Physical, chemical and kinetic factors affecting prion infectivity

The mouse-adapted scrapie prion strain RML is one of the most widely used in prion research. The introduction of a cell culture-based assay of RML prions, the scrapie cell assay (SCA) allows more rapid and precise prion titration. A semi-automated version of this assay (ASCA) was applied to explore a range of conditions that might influence the infectivity and properties of RML prions. These include resistance to freeze-thaw procedures; stability to endogenous proteases in brain homogenate despite prolonged exposure to varying temperatures; distribution of infective material between pellet and supernatant after centrifugation, the effect of reducing agents and the influence of detergent additives on the efficiency of infection. Apparent infectivity is increased significantly by interaction with cationic detergents. Importantly, we have also elucidated the relationship between the duration of exposure of cells to RML prions and the transmission of infection. We established that the infection process following contact of cells with RML prions is rapid and followed an exponential time course, implying a single rate-limiting process

Experimental sheep BSE prions generate the vCJD phenotype when serially passaged in transgenic mice expressing human prion protein

The epizootic prion disease of cattle, bovine spongiform encephalopathy (BSE), causes variant Creutzfeldt-Jakob disease (vCJD) in humans following dietary exposure. While it is assumed that all cases of vCJD attributed to a dietary aetiology are related to cattle BSE, sheep and goats are susceptible to experimental oral challenge with cattle BSE prions and farmed animals in the UK were undoubtedly exposed to BSE-contaminated meat and bone meal during the late 1980s and early 1990s. Although no natural field cases of sheep BSE have been identified, it cannot be excluded that some BSE-infected sheep might have entered the European human food chain. Evaluation of the zoonotic potential of sheep BSE prions has been addressed by examining the transmission properties of experimental brain isolates in transgenic mice that express human prion protein, however to-date there have been relatively few studies. Here we report that serial passage of experimental sheep BSE prions in transgenic mice expressing human prion protein with methionine at residue 129 produces the vCJD phenotype that mirrors that seen when the same mice are challenged with vCJD prions from patient brain. These findings are congruent with those reported previously by another laboratory, and thereby strongly reinforce the view that sheep BSE prions could have acted as a causal agent of vCJD within Europe

Evaluation of plasma tau and neurofilament light chain biomarkers in a 12-year clinical cohort of human prion diseases

Prion diseases are fatal neurodegenerative conditions with highly accurate CSF and imaging diagnostic tests, but major unmet needs for blood biomarkers. Using ultrasensitive immuno-assays, we measured tau and neurofilament light chain (NfL) protein concentrations in 709 plasma samples taken from 377 individuals with prion disease during a 12 year prospective clinical study, alongside healthy and neurological control groups. This provides an unprecedented opportunity to evaluate their potential as biomarkers. Plasma tau and NfL were increased across all prion disease types. For distinguishing sCJD from control groups including clinically-relevant "CJD mimics", both show considerable diagnostic value. In sCJD, NfL was substantially elevated in every sample tested, including during early disease with minimal functional impairment and in all follow-up samples. Plasma tau was independently associated with rate of clinical progression in sCJD, while plasma NfL showed independent association with severity of functional impairment. In asymptomatic PRNP mutation carriers, plasma NfL was higher on average in samples taken within 2 years of symptom onset than in samples taken earlier. We present biomarker trajectories for nine mutation carriers healthy at enrolment who developed symptoms during follow-up. NfL started to rise as early as 2 years before onset in those with mutations typically associated with more slowly progressive clinical disease. This shows potential for plasma NfL as a "proximity marker", but further work is needed to establish predictive value on an individual basis, and how this varies across different PRNP mutations. We conclude that plasma tau and NfL have potential to fill key unmet needs for biomarkers in prion disease: as a secondary outcome for clinical trials (NfL and tau); for predicting onset in at-risk individuals (NfL); and as an accessible test for earlier identification of patients that may have CJD and require more definitive tests (NfL). Further studies should evaluate their performance directly in these specific roles

Humanised transgenic mice are resistant to chronic wasting disease prions from Norwegian reindeer and moose

Chronic wasting disease (CWD) is the transmissible spongiform encephalopathy or prion disease affecting cervids. In 2016 the first cases of CWD were reported in Europe in Norwegian wild reindeer and moose. The origin and zoonotic potential of these new prion isolates remain unknown. In this study to investigate zoonotic potential we inoculated brain tissue from CWD-infected Norwegian reindeer and moose into transgenic mice overexpressing human prion protein. After prolonged post-inoculation survival periods no evidence for prion transmission was seen suggesting that the zoonotic potential of these isolates is low

A naturally occurring variant of the human prion protein completely prevents prion disease

