Detection of Chronic Wasting Disease Prions in Prairie Soils from Endemic Regions.

Chronic wasting disease (CWD) is a contagious prion disease that affects cervids in North America, Northern Europe, and South Korea. CWD is spread through direct and indirect horizontal transmission, with both clinical and preclinical animals shedding CWD prions in saliva, urine, and feces. CWD particles can persist in the environment for years, and soils may pose a risk for transmission to susceptible animals. Our study presents a sensitive method for detecting prions in the environmental samples of prairie soils. Soils were collected from CWD-endemic regions with high (Saskatchewan, Canada) and low (North Dakota, USA) CWD prevalence. Heat extraction with SDS-buffer, a serial protein misfolding cyclic amplification assay coupled with a real-time quaking-induced conversion assay was used to detect the presence of CWD prions in soils. In the prairie area of South Saskatchewan where the CWD prevalence rate in male mule deer is greater than 70%, 75% of the soil samples tested were positive, while in the low-prevalence prairie region of North Dakota (11% prevalence in male mule deer), none of the soils contained prion seeding activity. Soil-bound CWD prion detection has the potential to improve our understanding of the environmental spread of CWD, benefiting both surveillance and mitigation approaches.

Emergence of CWD strains.

Chronic wasting disease (CWD) strains present a novel challenge to defining and mitigating this contagious prion disease of deer, elk, moose, and reindeer. Similar to strains of other prion diseases (bovine spongiform encephalopathy, sheep scrapie), CWD strains can affect biochemical and neuropathological properties of the infectious agent, and importantly interspecies transmission. To date, ten CWD strains have been characterized. The expanding range of CWD in North America and its presence in South Korea as well as Scandinavian countries will potentially result in millions of cervids infected with CWD; thus, novel strains will continue to emerge. In this review, we will summarize the characteristics of known CWD strains and describe the impact of prion protein gene polymorphisms on the generation of strains. We will also discuss the evidence that individual cervids can harbor more than one CWD strain, complicating strain analysis, and affecting selection and adaptation of strains in new hosts.

In Vitro and In Vivo Evidence towards Fibronectin's Protective Effects against Prion Infection.

A distinctive signature of the prion diseases is the accumulation of the pathogenic isoform of the prion protein, PrPSc, in the central nervous system of prion-affected humans and animals. PrPSc is also found in peripheral tissues, raising concerns about the potential transmission of pathogenic prions through human food supplies and posing a significant risk to public health. Although muscle tissues are considered to contain levels of low prion infectivity, it has been shown that myotubes in culture efficiently propagate PrPSc. Given the high consumption of muscle tissue, it is important to understand what factors could influence the establishment of a prion infection in muscle tissue. Here we used in vitro myotube cultures, differentiated from the C2C12 myoblast cell line (dC2C12), to identify factors affecting prion replication. A range of experimental conditions revealed that PrPSc is tightly associated with proteins found in the systemic extracellular matrix, mostly fibronectin (FN). The interaction of PrPSc with FN decreased prion infectivity, as determined by standard scrapie cell assay. Interestingly, the prion-resistant reserve cells in dC2C12 cultures displayed a FN-rich extracellular matrix while the prion-susceptible myotubes expressed FN at a low level. In agreement with the in vitro results, immunohistopathological analyses of tissues from sheep infected with natural scrapie demonstrated a prion susceptibility phenotype linked to an extracellular matrix with undetectable levels of FN. Conversely, PrPSc deposits were not observed in tissues expressing FN. These data indicate that extracellular FN may act as a natural barrier against prion replication and that the extracellular matrix composition may be a crucial feature determining prion tropism in different tissues.

Neural transcriptomic signature of chronic wasting disease in white-tailed deer.

