Real-time Quaking-induced Conversion Assay Research Articles

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.

Characterisation of RT-QuIC negative cases from the UK National CJD Research and Surveillance programme.

Incorporation of the real-time quaking-induced conversion (RT-QuIC) assays for diagnosis of sporadic Creutzfeldt-Jakob disease (CJD) has transformed diagnosis largely related to its extremely high specificity. However, the test has a c.10% false-negative result and we aim to characterize the clinical features, investigation profile, and molecular subtype in this cohort of patients. 250 individuals diagnosed with definite sporadic CJD were identified from the UK National CJD Research and Surveillance Unit from 2012 to 2023. We compared the clinical features and investigation profile in those with a negative CSF RT-QuIC to those with a positive RT-QuIC. 27 individuals (10.8%) were CSF RT-QuIC negative. Median age of onset was younger (62years vs 68years, p = 0.002), median disease duration was longer (4.4months vs 10.5months, p < 0.001), and these individuals were less likely to present with gait difficulties (73% vs 93%, p = 0.003) or motor symptoms (62% vs 80%, p = 0.04). The sensitivity of electroencephalography and diffusion-weighted MRI were similar in both groups. In those who were RT-QuIC negative, there was an overrepresentation of the VV1 (32% vs 1%) and MM2 molecular subtypes (21% vs 3%). Co-occurring neurodegenerative disease was found in 33% (9/27) of those who were RT-QuIC negative. Individuals with sporadic CJD and a negative CSF RT-QuIC present with younger age of onset, different clinical features and are over-represented with the VV1 and MM2 subtypes of sporadic CJD. Further work is required to better understand the biochemical properties contributing to RT-QuIC negative results in these cases.

Detection of prions in matching post-mortem skin and cerebrospinal fluid samples using second-generation real-time quaking-induced conversion assay.

Real-time quaking-induced conversion assay (RT-QuIC) exploits templating activity of pathogenic prion protein for ultrasensitive detection of prions. We have utilized second generation RT-QuIC assay to analyze matching post-mortem cerebrospinal fluid and skin samples of 38 prion disease patients and of 30 deceased neurological controls. The analysis of cerebrospinal fluid samples led to 100% sensitivity and 100% specificity, but some samples had to be diluted before the analysis to alleviate the effect of present RT-QuIC inhibitors. The analysis of the corresponding skin samples provided 89.5% sensitivity and 100% specificity. The median seeding dose present in the skin was one order of magnitude higher than in the cerebrospinal fluid, despite the overall fluorescent signal of the skin samples was comparatively lower. Our data support the use of post-mortem cerebrospinal fluid for confirmation of prion disease diagnosis and encourage further studies of the potential of skin biopsy samples for intra-vitam prion diseases´ diagnostics.

Detection limitations of prion seeding activities in blood samples from patients with sporadic prion disease

BackgroundHuman prion diseases (HPDs) are fatal neurodegenerative disorders characterized by abnormal prion proteins (PrPSc). However, the detection of prion seeding activity in patients with high sensitivity remains challenging. Even though real-time quaking-induced conversion (RT-QuIC) assay is suitable for detecting prion seeding activity in a variety of specimens, it shows lower accuracy when whole blood, blood plasma, and blood-contaminated tissue samples are used. In this study, we developed a novel technology for the in vitro amplification of abnormal prion proteins in HPD to the end of enabling their detection with high sensitivity known as the enhanced quaking-induced conversion (eQuIC) assay.MethodsThree antibodies were used to develop the novel eQUIC method. Thereafter, SD50 seed activity was analyzed using brain tissue samples from patients with prion disease using the conventional RT-QUIC assay and the novel eQUIC assay. In addition, blood samples from six patients with solitary prion disease were analyzed using the novel eQuIC assay.ResultsThe eQuIC assay, involving the use of three types of human monoclonal antibodies, showed approximately 1000-fold higher sensitivity than the original RT-QuIC assay. However, when this assay was used to analyze blood samples from six patients with sporadic human prion disease, no prion activity was detected.ConclusionThe detection of prion seeding activity in blood samples from patients with sporadic prion disease remains challenging. Thus, the development of alternative methods other than RT-QuIC and eQuIC will be necessary for future research.

Improving protocols for α-synuclein seed amplification assays: analysis of preanalytical and analytical variables and identification of candidate parameters for seed quantification.

