High-resolution structure and strain comparison of infectious mammalian prions.

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 ß 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.

Sensitive detection of pathological seeds of α-synuclein, tau and prion protein on solid surfaces.

Prions or prion-like aggregates such as those composed of PrP, α-synuclein, and tau are key features of proteinopathies such as prion, Parkinson's and Alzheimer's diseases, respectively. Their presence on solid surfaces may be biohazardous under some circumstances. PrP prions bound to solids are detectable by ultrasensitive real-time quaking-induced conversion (RT-QuIC) assays if the solids can be immersed in assay wells or the prions transferred to pads. Here we show that prion-like seeds can remain detectable on steel wires for at least a year, or even after enzymatic cleaning and sterilization. We also show that contamination of larger objects with pathological seeds of α-synuclein, tau, and PrP can be detected by simply assaying a sampling medium that has been transiently applied to the surface. Human α-synuclein seeds in dementia with Lewy bodies brain tissue were detected by α-synuclein RT-QuIC after drying of tissue dilutions with concentrations as low as 10-6 onto stainless steel. Tau RT-QuIC detected tau seeding activity on steel exposed to Alzheimer's disease brain tissue diluted as much as a billion fold. Prion RT-QuIC assays detected seeding activity on plates exposed to brain dilutions as extreme as 10-5-10-8 from prion-affected humans, sheep, cattle and cervids. Sampling medium collected from surgical instruments used in necropsies of sporadic Creutzfeldt-Jakob disease-infected transgenic mice was positive down to 10-6 dilution. Sensitivity for prion detection was not sacrificed by omitting the recombinant PrP substrate from the sampling medium during its application to a surface and subsequent storage as long as the substrate was added prior to performing the assay reaction. Our findings demonstrate practical prototypic surface RT-QuIC protocols for the highly sensitive detection of pathologic seeds of α-synuclein, tau, and PrP on solid objects.

α-Synuclein seeding activity in duodenum biopsies from Parkinson's disease patients.

Abnormal deposition of α-synuclein is a key feature and biomarker of Parkinson's disease. α-Synuclein aggregates can propagate themselves by a prion-like seeding-based mechanism within and between tissues and are hypothesized to move between the intestine and brain. α-Synuclein RT-QuIC seed amplification assays have detected Parkinson's-associated α-synuclein in multiple biospecimens including post-mortem colon samples. Here we show intra vitam detection of seeds in duodenum biopsies from 22/23 Parkinson's patients, but not in 6 healthy controls by RT-QuICR. In contrast, no tau seeding activity was detected in any of the biopsies. Our seed amplifications provide evidence that the upper intestine contains a form(s) of α-synuclein with self-propagating activity. The diagnostic sensitivity and specificity for PD in this biopsy panel were 95.7% and 100% respectively. End-point dilution analysis indicated up to 106 SD50 seeding units per mg of tissue with positivity in two contemporaneous biopsies from individual patients suggesting widespread distribution within the superior and descending parts of duodenum. Our detection of α-synuclein seeding activity in duodenum biopsies of Parkinson's disease patients suggests not only that such analyses may be useful in ante-mortem diagnosis, but also that the duodenum may be a source or a destination for pathological, self-propagating α-synuclein assemblies.

Lack of Transmission of Chronic Wasting Disease Prions to Human Cerebral Organoids.

Chronic wasting disease (CWD) is a cervid prion disease with unknown zoonotic potential that might pose a risk to humans who are exposed. To assess the potential of CWD to infect human neural tissue, we used human cerebral organoids with 2 different prion genotypes, 1 of which has previously been associated with susceptibility to zoonotic prion disease. We exposed organoids from both genotypes to high concentrations of CWD inocula from 3 different sources for 7 days, then screened for infection periodically for up to 180 days. No de novo CWD propagation or deposition of protease-resistant forms of human prions was evident in CWD-exposed organoids. Some persistence of the original inoculum was detected, which was equivalent in prion gene knockout organoids and thus not attributable to human prion propagation. Overall, the unsuccessful propagation of CWD in cerebral organoids supports a strong species barrier to transmission of CWD prions to humans.

