Anti-prion drugs do not improve survival in novel knock-in models of inherited prion disease.

Prion diseases uniquely manifest in three distinct forms: inherited, sporadic, and infectious. Wild-type prions are responsible for the sporadic and infectious versions, while mutant prions cause inherited variants like fatal familial insomnia (FFI) and familial Creutzfeldt-Jakob disease (fCJD). Although some drugs can prolong prion incubation times up to four-fold in rodent models of infectious prion diseases, no effective treatments for FFI and fCJD have been found. In this study, we evaluated the efficacy of various anti-prion drugs on newly-developed knock-in mouse models for FFI and fCJD. These models express bank vole prion protein (PrP) with the pathogenic D178N and E200K mutations. We applied various drug regimens known to be highly effective against wild-type prions in vivo as well as a brain-penetrant compound that inhibits mutant PrPSc propagation in vitro. None of the regimens tested (Anle138b, IND24, Anle138b + IND24, cellulose ether, and PSCMA) significantly extended disease-free survival or prevented mutant PrPSc accumulation in either knock-in mouse model, despite their ability to induce strain adaptation of mutant prions. Our results show that anti-prion drugs originally developed to treat infectious prion diseases do not necessarily work for inherited prion diseases, and that the recombinant sPMCA is not a reliable platform for identifying compounds that target mutant prions. This work underscores the need to develop therapies and validate screening assays specifically for mutant prions, as well as anti-prion strategies that are not strain-dependent.

Anti-prion drugs do not improve survival in knock-in models of inherited prion disease.

Prion diseases uniquely manifest in three distinct forms: inherited, sporadic, and infectious. Wild-type prions are responsible for the sporadic and infectious versions, while mutant prions cause inherited variants like fatal familial insomnia (FFI) and familial Creutzfeldt-Jakob disease (fCJD). Although some drugs can prolong prion incubation times up to four-fold in rodent models of infectious prion diseases, no effective treatments for FFI and fCJD have been found. In this study, we evaluated the efficacy of various anti-prion drugs on newly-developed knock-in mouse models for FFI and fCJD. These models express bank vole prion protein (PrP) with the pathogenic D178N and E200K mutations. We applied various drug regimens known to be highly effective against wild-type prions in vivo as well as a brain-penetrant compound that inhibits mutant PrP Sc propagation in vitro . None of the regimens tested (Anle138b, IND24, Anle138b + IND24, cellulose ether, and PSCMA) significantly extended disease-free survival or prevented mutant PrP Sc accumulation in either knock-in mouse model, despite their ability to induce strain adaptation of mutant prions. Paradoxically, the combination of Anle138b and IND24 appeared to accelerate disease by 16% and 26% in kiBVI E200K and kiBVI D178N mice, respectively, and accelerated the aggregation of mutant PrP molecules in vitro . Our results show that anti-prion drugs originally developed to treat infectious prion diseases do not necessarily work for inherited prion diseases, and that the recombinant sPMCA is not a reliable platform for identifying compounds that target mutant prions. This work underscores the need to develop therapies and validate screening assays specifically for mutant prions.

Diagnostic reliability in teledermatology: a systematic review and a meta-analysis.

OBJECTIVES: To compare teledermatology and face-to-face (F2F) agreement in primary diagnoses of dermatological conditions. DESIGN: Systematic review and meta-analysis METHODS: MEDLINE, Embase, Cochrane Library (Wiley), CINAHL and medRxiv were searched between January 2010 and May 2022. Observational studies and randomised clinical trials that reported percentage agreement or kappa concordance for primary diagnoses between teledermatology and F2F physicians were included. Titles, abstracts and full-text articles were screened in duplicate. From 7173 citations, 44 articles were included. A random-effects meta-analysis was conducted to estimate pooled estimates. Primary outcome measures were mean percentage and kappa concordance for assessing diagnostic matches between teledermatology and F2F physicians. Secondary outcome measures included the agreement between teledermatologists, F2F dermatologists, and teledermatology and histopathology results. RESULTS: 44 studies were extracted and reviewed. The pooled agreement rate was 68.9%, and kappa concordance was 0.67. When dermatologists conducted F2F and teledermatology consults, the overall diagnostic agreement was significantly higher at 71% compared with 44% for non-specialists. Kappa concordance was 0.69 for teledermatologist versus specialist and 0.52 for non-specialists. Higher diagnostic agreements were also noted with image acquisition training and digital photography. The agreement rate was 76.4% between teledermatologists, 82.4% between F2F physicians and 55.7% between teledermatology and histopathology. CONCLUSIONS AND RELEVANCE: Teledermatology can be an attractive option particularly in resource-poor settings. Future efforts should be placed on incorporating image acquisition training and access to high-quality imaging technologies. TRIAL REGISTRATION NUMBER: 10.17605/OSF.IO/FJDVG.