The effect of preanalytical and analytical factors on the α-synuclein (α-syn) seed amplification assay's (SAA) performance has not been fully explored. Similarly, there is limited knowledge about the most suitable assay protocol and kinetic parameters for misfolded α-syn seed quantification. We studied the effect of centrifugation, repeated freeze-thaw cycles (up to seven), delayed freezing, detergent addition, and blood contamination on the performance of the cerebrospinal fluid (CSF) α-syn SAA real-time quaking-induced conversion (RT-QuIC). Moreover, we analysed the inter- and intra-plate variability, the recombinant protein batch effect, and the RT-QuIC parameters' variability when multiple samples were run in controlled conditions. Finally, we evaluated the assay potential of quantifying α-syn seed by assessing kinetic curves in serial CSF dilutions. Among tested preanalytical variables, a≥0.01 % blood contamination and adding detergents significantly affected the RT-QuIC kinetic parameters and the number of positive replicates. Increasing the number of replicates improved result reproducibility. The number of positive replicates in serially diluted CSF samples improved discrimination between samples with high and low seeding activity, and the time to threshold (LAG) was the most reliable kinetic parameter in multiple experiment settings. Preanalytical variables affecting α-syn RT-QuIC performance are limited to blood contamination and detergent addition. The number of positive replicates and the LAG are the most reliable variables for quantifying α-syn seeding activity. Their consistent measurement in serial dilution experiments, especially when associated with an increased number of sample replicates, will help to develop the α-syn RT-QuIC assay further into a quantitative test.

Detection of prions from spiked and free-ranging carnivore feces

Chronic wasting disease (CWD) is a highly contagious, fatal neurodegenerative disease caused by infectious prions (PrPCWD) affecting wild and captive cervids. Although experimental feeding studies have demonstrated prions in feces of crows (Corvus brachyrhynchos), coyotes (Canis latrans), and cougars (Puma concolor), the role of scavengers and predators in CWD epidemiology remains poorly understood. Here we applied the real-time quaking-induced conversion (RT-QuIC) assay to detect PrPCWD in feces from cervid consumers, to advance surveillance approaches, which could be used to improve disease research and adaptive management of CWD. We assessed recovery and detection of PrPCWD by experimental spiking of PrPCWD into carnivore feces from 9 species sourced from CWD-free populations or captive facilities. We then applied this technique to detect PrPCWD from feces of predators and scavengers in free-ranging populations. Our results demonstrate that spiked PrPCWD is detectable from feces of free-ranging mammalian and avian carnivores using RT-QuIC. Results show that PrPCWD acquired in natural settings is detectable in feces from free-ranging carnivores, and that PrPCWD rates of detection in carnivore feces reflect relative prevalence estimates observed in the corresponding cervid populations. This study adapts an important diagnostic tool for CWD, allowing investigation of the epidemiology of CWD at the community-level.

Optimization of RT-QuIC Assay Duration for Screening Chronic Wasting Disease in White-Tailed Deer.

Real-time quaking-induced conversion (RT-QuIC) assays have become a common tool to detect chronic wasting disease (CWD) and are very sensitive provided the assay duration is sufficient. However, a prolonged assay duration may lead to non-specific signal amplification. The wide range of pre-defined assay durations in current RT-QuIC applications presents a need for methods to optimize the RT-QuIC assay. In this study, receiver operating characteristic (ROC) analysis was applied to optimize the assay duration for CWD screening in obex and retropharyngeal lymph node (RLN) tissue specimens. Two different fluorescence thresholds were used: a fixed threshold based on background fluorescence (Tstdev) and a max-point ratio (maximum/background fluorescence) threshold (TMPR) to determine CWD positivity. The optimal assay duration was 33 h for obex and 30 h for RLN based on Tstdev, and 29 h for obex and 32 h for RLN based on TMPR. The optimized assay durations were then evaluated for screening CWD in white-tailed deer from an affected farm. At a replicate level, using the optimized assay durations with TStdev and TMPR, the level of agreement with enzyme-linked immunosorbent assay (ELISA) was significantly higher (p < 0.05) than that when using a 40 h assay duration. These findings demonstrate that the optimization of assay duration via a ROC analysis can improve RT-QuIC assays for screening CWD in white-tailed deer.

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.

Development of a sensitive real-time quaking-induced conversion (RT-QuIC) assay for application in prion-infected blood.

