Neighbouring modifications interfere with the detection of phosphorylated alpha-synuclein at Serine 129: Revisiting the specificity of pS129 antibodies

Abstract

Alpha-synuclein (aSyn) within Lewy bodies, Lewy neurites, and other pathological hallmarks of Parkinson's disease and synucleinopathies have consistently been shown to accumulate in aggregated and phosphorylated forms of the protein, predominantly at Serine 129 (S129). Antibodies against phosphorylated S129 (pS129) have emerged as the primary tools to investigate, monitor, and quantify aSyn pathology in the brain and peripheral tissues. However, most of the antibodies and immunoassays aimed at detecting pS129-aSyn were developed based on the assumption that neighbouring post-translational modifications (PTMs) either do not co-occur with pS129 or do not influence its detection. Herein, we demonstrate that the co-occurrence of multiple pathology-associated C-terminal PTMs (e.g., phosphorylation at Tyrosine 125 or truncation at residue 133 or 135) differentially influences the detection of pS129-aSyn species by pS129-aSyn antibodies. These observations prompted us to systematically reassess the specificity of the most commonly used pS129 antibodies against monomeric and aggregated forms of pS129-aSyn in mouse brain slices, primary neurons, mammalian cells and seeding models of aSyn pathology formation. We identified two antibodies that are insensitive to pS129 neighbouring PTMs. However, consistent with previous reports, most pS129 antibodies showed cross-reactivity towards other proteins and often detected low and high molecular weight bands in aSyn knock-out samples that could be easily mistaken for monomeric or High Molecular Weight aggregates of aSyn. Our observations suggest that the pS129 antibodies do not capture the biochemical and morphological diversity of aSyn pathology. They also underscore the need for more specific pS129 antibodies, more thorough characterization and validation of existing antibodies, and the use of the appropriate protein standards and controls in future studies.

Development and validation of an expanded antibody toolset that captures alpha-synuclein pathological diversity in Lewy body diseases

“…We have recently shown that the aSyn pS129 antibody binding could be affected by the presence of aSyn PTMs neighbouring S129 (Lashuel et al, 2022), and hypothesised that sensitivity to neighbouring PTMs could also be applicable to other C-terminal as well as N-terminal or NAC region antibodies. Therefore, we investigated the reactivity of the non-modified aSyn antibodies in the presence of PTMs nearby their epitopes by DB and WB, and the results are summarised in Table 1.…”

“…2F10-E12 110-115 and AB 134-138) of aSyn. Two additional antibodies were incorporated to cover the aSyn serine phosphorylations: AB EP1536Y pS129, as this antibody has been the most specific to aSyn pS129 species in our hands and in the literature (Delic et al, 2018; Lashuel et al, 2022), and LASH pS87, which was recently shown to detect LBs in PD and GCIs in MSA (Sonustun et al, 2022). For the N-terminal tyrosine phosphorylation modification, we opted for the monoclonal LASH-BL pY39 antibody.…”

“…We have recently shown that the aSyn pS129 antibody binding could be affected by the presence of aSyn PTMs neighbouring S129 (Lashuel et al, 2022), and hypothesised that sensitivity to neighbouring PTMs could also be applicable to other C-terminal as well as N-terminal or NAC region antibodies.…”

“…aSyn 1-119 and 1-122 (Anderson et al, 2006; Bhattacharjee et al, 2019; Kellie et al, 2015; Killinger et al, 2018; Ohrfelt et al, 2011), lack the epitope for aSyn pS129 antibodies. Furthermore, the occurrence of several modifications in close proximity to S129 suggests that they may interfere with the immunoreactivity of several aSyn C-terminal or pS129 antibodies (Lashuel et al, 2022; Mahul-Mellier et al, 2018).…”

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

“…The most likely reason for the selection of these antibody pairs is that the C-terminus of ␣SYN is the most immunogenic region and results in high affinity antibodies and thus better assay performance, as compared to N-terminal ␣SYN antibodies which exhibit in general a lower affinity thereby resulting in lower sensitivity assays. Nevertheless, it is possible that the multitude of C-terminal PTMs neighboring the epitopes of these antibodies could either eliminate or mask the epitope [42], or interfere with antibody binding to ␣SYN resulting in a biased analyte concentration. In addition, the great majority of ␣SYN immunoassays were developed based on the use of a single protein standard (full-length ␣SYN), which may have led to the downstream development of assays that do not necessarily capture the diversity of ␣SYN proteoforms.…”