Prion diseases are fatal neurodegenerative disorders with no approved therapies that halt or reverse disease progression. Given that cellular prion protein (PrP C ) expression is required for prion propagation and neurotoxicity, reducing its expression is a promising therapeutic strategy. However, complete PrP ablation, as seen in knockout models, causes subtle developmental and behavioral abnormalities, raising concerns about long-term safety. Here, we explore a complementary strategy that harnesses the dominant-negative effect of the naturally protective G127V PrP variant found in kuru-resistant individuals in Papua New Guinea. In CAD5 cell lines, we demonstrate that inducible expression of G126V PrP (the mouse equivalent of human G127V) along with WT PrP prevents and suppresses prion infection in a dose-dependent manner. Extending this approach to CAD5 cells that express bank vole PrP, we further show that the protective effect of G127V spans a wide range of naturally and artificially derived prion strains, highlighting the generality of the dominant-negative approach. Remarkably, prion resistance persists even after G126V expression had ceased, indicating a sustained protective effect that could obviate the need for continuous transgene expression in a therapeutic setting. Finally, we find that anchorless, recombinant G127V PrP retains a potent dominant-negative activity, suggesting the use of this protein as a biological therapeutic. Together, these findings define a framework for development of G127V, a naturally protective and evolutionarily selected PrP variant, as a therapeutic agent to treat or prevent prion diseases.

Prion diseases originate from the pathological misfolding of the cellular sialoglycoprotein prion protein (PrPC), universally found across mammalian species, into an aberrant conformation termed PrPSc, which exhibits high aggregation propensity and neurotoxicity. Distinct conformations of the misfolded and aggregated PrPSc, termed prion strains, can cause different disease phenotypes and transmission characteristics. Different prion strains exhibit well-defined and distinct glycoform preferences arising from two sialylated, N-linked glycans. Glycosylation, and in particular sialylation, have been demonstrated to modulate the replication rate of PrPSc, with profound implications for the propagation of prion diseases. In this work, we leverage high-resolution cryo-EM structural data and all-atom molecular dynamics simulations to elucidate the molecular basis of the glycoform preferences in mouse strains RML and ME7. We show that these preferences are determined by differential engagement of the major basic patch and palindromic region of PrP, shedding light on a long elusive, fundamental aspect of prion biology.

Abstract Protein misfolding and aggregation are critical in amyloidogenic diseases such as Alzheimer's disease, diabetes, and prion disorders. While aggregation has been widely studied in terms of extrinsic factors, the influence of intrinsic molecular features, particularly histidine tautomerism, remains poorly understood. In this mini‐review, we summarize recent computational studies elucidating how histidine tautomeric states regulate the structural changes, aggregation propensity, and intermolecular interactions of major amyloidogenic proteins, including amyloid‐β (Aβ40/42), Tau, amylin, prion protein, and profilin‐1, as well as their disease‐associated variants. We discuss tautomer‐dependent effects on monomer conformations, early oligomerization, fibril formation, and cross‐seeding behavior, and highlight the integration of molecular dynamics simulations and computational two‐dimensional infrared spectroscopy for resolving tautomer‐specific signatures. These findings emphasize histidine tautomerism as a critical but underestimated factor in amyloid aggregation mechanisms.