Efforts to prevent human-to-human transmission of variant Creutzfeldt-Jakob disease (vCJD) by contaminated blood would be aided by the development of a sensitive diagnostic test that could be routinely used to screen blood donations. As blood samples from vCJD patients are extremely rare, here we describe the optimisation of real-time quaking-induced conversion (RT-QuIC) for detection of PrPSc (misfolded prion protein, a marker of prion infection) in blood samples from an established large animal model of vCJD, sheep experimentally infected with bovine spongiform encephalopathy (BSE). Comparative endpoint titration experiments with RT-QuIC, miniaturized bead protein misfolding cyclic amplification (mb-PMCA) and intracerebral inoculation of a transgenic mouse line expressing sheep PrP (tgOvARQ), demonstrated highly sensitive detection of PrPSc by RT-QuIC in a reference sheep brain homogenate. Upon addition of a capture step with iron oxide beads, the RT-QuIC assay was able to detect PrPSc in whole blood samples from BSE-infected sheep up to two years before disease onset. Both RT-QuIC and mb-PMCA also demonstrated sensitive detection of PrPSc in a reference vCJD-infected human brain homogenate, suggesting that either assay may be suitable for application to human blood samples. Our results support the further development and evaluation of RT-QuIC as a diagnostic or screening test for vCJD.

Real-time quaking-induced conversion assay using a small-scale substrate production workflow for the diagnosis of Creutzfeldt-Jakob disease.

The lack of a dedicated surveillance program for prion disease, particularly in low- and middle-income countries (LMICs), has hindered the global effort to address this public health threat. Although cerebrospinal fluid (CSF) Real-time quaking-induced conversion (RT-QuIC) is considered the most reliable test for sporadic Creutzfeldt-Jakob disease (sCJD), its availability in LMICs is limited due to its cost and technical difficulty in generating the recombinant prion protein substrate (recPrP). This study aimed to evaluate the performance of RT-QuIC with recPrP produced in-house through a small-scale method - i.e. the application of reusable pre-packed chromatography columns and subsequent dialysis. Here, CSF specimens from patients suspected of having prion disease were consecutively collected and stored between October 2015 and January 2023. Electronic medical record data were reviewed to clinically classified participants as probable sCJD or non-sCJD. CSF RT-QuIC was performed using in-house recPrP. Its specificity and sensitivity for diagnosing probable sCJD were reported, along with details of other clinical data and investigations. We found that among 39 eligible participants, with a median (interquartile range) age of 64 (56-70) years and 16 (41%) female, 13 had probable sCJD and the remaining 26 unequivocally suffered from non-prion disorders. Magnetic resonance imaging and electroencephalogram were suggestive of sCJD in 100% (13/13) and 46.2% (6/13) of sCJD participants, respectively. RT-QuIC was positive in 12/13 sCJD participants (sensitivity 0.92, 95% confidence interval (CI) 0.67-0.99) and negative in all non-sCJD participants (specificity 1.00, 95%CI 0.87-1.00). CSF tau/p-tau ratio showed sensitivity and specificity of 0.62-1.0 and 0.85-1.0, respectively. In summary, RT-QuIC using recPrP generated through a small-scale workflow demonstrated great performance in detecting sCJD. Given its performance results along with its low cost, this technique could feasibly be implemented in LMICs and potentially be the first step towards establishing local prion disease surveillance programs.

Ticks harbor and excrete chronic wasting disease prions

Chronic wasting disease (CWD) is a fatal neurodegenerative disease caused by infectious prions (PrPCWD) affecting cervids. Circulating PrPCWD in blood may pose a risk for indirect transmission by way of hematophagous ectoparasites acting as mechanical vectors. Cervids can carry high tick infestations and exhibit allogrooming, a common tick defense strategy between conspecifics. Ingestion of ticks during allogrooming may expose naïve animals to CWD, if ticks harbor PrPCWD. This study investigates whether ticks can harbor transmission-relevant quantities of PrPCWD by combining experimental tick feeding trials and evaluation of ticks from free-ranging white-tailed deer (Odocoileus virginianus). Using the real-time quaking-induced conversion (RT-QuIC) assay, we show that black-legged ticks (Ixodes scapularis) fed PrPCWD-spiked blood using artificial membranes ingest and excrete PrPCWD. Combining results of RT-QuIC and protein misfolding cyclic amplification, we detected seeding activity from 6 of 15 (40%) pooled tick samples collected from wild CWD-infected white-tailed deer. Seeding activities in ticks were analogous to 10–1000 ng of CWD-positive retropharyngeal lymph node collected from deer upon which they were feeding. Estimates revealed a median infectious dose range of 0.3–42.4 per tick, suggesting that ticks can take up transmission-relevant amounts of PrPCWD and may pose a CWD risk to cervids.

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.

