A structural basis for prion strain diversity.

Recent cryogenic electron microscopy (cryo-EM) studies of infectious, ex vivo, prion fibrils from hamster 263K and mouse RML prion strains revealed a similar, parallel in-register intermolecular ß-sheet (PIRIBS) amyloid architecture. Rungs of the fibrils are composed of individual prion protein (PrP) monomers that fold to create distinct N-terminal and C-terminal lobes. However, disparity in the hamster/mouse PrP sequence precludes understanding of how divergent prion strains emerge from an identical PrP substrate. In this study, we determined the near-atomic resolution cryo-EM structure of infectious, ex vivo mouse prion fibrils from the ME7 prion strain and compared this with the RML fibril structure. This structural comparison of two biologically distinct mouse-adapted prion strains suggests defined folding subdomains of PrP rungs and the way in which they are interrelated, providing a structural definition of intra-species prion strain-specific conformations.

Prion strains viewed through the lens of cryo-EM.

Mammalian prions are lethal transmissible pathogens that cause fatal neurodegenerative diseases in humans and animals. They consist of fibrils of misfolded, host-encoded prion protein (PrP) which propagate through templated protein polymerisation. Prion strains produce distinct clinicopathological phenotypes in the same host and appear to be encoded by distinct misfolded PrP conformations and assembly states. Despite fundamental advances in our understanding of prion biology, key knowledge gaps remain. These include precise delineation of prion replication mechanisms, detailed explanation of the molecular basis of prion strains and inter-species transmission barriers, and the structural definition of neurotoxic PrP species. Central to addressing these questions is the determination of prion structure. While high-resolution definition of ex vivo prion fibrils once seemed unlikely, recent advances in cryo-electron microscopy (cryo-EM) and computational methods for 3D reconstruction of amyloids have now made this possible. Recently, near-atomic resolution structures of highly infectious, ex vivo prion fibrils from hamster 263K and mouse RML prion strains were reported. The fibrils have a comparable parallel in-register intermolecular ß-sheet (PIRIBS) architecture that now provides a structural foundation for understanding prion strain diversity in mammals. Here, we review these new findings and discuss directions for future research.

Overexpression of mouse prion protein in transgenic mice causes a non-transmissible spongiform encephalopathy.

Transgenic mice over-expressing human PRNP or murine Prnp transgenes on a mouse prion protein knockout background have made key contributions to the understanding of human prion diseases and have provided the basis for many of the fundamental advances in prion biology, including the first report of synthetic mammalian prions. In this regard, the prion paradigm is increasingly guiding the exploration of seeded protein misfolding in the pathogenesis of other neurodegenerative diseases. Here we report that a well-established and widely used line of such mice (Tg20 or tga20), which overexpress wild-type mouse prion protein, exhibit spontaneous aggregation and accumulation of misfolded prion protein in a strongly age-dependent manner, which is accompanied by focal spongiosis and occasional neuronal loss. In some cases a clinical syndrome developed with phenotypic features that closely resemble those seen in prion disease. However, passage of brain homogenate from affected, aged mice failed to transmit this syndrome when inoculated intracerebrally into further recipient animals. We conclude that overexpression of the wild-type mouse prion protein can cause an age-dependent protein misfolding disorder or proteinopathy that is not associated with the production of an infectious agent but can produce a phenotype closely similar to authentic prion disease.

2.7 Å cryo-EM structure of ex vivo RML prion fibrils.

Mammalian prions propagate as distinct strains and are composed of multichain assemblies of misfolded host-encoded prion protein (PrP). Here, we present a near-atomic resolution cryo-EM structure of PrP fibrils present in highly infectious prion rod preparations isolated from the brains of RML prion-infected mice. We found that prion rods comprise single-protofilament helical amyloid fibrils that coexist with twisted pairs of the same protofilaments. Each rung of the protofilament is formed by a single PrP monomer with the ordered core comprising PrP residues 94-225, which folds to create two asymmetric lobes with the N-linked glycans and the glycosylphosphatidylinositol anchor projecting from the C-terminal lobe. The overall architecture is comparable to that of recently reported PrP fibrils isolated from the brain of hamsters infected with the 263K prion strain. However, there are marked conformational variations that could result from differences in PrP sequence and/or represent distinguishing features of the distinct prion strains.