Cryo-EM of prion strains from the same genotype of host identifies conformational determinants.

Prion strains in a given type of mammalian host are distinguished by differences in clinical presentation, neuropathological lesions, survival time, and characteristics of the infecting prion protein (PrP) assemblies. Near-atomic structures of prions from two host species with different PrP sequences have been determined but comparisons of distinct prion strains of the same amino acid sequence are needed to identify purely conformational determinants of prion strain characteristics. Here we report a 3.2 Å resolution cryogenic electron microscopy-based structure of the 22L prion strain purified from the brains of mice engineered to express only PrP lacking glycophosphatidylinositol anchors [anchorless (a) 22L]. Comparison of this near-atomic structure to our recently determined structure of the aRML strain propagated in the same inbred mouse reveals that these two mouse prion strains have distinct conformational templates for growth via incorporation of PrP molecules of the same sequence. Both a22L and aRML are assembled as stacks of PrP molecules forming parallel in-register intermolecular ß-sheets and intervening loops, with single monomers spanning the ordered fibril core. Each monomer shares an N-terminal steric zipper, three major arches, and an overall V-shape, but the details of these and other conformational features differ markedly. Thus, variations in shared conformational motifs within a parallel in-register ß-stack fibril architecture provide a structural basis for prion strain differentiation within a single host genotype.

Large-scale validation of skin prion seeding activity as a biomarker for diagnosis of prion diseases.

Definitive diagnosis of sporadic Creutzfeldt-Jakob disease (sCJD) relies on the examination of brain tissues for the pathological prion protein (PrPSc). Our previous study revealed that PrPSc-seeding activity (PrPSc-SA) is detectable in skin of sCJD patients by an ultrasensitive PrPSc seed amplification assay (PrPSc-SAA) known as real-time quaking-induced conversion (RT-QuIC). A total of 875 skin samples were collected from 2 cohorts (1 and 2) at autopsy from 2-3 body areas of 339 cases with neuropathologically confirmed prion diseases and non-sCJD controls. The skin samples were analyzed for PrPSc-SA by RT-QuIC assay. The results were compared with demographic information, clinical manifestations, cerebrospinal fluid (CSF) PrPSc-SA, other laboratory tests, subtypes of prion diseases defined by the methionine (M) or valine (V) polymorphism at residue 129 of PrP, PrPSc types (#1 or #2), and gene mutations in deceased patients. RT-QuIC assays of the cohort #1 by two independent laboratories gave 87.3% or 91.3% sensitivity and 94.7% or 100% specificity, respectively. The cohort #2 showed sensitivity of 89.4% and specificity of 95.5%. RT-QuIC of CSF available from 212 cases gave 89.7% sensitivity and 94.1% specificity. The sensitivity of skin RT-QuIC was subtype dependent, being highest in sCJDVV1-2 subtype, followed by VV2, MV1-2, MV1, MV2, MM1, MM1-2, MM2, and VV1. The skin area next to the ear gave highest sensitivity, followed by lower back and apex of the head. Although no difference in brain PrPSc-SA was detected between the cases with false negative and true positive skin RT-QuIC results, the disease duration was significantly longer with the false negatives [12.0 ± 13.3 (months, SD) vs. 6.5 ± 6.4, p < 0.001]. Our study validates skin PrPSc-SA as a biomarker for the detection of prion diseases, which is influenced by the PrPSc types, PRNP 129 polymorphisms, dermatome sampled, and disease duration.

Seed amplification and neurodegeneration marker trajectories in individuals at risk of prion disease.

