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.

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.

Propagation of distinct CWD prion strains during peripheral and intracerebral challenges of gene-targeted mice.

Since prion diseases result from infection and neurodegeneration of the central nervous system (CNS), experimental characterizations of prion strain properties customarily rely on the outcomes of intracerebral challenges. However, natural transmission of certain prions, including those causing chronic wasting disease (CWD) in elk and deer, depends on propagation in peripheral host compartments prior to CNS infection. Using gene-targeted GtE and GtQ mice, which accurately control cellular elk or deer PrP expression, we assessed the impact that peripheral or intracerebral exposures play on CWD prion strain propagation and resulting CNS abnormalities. Whereas oral and intraperitoneal transmissions produced identical neuropathological outcomes in GtE and GtQ mice and preserved the naturally convergent conformations of elk and deer CWD prions, intracerebral transmissions generated CNS prion strains with divergent biochemical properties in GtE and GtQ mice that were changed compared to their native counterparts. While CWD replication kinetics remained constant during iterative peripheral transmissions and brain titers reflected those found in native hosts, serial intracerebral transmissions produced 10-fold higher prion titers and accelerated incubation times. Our demonstration that peripherally and intracerebrally challenged Gt mice develop dissimilar CNS diseases which result from the propagation of distinct CWD prion strains points to the involvement of tissue-specific cofactors during strain selection in different host compartments. Since peripheral transmissions preserved the natural features of elk and deer prions, whereas intracerebral propagation produced divergent strains, our findings illustrate the importance of experimental characterizations using hosts that not only abrogate species barriers but also accurately recapitulate natural transmission routes of native strains.

A rare case of Sporadic Creutzfeldt-Jakob disease at a remote mountain hospital in the Indian Himalayan Region.

Sporadic Creutzfeldt-Jakob disease (CJD) is a rare neurodegenerative spongiform encephalopathy that causes neuronal derangement secondary to prion protein. Its initial diagnosis is often complex and challenging due to non-specific clinical presentation, lack of awareness, and low clinical suspicion. This disease is invariably fatal, and most patients die within 12 months of presentation. Definite diagnosis of prion disease requires neuropathological analysis, usually done at autopsy. Here, we present the autopsy findings of a 57-year-old male patient, illustrating the complexity of diagnosing this disease early in the clinical course and the need for a broad differential diagnosis at the onset.

RNA-Seq Analysis of Mammalian Prion Disease.

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

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

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

Transmission of Norwegian reindeer CWD to sheep by intracerebral inoculation results in an unusual phenotype and prion distribution.

Chronic wasting disease (CWD), a prion disease affecting cervids, has been known in North America (NA) since the 1960s and emerged in Norway in 2016. Surveillance and studies have revealed that there are different forms of CWD in Fennoscandia: contagious CWD in Norwegian reindeer and sporadic CWD in moose and red deer. Experimental studies have demonstrated that NA CWD prions can infect various species, but thus far, there have been no reports of natural transmission to non-cervid species. In vitro and laboratory animal studies of the Norwegian CWD strains suggest that these strains are different from the NA strains. In this work, we describe the intracerebral transmission of reindeer CWD to six scrapie-susceptible sheep. Detection methods included immunohistochemistry (IHC), western blot (WB), enzyme-linked immunosorbent assay (ELISA), real-time quaking-induced conversion (RT-QuIC) and protein misfolding cyclic amplification (PMCA). In the brain, grey matter vacuolation was limited, while all sheep exhibited vacuolation of the white matter. IHC and WB conventional detection techniques failed to detect prions; however, positive seeding activity with the RT-QuIC and PMCA amplification techniques was observed in the central nervous system of all but one sheep. Prions were robustly amplified in the lymph nodes of all animals, mainly by RT-QuIC. Additionally, two lymph nodes were positive by WB, and one was positive by ELISA. These findings suggest that sheep can propagate reindeer CWD prions after intracerebral inoculation, resulting in an unusual disease phenotype and prion distribution with a low amount of detectable prions.

Protein folding, cellular stress and cancer.