Genetic prion diseases are caused by mutant prion protein (PrP) misfolding, eventually leading to the formation of PrPSc, the infectious prion isoform that propagates by inducing misfolding of native PrP. Different mutations are thought to generate distinct prion strains with unique self-replicating and neurotoxic properties, contributing to the phenotypic diversity of genetic prion diseases. We previously showed that transgenic mice expressing the mouse PrP homologs of the D178N-M129 and D178N-V129 mutations linked to fatal familial insomnia (FFI) and genetic Creutzfeldt-Jakob disease (CJD178) accumulate misfolded, mildly proteinase-K (PK)-resistant PrP in their brains. These mice develop spontaneous neurological illnesses resembling FFI and CJD178, but their diseases have not been found to be transmissible to various mouse lines. In this study, we further assessed their prion propagation potential by inoculating bank voles-shown here to be susceptible to human FFI and CJD178 prions-and by using RT-QuIC. Negative results from both approaches corroborate the idea that these mice do not generate infectious prions. However, when brain homogenates from Tg(FFI) and Tg(CJD) mice were subjected to protein misfolding cyclic amplification with RML PrPSc as a seed, they generated highly PK-resistant mutant prions (RMLFFI and RMLCJD) able to propagate in Tga20 mice overexpressing wild-type PrP. To determine whether these in vitro-converted prions modeled the human diseases better, we examined their transmissibility, biochemical traits, and neuropathological features. Despite successful serial propagation in Tga20 mice, RMLFFI and RMLCJD displayed long incubation times, poor transmissibility to C57BL/6 mice, identical PK-resistant PrP fragments, and distinctive neuropathological changes including large submeningeal and perivascular plaques enriched in endogenous proteolytically shed PrP lacking membrane anchorage. These findings indicate that, regardless of the M129V polymorphism, the D178N mutation imparts novel, stable strain properties to RML that do not recapitulate the features of FFI and CJD178. Our results offer new insights into how genetic PrP mutations influence prion strain characteristics and suggest that spontaneous and templated prionogenesis may follow distinct mechanistic pathways.

Prion diseases are fatal neurodegenerative disorders that can be idiopathic, associated with genetic mutations, or acquired by infection with misfolded prion protein. We developed two complementary transgenic mouse models to investigate how the L108I substitution in mouse prion protein (PrP) influences spontaneous prion formation and transmission characteristics. The transgenic mouse model overexpressing the variant at approximately three times wild-type (WT) PrP levels (TgMo(L108I)3x) consistently developed a spontaneous neurodegenerative disorder between 219 and 536 days of age with 100% penetrance. This spontaneous disease exhibited biochemical and neuropathological characteristics of atypical prion disorders, featuring a distinctive 7-10 kDa protease-resistant PrP fragment and pathology comparable to small ruminants' atypical scrapie and certain forms of Gerstmann-Sträussler-Scheinker syndrome (GSS). In contrast, the knock-in model expressing the same variant at physiological levels (TgMo(L108I)1x) showed no spontaneous disease beyond 600 days, demonstrating that both the specific amino acid substitution and elevated expression levels are necessary for spontaneous prion formation. The spontaneously generated prions transmitted efficiently to models expressing the I108 variant and to Tga20 mice overexpressing WT PrP but encountered a robust transmission barrier toward WT mice, indicating strain-specific replication requirements. The TgMo(L108I)3x model demonstrated exceptional versatility as a universal acceptor for heterogeneous prion isolates, demonstrating superior efficiency in propagating atypical variants like GSS A117V (57 ± 0.6 days) and rapid propagation of classical scrapie-derived mouse prion strains, including Rocky Mountains Laboratory mouse prion strain (RML) (85 ± 3.8 days) and 22L (95 ± 1 days). Comparative analysis revealed that the L108I substitution differentially impacts strain propagation, with greater acceleration of RML (~33% shorter incubation) than 22L (~0.5% shorter) compared to WT mice. These complementary systems offer powerful experimental platforms for investigating the molecular determinants of spontaneous prion formation, strain selection and transmission barriers, providing insights into idiopathic prion pathogenesis and developing therapeutic interventions.

Return of Mars samples is a high priority in the planetary science community and has remained an enduring goal of planetary exploration programs. Development of sterilization techniques to prevent potential contamination of Earth's biosphere with unknown life-forms that could exist on planetary bodies requires the use of the most robust biological indicators. We argue that self-seeding proteinaceous particles (prions) represent the most robust biological agents found on Earth. To evaluate the impact of various sterilization techniques on prion activity, we used derivatives of yeast prion proteins Sup35 and Ure2, which are not harmful to humans. Our study demonstrated that effective antimicrobial modalities, which include prolonged dry heat (up to 200°C), vapor hydrogen peroxide, gamma irradiation (up to 100 kGy), and ambient air or wet He/water plasma (deposited energy density of up to 6.3 kJ/cm2), did not eliminate the biological activity of yeast prions. However, ultraviolet C (UVC) irradiation at a wavelength of 260-270 nm for 16-24 days eliminated Ure2 prion detection and biological activity, and prolonged UVC irradiation eliminated detection of Sup35 prions and reduced, although did not eliminate, their biological activity. These data suggest that UVC could be an essential component of in-flight sterilization techniques for all future planetary missions.

