SIRT1 Regulates Mitochondrial Damage in N2a Cells Treated with the Prion Protein Fragment 106-126 via PGC-1α-TFAM-Mediated Mitochondrial Biogenesis.

Mitochondrial damage is an early and key marker of neuronal damage in prion diseases. As a process involved in mitochondrial quality control, mitochondrial biogenesis regulates mitochondrial homeostasis in neurons and promotes neuron health by increasing the number of effective mitochondria in the cytoplasm. Sirtuin 1 (SIRT1) is a NAD+-dependent deacetylase that regulates neuronal mitochondrial biogenesis and quality control in neurodegenerative diseases via deacetylation of a variety of substrates. In a cellular model of prion diseases, we found that both SIRT1 protein levels and deacetylase activity decreased, and SIRT1 overexpression and activation significantly ameliorated mitochondrial morphological damage and dysfunction caused by the neurotoxic peptide PrP106-126. Moreover, we found that mitochondrial biogenesis was impaired, and SIRT1 overexpression and activation alleviated PrP106-126-induced impairment of mitochondrial biogenesis in N2a cells. Further studies in PrP106-126-treated N2a cells revealed that SIRT1 regulates mitochondrial biogenesis through the PGC-1α-TFAM pathway. Finally, we showed that resveratrol resolved PrP106-126-induced mitochondrial dysfunction and cell apoptosis by promoting mitochondrial biogenesis through activation of the SIRT1-dependent PGC-1α/TFAM signaling pathway in N2a cells. Taken together, our findings further describe SIRT1 regulation of mitochondrial biogenesis and improve our understanding of mitochondria-related pathogenesis in prion diseases. Our findings support further investigation of SIRT1 as a potential target for therapeutic intervention of prion diseases.

Screening of Anti-Prion Compounds Using the Protein Misfolding Cyclic Amplification Technology.

Prion diseases are 100% fatal infectious neurodegenerative diseases affecting the brains of humans and other mammals. The disease is caused by the formation and replication of prions, composed exclusively of the misfolded prion protein (PrPSc). We invented and developed the protein misfolding cyclic amplification (PMCA) technology for in vitro prion replication, which allow us to replicate the infectious agent and it is commonly used for ultra-sensitive prion detection in biological fluids, tissues and environmental samples. In this article, we studied whether PMCA can be used to screen for chemical compounds that block prion replication. A small set of compounds previously shown to have anti-prion activity in various systems, mostly using cells infected with murine prions, was evaluated for their ability to prevent the replication of prions. Studies were conducted simultaneously with prions derived from 4 species, including human, cattle, cervid and mouse. Our results show that only one of these compounds (methylene blue) was able to completely inhibit prion replication in all species. Estimation of the IC50 for methylene blue inhibition of human prions causing variant Creutzfeldt-Jakob disease (vCJD) was 7.7 µM. Finally, we showed that PMCA can be used for structure-activity relationship studies of anti-prion compounds. Interestingly, some of the less efficient prion inhibitors altered the replication of prions in some species and not others, suggesting that PMCA is useful for studying the differential selectivity of potential drugs.

Temporal Characterization of Prion Shedding in Secreta of White-Tailed Deer in Longitudinal Study of Chronic Wasting Disease, United States.

Chronic wasting disease (CWD) affects cervids in North America, Asia, and Scandinavia. CWD is unique in its efficient spread, partially because of contact with infectious prions shed in secreta. To assess temporal profiles of CWD prion shedding, we collected saliva, urine, and feces from white-tailed deer for 66 months after exposure to low oral doses of CWD-positive brain tissue or saliva. We analyzed prion seeding activity by using modified amyloid amplification assays incorporating iron oxide bead extraction, which improved CWD detection and reduced false positives. CWD prions were detected in feces, urine, and saliva as early as 6 months postinfection. More frequent and consistent shedding was observed in deer homozygous for glycine at prion protein gene codon 96 than in deer expressing alternate genotypes. Our findings demonstrate that improved amplification methods can be used to identify early antemortem CWD prion shedding, which might aid in disease surveillance of cervids.

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.

Variation in the prion protein gene (PRNP) open reading frame sequence in French cervids.

