Isolation of Anti-Prion Compounds from Curcuma phaeocaulis Valeton Extract.

Prion diseases, known as a group of fatal neurodegenerative disorders caused by prions, remain incurable despite extensive research efforts. In a recent study, crude extract from Curcuma phaeocaulis Valeton (Cp) showed promising anti-prion efficacy in in vitro and in vivo models, prompting further investigation into their active compounds. We endeavored to identify the chemical constituents of the Cp extract and discover potential anti-prion agents. With the use of centrifugal partition chromatography (CPC), major constituents were isolated from the n-hexane (HX) fraction of the extract in a single step. Spectroscopic analysis confirmed the presence of curcumenone, curcumenol, and furanodienone. Subsequent efficacy testing in a cell culture model of prion disease identified curcumenol and furanodienone as active compounds. This study underscores the potential of natural products in the search for effective treatments against prion diseases.

The molecular determinants of a universal prion acceptor.

In prion diseases, the species barrier limits the transmission of prions from one species to another. However, cross-species prion transmission is remarkably efficient in bank voles, and this phenomenon is mediated by the bank vole prion protein (BVPrP). The molecular determinants of BVPrP's ability to function as a universal prion acceptor remain incompletely defined. Building on our finding that cultured cells expressing BVPrP can replicate both mouse and hamster prion strains, we systematically identified key residues in BVPrP that permit cross-species prion replication. We found that residues N155 and N170 of BVPrP, which are absent in mouse PrP but present in hamster PrP, are critical for cross-species prion replication. Additionally, BVPrP residues V112, I139, and M205, which are absent in hamster PrP but present in mouse PrP, are also required to enable replication of both mouse and hamster prions. Unexpectedly, we found that residues E227 and S230 near the C-terminus of BVPrP severely restrict prion accumulation following cross-species prion challenge, suggesting that they may have evolved to counteract the inherent propensity of BVPrP to misfold. PrP variants with an enhanced ability to replicate both mouse and hamster prions displayed accelerated spontaneous aggregation kinetics in vitro. These findings suggest that BVPrP's unusual properties are governed by a key set of amino acids and that the enhanced misfolding propensity of BVPrP may enable cross-species prion replication.

Aqueous extraction of formalin-fixed paraffin-embedded tissue and detection of prion disease using real-time quaking-induced conversion.

OBJECTIVE: The goal of the research presented here is to determine if methods previously developed for the aqueous extraction of PrPSc from formalin-fixed paraffin-embedded tissue (FFPET) are applicable to the detection PrPSc by real-time quaking induced conversion (RT-QuIC). Previous work has utilized aqueous extraction of FFPET for detection of transmissible spongiform encephalopathies (TSEs) utilizing western blot and ELISA. This research extends the range of suitable methods for detection of TSEs in FFPET to RT-QuIC, which is arguably the most sensitive method to detect TSEs. RESULTS: We found complete agreement between the TSE status and the results from RT-QuIC seeded with the aqueous extract of FFPET samples. The method affords the diagnostic assessment TSE status by RT-QuIC of FFPET without the use of organic solvents that would otherwise create a mixed chemical-biological waste for disposal.

The prion principle and Alzheimer's disease.

Similarities to molecular mechanisms underlying prion diseases may help to refine Alzheimer's disease therapies.

Protective role of cytosolic prion protein against virus infection in prion-infected cells.

Production of the amyloidogenic prion protein, PrPSc, which forms infectious protein aggregates, or prions, is a key pathogenic event in prion diseases. Functional prion-like protein aggregations, such as the mitochondrial adaptor protein MAVS and the inflammasome component protein ASC, have been identified to play a protective role in viral infections in mammalian cells. In this study, to investigate if PrPSc could play a functional role against external stimuli, we infected prion-infected cells with a neurotropic influenza A virus strain, IAV/WSN. We found that prion-infected cells were highly resistant to IAV/WSN infection. In these cells, NF-κB nuclear translocation was disturbed; therefore, mitochondrial superoxide dismutase (mtSOD) expression was suppressed, and mitochondrial reactive oxygen species (mtROS) was increased. The elevated mtROS subsequently activated NLRP3 inflammasomes, leading to the suppression of IAV/WSN-induced necroptosis. We also found that prion-infected cells accumulated a portion of PrP molecules in the cytosol, and that the N-terminal potential nuclear translocation signal of PrP impeded NF-κB nuclear translocation. These results suggest that PrPSc might play a functional role in protection against viral infections by stimulating the NLRP3 inflammasome-dependent antivirus mechanism through the cytosolic PrP-mediated disturbance of NF-κB nuclear translocation, which leads to suppression of mtSOD expression and consequently upregulation of the NLRP3 inflammasome activator mtROS. IMPORTANCE: Cytosolic PrP has been detected in prion-infected cells and suggested to be involved in the neurotoxicity of prions. Here, we also detected cytosolic PrP in prion-infected cells. We further found that the nuclear translocation of NF-κB was disturbed in prion-infected cells and that the N-terminal potential nuclear translocation signal of PrP expressed in the cytosol disturbed the nuclear translocation of NF-κB. Thus, the N-terminal nuclear translocation signal of cytosolic PrP might play a role in prion neurotoxicity. Prion-like protein aggregates in other protein misfolding disorders, including Alzheimer's disease were reported to play a protective role against various environmental stimuli. We here showed that prion-infected cells were partially resistant to IAV/WSN infection due to the cytosolic PrP-mediated disturbance of the nuclear translocation of NF-κB, which consequently activated NLRP3 inflammasomes after IAV/WSN infection. It is thus possible that prions could also play a protective role in viral infections.