Nanoparticle-Enhanced RT-QuIC (Nano-QuIC) Diagnostic Assay for Misfolded Proteins.

Misfolded proteins associated with various neurodegenerative diseases often accumulate in tissues or circulate in biological fluids years before the clinical onset, thus representing ideal diagnostic targets. Real-time quaking-induced conversion (RT-QuIC), a protein-based seeded-amplification assay, holds great potential for early disease detection, yet challenges remain for routine diagnostic application. Chronic Wasting Disease (CWD), associated with misfolded prion proteins of cervids, serves as an ideal model for evaluating new RT-QuIC methodologies. In this study, we investigate the previously untested hypothesis that incorporating nanoparticles into RT-QuIC assays can enhance their speed and sensitivity when applied to biological samples. We show that adding 50 nm silica nanoparticles to RT-QuIC experiments (termed Nano-QuIC) for CWD diagnostics greatly improves the performance by reducing detection times 2.5-fold and increasing sensitivity 10-fold by overcoming the effect of inhibitors in complex tissue samples. Crucially, no false positives were observed with these 50 nm silica nanoparticles, demonstrating the enhanced reliability and potential for diagnostic application of Nano-QuIC in detecting misfolded proteins.

Real-Time Quaking-Induced Conversion Assays for Prions: Applying a Sensitive but Imperfect Test in Clinical Practice.

Real-time quaking-induced conversion (RT-QuIC) assays offer a sensitive and specific means for detection of prions, although false negative results are recognized in clinical practice. We profile the clinical, laboratory, and pathologic features associated with false negative RT-QuIC assays and extend these to frame the diagnostic approach to patients with suspected prion disease. 113 patients with probable or definite prion disease were assessed at Mayo Clinic (Rochester, MN; Jacksonville, FL; Scottsdale, AZ) or Washington University School of Medicine (St. Louis, MO) from 2013-2021. RT-QuIC testing for prions was performed in CSF at the National Prion Disease Pathology Surveillance Center (Cleveland, OH). Initial RT-QuIC testing was negative in 13/113 patients (sensitivity 88.5%). RT-QuIC negative patients were younger (median 52.0 years vs 66.1 years, p<0.001). Other demographic and presenting features, and CSF cell count, protein, and glucose levels were similar in RT-QuIC negative and positive patients. Frequency of 14-3-3 positivity (4/13 vs 77/94, p<0.001) and median CSF total tau levels were lower in RT-QuIC negative patients (2517 vs 4001 pg/mL, p=0.020), while time from symptom onset to first presentation (153 vs 47 days, p=0.001) and symptomatic duration (710 vs 148 days, p=0.001) were longer. RT-QuIC is a sensitive yet imperfect measure necessitating incorporation of other test results when evaluating patients with suspected prion disease. Patients with negative RT-QuIC had lower markers of neuronal damage (CSF total-tau and protein 14-3-3) and longer symptomatic duration of disease suggesting that false negative RT-QuIC testing associates with a more indolent course.

369 Differentiating Dementia with Lewy Bodies and Alzheimer’s Disease Using Extracellular Vesicles

OBJECTIVES/GOALS: Extracellular vesicles (EVs) in biofluids can reflect pathology in the brain (like alpha-synuclein protein linked to Dementia with Lewy Bodies) making them attractive disease biomarkers to distinguish disease. Here, we will investigate their potential as non-invasive biomarkers to differentiate Dementia with Lewy Bodies &amp; Alzheimer's Disease. METHODS/STUDY POPULATION: We will leverage the collection of antemortem plasma from autopsy confirmed Dementia with Lewy Bodies (DLB), Alzheimer's Disease (AD) &amp; healthy controls (HC) through the Mayo Clinic Brain Bank. We will characterize antemortem brain-derived plasma EVs (bPEVs) &amp; post-mortem brain-derived EVs (bEVs) of the same patient using nanoparticle tracking, electron microscopy, &amp; western blotting for EV-associated proteins. We will then measure levels of total &amp; phosphorylated alpha-synuclein (asyn) using AlphaLisa. We will assess EVs' ability to initiate asyn aggregation using a real-time quaking-induced conversion (RT-QuIC) assay &amp; a cell-based Fluorescence Resonance Energy Transfer (FRET) assay. Once characterized, this will allow differentiation of bpEVs in DLB, AD, or healthy controls. RESULTS/ANTICIPATED RESULTS: We hypothesize that asyn levels and seeding capacity will discriminate DLB from AD. With AlphaLisa assay, we expect higher total and phosphorylated asyn levels in DLB bpEVs and their corresponding post-mortem bEVs. Using RT-QuIC and FRET assay, we expect EVs isolated from DLB patient samples to support seeded aggregation, whereas EVs from AD and HC will not. DISCUSSION/SIGNIFICANCE: To date, no diagnostic or less invasive biomarkers can distinguish DLB from AD. The successful completion of the aims outlined in this proposal will identify characteristics of bpEVs that differentiate DLB from AD or HC and support the development of bpEVs as a non-invasive, early biomarker to diagnose patients presenting with dementia from DLB.

