RT-QuIC Using C-Terminally Truncated α-Synuclein Forms Detects Differences in Seeding Propensity of Different Brain Regions from Synucleinopathies.
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ABSTRACT:
SUBMITTER: Poggiolini I
PROVIDER: S-EPMC8226794 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Rapid and sensitive detection of prions is important in managing prion diseases. The real-time quaking-induced conversion (RT-QuIC) assay for prion seeding activity has been applied to many prion diseases and provides for specific antemortem diagnostic testing. We evaluated RT-QuIC's long-term consistency and varied multiple reaction parameters. Repeated assays of a single scrapie sample using multiple plate readers and recombinant prion protein (rPrP(Sen)) substrates gave comparable results. N-terminal truncated hamster rPrP(Sen) (residues 90-231) hastened both prion-seeded and prion-independent reactions but maintained a clear kinetic distinction between the two. Raising temperatures or shaking speeds accelerated RT-QuIC reactions without compromising specificity. When applied to nasal brushings from Creutzfeldt-Jakob disease patients, higher temperatures accelerated RT-QuIC kinetics, and the use of hamster rPrP(Sen) (90-231) strengthened RT-QuIC responses. Elongation of shaking periods reduced scrapie-seeded reaction times, but continuous shaking promoted false-positive reactions. Furthermore, pH 7.4 provided for more rapid RT-QuIC reactions than more acidic pHs. Additionally, we show that small variations in the amount of sodium dodecyl sulfate (SDS) significantly impacted the assay. Finally, RT-QuIC performed in multiplate thermoshakers followed by fluorescence readings in separate plate readers enhanced assay throughput economically. Collectively, these results demonstrate improved speed, efficacy and practicality of RT-QuIC assays and highlight variables to be optimized for future applications.
| S-EPMC4885095 | biostudies-literature
Project description:The prion-like seeding of misfolded α-synuclein (αSyn) involved in the pathogenesis of Lewy body diseases (LBD) remains poorly understood at the molecular level. Using the real-time quaking-induced conversion (RT-QUIC) seeding assay, we investigated whether brain tissues from cases of dementia with Lewy bodies (DLB), which contain serine 129 (Ser129)-phosphorylated insoluble aggregates of αSyn, can convert Escherichia coli-derived recombinant αSyn (r-αSyn) to fibrils. Diffuse neocortical DLB yielded 50% seeding dose (SD50) values of 107~1010/g brain. Limbic DLB was estimated to have an SD50 value of ~105/g brain. Furthermore, RT-QUIC assay discriminated DLB from other neurological and neurodegenerative disorders. Unexpectedly, the prion-like seeding was reconstructed in reactions seeded with oligomer-like species, but not with insoluble aggregates of r-αSyn, regardless of Ser129 phosphorylation status. Our findings suggest that RT-QUIC using r-αSyn can be applied to detect seeding activity in LBD, and the culprit that causes prion-like seeding may be oligomeric forms of αSyn.
| S-EPMC5884914 | biostudies-literature
Project description:BackgroundParkinson's disease (PD) is a neurodegenerative disorder whose diagnosis is often challenging because symptoms may overlap with neurodegenerative parkinsonisms. PD is characterized by intraneuronal accumulation of abnormal ?-synuclein in brainstem while neurodegenerative parkinsonisms might be associated with accumulation of either ?-synuclein, as in the case of Multiple System Atrophy (MSA) or tau, as in the case of Corticobasal Degeneration (CBD) and Progressive Supranuclear Palsy (PSP), in other disease-specific brain regions. Definite diagnosis of all these diseases can be formulated only neuropathologically by detection and localization of ?-synuclein or tau aggregates in the brain. Compelling evidence suggests that trace-amount of these proteins can appear in peripheral tissues, including receptor neurons of the olfactory mucosa (OM).MethodsWe have set and standardized the experimental conditions to extend the ultrasensitive Real Time Quaking Induced Conversion (RT-QuIC) assay for OM analysis. In particular, by using human recombinant ?-synuclein as substrate of reaction, we have assessed the ability of OM collected from patients with clinical diagnoses of PD and MSA to induce ?-synuclein aggregation, and compared their seeding ability to that of OM samples collected from patients with clinical diagnoses of CBD and PSP.ResultsOur results showed that a significant percentage of MSA and PD samples induced ?-synuclein aggregation with high efficiency, but also few samples of patients with the clinical diagnosis of CBD and PSP caused the same effect. Notably, the final RT-QuIC aggregates obtained from MSA and PD samples owned peculiar biochemical and morphological features potentially enabling their discrimination.ConclusionsOur study provide the proof-of-concept that olfactory mucosa samples collected from patients with PD and MSA possess important seeding activities for ?-synuclein. Additional studies are required for (i) estimating sensitivity and specificity of the technique and for (ii) evaluating its application for the diagnosis of PD and neurodegenerative parkinsonisms. RT-QuIC analyses of OM and cerebrospinal fluid (CSF) can be combined with the aim of increasing the overall diagnostic accuracy of these diseases, especially in the early stages.
