animals showed neurologic signs and disease-associated prion protein accumulation in brain and lymphoid tissues. These findings highlight the importance of active surveillance and investigation of the epidemiology, transmission, and public health implications of this disease.

β2-microglobulin (B2M) is implicated in neuroinflammatory and neurodegenerative processes, but its role in prion diseases remains unclear. Here, the alterations in B2M expression and distribution as well as its correlation with PrPSc accumulation were investigated in several scrapie-infected rodent models and a prion-infected cell line. Markedly increased B2M levels were detected in the brains of prion-infected rodents at the terminal stage, as well as in the prion-infected SMB-S15 cells, with prominent colocalization with neurons and microglia. Transient overexpression of cytosolic abnormal PrP in cultured cells induced an increase in B2M, whereas the expression of wild-type PrP did not alter the B2M level. Resveratrol-induced clearance of PrPSc in SMB-S15 cells sufficiently reversed the increased level of B2M. Molecular interactions and histological spatial distributions between B2M and PrPSc were identified in scrapie-infected brains. The B2M upregulation during prion infection was independent of the MHC-I expression. Routine Western blot and ELISA failed to find significant differences in B2M levels between the cohorts of human prion disease (PrD) and non-PrD in cerebrospinal fluid (CSF), and between the cohorts of PrD and the normal population in serum. These findings suggest that prion infection and abnormal PrP accumulation induce remarkable B2M upregulation, and this interaction along with its MHC-I-independent upregulation highlights the potential of B2M in the pathological progression of prion diseases, providing a novel molecular basis for understanding prion-induced neuroinflammation.

Misfolding of the cellular prion protein (PRPC) is associated with fatal neurodegenerative prion diseases for which no treatments are currently available. Although the immune system is generally non-responsive (tolerant) to self-proteins such as PRPC, evidence of anti-prion antibodies suggests escape from self-tolerance in some individuals and supports the potential for the human immune system to be leveraged against prion disease. Human leukocyte antigen (HLA) plays a central role in rejecting endogenous non-self proteins (e.g. cancer neoantigens) by activating CD8+ cytolytic T cells via the Class I system (HLA-I) and CD4+ helper T cells via the Class II (HLA-II) system. Here we investigated the predicted binding affinity of 334 HLA molecules with all possible linear 9-mer (for HLA-I) and 15-, 18- and 22-mer (for HLA-II) PRPC peptides to identify peptide-HLA (pHLA) complexes with strong predicted binding (IC50 < 50 nM). We found that 12.4% of all prion peptides tested showed strong binding affinity to HLA molecules and that 20.2% of HLA alleles were able to bind strongly with PRPC peptides. These findings suggest that carriers of certain HLA alleles that are capable of binding strongly to PRPC peptides may have enhanced protection against prion disease, through reduction in the overall amount of PRPC available for conversion to the misfolded, infectious scrapie isoform (PRPSc) of PRPC and, potentially, by destroying it. These findings have implications for other disorders including common neurodegenerative diseases characterized by protein misfolding (e.g. α-synuclein, huntingtin, amyloid, tau, etc.).

Identifying reliable biomarkers for prion diseases remains a challenge, particularly for early detection in accessible body fluids. A recent mass spectrometry study on the cerebrospinal fluid proteome identified and validated five proteins significantly dysregulated in the preclinical stage of naturally scrapie-affected sheep. In this study, we quantified these proteins in serum and analysed their distribution and gene expression in the central nervous system, correlating these results with characteristic prion-related neuropathological features. Significantly elevated serum levels of synaptotagmin binding, cytoplasmic RNA interacting protein (SYNCRIP), phospholipase D3 (PLD3) and cathepsin D (CTSD) were detected in preclinical animals, mirroring previous cerebrospinal fluid findings. Central nervous system analyses on these three proteins together with osteopontin (SPP1) and complement component 4 (C4) revealed early and region-specific reduced immunoreactivity alongside upregulated gene expression in scrapie-affected animals, correlating significantly with prion-associated neuropathological features. Together, these findings highlight the potential of SYNCRIP, PLD3 and CTSD as promising minimally invasive biomarkers to diagnose prion diseases from the preclinical stage and provide new insights into the spatiotemporal regulation of the five proteins in the central nervous system throughout the progression of disease. Further research is needed to clarify the peripheral biomarker dynamics in relation to the neurodegenerative pathology.