Humanized 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 postinoculation survival periods no evidence for prion transmission was seen, suggesting that the zoonotic potential of these isolates is low.

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.

Highly infectious prions are not directly neurotoxic.

Prions are infectious agents which cause rapidly lethal neurodegenerative diseases in humans and animals following long, clinically silent incubation periods. They are composed of multichain assemblies of misfolded cellular prion protein. While it has long been assumed that prions are themselves neurotoxic, recent development of methods to obtain exceptionally pure prions from mouse brain with maintained strain characteristics, and in which defined structures-paired rod-like double helical fibers-can be definitively correlated with infectivity, allowed a direct test of this assertion. Here we report that while brain homogenates from symptomatic prion-infected mice are highly toxic to cultured neurons, exceptionally pure intact high-titer infectious prions are not directly neurotoxic. We further show that treatment of brain homogenates from prion-infected mice with sodium lauroylsarcosine destroys toxicity without diminishing infectivity. This is consistent with models in which prion propagation and toxicity can be mechanistically uncoupled.

Spontaneous generation of prions and transmissible PrP amyloid in a humanised transgenic mouse model of A117V GSS.

Inherited prion diseases are caused by autosomal dominant coding mutations in the human prion protein (PrP) gene (PRNP) and account for about 15% of human prion disease cases worldwide. The proposed mechanism is that the mutation predisposes to conformational change in the expressed protein, leading to the generation of disease-related multichain PrP assemblies that propagate by seeded protein misfolding. Despite considerable experimental support for this hypothesis, to-date spontaneous formation of disease-relevant, transmissible PrP assemblies in transgenic models expressing only mutant human PrP has not been demonstrated. Here, we report findings from transgenic mice that express human PrP 117V on a mouse PrP null background (117VV Tg30 mice), which model the PRNP A117V mutation causing inherited prion disease (IPD) including Gerstmann-Sträussler-Scheinker (GSS) disease phenotypes in humans. By studying brain samples from uninoculated groups of mice, we discovered that some mice (≥475 days old) spontaneously generated abnormal PrP assemblies, which after inoculation into further groups of 117VV Tg30 mice, produced a molecular and neuropathological phenotype congruent with that seen after transmission of brain isolates from IPD A117V patients to the same mice. To the best of our knowledge, the 117VV Tg30 mouse line is the first transgenic model expressing only mutant human PrP to show spontaneous generation of transmissible PrP assemblies that directly mirror those generated in an inherited prion disease in humans.

Recent Advances in Understanding Mammalian Prion Structure: A Mini Review.

Prions are lethal pathogens, which cause fatal neurodegenerative diseases in mammals. They are unique infectious agents and are composed of self-propagating multi-chain assemblies of misfolded host-encoded prion protein (PrP). Understanding prion structure is fundamental to understanding prion disease pathogenesis however to date, the high-resolution structure of authentic ex vivo infectious prions remains unknown. Advances in determining prion structure have been severely impeded by the difficulty in recovering relatively homogeneous prion particles from infected brain and definitively associating infectivity with the PrP assembly state. Recently, however, images of highly infectious ex vivo PrP rods that produce prion-strain specific disease phenotypes in mice have been obtained using cryo-electron microscopy and atomic force microscopy. These images have provided the most detailed description of ex vivo mammalian prions reported to date and have established that prions isolated from multiple strains have a common hierarchical structure. Misfolded PrP is assembled into 20 nm wide rods containing two fibers, each with double helical repeating substructure, separated by a characteristic central gap 8-10 nm in width. Irregularly structured material with adhesive properties distinct to that of the fibers is present within the central gap of the rod. Prions are clearly distinguishable from non-infectious recombinant PrP fibrils generated in vitro and from all other propagating protein structures so far described in other neurodegenerative diseases. The basic architecture of mammalian prions appears to be exceptional and fundamental to their lethal pathogenicity.

Structural features distinguishing infectious ex vivo mammalian prions from non-infectious fibrillar assemblies generated in vitro.