The aminoglycoside G418 hinders de novo prion infection in cultured cells.

The study of prions and the discovery of candidate therapeutics for prion disease have been facilitated by the ability of prions to replicate in cultured cells. Paradigms in which prion proteins from different species are expressed in cells with low or no expression of endogenous prion protein (PrP) have expanded the range of prion strains that can be propagated. In these systems, cells stably expressing a PrP of interest are typically generated via coexpression of a selectable marker and treatment with an antibiotic. Here, we report the unexpected discovery that the aminoglycoside G418 (Geneticin) interferes with the ability of stably transfected cultured cells to become infected with prions. In G418-resistant lines of N2a or CAD5 cells, the presence of G418 reduced levels of protease-resistant PrP following challenge with the RML or 22L strains of mouse prions. G418 also interfered with the infection of cells expressing hamster PrP with the 263K strain of hamster prions. Interestingly, G418 had minimal to no effect on protease-resistant PrP levels in cells with established prion infection, arguing that G418 selectively interferes with de novo prion infection. As G418 treatment had no discernible effect on cellular PrP levels or its localization, this suggests that G418 may specifically target prion assemblies or processes involved in the earliest stages of prion infection.

The utility of bank voles for studying prion disease.

The transmission of prions between species is typically an inefficient process due to the species barrier, which represents incompatibility between prion seed and substrate molecules. Bank voles (Myodes glareolus) are an exception to this rule, as they are susceptible to a diverse range of prion strains from many different animal species. In particular, bank voles can be efficiently infected with most types of human prions and have played a critical role in validating variably protease-sensitive prionopathy (VPSPr) and certain forms of Gerstmann-Sträussler-Scheinker (GSS) disease as bona fide prion disorders rather than non-transmissible proteinopathies. The bank vole prion protein (BVPrP) confers a "universal prion acceptor" phenotype when expressed in mice and when used as a substrate for in vitro prion amplification assays, indicating that the unique prion transmission properties of bank voles are mediated by BVPrP. Over-expression of BVPrP in mice can also promote the spontaneous development of prion disease, indicating that BVPrP is intrinsically prone to both spontaneous and template-directed misfolding. Here, we discuss the utility of bank voles and BVPrP for prion research and how they have provided new tools for establishing rapid animal bioassays, modeling spontaneous prion disease, standardizing prion diagnostics, and understanding the molecular basis of the species barrier.

Engineering a murine cell line for the stable propagation of hamster prions.

Prions are infectious protein aggregates that cause several fatal neurodegenerative diseases. Prion research has been hindered by a lack of cellular paradigms for studying the replication of prions from different species. Although hamster prions have been widely used to study prion replication in animals and within in vitro amplification systems, they have proved challenging to propagate in cultured cells. Because the murine catecholaminergic cell line CAD5 is susceptible to a diverse range of mouse prion strains, we hypothesized that it might also be capable of propagating nonmouse prions. Here, using CRISPR/Cas9-mediated genome engineering, we demonstrate that CAD5 cells lacking endogenous mouse PrP expression (CAD5-PrP-/- cells) can be chronically infected with hamster prions following stable expression of hamster PrP. When exposed to the 263K, HY, or 139H hamster prion strains, these cells stably propagated high levels of protease-resistant PrP. Hamster prion replication required absence of mouse PrP, and hamster PrP inhibited the propagation of mouse prions. Cellular homogenates from 263K-infected cells exhibited prion seeding activity in the RT-QuIC assay and were infectious to naïve cells expressing hamster PrP. Interestingly, murine N2a neuroblastoma cells ablated for endogenous PrP expression were susceptible to mouse prions, but not hamster prions upon expression of cognate PrP, suggesting that CAD5 cells either possess cellular factors that enhance or lack factors that restrict the diversity of prion strains that can be propagated. We conclude that transfected CAD5-PrP-/- cells may be a useful tool for assessing the biology of prion strains and dissecting the mechanism of prion replication.