The recent emergence of chronic wasting disease (CWD) in Europe has become a new public health risk for monitoring of wild and farmed cervids. This disease, due to prions, has proliferated in North America in a contagious manner. In several mammalian species, polymorphisms in the prion protein gene (PRNP) play a crucial role in the susceptibility to prions and their spread. To obtain a reliable picture of the distribution of PRNP polymorphisms in the two most common cervid species in France, we sequenced the open reading frame (ORF) of this gene in 2114 animals, 1116 roe deer (Capreolus capreolus) and 998 red deer (Cervus elaphus). Selection criteria such as historical origin, spatial distribution and sex ratio have been integrated to establish this sample collection. Except for one heterozygous animal with a non-synonymous mutation at codon 37 (G37A), all the 1116 French roe deer were monomorphic. Red deer showed greater variation with two non-synonymous substitutions (T98A; Q226E), three synonymous substitutions (codons 21, 78 and 136) and a new 24pb deletion (Δ69-77). We found significant regional variations between French regions in the frequency of the identified substitutions. After cloning of the PRNP ORF from animals presenting multiple non-synonymous polymorphisms, we identified six haplotypes and obtained a total of twelve genotypes. As in other European countries, we highlighted the apparent homogeneity of PRNP in the French roe deer and the existence of a greater diversity in the red deer. These results were in line with European phylogeographic studies on these two species.

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.

Expression of the cellular prion protein by mast cells in white-tailed deer carotid body, cervical lymph nodes and ganglia.

Chronic wasting disease (CWD) is a transmissible and fatal prion disease that affects cervids. While both oral and nasal routes of exposure to prions cause disease, the spatial and temporal details of how prions enter the central nervous system (CNS) are unknown. Carotid bodies (CBs) are structures that are exposed to blood-borne prions and are densely innervated by nerves that are directly connected to brainstem nuclei, known to be early sites of prion neuroinvasion. All CBs examined contained mast cells expressing the prion protein which is consistent with these cells playing a role in neuroinvasion following prionemia.

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.

Severe neurodegeneration in brains of transgenic rats producing human tau prions.

Both wild-type and mutant tau proteins can misfold into prions and self-propagate in the central nervous system of animals and people. To extend the work of others, we investigated the molecular basis of tau prion-mediated neurodegeneration in transgenic (Tg) rats expressing mutant human tau (P301S); this line of Tg rats is denoted Tg12099. We used the rat Prnp promoter to drive the overexpression of mutant tau (P301S) in the human 0N4R isoform. In Tg12099(+/+) rats homozygous for the transgene, ubiquitous expression of mutant human tau resulted in the progressive accumulation of phosphorylated tau inclusions, including silver-positive tangles in the frontal cortices and limbic system. Signs of central nervous system dysfunction were found in terminal Tg12099(+/+) rats exhibiting severe neurodegeneration and profound atrophy of the amygdala and piriform cortex. The greatest increases in tau prion activity were found in the corticolimbic structures. In contrast to the homozygous Tg12099(+/+) rats, we found lower levels of mutant tau in the hemizygous rats, resulting in few neuropathologic changes up to 2 years of age. Notably, these hemizygous rats could be infected by intracerebral inoculation with recombinant tau fibrils or precipitated tau prions from the brain homogenates of sick, aged homozygous Tg12099(+/+) rats. Our studies argue that the regional propagation of tau prions and neurodegeneration in the Tg12099 rats resembles that found in human primary tauopathies. These findings seem likely to advance our understanding of human tauopathies and may lead to effective therapeutics for Alzheimer's disease and other tau prion disorders.

Biodetection of an odor signature in white-tailed deer associated with infection by chronic wasting disease prions.

Chronic wasting disease (CWD) has become a major concern among those involved in managing wild and captive cervid populations. CWD is a fatal, highly transmissible spongiform encephalopathy caused by an abnormally folded protein, called a prion. Prions are present in a number of tissues, including feces and urine in CWD infected animals, suggesting multiple modes of transmission, including animal-to-animal, environmental, and by fomite. CWD management is complicated by the lack of practical, non-invasive, live-animal screening tests. Recently, there has been a focus on how the volatile odors of feces and urine can be used to discriminate between infected and noninfected animals in several different species. Such a tool may prove useful in identifying potentially infected live animals, carcasses, urine, feces, and contaminated environments. Toward this goal, dogs were trained to detect and discriminate CWD infected individuals from non-infected deer in a laboratory setting. Dogs were tested with novel panels of fecal samples demonstrating the dogs' ability to generalize a learned odor profile to novel odor samples based on infection status. Additionally, dogs were transitioned from alerting to fecal samples to an odor profile that consisted of CWD infection status with a different odor background using different sections of gastrointestinal tracts. These results indicated that canine biodetectors can discriminate the specific odors emitted from the feces of non-infected versus CWD infected white-tailed deer as well as generalizing the learned response to other tissues collected from infected individuals. These findings suggest that the health status of wild and farmed cervids can be evaluated non-invasively for CWD infection via monitoring of volatile metabolites thereby providing an effective tool for rapid CWD surveillance.

