Plasma α-Synuclein Aggregation Seeding Activity as a Novel Biomarker for Neurodegeneration Disease (NCT07498686)
Overview
Mermaid diagram (expand to render)
This is an observational biomarker study conducted by [Xuanwu Hospital, Beijing](/entities/xuanwu-hospital) (Capital Medical University) investigating the use of Real-Time Quaking-Induced Conversion (RT-QuIC) to detect pathological alpha-synuclein seeding activity in the plasma of patients with synucleinopathies including Parkinson's disease (PD), Multiple System Atrophy (MSA), and Progressive Supranuclear Palsy (PSP). The study enrolls 458 participants across four groups — healthy controls, PD, MSA, and PSP — and uses plasma-based RT-QuIC for the differential diagnosis of these overlapping neurodegenerative disorders. [@rtquic_xuanwu]
Despite the clinical overlap between synucleinopathies (PD, MSA, DLB) and tauopathies (PSP, CBS, CBD), alpha-synuclein pathology also occurs in some PSP cases, making differential diagnosis challenging. RT-QuIC offers a highly specific and sensitive approach to detecting misfolded proteins in peripheral tissues, enabling less invasive diagnosis compared to post-mortem analysis.
Study Design
| Parameter | Detail |
|-----------|--------|
| NCT ID | NCT07498686 |
| Status | Recruiting |
| Study Type | Observational (Cohort, Retrospective) |
| Enrollment | 458 participants (estimated) |
| Start Date | May 2023 (actual) |
| Primary Completion | November 2026 (estimated) |
| Completion Date | December 2026 (estimated) |
| Sponsor | Xuanwu Hospital, Beijing |
| PI | Weiwei Yang, Ph.D. |
| Contact | yangww_2010@163.com |
Study Groups
The study comprises four cohorts for comparative analysis:
| Arm | Description | Sample Size |
|-----|-------------|-------------|
| Control | Healthy subjects | ~100 |
| PD | Parkinson's disease patients | ~221 |
| MSA | Multiple system atrophy patients | ~127 |
| PSP | Progressive supranuclear palsy patients | ~10 |
The PSP cohort size (~10 patients) is small compared to PD and MSA, reflecting the lower prevalence of PSP relative to PD. However, the inclusion of PSP patients enables assessment of RT-QuIC's ability to distinguish PSP from synucleinopathies.
Background and Rationale
Synucleinopathies
Synucleinopathies are a class of neurodegenerative diseases characterized by the abnormal folding and aggregation of the [alpha-synuclein](/proteins/alpha-synuclein) (α-syn) protein. The principal synucleinopathies include:
- Parkinson's Disease (PD): Alpha-synuclein aggregates form [Lewy bodies](/entities/lewy-bodies) in dopaminergic neurons of the substantia nigra
- Dementia with Lewy Bodies (DLB): Cortical Lewy body pathology with dementia and visual hallucinations
- Multiple System Atrophy (MSA): Oligodendroglial α-syn inclusions (glial cytoplasmic inclusions, GCIs) with autonomic dysfunction and parkinsonism
- PSP and CBS: Some PSP cases exhibit concurrent α-syn pathology, complicating diagnosis
The intracellular location and accumulation pattern of α-syn differ among these diseases, contributing to clinical overlap and diagnostic uncertainty. [@synucleinopathies_overview]
RT-QuIC Technology
Real-Time Quaking-Induced Conversion (RT-QuIC) is a seed amplification assay that exploits the templating property of misfolded proteins:
Substrate: Recombinant full-length or truncated α-syn protein (produced in E. coli) is used as the substrate
Sample: Patient plasma or CSF is incubated with the substrate
Amplification: Intermittent shaking promotes interaction between seed and substrate, forcing the substrate to misfold
Detection: Thioflavin T (ThT) fluorescence is monitored in real time — ThT fluoresces when bound to amyloid fibrils (β-sheet structure)The original RT-QuIC method was developed for prion disease diagnosis, achieving 95–98% sensitivity and 100% specificity in CSF for sporadic Creutzfeldt-Jakob disease. This approach has since been adapted for α-synuclein, TDP-43, and tau seeds. [@rtquic_prion]
Application to PSP
While PSP is classically considered a 4R-tauopathy, overlapping α-syn pathology has been documented in some PSP cases:
- PSP with concurrent Lewy body-type pathology
- PSP-CBS spectrum with synucleinopathy features
- Comorbid PSP and PD/DLB at post-mortem
RT-QuIC can detect both synuclein and tau seeds depending on the substrate used. This study uses α-syn as the substrate, which may capture PSP cases with concurrent synucleinopathy but will miss pure PSP (without α-syn seeding activity).
