Early and accurate diagnosis of Creutzfeldt-Jakob disease (CJD) is a necessary to distinguish this untreatable disease from treatable rapidly progressive dementias, and to prevent iatrogenic ...transmission. Currently, definitive diagnosis of CJD requires detection of the abnormally folded, CJD-specific form of protease-resistant prion protein (PrP(CJD)) in brain tissue obtained postmortem or via biopsy; therefore, diagnosis of sporadic CJD in clinical practice is often challenging. Supporting investigations, including MRI, EEG and conventional analyses of cerebrospinal fluid (CSF) biomarkers, are helpful in the diagnostic work-up, but do not allow definitive diagnosis. Recently, novel ultrasensitive seeding assays, based on the amplified detection of PrP(CJD), have improved the diagnostic process; for example, real-time quaking-induced conversion (RT-QuIC) is a sensitive method to detect prion-seeding activity in brain homogenate from humans with any subtype of sporadic CJD. RT-QuIC can also be used for in vivo diagnosis of CJD: its diagnostic sensitivity in detecting PrP(CJD) in CSF samples is 96%, and its specificity is 100%. Recently, we provided evidence that RT-QuIC of olfactory mucosa brushings is a 97% sensitive and 100% specific for sporadic CJD. These assays provide a basis for definitive antemortem diagnosis of prion diseases and, in doing so, improve prospects for reducing the risk of prion transmission. Moreover, they can be used to evaluate outcome measures in therapeutic trials for these as yet untreatable infections.
Within the extensive range of self-propagating pathologic protein aggregates of mammals, prions are the most clearly infectious (e.g., ∼109 lethal doses per milligram). The structures of such lethal ...assemblies of PrP molecules have been poorly understood. Here we report a near-atomic core structure of a brain-derived, fully infectious prion (263K strain). Cryo-electron microscopy showed amyloid fibrils assembled with parallel in-register intermolecular β sheets. Each monomer provides one rung of the ordered fibril core, with N-linked glycans and glycolipid anchors projecting outward. Thus, single monomers form the templating surface for incoming monomers at fibril ends, where prion growth occurs. Comparison to another prion strain (aRML) revealed major differences in fibril morphology but, like 263K, an asymmetric fibril cross-section without paired protofilaments. These findings provide structural insights into prion propagation, strains, species barriers, and membrane pathogenesis. This structure also helps frame considerations of factors influencing the relative transmissibility of other pathologic amyloids.
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•Cryo-EM reveals parallel in-register structure for an infectious brain-derived prion•N-linked glycans and GPI anchor project outward from the fibril core•Comparison to another prion strain reveals distinct conformational templates•In silico modeling suggests a structural basis for a prion transmission barrier
Kraus et al. provide a near-atomic-resolution structure of a brain-derived prion solved by cryo-electron microscopy. The parallel in-register assembly of prion protein monomers within the prion fibril, together with observed strain-dependent differences in fibril morphology, provide a structural foundation for understanding prion replication, strains, species barriers, and membrane pathogenesis.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
...whereas approximately 10‒15 g of brain-derived prions can be lethal, inoculation of a >109-fold larger amount of synthetic recombinant PrP fibrils can be innocuous 2. ...in evaluating and ...understanding the risks posed by various self-replicative protein assemblies, it is crucial to consider conformational details. (D) Extended lateral views of fibril density maps, with brackets indicating the relative cross-over distances. cryo-EM, cryogenic electron microscopy; PrP, prion protein. https://doi.org/10.1371/journal.ppat.1010594.g001 Figure omitted. ...there are more than 30 different types of PrP-based prions in mammals including various strains, sequences, and combinations thereof. ...much remains to be done to explain the full range of prion structures and how those structures underpin strain-specific disease phenotypes. Each of these PIRIBS-based structures has tightly packed regions in which the substitution of a heterologous residue might cause unfavorable steric clashes, electrostatic interactions, changes in hydrogen bonding, or other perturbations that could slow or prevent full conformational conversion of incoming monomers.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Blood-borne transmission of infectious prions during the symptomatic and asymptomatic stages of disease occurs for both human and animal transmissible spongiform encephalopathies (TSEs). The ...geographical distribution of the cervid TSE, chronic wasting disease (CWD), continues to spread across North America and the prospective number of individuals harboring an asymptomatic infection of human variant Creutzfeldt-Jakob Disease (vCJD) in the United Kingdom has been projected to be ~1 in 3000 residents. Thus, it is important to monitor cervid and human blood products to ensure herd health and human safety. Current methods for detecting blood-associated prions rely primarily upon bioassay in laboratory animals. While bioassay provides high sensitivity and specificity, it requires many months, animals, and it is costly. Here we report modification of the real time quaking-induced conversion (RT-QuIC) assay to detect blood-borne prions in whole blood from prion-infected preclinical white-tailed deer, muntjac deer, and Syrian hamsters, attaining sensitivity of >90% while maintaining 100% specificity. Our results indicate that RT-QuIC methodology as modified can provide consistent and reliable detection of blood-borne prions in preclinical and symptomatic stages of two animal TSEs, offering promise for prionemia detection in other species, including humans.