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

Abstract

ABSTRACTThe abnormal aggregation and accumulation of alpha-synuclein (aSyn) in the brain is a defining hallmark of synucleinopathies. An increasing number of studies show that aSyn in pathological aggregates exists as a complex mixture of various post-translationally modified forms and conformations. The distribution of these different species changes during disease progression and varies among the different synucleinopathies. Current approaches for detecting aSyn in human tissues and disease models rely on a limited set of antibodies that have not been thoroughly assessed for their ability to capture the diversity of aSyn proteoforms and aggregation states, and thus are unlikely to provide a complete estimation of aSyn pathology in the brain. To address these challenges, we developed, validated, and characterized an expanded set of antibodies that target different sequences and post-translational modifications (PTMs) along the entire length of aSyn, and recognize all conformations of the protein (monomers, oligomers and fibrils). We demonstrate that the use of multiple antibodies targeting different regions on aSyn is necessary to reveal the heterogeneity of aSyn pathology in human Lewy body disease (LBD) brains, and in neuronal and animal models of aSyn aggregation and inclusion formation. We also present, for the first time, the profiling of aSyn pathology using antibodies against all its key post-translationally modified forms across sporadic and familial LBDs. The antibody validation pipeline we describe here paves the way for a more systematic investigation of the diversity of aSyn pathology in the human brain and peripheral tissues, and in cellular and animal models of synucleinopathies.