Streamlined alpha-synuclein RT-QuIC assay for various biospecimens in Parkinson’s disease and dementia with Lewy bodies (original) (raw)
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JAMA Neurology, 2017
IMPORTANCE Parkinson disease (PD) is a highly prevalent and incurable neurodegenerative disease associated with the accumulation of misfolded α-synuclein (αSyn) aggregates. An important problem in this disease is the lack of a sensitive, specific, and noninvasive biochemical diagnosis to help in clinical evaluation, monitoring of disease progression, and early differential diagnosis from related neurodegenerative diseases. OBJECTIVE To develop a novel assay with high sensitivity and specificity to detect small quantities of αSyn aggregates circulating in cerebrospinal fluid (CSF) of patients affected by PD and related synucleinopathies. DESIGN, SETTING, AND PARTICIPANTS The strategy evaluated in this proof-of-concept study uses the protein misfolding cyclic amplification (PMCA) technology that detects minute amounts of misfolded oligomers by taking advantage of their ability to nucleate further aggregation, enabling a very high amplification of the signal. The technology was first adapted with synthetic αSyn oligomers prepared in vitro and used to screen in 2 blinded cohorts of CSF samples from German and Japanese patients with PD (n = 76) and individuals serving as controls affected by other neurologic disorders (n = 65), neurodegenerative diseases (n = 18), and Alzheimer disease (n = 14). The kinetics of αSyn aggregation were measured by αSyn-PMCA in the presence of CSF samples from the participants to detect αSyn oligomeric seeds present in this biological fluid. The assays were conducted from November 15, 2013, to August 28, 2015. MAIN OUTCOMES AND MEASURES Kinetic parameters correlated with disease severity at the time of sample collection, measured by the Hoehn and Yahr scale, with the lowest grade indicating unilateral involvement with minimal or no functional impairment, and the highest grade defining patients with complete confinement to wheelchair or bed. RESULTS Studies with synthetic αSyn aggregates showed that αSyn-PMCA enabled to detect as little as 0.1 pg/mL of αSyn oligomers. The αSyn-PMCA signal was directly proportional to the amount of αSyn oligomers added to the reaction. A blinded study of CSF samples correctly identified patients affected by PD with an overall sensitivity of 88.5% (95% CI, 79.2%-94.6%) and specificity of 96.9% (95% CI, 89.3%-99.6%). The αSyn-PMCA results for different patients correlated with the severity of the clinical symptoms of PD (Japanese cohort: r s = −0.54, P = .006; German cohort: r s = −0.36, P = .02). CONCLUSIONS AND RELEVANCE The findings suggest that detection of αSyn oligomers by αSyn-PMCA in the CSF of patients affected by PD may offer a good opportunity for a sensitive and specific biochemical diagnosis of the disease. Further studies are needed to investigate the usefulness of αSyn-PMCA to monitor disease progression and for preclinical identification of patients who may develop PD.
Acta Neuropathologica
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) an...