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The prion-like spreading and accumulation of specific protein aggregates appear to be central to the pathogenesis of many human diseases, including Alzheimer's and Parkinson's. Accumulating evidence ...indicates that inoculation of tissue extracts from diseased individuals into suitable experimental animals can in many cases induce the aggregation of the disease-associated protein, as well as related pathological lesions. These findings, together with the history of the prion field, have raised the questions about whether such disease-associated protein aggregates are transmissible between humans by casual or iatrogenic routes, and, if so, do they propagate enough in the new host to cause disease? These practical considerations are important because real, and perhaps even only imagined, risks of human-to-human transmission of diseases such as Alzheimer's and Parkinson's may force costly changes in clinical practice that, in turn, are likely to have unintended consequences. The prion field has taught us that a single protein, PrP, can aggregate into forms that can propagate exponentially in vitro, but range from being innocuous to deadly when injected into experimental animals in ways that depend strongly on factors such as conformational subtleties, routes of inoculation, and host responses. In assessing the hazards posed by various disease-associated, self-propagating protein aggregates, it is imperative to consider both their actual transmissibilities and the pathological consequences of their propagation, if any, in recipient hosts.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
An early and accurate in vivo diagnosis of rapidly progressive dementia remains challenging, despite its critical importance for the outcome of treatable forms, and the formulation of prognosis. ...Real-Time Quaking-Induced Conversion (RT-QuIC) is an in vitro assay that, for the first time, specifically discriminates patients with prion disease. Here, using cerebrospinal fluid (CSF) samples from 239 patients with definite or probable prion disease and 100 patients with a definite alternative diagnosis, we compared the performance of the first (PQ-CSF) and second generation (IQ-CSF) RT-QuIC assays, and investigated the diagnostic value of IQ-CSF across the broad spectrum of human prions. Our results confirm the high sensitivity of IQ-CSF for detecting human prions with a sub-optimal sensitivity for the sporadic CJD subtypes MM2C and MM2T, and a low sensitivity limited to variant CJD, Gerstmann-Sträussler-Scheinker syndrome and fatal familial insomnia. While we found no difference in specificity between PQ-CSF and IQ-CSF, the latter showed a significant improvement in sensitivity, allowing prion detection in about 80% of PQ-CSF negative CJD samples. Our results strongly support the implementation of IQ-CSF in clinical practice. By rapidly confirming or excluding CJD with high accuracy the assay is expected to improve the outcome for patients and their enrollment in therapeutic trials.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Alzheimer disease (AD) and chronic traumatic encephalopathy (CTE) involve the abnormal accumulation in the brain of filaments composed of both three-repeat (3R) and four-repeat (4R) (3R/4R) tau ...isoforms. To probe the molecular basis for AD’s tau filament propagation and to improve detection of tau aggregates as potential biomarkers, we have exploited the seeded polymerization growth mechanism of tau filaments to develop a highly selective and ultrasensitive cell-free tau seed amplification assay optimized for AD (AD real-time quaking-induced conversion or AD RT-QuIC). The reaction is based on the ability of AD tau aggregates to seed the formation of amyloid fibrils made of certain recombinant tau fragments. AD RT-QuIC detected seeding activity in AD (
n
= 16) brains at dilutions as extreme as 10
7
–10
10
-fold, but was 10
2
–10
6
-fold less responsive when seeded with brain from most cases of other types of tauopathy with comparable loads of predominant 3R or 4R tau aggregates. For example, AD brains had average seeding activities that were orders of magnitude higher than Pick disease brains with predominant 3R tau deposits, but the opposite was true using our previously described Pick-optimized tau RT-QuIC assay. CTE brains (
n
= 2) had seed concentrations comparable to the weakest of the AD specimens, and higher than 3 of 4 specimens with 3R/4R primary age-related tauopathy. AD seeds shared properties with the tau filaments found in AD brains, as AD seeds were sarkosyl-insoluble, protease resistant, and reactive with tau antibodies. Moreover, AD RT-QuIC detected as little as 16 fg of pure synthetic tau fibrils. The distinctive seeding activity exhibited by AD and CTE tau filaments compared to other types of tauopathies in these seeded polymerization reactions provides a mechanistic basis for their consistent propagation as specific conformers in patients with 3R/4R tau diseases. Importantly, AD RT-QuIC also provides rapid ultrasensitive quantitation of 3R/4R tau-seeding activity as a biomarker.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Objective
Several prion amplification systems have been proposed for detection of prions in cerebrospinal fluid (CSF), most recently, the measurements of prion seeding activity with second‐generation ...real‐time quaking‐induced conversion (RT‐QuIC). The objective of this study was to investigate the diagnostic performance of the RT‐QuIC prion test in the broad phenotypic spectrum of prion diseases.
