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.

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.

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.

Innate Immune Status of Glia Modulates Prion Propagation in Early Stage of Infection.

Prion diseases are progressive neurodegenerative disorders affecting humans and various mammals. The prominent neuropathological change in prion-affected brains is neuroinflammation, histopathologically characterized by reactive gliosis surrounding prion deposition. The cause and effect of these cellular responses are still unclear. Here we investigate the impact of innate immune responses on prion replication using in vitro cell culture models. Hamster-adapted transmissible mink encephalopathy prions, hyper (HY) and drowsy (DY) strains, were assayed for accumulation of pathogenic prion protein (PrPSc) in primary glial cultures derived from 8-day-old hamster pups. The kinetics of PrPSc accumulation largely depended on prion strain and brain regions from where glial cells originated. Glial cells derived from the cerebellum were susceptible to HY, but resistant to DY strain as determined by western blot analysis, immunocytochemistry, and animal bioassay. Glial cells from the cerebral cortex were, however, refractory to both strains. PrPSc accumulation was affected by innate immune modulators. Priming glial cells with lipopolysaccharide decreased prion replication, whereas pre-treatment with dexamethasone, inhibiting innate immunity, increased susceptibility to DY infection. Our results suggest that neuroinflammation resulting from prion infection is a response to resolve and/or prevent prion propagation in the brain. It implies a therapeutic potential of innate immune modulation in the early stages of prion disease.

Nanopore sequencing identifies a higher frequency and expanded spectrum of mitochondrial DNA deletion mutations in human aging.

Mitochondrial DNA (mtDNA) deletion mutations cause many human diseases and are linked to age-induced mitochondrial dysfunction. Mapping the mutation spectrum and quantifying mtDNA deletion mutation frequency is challenging with next-generation sequencing methods. We hypothesized that long-read sequencing of human mtDNA across the lifespan would detect a broader spectrum of mtDNA rearrangements and provide a more accurate measurement of their frequency. We employed nanopore Cas9-targeted sequencing (nCATS) to map and quantitate mtDNA deletion mutations and develop analyses that are fit-for-purpose. We analyzed total DNA from vastus lateralis muscle in 15 males ranging from 20 to 81 years of age and substantia nigra from three 20-year-old and three 79-year-old men. We found that mtDNA deletion mutations detected by nCATS increased exponentially with age and mapped to a wider region of the mitochondrial genome than previously reported. Using simulated data, we observed that large deletions are often reported as chimeric alignments. To address this, we developed two algorithms for deletion identification which yield consistent deletion mapping and identify both previously reported and novel mtDNA deletion breakpoints. The identified mtDNA deletion frequency measured by nCATS correlates strongly with chronological age and predicts the deletion frequency as measured by digital PCR approaches. In substantia nigra, we observed a similar frequency of age-related mtDNA deletions to those observed in muscle samples, but noted a distinct spectrum of deletion breakpoints. NCATS-mtDNA sequencing allows the identification of mtDNA deletions on a single-molecule level, characterizing the strong relationship between mtDNA deletion frequency and chronological aging.

Movement of Chronic Wasting Disease Prions in Prairie, Boreal and Alpine Soils.

Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy negatively impacting cervids on three continents. Soil can serve as a reservoir for horizontal transmission of CWD by interaction with the infectious prion protein (PrPCWD) shed by diseased individuals and from infected carcasses. We investigated the pathways for PrPCWD migration in soil profiles using lab-scale soil columns, comparing PrPCWD migration through pure soil minerals (quartz, illite and montmorillonite), and diverse soils from boreal (Luvisol, Brunisol) and prairie (Chernozem) regions. We analyzed the leachate of the soil columns by immunoblot and protein misfolding cyclic amplification (PMCA) and detected PrP in the leachates of columns composed of quartz, illite, Luvisol and Brunisol. Animal bioassay confirmed the presence of CWD infectivity in the leachates from quartz, illite and Luvisol columns. Leachates from columns with montmorillonite and prairie Chernozems did not contain PrP detectable by immunoblotting or PMCA; bioassay confirmed that the Chernozemic leachate was not infectious. Analysis of the solid phase of the columns confirmed the migration of PrP to lower layers in the illite column, while the strongest signal in the montmorillonite column remained close to the surface. Montmorillonite, the prevalent clay mineral in prairie soils, has the strongest prion binding ability; by contrast, illite, the main clay mineral in northern boreal and tundra soils, does not bind prions significantly. This suggests that in soils of North American CWD-endemic regions (Chernozems), PrPCWD would remain on the soil surface due to avid binding to montmorillonite. In boreal Luvisols and mountain Brunisols, prions that pass through the leaf litter will continue to move through the soil mineral horizon, becoming less bioavailable. In light-textured soils where quartz is a dominant mineral, the majority of the infectious prions will move through the soil profile. Local soil properties may consequently determine the efficiency of environmental transmission of CWD.

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.

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.

Age- and time-dependent mitochondrial genotoxic and myopathic effects of beta-guanidinopropionic acid, a creatine analog, on rodent skeletal muscles.

Beta-guanidinopropionic acid (GPA) is a creatine analog suggested as a treatment for hypertension, diabetes, and obesity, which manifest primarily in older adults. A notable side effect of GPA is the induction of mitochondrial DNA deletion mutations. We hypothesized that mtDNA deletions contribute to muscle aging and used the mutation promoting effect of GPA to examine the impact of mtDNA deletions on muscles with differential vulnerability to aging. Rats were treated with GPA for up to 4 months starting at 14 or 30 months of age. We examined quadriceps and adductor longus muscles as the quadriceps exhibits profound age-induced deterioration, while adductor longus is maintained. GPA decreased body and muscle mass and mtDNA copy number while increasing mtDNA deletion frequency. The interactions between age and GPA treatment observed in the quadriceps were not observed in the adductor longus. GPA had negative mitochondrial effects in as little as 4 weeks. GPA treatment exacerbated mtDNA deletions and muscle aging phenotypes in the quadriceps, an age-sensitive muscle, while the adductor longus was spared. GPA has been proposed for use in age-associated diseases, yet the pharmacodynamics of GPA differ with age and include the detrimental induction of mtDNA deletions, a mitochondrial genotoxic stress that is pronounced in muscles that are most vulnerable to aging. Further research is needed to determine if the proposed benefits of GPA on hypertension, diabetes, and obesity outweigh the detrimental mitochondrial and myopathic side effects.

Chronic wasting disease prions in mule deer interdigital glands.

Chronic wasting disease (CWD) is a geographically expanding, fatal neurodegenerative disease in cervids. The disease can be transmitted directly (animal-animal) or indirectly via infectious prions shed into the environment. The precise mechanisms of indirect CWD transmission are unclear but known sources of the infectious prions that contaminate the environment include saliva, urine and feces. We have previously identified PrPC expression in deer interdigital glands, sac-like exocrine structures located between the digits of the hooves. In this study, we assayed for CWD prions within the interdigital glands of CWD infected deer to determine if they could serve as a source of prion shedding and potentially contribute to CWD transmission. Immunohistochemical analysis of interdigital glands from a CWD-infected female mule deer identified disease-associated PrPCWD within clusters of infiltrating leukocytes adjacent to sudoriferous and sebaceous glands, and within the acrosyringeal epidermis of a sudoriferous gland tubule. Proteinase K-resistant PrPCWD material was amplified by serial protein misfolding cyclic amplification (sPMCA) from soil retrieved from between the hoof digits of a clinically affected mule deer. Blinded testing of interdigital glands from 11 mule deer by real-time quake-induced conversion (RT-QuIC) accurately identified CWD-infected animals. The data described suggests that interdigital glands may play a role in the dissemination of CWD prions into the environment, warranting future investigation.

Remdesivir does not affect mitochondrial DNA copy number or deletion mutation frequency in aged male rats: A short report.