Human prion diseases are remarkable for long incubation times followed typically by rapid clinical decline. Seed amplification assays and neurodegeneration biofluid biomarkers are remarkably useful in the clinical phase, but their potential to predict clinical onset in healthy people remains unclear. This is relevant not only to the design of preventive strategies in those at-risk of prion diseases, but more broadly, because prion-like mechanisms are thought to underpin many neurodegenerative disorders. Here, we report the accrual of a longitudinal biofluid resource in patients, controls and healthy people at risk of prion diseases, to which ultrasensitive techniques such as real-time quaking-induced conversion (RT-QuIC) and single molecule array (Simoa) digital immunoassays were applied for preclinical biomarker discovery. We studied 648 CSF and plasma samples, including 16 people who had samples taken when healthy but later developed inherited prion disease (IPD) ('converters'; range from 9.9 prior to, and 7.4 years after onset). Symptomatic IPD CSF samples were screened by RT-QuIC assay variations, before testing the entire collection of at-risk samples using the most sensitive assay. Glial fibrillary acidic protein (GFAP), neurofilament light (NfL), tau and UCH-L1 levels were measured in plasma and CSF. Second generation (IQ-CSF) RT-QuIC proved 100% sensitive and specific for sporadic Creutzfeldt-Jakob disease (CJD), iatrogenic and familial CJD phenotypes, and subsequently detected seeding activity in four presymptomatic CSF samples from three E200K carriers; one converted in under 2 months while two remain asymptomatic after at least 3 years' follow-up. A bespoke HuPrP P102L RT-QuIC showed partial sensitivity for P102L disease. No compatible RT-QuIC assay was discovered for classical 6-OPRI, A117V and D178N, and these at-risk samples tested negative with bank vole RT-QuIC. Plasma GFAP and NfL, and CSF NfL levels emerged as proximity markers of neurodegeneration in the typically slow IPDs (e.g. P102L), with significant differences in mean values segregating healthy control from IPD carriers (within 2 years to onset) and symptomatic IPD cohorts; plasma GFAP appears to change before NfL, and before clinical conversion. In conclusion, we show distinct biomarker trajectories in fast and slow IPDs. Specifically, we identify several years of presymptomatic seeding positivity in E200K, a new proximity marker (plasma GFAP) and sequential neurodegenerative marker evolution (plasma GFAP followed by NfL) in slow IPDs. We suggest a new preclinical staging system featuring clinical, seeding and neurodegeneration aspects, for validation with larger prion at-risk cohorts, and with potential application to other neurodegenerative proteopathies.

Tau seeds occur before earliest Alzheimer's changes and are prevalent across neurodegenerative diseases.

Tau neurofibrillary tangles are a hallmark of Alzheimer's disease neuropathological change. However, it remains largely unclear how distinctive Alzheimer's disease tau seeds (i.e. 3R/4R) correlate with histological indicators of tau accumulation. Furthermore, AD tau co-pathology is thought to influence features and progression of other neurodegenerative diseases including Lewy body disease; yet measurements of different types of tau seeds in the setting of such diseases is an unmet need. Here, we use tau real-time quaking-induced conversion (RT-QuIC) assays to selectively quantitate 3R/4R tau seeds in the frontal lobe which accumulates histologically identifiable tau pathology at late disease stages of AD neuropathologic change. Seed quantitation across a spectrum of neurodegenerative disease cases and controls indicated tau seeding activity can be detected well before accompanying histopathological indication of tau deposits, and even prior to the earliest evidence of Alzheimer's-related tau accumulation anywhere in the brain. In later stages of AD, 3R/4R tau RT-QuIC measures correlated with immunohistochemical tau burden. In addition, Alzheimer's tau seeds occur in the vast majority of cases evaluated here inclusive of primary synucleinopathies, frontotemporal lobar degeneration and even controls albeit at multi-log lower levels than Alzheimer's cases. α-synuclein seeding activity confirmed synucleinopathy cases and further indicated the co-occurrence of α-synuclein seeds in some Alzheimer's disease and primary tauopathy cases. Our analysis indicates that 3R/4R tau seeds in the mid-frontal lobe correlate with the overall Braak stage and Alzheimer's disease neuropathologic change, supporting the quantitative predictive value of tau RT-QuIC assays. Our data also indicate 3R/4R tau seeds are elevated in females compared to males at high (≥ IV) Braak stages. This study suggests 3R/4R tau seeds are widespread even prior to the earliest stages of Alzheimer's disease changes, including in normal, and even young individuals, with prevalence across multiple neurodegenerative diseases to further define disease subtypes.

