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.

Primary Nucleation of Polymorphic α-Synuclein Dimers Depends on Copper Concentrations and Definite Copper-Binding Site

“…The primary nucleation of AS and the total aggregation process of AS are affected by the presence of copper ions [ 9 , 11 , 12 , 13 , 14 , 15 ]. It is well-known that AS fibrils and oligomers are polymorphic [ 26 , 27 , 28 , 31 , 32 ]. The effect of copper ions on polymorphic AS fibrils has been investigated at the molecular level [ 19 ].…”

“…Obviously, polymorphic AS fibrils are derived from polymorphic AS oligomers; thus, it is expected that distinct copper-binding sites also increase polymorphic AS oligomers. Indeed, recently, it has been reported that AS oligomers are polymorphic [ 31 , 32 ]. To date, the copper-binding sites in polymorphic AS oligomers have not been investigated either by experimental or by computational methods.…”

Flanking regions, amyloid cores, and polymorphism: the potential interplay underlying structural diversity

“…Considering our growing understanding of polymorphic amyloid structures, it seems likely that antibodies targeting generic aggregates of amyloid proteins will continue to perform poorly in therapeutic applications. Accordingly, focus has shifted to investigating conformation-specific antibodies that target polymorphic structures ( 302 , 303 , 304 , 305 , 306 , 307 ). Exciting results from in vitro experiments discussed thus far suggest that the conformational ensemble of monomers can be modulated by various additives and environmental condition ( 308 , 309 ).…”