Seeded polymerisation of proteins forming amyloid fibres and their spread in tissues has been implicated in the pathogenesis of multiple neurodegenerative diseases: so called "prion-like" mechanisms. While ex vivo mammalian prions, composed of multichain assemblies of misfolded host-encoded prion protein (PrP), act as lethal infectious agents, PrP amyloid fibrils produced in vitro generally do not. The high-resolution structure of authentic infectious prions and the structural basis of prion strain diversity remain unknown. Here we use cryo-electron microscopy and atomic force microscopy to examine the structure of highly infectious PrP rods isolated from mouse brain in comparison to non-infectious recombinant PrP fibrils generated in vitro. Non-infectious recombinant PrP fibrils are 10 nm wide single fibres, with a double helical repeating substructure displaying small variations in adhesive force interactions across their width. In contrast, infectious PrP rods are 20 nm wide and contain two fibres, each with a double helical repeating substructure, separated by a central gap of 8-10 nm in width. This gap contains an irregularly structured material whose adhesive force properties are strikingly different to that of the fibres, suggestive of a distinct composition. The structure of the infectious PrP rods, which cause lethal neurodegeneration, readily differentiates them from all other protein assemblies so far characterised in other neurodegenerative diseases.

Evaluating the causality of novel sequence variants in the prion protein gene by example.

The estimation of pathogenicity and penetrance of novel prion protein gene (PRNP) variants presents significant challenges, particularly in the absence of family history, which precludes the application of Mendelian segregation. Moreover, the ambiguities of prion disease pathophysiology renders conventional in silico predictions inconclusive. Here, we describe 2 patients with rapid cognitive decline progressing to akinetic mutism and death within 10 weeks of symptom onset, both of whom possessed the novel T201S variant in PRNP. Clinically, both satisfied diagnostic criteria for probable sporadic Creutzfeldt-Jakob disease and in one, the diagnosis was confirmed by neuropathology. While computational analyses predicted that T201S was possibly deleterious, molecular strain typing, prion protein structural considerations, and calculations leveraging large-scale population data (gnomAD) indicate that T201S is at best either of low penetrance or nonpathogenic. Thus, we illustrate the utility of harnessing multiple lines of prion disease-specific evidence in the evaluation of the T201S variant, which may be similarly applied to assess other novel variants in PRNP.

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.

Soluble Aß aggregates can inhibit prion propagation.

Mammalian prions cause lethal neurodegenerative diseases such as Creutzfeldt-Jakob disease (CJD) and consist of multi-chain assemblies of misfolded cellular prion protein (PrPC). Ligands that bind to PrPC can inhibit prion propagation and neurotoxicity. Extensive prior work established that certain soluble assemblies of the Alzheimer's disease (AD)-associated amyloid ß-protein (Aß) can tightly bind to PrPC, and that this interaction may be relevant to their toxicity in AD. Here, we investigated whether such soluble Aß assemblies might, conversely, have an inhibitory effect on prion propagation. Using cellular models of prion infection and propagation and distinct Aß preparations, we found that the form of Aß assemblies which most avidly bound to PrP in vitro also inhibited prion infection and propagation. By contrast, forms of Aß which exhibit little or no binding to PrP were unable to attenuate prion propagation. These data suggest that soluble aggregates of Aß can compete with prions for binding to PrPC and emphasize the bidirectional nature of the interplay between Aß and PrPC in Alzheimer's and prion diseases. Such inhibitory effects of Aß on prion propagation may contribute to the apparent fall-off in the incidence of sporadic CJD at advanced age where cerebral Aß deposition is common.

Methods for Molecular Diagnosis of Human Prion Disease.

Human prion diseases are associated with a range of clinical presentations, and they are classified by both clinicopathological syndrome and etiology, with subclassification according to molecular criteria. Here, we describe updated procedures that are currently used within the MRC Prion Unit at UCL to determine a molecular diagnosis of human prion disease. Sequencing of the PRNP open reading frame to establish the presence of pathogenic mutations is described, together with detailed methods for immunoblot or immunohistochemical determination of the presence of abnormal prion protein in the brain or peripheral tissues.

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.

Ex vivo mammalian prions are formed of paired double helical prion protein fibrils.

Mammalian prions are hypothesized to be fibrillar or amyloid forms of prion protein (PrP), but structures observed to date have not been definitively correlated with infectivity and the three-dimensional structure of infectious prions has remained obscure. Recently, we developed novel methods to obtain exceptionally pure preparations of prions from mouse brain and showed that pathogenic PrP in these high-titre preparations is assembled into rod-like assemblies. Here, we have used precise cell culture-based prion infectivity assays to define the physical relationship between the PrP rods and prion infectivity and have used electron tomography to define their architecture. We show that infectious PrP rods isolated from multiple prion strains have a common hierarchical assembly comprising twisted pairs of short fibres with repeating substructure. The architecture of the PrP rods provides a new structural basis for understanding prion infectivity and can explain the inability to systematically generate high-titre synthetic prions from recombinant PrP.