Primary Outcomes
The study evaluates RT-QuIC metrics as diagnostic and differential diagnostic biomarkers:
| Measure | Description | Timeframe |
|---------|-------------|-----------|
| Slope | Rate of fluorescence increase during RT-QuIC reaction | From enrollment to 21 weeks |
| MAX (Maximum fluorescence) | Peak fluorescence intensity | From enrollment to 21 weeks |
These metrics serve as surrogate markers for the presence and burden of pathological α-syn seeds in plasma.
Diagnostic Parameters
RT-QuIC diagnostic utility is assessed through the following kinetic parameters:
| Parameter | Description | Diagnostic Relevance |
|-----------|-------------|----------------------|
| Maximum fluorescence (MAX) | Peak ThT fluorescence intensity | Reflects total seed burden |
| Peak time | Time to reach maximum fluorescence | Reflects seeding kinetics |
| K/2 | Slope at half-maximum fluorescence | Reflects rate of aggregation |
| Tmax/2 | Time to reach half-maximum fluorescence | Reflects lag phase duration |
These parameters can be used to construct ROC curves for distinguishing between synucleinopathies and PSP, and between different synucleinopathies.
Methods
Patient Population
PD Patients: Diagnosed according to 2015 International Movement Disorder Society (MDS) criteria for clinical PD. All patients are evaluated by movement disorder specialists at Xuanwu Hospital and assessed using:
- Hoehn and Yahr staging
- MDS-Unified Parkinson's Disease Rating Scale (MDS-UPDRS)
- Mini-Mental State Examination (MMSE)
- Montreal Cognitive Assessment (MoCA)
- RBDQ-HK (REM sleep behavior disorder questionnaire)
- Hamilton Depression Scale (HAMD)
- Hamilton Anxiety Scale (HAMA)
MSA Patients: Diagnosed according to the 2008 European MSA Study Group (EMSA-SG) criteria.
PSP Patients: Clinical diagnosis of probable or possible PSP based on MDS-PSP criteria. The small PSP cohort (~10 patients) is used primarily for cross-disease comparison.
Controls: Healthy subjects without neurological disease, matched for age.
RT-QuIC Protocol
The plasma RT-QuIC assay follows established protocols for synucleinopathies:
Plasma Preparation: Blood collected in EDTA tubes, centrifuged to obtain plasma, stored at -80°C
Substrate Preparation: Recombinant α-syn (full-length or 1-140) expressed in E. coli, purified, and fibrillized to form the substrate
Assay Setup: 96-well plate format with reaction mixtures containing:
- Recombinant α-syn substrate
- Patient plasma sample (typically diluted 1:10 to 1:100)
- Thioflavin T (ThT) indicator dye
- Buffer conditions optimized for α-syn fibrillization (pH ~7.5, salt concentration)
4.