Parkinson’s disease biomarkers based on α‐synuclein
Journal of Neurochemistry, 2019
Parkinson's disease is the second most common neurodegenerative disorder after Alzheimer's disease and is estimated to affect approximately 1-4% of individuals aged over 60 years old. Although considerable efforts have been invested into developing disease-modifying therapies for Parkinson's disease, such efforts have been confounded by the difficulty in accurately diagnosing Parkinson's disease during life to enable accurate patient stratification for clinical trialling of candidate therapeutics. Therefore, the search for effective biomarkers that can be accurately evaluated during life with non-invasive means is a pressing issue in the field. Since the discovery of α-synuclein as a protein linked to a familial form of Parkinson's disease, later identified as the major protein component of the neuropathological hallmark of idiopathic Parkinson's disease, considerable interest has focused on this protein and its distinct conformers. We describe here the progress that has been made in the area of Parkinson's disease biomarker discovery with a focus on α-synuclein. In particular, we highlight the novel assays that have been employed and the increasing complexity in evaluating α-synuclein with regard to the considerable diversity of conformers that exist in the biofluids and peripheral tissues under disease conditions. Abbreviations used α-syn-alpha-synuclein CSF-cerebrospinal fluid DATATOP-Deprenyl and tocopherol antioxidative therapy of parkinsonism DeNoPA-De novo parkinson's disease DLB-dementia with lewy bodies ELISA-enzyme linked immunosorbent assay LB-Lewy body o-α-syn-oligomeric α-syn PD-Parkinson's disease PMCA-protein misfolding cyclic amplification PPMI-Parkinson's progression markers initiative pS129 α-syn-phosphorylated α-syn at S129 PSP-progressive supranuclear palsy RT-QuIC-real-time quaking-induced conversion
Movement Disorders, 2019
A BS TRACT: Background: PD diagnosis is based primarily on clinical criteria and can be inaccurate. Biological markers, such as α-synuclein aggregation, that reflect ongoing pathogenic processes may increase diagnosis accuracy and allow disease progression monitoring. Though α-synuclein aggregation assays have been published, reproducibility, standardization, and validation are key challenges for their development as clinical biomarkers. Objective: To cross-validate two α-synuclein seeding aggregation assays developed to detect pathogenic oligomeric α-synuclein species in CSF using samples from the same PD patients and healthy controls from the BioFIND cohort. Methods: CSF samples were tested by two independent laboratories in a blinded fashion. BioFIND features standardized biospecimen collection of clinically typical moderate PD patients and nondisease controls. α-synuclein aggregation was measured by protein misfolding cyclic amplification (Soto lab) and real-time quaking-induced conversion (Green lab). Results were analyzed by an independent statistician. Results: Measuring 105 PD and 79 healthy control CSF samples, these assays showed 92% concordance. The areas under the curve from receiver operating characteristic curve analysis for the diagnosis of PD versus healthy controls were 0.93 for protein misfolding cyclic amplification, 0.89 for real-time quaking-induced conversion, and 0.95 when considering only concordant assay results. Clinical characteristics of false-positive and-negative subjects were not different from true-negative and-positive subjects, respectively. Conclusions: These α-synuclein seeding aggregation assays are reliable and reproducible for PD diagnosis. Assay parameters did not correlate with clinical parameters, including disease severity or duration. This assay is highly accurate for PD diagnosis and may impact clinical practice and clinical trials.
2014
The quantification of a-synuclein (aSyn) in cerebrospinal fluid (CSF) has been proposed as a diagnostic biomarker for Parkinson's disease and other aSyn-related diseases, such as multiple system atrophy and dementia with Lewy bodies. Most studies show decreased levels of aSyn in diseased CSF samples compared to control samples, but discrepant findings and overlapping values have been a major limitation for the use of CSF aSyn as a biomarker. This review addresses the current knowledge and investigates whether CSF aSyn is an ideal biomarker that can detect fundamental neuropathology features. It will also discuss whether CSF aSyn has been validated in neuropathologically confirmed cases, whether it shows a diagnostic sensitivity and whether it has a specificity above 80%. The review of current literature will also determine if sampling CSF aSyn is reliable, reproducible, noninvasive, simple to perform, inexpensive, and whether it has been investigated by at least two independent studies. CSF aSyn appears to meet most of these criteria, which have been proposed for ideal biomarkers, but further validation of this and other markers is needed to best introduce a panel of biomarkers in the early and differential diagnosis of Parkinson's disease.
Disease-Associated α-Synuclein Aggregates as Biomarkers of Parkinson Disease Clinical Stage
Neurology
Background and objectives:There’s an unmet need to identify robust diagnostic biomarker that can mirror Parkinson’s disease (PD) clinical course. Here we present a novel approach to investigate disease associated αSyn aggregates as biomarkers of PD clinical stage.Methods:We combined both seed amplification assay (SAA) and enzyme-linked immunosorbent assay (ELISA) to provide a quantitative test readout that reflects the clinical severity of PD patients. To attain this goal, we initially explored the potential of our test using two sets of human brain homogenates (pilot and validation sets), and then verified it with two independent human CSF cohorts; discovery (62 PD, and 34 control) and validation (49 PD and 48 control).Results:We showed that oligomers-specific ELISA robustly quantified SAA end product from subjects with PD or DLB with high sensitivity and specificity scores (100%). Analysis also demonstrated that seeding activity could be detected earlier with oligomeric ELISA as t...
