Suppression, disaggregation, and modulation of γ-Synuclein fibrillation pathway by green tea polyphenol EGCG

Oligomerization of gamma-Synuclein is known to have implications for both neurodegeneration and cancer. Although it is known to co-exist with the fibrillar deposits of alpha-Synuclein (Lewy bodies), a hallmark in Parkinson's disease (PD), the effect of potential therapeutic modulators on the fibrillation pathway of gamma-Syn remains unexplored. By a combined use of various biophysical tools and cytotoxicity assays we demonstrate that the flavonoid epigallocatechin-3-gallate (EGCG) significantly suppresses gamma-Syn fibrillation by affecting its nucleation and binds with the unstructured, nucleus forming oligomers of gamma-Syn to modulate the pathway to form alpha-helical containing higher-order oligomers (similar to 158 kDa and similar to 670 kDa) that are SDS-resistant and conformationally restrained in nature. Seeding studies reveal that these oligomers although on-pathway in nature, are kinetically retarded and rate-limiting species that slows down fibril elongation. We observe that EGCG also disaggregates the protofibrils and mature gamma-Syn fibrils into similar SDS-resistant oligomers. Steady-state and time-resolved fluorescence spectroscopy and isothermal titration calorimetry (ITC) reveal a weak non-covalent interaction between EGCG and gamma-Syn with the dissociation constant in the mM range (K-d similar to 2-10 mM). Interestingly, while EGCG-generated oligomers completely rescue the breast cancer (MCF-7) cells from gamma-Syn toxicity, it reduces the viability of neuroblastoma (SH-SY5Y) cells. However, the disaggregated oligomers of gamma-Syn are more toxic than the disaggregated fibrils for MCF-7cells. These findings throw light on EGCG-mediated modulation of gamma-Syn fibrillation and suggest that investigation on the effects of such modulators on gamma-Syn fibrillation is critical in identifying effective therapeutic strategies using small molecule modulators of synucleopathies.