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

Abstract

The 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.

Artificial intelligence–coupled plasmonic infrared sensor for detection of structural protein biomarkers in neurodegenerative diseases

“…Next, we proceed to the second step—antibody immobilization. Here, we chose a sequence-specific antibody targeting the flexible C-terminal domain of the protein: SYN-211, as the capturing agent that binds to full-length aSyn irrespective of its conformation, thereby enabling unbiased structural analysis of the protein ( 55 ). The antibody solution is injected at ~30 min, and we note an increase in the sensorgram, indicating the binding of the antibody on the thiolated sensing element ( Fig.…”

Prominent astrocytic alpha-synuclein pathology with unique post-translational modification signatures unveiled across Lewy body disorders

“…We have previously shown that the use of antibodies against different regions and post-translational modifications of aSyn enables revealing the pathological diversity across synucleinopathies (Altay et al, 2022). Therefore, we sought to use an expanded antibody tool box to characterize the aSyn astrocytic pathology.…”

Large-scale visualisation of α-synuclein oligomers in Parkinson's disease brain tissue

“…Indeed, antibodies to multiple epitopes, including those targeting the N terminus, have been shown to provide more detailed information on other oligomer subtypes and locations in the brain. 72,73 (iii) Detailed characterisation of the disease-specific oligomer population: this will require super-resolution methods or electron microscopy to visualise nanoscale structures. To use super-resolution methods, further signal-to-noise ratio improvement is needed.…”

“…Indeed, antibodies to multiple epitopes, including those targeting the N terminus, have been shown to provide more detailed information on other oligomer subtypes and locations in the brain. 72,73…”