Background: Prion diseases represent a group of rare, progressive, and invariably fatal neurodegenerative disorders. Their hallmark is the infectious nature of the misfolded prion protein (PrP^Sc), which propagates by inducing conformational changes in the physiological form (PrP^C). Despite advances in basic science, these disorders still pose major clinical and therapeutic challenges. Methods: A narrative review of the scientific literature was conducted across major biomedical databases, including PubMed, Scopus, Web of Science, and Google Scholar, covering publications up to January 2025. In addition, we describe an illustrative clinical case of a young patient with probable iatrogenic Creutzfeldt-Jakob disease following corneal transplantation, used to highlight diagnostic uncertainty and infection-control implications. Findings: Evidence confirms that PrP^Sc drives neurodegenerative processes and transmissibility, with phenotypic and genetic variants influencing clinical course and prognosis. From a diagnostic perspective, neuroimaging techniques and cerebrospinal fluid biomarkers have undergone substantial refinement, with RT-QuIC emerging as a highly specific and sensitive assay. Therapeutic options remain unsatisfactory: no treatment has shown a significant impact on survival. However, innovative strategies (including monoclonal antibodies, gene-based interventions, and modulation of PrP^C) represent promising avenues of investigation. Conclusions: Prion diseases remain an unresolved challenge at the intersection of neurology and infectious diseases. Earlier diagnosis through advanced biomarkers and continued development of targeted therapies are essential to improve patient management, while the persistence of iatrogenic cases underscores the ongoing relevance of surveillance and preventive strategies in clinical practice.

Genetic causes and modifiers of prion diseases.

Ensuring structural integrity of Hib capsular polysaccharide in conjugate vaccine manufacturing by 1H NMR and HPSEC-MALLS/RI.

Prion diseases manifest clinically in three different forms. Sporadic and infectious forms of prion disease are caused by the conversion of WT, cellular prion protein (PrPC) into its pathogenic conformer (PrPSc). In contrast, genetic forms of prion diseases are caused by mutations in the PrP sequence that promote mutant PrPSc formation. When reconstituted with either polyanionic or lipid cofactors, purified PrPC substrate can be converted in vitro into PrPSc products that display high levels of specific infectivity when inoculated in WT hosts. In contrast, various protein-only PrPSc molecules formed in the absence of cofactors display much lower levels of specific infectivity. Here, we report that protein-only PrPSc molecules with different sequences can induce the formation of proteinase K-resistant PrPSc molecules and spongiform degeneration in the brains of knock-in mice expressing PrP harbouring the pathogenic E200K mutation, but not in hosts expressing WT PrP. These results indicate that the E200K mutation enhances host susceptibility to various protein-only PrPSc fibrils, suggesting fundamental differences in the replication mechanisms of WT versus mutant prions.

Influence of Methionine Oxidation on Protein Stability and Association Studied by Free Energy Simulations.

