described the generation of three novel pS129-aSyn antibodies 5. evaluated the specificity of MJF-R13, pSyn#64, and EP1536Y, and reported that all three showed cross-reactivity and lack of specificity towards endogenous aSyn in wildtype (WT) or SNCA KO brain slices or primary cultures 11. However, in a recent study Arlinghaus et al. EP1536Y showed the highest sensitivity and specificity for detecting pS129-aSyn 4, 9. Consistent with previous studies by Rutherford et al. Three of the four clones (MJF-R13, 81 A, and pSyn#64) showed non-specific staining in tissues from aSyn knock-out (KO) mice and all four antibodies cross-reacted with other proteins. compared the specificity of four of the most commonly used pS129 monoclonal antibodies (clones EP1536Y, MJF-R13, 81A, and pSyn#64) in brain slices and protein lysates from mice and rats 4. Several pS129 antibodies have also been shown to cross-react with cellular nuclei under conditions where aSyn is either not expressed or could not be detected in nuclear fractions by Western blotting (WB) 9, 10. reported that some of their in-house generated monoclonal pS129 antibodies cross-reacted with neurofilament subunits (NFL) phosphorylated at Serine 473 while others cross-reacted with other proteins 8. However, most of these studies have focused primarily on the cross-reactivity of the pS129-aSyn antibodies with other proteins 4, 6, 7. This has prompted several studies to assess the sensitivity and specificity of pS129-aSyn antibodies. ![]() These observations, combined with the development of a large number of antibodies against pS129-aSyn, have led to an increased reliance on pS129 antibodies as the primary tools for monitoring and quantifying aSyn pathology formation and spreading in human brains, peripheral tissues, and in cellular and animal models of synucleinopathies 5. Finally, pS129 immunoreactivity has been observed in peripheral tissues and organs associated with non-motor symptoms of PD. Furthermore, an increase in pS129 levels in both cellular models and brains of animal models of synucleinopathies is usually associated with the appearance of aSyn aggregates, and the majority of pS129-aSyn species are typically found in the insoluble fractions of brains and cell extracts 4. Subsequent studies on the biochemical composition of Lewy bodies (LB) revealed that pS129 is the dominant aSyn post-translational modification (PTM) in LB 2, 3. Initial studies suggested that the majority (>90%) of aSyn in PD and dementia with Lewy bodies (DLB) brains is phosphorylated at S129 1. Phosphorylation of alpha-synuclein (aSyn) at serine 129 (pS129) has become the most commonly used marker of aSyn pathology formation and propagation in Parkinson’s disease (PD) and other synucleinopathies. ![]() Finally, our work underscores the need for more pS129 antibodies that are not sensitive to neighboring PTMs and more thorough characterization and validation of existing and new antibodies. Our observations suggest that not all pS129 antibodies capture the biochemical and morphological diversity of aSyn pathology, and all should be used with the appropriate protein standards and controls when investigating aSyn under physiological conditions. Although most pS129 antibodies showed good performance in detecting aSyn aggregates in cells, neurons and mouse brain tissue containing abundant aSyn pathology, they also showed cross-reactivity towards other proteins and often detected non-specific low and high molecular weight bands in aSyn knock-out samples that could be easily mistaken for monomeric or high molecular weight aSyn species. We identified two antibodies that are insensitive to pS129 neighboring PTMs. ![]() 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. Herein, we demonstrate that the co-occurrence of multiple pathology-associated C-terminal post-translational modifications (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. Antibodies against phosphorylated alpha-synuclein (aSyn) at S129 have emerged as the primary tools to investigate, monitor, and quantify aSyn pathology in the brain and peripheral tissues of patients with Parkinson’s disease and other neurodegenerative diseases.
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