Methods
We performed CSF RT‐QuIC testing in 2,141 patients who had rapidly progressive neurological disorders, determined diagnostic sensitivity and specificity in 272 cases that were autopsied, and evaluated the impact of mutations and polymorphisms in the PRNP gene, and type 1 or type 2 human prions on diagnostic performance.
Results
The 98.5% diagnostic specificity and 92% sensitivity of CSF RT‐QuIC in a blinded retrospective analysis matched the 100% specificity and 95% sensitivity of a blind prospective study. The CSF RT‐QuIC differentiated 94% of cases of sporadic Creutzfeldt–Jakob disease (sCJD) MM1 from the sCJD MM2 phenotype, and 80% of sCJD VV2 from sCJD VV1. The mixed prion type 1‐2 and cases heterozygous for codon 129 generated intermediate CSF RT‐QuIC patterns, whereas genetic prion diseases revealed distinct profiles for each PRNP gene mutation.
Interpretation
The diagnostic performance of the improved CSF RT‐QuIC is superior to surrogate marker tests for prion diseases such as 14‐3‐3 and tau proteins, and together with PRNP gene sequencing the test allows the major prion subtypes to be differentiated in vivo. This differentiation facilitates prediction of the clinicopathological phenotype and duration of the disease—two important considerations for envisioned therapeutic interventions. ANN NEUROL 2017;81:79–92
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
IMPORTANCE: Early and accurate in vivo diagnosis of Creutzfeldt-Jakob disease (CJD) is necessary for quickly distinguishing treatable from untreatable rapidly progressive dementias and for future ...therapeutic trials. This early diagnosis is becoming possible using the real-time quaking-induced conversion (RT-QuIC) seeding assay, which detects minute amounts of the disease-specific pathologic prion protein in cerebrospinal fluid (CSF) or olfactory mucosa (OM) samples. OBJECTIVE: To develop an algorithm for accurate and early diagnosis of CJD by using the RT-QuIC assay on CSF samples, OM samples, or both. DESIGN, SETTING, AND PARTICIPANTS: In this case-control study, samples of CSF and OM were collected from 86 patients with a clinical diagnosis of probable (n = 51), possible (n = 24), or suspected (n = 11) CJD and 104 negative control samples (54 CSF and 50 OM). The CSF and OM samples were analyzed using conventional RT-QuIC. The CSF samples underwent further testing using improved RT-QuIC conditions. In addition, the diagnostic performance of a novel, easy-to-use, gentle flocked swab for sampling of OM was evaluated. Data were collected from January 1 to June 30, 2015. MAIN OUTCOME AND MEASURES: Correlations between RT-QuIC results and the final diagnosis of recruited patients. RESULTS: Among the 86 patients (37 men 43% and 49 women 57%; mean SD age, 65.7 11.5 years) included for analysis, all 61 patients with sporadic CJD had positive RT-QuIC findings using OM or CSF samples or both for an overall RT-QuIC diagnostic sensitivity of 100% (95% CI, 93%-100%). All patients with a final diagnosis of non–prion disease (71 CSF and 67 OM samples) had negative RT-QuIC findings for 100% specificity (95% CI, 94%-100%). Of 8 symptomatic patients with various mutations causing CJD or Gerstmann-Sträussler-Scheinker syndrome, 6 had positive and 2 had negative RT-QuIC findings for a sensitivity of 75% (95% CI, 36%-96%). CONCLUSIONS AND RELEVANCE: A proposed diagnostic algorithm for sporadic CJD combines CSF and OM RT-QuIC testing to provide virtually 100% diagnostic sensitivity and specificity in the clinical phase of the disease.
The diagnosis and treatment of synucleinopathies such as Parkinson disease and dementia with Lewy bodies would be aided by the availability of assays for the pathogenic disease-associated forms of ...α-synuclein (αSyn
) that are sufficiently sensitive, specific, and practical for analysis of accessible diagnostic specimens. Two recent αSyn
seed amplification tests have provided the first prototypes for ultrasensitive and specific detection of αSyn
in patients' cerebrospinal fluid. These prototypic assays require 5-13 days to perform. Here, we describe an improved α-synuclein real time quaking-induced conversion (αSyn RT-QuIC) assay that has similar sensitivity and specificity to the prior assays, but can be performed in 1-2 days with quantitation. Blinded analysis of cerebrospinal fluid from 29 synucleinopathy cases 12 Parkinson's and 17 dementia with Lewy bodies and 31 non-synucleinopathy controls, including 16 Alzheimer's cases, yielded 93% diagnostic sensitivity and 100% specificity for this test so far. End-point dilution analyses allowed quantitation of relative amounts of αSyn
seeding activity in cerebrospinal fluid samples, and detection in as little as 0.2 μL. These results confirm that αSyn
seeding activity is present in cerebrospinal fluid. We also demonstrate that it can be rapidly detected, and quantitated, even in early symptomatic stages of synucleinopathy.
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