Proteins are acknowledged as the phenotypical manifestation of the genotype, because protein-coding genes carry the information for the strings of amino acids that constitute the proteins. It is widely accepted that protein function depends on the corresponding "native" structure or folding achieved within the cell, and that native protein folding corresponds to the lowest free energy minimum for a given protein. However, protein folding within the cell is a non-deterministic dissipative process that from the same input may produce different outcomes, thus conformational heterogeneity of folded proteins is the rule and not the exception. Local changes in the intracellular environment promote variation in protein folding. Hence protein folding requires "supervision" by a host of chaperones and co-chaperones that help their client proteins to achieve the folding that is most stable according to the local environment. Such environmental influence on protein folding is continuously transduced with the help of the cellular stress responses (CSRs) and this may lead to changes in the rules of engagement between proteins, so that the corresponding protein interactome could be modified by the environment leading to an alternative cellular phenotype. This allows for a phenotypic plasticity useful for adapting to sudden and/or transient environmental changes at the cellular level. Starting from this perspective, hereunder we develop the argument that the presence of sustained cellular stress coupled to efficient CSRs may lead to the selection of an aberrant phenotype as the resulting adaptation of the cellular proteome (and the corresponding interactome) to such stressful conditions, and this can be a common epigenetic pathway to cancer.

Cleavage site-directed antibodies reveal the prion protein in humans is shed by ADAM10 at Y226 and associates with misfolded protein deposits in neurodegenerative diseases.

Proteolytic cell surface release ('shedding') of the prion protein (PrP), a broadly expressed GPI-anchored glycoprotein, by the metalloprotease ADAM10 impacts on neurodegenerative and other diseases in animal and in vitro models. Recent studies employing the latter also suggest shed PrP (sPrP) to be a ligand in intercellular communication and critically involved in PrP-associated physiological tasks. Although expectedly an evolutionary conserved event, and while soluble forms of PrP are present in human tissues and body fluids, for the human body neither proteolytic PrP shedding and its cleavage site nor involvement of ADAM10 or the biological relevance of this process have been demonstrated thus far. In this study, cleavage site prediction and generation (plus detailed characterization) of sPrP-specific antibodies enabled us to identify PrP cleaved at tyrosin 226 as the physiological and apparently strictly ADAM10-dependent shed form in humans. Using cell lines, neural stem cells and brain organoids, we show that shedding of human PrP can be stimulated by PrP-binding ligands without targeting the protease, which may open novel therapeutic perspectives. Site-specific antibodies directed against human sPrP also detect the shed form in brains of cattle, sheep and deer, hence in all most relevant species naturally affected by fatal and transmissible prion diseases. In human and animal prion diseases, but also in patients with Alzheimer`s disease, sPrP relocalizes from a physiological diffuse tissue pattern to intimately associate with extracellular aggregated deposits of misfolded proteins characteristic for the respective pathological condition. Findings and research tools presented here will accelerate novel insight into the roles of PrP shedding (as a process) and sPrP (as a released factor) in neurodegeneration and beyond.

Structural Variations of Prions and Prion-like Proteins Associated with Neurodegeneration.

Neurodegeneration is becoming one of the leading causes of death worldwide as the population expands and grows older. There is a growing desire to understand the mechanisms behind prion proteins as well as the prion-like proteins that make up neurodegenerative diseases (NDs), including Alzheimer's disease (AD) and Parkinson's disease (PD). Both amyloid-ß (Aß) and hyperphosphorylated tau (p-tau) proteins behave in ways similar to those of the infectious form of the prion protein, PrPSc, such as aggregating, seeding, and replicating under not yet fully understood mechanisms, thus the designation of prion-like. This review aims to highlight the shared mechanisms between prion-like proteins and prion proteins in the structural variations associated with aggregation and disease development. These mechanisms largely focus on the dysregulation of protein homeostasis, self-replication, and protein aggregation, and this knowledge could contribute to diagnoses and treatments for the given NDs.

Scrapie versus Chronic Wasting Disease in White-Tailed Deer.

White-tailed deer are susceptible to scrapie (WTD scrapie) after oronasal inoculation with the classical scrapie agent from sheep. Deer affected by WTD scrapie are difficult to differentiate from deer infected with chronic wasting disease (CWD). To assess the transmissibility of the WTD scrapie agent and tissue phenotypes when further passaged in white-tailed deer, we oronasally inoculated wild-type white-tailed deer with WTD scrapie agent. We found that WTD scrapie and CWD agents were generally similar, although some differences were noted. The greatest differences were seen in bioassays of cervidized mice that exhibited significantly longer survival periods when inoculated with WTD scrapie agent than those inoculated with CWD agent. Our findings establish that white-tailed deer are susceptible to WTD scrapie and that the presence of WTD scrapie agent in the lymphoreticular system suggests the handling of suspected cases should be consistent with current CWD guidelines because environmental shedding may occur.

In vivo detection of Alzheimer's and Lewy body disease concurrence: Clinical implications and future perspectives.

