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.

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.

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.

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.

Evidence for human transmission of amyloid-ß pathology and cerebral amyloid angiopathy.

More than two hundred individuals developed Creutzfeldt-Jakob disease (CJD) worldwide as a result of treatment, typically in childhood, with human cadaveric pituitary-derived growth hormone contaminated with prions. Although such treatment ceased in 1985, iatrogenic CJD (iCJD) continues to emerge because of the prolonged incubation periods seen in human prion infections. Unexpectedly, in an autopsy study of eight individuals with iCJD, aged 36-51 years, in four we found moderate to severe grey matter and vascular amyloid-ß (Aß) pathology. The Aß deposition in the grey matter was typical of that seen in Alzheimer's disease and Aß in the blood vessel walls was characteristic of cerebral amyloid angiopathy and did not co-localize with prion protein deposition. None of these patients had pathogenic mutations, APOE ε4 or other high-risk alleles associated with early-onset Alzheimer's disease. Examination of a series of 116 patients with other prion diseases from a prospective observational cohort study showed minimal or no Aß pathology in cases of similar age range, or a decade older, without APOE ε4 risk alleles. We also analysed pituitary glands from individuals with Aß pathology and found marked Aß deposition in multiple cases. Experimental seeding of Aß pathology has been previously demonstrated in primates and transgenic mice by central nervous system or peripheral inoculation with Alzheimer's disease brain homogenate. The marked deposition of parenchymal and vascular Aß in these relatively young patients with iCJD, in contrast with other prion disease patients and population controls, is consistent with iatrogenic transmission of Aß pathology in addition to CJD and suggests that healthy exposed individuals may also be at risk of iatrogenic Alzheimer's disease and cerebral amyloid angiopathy. These findings should also prompt investigation of whether other known iatrogenic routes of prion transmission may also be relevant to Aß and other proteopathic seeds associated with neurodegenerative and other human diseases.

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). 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 state. 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.

Transmission Properties of Human PrP 102L Prions Challenge the Relevance of Mouse Models of GSS.

Inherited prion disease (IPD) is caused by autosomal-dominant pathogenic mutations in the human prion protein (PrP) gene (PRNP). A proline to leucine substitution at PrP residue 102 (P102L) is classically associated with Gerstmann-Sträussler-Scheinker (GSS) disease but shows marked clinical and neuropathological variability within kindreds that may be caused by variable propagation of distinct prion strains generated from either PrP 102L or wild type PrP. To-date the transmission properties of prions propagated in P102L patients remain ill-defined. Multiple mouse models of GSS have focused on mutating the corresponding residue of murine PrP (P101L), however murine PrP 101L, a novel PrP primary structure, may not have the repertoire of pathogenic prion conformations necessary to accurately model the human disease. Here we describe the transmission properties of prions generated in human PrP 102L expressing transgenic mice that were generated after primary challenge with ex vivo human GSS P102L or classical CJD prions. We show that distinct strains of prions were generated in these mice dependent upon source of the inoculum (either GSS P102L or CJD brain) and have designated these GSS-102L and CJD-102L prions, respectively. GSS-102L prions have transmission properties distinct from all prion strains seen in sporadic and acquired human prion disease. Significantly, GSS-102L prions appear incapable of transmitting disease to conventional mice expressing wild type mouse PrP, which contrasts strikingly with the reported transmission properties of prions generated in GSS P102L-challenged mice expressing mouse PrP 101L. We conclude that future transgenic modeling of IPDs should focus exclusively on expression of mutant human PrP, as other approaches may generate novel experimental prion strains that are unrelated to human disease.

A novel prion disease associated 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.).

Iatrogenic CJD due to pituitary-derived growth hormone with genetically determined incubation times of up to 40 years.