Remdesivir is a leading therapy in patients with moderate to severe coronavirus 2 (SARS-CoV-2) infection; the majority of whom are older individuals. Remdesivir is a nucleoside analog that incorporates into nascent viral RNA, inhibiting RNA-directed RNA polymerases, including that of SARS-CoV-2. Less is known about remdesivir's effects on mitochondria, particularly in older adults where mitochondria are known to be dysfunctional. Furthermore, its effect on age-induced mitochondrial mutations and copy number has not been previously studied. We hypothesized that remdesivir adversely affects mtDNA copy number and deletion mutation frequency in aged rodents. To test this hypothesis, 30-month-old male F333BNF1 rats were treated with remdesivir for three months. To determine if remdesivir adversely affects mtDNA, we measured copy number and mtDNA deletion frequency in rat hearts, kidneys, and skeletal muscles using digital PCR. We found no effects from three months of remdesivir treatment on mtDNA copy number or deletion mutation frequency in 33-month-old rats. These data support the notion that remdesivir does not compromise mtDNA quality or quantity at old age in mammals. Future work should focus on examining additional tissues such as brain and liver, and extend testing to human clinical samples.

Susceptibility of Beavers to Chronic Wasting Disease.

Chronic wasting disease (CWD) is a contagious, fatal, neurodegenerative prion disease of cervids. The expanding geographical range and rising prevalence of CWD are increasing the risk of pathogen transfer and spillover of CWD to non-cervid sympatric species. As beavers have close contact with environmental and food sources of CWD infectivity, we hypothesized that they may be susceptible to CWD prions. We evaluated the susceptibility of beavers to prion diseases by challenging transgenic mice expressing beaver prion protein (tgBeaver) with five strains of CWD, four isolates of rodent-adapted prions and one strain of Creutzfeldt-Jakob disease. All CWD strains transmitted to the tgBeaver mice, with attack rates highest from moose CWD and the 116AG and H95+ strains of deer CWD. Mouse-, rat-, and especially hamster-adapted prions were also transmitted with complete attack rates and short incubation periods. We conclude that the beaver prion protein is an excellent substrate for sustaining prion replication and that beavers are at risk for CWD pathogen transfer and spillover.

Cellular prion protein distribution in the vomeronasal organ, parotid, and scent glands of white-tailed deer and mule deer.

Chronic wasting disease (CWD) is a contagious and fatal transmissible spongiform encephalopathy affecting species of the cervidae family. CWD has an expanding geographic range and complex, poorly understood transmission mechanics. CWD is disproportionately prevalent in wild male mule deer and male white-tailed deer. Sex and species influences on CWD prevalence have been hypothesized to be related to animal behaviours that involve deer facial and body exocrine glands. Understanding CWD transmission potential requires a foundational knowledge of the cellular prion protein (PrPC) in glands associated with cervid behaviours. In this study, we characterized the presence and distribution of PrPC in six integumentary and two non-integumentary tissues of hunter-harvested mule deer (Odocoileus hemionus) and white-tailed deer (O. virginianus). We report that white-tailed deer expressed significantly more PrPC than their mule deer in the parotid, metatarsal, and interdigital glands. Females expressed more PrPC than males in the forehead and preorbital glands. The distribution of PrPC within the integumentary exocrine glands of the face and legs were localized to glandular cells, hair follicles, epidermis, and immune cell infiltrates. All tissues examined expressed sufficient quantities of PrPC to serve as possible sites of prion initial infection, propagation, and shedding.

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.

Metformin Treatment in Old Rats and Effects on Mitochondrial Integrity.

