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

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.

MJF-14 proximity ligation assay detects early non-inclusion alpha-synuclein pathology with enhanced specificity and sensitivity

“…This allows a confident examination of endogenous a-synuclein aggregation without the risk of cross-detection of the exogenous PFFs. As the stealth PFFs were also engineered to be S129A-mutated, and thereby non-phosphorylatable at serine-129, pS129-a-synuclein-specific antibodies will also only stain endogenous asynuclein in this setup 48 . Thereby, these tools enable detailed studies into the early aggregation and phosphorylation processes and can be combined with a range of model systems.…”

“…Full-length S129A-mutated human a-synuclein and S129A-mutated human a-synuclein with an added glycine residue at the C-terminal (S129A-a-synuclein-141G) was expressed in BL21(DE3)-competent cells and purified by reverse phase chromatography as previously described [46][47][48] . To prepare pre-formed fibrils (PFFs), soluble monomeric a-synuclein (4 mg/mL) was incubated in PBS at 37°C with 1050 rpm shaking for 72 hours, before collection, characterization, and storage as previously described [48][49][50] . Immediately before use, aliquots of PFFs were quickly thawed, sonicated briefly using a Branson SFX 250 sonifier (0.3 s on, 0.7 s off, 30% power, for 45 pulses).…”

“…In order to test the MJF-14 PLA in a different, likely more physiologically relevant, model, we cultured human cortical neurons in chambered slides and treated them with pre-formed fibrils (PFFs) to induce a-synuclein aggregation. Initial experiments showed a potential detection of the added PFFs by the MJF-14 PLA, especially at early timepoints, and we therefore included the novel stealth PFFs in our setup, invisible to the MJF-14 antibody due to a C-terminally added glycine, as recently described 48 . Thus, cultured neurons were treated with either standard S129A-mutated PFFs or stealth PFFs (S129A-mutated a-synuclein-141G) and fixed at 2 hours or 7 days post treatment (Fig.…”