Prion protein gene (PRNP) variation in German and Danish cervids.

The structure of cellular prion proteins encoded by the prion protein gene (PRNP) impacts susceptibility to transmissible spongiform encephalopathies, including chronic wasting disease (CWD) in deer. The recent emergence of CWD in Northern European reindeer (Rangifer tarandus), moose (Alces alces alces) and red deer (Cervus elaphus), in parallel with the outbreak in North America, gives reason to investigate PRNP variation in European deer, to implement risk assessments and adjust CWD management for deer populations under threat. We here report PRNP-sequence data from 911 samples of German red, roe (Capreolus capreolus), sika (Cervus nippon) and fallow deer (Dama dama) as well as additional data from 26 Danish red deer close to the German border and four zoo species not native to Germany. No PRNP sequence variation was observed in roe and fallow deer, as previously described for populations across Europe. In contrast, a broad PRNP variation was detected in red deer, with non-synonymous polymorphisms at codons 98, 226 and 247 as well as synonymous mutations at codons 21, 78, 136 and 185. Moreover, a novel 24 bp deletion within the octapeptide repeat was detected. In summary, 14 genotypes were seen in red deer with significant differences in their geographical distribution and frequencies, including geographical clustering of certain genotypes, suggesting "PRNP-linages" in this species. Based on data from North American CWD and the genotyping results of the European CWD cases, we would predict that large proportions of wild cervids in Europe might be susceptible to CWD once introduced to naive populations.

Involvement of the nigrostriatal system in Gerstman-Sträussler-Scheinker disease with the PRNP-P102L mutation.

INTRODUCTION: Gerstmann-Sträussler-Scheinker disease (GSS) is an autosomal-dominant inherited prion disease most often associated with the human prion protein gene (PRNP)-P102L mutation. Although patients manifest considerable phenotypic heterogeneity, the involvement of the nigrostriatal system has not been well-studied. METHODS: We performed dopamine transporter single-photon emission computed tomography (DAT-SPECT) using 123I-ioflupane to investigate the nigrostriatal system function in nine patients with the PRNP-P102L mutation. We also examined the pathological findings in another patient whose predominant feature was ataxia and who died 5 years after disease onset. RESULTS: Striatum uptake of 123I-ioflupane indicated by specific binding ratio (SBR) values was significantly reduced in two patients. The DAT-SPECT examination was performed 6 months after disease onset in one of these patients who manifested rapidly developing cognitive decline mimicking Creutzfeldt-Jakob disease. DAT-SPECT was also performed 9 years after disease onset in another patient who manifested the conventional features of GSS involving ataxia and dementia in the initial phase but showed akinetic mutism at the examination time. Another patient examined 2 years after disease onset who predominantly manifested ataxia showed marginally abnormal SBR values. An autopsy case showed moderate neuronal loss in the substantia nigra, and the degree of neuronal loss was similar in most other parts of the brain. CONCLUSION: Nigrostriatal system involvement may occur in patients with GSS associated with the PRNP-P102L mutation, even though parkinsonism is not the predominant feature.

Wet Conformation of Prion-Like Domain and Intimate Correlation of Hydration and Conformational Fluctuations.

Proteins with prion-like domains (PLDs) are involved in neurodegeneration-associated aggregation and are prevalent in liquid-like membrane-less organelles. These PLDs contain amyloidogenic stretches but can maintain dynamic disordered conformations, even in the condensed phase. However, the molecular mechanism underlying such intricate conformational properties of PLDs remains elusive. Here we employed molecular dynamics simulations to investigate the conformational properties of a prototypical PLD system (i.e., FUS PLD). According to our simulation results, PLD adopts a wet collapsed conformation, wherein most residues maintain sufficient hydration with the abundance of internal water. These internal water molecules can rapidly exchange between the protein interior and the bulk, enabling intensive coupling of the entire protein with its hydration environment. The dynamic exchange of water molecules is intimately correlated to the overall conformational fluctuations of PLD. Furthermore, the abundance of dynamic internal water suppresses the formation of aggregation-prone ordered structures. These results collectively elucidate the crucial role of internal water in sustaining the dynamic disordered conformation of the PLD and inhibiting its aggregation propensity.