| S-EPMC6686411 | biostudies-literature
Project description:Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy (TSE) that causes a fatal neurodegenerative disease in cervids. Cases of CWD are rapidly increasing in North America among wild and farmed cervid populations, and potential for zoonotic transmission is not yet determined. Therefore, in order to manage the disease, it is imperative to devise a system that can detect CWD during its early phases to prevent spread to new captive herds through introduction of CWD-affected animals into otherwise CWD-free herds. Real-time quaking-induced conversion (RT-QuIC) assays have been applied to detect the presence of disease-associated prions from various samples in both animals and humans. In this study, we have tested the use of five Hofmeister anions that range from weakly hydrating to strongly hydrating: Na3citrate, Na2SO4, NaCl, NaI, and NaClO4 in RT-QuIC reactions for CWD seeding activity using different recombinant prion proteins as substrates. This work shows how the ionic environment of the RT-QuIC reaction can enhance or diminish the seeding activity. The use of Na2SO4 or NaI as the sodium salt for RT-QuIC using bank vole recombinant prion substrate for the detection of CWD using brain samples reduces the lag time to detect with reasonable specificity. For detection of the CWD in fecal samples, only NaI showed comparable reduction in lag time relative to NaCl but required reduced temperature to alleviate spontaneous fibril formation in negative control samples. Selection of the proper ion environment and recombinant prion protein substrate will make RT-QuIC a powerful diagnostic tool for early detection of CWD prions, further supporting CWD surveillance in wild and captive cervids.
| S-EPMC8515057 | biostudies-literature
Project description: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 ?-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 ?-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 ?-synuclein seeding activity in the applied experimental setting. The analysis of the fluorescence response reflecting the amount of ?-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, ?-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 ?-synuclein seeding activity in MSA seems to support the current view that MSA and LBD are associated with different conformational strains of ?-synuclein.
| S-EPMC7299922 | biostudies-literature
Project description:α-Synuclein is a key protein of the nervous system, which regulates the release and recycling of neurotransmitters in the synapses. It is also involved in several neurodegenerative conditions, including Parkinson's disease and Multiple System Atrophy, where it forms toxic aggregates. The N-terminus of α-synuclein is of particular interest as it has been linked to both the physiological and pathological functions of the protein and undergoes post-translational modification. One such modification, N-terminal truncation, affects the aggregation propensity of the protein in vitro and is also found in aggregates from patients' brains. To date, our understanding of the role of this modification has been limited by the many challenges of introducing biologically relevant N-terminal truncations with no overhanging starting methionine. Here, we present a method to produce N-terminally truncated variants of α-synuclein that do not carry extra terminal residues. We show that our method can generate highly pure protein to facilitate the study of this modification and its role in physiology and disease. Thanks to this method, we have determined that the first six residues of α-synuclein play an important role in the formation of the amyloids.
| S-EPMC9184721 | biostudies-literature
Project description:Chronic wasting disease (CWD) is a prion disease circulating in wild and farmed cervid populations throughout North America (United States and Canada), Europe (Finland, Norway, Sweden), and South Korea. CWD is a long-term threat to all cervid populations and to cervid hunting heritage, with the potential to cause substantial economic losses across multiple sectors. In North America, hunting and farming industries focused on the processing and consumption of white-tailed deer (WTD) venison are particularly vulnerable to CWD prion contamination, as millions of WTD are consumed annually. Real-time quaking-induced conversion (RT-QuIC) is a highly sensitive assay amplifying misfolded CWD prions in vitro and has facilitated CWD prion detection in a variety of tissues and excreta. To date, no study has comprehensively examined CWD prion content across bulk skeletal muscle tissues harvested from individual CWD infected WTD. Here, we use RT-QuIC to characterize prion-seeding activity in a variety of skeletal muscles from both wild and farmed CWD-positive WTD. We successfully detected CWD prions in muscles commonly used for consumption (e.g., backstrap, tenderloin, etc.) as well as within tongue and neck samples of WTD. Our results suggest that CWD prions are distributed across the skeletal muscles of infected WTD. We posit that RT-QuIC will be a useful tool for monitoring CWD prions in venison and that the method (with additional protocol optimization and high-throughput functionality) could be used to reduce and/or prevent CWD prions from entering animal and human food chains.
