Blood α-Synuclein Separates Parkinson's Disease from Dementia with Lewy Bodies

Abstract

Objective: Aggregation of misfolded α-synuclein (aSyn) within the brain is the pathologic hallmark of Lewy body diseases (LBDs), including Parkinson's disease (PD), and dementia with Lewy bodies (DLB) disease. Although evidence exists for aSyn "strains," conformations with distinct biological properties, biomarkers for PD versus DLB are lacking. Here, we used monoclonal antibodies selective for two different in vitro aSyn species - termed strain A and B - to evaluate human brain tissue, cerebrospinal fluid (CSF), and plasma.

Methods: Using these antibodies, we characterized specific aSyn species in human specimens from neurologically normal individuals and individuals with LBD using enzyme-linked immunosorbent assay (ELISA), Western blot, and immunohistochemistry. We also characterized aSyn species immunoprecipitated from brain lysate or plasma with these antibodies using seed amplification assays (SAAs) and a cellular model.

Results: Surprisingly, levels of strain A and B aSyn species were higher in plasma from individuals with PD compared to DLB in 2 independent cohorts. Lower levels of plasma aSyn strain A species predicted a faster rate of cognitive decline in individuals with PD. Furthermore, strain A and strain B aSyn species were undetectable in CSF, and their levels in brain versus plasma did not correlate. Moreover, plasma aSyn species isolated by aSyn strain antibodies could template aSyn fibrillization, and they could seed formation of aSyn inclusions in cells.

Interpretation: Our findings suggest that circulating plasma aSyn strains may impact LBD clinical presentation, particularly cognition. The enrichment of these aSyn species in plasma but not CSF also suggests a potential source outside the brain. ANN NEUROL 2025;98:682-698.

Figure 1

Strain A (7015) and Strain B (9027) antibodies selectively and differentially recognize aSyn pathology in human brain tissue. Mice were immunized with 2 different in vitro generated strains of aSyn to produce 7015 (strain A) and 9027 (strain B) monoclonal antibodies (A). Brain regions (B) known to associate with diffuse spread of Lewy pathology from 10 NCs and 10 individuals with LBD were immunostained with strain A (C) and strain B (D) antibodies with representative images showing preferential recognition of thread‐like, “neuritic” (arrowheads) versus dot‐like, “aggregate” (arrows) pathology, respectively. Compared to strain A‐associated pathology (E), strain B‐associated aSyn pathology (F) more strongly correlated with p‐aSyn, beta‐amyloid, and p‐tau staining across all 3 examined brain regions. The p value and r statistic for Pearson correlation are reported in E and F. AO = area occupied; LBD = Lewy body disease; NC = normal control; p‐aSyn = phosphorylated aSyn; PDD = Parkinson's disease dementia; p‐tau = phosphorylated tau; SMTC = superior middle temporal cortex. [Color figure can be viewed at www.annalsofneurology.org]

Figure 2

The aSyn strains can be detected by ELISA and are uniquely enriched in plasma over CSF. Sandwich ELISAs using MJFR1 (total aSyn) antibody for capture and strain A or strain B biotinylated antibodies for detection show differential selectivity for recombinant a‐syn PFFs versus monomer (A). The WB with strain A (B) and with strain B (C) antibodies detect HMW (arrows) aSyn species only in brain lysates from individuals with PD or DLB. GAPDH is displayed as a loading control (D). Strain A aSyn levels measured by ELISA (E) correspond at the individual level with levels of HMW aSyn species detected by immunoblot with strain A antibody (B), whereas strain B aSyn levels measured by ELISA (F) show less individual‐level correspondence to quantities detected by immunoblot (C). Strain A and B aSyn species are not readily detectable in CSF (n = 44 individuals tested), but robustly detected in plasma (G). For matched brain and plasma samples (plasma collected within 2 years of autopsy for 10 individuals), plasma aSyn strain levels do not correlate with brain aSyn levels (RIPA extracts shown) (H). For B to F, each column/lane corresponds to brain lysate from one individual. DLB = dementia with Lewy bodies; ELISA = enzyme‐linked immunosorbent assay; CSF = cerebrospinal fluid; HMW = high molecular weight; NC = neurologically normal control; PD = Parkinson's disease; PFFs = preformed fibrils; RIPA = radioimmunoprecipitation assay; SMTC = superior middle temporal cortex; WB = Western blot. [Color figure can be viewed at www.annalsofneurology.org]

Figure 3

Plasma levels of aSyn strains differentiate individuals with DLB from individuals with PD. Both aSyn strain A and B levels are significantly elevated in PD plasma (red diamonds) compared to DLB (blue triangles), NC (grey circles), and AD (grey squares) in the UPenn cohort, n = 235 (A), whereas strain B levels are significantly elevated, and strain A levels shows a trend toward elevation, in PD relative to DLB in the PDBP cohort, n = 200. (B). Differentiation of PD versus DLB (C) but not PD versus NC (D) in both cohorts is significantly improved (as captured in the area under the ROC curve, AUC) when plasma aSyn strain levels are incorporated into predictors, compared with predictors using only age, sex, and plate to predict disease class. Thresholds, sensitivity, and specificity are established using Youden's method. Error bars represent SEM. For differences among groups, the p values (corrected for multiple comparisons for Kruskal‐Wallis post hoc tests) for one‐way ANOVA and for the LME model are shown in A and B, respectively. Significance testing for differences between ROC curves was performed using the DeLong method. **p < 0.01, ***p < 0.001. AD = Alzheimer's disease; ANOVA = analysis of variance; AU = arbitrary units for plate and batch normalized measures; AUC = area under the curve; DLB = dementia with Lewy bodies; LME = linear mixed‐effect; NC = normal control; PD = Parkinson's disease; PDBP = NIH Parkinson's Disease Biomarker Project; ROC = receiver‐operator characteristic; Sens., sensitivity; Spec., specificity; UPenn = University of Pennsylvania. [Color figure can be viewed at www.annalsofneurology.org]

