Cyclic-NDGA effectively inhibits human gamma-synuclein fibrillation, forms off-pathway species, and disintegrates pre-formed mature fibrils.

Abstract

Parkinson's disease is one of the various diseases resulting from protein misfolding, aggregation and fibrillation and is characterized by Lewy body deposits which contain the protein α-synuclein (α-syn) as their major component. γ-synuclein (γ-syn), one of the three synucleins is also present in the Lewy bodies along with α-syn and is also implicated in various types of cancers especially breast cancer. It is known to seed the fibrillation of α-syn after its oxidation at methionine residue leading to synucleinopathy. Despite its involvement in synucleinopathy, the search for small molecule therapeutics against γ-syn fibrillation remains largely unexplored. We have investigated the modulatory properties of cyclic-Nordihydroguaiaretic acid (cNDGA), a plant polyphenol, on the structural and aggregational properties of human γ-syn employing various techniques namely ThT fluorescence, Rayleigh Light scattering, ANS binding, far-UV CD and FTIR spectroscopy, atomic force microscopy and MTT assay. cNDGA inhibits the fibrillation of γ-syn and unlike mature control fibrils, γ-syn forms large aggregates with no ThT binding property, negligible hydrophobicity, with lesser β-sheet content and reduced cytotoxicity when incubated with cNDGA. These species formed as a result of weak interaction of γ-syn with cNDGA (Kd ∼10−5M) as investigated by isothermal titration calorimetry are off pathway in nature suggested by seeding experiments. Increasing concentration of cNDGA resulted in an increased recovery of monomeric γ-syn as shown by SDS-PAGE and Native-PAGE. cNDGA was most effective in suppression of fibrillation when added at the beginning of the fibrillation kinetics and was also capable of disintegrating the pre-formed mature fibrils. It can be concluded that cNDGA effectively suppresses γ-syn fibrillation and forms species that are less toxic in nature. These findings could be further explored for the use of cNDGA and similar flavonoids as therapeutics against γ-synucleinopathies.