Polymorphic Alpha-Synuclein Oligomers: Characterization and Differential Detection with Novel Corresponding Antibodies

Abstract

The pathological hallmark of many neurodegenerative diseases is the accumulation of characteristic proteinaceous aggregates. Parkinson's disease and dementia with Lewy bodies can be characterized as synucleinopathies due to the abnormal accumulation of the protein alpha-synuclein (α-Syn). Studies have shown amyloidogenic proteins such as α-Syn and tau can exist as polymorphic aggregates, a theory widely studied mostly in their fibrillar morphology. It is now well understood that an intermediate state of aggregates, oligomers, are the most toxic species. We have shown α-Syn, when modified by different physiological inducers, result in distinct oligomeric conformations of α-Syn. Polymorphic α-Syn oligomers exhibit distinct properties such as aggregate size, conformation, and differentially interact with tau. In this study, we confirm α-Syn oligomeric polymorphs furthermore using in-house novel α-Syn toxic conformation monoclonal antibodies (SynTCs). It is unclear the biological relevance of α-Syn oligomeric polymorphisms. Utilizing a combination of biochemical, biophysical, and cell-based assays, we characterize α-Syn oligomeric polymorphs. We found α-Syn oligomeric polymorphs exhibit distinct immunoreactivity and SynTCs exhibit differential selectivity and binding affinity for α-Syn species. Isothermal titration calorimetry experiments suggest distinct α-Syn:SynTC binding enthalpies in a species-specific manner. Additionally, we found SynTCs differentially reduce α-Syn oligomeric polymorph-mediated neurotoxicity and propagation in primary cortical neurons in a polymorph-specific manner. These studies demonstrate the biological significance of polymorphic α-Syn oligomers along with the importance of polymorph-specific antibodies that target toxic α-Syn aggregates. Monoclonal antibodies that can target the conformational heterogeneity of α-Syn oligomeric species and reduce their mediated toxicity have promising immunotherapeutic potential.

Keywords: Alpha-synuclein; Amyloid; Monoclonal antibodies; Neurotoxicity; Oligomers; Protein aggregation.

Fig. 1

Schematic of experimental overview. Three different α-Syn oligomeric polymorphs were systematically characterized biochemically and biophysically and evaluated by cellular spreading. In addition to traditional methods, we used 3 novel α-Syn toxic conformation antibodies

Fig. 2

Biochemical characterization of α-Syn oligomeric polymorphs. α-Syn oligomeric polymorphs were characterized by dot blotting, quantification provided in supplementary Fig. 1 (top), western blotting (middle), and indirect enzyme-linked immunosorbent assay (ELISA) (bottom). α-Syn monomer, different α-Syn oligomer preparations, α-Syn fibrils, and amyloidogenic proteins, tau and amyloid β, were characterized with primary antibodies: SynTC1 (a, e, i), SynTC2 (b, f, j), SynTC3 (c, g, k). Results revealed the selectivity of the SynTCs for α-synuclein, confirmed by total Syn antibody, LB509 (d, h, i). Analyses confirm differences in immunoreactivity for α-Syn oligomeric conformers. Indirect ELISA: data represented as mean ± SD

Fig. 3

Isothermal titration calorimetry (ITC) confirms distinct binding profiles of SynTCs and α-Syn oligomers. Integrated binding curves of the isothermal titration calorimetry (ITC) experiment of 2 μM α-Syn oligomer titrated with 8 μM SynTC at 25 °C. A one set of sites binding model was used for all experiments. Binding curves were fitted with a nonlinear regression model. Thermodynamic and stoichiometric parameters obtained from the fitting of the binding curve are shown

Fig. 4

SynTC immunodepletion of α-Syn oligomeric polymorphs reduces α-Syn seeding and endogenous aggregation. Primary neurons were treated with α-Syn oligomer or α-Syn oligomer preincubated with a SynTC for 30 min at RT. Immunocytochemistry was done following 24-h incubation. Syn10842 (green), total α-Syn polyclonal antibody (rabbit), LB509 (red), total α-Syn monoclonal antibody (mouse), and BIIITubulin (magenta), neuronal marker, were used to stain cells. White arrowheads indicate colocalization of Anti-Syn10842 and Anti-LB509 in neurons. Quantification of average fluorescence intensity of α-Syn aggregates calculated from five different regions of interest (ROI). Bar graph showed as mean ± SD (****p < 0.0001). Scale bar = 10 µm

Fig. 5

α-Syn oligomeric polymorphs are cytotoxic and are differentially neutralized by SynTCs. SynTCs inhibit cytotoxicity exerted by α-synuclein oligomers in human neuroblastoma SH-SY5Y cells (a–f) and primary cortical neurons isolated from mice overexpressing human α-Syn (g–l). α-Syn oligomers (0.5uM) were preincubated with a SynTC (2 uM) at a ratio of 1:4 for 30 min at RT and added to the cells for 24 h. Cytotoxicity was analyzed by LDH (gray bars) and MTS (blue bars) cell-based assays. Bars and error bars represent means and standard deviations, respectively (***P < 0.001).