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.

Synthesis and anti-prion aggregation activity of acylthiosemicarbazide analogues

Prions are infectious protein particles known to cause prion diseases. The biochemical entity of the pathogen is the misfolded prion protein (PrPSc) that forms insoluble amyloids to impair brain function. PrPSc interacts with the non-pathogenic, cellular prion protein (PrPC) and facilitates conversion into a nascent misfolded isoform. Several small molecules have been reported to inhibit the aggregation of PrPSc but no pharmacological intervention was well established thus far. We, here, report that acylthiosemicarbazides inhibit the prion aggregation. Compounds 7x and 7y showed almost perfect inhibition (EC50 = 5 µM) in prion aggregation formation assay. The activity was further confirmed by atomic force microscopy, semi-denaturing detergent agarose gel electrophoresis and real-time quaking induced conversion assay (EC50 = 0.9 and 2.8 µM, respectively). These compounds also disaggregated pre-existing aggregates in vitro and one of them decreased the level of PrPSc in cultured cells with permanent prion infection, suggesting their potential as a treatment platform. In conclusion, hydroxy-2-naphthoylthiosemicarbazides can be an excellent scaffold for the discovery of anti-prion therapeutics.

Misfolded α-Synuclein Assessment in the Skin and CSF by RT-QuIC in Isolated REM Sleep Behavior Disorder.

Real-time quaking-induced conversion (RT-QuIC) assay detects misfolded α-synuclein (AS) in the skin and CSF of patients with the synucleinopathies Parkinson disease and dementia with Lewy bodies. Isolated REM sleep behavior disorder (IRBD) constitutes the prodromal stage of these synucleinopathies. We aimed to compare the ability of RT-QuIC to identify AS in the skin and CSF of patients with IRBD. This was a cross-sectional study where consecutive patients with polysomnographic-confirmed IRBD and age-matched controls without RBD underwent skin biopsy and lumbar puncture the same day. Three-millimeter skin punch biopsies were obtained bilaterally in the cervical region from dorsal C7 and C8 dermatomes and in distal legs. RT-QuIC assessed AS in these 6 skin sites and the CSF. We recruited 91 patients with IRBD and 41 controls. In the skin, sensitivity to detect AS was 76.9% (95% CI 66.9-85.1), specificity 97.6% (95% CI 87.1-99.9) positive predictive value 98.6% (95% CI 91.0-99.8), negative predictive value 65.6% (95% CI 56.6-73.6), and accuracy 83.3% (95% CI 75.9-89.3). In the CSF, the sensitivity was 75.0% (95% CI 64.6-83.6), the specificity was 97.5% (95% CI 86.8-99.9), the positive predictive value was 98.5% (95% CI 90.5-99.8), the negative predictive value was 63.9% (95% CI 55.2-71.9), and the accuracy was 82.0% (95% CI 74.3-88.3). Results in the skin and CSF samples showed 99.2% agreement. Compared with negative patients, RT-QuIC AS-positive patients had a higher likelihood ratio of prodromal Parkinson disease (p < 0.001) and showed more frequently hyposmia (p < 0.001), dopamine transporter imaging single-photon emission CT deficit (p = 0.002), and orthostatic hypotension (p = 0.014). No severe or moderate adverse effects were reported. There was no difference between the percentage of participants reporting mild adverse events secondary to skin biopsy or lumbar puncture (9.1% vs 17.2%; p = 0.053). One hundred and ten (83%) and 104 (80%) participants, respectively, stated they would accept to undergo skin biopsy and lumbar puncture again for research purposes. Our study in IRBD shows that (1) RT-QuIC detects AS in the skin and CSF with similar high sensitivity, specificity, and agreement, (2) AS RT-QuIC positivity is associated with supportive features and biomarkers of synucleinopathy, and (3) skin punch biopsy and lumbar puncture have comparable mild adverse effects, tolerance, and acceptance. RT-QuIC in the skin or CSF might represent a patient selection strategy for future neuroprotective trials targeting AS in IRBD. This study provides Class III evidence that RT-QuIC-detected AS in the skin and CSF distinguishes patients with IRBD from controls.