ABSTRACTPrion propagation, in which the cellular prion protein (PrPC) is conformationally converted into an infectious structure (PrPSc), is now well understood. However, the molecular mechanism responsible for the neurotoxicity of prions remains unclear. Synaptic loss is one of the earliest events in bothin vivoandin vitromodels of prion disease. We previously developed a neuronal cell culture model to analyze the mechanisms of prion-induced synaptic degeneration in a physiologically relevant setting. Using this system, we showed that exposure of hippocampal neurons to PrPScengages a NMDAR/p38 mitogen-activated protein kinase (MAPK) signaling pathway that results in rapid, PrPC-dependent loss of synaptic transmission and retraction of dendritic spines. To comprehensively identify the components of this synaptotoxic signaling pathway, we measured changes in the phosphoproteome and transcriptome of hippocampal neurons exposed to PrPScwhile they were undergoing the process of dendritic spine retraction. We then used these data as input into the L1000 and P100 databases of transcriptomic and proteomic drug signatures, leading to the discovery of 17 compounds that were able to prevent PrPSc-induced spine retraction. These compounds converged on three protein kinase targets: Ca2+/calmodulin-dependent protein kinase II (CaMKII), protein kinase C (PKC), and glycogen synthase kinase 3β (GSK3β). Using immunocytochemical staining, we confirmed that PrPSctreatment of hippocampal neurons induced phosphorylation of the three kinases and caused their rapid translocation to dendritic spines. Along with N-methyl-D-aspartate receptors (NMDARs) on the neuronal surface, which trigger an initial influx of Ca2+in response to PrPSc, these kinases constitute key nodes in a signaling network that mediates prion synaptotoxicity. Taken together, our results provide new insights into the mechanisms of prion neurotoxicity, and they identify novel molecular targets and inhibitory compounds that can be utilized for therapy of prion diseases.AUTHOR SUMMARYThe mechanism by which prions propagate is now well established, but how they cause neurodegenerative changes is still uncertain. The earliest effects of prion infection occur at the level of the synapse, and we previously established an experimental system using cultured hippocampal neurons to assay prion synaptotoxicity. To search comprehensively for components of the synaptotoxic signaling pathway, we employed a novel, small-molecule discovery pipeline based on the transcriptomic and phosphoproteomic profiles of prion-treated neurons. This approach converged on inhibitors of three different protein kinases (Ca2+/calmodulin-dependent protein kinase II, protein kinase C, and glycogen synthase kinase 3β), which, along with N-methyl-D-aspartate receptors, constitute key nodes in a prion synaptotoxic signaling network that can be targeted for therapeutic benefit.

The study of prion biology has traditionally relied on transgenic mouse models, which, while valuable, require significant time and resources to develop. Here, we present a rapid and flexible alternative using adeno-associated virus (AAV) vectors to express modified prion proteins in PrP-knockout (PrP-KO) mice. Through systematic evaluation of multiple AAV constructs, we optimized vector design by comparing different CNS-specific promoters and regulatory elements to generate prion disease models capable of faithfully propagating the inoculated prion strain. We identified an optimized AAV construct incorporating the human synapsin promoter, MVM enhancer, and WPRE posttranscriptional regulatory element encapsidated in the AAV9P31 serotype to drive neuron-specific expression of modified mouse PrP (W144Y epitope) and bank vole I109 PrP (W145Y epitope). Following intravenous administration, we achieved brain-wide expression at levels comparable to or even exceeding endogenous PrP in some regions. When challenged with mouse-adapted RML prions or human Gerstmann-Sträussler-Scheinker (GSS-A117V) disease-causing prions, AAV-PrP mice developed characteristic signs of prion disease with accelerated kinetics (58-106 days post-inoculation for RML; 105-112 dpi for GSS-A117V), displaying features typical of each strain. Serial transmission of AAV-generated RML prions to wild-type mice confirmed preservation of strain-specific properties (165 ± 4 dpi), validating the authenticity of prion propagation in this system. This approach provides a versatile platform for rapidly generating and studying prion variants in an authentic brain environment. By reducing model generation time from months to weeks, this system enables accelerated investigation of prion structure-function relationships, strain properties, and therapeutic strategies, with potential applications extending to other protein misfolding diseases.