Convergent generation of atypical prions in knockin mouse models of genetic prion disease.

Most cases of human prion disease arise due to spontaneous misfolding of WT or mutant prion protein, yet recapitulating this event in animal models has proven challenging. It remains unclear whether spontaneous prion generation can occur within the mouse lifespan in the absence of protein overexpression and how disease-causing mutations affect prion strain properties. To address these issues, we generated knockin mice that express the misfolding-prone bank vole prion protein (BVPrP). While mice expressing WT BVPrP (I109 variant) remained free from neurological disease, a subset of mice expressing BVPrP with mutations (D178N or E200K) causing genetic prion disease developed progressive neurological illness. Brains from spontaneously ill knockin mice contained prion disease-specific neuropathological changes as well as atypical protease-resistant BVPrP. Moreover, brain extracts from spontaneously ill D178N- or E200K-mutant BVPrP-knockin mice exhibited prion seeding activity and transmitted disease to mice expressing WT BVPrP. Surprisingly, the properties of the D178N- and E200K-mutant prions appeared identical before and after transmission, suggesting that both mutations guide the formation of a similar atypical prion strain. These findings imply that knockin mice expressing mutant BVPrP spontaneously develop a bona fide prion disease and that mutations causing prion diseases may share a uniform initial mechanism of action.

Extraneural infection route restricts prion conformational variability and attenuates the impact of quaternary structure on infectivity.

Prions can exist as different strains that consist of conformational variants of the misfolded, pathogenic prion protein isoform PrPSc. Defined by stably transmissible biological and biochemical properties, strains have been identified in a spectrum of prion diseases, including chronic wasting disease (CWD) of wild and farmed cervids. CWD is highly contagious and spreads via direct and indirect transmission involving extraneural sites of infection, peripheral replication and neuroinvasion of prions. Here, we investigated the impact of infection route on CWD prion conformational selection and propagation. We used gene-targeted mouse models expressing deer PrP for intracerebral or intraperitoneal inoculation with fractionated or unfractionated brain homogenates from white-tailed deer, harboring CWD strains Wisc-1 or 116AG. Upon intracerebral inoculation, Wisc-1 and 116AG-inoculated mice differed in conformational stability of PrPSc. In brains of mice infected intraperitoneally with either inoculum, PrPSc propagated with identical conformational stability and fewer PrPSc deposits in most brain regions than intracerebrally inoculated animals. For either inoculum, PrPSc conformational stability in brain and spinal cord was similar upon intracerebral infection but significantly higher in spinal cords of intraperitoneally infected animals. Inoculation with fractionated brain homogenates resulted in lower variance of survival times upon intraperitoneal compared to intracerebral infection. In summary, we demonstrate that extraneural infection mitigates the impact of PrPSc quaternary structure on infection and reduces conformational variability of PrPSc propagated in the brain. These findings provide new insights into the evolution of stable CWD strains in natural, extraneural transmissions.

Genetic characterization of the prion protein gene in camels (Camelus) with comments on the evolutionary history of prion disease in Cetartiodactyla.