Chronic wasting disease (CWD) and scrapie are transmissible spongiform encephalopathy diseases caused by prions, infectious forms of the prion protein. Currently, immunohistochemistry (IHC) is the sole approved diagnostic method for confirming these prion infections in formalin-fixed tissues. Evaluation of prion IHC requires specially trained veterinary pathologists to assess multiple quality control parameters as well as characteristic chromogen immunolabeling patterns. This manual slide review creates a significant bottleneck for laboratories needing to rapidly scale up surveillance during periods of increased testing demand. Given the repetitive and standardized nature of prion IHC slide review, this assay represents an ideal candidate for computer-assisted diagnostics. To address this challenge, we developed a deep learning-based image analysis approach tailored to review slides from large-scale veterinary prion disease surveillance. Our training dataset included 143 prion IHC whole-slide images containing a total of 3296 annotations. Annotated images were segmented into nonoverlapping tiles and used to fine-tune a pretrained convolutional neural network, enhancing the model's ability to recognize prion-specific quality control parameters and labeling features. When tested on a separate, blinded testing dataset of 50 CWD IHC slides, the model achieved 100% concordance for chromogenic labeling when compared with evaluation by a trained veterinary pathologist. The overarching objective of this project is to automate the initial review of prion IHC slides using deep learning-based image analysis to substantially reduce the time needed for evaluation. Implementation of this technology should enhance diagnostic consistency, improve efficiency, and provide scalable capabilities essential for comprehensive prion surveillance throughout the veterinary diagnostic laboratory network.

Mutational analysis of the cellular prion protein (PrP[C]) has revealed various regions of the protein that modulate prion propagation. However, these approaches involve deletions, insertions, or replacements in the presence of the WT PrP[C], which may mask the true phenotype. Here, site-directed alanine mutagenesis of the prion protein (PrP) was conducted to identify sites, particularly potential "surface patches" of PrP[C] required for prion propagation. Mutations were targeted to the helical, sheet, and loop regions of PrP[C], or a combination thereof and the mutated proteins expressed in mouse neuroblastoma cells in which the endogenous PrP[C] had been silenced. Using the scrapie cell assay, a highly sensitive cell culture-based bioassay for quantifying infectious titers of Rocky Mountain Laboratory (RML) prions, we found that all mutations within the structured 119 to 231 domain, irrespective of secondary structure, severely reduced prion propagation. We further analyzed four strongly inhibiting mutations within conformationally variable loop regions of PrP[C] - residues 123 to 125 (PRP1); 134 to 135 (PRP4); 139,141 (PRP5), and connecting helix residues 146 and 153, respectively, as well as residues 188,191 to 192 (PRP13). Mutations in PRP1, 4 and 5 dominantly inhibited prion propagation even in the presence of WT PrP[C], while mutations in PRP13 had no comparable effect, suggesting that the former disrupt prion fibril structure or its replication mechanism. None of the most inhibitory mutations substantially altered PrP stability, and mutant PrPs were able to both form amyloid in vitro and seed fibril formation of WT PrP.

The pathogenic conversion of the cellular prion protein (PrP[C]) into the β-sheet-rich isoform PrP[Sc] is the pivotal pathogenic event in prion disease, yet the molecular steps that govern this structural transition remain elusive. In this study, we introduce a new approach to monitor site-specific conformational transitions that occur during infectious prion formation. The method relies on genetically encoded substitution of a fluorescent, environmentally sensitive non-canonical amino acid, L-(7-hydroxycoumarin-4-yl)ethylglycine (7-HCAA), into recombinant PrP substate molecules, allowing real-time monitoring of structural changes in high-efficiency in vitro PrP[Sc] conversion reactions. As proof of principle, we show that the W99 7-HCAA recPrP substate efficiently propagates two different PrP[Sc] conformers (infectious cofactor PrP[Sc] and non-infectious protein-only PrP[Sc]). Bioassays in knock-in mice expressing bank vole (BV) PrP confirm that W99 7-HCAA cofactor PrP[Sc] produced by serial propagation is infectious, causing scrapie with an incubation period and neuropathological profile like those induced by wild-type cofactor PrP[Sc]. Marked differences in fluorescence intensity were observed between native, misfolded, and denatured states of W99 7-HCAA PrP, confirming that 7-HCAA reports on local changes in PrP conformation. Together, these findings establish 7-HCAA as a site-specific and sensitive probe of local PrP conformation. Moreover, the results suggest a new and broadly applicable strategy for studying conformational dynamics in amyloid-forming proteins.