BACKGROUND: The increasing prevalence and expanding geographical range of the chronic wasting disease (CWD) panzootic in cervids is threatening human, animal, environmental and economic health. The pathogenesis of CWD in cervids is, however, not well understood. We used RNA sequencing (RNA-seq) to compare the brain transcriptome from white-tailed deer (WTD; Odocoileus virginianus) clinically affected with CWD (n = 3) to WTD that tested negative (n = 8) for CWD. In addition, one preclinical CWD+ brain sample was analyzed by RNA-seq. RESULTS: We found 255 genes that were significantly deregulated by CWD, 197 of which were upregulated. There was a high degree of overlap in differentially expressed genes (DEGs) identified when using either/both the reference genome assembly of WTD for mapping sequenced reads to or the better characterized genome assembly of a closely related model species, Bos taurus. Quantitative PCR of a subset of the DEGs confirmed the RNA-seq data. Gene ontology term enrichment analysis found a majority of genes involved in immune activation, consistent with the neuroinflammatory pathogenesis of prion diseases. A metagenomic analysis of the RNA-seq data was conducted to look for the presence of spiroplasma and other bacteria in CWD infected deer brain tissue. CONCLUSIONS: The gene expression changes identified highlight the role of innate immunity in prion infection, potential disease associated biomarkers and potential targets for therapeutic agents. An association between CWD and spiroplasma infection was not found.

Prion protein lowering is a disease-modifying therapy across prion disease stages, strains and endpoints.

Lowering of prion protein (PrP) expression in the brain is a genetically validated therapeutic hypothesis in prion disease. We recently showed that antisense oligonucleotide (ASO)-mediated PrP suppression extends survival and delays disease onset in intracerebrally prion-infected mice in both prophylactic and delayed dosing paradigms. Here, we examine the efficacy of this therapeutic approach across diverse paradigms, varying the dose and dosing regimen, prion strain, treatment timepoint, and examining symptomatic, survival, and biomarker readouts. We recapitulate our previous findings with additional PrP-targeting ASOs, and demonstrate therapeutic benefit against four additional prion strains. We demonstrate that <25% PrP suppression is sufficient to extend survival and delay symptoms in a prophylactic paradigm. Rise in both neuroinflammation and neuronal injury markers can be reversed by a single dose of PrP-lowering ASO administered after the detection of pathological change. Chronic ASO-mediated suppression of PrP beginning at any time up to early signs of neuropathology confers benefit similar to constitutive heterozygous PrP knockout. Remarkably, even after emergence of frank symptoms including weight loss, a single treatment prolongs survival by months in a subset of animals. These results support ASO-mediated PrP lowering, and PrP-lowering therapeutics in general, as a promising path forward against prion disease.

Chronic wasting disease (CWD) prion strains evolve via adaptive diversification of conformers in hosts expressing prion protein polymorphisms.

Chronic wasting disease (CWD) is caused by an unknown spectrum of prions and has become enzootic in populations of cervid species that express cellular prion protein (PrPC) molecules varying in amino acid composition. These PrPC polymorphisms can affect prion transmission, disease progression, neuropathology, and emergence of new prion strains, but the mechanistic steps in prion evolution are not understood. Here, using conformation-dependent immunoassay, conformation stability assay, and protein-misfolding cyclic amplification, we monitored the conformational and phenotypic characteristics of CWD prions passaged through deer and transgenic mice expressing different cervid PrPC polymorphisms. We observed that transmission through hosts with distinct PrPC sequences diversifies the PrPCWD conformations and causes a shift toward oligomers with defined structural organization, replication rate, and host range. When passaged in host environments that restrict prion replication, distinct co-existing PrPCWD conformers underwent competitive selection, stabilizing a new prion strain. Nonadaptive conformers exhibited unstable replication and accumulated only to low levels. These results suggest a continuously evolving diversity of CWD conformers and imply a critical interplay between CWD prion plasticity and PrPC polymorphisms during prion strain evolution.

Chronic wasting disease: a cervid prion infection looming to spillover.

The spread of chronic wasting disease (CWD) during the last six decades has resulted in cervid populations of North America where CWD has become enzootic. This insidious disease has also been reported in wild and captive cervids from other continents, threatening ecosystems, livestock and public health. These CWD "hot zones" are particularly complex given the interplay between cervid PRNP genetics, the infection biology, the strain diversity of infectious prions and the long-term environmental persistence of infectivity, which hinder eradication efforts. Here, we review different aspects of CWD including transmission mechanisms, pathogenesis, epidemiology and assessment of interspecies infection. Further understanding of these aspects could help identify "control points" that could help reduce exposure for humans and livestock and decrease CWD spread between cervids.

Mitochondrial DNA deletion mutations increase exponentially with age in human skeletal muscle.