Metformin, a commonly used well-tolerated treatment for type 2 diabetes, is being deployed in clinical trials to ameliorate aging in older nondiabetic humans. Concerningly, some experiments in model organisms have suggested that metformin use at old ages shortens life span and is toxic to mitochondria. The demonstrated safety of metformin therapy in humans and the conflicting data from model organisms compelled us to test the hypothesis that metformin treatment would be toxic to older rats. To define an effective dose in 30-month-old hybrid rats, we evaluated two doses of metformin (0.1%, 0.75% of the diet) and treated the rats for 4 months. Body mass decreased at the 0.75% dose. Neither dose affected mortality between 30 and 34 months of age. We assessed mitochondrial integrity by measuring mitochondrial DNA (mtDNA) copy number and deletion mutation frequency, and mitochondrial respiration in skeletal muscle and the heart. In skeletal muscle, we observed no effect of metformin on quadriceps mass, mtDNA copy number, or deletion frequency. In the heart, metformin-treated rats had higher mtDNA copy number, lower cardiac mass, with no change in mtDNA deletion frequency. Metformin treatment resulted in lower mitochondrial complex I-dependent respiration in the heart. We found that, in old rats, metformin did not compromise mtDNA integrity, did not affect mortality, and may have cardiac benefits. These data provide some reassurance that a metformin intervention in aged mammals is not toxic at appropriate doses.

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.

White-tailed deer S96 prion protein does not support stable in vitro propagation of most common CWD strains.

PrPC variation at residue 96 (G/S) plays an important role in the epidemiology of chronic wasting disease (CWD) in exposed white-tailed deer populations. In vivo studies have demonstrated the protective effect of serine at codon 96, which hinders the propagation of common CWD strains when expressed in homozygosis and increases the survival period of S96/wt heterozygous deer after challenge with CWD. Previous in vitro studies of the transmission barrier suggested that following a single amplification step, wt and S96 PrPC were equally susceptible to misfolding when seeded with various CWD prions. When we performed serial prion amplification in vitro using S96-PrPC, we observed a reduction in the efficiency of propagation with the Wisc-1 or CWD2 strains, suggesting these strains cannot stably template their conformations on this PrPC once the primary sequence has changed after the first round of replication. Our data shows the S96-PrPC polymorphism is detrimental to prion conversion of some CWD strains. These data suggests that deer homozygous for S96-PrPC may not sustain prion transmission as compared to a deer expressing G96-PrPC.

Skeletal muscle mitochondrial DNA copy number and mitochondrial DNA deletion mutation frequency as predictors of physical performance in older men and women.

Mitochondrial DNA (mtDNA) quality and quantity relate to two hallmarks of aging-genomic instability and mitochondrial dysfunction. Physical performance relies on mitochondrial integrity and declines with age, yet the interactions between mtDNA quantity, quality, and physical performance are unclear. Using a validated digital PCR assay specific for mtDNA deletions, we tested the hypothesis that skeletal muscle mtDNA deletion mutation frequency (i.e., a measure of mtDNA quality) or mtDNA copy number predicts physical performance in older adults. Total DNA was isolated from vastus lateralis muscle biopsies and used to quantitate mtDNA copy number and mtDNA deletion frequency by digital PCR. The biopsies were obtained from a cross-sectional cohort of 53 adults aged 50 to 86 years. Before the biopsy procedure, physical performance measurements were collected, including VO2max, modified physical performance test score, 6-min walk distance, gait speed, grip strength, and total lean and leg mass. Linear regression models were used to evaluate the relationships between age, sex, and the outcomes. We found that mtDNA deletion mutation frequency increased exponentially with advancing age. On average from ages 50 to 86, deletion frequency increased from 0.008 to 0.15%, an 18-fold increase. Females may have lower deletion frequencies than males at older ages. We also measured declines in VO2max and mtDNA copy number with age in both sexes. The mtDNA deletion frequency measured from single skeletal muscle biopsies predicted 13.3% of the variation in VO2max. Copy number explained 22.6% of the variation in mtDNA deletion frequency and 10.4% of the lean mass variation. We found predictive relationships between age, mtDNA deletion mutation frequency, mtDNA copy number, and physical performance. These data are consistent with a role for mitochondrial function and genome integrity in maintaining physical performance with age. Analyses of mtDNA quality and quantity in larger cohorts and longitudinal studies could extend our understanding of the importance of mitochondrial DNA in human aging and longevity.

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.

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.