Figure 4

Strain A levels predict the rate of cognitive decline in PD. Plasma measures of strain A and B aSyn species were used to predict subsequent cognitive and motor course over 10 years for 95 longitudinally followed individuals with PD in LME models. Disease trajectory for individuals with PD with different levels of strain A levels at baseline are shown in panels A (cognitive disease course) and B (motor disease course). Disease trajectory for individuals with PD with different levels of strain B levels at baseline are shown in panels C (cognitive disease course) and D (motor disease course). For all panels, the colors indicate the modeled disease course for each tertile of aSyn strain measure, from highest (red), to middle (green), to lowest (blue), with bands representing 95% confidence intervals. Cognitive performance is reflected in the age‐adjusted DRS score, where higher values represent better performance, and motor impairment is reflected in the UPDRS part III score, where higher values represent greater impairment. DRS = dementia rating scale; LME = linear mixed‐effect; PD = Parkinson's disease; UPDRS = Unified Parkinson's Disease Rating Scale [Color figure can be viewed at www.annalsofneurology.org]

Figure 5

The aSyn species isolated from pooled plasma by strain‐selective antibodies can seed aSyn fibrillization by SAA. (A) Then, 0.05 μg/ml of protein isolated by IP using strain A, strain B, or total aSyn (syn211) antibodies, or negative control isotype antibody, from plasma or brain lysates (caudate) was tested for ability to seed fibrillization of aSyn monomer substrate in SAA. (B) In control conditions, addition of aSyn PFFs readily induced aSyn fibrillization, whereas the addition of monomeric aSyn did not. (C and D) Representative SAA curves from plasma (C) and brain (D) IPs in material from individuals with PD. (E and F) Representative SAA curves from plasma IPs in material from individuals with PD (E), compared with individuals with DLB (F). (G) Quantification of AUC, normalized to PFF‐seeded condition, for 4 replicate SAAs seeded with IP material from 2 different plasma and 3 different brain lysate pools from individuals with PD. For plasma, strain A aSyn (green) was significantly more potent in inducing aSyn fibrillization, compared to the isotype control IP condition (yellow). For the brain, total aSyn IP (purple) was significantly more potent in inducing aSyn fibrillization, compared to the isotype control IP condition (yellow). One‐way ANOVA (factor: type of IP seed) with post hoc Tukey test was used to assess whether AUC was significantly different comparing negative control isotype IgG IP versus aSyn antibody IP seed material. (H) Quantification of AUC, normalized to PFF‐seeded condition, for 3 replicate SAAs seeded with 3 PD, 3 NC, and 3 DLB plasma pools. Plasma aSyn IP with strain A antibody (green) from PD was significantly more potent in inducing aSyn fibrillization, compared to plasma aSyn IP with strain A antibody from individuals with DLB or NCs. Two‐way ANOVA (factors: IP seed and disease group) with post hoc Tukey test was used to assess for significant differences between SAA conditions. *p < 0.05, error bars represent SEM. ANOVA = analysis of variance; AUC = area under the curve; DLB = dementia with Lewy bodies; IP = immunoprecipitation; NCs = normal controls; PD = Parkinson's disease; PFFs = pre‐formed fibrils; SAA = seed amplification assay. [Color figure can be viewed at www.annalsofneurology.org]

Figure 6

Strain A aSyn species isolated from individual plasma samples by strain‐selective antibodies can seed aSyn fibrillization in SAA. (A) Then, 0.05 μg/ml of protein isolated by IP using strain A antibodies (green) or negative control isotype antibody (yellow) from plasma of 10 individuals with PD was tested for ability to seed fibrillization of aSyn monomer substrate in SAA. Average SAA curves with SEM error bars from assay quadruplicate wells are shown for each individual sample. (B) Quantification of AUC, normalized to PFF‐seeded condition, for all 10 individual PD plasma sample SAAs seeded with IPs with isotype or strain A antibody are displayed. (C) Time to reach fluorescence threshold of 2,500 units is expressed as 1/hours for all 10 individual PD plasma samples SAAs seeded with IPs with isotype or strain A antibody are displayed. Kruskal‐Wallis test was used to assess for significant differences between isotype and strain A IP seeded SAAs. ***p < 0.001, error bars represent SEM. AUC = area under the curve; IP = immunoprecipitation; PFF = pre‐formed fibril; SAA = seed amplification assay; [Color figure can be viewed at www.annalsofneurology.org]

Figure 7

Strain A species from PD plasma induce aSyn inclusions in the SK‐MEL‐28 cell line. (A) Application of recombinant fibrillar aSyn (PFF) but not monomeric aSyn induces detergent‐insoluble inclusions (arrowheads) in SK‐MEL‐28 cells. (B) Representative images of SK‐MEL‐28 cells treated with material derived from PD plasma via IP using strain A, strain B, total aSyn, or negative control (isotype IgG) antibody. Cells were stained for total aSyn (red), alpha‐tubulin (green), and nuclei (blue). The Cell Profiler was used to quantify number of inclusions (C) and total aSyn intensity in inclusions (D) per cell (normalized to number of nuclei per field). Individual points in graphs represent quantification for unique fields of view. Points with the same color are from the same experimental replicate (n = 3). **p < 0.01, ***p < 0.001, error bars represent SEM. IP = immunoprecipitation; PD = Parkinson's disease; PFF = pre‐formed fibril. [Color figure can be viewed at www.annalsofneurology.org]