Second passage experiments of chronic wasting disease in transgenic mice overexpressing human prion protein.

Chronic wasting disease (CWD) is a prion disease of cervids including deer, elk, reindeer, and moose. Human consumption of cervids is common, therefore assessing the risk potential of CWD transmission to humans is critical. In a previous study, we tested CWD transmission via intracerebral inoculation into transgenic mice (tg66 and tgRM) that over-expressed human prion protein. Mice screened by traditional prion detection assays were negative. However, in a group of 88 mice screened by the ultrasensitive RT-QuIC assay, we identified 4 tg66 mice that produced inconsistent positive RT-QuIC reactions. These data could be false positive reactions, residual input inoculum or indicative of subclinical infections suggestive of cross species transmission of CWD to humans. Additional experiments were required to understand the nature of the prion seeding activity in this model. In this manuscript, second passage experiments using brains from mice with weak prion seeding activity showed they were not infectious to additional recipient tg66 mice. Clearance experiments showed that input CWD prion seeding activity was eliminated by 180 days in tg66 mice and PrPKO mice, which are unable to replicate prion protein, indicating that the weak positive levels of seeding activity detected at later time points was not likely residual inoculum. The failure of CWD prions to cause disease in tg66 after two sequential passages suggested that a strong species barrier prevented CWD infection of mice expressing human prion protein.

Million-fold sensitivity enhancement in proteopathic seed amplification assays for biospecimens by Hofmeister ion comparisons.

Recent work with prion diseases and synucleinopathies indicates that accurate diagnostic methods for protein-folding diseases can be based on the ultrasensitive, amplified measurement of pathological aggregates in biospecimens. A better understanding of the physicochemical factors that control the seeded polymerization of such aggregates, and their amplification in vitro, should allow improvements in existing assay platforms, as well as the development of new assays for other proteopathic aggregates. Here, we systematically investigated the effects of the ionic environment on the polymerization of tau, α-synuclein, and the prion protein (PrP) induced by aggregates in biospecimens. We screened salts of the Hofmeister series, a relative ordering of strongly and weakly hydrated salts that tend to precipitate or solubilize proteins. We found that sensitivities of tau-based assays for Alzheimer's seeds and PrP-based assays for prions were best in weakly hydrated anions. In contrast, we saw an inverse trend with different tau-based assays, improving detection sensitivity for progressive supranuclear palsy seeds by ≈106 Hofmeister analysis also improved detection of sporadic Creutzfeldt-Jakob disease prions in human nasal brushings and chronic wasting disease prions in deer-ear homogenates. Our results demonstrate strong and divergent influences of ionic environments on the amplification and detection of proteopathic seeds as biomarkers for protein-folding diseases.

Ultrasensitive RT-QuIC assay with high sensitivity and specificity for Lewy body-associated synucleinopathies.

