Suppression and modulation mechanism underlying the flavonoid-induced inhibition of fibrillation of α-synuclein

Abstract

The accumulation of -synuclein (Syn) aggregates is a hallmark of synucleinopathies, including Parkinsons disease (PD). However, the progression from harmless monomeric Syn to misfolded oligomers and fibrillar species remains largely unclear. In this study, we examined the effect of Cyanidin, a naturally occurring neuroprotective compound, on the aggregation properties of -syn using a combination of various biophysical tools. Thioflavin T fluorescence measurements revealed the inhibition of -syn fibrillation in the presence of sub-stoichiometric concentration of Cyanidin, indicating its effect on -syn nucleation. Ultracentrifugation and size exclusion chromatographic analyses demonstrated that increasing concentrations of Cyanidin reduced the conversion of monomers to aggregated forms of -syn. Analysis of the aggregation reaction of -syn based on monomer concentration suggested that Cyanidin reduces the in-vitro conversion of monomers to amyloid nuclei at sub-stoichiometric micromolar concentrations. Cyanidin was observed to bind weakly with the unstructured, monomeric -syn, limiting the nucleation step and modulating the pathway to form conformationally restrained, SDS-resistant -helical higher-order oligomers ([~]670 kDa), which are less-hydrophobic. AFM and TEM images show that Cyanidin also possesses strong fibril disaggregation activity. Furthermore, seeding studies reveal that the higher-order oligomers, which are otherwise stable, destabilise upon sonication and attain seeding capability. Steady-state fluorescence spectroscopy and isothermal titration calorimetry (ITC) reveal weak interactions between Cyanidin and Syn, with a dissociation constant in the mM range. Interestingly, Cyanidin-generated oligomers increase the viability of neuroblastoma (SH-SY5Y) cells, thereby protecting the neuronal cells from degeneration. Our study suggests that stabilization of structured oligomers by small molecule modulators like Cyanidin provides a viable strategy to interfere with Syn fibrillization.