Transmissible spongiform encephalopathies (TSEs) are a fatal neurogenerative disease that include Creutzfeldt-Jakob disease in humans, scrapie in sheep and goats, bovine spongiform encephalopathy (BSE), and several others as well as the recently described camel prion disease (CPD). CPD originally was documented in 3.1% of camels examined during an antemortem slaughterhouse inspection in the Ouargla region of Algeria. Of three individuals confirmed for CPD, two were sequenced for the exon 3 of the prion protein gene (PRNP) and were identical to sequences previously reported for Camelus dromedarius. Given that other TSEs, such as BSE, are known to be capable of cross-species transmission and that there is household consumption of meat and milk from Camelus, regulations to ensure camel and human health should be a One Health priority in exporting countries. Although the interspecies transmissibility of CPD currently is unknown, genotypic characterization of Camelus PRNP may be used for predictability of predisposition and potential susceptibility to CPD. Herein, eight breeds of dromedary camels from a previous genetic (mitochondrial DNA and microsatellites) and morphological study were genotyped for PRNP and compared to genotypes from CPD-positive Algerian camels. Sequence data from PRNP indicated that Ethiopian camels possessed 100% sequence identity to CPD-positive camels from Algeria. In addition, the camel PRNP genotype is unique compared to other members of the Orders Cetartiodactyla and Perissodactyla and provides an in-depth phylogenetic analysis of families within Cetartiodactyla and Perissodactyla that was used to infer the evolutionary history of the PRNP gene.

RNA-Seq Analysis of Mammalian Prion Disease.

A protein, which can attain a prion state, differs from standard proteins in terms of structural conformation and aggregation propensity. High-throughput sequencing technology provides an opportunity to gain insight into the prion disease condition when coupled with single-cell RNA-Seq analysis to reveal transcriptional changes during prion-based pathogenicity. In this chapter, we present a protocol for RNA-Seq analysis of mammalian prion disease using a single-cell RNA sequencing dataset procured from the NCBI GEO database. This protocol is a tool that can assist researchers in characterizing mammalian prion disease in a reproducible and reusable manner. Further, the resulting output has the potential to provide transcript biomarkers for mammalian prion diseases, which can be employed for diagnostic and prognostic purposes.

Adaptation of the protein misfolding cyclic amplification (PMCA) technique for the screening of anti-prion compounds.

Prion diseases result from the misfolding of the physiological prion protein (PrPC) to a pathogenic conformation (PrPSc). Compelling evidence indicates that prevention and/or reduction of PrPSc replication are promising therapeutic strategies against prion diseases. However, the existence of different PrPSc conformations (or strains) associated with disease represents a major problem when identifying anti-prion compounds. Efforts to identify strain-specific anti-prion molecules are limited by the lack of biologically relevant high-throughput screening platforms to interrogate compound libraries. Here, we describe adaptations to the protein misfolding cyclic amplification (PMCA) technology (able to faithfully replicate PrPSc strains) that increase its throughput to facilitate the screening of anti-prion molecules. The optimized PMCA platform includes a reduction in sample and reagents, as well as incubation/sonication cycles required to efficiently replicate and detect rodent-adapted and cervid PrPSc strains. The visualization of PMCA products was performed via dot blots, a method that contributed to reduced processing times. These technical changes allowed us to evaluate small molecules with previously reported anti-prion activity. This proof-of-principle screening was evaluated for six rodent-adapted prion strains. Our data show that these compounds targeted either none, all or some PrPSc strains at variable concentrations, demonstrating that this PMCA system is suitable to test compound libraries for putative anti-prion molecules targeting specific PrPSc strains. Further analyses of a small compound library against deer prions demonstrate the potential of this new PMCA format to identify strain-specific anti-prion molecules. The data presented here demonstrate the use of the PMCA technique in the selection of prion strain-specific anti-prion compounds.

Neuropathologically directed profiling of PRNP somatic and germline variants in sporadic human prion disease.

