Generation of monoclonal antibodies against phosphorylated α-Synuclein at serine 129: Research tools for synucleinopathies

The majority of alpha-synuclein (alpha-syn) within Lewy bodies (LBs) has been reported to be phosphorylated at serine 129 (pS129-alpha-syn), suggesting a central role for phosphorylation in the pathogenesis of Parkinson's disease (PD) and related synucleinopathies. Various studies have investigated the effect of alpha-syn phosphorylation but have failed to reach a consensus as to whether this modification accelerates or inhibits alpha-syn aggregation. Nevertheless, pS129-alpha-syn is a reliable marker of alpha-syn aggregates and is widely evaluated in biomarkers and post-mortem studies. While several antibodies specific for pS129-alpha-syn exist, their reactivity with non-specific antigens appears to be a common challenge. To gain valuable insights into the role of alpha-syn phosphorylation in disease pathogenesis, antibodies that are highly specific to pS129-alpha-syn are necessary. In this study, we describe the generation of three mouse monoclonal antibodies (mAbs; 5B9, 6H5 and 9G1) using hybridoma technology. These were thoroughly characterized and validated in combination with our previously generated mAb (PS129), and the commercial ab51253 (Abcam). We demonstrated that our mAbs are highly specific for pS129-alpha-syn and do not cross react with wild-type alpha-syn. Results from staining of post-mortem human brain tissue showed that our mAbs detect pS129-alpha-syn pathology in patients with synucleinopathies. This study highlights three new antibodies as excellent and highly specific research tools to explore the role of pS129-alpha-syn inclusions in synucleinopathies.