Specific Detection of Endogenous S129 Phosphorylated α-Synuclein in Tissue Using Proximity Ligation Assay

BACKGROUNDSynucleinopathies are a group of neurodegenerative disorders that are pathologically characterized by the accumulation of protein aggregates called Lewy Bodies. Lewy bodies are primarily composed of -synuclein (asyn) protein, which is phosphorylated at serine 129 (pS129) when aggregated. Currently available commercial antibodies used to stain for pS129 asyn can cross react with other proteins, thus making it difficult to specifically detect endogenous pS129 asyn and to interpret pS129 asyn staining. OBJECTIVETo develop a staining procedure that detects pS129 asyn with high specificity and low background. METHODSWe use the fluorescent and brightfield in situ Proximity Ligation Assay (PLA) to specifically detect pS129 asyn in cell culture, mouse and human brain sections. RESULTSThe pS129 asyn PLA specifically stained for endogenous, soluble pS129 asyn in cell culture, mouse brain sections, and human brain tissue without significant cross-reactivity or background signal. CONCLUSIONSThis PLA method can be used to specifically detect pS129 asyn in order to further explore and understand its role in health and disease.