A novel approach to detecting plasma synuclein aggregates for Parkinson's disease diagnosis

Abstract

Alpha-synuclein (αSyn) aggregates are pathognomonic of Parkinson's disease (PD) and play a critical role in its pathogenesis. However, existing diagnostic approaches rely on invasive cerebrospinal fluid (CSF) sampling or tissue biopsies, limiting their accessibility and scalability in clinical practice. Here, we present the Constant Shake-Induced Conversion (CSIC) assay, a novel plasma-based technique for the detection of αSyn aggregates. A total of 102 participants, comprising 42 PD patients and 60 healthy controls (HCs), were enrolled. Plasma samples were subjected to CSIC and validated via αSyn depletion, enzyme-linked immunosorbent assay (ELISA), and Western blotting. Diagnostic performance was assessed using receiver operating characteristic (ROC) analysis, and clinical associations were examined using Spearman's correlation. The CSIC assay achieved an area under the curve (AUC) of 0.91, with 81% sensitivity and 85% specificity in distinguishing PD from HCs. Assay specificity was confirmed through αSyn depletion, and reproducibility assessments yielded intra- and inter-assay coefficients of variation below 10% and ~20%, respectively. Notably, plasma αSyn aggregate levels correlated with Hoehn and Yahr (H&Y) stage (r = 0.69), Unified Parkinson's Disease Rating Scale (UPDRS) (r = 0.68), and Montreal Cognitive Assessment scores (r = -0.47). These findings establish CSIC as a robust, non-invasive diagnostic method with strong potential for clinical implementation in PD.

Fig. 1. Performance analysis of CSIC.

a Schematic diagram illustrating the proposed CSIC assay. αSyn seeds present in blood are amplified through constant shaking. His-αSyn is used as the spiking material, and ThT fluorescence is measured to confirm the presence of amplified aggregates. b, c Dose-dependent αSyn aggregation kinetics measured by CSIC in normal plasma. Synthetic αSyn seeds at varying concentrations (1000 pg, 100 pg, 10 pg, 1 pg, 0.1 pg, and 0.01 pg) were spiked into normal plasma along with monomeric His-tagged αSyn. Each seed concentration was tested in duplicate (n = 2). The seed-free condition contained only monomeric His-αSyn. Colors indicate synuclein seed concentrations, ranging from 1000 pg (dark red) to 0.01 pg (gray). The black line represents the no-seed control. Aggregation kinetics were monitored in real time (b), with data shown as mean ± standard error of the mean (S.E.M.). T₅₀ was determined for each seed concentration (c).

Fig. 2. Verification of CSIC in plasma.

a Schematic representation of the experimental design. HC and PD plasma samples were subjected to αSyn depletion, followed by verification using b ELISA, c Western blotting, and d CSIC. b The αSyn-depleted supernatant was analyzed using sandwich ELISA to detect total αSyn. The red and blue bars represent PD and HC plasma, respectively. c αSyn was immunoprecipitated from HC and PD plasma samples using an anti-αSyn antibody (MJFR1). The presence of immunoprecipitated αSyn was verified by Western blotting using an anti-αSyn (211)-HRP antibody. The immunoprecipitated αSyn is indicated by an arrow. d CSIC was used to amplify αSyn aggregates in αSyn-depleted and untreated HC and PD plasma samples. The bar graph shows αSyn aggregates derived from CSIC. The red bar represents PD plasma, and the blue bar represents HC plasma. e CSIC was used to amplify αSyn aggregates in plasma samples from HCs (n = 3) and PD patients (n = 3). The red and blue lines represent amplified αSyn aggregates from PD and HC plasma, respectively. f Following CSIC, the final product was subjected to a sedimentation assay. The supernatant (sup) and pellet were separated, and equal volumes were analyzed using Western blotting. Immunoblotting was performed using an anti-αSyn (211)-HRP antibody. Arrows indicate the αSyn bands.

Fig. 3. Comparison of plasma αSyn aggregate levels using CSIC between groups.

a Box and whisker plots showing the range and average distribution of αSyn aggregates detected using CSIC. Statistical analysis was conducted using Mann-Whitney U tests due to the non-parametric distribution, with significant differences between the HC and PD groups at p < 0.0001 (****) indicated. b ROC curve of αSyn aggregates in accordance with CSIC parameters for discriminating patients with PD from HC. c Repeatability and reproducibility tests. Repeatability assays were conducted on plasma samples from healthy individuals (n = 3) and patients with PD (n = 3). Each sample was tested three times by three different experimenters to assess the reproducibility of the assay.

Fig. 4. Correlations between CSIC parameters and clinical scores.

a Heatmap representation of spearman correlation coefficients (r) between CSIC and clinical scales. The correlation coefficients are color-coded as shown in the vertical bar. 0 indicates no linear relationship, +1/−1 indicates a perfect linear positive/negative relationship. Correlations between CSIC parameters and clinical scores: b H&Y, c UPDRS, and d MoCA. All r values were calculated using raw data.