The mechanism by which prions composed of PrPSc cause the neuropathological aberrations characteristic of prion diseases remains elusive. Previous studies have defined a synaptotoxic signaling pathway in which extracellular PrPSc stimulates NMDA receptor-mediated Ca2+ influx, activation of p38 MAPK, and collapse of the actin cytoskeleton in dendritic spines, resulting in functional decrements in synaptic transmission. However, these studies did not determine whether synaptotoxic signaling is directly linked to conversion of cell-surface PrPC to PrPSc, or whether it can be initiated by extracellular PrPSc independently of PrP conversion. To address this question, we employed two different experimental strategies, both of which interfere with PrPC-PrPSc conversion: (1) neuronal expression of PrPC mutants that are locked in the PrPC conformation (G126V and V208M); and (2) application of extracellular PrPSc from a species (mouse or hamster) that is unable to convert neuronal PrPC of the other species. We first confirmed that both of these strategies resulted in impaired PrPC-PrPSc conversion in cultured N2a and CAD5 cell lines. To assay synaptotoxicity, we then used lentiviral transduction to express the PrPC variants in primary cultures of hippocampal neurons from PrP-null mice, and quantitated dendritic spine density after exposure to purified prions. Expression of G126V PrP completely prevented spine retraction in response to three different murine prion strains (RML, 22L, and ME7), while the effect of V208M PrP was strain-dependent, consistent with partial stabilization of PrP structure by this mutation. Expression of hamster PrPC or mouse PrPC greatly attenuated spine retraction in response to murine 22L and hamster 263K prions, respectively. These findings support a model in which newly formed PrPSc at the neuronal surface is required to initiate prion-mediated synaptotoxic signaling. This work also suggests use of the G126V mutation as part of a therapeutic strategy to reduce PrPSc conversion in prion diseases.

The mechanism by which prions composed of PrPSc cause the neuropathological aberrations characteristic of prion diseases remains elusive. Previous studies have defined a synaptotoxic signaling pathway in which extracellular PrPSc stimulates NMDA receptor-mediated Ca2+ influx, activation of p38 MAPK, and collapse of the actin cytoskeleton in dendritic spines, resulting in functional decrements in synaptic transmission. However, these studies did not determine whether synaptotoxic signaling is directly linked to conversion of cell-surface PrPC to PrPSc, or whether it can be initiated by extracellular PrPSc independently of PrP conversion. To address this question, we employed two different experimental strategies, both of which interfere with PrPC-PrPSc conversion: (1) neuronal expression of PrPC mutants that are locked in the PrPC conformation (G126V and V208M); and (2) application of extracellular PrPSc from a species (mouse or hamster) that is unable to convert neuronal PrPC of the other species. We first confirmed that both of these strategies resulted in impaired PrPC-PrPSc conversion in cultured N2a and CAD5 cell lines. To assay synaptotoxicity, we then used lentiviral transduction to express the PrPC variants in primary cultures of hippocampal neurons from PrP-null mice, and quantitated dendritic spine density after exposure to purified prions. Expression of G126V PrP completely prevented spine retraction in response to three different murine prion strains (RML, 22L, and ME7), while the effect of V208M PrP was strain-dependent, consistent with partial stabilization of PrP structure by this mutation. Expression of hamster PrPC or mouse PrPC greatly attenuated spine retraction in response to murine 22L and hamster 263K prions, respectively. These findings support a model in which newly formed PrPSc at the neuronal surface is required to initiate prion-mediated synaptotoxic signaling. This work also suggests use of the G126V mutation as part of a therapeutic strategy to reduce PrPSc conversion in prion diseases.

Quaking-induced conversion (QuIC) tests, which detect prion-seeding activity in cerebrospinal fluid (CSF), have markedly advanced the antemortem diagnosis of prion diseases such as Creutzfeldt-Jakob disease (CJD). These tests provide high diagnostic accuracy and enable timely differentiation from other rapidly progressive neurodegenerative disorders. However, a key limitation of current QuIC tests are the reduced sensitivity in detecting inherited prion diseases and rare sporadic subtypes, including variably protease-sensitive prionopathy (VPSPr). To address this gap, we evaluated a simplified QuIC test, end-point QuIC (EP-QuIC), incorporating a novel recombinant prion protein substrate derived from the North American deer mouse (Peromyscus maniculatus). The diagnostic performance of the modified QuIC test was evaluated using CSF samples from 61 sporadic CJD, 50 inherited prion disease, and 5 VPSPr cases. EP-QuIC with the deer mouse substrate achieved 96.6% sensitivity (111/116) and 100% specificity (35/35), outperforming both standard EP-QuIC (87.1%) and next-generation (IQ-CSF) real-time-QuIC (72.4%) across the same cohort. Notably, this enhanced assay detected inherited mutations, such as D178N, that were previously undetectable with existing diagnostic tests. These findings demonstrate that adapting EP-QuIC with an optimized substrate, termed enhanced sensitivity QuIC (ES-QuIC), significantly improves diagnostic performance for inherited and atypical prion diseases. By expanding the diagnostic reach of QuIC tests, this study strengthens antemortem surveillance, reduces reliance on postmortem confirmation, and improves opportunities for early intervention and clinical trial enrollment, particularly for genetic cases most likely to benefit from emerging therapeutic strategies. ANN NEUROL 2026.