BACKGROUND: Mitochondrial DNA (mtDNA) deletion mutations lead to electron transport chain-deficient cells and age-induced cell loss in multiple tissues and mammalian species. Accurate quantitation of somatic mtDNA deletion mutations could serve as an index of age-induced cell loss. Quantitation of mtDNA deletion molecules is confounded by their low abundance in tissue homogenates, the diversity of deletion breakpoints, stochastic accumulation in single cells, and mosaic distribution between cells. AIMS: Translate a pre-clinical assay to quantitate mtDNA deletions for use in human DNA samples, with technical and biological validation, and test this assay on human subjects of different ages. METHODS: We developed and validated a high-throughput droplet digital PCR assay that quantitates human mtDNA deletion frequency. RESULTS: Analysis of human quadriceps muscle samples from 14 male subjects demonstrated that mtDNA deletion frequency increases exponentially with age-on average, a 98-fold increase from age 20-80. Sequence analysis of amplification products confirmed the specificity of the assay for human mtDNA deletion breakpoints. Titration of synthetic mutation mixtures found a lower limit of detection of at least 0.6 parts per million. Using muscle DNA from 6-month-old mtDNA mutator mice, we measured a 6.4-fold increase in mtDNA deletion frequency (i.e., compared to wild-type mice), biologically validating the approach. DISCUSSION/CONCLUSIONS: The exponential increase in mtDNA deletion frequency is concomitant with the known muscle fiber loss and accelerating mortality that occurs with age. The improved assay permits the accurate and sensitive quantification of deletion mutations from DNA samples and is sufficient to measure changes in mtDNA deletion mutation frequency in healthy individuals across the lifespan and, therefore, patients with suspected mitochondrial diseases.

A General Mass Spectrometry-Based Method of Quantitating Prion Polymorphisms from Heterozygous Chronic Wasting Disease-Infected Cervids.

Chronic wasting disease (CWD) is the only prion disease naturally transmitted among farmed and free-ranging cervids (deer, elk, moose, etc.). These diseases are always fatal and have long asymptomatic incubation periods. By 2019, CWD-infected cervids had been detected in 26 states, three Canadian provinces, South Korea, Norway, Finland, and Sweden. Prions (PrPSc) replicate by inducing a normal cellular prion protein (PrPC) to adopt the prion conformation. This prion templated conformational conversion is influenced by PrPC polymorphisms. Cervid PrPC contains at least 20 different polymorphic sites. By using chymotrypsin, trypsin, or trypsin followed by chymotrypsin to digest denatured cervid PrP, 19 peptides suitable for multiple reaction monitoring (MRM)-based analysis and spanning positions 30-51, 61-112, and 114-231 of cervid PrP were identified. Ten of these peptides span polymorphism-containing regions of cervid PrP. The other nine contain no polymorphisms, so they can be used as internal standards. Calibration curves relating the area ratios of MRM signals from polymorphism-containing peptides to appropriate internal standard peptides were linear and had excellent correlation coefficients. Samples from heterozygous (G96/S96) white-tailed deer orally dosed with CWD from homozygous (G96/G96) deer were analyzed. The G96 polymorphism comprised 75 ± 5% of the total PrP from the G96/S96 heterozygotes. Heterozygous animals facilitate conversion of different PrPC polymorphisms into PrPSc. This approach can be used to quantitate the relative amounts of the polymorphisms present in other animal species and even humans.

Soil humic acids degrade CWD prions and reduce infectivity.

Chronic wasting disease (CWD), an environmentally transmissible, fatal prion disease is endemic in North America, present in South Korea and has recently been confirmed in northern Europe. The expanding geographic range of this contagious disease of free-ranging deer, moose, elk and reindeer has resulted in increasing levels of prion infectivity in the environment. Soils are involved in CWD horizontal transmission, acting as an environmental reservoir, and soil mineral and organic compounds have the ability to bind prions. Upper horizons of soils are usually enriched with soil organic matter (SOM), however, the role of SOM in prion conservation and mobility remains unclear. In this study, we show that incubation of PrPCWD with humic acids (HA), a major SOM compound, affects both the molecular weight and recovery of PrPCWD. Detection of PrPCWD is reduced as HA concentration increases. Native HA extracted from pristine soils also reduces or entirely eliminates PrPCWD signal. Incubation of CWD prions with HA significantly increased incubation periods in tgElk mice demonstrating that HA can reduce CWD infectivity.