Patients with iatrogenic Creutzfeldt-Jakob disease due to administration of cadaver-sourced growth hormone during childhood are still being seen in the UK 30 years after cessation of this treatment. Of the 77 patients who have developed iatrogenic Creutzfeldt-Jakob disease, 56 have been genotyped. There has been a marked change in genotype profile at polymorphic codon 129 of the prion protein gene (PRNP) from predominantly valine homozygous to a mixed picture of methionine homozygous and methionine-valine heterozygous over time. The incubation period of iatrogenic Creutzfeldt-Jakob disease is significantly different between all three genotypes. This experience is a striking contrast with that in France and the USA, which may relate to contamination of different growth hormone batches with different strains of human prions. We describe the clinical, imaging, molecular and autopsy features in 22 of 24 patients who have developed iatrogenic Creutzfeldt-Jakob disease in the UK since 2003. Mean age at onset of symptoms was 42.7 years. Gait ataxia and lower limb dysaesthesiae were the most frequent presenting symptoms. All had cerebellar signs, and the majority had myoclonus and lower limb pyramidal signs, with relatively preserved cognitive function, when first seen. There was a progressive decline in neurological and cognitive function leading to death after 5-32 (mean 14) months. Despite incubation periods approaching 40 years, the clinical duration in methionine homozygote patients appeared to be shorter than that seen in heterozygote patients. MRI showed restricted diffusion in the basal ganglia, thalamus, hippocampus, frontal and the paracentral motor cortex and cerebellar vermis. The electroencephalogram was abnormal in 15 patients and cerebrospinal fluid 14-3-3 protein was positive in half the patients. Neuropathological examination was conducted in nine patients. All but one showed synaptic prion deposition with numerous kuru type plaques in the basal ganglia, anterior frontal and parietal cortex, thalamus, basal ganglia and cerebellum. The patient with the shortest clinical duration had an atypical synaptic deposition of abnormal prion protein and no kuru plaques. Taken together, these data provide a remarkable example of the interplay between the strain of the pathogen and host prion protein genotype. Based on extensive modelling of human prion transmission barriers in transgenic mice expressing human prion protein on a mouse prion protein null background, the temporal distribution of codon 129 genotypes within the cohort of patients with iatrogenic Creutzfeldt-Jakob disease in the UK suggests that there was a point source of infecting prion contamination of growth hormone derived from a patient with Creutzfeldt-Jakob disease expressing prion protein valine 129.

Inherited prion disease A117V is not simply a proteinopathy but produces prions transmissible to transgenic mice expressing homologous prion protein.

Prions are infectious agents causing fatal neurodegenerative diseases of humans and animals. In humans, these have sporadic, acquired and inherited aetiologies. The inherited prion diseases are caused by one of over 30 coding mutations in the human prion protein (PrP) gene (PRNP) and many of these generate infectious prions as evidenced by their experimental transmissibility by inoculation to laboratory animals. However, some, and in particular an extensively studied type of Gerstmann-Sträussler-Scheinker syndrome (GSS) caused by a PRNP A117V mutation, are thought not to generate infectious prions and instead constitute prion proteinopathies with a quite distinct pathogenetic mechanism. Multiple attempts to transmit A117V GSS have been unsuccessful and typical protease-resistant PrP (PrP(Sc)), pathognomonic of prion disease, is not detected in brain. Pathogenesis is instead attributed to production of an aberrant topological form of PrP, C-terminal transmembrane PrP ((Ctm)PrP). Barriers to transmission of prion strains from one species to another appear to relate to structural compatibility of PrP in host and inoculum and we have therefore produced transgenic mice expressing human 117V PrP. We found that brain tissue from GSS A117V patients did transmit disease to these mice and both the neuropathological features of prion disease and presence of PrP(Sc) was demonstrated in the brains of recipient transgenic mice. This PrP(Sc) rapidly degraded during laboratory analysis, suggesting that the difficulty in its detection in patients with GSS A117V could relate to post-mortem proteolysis. We conclude that GSS A117V is indeed a prion disease although the relative contributions of (Ctm)PrP and prion propagation in neurodegeneration and their pathogenetic interaction remains to be established.

Atypical scrapie prions from sheep and lack of disease in transgenic mice overexpressing human prion protein.

Public and animal health controls to limit human exposure to animal prions are focused on bovine spongiform encephalopathy (BSE), but other prion strains in ruminants may also have zoonotic potential. One example is atypical/Nor98 scrapie, which evaded statutory diagnostic methods worldwide until the early 2000s. To investigate whether sheep infected with scrapie prions could be another source of infection, we inoculated transgenic mice that overexpressed human prion protein with brain tissue from sheep with natural field cases of classical and atypical scrapie, sheep with experimental BSE, and cattle with BSE. We found that these mice were susceptible to BSE prions, but disease did not develop after prolonged postinoculation periods when mice were inoculated with classical or atypical scrapie prions. These data are consistent with the conclusion that prion disease is less likely to develop in humans after exposure to naturally occurring prions of sheep than after exposure to epizootic BSE prions of ruminants.

