Novel antibodies to phosphorylated α-synuclein serine 129 and NFL serine 473 demonstrate the close molecular homology of these epitopes

Pathological inclusions containing aggregated, highly phosphorylated (at serine129) alpha-synuclein (alpha S pSer129) are characteristic of a group of neurodegenerative diseases termed synucleinopathies. Antibodies to the pSer129 epitope can be highly sensitive in detecting alpha S inclusions in human tissue and experimental models of synucleinopathies. However, the generation of extensively specific pSer129 antibodies has been problematic, in some cases leading to the misinterpretation of alpha S inclusion pathology. One common issue is cross-reactivity to the low molecular mass neurofilament subunit (NFL) phosphorylated at Ser473. Here, we generated a series of monoclonal antibodies to the pSer129 alpha S and pSer473 NFL epitopes. We determined the relative abilities of the known aS kinases, polo-like kinases (PLK) 1, 2 and 3 and casein kinase (CK) II in phosphorylating NFL and alpha S, while using this information to characterize the specificity of the new antibodies. NFL can be phosphorylated by PLK1, 2 and 3 at Ser473; however CKII shows the highest phosphorylation efficiency and specificity for this site. Conversely, PLK3 is the most efficient kinase at phosphorylating alpha S at Ser129, but there is overlay in the ability of these kinases to phosphorylate both epitopes. Antibody 4F8, generated to the pSer473 NFL epitope, was relatively specific for phosphorylated NFL, however it could uniquely cross-react with pSer129 alpha S when highly phosphorylated, further showing the structural similarity between these phospho-epitopes. All of the new pSer129 antibodies detected pathological alpha S inclusions in human brains and mouse and cultured cell experimental models of induced synucleinopathies. Several of these pSer129 alpha S antibodies reacted with the pSer473 NFL epitope, but 2 clones (LS3-2C2 and LS4-2G12) did not. However, LS3-2C2 demonstrated cross-reactivity with other proteins. Our findings further demonstrate the difficulties in generating specific pSer129 alpha S antibodies, but highlights that the use of multiple antibodies, such as those generated here, can provide a sensitive and accurate assessment of aS pathology.