Novel Sarcopenia-related Alterations in Sarcomeric Protein Post-translational Modifications (PTMs) in Skeletal Muscles Identified by Top-down Proteomics.

Sarcopenia, the age-related loss of skeletal muscle mass and strength, is a significant cause of morbidity in the elderly and is a major burden on health care systems. Unfortunately, the underlying molecular mechanisms in sarcopenia remain poorly understood. Herein, we utilized top-down proteomics to elucidate sarcopenia-related changes in the fast- and slow-twitch skeletal muscles of aging rats with a focus on the sarcomeric proteome, which includes both myofilament and Z-disc proteins-the proteins that constitute the contractile apparatuses. Top-down quantitative proteomics identified significant changes in the post-translational modifications (PTMs) of critical myofilament proteins in the fast-twitch skeletal muscles of aging rats, in accordance with the vulnerability of fast-twitch muscles to sarcopenia. Surprisingly, age-related alterations in the phosphorylation of Cypher isoforms, proteins that localize to the Z-discs in striated muscles, were also noted in the fast-twitch skeletal muscle of aging rats. This represents the first report of changes in the phosphorylation of Z-disc proteins in skeletal muscle during aging. In addition, increased glutathionylation of slow skeletal troponin I, a novel modification that may help protect against oxidative damage, was observed in slow-twitch skeletal muscles. Furthermore, we have identified and characterized novel muscle type-specific proteoforms of myofilament proteins and Z-disc proteins, including a novel isoform of the Z-disc protein Enigma. The finding that the phosphorylation of Z-disc proteins is altered in response to aging in the fast-twitch skeletal muscles of aging rats opens new avenues for the investigation of the role of Z-discs in age-related muscle dysfunction.

Prion protein polymorphisms associated with reduced CWD susceptibility limit peripheral PrPCWD deposition in orally infected white-tailed deer.

BACKGROUND: Chronic wasting disease (CWD) is a prion disease affecting members of the Cervidae family. PrPC primary structures play a key role in CWD susceptibility resulting in extended incubation periods and regulating the propagation of CWD strains. We analyzed the distribution of abnormal prion protein (PrPCWD) aggregates in brain and peripheral organs from orally inoculated white-tailed deer expressing four different PRNP genotypes: Q95G96/Q95G96 (wt/wt), S96/wt, H95/wt and H95/S96 to determine if there are substantial differences in the deposition pattern of PrPCWD between different PRNP genotypes. RESULTS: Although we detected differences in certain brain areas, globally, the different genotypes showed similar PrPCWD deposition patterns in the brain. However, we found that clinically affected deer expressing H95 PrPC, despite having the longest survival periods, presented less PrPCWD immunoreactivity in particular peripheral organs. In addition, no PrPCWD was detected in skeletal muscle of any of the deer. CONCLUSIONS: Our data suggest that expression of H95-PrPC limits peripheral accumulation of PrPCWD as detected by immunohistochemistry. Conversely, infected S96/wt and wt/wt deer presented with similar PrPCWD peripheral distribution at terminal stage of disease, suggesting that the S96-PrPC allele, although delaying CWD progression, does not completely limit the peripheral accumulation of the infectious agent.

Chronic Wasting Disease Prion Strain Emergence and Host Range Expansion.

Human and mouse prion proteins share a structural motif that regulates resistance to common chronic wasting disease (CWD) prion strains. Successful transmission of an emergent strain of CWD prion, H95+, into mice resulted in infection. Thus, emergent CWD prion strains may have higher zoonotic potential than common strains.

Top-Down Targeted Proteomics Reveals Decrease in Myosin Regulatory Light-Chain Phosphorylation That Contributes to Sarcopenic Muscle Dysfunction.