Clinical Trial Simulations Based on Genetic Stratification and the Natural History of a Functional Outcome Measure in Creutzfeldt-Jakob Disease.

IMPORTANCE: A major challenge for drug development in neurodegenerative diseases is that adequately powered efficacy studies with meaningful end points typically require several hundred participants and long durations. Prion diseases represent the archetype of brain diseases caused by protein misfolding, the most common subtype being sporadic Creutzfeldt-Jakob disease (sCJD), a rapidly progressive dementia. There is no well-established trial method in prion disease. OBJECTIVE: To establish a more powerful and meaningful clinical trial method in sCJD. DESIGN, SETTING, AND PARTICIPANTS: A stratified medicine and simulation approach based on a prospective interval-cohort study conducted from October 2008 to June 2014. This study involved 598 participants with probable or definite sCJD followed up over 470 patient-years at a specialist national referral service in the United Kingdom with domiciliary, care home, and hospital patient visits. We fitted linear mixed models to the outcome measurements, and simulated clinical trials involving 10 to 120 patients (no dropouts) with early to moderately advanced prion disease using model parameters to compare the power of various designs. MAIN OUTCOMES AND MEASURES: A total of 2681 assessments were done using a functionally orientated composite end point (Medical Research Council Scale) and associated with clinical investigations (brain magnetic resonance imaging, electroencephalography, and cerebrospinal fluid analysis) and molecular data (prion protein [PrP] gene sequencing, PrPSc type). RESULTS: Of the 598 participants, 273 were men. The PrP gene sequence was significantly associated with decline relative to any other demographic or investigation factors. Patients with sCJD and polymorphic codon 129 genotypes MM, VV, and MV lost 10% of their function in 5.3 (95% CI, 4.2-6.9), 13.2 (95% CI, 10.9-16.6), and 27.8 (95% CI, 21.9-37.8) days, respectively (P < .001). Simulations indicate that an adequately powered (80%; 2-sided α = .05) open-label randomized trial using 50% reduction in Medical Research Council Scale decline as the primary outcome could be conducted with only 120 participants assessed every 10 days and only 90 participants assessed daily, providing considerably more power than using survival as the primary outcome. Restricting to VV or MV codon 129 genotypes increased power even further. Alternatively, single-arm intervention studies (half the total sample size) could provide similar power in comparison to the natural history cohort. CONCLUSIONS AND RELEVANCE: Functional end points in neurodegeneration need not require long and very large clinical studies to be adequately powered for efficacy. Patients with sCJD may be an efficient and cost-effective group for testing disease-modifying therapeutics. Stratified medicine and natural history cohort approaches may transform the feasibility of clinical trials in orphan diseases.

A systematic investigation of production of synthetic prions from recombinant prion protein.

According to the protein-only hypothesis, infectious mammalian prions, which exist as distinct strains with discrete biological properties, consist of multichain assemblies of misfolded cellular prion protein (PrP). A critical test would be to produce prion strains synthetically from defined components. Crucially, high-titre 'synthetic' prions could then be used to determine the structural basis of infectivity and strain diversity at the atomic level. While there have been multiple reports of production of prions from bacterially expressed recombinant PrP using various methods, systematic production of high-titre material in a form suitable for structural analysis remains a key goal. Here, we report a novel high-throughput strategy for exploring a matrix of conditions, additives and potential cofactors that might generate high-titre prions from recombinant mouse PrP, with screening for infectivity using a sensitive automated cell-based bioassay. Overall, approximately 20,000 unique conditions were examined. While some resulted in apparently infected cell cultures, this was transient and not reproducible. We also adapted published methods that reported production of synthetic prions from recombinant hamster PrP, but again did not find evidence of significant infectious titre when using recombinant mouse PrP as substrate. Collectively, our findings are consistent with the formation of prion infectivity from recombinant mouse PrP being a rare stochastic event and we conclude that systematic generation of prions from recombinant PrP may only become possible once the detailed structure of authentic ex vivo prions is solved.