Creutzfeldt-Jakob Disease (CJD), the most common human prion disease, is associated with pathologic misfolding of the prion protein (PrP), encoded by the PRNP gene. Of human prion disease cases, < 1% were transmitted by misfolded PrP, ~ 15% are inherited, and ~ 85% are sporadic (sCJD). While familial cases are inherited through germline mutations in PRNP, the cause of sCJD is unknown. Somatic mutations have been hypothesized as a cause of sCJD, and recent studies have revealed that somatic mutations accumulate in neurons during aging. To investigate the hypothesis that somatic mutations in PRNP may underlie sCJD, we performed deep DNA sequencing of PRNP in 205 sCJD cases and 170 age-matched non-disease controls. We included 5 cases of Heidenhain variant sporadic CJD (H-sCJD), where visual symptomatology and neuropathology implicate localized initiation of prion formation, and examined multiple regions across the brain including in the affected occipital cortex. We employed Multiple Independent Primer PCR Sequencing (MIPP-Seq) with a median depth of > 5000× across the PRNP coding region and analyzed for variants using MosaicHunter. An allele mixing experiment showed positive detection of variants in bulk DNA at a variant allele fraction (VAF) as low as 0.2%. We observed multiple polymorphic germline variants among individuals in our cohort. However, we did not identify bona fide somatic variants in sCJD, including across multiple affected regions in H-sCJD, nor in control individuals. Beyond our stringent variant-identification pipeline, we also analyzed VAFs from raw sequencing data, and observed no evidence of prion disease enrichment for the known germline pathogenic variants P102L, D178N, and E200K. The lack of PRNP pathogenic somatic mutations in H-sCJD or the broader cohort of sCJD suggests that clonal somatic mutations may not play a major role in sporadic prion disease. With H-sCJD representing a localized presentation of neurodegeneration, this serves as a test of the potential role of clonal somatic mutations in genes known to cause familial neurodegeneration.

Prion diseases, always a threat?

Prion diseases are caused by prions, which are proteinaceous infectious particles that have been identified as causative factors of transmissible spongiform encephalopathies such as Creutzfeldt-Jakob disease (CJD). Prion diseases are devastating neurodegenerative disorders in humans and many animals, including sheep, cows, deer, cats, and camels. Prion diseases are classified into sporadic and genetic forms. Additionally, a third, environmentally acquired category exists. This type includes kuru, iatrogenic CJD caused by human dura mater grafts or human pituitary-derived hormones, and variant CJD transmitted through food contaminated with bovine spongiform encephalopathy prions. Bovine spongiform encephalopathy and variant CJD have nearly been controlled, but chronic wasting disease, a prion disease affecting deer, is spreading widely in North America and South Korea and recently in Northern Europe. Recently, amyloid-beta, alpha-synuclein, and other proteins related to Alzheimer's disease, Parkinson's disease, and other neurodegenerative diseases were reported to have prion features such as transmission to animals. Amyloid-beta transmission to humans has been suggested in iatrogenic CJD cases and in cerebral amyloid angiopathy cases with cerebral bleeding occurring long after childhood neurosurgery with or without cadaveric dura mater transplantation. These findings indicate that diseases caused by various prions, namely various transmissible proteins, appear to be a threat, particularly in the current longevity society. Prion disease represented by CJD has obvious transmissibility and is considered to be an "archetype of various neurodegenerative diseases". Overcoming prion diseases is a top priority currently in our society, and this strategy will certainly contribute to elucidating pathomechanism of other neurodegenerative diseases and developing new therapies for them.

Modulation of prion protein expression through cryptic splice site manipulation.

Lowering expression of prion protein (PrP) is a well-validated therapeutic strategy in prion disease, but additional modalities are urgently needed. In other diseases, small molecules have proven capable of modulating pre-mRNA splicing, sometimes by forcing inclusion of cryptic exons that reduce gene expression. Here, we characterize a cryptic exon located in human PRNP's sole intron and evaluate its potential to reduce PrP expression through incorporation into the 5' untranslated region. This exon is homologous to exon 2 in nonprimate species but contains a start codon that would yield an upstream open reading frame with a stop codon prior to a splice site if included in PRNP mRNA, potentially downregulating PrP expression through translational repression or nonsense-mediated decay. We establish a minigene transfection system and test a panel of splice site alterations, identifying mutants that reduce PrP expression by as much as 78%. Our findings nominate a new therapeutic target for lowering PrP.

Updated global epidemiology atlas of human prion diseases.

Introduction: Human prion disease (PrD), a group of fatal and transmissible neurodegenerative diseases, consists of Creutzfeldt-Jakob disease (CJD), kuru, fatal familial insomnia (FFI), Gerstmann-Sträussler-Scheinker disease (GSS), and variably protease-sensitive prionopathy (VPSPr). The emergence of bovine spongiform encephalopathy (BSE) in cattle and variant CJD (vCJD) has greatly threatened public health, both in humans and animals. Since the 1990's, dozens of countries and territories have conducted PrD surveillance programs. Methods: In this study, the case numbers and alternative trends of different types of PrD globally and in various countries or territories from 1993 to 2020 were collected and analyzed based on the data from the websites of the international and national PrD surveillance programs, as well as from relevant publications. Results: The total numbers of the reported PrD and sporadic CJD (sCJD) cases in 34 countries with accessible annual case numbers were 27,872 and 24,623, respectively. The top seven countries in PrD cases were the USA (n = 5,156), France (n = 3,276), Germany (n = 3,212), Italy (n = 2,995), China (n = 2,662), the UK (n = 2,521), Spain (n = 1,657), and Canada (n = 1,311). The annual PrD case numbers and mortalities, either globally or in the countries, showed an increased trend in the past 27 years. Genetic PrD cases accounted for 10.83% of all reported PrD cases; however, the trend varied largely among the different countries and territories. There have been 485 iatrogenic CJD (iCJD) cases and 232 vCJD cases reported worldwide. Discussion: The majority of the countries with PrD surveillance programs were high- and upper-middle-income countries. However, most low- and lower-middle-income countries in the world did not conduct PrD surveillance or even report PrD cases, indicating that the number of human PrD cases worldwide is markedly undervalued. Active international PrD surveillance for both humans and animals is still vital to eliminate the threat of prion disease from a public health perspective.