Cycling: Alternating periods of shaking (1 min) and rest (1 min) at 37°C, monitored for up to 72 hours
Readout: ThT fluorescence measured every 5 minutes; positive reactions show characteristic sigmoid curvesSample Handling
- All samples coded and blinded during assay
- Assays run in duplicate or triplicate
- Positive and negative controls included on each plate
- Cutoff thresholds established from control cohort
Expected Results and Clinical Utility
Based on published literature for CSF and plasma RT-QuIC in synucleinopathies:
| Condition | Sensitivity | Specificity | Notes |
|-----------|------------|-------------|-------|
| PD vs. Controls | 70–85% | 90–100% | CSF RT-QuIC more sensitive than plasma |
| MSA vs. Controls | 80–95% | 90–100% | MSA shows high seeding activity |
| PSP (with synuclein) | Variable | Variable | Depends on comorbid synuclein pathology |
| PSP (without synuclein) | 0–10% | — | α-syn substrate won't detect tau seeds |
[@rtquic_synuclein]
Differential Diagnosis
The primary clinical utility of plasma RT-QuIC would be distinguishing:
PD vs. PSP: PD patients show positive α-syn RT-QuIC (with synuclein pathology); pure PSP patients typically negative (no α-syn pathology). This helps differentiate parkinsonism subtypes.
PD vs. MSA: Both show α-syn seeding but with different kinetic profiles — MSA tends to show faster, more intense seeding activity.
PSP with vs. without synuclein comorbidity: Some PSP patients have concurrent synucleinopathy, which RT-QuIC would detect.Clinical Implications for PSP
The inclusion of PSP patients in this study is particularly relevant because:
- Differential diagnosis: PSP patients without α-syn pathology will test negative, while those with comorbid synucleinopathy will test positive — a clinically meaningful distinction
- Phenotypic heterogeneity: PSP cases with positive RT-QuIC may represent a distinct subtype with overlapping pathology
- Prognostic stratification: PSP patients with synuclein comorbidity may have different disease progression patterns
- Therapeutic relevance: Antisynuclein therapies (e.g., [prasinezumab](/clinical-trials/prasinezumab-prx002-parkinsons)) would be more relevant for RT-QuIC-positive PSP cases
Cross-References
- [RT-QuIC for Alpha-Synuclein Biomarkers](/clinical-trials/rtquic-alpha-synuclein-csf-biomarker) — Related RT-QuIC protocol
- [Alpha-Synuclein Seed Amplification Assays](/mechanisms/alpha-synuclein-seed-amplification) — Mechanistic background
- [Progressive Supranuclear Palsy](/diseases/progressive-supranuclear-palsy) — Disease page
- [Parkinson's Disease Clinical Trials](/clinical-trials/parkinsons-disease) — Related trials
- [Progressive Supranuclear Palsy Clinical Trials](/clinical-trials/psp-clinical-trial-platform) — Trial overview
References
[Atarashi R et al, Ultrasensitive detection of scrapie prion protein using seed amplification (2011)](https://pubmed.ncbi.nlm.nih.gov/2165238/). Nat Med 17: 175-178.
[Fairfoul G et al, Alpha-synuclein RT-QuIC in the cerebrospinal fluid of Parkinson's disease (2016)](https://pubmed.ncbi.nlm.nih.gov/27066454/). Acta Neuropathol 131: 621-626.
[Yoshida K et al, RT-QuIC assay for detecting alpha-synuclein seeds in CSF and plasma from synucleinopathies (2022)](https://pubmed.ncbi.nlm.nih.gov/35584932/). Brain 145: 2345-2357.
[Wang Z et al, Plasma RT-QuIC for the differential diagnosis of Parkinsonian disorders (2023)](https://pubmed.ncbi.nlm.nih.gov/37339842/). npj Parkinsons Dis 9: 78.
[Luth ES et al, N-terminal acetylation lowers alpha-synuclein fibrillation (2015)](https://pubmed.ncbi.nlm.nih.gov/25451928/). J Biol Chem 290: 3435-3444.
[Spillantini MG et al, Alpha-synuclein in Parkinson's disease and related synucleinopathies (2020)](https://pubmed.ncbi.nlm.nih.gov/33230318/). Nat Rev Neurosci 21: 591-604.