Disease-Associated α-Synuclein Aggregates as Biomarkers of Parkinson Disease Clinical Stage

Abstract

Background and objectives: Robust biomarkers that can mirror Parkinson disease (PD) are of great significance. In this study, we present a novel approach to investigate disease-associated α-synuclein (αSyn) aggregates as biomarkers of PD clinical stage.

Methods: We combined both seed amplification assay (SAA) and ELISA to provide a quantitative test readout that reflects the clinical severity of patients with PD. To attain this goal, we initially explored the potential of our test using 2 sets of human brain homogenates (pilot and validation sets) and then verified it with 2 independent human CSF cohorts; discovery (62 patients with PD and 34 controls) and validation (49 patients with PD and 48 controls) cohorts.

Results: We showed that oligomers-specific ELISA robustly quantified SAA end product from patients with PD or dementia with Lewy bodies with high sensitivity and specificity scores (100%). Analysis also demonstrated that seeding activity could be detected earlier with oligomeric ELISA as the test readout rather than SAA alone. Of more importance, multiplexing the assays provided robust information about the patients' clinical disease stage. In the discovery cohort, levels of CSF-seeded αSyn oligomers correlated with the severity of the clinical symptoms of PD as measured by the Unified Parkinson Disease Rating Scale (UPDRS) motor (r = 0.58, p < 0.001) and Hoehn and Yahr (H&Y) scores (r = 0.43, p < 0.01). Similar correlations were observed in the validation cohort between the concentrations of CSF-seeded αSyn oligomers and both UPDRS motor (r = 0.50, p < 0.01) and H&Y scores (r = 0.49, p < 0.01). At 20 hours, receiver operating characteristic curves analysis yielded a sensitivity of 91.9% (95% CI 82.4%-96.5%) and a specificity of 85.3% (95% CI 69.8%-93.5%), with an area under the curve of 0.969 for CSF-seeded αSyn oligomers differentiating those with PD from controls in the discovery CSF cohort, whereas, a sensitivity of 80.7% (95% CI 69.1%-88.5%), a specificity of 76.5% (95% CI 60.0%-87.5%), and area under the curve of 0.860 were generated with thioflavin T maximum intensity of fluorescence at the same time point.

Discussion: We showed that combining SAA and ELISA assays is a more promising diagnostic tool than SAA alone, providing information about the disease stage by correlating with clinical measures of disease severity.

Classification of evidence: This study provides Class III evidence that CSF-seeded αSyn oligomers can accurately discriminate patients with PD and normal controls and CSF-seeded αSyn oligomers levels correlate with PD severity.

Figure 1. Flowchart of the Study Cohorts

Flowchart presenting the number of cases per cohort and the primary question answered by each samples' set. αSyn = α-synuclein; DLB = dementia with Lewy bodies; HC = healthy control; PD = Parkinson disease.

Figure 2. SAA and ELISA Analysis of the Pilot BH Samples Set

RT-QuIC reactions seeded in triplicate with brain homogenate (BH) from controls (Ctrl, blue), Parkinson disease (PD, red) cases, or dementia with Lewy bodies (DLB, green) cases. The solid line of each sample trace represents the average ThT signal of triplicate wells. The colored ribbon represents the standard error (±SD) (A). Comparison of Imax, area under the ThT curve (ThT AUC), and seeded αSyn oligomers for each group. The dots represent the single cases, and the lines reflect the group's average. Statistical analysis was not conducted at this stage because of the small sample size; however, the differences between the groups are clearly pronounced (B, C). αSyn = α-synuclein; SAA = seed amplification assay.

Figure 3. SAA and ELISA Analysis of the Validation BH Samples Set

RT-QuIC reactions seeded in triplicate with brain homogenate (BH) from subjects with αSyn pathology (PD, n = 1 and DLB, n = 4) or without αSyn pathology (AD, n = 3 and Ctrl, n = 2). The solid line represents the average ThT signal per group (A). Monitoring the changes of αSyn oligomers' levels over multiple time points (0, 48, 72, 96, and 120 hours) for each group for ELISA (B), and SAA (C). αSyn = α-synuclein; SAA = seed amplification assay.

Figure 4. RT-QuIC Reactions Seeded in Triplicate With CSF From Controls and Patients With PD

RT-QuIC reactions seeded in triplicate with CSF samples from Ctrls (blue) and patients with PD (red). The solid line represents the average ThT signal of triplicate wells. The colored ribbon represents the standard error (A). Comparison of the mean maximum fluorescence at SAA end point and at 20 hours of the assay run for each diagnostic group, respectively (B). Comparison of seeded CSF αSyn oligomers and total, respectively, at 20 hours of the assay run for each diagnostic group (C). Floating bars show the min to max with the line at the mean. αSyn = α-synuclein.

Figure 5. Correlation Analysis in Discovery and Validation Cohorts

Scatterplots showing the correlation analysis in the discovery cohort between CSF-seeded αSyn oligomers and UPDRS motor scores and H&Y scores, respectively (A, B). Scatterplots showing the correlation analysis in the validation cohort between UPDRS motor and H&Y scores with seeded CSF αSyn oligomers (C, D). The subplots present the same dataset excluding extreme data points highlighted with the red square. The solid line highlights the calculated regression line. p Values and Spearman r_s are displayed for each correlation. H&Y = Hoehn and Yahr; UPDRS-III = Unified Parkinson Disease Rating Scale Part-III.