Abstract
Pathological inclusions containing aggregated, highly phosphorylated (at serine129) α-synuclein (αS pSer129) are characteristic of a group of neurodegenerative diseases termed synucleinopathies. Antibodies to the pSer129 epitope can be highly sensitive in detecting αS 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 αS 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 αS and pSer473 NFL epitopes. We determined the relative abilities of the known αS kinases, polo-like kinases (PLK) 1, 2 and 3 and casein kinase (CK) II in phosphorylating NFL and αS, 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 αS 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 αS when highly phosphorylated, further showing the structural similarity between these phospho-epitopes. All of the new pSer129 antibodies detected pathological αS inclusions in human brains and mouse and cultured cell experimental models of induced synucleinopathies. Several of these pSer129 αS 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 αS antibodies, but highlights that the use of multiple antibodies, such as those generated here, can provide a sensitive and accurate assessment of αS pathology.Electronic supplementary materialThe online version of this article (doi:10.1186/s40478-016-0357-9) contains supplementary material, which is available to authorized users.
Highlights
Synucleinopathies are a group of neurodegenerative diseases that are characterized by the presence of proteinaceous inclusions containing aggregated α-synuclein [1,2,3,4]
Assessment of neurofilament subunit (NFL) and αS phosphorylation by casein kinase II and polo-like kinases CKII was previously reported as a major kinase responsible for the phosphorylation of αS, at Ser129 [19, 36, 37], and NFL [38,39,40]
The presence of the serine 473 mutated to alanine (S473A) mutation in NFL only reduced the amount of phosphate incorporation by PLK1 and PLK3 by 0.45 and 0.37 mol phosphate/mol protein respectively, indicating that Ser473 is not the major site targeted by these kinases
Summary
Synucleinopathies are a group of neurodegenerative diseases that are characterized by the presence of proteinaceous inclusions containing aggregated α-synuclein (αS) [1,2,3,4]. PSer129 αS antibody, 81A [19] that had been used in many studies to document the formation of pathological αS inclusions in model systems of induced inclusion formation [20,21,22,23], was later shown to lead to the over-representation or misinterpretation of the inclusion formation due to its strong cross-reaction to the low molecular mass neurofilament subunit (NFL) phosphorylated at serine 473 [18] This issue underscores the importance of using highly specific and well characterized antibodies
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