Abstract Intraneuronal α-synuclein inclusions in the brain are hallmarks of so-called Lewy body diseases - Parkinson’s disease and Dementia with Lewy bodies. Lewy bodies are cytoplasmic inclusions, containing mainly aggregated α-synuclein together with some other proteins including ubiquitin, neurofilament protein, and alpha B crystallin. In its monomeric form, α-synuclein is predominantly localized in nerve terminals, regulating neuronal transmission and synaptic vesicle trafficking. Monomeric α-synuclein lacks a well-defined three-dimensional structure and is considered an intrinsically disordered protein. However, in diseased cells α-synuclein aggregates into oligomeric and fibrillar amyloid species, which can be detected using aggregate-specific antibodies. Here we investigate the aggregate specificity of rabbit monoclonal MJFR14-6-4-2 antibodies, preferentially recognizing aggregated α-synuclein species. We conclude that partial masking of epitope in unstructured monomer in combination with a high local concentration of epitopes instead of distinct epitope conformation is the main reason for apparent selectivity towards various aggregates, including oligomers, fibrils, and artificial virus-like particle constructs bearing multiple copies of the MJFR14-6-4-2 epitope. Based on the structural insight, we were able to express mutant α-synuclein that when fibrillated are unable to bind MJFR14-6-4-2. Using these “stealth” fibrils as a tool for seeding cellular α-synuclein aggregation, provides superior signal/noise ratio for detection of cellular α-synuclein aggregates by MJFR14-6-4-2 immunocytochemistry. Our data provide a molecular level understanding of specific recognition of toxic amyloid oligomers, which is critical for the development of inhibitors against synucleinopathies.

Structural basis of epitope recognition by anti-alpha synuclein antibodies MJFR14-6-4-2