Disruption of the nascent polypeptide-associated complex leads to reduced polyglutamine aggregation and toxicity.

The nascent polypeptide-associate complex (NAC) is a heterodimeric chaperone complex that binds near the ribosome exit tunnel and is the first point of chaperone contact for newly synthesized proteins. Deletion of the NAC induces embryonic lethality in many multi-cellular organisms. Previous work has shown that the deletion of the NAC rescues cells from prion-induced cytotoxicity. This counterintuitive result led us to hypothesize that NAC disruption would improve viability in cells expressing human misfolding proteins. Here, we show that NAC disruption improves viability in cells expressing expanded polyglutamine and also leads to delayed and reduced aggregation of expanded polyglutamine and changes in polyglutamine aggregate morphology. Moreover, we show that NAC disruption leads to changes in de novo yeast prion induction. These results indicate that the NAC plays a critical role in aggregate organization as a potential therapeutic target in neurodegenerative disorders.

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.

Rapid and sensitive determination of residual prion infectivity from prion-decontaminated surfaces.

Prion diseases are untreatable fatal transmissible neurodegenerative diseases that affect a wide range of mammals, including humans, and are caused by PrPSc, the infectious self-templating conformation of the host-encoded protein, PrPC. Prion diseases can be transmitted via surfaces (e.g., forceps, EEG electrodes) in laboratory and clinical settings. Here, we use a combination of surface swabbing and real-time quaking-induced conversion (RT-QuIC) to test for residual surface-associated prions following prion disinfection. We found that treatment of several prion-contaminated laboratory and clinically relevant surfaces with either water or 70% EtOH resulted in robust detection of surface-associated prions. In contrast, treatment of surfaces with sodium hypochlorite resulted in a failure to detect surface-associated prions. RT-QuIC analysis of prion-contaminated stainless steel wires paralleled the findings of the surface swab studies. Importantly, animal bioassay and RT-QuIC analysis of the same swab extracts are in agreement. We report on conditions that may interfere with the assay that need to be taken into consideration before using this technique. Overall, this method can be used to survey laboratory and clinical surfaces for prion infectivity following prion decontamination protocols.IMPORTANCEPrion diseases can be accidentally transmitted in clinical and occupational settings. While effective means of prion decontamination exist, methods for determining the effectiveness are only beginning to be described. Here, we analyze surface swab extracts using real-time quaking-induced conversion (RT-QuIC) to test for residual prions following prion disinfection of relevant clinical and laboratory surfaces. We found that this method can rapidly determine the efficacy of surface prion decontamination. Importantly, examination of surface extracts with RT-QuIC and animal bioassay produced similar findings, suggesting that this method can accurately assess the reduction in prion titer. We identified surface contaminants that interfere with the assay, which may be found in clinical and laboratory settings. Overall, this method can enhance clinical and laboratory prion safety measures.

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.

Norwegian moose CWD induces clinical disease and neuroinvasion in gene-targeted mice expressing cervid S138N prion protein.

Chronic wasting disease (CWD) is a prion disease affecting deer, elk and moose in North America and reindeer, moose and red deer in Northern Europe. Pathogenesis is driven by the accumulation of PrPSc, a pathological form of the host's cellular prion protein (PrPC), in the brain. CWD is contagious among North American cervids and Norwegian reindeer, with prions commonly found in lymphatic tissue. In Nordic moose and red deer CWD appears exclusively in older animals, and prions are confined to the CNS and undetectable in lymphatic tissues, indicating a sporadic origin. We aimed to determine transmissibility, neuroinvasion and lymphotropism of Nordic CWD isolates using gene-targeted mice expressing either wild-type (138SS/226QQ) or S138N (138NN/226QQ) deer PrP. When challenged with North American CWD strains, mice expressing S138N PrP did not develop clinical disease but harbored prion seeding activity in brain and spleen. Here, we infected these models intracerebrally or intraperitoneally with Norwegian moose, red deer and reindeer CWD isolates. The moose isolate was the first CWD type to cause full-blown disease in the 138NN/226QQ model in the first passage, with 100% attack rate and shortened survival times upon second passage. Furthermore, we detected prion seeding activity or PrPSc in brains and spinal cords, but not spleens, of 138NN/226QQ mice inoculated intraperitoneally with the moose isolate, providing evidence of prion neuroinvasion. We also demonstrate, for the first time, that transmissibility of the red deer CWD isolate was restricted to transgenic mice overexpressing elk PrPC (138SS/226EE), identical to the PrP primary structure of the inoculum. Our findings highlight that susceptibility to clinical disease is determined by the conformational compatibility between prion inoculum and host PrP primary structure. Our study indicates that neuroinvasion of Norwegian moose prions can occur without, or only very limited, replication in the spleen, an unprecedented finding for CWD.