INTRODUCTION: The recent introduction of seed amplification assays (SAAs) detecting misfolded α-synuclein, a pathology-specific marker for Lewy body disease (LBD), has allowed the in vivo identification and phenotypic characterization of patients with co-occurring Alzheimer's disease (AD) and LBD since the early clinical or even preclinical stage. METHODS: We reviewed studies with an in vivo biomarker-based diagnosis of AD-LBD copathology. RESULTS: Studies in large cohorts of cognitively impaired individuals have shown that cerebrospinal fluid (CSF) biomarkers detect the coexistence of AD and LB pathology in approximately 20%-25% of them, independently of the primary clinical diagnosis. Compared to those with pure AD, AD-LBD patients showed worse global cognition, especially in attentive/executive and visuospatial functions, and worse motor functions. In cognitively unimpaired individuals, concurrent AD-LBD pathologies predicted longitudinal cognitive progression with faster worsening of global cognition, memory, and attentive/executive functions. DISCUSSION: Future research studies aiming for a better precision medicine approach should develop SAAs further to reach a quantitative evaluation or staging of each underlying pathology using a single biofluid sample. HIGHLIGHTS: α-Synuclein seed amplification assays (SAAs) provide a specific marker for Lewy body disease (LBD). SAAs allow for the in vivo identification of co-occurring LBD in patients with Alzheimer's disease (AD). AD-LBD coexist in 20-25% of cognitively impaired elderly individuals, and ∼8% of those asymptomatic. Compared to pure AD, AD-LBD causes a faster worsening of cognitive functions. AD-LBD is associated with worse attentive/executive, memory, visuospatial and motor functions.

Evaluating the inter-species transmission risk of amyloid beta peptide aggregates via ingestion.

BACKGROUND: Recent reports suggest that amyloid beta (Aß) peptides can exhibit prion-like pathogenic properties. Transmission of Aß peptide and the development of associated pathologies after surgeries with contaminated instruments and intravenous or intracerebral inoculations have now been reported across fish, rodents, primates, and humans. This raises a worrying prospect of Aß peptides also having other characteristics typical of prions, such as evasion of the digestive process. We asked if such transmission of Aß aggregates via ingestion was possible. METHODS: We made use of a transgenic Drosophila melanogaster line expressing human Aß peptide prone to aggregation. Fly larvae were fed to adult zebrafish under two feeding schemes. The first was a short-term, high-intensity scheme over 48 h to determine transmission and retention in the gut. The second, long-term scheme specifically examined retention and accumulation in the brain. The gut and brain tissues were examined by histology, western blotting, and mass spectrometric analyses. RESULTS: None of the analyses could detect Aß aggregates in the guts of zebrafish following ingestion, despite being easily detectable in the feed. Additionally, there was no detectable accumulation of Aß in the brain tissue or development of associated pathologies after prolonged feeding. CONCLUSIONS: While human Aß aggregates do not appear to be readily transmissible by ingestion across species, two prospects remain open. First, this mode of transmission, if occurring, may stay below a detectable threshold and may take much longer to manifest. A second possibility is that the human Aß peptide is not able to trigger self-propagation or aggregation in other species. Either possibility requires further investigation, taking into account the possibility of such transmission from agricultural species used in the food industry.

Prion Seeding Activity in Plant Tissues Detected by RT-QuIC.

Prion diseases such as scrapie, bovine spongiform encephalopathy (BSE), and chronic wasting disease (CWD) affect domesticated and wild herbivorous mammals. Animals afflicted with CWD, the transmissible spongiform encephalopathy of cervids (deer, elk, and moose), shed prions into the environment, where they may persist and remain infectious for years. These environmental prions may remain in soil, be transported in surface waters, or assimilated into plants. Environmental sampling is an emerging area of TSE research and can provide more information about prion fate and transport once shed by infected animals. In this study, we have developed the first published method for the extraction and detection of prions in plant tissue using the real-time quaking-induced conversion (RT-QuIC) assay. Incubation with a zwitterionic surfactant followed by precipitation with sodium phosphotungstate concentrates the prions within samples and allows for sensitive detection of prion seeding activity. Using this protocol, we demonstrate that prions can be detected within plant tissues and on plant surfaces using the RT-QuIC assay.

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.

Characterization of the 263K-derived microsomal fraction: a source of prions for nanofiltration validation studies.