The clinical diagnosis of synucleinopathies, including Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA), is challenging, especially at an early disease stage, due to the heterogeneous and often non-specific clinical manifestations. The discovery of reliable specific markers for synucleinopathies would consequently be of great aid to the diagnosis and management of these disorders. Real-Time Quaking-Induced Conversion (RT-QuIC) is an ultrasensitive technique that has been previously used to detect self-templating amyloidogenic proteins in the cerebrospinal fluid (CSF) and other biospecimens in prion disease and synucleinopathies. Using a wild-type recombinant α-synuclein as a substrate, we applied RT-QuIC to a large cohort of 439 CSF samples from clinically well-characterized, or post-mortem verified patients with parkinsonism or dementia. Of significance, we also studied patients with isolated REM sleep behavior disorder (iRBD) (n = 18) and pure autonomic failure (PAF) (n = 28), representing clinical syndromes that are often caused by a synucleinopathy, and may precede the appearance of parkinsonism or cognitive decline. The results show that our RT-QuIC assay can accurately detect α-synuclein seeding activity across the spectrum of Lewy Body (LB)-related disorders (LBD), including DLB, PD, iRBD, and PAF, with an overall sensitivity of 95.3%. In contrast, all but two patients with MSA showed no α-synuclein seeding activity in the applied experimental setting. The analysis of the fluorescence response reflecting the amount of α-synuclein seeds revealed no significant differences between the clinical syndromes associated with LB pathology. Finally, the assay demonstrated 98% specificity in a neuropathological cohort of 101 cases lacking LB pathology. In conclusion, α-synuclein RT-QuIC provides an accurate marker of synucleinopathies linked to LB pathology and may have a pivotal role in the early discrimination and management of affected patients. The finding of no α-synuclein seeding activity in MSA seems to support the current view that MSA and LBD are associated with different conformational strains of α-synuclein.

Correction to: Ultrasensitive RT-QuIC assay with high sensitivity and specificity for Lewy body-associated synucleinopathies.

he article Ultrasensitive RT­QuIC assay with high sensitivity and specificity for Lewy body­.

Lack of Transmission of Chronic Wasting Disease to Cynomolgus Macaques.

Chronic wasting disease (CWD) is a fatal prion disease that can infect deer, elk, and moose. CWD was first recognized in captive deer kept in wildlife facilities in Colorado from 1967 to 1979. CWD has now been detected in 25 U.S. states, 2 Canadian provinces, South Korea, Norway, and Finland. It is currently unknown if humans are susceptible to CWD infection. Understanding the health risk from consuming meat and/or products from CWD-infected cervids is a critical human health concern. Previous research using transgenic mouse models and in vitro conversion assays suggests that a significant species barrier exists between CWD and humans. To date, reported epidemiologic studies of humans consuming cervids in areas where CWD is endemic have found no evidence to confirm CWD transmission to humans. Previously, we reported data from ongoing cross-species CWD transmission studies using two species of nonhuman primates as models. Squirrel monkeys (SM) and cynomolgus macaques (CM) were inoculated by either the intracerebral or oral route with brain homogenates from CWD-infected deer and elk containing high levels of infectivity. SM were highly susceptible to CWD infection, while CM were not. In the present study, we present new data for seven CWD-inoculated CM euthanized 11 to 13 years after CWD inoculation and eight additional uninoculated control CM. New and archival CM tissues were screened for prion infection by using the ultrasensitive real-time quaking-induced conversion (RT-QuIC) assay, immunohistochemistry, and immunoblotting. In this study, there was no clinical, pathological, or biochemical evidence suggesting that CWD was transmitted from cervids to CM.IMPORTANCE Chronic wasting disease (CWD) is a fatal prion disease found in deer, elk, and moose. Since it was first discovered in the late 1960s, CWD has now spread to at least 25 U.S. states, 2 Canadian provinces, South Korea, Norway, and Finland. Eradication of CWD from areas of endemicity is very unlikely, and additional spread will occur. As the range and prevalence of CWD increase, so will the potential for human exposure to CWD prions. It is currently unknown if CWD poses a risk to human health. However, determining this risk is critical to preventing a scenario similar to that which occurred when mad cow disease was found to be transmissible to humans. In the present study, we used cynomolgus macaque monkeys as a surrogate model for CWD transmission to humans. After 13 years, no evidence for CWD transmission to macaques was detected clinically or by using highly sensitive prion disease-screening assays.

Role of the central lysine cluster and scrapie templating in the transmissibility of synthetic prion protein aggregates.