Among small ruminants, natural bovine spongiform encephalopathy (BSE) has been confirmed only in goats, raising concern about the possible entry of caprine BSE into the food chain. Genetic selection for PRNP alleles associated with resistance to classical scrapie, particularly K222 and S127, has been proposed as a control strategy, but their effect on caprine BSE is unclear. Nine Alpine goats carrying different PRNP genotypes at codons 222 (Q/Q, Q/K, K/K) and 127 (G/G, G/S) were intracerebrally inoculated with a second-passage caprine BSE isolate and monitored until terminal disease or censoring. Brain and selected peripheral tissues were examined for PrP[Sc] by immunohistochemistry, western blotting, and conformation-dependent immunoassay, and lesion severity and PrP[Sc] distribution were compared among genotypes. All goats lacking the S127 allele developed clinical disease with widespread PrP[Sc] accumulation in the central nervous system. Compared with Q/Q222 goats, Q/K222 and K/K222 goats showed longer incubation periods and lower overall PrP[Sc] and vacuolation scores, although disease still occurred in both genotypes after intracerebral challenge. Two G/S127 goats, one Q/Q222 and one Q/K222, remained clinically healthy and biopsy-negative at > 1400 days post-inoculation, but were right-censored because they were alive at last follow-up and had not undergone post mortem examination. PrP[Sc] was also variably detected in lymphoreticular tissues and in the enteric and peripheral nervous systems, irrespective of genotype. Under these stringent challenge conditions, K222 delays disease onset but does not confer complete resistance. The prolonged survival of S127 carriers remains a preliminary, censored observation requiring post mortem confirmation.

Among senescence-accelerated prone mice (SAMP), the SAMP8 and SAMP10 strains exhibit significant age-related deteriorations in learning and memory. Previous studies have reported that SAMP strains carry high level of Akv-type endogenous ecotropic murine leukemia virus (E-MuLV), whereas the senescence-accelerated resistant strain SAMR1 contains very low or undetectable levels of the virus. Retroviral infections, including MuLV, have been implicated in the acceleration of prion pathogenesis. Prion diseases are characterized by neuronal loss, spongiform degeneration, and astrogliosis and are caused by the infectious prion protein (PrP[Sc]), which arises from the misfolding of the normal cellular prion protein (PrP[C]). Notably SAMP8 mice infected with scrapie exhibit shortened survival and increased PrP[Sc] accumulation compared with SAMR1 mice. In this study, we investigated the role of endogenous E-MuLV to prion disease progression using senescence-accelerated mouse models. Following infection with the 22L scrapie strain, SAMP10 mice displayed significantly shortened survival compared with SAMR1 mice after both intracerebral (IC: 121.8±1.3 vs. 141.0±2.2 days) and intraperitoneal (IP: 184.4±1.3 vs. 216.2±2.7 days) inoculation. SAMP10 mice also showed earlier and more pronounced PrP[Sc] accumulation during the clinical phase, along with enhanced vacuolation. Furthermore, in a cerebellar slice culture model of 22L scrapie infection, treatment with the antiretroviral drug zidovudine significantly reduced PrP[Sc] accumulation in SAMP10 mice. Taken together, these findings suggest that endogenous E-MuLV contribute to the accelerated prion pathogenesis by promoting early and elevated PrP[Sc] accumulation, leading to shortened survival.

Neurodegenerative diseases (NDs), such as Alzheimer's, Parkinson's, and prion diseases, are characterized by the dynamical spread of toxic proteins through the brain. In prion diseases, cellular prion protein (PrP C), produced by neurons, misfolds into a toxic form, known as scrapie prion protein (PrP Sc). PrP Sc induces neuronal stress which ultimately leads to cell death. In this paper, we develop mathematical models for the progression of prion diseases, incorporating a cellular defense mechanism that introduces a delay term affecting protein translation and a volatility term accounting for unaccounted biological factors influencing the system. We also extend the model to capture the spatial spread of toxic proteins over the brain connectome. Our first objective is to establish the existence and uniqueness of a global positive solution to the prion disease models. Afterwards, we analyze the asymptotic behavior of the models by identifying regimes of persistence and extinction of toxic proteins. For the deterministic delayed systems, we perform a stability analysis for the persistence and demonstrate that the system undergoes a Hopf bifurcation. We also study the intensity of fluctuations of the equilibrium state of the stochastic model. Additionally, we present numerical simulations to illustrate the model dynamics using biologically relevant parameters.