Comparative Analysis of Total Alpha-Synuclein (αSYN) Immunoassays Reveals That They Do Not Capture the Diversity of Modified αSYN Proteoforms

Abstract

Background: The development of therapeutics for Parkinson’s disease (PD) requires the establishment of biomarker assays to enable stratifying patients, monitoring disease progression, and assessing target engagement. Attempts to develop diagnostic assays based on detecting levels of the α-synuclein (αSYN) protein, a central player in the pathogenesis of PD, have yielded inconsistent results. Objective: To determine whether the three commercial kits that have been extensively used for total αSYN quantification in human biological fluids (from Euroimmun, MSD, and Biolegend) are capable of capturing the diversity and complexity of relevant αSYN proteoforms. Methods: We investigated and compared the ability of the different assays to detect the diversity of αSYN proteoforms using a library of αSYN proteins that comprise the majority of disease-relevant αSYN variants and post-translational modifications (PTMs). Results: Our findings showed that none of the three tested immunoassays accurately capture the totality of relevant αSYN species, and that these assays are unable to recognize most disease-associated C-terminally truncated variants of αSYN. Moreover, several N-terminal truncations and phosphorylation/nitration PTMs differentially modify the level of αSYN detection and recovery by different immunoassays, and a CSF matrix effect was observed for most of the αSYN proteoforms analyzed by the three immunoassays. Conclusion: Our results show that the tested immunoassays do not capture the totality of the relevant αSYN species and therefore may not be appropriate tools to provide an accurate measure of total αSYN levels in samples containing modified forms of the protein. This highlights the need for next generation αSYN immunoassays that capture the diversity of αSYN proteoforms.

Revisiting the specificity and ability of phospho-S129 antibodies to capture alpha-synuclein biochemical and pathological diversity

“…Therefore, when additional aSyn PTMs are detected in LBs, it is plausible to assume that a significant percentage of aSyn within LBs bears multiple PTMs. Our results demonstrate that the presence of other PTMs in close proximity to pS129 dramatically influences antibody-based detection of monomeric 77 and aggregated forms of pS129-aSyn in a site, PTM type, and PTM number dependent manner. These findings, combined with increasing evidence demonstrating that aSyn aggregates bear multiple modifications and are enriched in C-terminally truncated species, suggest that it is unlikely that antibodies against pS129 or any single C-terminal (a.a. 115-140) targeting antibody are capable of capturing the diversity of aSyn species or aSyn pathology in the brain.…”

Central and peripheral α‐synuclein in Parkinson disease detected by seed amplification assay

“…While the relationship is conceptually intuitive, any significant relationship between the two measures would be expected to be of small effect size, given the aforementioned considerations related to measures of total aSyn by antibodies, whereby only a small percentage of the overall measure likely reflects pathologic forms of aSyn. 22 In addition, the SAA does not quantitate the absolute amount of pathologic aSyn, and we cannot exclude the possibility that there are pathologic components of total aSyn not amplified by the SAA. Our findings are, however, in contrast to Kang et al 26 as well as Poggiolini et al, 3 where even in a larger number of samples, a significant relationship between CSF aSyn and F max and/or T 50 was not found.…”

“…Further complicating the interpretation of total αSyn values is that while the full‐length protein is the most common form in the human body, there are at least three isoforms produced by alternative splicing. αSyn also undergoes several post‐translational modifications (PTMs), some of which are disease‐specific, 19 but are not measured by the assay applied to S4 specimens 22 . Another factor hampering the comparability of immunoassays are the antibodies used and the respective binding sites that leads to variability 18,22 .…”

“…αSyn also undergoes several post‐translational modifications (PTMs), some of which are disease‐specific, 19 but are not measured by the assay applied to S4 specimens 22 . Another factor hampering the comparability of immunoassays are the antibodies used and the respective binding sites that leads to variability 18,22 . As different assays for αSyn are being developed, relating them to total αSyn values remains important, especially since ratios of different measures of the protein may improve diagnostic accuracy 18,23 .…”

Opportunities and challenges of alpha-synuclein as a potential biomarker for Parkinson’s disease and other synucleinopathies

“…A recent comparative study demonstrated that three of the most commonly used immunoassays (ELISA) to measure total aSyn levels failed to detect the most disease-associated C-terminally truncated forms of aSyn. It showed that several N-terminal truncations and phosphorylation differentially influence aSyn detection and recovery by the three immunoassays 211 . Overall, the majority of published or commercially available immunoassays relied on antibodies that were not thoroughly validated, or the validation data were not reported.…”