Mammalian prion structures and replication mechanisms are poorly understood. Most synthetic recombinant prion protein (rPrP) amyloids prepared without cofactors are non-infectious or much less infectious than bona fide tissue-derived PrPSc. This effect has been associated with differences in folding of the aggregates, manifested in part by reduced solvent exclusion and protease-resistance in rPrP amyloids, especially within residues ~90-160. Substitution of 4 lysines within residues 101-110 of rPrP (central lysine cluster) with alanines (K4A) or asparagines (K4N) allows formation of aggregates with extended proteinase K (PK) resistant cores reminiscent of PrPSc, particularly when seeded with PrPSc. Here we have compared the infectivity of rPrP aggregates made with K4N, K4A or wild-type (WT) rPrP, after seeding with scrapie brain homogenate (ScBH) or normal brain homogenate (NBH). None of these preparations caused clinical disease on first passage into rodents. However, the ScBH-seeded fibrils (only) led to a subclinical pathogenesis as indicated by increases in prion seeding activity, neuropathology, and abnormal PrP in the brain. Seeding activities usually accumulated to much higher levels in animals inoculated with ScBH-seeded fibrils made with the K4N, rather than WT, rPrP molecules. Brain homogenates from subclinical animals induced clinical disease on second passage into "hamsterized" Tg7 mice, with shorter incubation times in animals inoculated with ScBH-seeded K4N rPrP fibrils. On second passage from animals inoculated with ScBH-seeded WT fibrils, we detected an additional PK resistant PrP fragment that was similar to that of bona fide PrPSc. Together these data indicate that both the central lysine cluster and scrapie seeding of rPrP aggregates influence the induction of PrP misfolding, neuropathology and clinical manifestations upon passage in vivo. We confirm that some rPrP aggregates can initiate further aggregation without typical pathogenesis in vivo. We also provide evidence that there is little, if any, biohazard associated with routine RT-QuIC assays.

PrP Knockout Cells Expressing Transmembrane PrP Resist Prion Infection.

Glycosylphosphatidylinositol (GPI) anchoring of the prion protein (PrPC) influences PrPC misfolding into the disease-associated isoform, PrPres, as well as prion propagation and infectivity. GPI proteins are found in cholesterol- and sphingolipid-rich membrane regions called rafts. Exchanging the GPI anchor for a nonraft transmembrane sequence redirects PrPC away from rafts. Previous studies showed that nonraft transmembrane PrPC variants resist conversion to PrPres when transfected into scrapie-infected N2a neuroblastoma cells, likely due to segregation of transmembrane PrPC and GPI-anchored PrPres in distinct membrane environments. Thus, it remained unclear whether transmembrane PrPC might convert to PrPres if seeded by an exogenous source of PrPres not associated with host cell rafts and without the potential influence of endogenous expression of GPI-anchored PrPC To further explore these questions, constructs containing either a C-terminal wild-type GPI anchor signal sequence or a nonraft transmembrane sequence containing a flexible linker were expressed in a cell line derived from PrP knockout hippocampal neurons, NpL2. NpL2 cells have physiological similarities to primary neurons, representing a novel and advantageous model for studying transmissible spongiform encephalopathy (TSE) infection. Cells were infected with inocula from multiple prion strains and in different biochemical states (i.e., membrane bound as in brain microsomes from wild-type mice or purified GPI-anchorless amyloid fibrils). Only GPI-anchored PrPC supported persistent PrPres propagation. Our data provide strong evidence that in cell culture GPI anchor-directed membrane association of PrPC is required for persistent PrPres propagation, implicating raft microdomains as a location for conversion. IMPORTANCE: Mechanisms of prion propagation, and what makes them transmissible, are poorly understood. Glycosylphosphatidylinositol (GPI) membrane anchoring of the prion protein (PrPC) directs it to specific regions of cell membranes called rafts. In order to test the importance of the raft environment on prion propagation, we developed a novel model for prion infection where cells expressing either GPI-anchored PrPC or transmembrane-anchored PrPC, which partitions it to a different location, were treated with infectious, misfolded forms of the prion protein, PrPres We show that only GPI-anchored PrPC was able to convert to PrPres and able to serially propagate. The results strongly suggest that GPI anchoring and the localization of PrPC to rafts are crucial to the ability of PrPC to propagate as a prion.

