How epigallocatechin gallate can inhibit α-synuclein oligomer toxicity in vitro

Abstract

Oligomeric species of various proteins are linked to the pathogenesis of different neurodegenerative disorders. Consequently, there is intense focus on the discovery of novel inhibitors, e.g. small molecules and antibodies, to inhibit the formation and block the toxicity of oligomers. In Parkinson disease, the protein α-synuclein (αSN) forms cytotoxic oligomers. The flavonoid epigallocatechin gallate (EGCG) has previously been shown to redirect the aggregation of αSN monomers and remodel αSN amyloid fibrils into disordered oligomers. Here, we dissect EGCG's mechanism of action. EGCG inhibits the ability of preformed oligomers to permeabilize vesicles and induce cytotoxicity in a rat brain cell line. However, EGCG does not affect oligomer size distribution or secondary structure. Rather, EGCG immobilizes the C-terminal region and moderately reduces the degree of binding of oligomers to membranes. We interpret our data to mean that the oligomer acts by destabilizing the membrane rather than by direct pore formation. This suggests that reduction (but not complete abolition) of the membrane affinity of the oligomer is sufficient to prevent cytotoxicity.

Keywords: Dynamic Light Scattering; EGCG; Isothermal Titration Calorimetry (ITC); Membrane; Nuclear Magnetic Resonance (NMR); Oligomer; Toxicity; X-ray Scattering; α-Synuclein.

FIGURE 1.

EGCG inhibits oligomer toxicity. A, inhibition of the ability of oligomers to permeabilize membranes at 0.2 μm oligomer (monomer equivalents) as measured with the calcein release assay. Inset, structure of EGCG. B, EGCG inhibits oligomer cytotoxicity. OLN-93 cells were exposed to 5 μm αSN oligomer or monomer and 0–5 μm EGCG for 24 h. Cell viability was measured with the MTT assay and trypan nuclei staining. Both assays are given as triplicates with standard deviation. The curves on A and B are fitted to a simple binding isotherm.

FIGURE 2.

Fluorescence confocal microscopy of the effect of αSN oligomers on DOPG vesicles. A–D show vesicles before (A and C) and after (B and D) disruption by EGCG-free oligomers. E and F show images double-stained with Alexa 633-labeled oligomers (green) preincubated with equimolar amounts of EGCG and subsequently added to vesicles containing encapsulated Alexa 488 (red). G, combines E and F. Bar in E–G is 200 μm.

FIGURE 3.

NMR analysis of oligomer-EGCG interactions. A, ¹H-¹⁵N HSQC spectrum of αSN monomer. B, ¹H-¹⁵N HSQC spectrum of αSN oligomer where the signal contribution from monomeric αSN is subtracted. C, zoomed region of the superimposed spectra of αSN monomer (black) and oligomer where the signal contribution from monomeric αSN is subtracted (red). D, loss of the NMR signals from the αSN oligomer upon addition of different amounts of EGCG (molar ratios in terms of monomer αSN concentrations). a.u., arbitrary units.

FIGURE 4.

TEM images show no effect of EGCG on oligomer structure. A, free oligomers. B, oligomers incubated with EGCG at an EGCG/αSN_monomer molar ratio of 5:1.

FIGURE 5.

EGCG does not induce any major structural changes in the oligomer. A, SEC-refraction index analysis of oligomers incubated alone (black) and with EGCG (red). B, SAXS spectra of oligomers (black) and oligomers preincubated with EGCG (red) with the best fit to data of the models described in the text. For visual comparison, the EGCG data and fit have been displaced down by a factor of 10. C, far-UV CD spectra of monomer, oligomer, and oligomer and EGCG at a molar ratio of 1:1 (□ and gray line).

FIGURE 6.

Inhibition of oligomer-membrane interactions. A, AF4-UV size separation of monomer and oligomer from an oligomer preparation incubated without (black) and with (red) DOPG vesicles. B, as A, but here oligomers have been preincubated with EGCG. mAU, milli-absorption units.

FIGURE 7.

Calorimetric analysis of the interactions between vesicles and either αSN monomer or αSN oligomer, which have been incubated with or without EGCG. A, ITC analysis of the interaction energy between protein samples and DOPG vesicles. B, DSC analysis of the effect of protein samples in the phase transition of DMPG vesicles.

FIGURE 8.

Schematic representation of EGCG inhibition of oligomer toxicity. Illustration courtesy of Simon Lykkemark.