Development and validation of an expanded antibody toolset that captures alpha-synuclein pathological diversity in Lewy body diseases

The abnormal aggregation and accumulation of alpha-synuclein (aSyn) in the brain is a defining hallmark of synucleinopathies. Different aSyn conformations and post-translationally modified forms accumulate in pathological inclusions and vary in abundance across synucleinopathies. Relying on antibodies that have not been assessed for their ability to capture the diversity of aSyn species may not provide an accurate estimation of aSyn pathology in human brains or disease models. To address this challenge, we developed and characterised an expanded antibody panel that targets different sequences and post-translational modifications along the length of aSyn and recognises all three aSyn conformations (monomeric, oligomeric, fibrillar). Next, we profiled aSyn pathology across sporadic and familial Lewy body diseases (LBDs) and reveal heterogeneously modified aSyn pathologies rich in Serine 129 phosphorylation but also in Tyrosine 39 nitration and N- and C-terminal tyrosine phosphorylations, scattered to neurons and glia. We also show that aSyn may become hyperphosphorylated during the aggregation and inclusion maturation processes in neuronal and animal models of aSyn aggregation and spreading. The antibody validation pipeline we describe here paves the way for more systematic investigations of aSyn pathological diversity in the human brain and peripheral tissues, and in cellular and animal models of synucleinopathies. ### Competing Interest Statement Hilal A. Lashuel is the co-founder and chief scientific officer of ND BioSciences, Epalinges, Switzerland, a company that develops diagnostics and treatments for neurodegenerative diseases (NDs) based on platforms that reproduce the complexity and diversity of proteins implicated in NDs and their pathologies. HAL has received funding from industry to support research on neurodegenerative diseases, including Merck Serono, UCB, and Abbvie. These companies had no specific role in the conceptualization, preparation of and decision to publish this manuscript.