Colorectal cancer (CRC) remains a major cause of cancer-related deaths in advanced disease, and activating KRAS/NRAS mutations limit the use of anti-EGFR antibodies to RAS-wild-type tumors. The cellular prion protein (PrPC) has been linked to aggressive and chemoresistant CRC, but its extracellular partners and functional relevance in KRAS-mutant disease are not fully defined. Here, we examined extracellular PrPC complexes and PrPC-associated signaling in CRC cell lines and xenografts using a neutralizing PrPC monoclonal antibody. Across a CRC panel that included SNU-C5/WT and its 5-fluorouracil- and oxaliplatin-resistant derivatives, HT-29 (KRAS-wild-type), and HCT-8 and LoVo (KRAS-mutant), co-immunoprecipitation showed that PrPC forms complexes with the 37/67 kDa laminin receptor (RPSA), with PrPC-RPSA association particularly increased in KRAS-mutant HCT-8 and LoVo cells. PrPC protein levels were higher in KRAS-mutant HCT-8, SW620, and SNU-407 cells than in HT-29, and PrPC neutralization reduced viability in all four lines. Accordingly, we assessed upstream RAS activity and found that active RAS (RAS-GTP) was higher in KRAS-mutant cells than in HT-29, and PrPC treatment was associated with reduced RAS-GTP levels. In the same KRAS-mutant setting, basal AKT phosphorylation exceeded that in HT-29, and PrPC treatment lowered AKT phosphorylation without changing total AKT. Moreover, PrPC treatment was associated with reduced ERK1/2 phosphorylation in KRAS-mutant cells, suggesting attenuation of downstream RAS pathway output. These signaling changes coincided with a decrease in the S-phase fraction and an increase in G1. In an HCT-8 (KRAS G13D) xenograft model, PrPC monotherapy inhibited tumor growth in a dose-dependent manner, and 5-fluorouracil (5-FU) monotherapy produced an intermediate effect. The combination of PrPC (10 mg/kg) and 5-FU (20 mg/kg) yielded the greatest tumor growth inhibition among the tested regimens. Consistent with this enhanced tumor control, immunofluorescence of xenograft tissues showed that PrPC, particularly with 5-FU, reduced intratumoral PrPC and PCNA and decreased CD31-positive microvessels and α-SMA-positive vessel structures. Taken together, these findings suggest that extracellular PrPC supports RAS-AKT signaling, proliferation, and tumor-associated angiogenesis in KRAS-mutant colorectal cancer, and that PrPC neutralization additively enhances 5-fluorouracil activity in KRAS-mutant models. The data provide a preclinical basis for evaluating PrPC antibodies in combination with fluoropyrimidine-based regimens in patients with KRAS-mutant CRC.

