Objective: To identify α-synuclein (α-syn) epitopes bound by endogenous auto-antibodies in CSF from healthy controls (HC) and Parkinson disease (PD) patients.

Background: In PD, endogenous auto-antibodies responses to the amyloidogenic protein α-syn might reflect the underlying neuropathological disease burden. We previously developed an enzyme-linked immunosorbent assay (ELISA) to measure α-syn auto-antibodies in human biological fluids. However, the specific α-syn epitopes recognized by auto-antibodies are unknown. We hypothesized that endogenous auto-antibodies would recognize a portion of α-syn more amenable to immunogenic responses, and thereby could influence the pathogenicity of α-syn.

Methods: Wild-type (α-syn_1-140), N-terminal truncated (α-syn_58-140), or C-terminal truncated (α-syn_1-120 and α-syn_1-99) human α-syn was expressed and purified in BL21(DE3)-RIL cells using PRK172 expression vectors. 100 ng wild-type or truncated α-syn per well was affixed to 384-well ELISA plates. After blocking, CSF (1:5 in PBS) was incubated overnight followed by incubation with a horseradish peroxidase(HRP)-conjugated anti-human IgG secondary antibody and detected using a chromogenic assay. Mouse monoclonal antibody Syn211 and rabbit polyclonal antibodies SNL-1 and SNL-4 with known α-syn binding epitopes were used as positive controls, along with species appropriate HRP-conjugates. Univariate comparisons were performed using two-sample t-test for α-syn truncations.

Results: CSF samples from 52 HC and 93 PD patients were analyzed. PD CSF had significantly higher levels of auto-antibody to α-syn_1-140 as compared to HC CSF (p = 0.003). ELISA using α-syn_58-140 or α-syn_1-120 did not alter binding of PD auto-antibodies, which remained significantly higher than HC participants (p = 0.004 and p = 0.005). However, ELISA using α-syn_1-99 significantly reduced recruitment of PD auto-antibodies and levels were no longer significantly different than HC participants (p = 0.07). In keeping with their known binding epitopes, Syn211 did not detect either α-syn_1-120 or α-syn_1-99, SNL-1 did not detect α-syn_1-99, and SNL-4 did not detect α-syn_58-140.

Conclusions: Endogenous CSF auto-antibodies to α-syn appear to detect a portion of the C-terminal between amino acids 100 and 120. These interactions in this region could have implications for α-syn pathogenicity.

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MDS Abstracts - https://www.mdsabstracts.org/abstract/epitope-mapping-of-csf-auto-antibodies-to-alpha-synuclein/