| S-EPMC8373970 | biostudies-literature
Project description:Misfolding, aggregation and deposition of α-synuclein (α-syn) are major pathologic characteristics of Parkinson's disease (PD) and the related synucleinopathy, multiple system atrophy (MSA). The spread of α-syn pathology across brain regions is thought to play a key role in the onset and progression of clinical phenotypes. Thus, there is increasing interest in developing strategies that target and attenuate α-syn aggregation and spread. Recent studies of brain-penetrating polyphenolic acids, namely, 3-hydroxybenzoic acid (3-HBA), 3,4-dihydroxybenzoic acid (3,4-diHBA), and 3-(3-hydroxyphenyl)propionic acid (3-HPPA) that are derived from gut microbiota metabolism of dietary polyphenols, show in vitro ability to effectively modulate α-syn misfolding, oligomerization, and mediate aggregated α-syn neurotoxicity. Here we investigate whether 3-HBA, 4-hydroxybenzoic acid (4-HBA), 3,4-diHBA, or 3-HPPA interfere with α-syn spreading in a cell-based system. Using HEK293 cells overexpressing α-syn-A53T-CFP/YFP, we assessed α-syn seeding activity using Fluorescence Resonance Energy Transfer (FRET) to detect and quantify α-syn aggregation. We demonstrated that 3-HPPA, 3,4-diHBA, 3-HBA, and 4-HBA significantly attenuated intracellular α-syn seeding aggregation. To determine whether our compounds could inhibit brain-derived seeding activity, we utilized insoluble α-syn extracted from post-mortem MSA or PD brain specimens. We found that 3-HPPA effectively attenuated MSA-induced aggregation of monomer into high molecular weight aggregates capable of inducing intracellular aggregation. Outcomes from our studies suggest interactions between gut microbiome and certain dietary factors may form the basis for effective therapies that modulate pathologic α-syn propagation. Collectively, our findings provide the basis for future developments of probiotic, prebiotic, or synbiotic approaches for modulating the onset and/or progression of α-synucleinopathies.
| S-EPMC7212829 | biostudies-literature
Project description:BackgroundLeucine-rich kinase 2 (LRRK2)-linked Parkinson's disease (PD) is clinically indistinguishable from idiopathic PD (IPD). A pleiotropic neuropathology has been recognized but the majority of studies in LRRK2 p.G2019S patients reveal Lewy-type synucleinopathy as its principal histological substrate. To date no in vivo biomarkers of synucleinopathy have been found in LRRK2 mutation carriers.ObjectivesWe used real-time quaking-induced conversion (RT-QuIC) technique to assess the presence of alpha-synuclein (a-syn) aggregates in cerebrospinal fluid (CSF) of LRRK2 p.G2019S carriers.MethodsCSF samples of 51 subjects were analyzed: 15 LRRK2 p.G2019S PD, 10 IPD, 16 LRRK2 p.G2019S nonmanifesting carriers (NMC) and 10 healthy controls. The presence of parkinsonism and prodromal symptoms was assessed in all study subjects.ResultsForty percent (n = 6) LRRK2-PD, and 18.8% (n = 3) LRRK2-NMC had a positive a-syn RT-QuIC response. RT-QuIC detected IPD with 90% sensitivity and 80% specificity. No clinical differences were detected between LRRK2-PD patients with positive and negative RT-QuIC. A positive RT-QuIC result in LRRK2-NMC occurred in a higher proportion of subjects meeting the Movement Disorder Society research criteria for prodromal PD.InterpretationRT-QuIC detects a-syn aggregation in CSF in a significant number of patients with LRRK2-PD, but less frequently than in IPD. A small percentage of LRRK2-NMC tested also positive. If appropriately validated in long-term studies with large number of mutation carriers, and hopefully, postmortem or in vivo confirmation of histopathology, RT-QuIC could contribute to the selection of candidates to receive disease modifying drugs, in particular treatments targeting a-syn deposition.
| S-EPMC6562027 | biostudies-literature
Project description:We applied RT-QuIC assay to detect α-synuclein aggregates in cerebrospinal fluid (CSF) of patients with suspected Creutzfeldt-Jakob disease who had a neuropathological diagnosis of dementia with Lewy bodies (DLB) (n = 7), other neurodegenerative diseases with α-synuclein mixed pathology (n = 20), or without Lewy-related pathology (n = 49). The test had a sensitivity of 92.9% and specificity of 95.9% in distinguishing α-synucleinopathies from non-α-synucleinopathies. When performed in the CSF of patients with DLB (n = 36), RT-QuIC was positive in 17/20 with probable DLB, 0/6 with possible DLB, and 0/10 with Alzheimer disease. These results indicate that RT-QuIC for α-synuclein is an accurate test for DLB diagnosis.
| S-EPMC6801172 | biostudies-literature