Simple 3D spheroid cell culture model for studies of prion infection.

Mouse neuronal CAD 5 cell line effectively propagates various strains of prions. Previously, we have shown that it can also be differentiated into the cells morphologically resembling neurons. Here, we demonstrate that CAD 5 cells chronically infected with prions undergo differentiation under the same conditions. To make our model more realistic, we triggered the differentiation in the 3D culture created by gentle rocking of CAD 5 cell suspension. Spheroids formed within 1 week and were fully developed in less than 3 weeks of culture. The mature spheroids had a median size of ~300 µm and could be cultured for up to 12 weeks. Increased expression of differentiation markers GAP 43, tyrosine hydroxylase, ß-III-tubulin and SNAP 25 supported the differentiated status of the spheroid cells. The majority of them were found in the G0/G1 phase of the cell cycle, which is typical for differentiated cells. Moreover, half of the PrPC on the cell membrane was N-terminally truncated, similarly as in differentiated CAD 5 adherent cells. Finally, we demonstrated that spheroids could be created from prion-infected CAD 5 cells. The presence of prions was verified by immunohistochemistry, western blot and seed amplification assay. We also confirmed that the spheroids can be infected with the prions de novo. Our 3D culture model of differentiated CAD 5 cells is low cost, easy to produce and cultivable for weeks. We foresee its possible use in the testing of anti-prion compounds and future studies of prion formation dynamics.

Fluid Biomarkers in Individuals at Risk for Genetic Prion Disease up to Disease Conversion.

OBJECTIVES: To longitudinally characterize disease-relevant CSF and plasma biomarkers in individuals at risk for genetic prion disease up to disease conversion. METHODS: This single-center longitudinal cohort study has followed known carriers of PRNP pathogenic variants at risk for prion disease, individuals with a close relative who died of genetic prion disease but who have not undergone predictive genetic testing, and controls. All participants were asymptomatic at first visit and returned roughly annually. We determined PRNP genotypes, measured NfL and GFAP in plasma, and RT-QuIC, total PrP, NfL, T-tau, and beta-synuclein in CSF. RESULTS: Among 41 carriers and 21 controls enrolled, 28 (68%) and 15 (71%) were female, and mean ages were 47.5 and 46.1. At baseline, all individuals were asymptomatic. We observed RT-QuIC seeding activity in the CSF of 3 asymptomatic E200K carriers who subsequently converted to symptomatic and died of prion disease. 1 P102L carrier remained RT-QuIC negative through symptom conversion. No other individuals developed symptoms. The prodromal window from detection of RT-QuIC positivity to disease onset was 1 year long in an E200K individual homozygous (V/V) at PRNP codon 129 and 2.5 and 3.1 years in 2 codon 129 heterozygotes (M/V). Changes in neurodegenerative and neuroinflammatory markers were variably observed prior to onset, with increases observed for plasma NfL in 4/4 converters, and plasma GFAP, CSF NfL, CSF T-tau, and CSF beta-synuclein each in 2/4 converters, although values relative to age and fold changes relative to individual baseline were not remarkable for any of these markers. CSF PrP was longitudinally stable with mean coefficient of variation 9.0% across all individuals over up to 6 years, including data from converting individuals at RT-QuIC-positive timepoints. DISCUSSION: CSF prion seeding activity may represent the earliest detectable prodromal sign in E200K carriers. Neuronal damage and neuroinflammation markers show limited sensitivity in the prodromal phase. CSF PrP levels remain stable even in the presence of RT-QuIC seeding activity. CLINICAL TRIALS REGISTRATION: ClinicalTrials.gov NCT05124392 posted 2017-12-01, updated 2023-01-27.