EGCG-mediated Protection of the Membrane Disruption and Cytotoxicity Caused by the 'Active Oligomer' of α-Synuclein

Abstract

(-)-Epigallocatechin gallate (EGCG), the major component of green tea, has been re-evaluated with α-synuclein (αS), a pathological constituent of Parkinson's disease, to elaborate its therapeutic value. EGCG has been demonstrated to not only induce the off-pathway 'compact' oligomers of αS as suggested previously, but also drastically enhance the amyloid fibril formation of αS. Considering that the EGCG-induced amyloid fibrils could be a product of on-pathway SDS-sensitive 'transient' oligomers, the polyphenol effect on the transient 'active' oligomers (AOs) was investigated. By facilitating the fibril formation and thus eliminating the toxic AOs, EGCG was shown to suppress the membrane disrupting radiating amyloid fibril formation on the surface of liposomal membranes and thus protect the cells which could be readily affected by AOs. Taken together, EGCG has been suggested to exhibit its protective effect against the αS-mediated cytotoxicity by not only producing the off-pathway 'compact' oligomers, but also facilitating the conversion of 'active' oligomers into amyloid fibrils.

Figure 1

Analyses of molecular interaction between αS and EGCG. (A) Time-resolved SDS-PAGE analyses of the αS fibrillation carried out in the absence or presence of EGCG. The SDS-resistant aggregates of αS were revealed as the protein bands indicated with black arrows. Full-length gels are presented in Supplementary Information Figure 6. (B) Fibrillations of αS (70 μM) in the presence of various concentrations of EGCG at molar ratios indicated were monitored with Th-T binding fluorescence assay. (C) TEM images of αS amyloid fibrils prepared without and with EGCG. Scale bars represent 0.5 μm. αS and EGCG were co-incubated for 130 hr at 37 °C with agitation. (D) Interference of EGCG with the Th-T binding fluorescence of amyloid fibrils. The Th-T treated amyloid fibrils were co-incubated with various concentrations of EGCG (0 μM for black dots, 70 μM for red dots, 210 μM for green dots, 350 μM for blue dots) for 5 min. Inset shows the Th-T binding fluorescence intensity monitored at 482 nm in the presence of various EGCG concentrations.

Figure 2

Spectroscopic evaluation of molecular interaction between αS and EGCG with intrinsic fluorescence, CD spectroscopy, and FTIR spectroscopy. (A) Dissociation constant between αS and EGCG assessed with the intrinsic fluorescence of tyrosine residues of αS. Differences in the tyrosine intrinsic fluorescence emitted at 308 nm in the presence (Ii) and absence (Io) of EGCG were plotted as a function of EGCG concentration. Dissociation constant (K_d) of 100 μM was calculated from a double-reciprocal plot of the saturation curve (Inset). (B) Circular dichroism (CD) spectra of the amyloid fibrils of αS prepared without (black dots) and with (red dots) EGCG following co-incubation for 130 hr at 37 °C with agitation. (C) FTIR spectra and their second derivative spectra of the amyloid fibrils of αS prepared without (left) and with (right) EGCG after 96hr incubation at 37 °C with agitation. The FTIR absorption spectra have been deconvolved according to the peaks found in the second derivative spectra.

Figure 3

Co-existence of the two distinctive types of αS aggregates produced by EGCG. (A) The oligomers (yellow arrow heads) and amyloid fibrils (red or white arrows) of αS (70 μM) obtained after incubation with and without EGCG (350 μM) at 37 °C for 24 hr with agitation were revealed with TEM (upper) and AFM (lower). Scale bars represent 200 nm. (B) Separation of the oligomers and amyloid fibrils with a size exclusion chromatography (Sephacryl S-200). (C) TEM images of the aggregates present in the fractions numbered 15 and 23 from the chromatography. The granular forms of oligomeric species are indicated with black arrows. Scale bars indicate 0.5 μm. (D) SDS-PAGE analysis of the fractions from the size exclusion chromatography. The gels were stained with CBB. The SDS-resistant aggregates of αS were revealed as the high molecular weight protein bands indicated by black arrows. (E) The αS aggregation processes occurring in the absence (upper) and presence (lower) of EGCG at 1:5 molar ratio were followed with the images of TEM and AFM. Scale bars represent 200 nm.

Figure 4

EGCG effects on the ‘active’ oligomers (AOs) of αS. (A) Production of CAFs from AOs (70 μM) via the repetitive centrifugal membrane filtration in the presence or absence of EGCG (350 μM) was assessed with TEM. Scale bars represent 0.5 μm. (B) Membrane disruption by AOs. Following the pre-incubation of AOs (70 μM) with EGCG at various molar ratios for 3 hr at 37 °C, the DOPC-liposomes were treated with the AOs for 0 min, 15 min, and 30 min. The membrane disruption and RAF formation were examined with TEM. Percentages of the disrupted liposomes are indicated. Scale bars represent 200 nm.

Figure 5

EGCG effect on the cytotoxicity caused by AOs. (A) Cytotoxicity caused by AOs. SH-SY5Y cells were treated with AOs at various concentrations for 4 hr at 37 °C. The cell viability was assessed with colorimetric MTS assay. (B) Cell viability of SH-SY5Y cells after 4 hr incubation with the AOs (7 μM) pre-treated with EGCG at various concentrations. (C) The cells attached on a glass slide were treated with AOs at a high concentration of 70 μM for 10 min at room temperature. The arrows indicate the morphologically altered cells on the surface. (D) Cell detachment from a glass surface. After treating SH-SY5Y cells with the AOs (70 μM) pre-treated with and without EGCG (350 μM) for 3 hr, the cells detached from the surface before and after the 10 min incubation were counted and compared. *and **indicate t-test with p ≤ 0.05 and p ≤ 0.01, respectively, between the groups compared.