Defining the phenotypic spectrum of sporadic Creutzfeldt-Jakob disease MV2K: the kuru plaque type.

The current classification of sporadic Creutzfeldt-Jakob disease identifies six major subtypes mainly defined by the combination of the genotype at polymorphic codon 129 (methionine/M or valine/V) of the prion protein gene and the type (1 or 2) of misfolded prion protein accumulating in the brain (e.g. MM1, MM2, MV1, MV2, etc.). Here, we systematically characterized the clinical and histo-molecular features associated with the third prevalent subtype, the MV2 subtype with kuru plaques (MV2K), in the most extensive series collected to date. We evaluated neurological histories, cerebrospinal biomarkers, brain MRI and EEG results in 126 patients. The histo-molecular assessment included misfolded prion protein typing, standard histologic staining and immunohistochemistry for prion protein in several brain areas. We also investigated the prevalence and topographic extent of coexisting MV2-cortical features, the number of cerebellar kuru plaques and their effect on clinical phenotype. Systematic regional typing revealed a western blot profile of misfolded prion protein comprising a doublet of 19 and 20 kDa unglycosylated fragments, with the former more prominent in neocortices and the latter in the deep grey nuclei. The 20/19 kDa fragment ratio positively correlated with the number of cerebellar kuru plaques. The mean disease duration was exceedingly longer than in the typical MM1 subtype (18.0 versus 3.4 months). Disease duration correlated positively with the severity of pathologic change and the number of cerebellar kuru plaques. At the onset and early stages, patients manifested prominent, often mixed, cerebellar symptoms and memory loss, variably associated with behavioural/psychiatric and sleep disturbances. The cerebrospinal fluid prion real-time quaking-induced conversion assay was positive in 97.3% of cases, while 14-3-3 protein and total-tau positive tests were 52.6 and 75.9%. Brain diffusion-weighted MRI showed hyperintensity of the striatum, cerebral cortex and thalamus in 81.4, 49.3 and 33.8% of cases, and a typical profile in 92.2%. Mixed histotypes (MV2K + MV2-cortical) showed an abnormal cortical signal more frequently than the pure MV2K (64.7 versus 16.7%, P = 0.007). EEG revealed periodic sharp-wave complexes in only 8.7% of participants. These results further establish MV2K as the most common 'atypical' subtype of sporadic Creutzfeldt-Jakob disease, showing a clinical course that often challenges the early diagnosis. The plaque-type aggregation of the misfolded prion protein accounts for most of the atypical clinical features. Nonetheless, our data strongly suggest that the consistent use of the real-time quaking-induced conversion assay and brain diffusion-weighted MRI allows an accurate early clinical diagnosis in most patients.

Diagnostic performance of CSF biomarkers in a well-characterized Australian cohort of sporadic Creutzfeldt-Jakob disease.

The most frequently utilized biomarkers to support a pre-mortem clinical diagnosis of sporadic Creutzfeldt-Jakob disease (sCJD) include concentrations of the 14-3-3 and total tau (T-tau) proteins, as well as the application of protein amplification techniques, such as the real time quaking-induced conversion (RT-QuIC) assay, in cerebrospinal fluid (CSF). Utilizing CSF from a cohort of neuropathologically confirmed (definite) sCJD (n = 50) and non-CJD controls (n = 48), we established the optimal cutpoints for the fully automated Roche Elecsys® immunoassay for T-tau and the CircuLexTM 14-3-3 Gamma ELISA and compared these to T-tau protein measured using a commercially available assay (INNOTEST hTAU Ag) and 14-3-3 protein detection by western immunoblot (WB). These CSF specimens were also assessed for presence of misfolded prion protein using the RT-QuIC assay. T-tau showed similar diagnostic performance irrespective of the assay utilized, with ~90% sensitivity and specificity. The 14-3-3 protein detection by western blot (WB) has 87.5% sensitivity and 66.7% specificity. The 14-3-3 ELISA demonstrated 81.3% sensitivity and 84.4% specificity. RT-QuIC was the single best performing assay, with a sensitivity of 92.7% and 100% specificity. Our study indicates that a combination of all three CSF biomarkers increases sensitivity and offers the best chance of case detection pre-mortem. Only a single sCJD case in our cohort was negative across the three biomarkers, emphasizing the value of autopsy brain examination on all suspected CJD cases to ensure maximal case ascertainment.