PrP P102L and Nearby Lysine Mutations Promote Spontaneous In Vitro Formation of Transmissible Prions.

Accumulation of fibrillar protein aggregates is a hallmark of many diseases. While numerous proteins form fibrils by prion-like seeded polymerization in vitro, only some are transmissible and pathogenic in vivo To probe the structural features that confer transmissibility to prion protein (PrP) fibrils, we have analyzed synthetic PrP amyloids with or without the human prion disease-associated P102L mutation. The formation of infectious prions from PrP molecules in vitro has required cofactors and/or unphysiological denaturing conditions. Here, we demonstrate that, under physiologically compatible conditions without cofactors, the P102L mutation in recombinant hamster PrP promoted prion formation when seeded by minute amounts of scrapie prions in vitro Surprisingly, combination of the P102L mutation with charge-neutralizing substitutions of four nearby lysines promoted spontaneous prion formation. When inoculated into hamsters, both of these types of synthetic prions initiated substantial accumulation of prion seeding activity and protease-resistant PrP without transmissible spongiform encephalopathy (TSE) clinical signs or notable glial activation. Our evidence suggests that PrP's centrally located proline and lysine residues act as conformational switches in the in vitro formation of transmissible PrP amyloids.IMPORTANCE Many diseases involve the damaging accumulation of specific misfolded proteins in thread-like aggregates. These threads (fibrils) are capable of growing on the ends by seeding the refolding and incorporation of the normal form of the given protein. In many cases such aggregates can be infectious and propagate like prions when transmitted from one individual host to another. Some transmitted aggregates can cause fatal disease, as with human iatrogenic prion diseases, while other aggregates appear to be relatively innocuous. The factors that distinguish infectious and pathogenic protein aggregates from more innocuous ones are poorly understood. Here we have compared the combined effects of prion seeding and mutations of prion protein (PrP) on the structure and transmission properties of synthetic PrP aggregates. Our results highlight the influence of specific sequence features in the normally unstructured region of PrP that influence the infectious and neuropathogenic properties of PrP-derived aggregates.

Inactivation of Prions and Amyloid Seeds with Hypochlorous Acid.

Hypochlorous acid (HOCl) is produced naturally by neutrophils and other cells to kill conventional microbes in vivo. Synthetic preparations containing HOCl can also be effective as microbial disinfectants. Here we have tested whether HOCl can also inactivate prions and other self-propagating protein amyloid seeds. Prions are deadly pathogens that are notoriously difficult to inactivate, and standard microbial disinfection protocols are often inadequate. Recommended treatments for prion decontamination include strongly basic (pH ≥~12) sodium hypochlorite bleach, ≥1 N sodium hydroxide, and/or prolonged autoclaving. These treatments are damaging and/or unsuitable for many clinical, agricultural and environmental applications. We have tested the anti-prion activity of a weakly acidic aqueous formulation of HOCl (BrioHOCl) that poses no apparent hazard to either users or many surfaces. For example, BrioHOCl can be applied directly to skin and mucous membranes and has been aerosolized to treat entire rooms without apparent deleterious effects. Here, we demonstrate that immersion in BrioHOCl can inactivate not only a range of target microbes, including spores of Bacillus subtilis, but also prions in tissue suspensions and on stainless steel. Real-time quaking-induced conversion (RT-QuIC) assays showed that BrioHOCl treatments eliminated all detectable prion seeding activity of human Creutzfeldt-Jakob disease, bovine spongiform encephalopathy, cervine chronic wasting disease, sheep scrapie and hamster scrapie; these findings indicated reductions of ≥103- to 106-fold. Transgenic mouse bioassays showed that all detectable hamster-adapted scrapie infectivity in brain homogenates or on steel wires was eliminated, representing reductions of ≥~105.75-fold and >104-fold, respectively. Inactivation of RT-QuIC seeding activity correlated with free chlorine concentration and higher order aggregation or destruction of proteins generally, including prion protein. BrioHOCl treatments had similar effects on amyloids composed of human α-synuclein and a fragment of human tau. These results indicate that HOCl can block the self-propagating activity of prions and other amyloids.