Mammalian prions, transmissible agents causing lethal neurodegenerative diseases, are composed of assemblies of misfolded cellular prion protein (PrP)1. A novel PrP variant, G127V, was under positive evolutionary selection during the epidemic of kuru—an acquired prion disease epidemic of the Fore population in Papua New Guinea—and appeared to provide strong protection against disease in the heterozygous state2. Here we have investigated the protective role of this variant and its interaction with the common, worldwide M129V PrP polymorphism. V127 was seen exclusively on a M129 PRNP allele. We demonstrate that transgenic mice expressing both variant and wild-type human PrP are completely resistant to both kuru and classical Creutzfeldt–Jakob disease (CJD) prions (which are closely similar) but can be infected with variant CJD prions, a human prion strain resulting from exposure to bovine spongiform encephalopathy prions to which the Fore were not exposed. Notably, mice expressing only PrP V127 were completely resistant to all prion strains, demonstrating a different molecular mechanism to M129V, which provides its relative protection against classical CJD and kuru in the heterozygous state. Indeed, this single amino acid substitution (G→V) at a residue invariant in vertebrate evolution is as protective as deletion of the protein. Further study in transgenic mice expressing different ratios of variant and wild-type PrP indicates that not only is PrP V127 completely refractory to prion conversion but acts as a potent dose-dependent inhibitor of wild-type prion propagation

Protease-Sensitive Conformers in Broad Spectrum of Distinct PrPSc Structures in Sporadic Creutzfeldt-Jakob Disease Are Indicator of Progression Rate

The origin, range, and structure of prions causing the most common human prion disease, sporadic Creutzfeldt-Jakob disease (sCJD), are largely unknown. To investigate the molecular mechanism responsible for the broad phenotypic variability of sCJD, we analyzed the conformational characteristics of protease-sensitive and protease-resistant fractions of the pathogenic prion protein (PrPSc) using novel conformational methods derived from a conformation-dependent immunoassay (CDI). In 46 brains of patients homozygous for polymorphisms in the PRNP gene and exhibiting either Type 1 or Type 2 western blot pattern of the PrPSc, we identified an extensive array of PrPSc structures that differ in protease sensitivity, display of critical domains, and conformational stability. Surprisingly, in sCJD cases homozygous for methionine or valine at codon 129 of the PRNP gene, the concentration and stability of protease-sensitive conformers of PrPSc correlated with progression rate of the disease. These data indicate that sCJD brains exhibit a wide spectrum of PrPSc structural states, and accordingly argue for a broad spectrum of prion strains coding for different phenotypes. The link between disease duration, levels, and stability of protease-sensitive conformers of PrPSc suggests that these conformers play an important role in the pathogenesis of sCJD

A Novel Prion Disease Presenting with Diarrhea and Autonomic Neuropathy

Background. Human prion diseases, although variable in clinicopathological phenotype, generally present as neurologic or neuropsychiatric conditions associated with rapid multifocal central nervous system degeneration that is usually dominated by dementia and cerebellar ataxia. Approximately 15% of cases of recognized prion disease are inherited and associated with coding mutations in the gene encoding prion protein (PRNP). The availability of genetic diagnosis has led to a progressive broadening of the recognized spectrum of disease. Methods. We used longitudinal clinical assessments over a period of 20 years at one hospital combined with genealogical, neuropsychological, neurophysiological, neuroimaging, pathological, molecular genetic, and biochemical studies, as well as studies of animal transmission, to characterize a novel prion disease in a large British kindred. We studied 6 of 11 affected family members in detail, along with autopsy or biopsy samples obtained from 5 family members. Results. We identified a PRNP Y163X truncation mutation and describe a distinct and consistent phenotype of chronic diarrhea with autonomic failure and a length-dependent axonal, predominantly sensory, peripheral polyneuropathy with an onset in early adulthood. Cognitive decline and seizures occurred when the patients were in their 40s or 50s. The deposition of prion protein amyloid was seen throughout peripheral organs, including the bowel and peripheral nerves. Neuropathological examination during end-stage disease showed the deposition of prion protein in the form of frequent cortical amyloid plaques, cerebral amyloid angiopathy, and tauopathy. A unique pattern of abnormal prion protein fragments was seen in brain tissue. Transmission studies in laboratory mice were negative. Conclusions. Abnormal forms of prion protein that were found in multiple peripheral tissues were associated with diarrhea, autonomic failure, and neuropathy. (Funded by the U.K. Medical Research Council and others.

Biochemical Properties of Highly Neuroinvasive Prion Strains

Infectious prions propagate from peripheral entry sites into the central nervous system (CNS), where they cause progressive neurodegeneration that ultimately leads to death. Yet the pathogenesis of prion disease can vary dramatically depending on the strain, or conformational variant of the aberrantly folded and aggregated protein, PrPSc. Although most prion strains invade the CNS, some prion strains cannot gain entry and do not cause clinical signs of disease. The conformational basis for this remarkable variation in the pathogenesis among strains is unclear. Using mouse-adapted prion strains, here we show that highly neuroinvasive prion strains primarily form diffuse aggregates in brain and are noncongophilic, conformationally unstable in denaturing conditions, and lead to rapidly lethal disease. These neuroinvasive strains efficiently generate PrPSc over short incubation periods. In contrast, the weakly neuroinvasive prion strains form large fibrillary plaques and are stable, congophilic, and inefficiently generate PrPSc over long incubation periods. Overall, these results indicate that the most neuroinvasive prion strains are also the least stable, and support the concept that the efficient replication and unstable nature of the most rapidly converting prions may be a feature linked to their efficient spread into the CNS