Spontaneous generation of mammalian prions.

Prions are transmissible agents that cause lethal neurodegeneration in humans and other mammals. Prions bind avidly to metal surfaces such as steel wires and, when surface-bound, can initiate infection of brain or cultured cells with remarkable efficiency. While investigating the properties of metal-bound prions by using the scrapie cell assay to measure infectivity, we observed, at low frequency, positive assay results in control groups in which metal wires had been coated with uninfected mouse brain homogenate. This phenomenon proved to be reproducible in rigorous and exhaustive control experiments designed to exclude prion contamination. The infectivity generated in cell culture could be readily transferred to mice and had strain characteristics distinct from the mouse-adapted prion strains used in the laboratory. The apparent "spontaneous generation" of prions from normal brain tissue could result if the metal surface, possibly with bound cofactors, catalyzed de novo formation of prions from normal cellular prion protein. Alternatively, if prions were naturally present in the brain at levels not detectable by conventional methods, metal surfaces might concentrate them to the extent that they become quantifiable by the scrapie cell assay.

Effect of fixation on brain and lymphoreticular vCJD prions and bioassay of key positive specimens from a retrospective vCJD prevalence study.

Anonymous screening of lymphoreticular tissues removed during routine surgery has been applied to estimate the UK population prevalence of asymptomatic vCJD prion infection. The retrospective study of Hilton et al (J Pathol 2004; 203: 733-739) found accumulation of abnormal prion protein in three formalin-fixed appendix specimens. This led to an estimated UK prevalence of vCJD infection of ∼1 in 4000, which remains the key evidence supporting current risk reduction measures to reduce iatrogenic transmission of vCJD prions in the UK. Confirmatory testing of these positives has been hampered by the inability to perform immunoblotting of formalin-fixed tissue. Animal transmission studies offer the potential for 'gold standard' confirmatory testing but are limited by both transmission barrier effects and known effects of fixation on scrapie prion titre in experimental models. Here we report the effects of fixation on brain and lymphoreticular human vCJD prions and comparative bioassay of two of the three prevalence study formalin-fixed, paraffin-embedded (FFPE) appendix specimens using transgenic mice expressing human prion protein (PrP). While transgenic mice expressing human PrP 129M readily reported vCJD prion infection after inoculation with frozen vCJD brain or appendix, and also FFPE vCJD brain, no infectivity was detected in FFPE vCJD spleen. No prion transmission was observed from either of the FFPE appendix specimens. The absence of detectable infectivity in fixed, known positive vCJD lymphoreticular tissue precludes interpreting negative transmissions from vCJD prevalence study appendix specimens. In this context, the Hilton et al study should continue to inform risk assessment pending the outcome of larger-scale studies on discarded surgical tissues and autopsy samples.

Inherited prion disease with 4-octapeptide repeat insertion: disease requires the interaction of multiple genetic risk factors.

Genetic factors are implicated in the aetiology of sporadic late-onset neurodegenerative diseases. Whether these genetic variants are predominantly common or rare, and how multiple genetic factors interact with each other to cause disease is poorly understood. Inherited prion diseases are highly heterogeneous and may be clinically mistaken for sporadic Creutzfeldt-Jakob disease because of a negative family history. Here we report our investigation of patients from the UK with four extra octapeptide repeats, which suggest that the risk of clinical disease is increased by a combination of the mutation and a susceptibility haplotype on the wild-type chromosome. The predominant clinical syndrome is a progressive cortical dementia with pyramidal signs, myoclonus and cerebellar abnormalities that closely resemble sporadic Creutzfeldt-Jakob disease. Autopsy shows perpendicular deposits of prion protein in the molecular layer of the cerebellum. Identity testing, PRNP microsatellite haplotyping and genealogical work confirm no cryptic close family relationships and suggests multiple progenitor disease haplotypes. All patients were homozygous for methionine at polymorphic codon 129. In addition, at a single nucleotide polymorphism upstream of PRNP thought to confer susceptibility to sporadic Creutzfeldt-Jakob disease (rs1029273), all patients were homozygous for the risk allele (combined P=5.9×10(-5)). The haplotype identified may also be a risk factor in other partially penetrant inherited prion diseases although it does not modify age of onset. Blood expression of PRNP in healthy individuals was modestly higher in carriers of the risk haplotype. These findings may provide a precedent for understanding apparently sporadic neurodegenerative diseases caused by rare high-risk mutations.

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.