BBC pay: we need equality all the way down - not just for the elite

Firms like the BBC can only afford to offer 'top talent' huge financial rewards by controlling costs down below, writes Mark Bank

Region-specific protein misfolding cyclic amplification reproduces brain tropism of prion strains

Human prion diseases such as Creutzfeldt-Jakob disease are transmissible brain proteinopathies, characterized by the accumulation of a misfolded isoform of the host cellular prion protein (PrP) in the brain. According to the prion model, prions are defined as proteinaceous infectious particles composed solely of this abnormal isoform of PrP (PrPSc). Even in the absence of genetic material, various prion strains can be propagated in experimental models. They can be distinguished by the pattern of disease they produce and especially by the localization of PrPSc deposits within the brain and the spongiform lesions they induce. The mechanisms involved in this strain-specific targeting of distinct brain regions still are a fundamental, unresolved question in prion research. To address this question, we exploited a prion conversion in vitro assay, protein misfolding cyclic amplification (PMCA), by using experimental scrapie and human prion strains as seeds and specific brain regions from mice and humans as substrates. We show here that region-specific PMCA in part reproduces the specific brain targeting observed in experimental, acquired, and sporadic Creutzfeldt-Jakob diseases. Furthermore, we provide evidence that, in addition to cellular prion protein, other region-and species-specific molecular factors influence the strain-dependent prion conversion process. This important step toward understanding prion strain propagation in the human brain may impact research on the molecular factors involved in protein misfolding and the development of ultrasensitive methods for diagnosing prion disease

Transmission of cervid prions to humanized mice demonstrates the zoonotic potential of CWD

International audiencePrions cause infectious and fatal neurodegenerative diseases in mammals. Chronic wasting disease (CWD), a prion disease of cervids, spreads efficiently among wild and farmed animals. Potential transmission to humans of CWD is a growing concern due to its increasing prevalence. Here, we provide evidence for a zoonotic potential of CWD prions, and its probable signature using mice expressing human prion protein (PrP) as an infection model. Inoculation of these mice with deer CWD isolates resulted in atypical clinical manifestation with prion seeding activity and efficient transmissible infectivity in the brain and, remarkably, in feces, but without classical neuropathological or Western blot appearances of prion diseases. Intriguingly, the protease-resistant PrP in the brain resembled that found in a familial human prion disease and was transmissible upon second passage. Our results suggest that CWD might infect humans, although the transmission barrier is likely higher compared to zoonotic transmission of cattle prions. Notably, our data suggest a different clinical presentation, prion signature, and tissue tropism, which causes challenges for detection by current diagnostic assays. Furthermore, the presence of infectious prions in feces is concerning because if this occurs in humans, it is a source for human-to-human transmission. These findings have strong implications for public health and CWD management

Early and Non-Invasive Detection of Chronic Wasting Disease Prions in Elk Feces by Real-Time Quaking Induced Conversion

<div><p>Chronic wasting disease (CWD) is a fatal prion disease of wild and captive cervids in North America. Prions are infectious agents composed of a misfolded version of a host-encoded protein, termed PrP^Sc. Infected cervids excrete and secrete prions, contributing to lateral transmission. Geographical distribution is expanding and case numbers in wild cervids are increasing. Recently, the first European cases of CWD have been reported in a wild reindeer and two moose from Norway. Therefore, methods to detect the infection early in the incubation time using easily available samples are desirable to facilitate effective disease management. We have adapted the real-time quaking induced conversion (RT-QuIC) assay, a sensitive <i>in vitro</i> prion amplification method, for pre-clinical detection of prion seeding activity in elk feces. Testing fecal samples from orally inoculated elk taken at various time points post infection revealed early shedding and detectable prion seeding activity throughout the disease course. Early shedding was also found in two elk encoding a PrP genotype associated with reduced susceptibility for CWD. In summary, we suggest that detection of CWD prions in feces by RT-QuIC may become a useful tool to support CWD surveillance in wild and captive cervids. The finding of early shedding independent of the elk’s prion protein genotype raises the question whether prolonged survival is beneficial, considering accumulation of environmental prions and its contribution to CWD transmission upon extended duration of shedding.</p></div

Mouse rPrP is a more efficient substrate for amplification of CWD seeding activity.