Diagnostic and prognostic value of human prion detection in cerebrospinal fluid.

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.

A test for Creutzfeldt-Jakob disease using nasal brushings.

BACKGROUND: Definite diagnosis of sporadic Creutzfeldt-Jakob disease in living patients remains a challenge. A test that detects the specific marker for Creutzfeldt-Jakob disease, the prion protein (PrP(CJD)), by means of real-time quaking-induced conversion (RT-QuIC) testing of cerebrospinal fluid has a sensitivity of 80 to 90% for the diagnosis of sporadic Creutzfeldt-Jakob disease. We have assessed the accuracy of RT-QuIC analysis of nasal brushings from olfactory epithelium in diagnosing sporadic Creutzfeldt-Jakob disease in living patients. METHODS: We collected olfactory epithelium brushings and cerebrospinal fluid samples from patients with and patients without sporadic Creutzfeldt-Jakob disease and tested them using RT-QuIC, an ultrasensitive, multiwell plate-based fluorescence assay involving PrP(CJD)-seeded polymerization of recombinant PrP into amyloid fibrils. RESULTS: The RT-QuIC assays seeded with nasal brushings were positive in 30 of 31 patients with Creutzfeldt-Jakob disease (15 of 15 with definite sporadic Creutzfeldt-Jakob disease, 13 of 14 with probable sporadic Creutzfeldt-Jakob disease, and 2 of 2 with inherited Creutzfeldt-Jakob disease) but were negative in 43 of 43 patients without Creutzfeldt-Jakob disease, indicating a sensitivity of 97% (95% confidence interval [CI], 82 to 100) and specificity of 100% (95% CI, 90 to 100) for the detection of Creutzfeldt-Jakob disease. By comparison, testing of cerebrospinal fluid samples from the same group of patients had a sensitivity of 77% (95% CI, 57 to 89) and a specificity of 100% (95% CI, 90 to 100). Nasal brushings elicited stronger and faster RT-QuIC responses than cerebrospinal fluid (P<0.001 for the between-group comparison of strength of response). Individual brushings contained approximately 10(5) to 10(7) prion seeds, at concentrations several logs10 greater than in cerebrospinal fluid. CONCLUSIONS: In this preliminary study, RT-QuIC testing of olfactory epithelium samples obtained from nasal brushings was accurate in diagnosing Creutzfeldt-Jakob disease and indicated substantial prion seeding activity lining the nasal vault. (Funded by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases and others.).

PrPSc-Specific Antibody Reveals C-Terminal Conformational Differences between Prion Strains.

UNLABELLED: Understanding the structure of PrP(Sc) and its strain variation has been one of the major challenges in prion disease biology. To study the strain-dependent conformations of PrP(Sc), we purified proteinase-resistant PrP(Sc) (PrP(RES)) from mouse brains with three different murine-adapted scrapie strains (Chandler, 22L, and Me7) and systematically tested the accessibility of epitopes of a wide range of anti-PrP and anti-PrP(Sc) specific antibodies by indirect enzyme-linked immunosorbent assay (ELISA). We found that epitopes of most anti-PrP antibodies were hidden in the folded structure of PrP(RES), even though these epitopes are revealed with guanidine denaturation. However, reactivities to a PrP(Sc)-specific conformational C-terminal antibody showed significant differences among the three different prion strains. Our results provide evidence for strain-dependent conformational variation near the C termini of molecules within PrP(Sc) multimers. IMPORTANCE: It has long been apparent that prion strains can have different conformations near the N terminus of the PrP(Sc) protease-resistant core. Here, we show that a C-terminal conformational PrP(Sc)-specific antibody reacts differently to three murine-adapted scrapie strains. These results suggest, in turn, that conformational differences in the C terminus of PrP(Sc) also contribute to the phenotypic distinction between prion strains.