BACKGROUND: The manufacturing processes of plasma products include steps that can remove prions. The efficacy of these steps is measured in validation studies using animal brain-derived prion materials called spikes. Because the nature of the prion agent in blood is not known, the relevance of these spikes, particularly with steps that are based on retention mechanisms such as nanofiltration, is important to investigate. STUDY DESIGN AND METHODS: The aggregation and sizes of PrPres assemblies of microsomal fractions (MFs) extracted from 263K-infected hamster brains were analyzed using velocity gradients. The separated gradient fractions were either inoculated to Tg7 mice expressing hamster-PrPc to measure infectivity or used in Protein Misfolding Cyclic Amplification for measuring seeding activity. The collected data allowed for reanalyzing results from previous nanofiltration validation studies. RESULTS: A significant portion of MFs was found to be composed of small PrPres assemblies, estimated to have a size ≤24 mers (~22-528 kDa), and to contain a minimum of 20% of total prion infectivity. With this data we could calculate reductions of 4.10 log (15 N), 2.53 log (35 N), and 1.77 log (35 N) from validation studies specifically for these small PrPres objects. CONCLUSION: Our gradient data provided evidence that nanofilters can remove the majority of the smallest PrPres entities within microsomes spikes, estimated to be in a size below 24 mers, giving insight about the fact that, in our conditions, size exclusion may not be the only mechanism for retention nanofiltration.

Functional amyloids.

While amyloid has traditionally been viewed as a harmful formation, emerging evidence suggests that amyloids may also play a functional role in cell biology, contributing to normal physiological processes that have been conserved throughout evolution. Functional amyloids have been discovered in several creatures, spanning from bacteria to mammals. These amyloids serve a multitude of purposes, including but not limited to, forming biofilms, melanin synthesis, storage, information transfer, and memory. The functional role of amyloids has been consistently validated by the discovery of more functional amyloids, indicating a conceptual convergence. The biology of amyloids is well-represented by non-pathogenic amyloids, given the numerous ones already identified and the ongoing rate of new discoveries. In this chapter, functional amyloids in microorganisms, animals, and plants are described.

Lack of Transmission of Chronic Wasting Disease Prions to Human Cerebral Organoids.

Chronic wasting disease (CWD) is a cervid prion disease with unknown zoonotic potential that might pose a risk to humans who are exposed. To assess the potential of CWD to infect human neural tissue, we used human cerebral organoids with 2 different prion genotypes, 1 of which has previously been associated with susceptibility to zoonotic prion disease. We exposed organoids from both genotypes to high concentrations of CWD inocula from 3 different sources for 7 days, then screened for infection periodically for up to 180 days. No de novo CWD propagation or deposition of protease-resistant forms of human prions was evident in CWD-exposed organoids. Some persistence of the original inoculum was detected, which was equivalent in prion gene knockout organoids and thus not attributable to human prion propagation. Overall, the unsuccessful propagation of CWD in cerebral organoids supports a strong species barrier to transmission of CWD prions to humans.

The Role of Glial Cells in Neurobiology and Prion Neuropathology.

Prion diseases are rare and neurodegenerative diseases that are characterized by the misfolding and infectious spread of the prion protein in the brain, causing progressive and irreversible neuronal loss and associated clinical and behavioral manifestations in humans and animals, ultimately leading to death. The brain has a complex network of neurons and glial cells whose crosstalk is critical for function and homeostasis. Although it is established that prion infection of neurons is necessary for clinical disease to occur, debate remains in the field as to the role played by glial cells, namely astrocytes and microglia, and whether these cells are beneficial to the host or further accelerate disease. Here, we review the current literature assessing the complex morphologies of astrocytes and microglia, and the crosstalk between these two cell types, in the prion-infected brain.

The 23rd Annual Meeting of the Rocky Mountain Virology Association.

Located 50 miles west of Fort Collins, Colorado, Colorado State University's Mountain Campus in Pingree Park hosted the 23rd annual Rocky Mountain Virology Association meeting in 2023 with 116 participants. The 3-day event at the end of September consisted of 28 talks and 43 posters that covered the topics of viral evolution and surveillance, developments in prion research, arboviruses and vector biology, host-virus interactions, and viral immunity and vaccines. This year's Randall Jay Cohrs keynote presentation covered the topic of One Health and emerging coronaviruses. This timely discussion covered the importance of global disease surveillance, international collaboration, and trans-disciplinary research teams to prevent and control future pandemics. Peak fall colors flanked the campus and glowed along the multiple mountain peaks, allowing for pristine views while discussing science and networking, or engaging in mountain activities like fly fishing and hiking. On behalf of the Rocky Mountain Virology Association, this report summarizes select presentations from the 23rd annual meeting.