<p>Serial dilutions of CWD-positive <b>(a)</b> or negative <b>(b)</b> elk brain homogenates were used as seeds for RT-QuIC reactions. As a substrate, either mouse (left panel) or deer rPrP (right panel) were used. Y-axes indicate the relative ThT fluorescence units, x-axes show the reaction time. A threshold to determine positive reactions was calculated by using the average baseline fluorescence plus 5 standard deviations, which is indicated as a solid line at app. 50,000 RFU. <b>(c)</b> The time to reach the threshold (y-axis) was determined for each individual reaction. Dilutions of 2x10^-2 to 2x10^-5 of reactions with either mouse or deer rPrP were included, and the averages of the time to reach the threshold are shown. Bars represent standard deviations. For each dilution mouse and deer rPrP were compared and the difference between the two groups was statistically evaluated using unpaired student’s t-test (GraphPad Prism software; ** = p-value < 0.01; ns = not significant).</p

NaPTA precipitation and substrate replacement enable detection of CWD seeding activity in feces.

<p><b>(a)</b> Fecal homogenates (10% w/v) of non-infected elk or mule deer were subjected to NaPTA precipitation and 10fold concentration (lower panel) or not (upper panel). These samples were either used undiluted or in dilutions as indicated for seeding RT-QuIC reactions with mouse rPrP as a substrate. The y-axes show relative ThT fluorescence units, the x-axes depict the reaction time. <b>(b)</b> Fecal homogenates of two individual elk which were orally infected with CWD were subjected to NaPTA precipitation and 10fold concentration. RT-QuIC reactions with mouse rPrP as a substrate were run for 50 hours (upper panel), or for 75 hours (lower panel). For the latter, the reaction was stopped after 25 hours, and 90% of the reaction volume was removed and replaced by freshly prepared RT-QuIC mix containing rPrP substrate and ThT. Then the RT-QuIC assay was continued.</p

Oral administration of repurposed drug targeting Cyp46A1 increases survival times of prion infected mice

Abstract Prion diseases are fatal, infectious, and incurable neurodegenerative disorders caused by misfolding of the cellular prion protein (PrPC) into the infectious isoform (PrPSc). In humans, there are sporadic, genetic and infectious etiologies, with sporadic Creutzfeldt-Jakob disease (sCJD) being the most common form. Currently, no treatment is available for prion diseases. Cellular cholesterol is known to impact prion conversion, which in turn results in an accumulation of cholesterol in prion-infected neurons. The major elimination of brain cholesterol is achieved by the brain specific enzyme, cholesterol 24-hydroxylase (CYP46A1). Cyp46A1 converts cholesterol into 24(S)-hydroxycholesterol, a membrane-permeable molecule that exits the brain. We have demonstrated for the first time that Cyp46A1 levels are reduced in the brains of prion-infected mice at advanced disease stage, in prion-infected neuronal cells and in post-mortem brains of sCJD patients. We have employed the Cyp46A1 activator efavirenz (EFV) for treatment of prion-infected neuronal cells and mice. EFV is an FDA approved anti-HIV medication effectively crossing the blood brain barrier and has been used for decades to chronically treat HIV patients. EFV significantly mitigated PrPSc propagation in prion-infected cells while preserving physiological PrPC and lipid raft integrity. Notably, oral administration of EFV treatment chronically at very low dosage starting weeks to months after intracerebral prion inoculation of mice significantly prolonged the lifespan of animals. In summary, our results suggest that Cyp46A1 as a novel therapeutic target and that its activation through repurposing the anti-retroviral medication EFV might be valuable treatment approach for prion diseases