[Investigation of the inhibitory potential of caffeic acid phenethyl ester on prion replication, amplification, and fibril formation in vitro].

Objective: To investigate the effects and possible mechanisms of caffeic acid phenethyl ester (CAPE) on the replication, amplification, and fibre formation of prions (PrPSc). Methods: The CCK8 assay was used to detect the cell viability of the prion-infected cell model SMB-S15 after CAPE treatment for 3 days and 7 days and the maximum safe concentration of CAPE for SMB-S15 was obtained. The cells were treated with a concentration within a safe range, and the content of PrPSc in the cells before and after CAPE treatment was analyzed by western blot. Protein misfolding cycle amplification (PMCA) and western blot were used to assess changes in PrPSc level in amplification products following CAPE treatment. Real-time-quaking induced conversion assay (RT-QuIC) technology was employed to explore the changes in fibril formation before and after CAPE treatment. The binding affinity between CAPE and murine recombinant full-length prion protein was determined using a molecular interaction assay. Results: CCK8 cell viability assay results demonstrated that treatment with 1 µmol/L CAPE for 3 and 7 days did not exhibit statistically significant differences in cell viability compared to the control group (all P<0.05). However, when the concentration of CAPE exceeded 1 µmol/L, a significant reduction in cell viability was observed in cells treated with CAPE for 3 and 7 days, compared to the control group (all P<0.05). Thus, 1 µmol/L was determined as the maximum safe concentration of CAPE treatment for SMB-S15 cells. The western blot results revealed that treatment with CAPE for both 3 and 7 days led to a detectable reduction in the levels of PrPSc in SMB-S15 cells (all P<0.05). The products of PMCA experiments were assessed using western blot. The findings revealed a significant decrease in the levels of PrPSc (relative grey value) in the PMCA amplification products of adapted-strains SMB-S15, 139A, and ME7 following treatment with CAPE, as compared to the control group (all P<0.05). The RT-QuIC experimental results demonstrated a reduction in fibril formation (as indicated by ThT peak values) in CAPE-treated mouse-adapted strains 139A, ME7, and SMB-S15, as well as in SMB-S15 cells infected with prions. Furthermore, CAPE exhibited varying degrees of inhibition towards different seed fibrils formation, with statistically significant differences observed (all P<0.05). Notably, CAPE exhibited a more pronounced inhibitory effect on ME7 seed fibrils. Molecular interaction analyses demonstrated significant binding between CAPE and murine recombinant prion protein, and the association constant was (2.92±0.41)×10-6 mol/L. Conclusions: CAPE inhibits PrPSc replication, amplification, and fibril formation in vitro possibly due to specific interactions with the prion protein at the molecular level.

Assessment of the Zoonotic Potential of Atypical Scrapie Prions in Humanized Mice Reveals Rare Phenotypic Convergence but Not Identity With Sporadic Creutzfeldt-Jakob Disease Prions.

BACKGROUND: Atypical/Nor98 scrapie (AS) is an idiopathic infectious prion disease affecting sheep and goats. Recent findings suggest that zoonotic prions from classical bovine spongiform encephalopathy (C-BSE) may copropagate with atypical/Nor98 prions in AS sheep brains. Investigating the risk AS poses to humans is crucial. METHODS: To assess the risk of sheep/goat-to-human transmission of AS, we serially inoculated brain tissue from field and laboratory isolates into transgenic mice overexpressing human prion protein (Met129 allele). We studied clinical outcomes as well as presence of prions in brains and spleens. RESULTS: No transmission occurred on the primary passage, with no clinical disease or pathological prion protein in brains and spleens. On subsequent passages, 1 isolate gradually adapted, manifesting as prions with a phenotype resembling those causing MM1-type sporadic Creutzfeldt-Jakob disease in humans. However, further characterization using in vivo and in vitro techniques confirmed both prion agents as different strains, revealing a case of phenotypic convergence. Importantly, no C-BSE prions emerged in these mice, especially in the spleen, which is more permissive than the brain for C-BSE cross-species transmission. CONCLUSIONS: The results obtained suggest a low zoonotic potential for AS. Rare adaptation may allow the emergence of prions phenotypically resembling those spontaneously forming in humans.