The FASEB journal, 2006
To date there is no accepted clinical diagnostic test for Parkinson's disease (PD) based on biochemical analysis of blood or cerebrospinal fluid (CSF). ␣-Synuclein (␣-syn) protein has been linked to the pathogenesis of PD with the discovery of mutations in the gene encoding ␣-syn in familial cases with early-onset PD. Lewy bodies and Lewy neurites, which constitute the main pathological features in the brains of patients with sporadic PD and dementia with Lewy bodies, are formed by the conversion of soluble monomers of ␣-syn into insoluble aggregates. We recently reported the presence of ␣-syn in normal human blood plasma and in postmortem CSF. Here, we investigated whether ␣-syn can be used as a biomarker for PD. We have developed a novel ELISA method that detects only oligomeric "soluble aggregates" of ␣-syn. Using this ELISA, we report the presence of significantly elevated
The FASEB Journal, 2006
To date there is no accepted clinical diagnostic test for Parkinson's disease (PD) based on biochemical analysis of blood or cerebrospinal fluid (CSF). ␣-Synuclein (␣-syn) protein has been linked to the pathogenesis of PD with the discovery of mutations in the gene encoding ␣-syn in familial cases with early-onset PD. Lewy bodies and Lewy neurites, which constitute the main pathological features in the brains of patients with sporadic PD and dementia with Lewy bodies, are formed by the conversion of soluble monomers of ␣-syn into insoluble aggregates. We recently reported the presence of ␣-syn in normal human blood plasma and in postmortem CSF. Here, we investigated whether ␣-syn can be used as a biomarker for PD. We have developed a novel ELISA method that detects only oligomeric "soluble aggregates" of ␣-syn. Using this ELISA, we report the presence of significantly elevated (P)200.0؍ levels of oligomeric forms of ␣-syn in plasma samples obtained from 34 PD patients compared with 27 controls; 52% (95% confidence intervals 0.353-0.687) of the PD patients displayed signals >0.5 OD with our ELISA assay in comparison to only 14.8% (95% confidence intervals 0.014-0.281) for the control cases. An analysis of the test's diagnostic value revealed a specificity of 0.852 (95% confidence intervals 0.662-0.958), sensitivity of 0.529 (95% confidence intervals 0.351-0.702) and a positive predictive value of 0.818 (95% confidence intervals 0.597-0.948). These observations offer new opportunities for developing diagnostic tests for PD and related diseases and for testing therapeutic agents aimed at preventing or reversing the aggregation of ␣-syn.-El-Agnaf, O. M. A., Salem, S. A.,
In vivo distribution of α-synuclein in multiple tissues and biofluids in Parkinson disease
Neurology, 2020
ObjectiveThe Systemic Synuclein Sampling Study (S4) measured α-synuclein in multiple tissues and biofluids within the same patients with Parkinson disease (PD) vs healthy controls (HCs).MethodsS4 was a 6-site cross-sectional observational study of participants with early, moderate, or advanced PD and HCs. Motor and nonmotor measures and dopamine transporter SPECT were obtained. Biopsies of skin, colon, submandibular gland (SMG), CSF, saliva, and blood were collected. Tissue biopsy sections were stained with 5C12 monoclonal antibody against pathologic α-synuclein; digital images were interpreted by neuropathologists blinded to diagnosis. Biofluid total α-synuclein was quantified using ELISA.ResultsThe final cohort included 59 patients with PD and 21 HCs. CSF α-synuclein was lower in patients with PD vs HCs; sensitivity/specificity of CSF α-synuclein for PD diagnosis was 87.0%/63.2%, respectively. Sensitivity of α-synuclein immunoreactivity for PD diagnosis was 56.1% for SMG and 24.1%...