Complex of EGCG with Cu(II) Suppresses Amyloid Aggregation and Cu(II)-Induced Cytotoxicity of α-Synuclein

Abstract

Accumulation of α-synuclein (α-Syn) is a remarkable pathology for Parkinson's disease (PD), therefore clearing it is possibly a promising strategy for treating PD. Aberrant copper (Cu(II)) homeostasis and oxidative stress play critical roles in the abnormal aggregation of α-Syn in the progress of PD. It is reported that the polyphenol (-)-epi-gallocatechin gallate (EGCG) can inhibit α-Syn fibrillation and aggregation, disaggregate α-Syn mature fibrils, as well as protect α-Syn overexpressed-PC12 cells against damage. Also, previous studies have reported that EGCG can chelate many divalent metal ions. What we investigate here is whether EGCG can interfere with the Cu(II) induced fibrillation of α-Syn and protect the cell viability. In this work, on a molecular and cellulaire basis, we demonstrated that EGCG can form a Cu(II)/EGCG complex, leading to the inhibition of Cu(II)-induced conformation transition of α-Syn from random coil to β-sheet, which is a dominant structure in α-Syn fibrils and aggregates. Moreover, we found that the mixture of Cu(II) and EGCG in a molar ratio from 0.5 to 2 can efficiently inhibit this process. Furthermore, we demonstrated that in the α-Syn transduced-PC12 cells, EGCG can inhibit the overexpression and fibrillation of α-Syn in the cells, and reduce Cu(II)-induced reactive oxygen species (ROS), protecting the cells against Cu(II)-mediated toxicity.

Keywords: EGCG; Parkinson’s disease; complex; copper; α-synuclein.

Figure 1

(A) Sequences of wild-type human α-Syn. (B) Structure of EGCG, and the potential Cu(II) binding sites in EGCG [19].

Figure 2

ThT fluorescence of α-Syn with Cu(II) and EGCG presence. (A) The fibrillation kinetics of 50 μM α-Syn in the absence or presence of 10, 20, and 40 μM Cu(II), respectively. (B) The fibrillation kinetics of 50 μM α-Syn incubated with Cu(II) concentration of 0, 10, 20, and 40 μM and EGCG constant concentration of 20 μM. The maximum fluorescence intensity of control group (50 μM α-Syn) was normalized as 1.0, the intensities for other groups were referred to it.

Figure 3

CD spectra of 50 μM α-Syn incubated in the absence (A) or presence of (B) 40 μM Cu(II), (C) 20 μM EGCG, (D) the mixture of 20 μM EGCG and 40 μM Cu(II) after incubation for 0 h (line in black), 10 h (dash in red), and 29 h (dot in blue).

Figure 4

TEM analysis of morphologies of α-Syn aggregates. 50 μM α-Syn incubated alone for 0 h (A), 29 h (B). 50 μM α-Syn incubated in the presence of (C) 20 μM Cu(II), (D) 40 μM Cu(II), (E) 20 μM EGCG, (F) the mixture of 10 μM Cu(II) and 20 μM EGCG, (G) the mixture of 20 μM Cu(II) and 20 μM EGCG, and (H) a mixture of 40 μM Cu(II) and 20 μM EGCG, respectively.

Figure 5

¹H-NMR of α-Syn treated with Cu(II), EGCG, or their mixture. (A) The aromatic region of α-Syn within 6.55 and 8.65 ppm; (B) high field region of α-Syn and EGCG within 0.55 and 3.15 ppm. Samples are 50 μM α-Syn incubated for 0 h (1) and 29 h (2) alone, in the presence of 40 μM Cu(II) (3), 20 μM EGCG (4), the mixture of 10 (5), 20 (6), 40 (7) μM Cu(II) with 20 μM EGCG, respectively, for 58 h in PBS buffer (20 mM, pH 7.4).

Figure 6

Western blot analyses for the α-Syn expression in the transduced PC12cells. The β-actin was used as an endogenous quantitative reference. Cells were treated with (A) Cu(II) (0, 0.01, 0.1, 1, 10, and 100 μM), (B) EGCG (0, 0.01, 0.1, 1, 10, and 50 μM), and (C) the mixture of 10 μM Cu(II) with EGCG in ratio of Cu(II)/EGCG as 10:1, 2:1, 1:1, 1:2, and 1:3 for 48 h, respectively. Error bars = SD, n = 3; *: p < 0.05, **: p < 0.01, ***: p < 0.001 compared with the control group (protein only).

Figure 7

LSCM images of α-Syn aggregates in the transduced PC12 cells treated without and with 10 μM Cu(II), 10 μM EGCG, or the mixture of 10 μM Cu(II) (symbol of C) with EGCG (symbol of E) in ratio of Cu(II)/EGCG (C:E) as 2:1, 1:1, and 1:2 for 48 h, respectively. α-Syn in the red, α-Syn fibrils in the ThT green, and the cell nuclei in the DAPI blue. Scale bars = 200 μm. The pictures shown are representative of three individual experiments.

Figure 8

EGCG effect on the cells activity against Cu(II)-induced cytotoxicity in the transduced PC12 cells. (A) FACS analysis of the cells treated without (1) (control) and with 10 μM EGCG (2), 10 μM Cu(II) (3), the mixture of 10 μM Cu(II) with 5 (4), 10 (5), and 20 (6) μM EGCG for 48 h, respectively. (B) The cell apoptotic percentage in D2 and D4 phase together with FACS determined by PI and Annexin V-FITC fluorescence intensity upon various indicated treatments. Error bars = SD, n = 3; *: p < 0.05, ***: p < 0.001 vs. control group; ###: p < 0.001 vs. [Cu(II)] = 10 μM.

Figure 9

ROS in the α-Syn transduced PC12cells treated by H₂O₂ for 15 min, Cu(II), and EGCG for 24 h. Error bars = SD, n = 3; ***: p < 0.001 vs. control group; ##: p < 0.01, ###: p < 0.001 vs. [Cu(II)] = 10 μM.