Sarcopenia, the loss of skeletal muscle mass and function with advancing age, is a significant cause of disability and loss of independence in the elderly and thus, represents a formidable challenge for the aging population. Nevertheless, the molecular mechanism(s) underlying sarcopenia-associated muscle dysfunction remain poorly understood. In this study, we employed an integrated approach combining top-down targeted proteomics with mechanical measurements to dissect the molecular mechanism(s) in age-related muscle dysfunction. Top-down targeted proteomic analysis uncovered a progressive age-related decline in the phosphorylation of myosin regulatory light chain (RLC), a critical protein involved in the modulation of muscle contractility, in the skeletal muscle of aging rats. Top-down tandem mass spectrometry analysis identified a previously unreported bis-phosphorylated proteoform of fast skeletal RLC and localized the sites of decreasing phosphorylation to Ser14/15. Of these sites, Ser14 phosphorylation represents a previously unidentified site of phosphorylation in RLC from fast-twitch skeletal muscle. Subsequent mechanical analysis of single fast-twitch fibers isolated from the muscles of rats of different ages revealed that the observed decline in RLC phosphorylation can account for age-related decreases in the contractile properties of sarcopenic fast-twitch muscles. These results strongly support a role for decreasing RLC phosphorylation in sarcopenia-associated muscle dysfunction and suggest that therapeutic modulation of RLC phosphorylation may represent a new avenue for the treatment of sarcopenia.

Toll-like receptor-mediated immune response inhibits prion propagation.

Prion diseases are progressive neurodegenerative disorders affecting humans and various mammals. The prominent neuropathological change in prion diseases is neuroinflammation characterized by activation of neuroglia surrounding prion deposition. The cause and effect of this cellular response, however, is unclear. We investigated innate immune defenses against prion infection using primary mixed neuronal and glial cultures. Conditional prion propagation occurred in glial cultures depending on their immune status. Preconditioning of the cells with the toll-like receptor (TLR) ligand, lipopolysaccharide, resulted in a reduction in prion propagation, whereas suppression of the immune responses with the synthetic glucocorticoid, dexamethasone, increased prion propagation. In response to recombinant prion fibrils, glial cells up-regulated TLRs (TLR1 and TLR2) expression and secreted cytokines (tumor necrosis factor-α, interleukin-1ß, interleukin-6, granulocyte-macrophage colony-stimulating factor, and interferon-ß). Preconditioning of neuronal and glial cultures with recombinant prion fibrils inhibited prion replication and altered microglial and astrocytic populations. Our results provide evidence that, in early stages of prion infection, glial cells respond to prion infection through TLR-mediated innate immunity.

Deer Prion Proteins Modulate the Emergence and Adaptation of Chronic Wasting Disease Strains.

UNLABELLED: Transmission of chronic wasting disease (CWD) between cervids is influenced by the primary structure of the host cellular prion protein (PrP(C)). In white-tailed deer, PRNP alleles encode the polymorphisms Q95 G96 (wild type [wt]), Q95 S96 (referred to as the S96 allele), and H95 G96 (referred to as the H95 allele), which differentially impact CWD progression. We hypothesize that the transmission of CWD prions between deer expressing different allotypes of PrP(C) modifies the contagious agent affecting disease spread. To evaluate the transmission properties of CWD prions derived experimentally from deer of four PRNP genotypes (wt/wt, S96/wt, H95/wt, or H95/S96), transgenic (tg) mice expressing the wt allele (tg33) or S96 allele (tg60) were challenged with these prion agents. Passage of deer CWD prions into tg33 mice resulted in 100% attack rates, with the CWD H95/S96 prions having significantly longer incubation periods. The disease signs and neuropathological and protease-resistant prion protein (PrP-res) profiles in infected tg33 mice were similar between groups, indicating that a prion strain (Wisc-1) common to all CWD inocula was amplified. In contrast, tg60 mice developed prion disease only when inoculated with the H95/wt and H95/S96 CWD allotypes. Serial passage in tg60 mice resulted in adaptation of a novel CWD strain (H95(+)) with distinct biological properties. Transmission of first-passage tg60CWD-H95(+) isolates into tg33 mice, however, elicited two prion disease presentations consistent with a mixture of strains associated with different PrP-res glycotypes. Our data indicate that H95-PRNP heterozygous deer accumulated two CWD strains whose emergence was dictated by the PrP(C) primary structure of the recipient host. These findings suggest that CWD transmission between cervids expressing distinct PrP(C) molecules results in the generation of novel CWD strains. IMPORTANCE: CWD prions are contagious among wild and captive cervids in North America and in South Korea. We present data linking the amino acid variant Q95H in white-tailed deer cellular prion protein (PrP(C)) to the emergence of a novel CWD strain (H95(+)). We show that, upon infection, deer expressing H95-PrP(C) molecules accumulated a mixture of CWD strains that selectively propagated depending on the PRNP genotype of the host in which they were passaged. Our study also demonstrates that mice expressing the deer S96-PRNP allele, previously shown to be resistant to various cervid prions, are susceptible to H95(+) CWD prions. The potential for the generation of novel strains raises the possibility of an expanded host range for CWD.