The prion protein is embedded in a molecular environment that modulates transforming growth factor ß and integrin signaling.

At times, it can be difficult to discern if a lack of overlap in reported interactions for a protein-of-interest reflects differences in methodology or biology. In such instances, systematic analyses of protein-protein networks across diverse paradigms can provide valuable insights. Here, we interrogated the interactome of the prion protein (PrP), best known for its central role in prion diseases, in four mouse cell lines. Analyses made use of identical affinity capture and sample processing workflows. Negative controls were generated from PrP knockout lines of the respective cell models, and the relative levels of peptides were quantified using isobaric labels. The study uncovered 26 proteins that reside in proximity to PrP. All of these proteins are predicted to have access to the outer face of the plasma membrane, and approximately half of them were not reported to interact with PrP before. Strikingly, although several proteins exhibited profound co-enrichment with PrP in a given model, except for the neural cell adhesion molecule 1, no protein was highly enriched in all PrP-specific interactomes. However, Gene Ontology analyses revealed a shared association of the majority of PrP candidate interactors with cellular events at the intersection of transforming growth factor ß and integrin signaling.

The function of the cellular prion protein in health and disease.

The essential role of the cellular prion protein (PrPC) in prion disorders such as Creutzfeldt-Jakob disease is well documented. Moreover, evidence is accumulating that PrPC may act as a receptor for protein aggregates and transduce neurotoxic signals in more common neurodegenerative disorders, such as Alzheimer's disease. Although the pathological roles of PrPC have been thoroughly characterized, a general consensus on its physiological function within the brain has not yet been established. Knockout studies in various organisms, ranging from zebrafish to mice, have implicated PrPC in a diverse range of nervous system-related activities that include a key role in the maintenance of peripheral nerve myelination as well as a general ability to protect against neurotoxic stimuli. Thus, the function of PrPC may be multifaceted, with different cell types taking advantage of unique aspects of its biology. Deciphering the cellular function(s) of PrPC and the consequences of its absence is not simply an academic curiosity, since lowering PrPC levels in the brain is predicted to be a powerful therapeutic strategy for the treatment of prion disease. In this review, we outline the various approaches that have been employed in an effort to uncover the physiological and pathological functions of PrPC. While these studies have revealed important clues about the biology of the prion protein, the precise reason for PrPC's existence remains enigmatic.

Towards authentic transgenic mouse models of heritable PrP prion diseases.

Attempts to model inherited human prion disorders such as familial Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker (GSS) disease, and fatal familial insomnia (FFI) using genetically modified mice have produced disappointing results. We recently demonstrated that transgenic (Tg) mice expressing wild-type bank vole prion protein (BVPrP) containing isoleucine at polymorphic codon 109 develop a spontaneous neurodegenerative disorder that exhibits many of the hallmarks of prion disease. To determine if mutations causing inherited human prion disease alter this phenotype, we generated Tg mice expressing BVPrP containing the D178N mutation, which causes FFI; the E200K mutation, which causes familial CJD; or an anchorless PrP mutation similar to mutations that cause GSS. Modest expression levels of mutant BVPrP resulted in highly penetrant spontaneous disease in Tg mice, with mean ages of disease onset ranging from ~120 to ~560 days. The brains of spontaneously ill mice exhibited prominent features of prion disease-specific neuropathology that were unique to each mutation and distinct from Tg mice expressing wild-type BVPrP. An ~8-kDa proteinase K-resistant PrP fragment was found in the brains of spontaneously ill Tg mice expressing either wild-type or mutant BVPrP. The spontaneously formed mutant BVPrP prions were transmissible to Tg mice expressing wild-type or mutant BVPrP as well as to Tg mice expressing mouse PrP. Thus, Tg mice expressing mutant BVPrP exhibit many of the hallmarks of heritable prion disorders in humans including spontaneous disease, protease-resistant PrP, and prion infectivity.