Creutzfeldt-Jakob disease (CJD) is a rare transmissible neurodegenerative disease caused by misfolded prion proteins leading to rapid mental deterioration and death. Misfolded prion proteins form insoluble aggregates that cause irreversible neurological damage. While typically presenting with symptoms such as cognitive decline and behavioral changes, atypical presentations of CJD include symptoms such as sensorineural hearing loss or balance loss. The goal of this study is to systematically characterize atypical presentations of CJD with hearing and balance loss following Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines. The PubMed database was utilized to analyze all case reports from the years 1990 to 2024 using the keywords “Creutzfeldt-Jakob disease”, “hearing loss”, and “balance”. A total of 16 cases were retrieved, and 11 were included in the study. Among cases retrieved, we identified eight patients with hearing loss and three with balance loss. Of hearing loss patients, four (50%) were male and four (50%) were female. The average age was 63.75 years (SD=11.17 years). The most common symptoms that accompanied hearing loss were analyzed (N, %), and include gait disturbances (8, 100%), myoclonus (6, 75%), cognitive impairment (4, 50%), akinetic mutism (4, 50%), and vision disturbances (4, 50%). The tests utilized to diagnose CJD in patients with hearing loss included MRI (5, 63%), EEG (5, 63%), and 14-3-3 protein (4, 50%). These findings highlight the importance of recognizing hearing and balance loss as potential early symptoms of CJD to aid in earlier diagnosis and a better understanding of disease progression.

Expression of the cellular prion protein, PrPC, on the surface of neurons plays an important role in the pathogenesis of prion disease. We performed genome-wide CRISPR/Cas9 knockout screens in prion-infectible cells of neuronal origin (CAD5) to identify regulators of cell surface PrPC expression. We identified and validated 46 positive and 21 negative regulators of cell surface PrPC expression in undifferentiated CAD5 cells. Pathway analysis of the screening dataset showed that genes involved in the glycophosphatidylinositol (GPI) anchor and N-glycosylation biosynthetic pathways were overrepresented as positive regulators of cell surface PrPC. We also sought to determine whether the same or different genes regulate cell surface PrPC in CAD5 cells that have been differentiated to a more neuronal state and validated 41 positive and 13 negative regulators of CAD5 cell surface PrPC expression in the differentiated state. We identified 23 core genes as shared between the undifferentiated and differentiated cell states, including many positive regulators involved in GPI anchor biosynthesis. Intriguingly, unique regulators were also identified in the undifferentiated and differentiated cell states, suggesting that some mechanisms regulating cell surface PrPC expression in CAD5 cells are dependent on cell state. This list of core genes involved in regulating cell surface PrPC expression in a prion-susceptible, neuron-like cell type offers a valuable guide for future research and may help identify potential therapeutic targets for prion disease and other neurodegenerative diseases.

Transgenic mice overexpressing bank vole prion protein with the isoleucine 109 polymorphism, TgVole(I109)4x, develop spontaneous neurodegenerative disease with sex-dependent onset, averaging 170 days in females and 200 days in males at terminal stage. The clinical and pathological features closely resemble Gerstmann-Sträussler-Scheinker syndrome (GSS), with characteristic ataxia, dysmetria, kyphosis, and prominent PrP plaques. Biochemical analysis reveals an atypical prion protein banding pattern with a distinctive low molecular weight band (7–10 kDa) following proteinase K digestion, similar to other atypical prion diseases such as small ruminants atypical scrapie (AS). Importantly, these spontaneously generated prions are highly infectious when passaged to mice expressing the same I109 polymorphism as well as to wild bank voles carrying the I109 polymorphism, but not to models expressing the methionine variant at this position, demonstrating the critical role of this specific polymorphism in atypical prion propagation. Temporal analysis reveals that infectious prions emerge significantly (2–3 months) before clinical signs appear, offering important insights into the pre-clinical phase of prion diseases. Serum neurofilament light chain levels increase significantly at 80 days of age, approximately 100 days before clinical onset, providing a wide therapeutic window with a reliable biomarker. The TgVole(I109)4× model exhibits extraordinary versatility in propagating diverse prion strains, showing remarkable susceptibility to atypical prions (including GSS and AS) with exceptionally short incubation periods, while maintaining the ability to efficiently propagate classical and recombinant prion strains. We present here a thoroughly characterized transgenic mouse model that spontaneously develops an atypical, bona fide prion disease with sex-related differences in disease onset. This model offers valuable insights into spontaneous and atypical prionopathies while demonstrating exceptional versatility for studying diverse prion strains and potential utility for evaluating therapeutic interventions when used with appropriate study designs that account for individual variability.Supplementary InformationThe online version contains supplementary material available at 10.1186/s40478-025-02213-7.