Transcriptomic responses to prion disease in rats.

BACKGROUND: Prions diseases are fatal neurodegenerative diseases of mammals. While the molecular responses to prion infection have been extensively characterized in the laboratory mouse, little is known in other rodents. To explore these responses and make comparisons, we generated a prion disease in the laboratory rat by successive passage beginning with mouse RML prions. RESULTS: We describe the accumulation of rat prions, associated pathology and the transcriptional impact throughout the disease course. Comparative transcriptional profiling between laboratory mice and rats suggests that similar molecular and cellular processes are unfolding in response to prion infection. At the level of individual transcripts, however, variability exists between mice and rats and many genes deregulated by prion infection in mice are not affected in rats. CONCLUSION: Our findings detail the molecular responses to prion disease in the rat and highlight the usefulness of comparative approaches to understanding neurodegeneration and prion diseases in particular.

Infectious prions accumulate to high levels in non proliferative C2C12 myotubes.

Prion diseases are driven by the strain-specific, template-dependent transconformation of the normal cellular prion protein (PrP(C)) into a disease specific isoform PrP(Sc). Cell culture models of prion infection generally use replicating cells resulting in lower levels of prion accumulation compared to animals. Using non-replicating cells allows the accumulation of higher levels of PrP(Sc) and, thus, greater amounts of infectivity. Here, we infect non-proliferating muscle fiber myotube cultures prepared from differentiated myoblasts. We demonstrate that prion-infected myotubes generate substantial amounts of PrP(Sc) and that the level of infectivity produced in these post-mitotic cells, 10(5.5) L.D.50/mg of total protein, approaches that observed in vivo. Exposure of the myotubes to different mouse-adapted agents demonstrates strain-specific replication of infectious agents. Mouse-derived myotubes could not be infected with hamster prions suggesting that the species barrier effect is intact. We suggest that non-proliferating myotubes will be a valuable model system for generating infectious prions and for screening compounds for anti-prion activity.

Gustav Oppenheim (1882-1937) and the Discovery of Cerebral Amyloid Angiopathy.

The discovery of cerebral amyloid angiopathy (CAA) is frequently attributed to Dr. Gustav Oppenheim-a man who has been largely passed over in history. Oppenheim's clinical and neuropathologic research covered a variety of disorders, but his name is best known for his work on senile dementia and CAA. Although Oppenheim was in fact not the first to discover CAA, his neuropathologic observations and inferences on neurodegenerative disease proved to be remarkably faithful to our modern understanding of neurodegenerative diseases. As a neurologist, he served in the First World War and was later subjected to religious persecutions in the leadup to the Holocaust but was not fortunate enough to emigrate before his death. The life, social impact, and previously overlooked contributions to science and medicine by Oppenheim are detailed.

Sheep scrapie and deer rabies in England prior to 1800.

Eighteenth-century England witnessed the emergence of two neurological diseases in animals. Scrapie, a transmissible spongiform encephalopathy, is a fatal neurodegenerative disease of sheep and goats that appears in classical and atypical forms. Reports of classical scrapie in continental Europe with described symptoms date back to 1750 in what is now western Poland. However, two major outbreaks of scrapie appeared in England prior to the 1800s. References to a sheep disease with a resemblance to scrapie first appear in Southwestern England between 1693 and 1722 and in the East Midlands between 1693 and 1706. Concurrent with the descriptions of scrapie in sheep was a neurological disease of deer first appearing in the East of England. Two 18th-century writers remarked on the symptomatic similarities between the sheep and deer neurological diseases. Multiple outbreaks of the unknown deer disease existing as early as 1772 are examined and are identified as rabies.