The Aggregation Conditions Define Whether EGCG is an Inhibitor or Enhancer of α-Synuclein Amyloid Fibril Formation

Abstract

The amyloid fibril formation by α -synuclein is a hallmark of various neurodegenerative disorders, most notably Parkinson's disease. Epigallocatechin gallate (EGCG) has been reported to be an efficient inhibitor of amyloid formation by numerous proteins, among them α -synuclein. Here, we show that this applies only to a small region of the relevant parameter space, in particular to solution conditions where EGCG readily oxidizes, and we find that the oxidation product is a much more potent inhibitor compared to the unmodified EGCG. In addition to its inhibitory effects, EGCG and its oxidation products can under some conditions even accelerate α -synuclein amyloid fibril formation through facilitating its heterogeneous primary nucleation. Furthermore, we show through quantitative seeding experiments that, contrary to previous reports, EGCG is not able to re-model α -synuclein amyloid fibrils into seeding-incompetent structures. Taken together, our results paint a complex picture of EGCG as a compound that can under some conditions inhibit the amyloid fibril formation of α -synuclein, but the inhibitory action is not robust against various physiologically relevant changes in experimental conditions. Our results are important for the development of strategies to identify and characterize promising amyloid inhibitors.

Keywords: EGCG; Parkinson disease; amyloid; inhibition; kinetics; nucleation; seeding.

Figure 1

The effects of different ratios (1:1 and 5:1 with respect to protein) of EGCG and EGCG_ox on the aggregation kinetics of $\alpha$-synuclein at different pH values (pH 3 to pH 7), monitored in high-binding surface plates in the presence (A) and absence (B) of glass beads.

Figure 2

Overview of the effects of EGCG and EGCG_ox on $\alpha$-synuclein aggregation monitored in high-binding surface plates assayed by (A) maximum ThT fluorescence intensity and (B) t₅₀ of the aggregation time course. Filled bars represent aggregation in the presence of glass beads and striped bars in the absence of glass beads. Error bars are standard deviations. The data are normalized to the control of the corresponding condition, i.e., the aggregation in the absence of EGCG or EGCG_ox, the kinetic parameters of which are indicated with the horizontal dashed line at a factor of one. Relative t₅₀ values are only displayed, if any fibril formation is detected by an increase in ThT fluorescence intensity.

Figure 3

The effects of EGCG and EGCG_ox on the aggregation kinetics of $\alpha$-synuclein at different pH values (pH 3 to pH 7) monitored in a non-binding surface plate in the presence (A) and absence (B) of glass beads.

Figure 4

Overview of the effects of EGCG and EGCG_ox on $\alpha$-synuclein aggregation monitored in a non-binding surface plate assayed by (A) maximum ThT fluorescence intensity and (B) t₅₀ of the aggregation. Filled bars represent aggregation in the presence of glass beads and striped bars without glass beads. Error bars are standard deviations. The data are normalized to the control of the corresponding condition, and the comparison is outlined with the dashed line (at 1).

Figure 5

(A) Soluble $\alpha$-synuclein concentration measured in the supernatant after centrifuging the end product of the aggregation reactions in a non-binding surface plate in the presence of glass beads. Radius in nm and concentration in μM of the three replicates of $\alpha$-synuclein at pH 6 (control) (left), concentration in μM (middle), and radius in nm (right) of the end product of the aggregation reactions at pH 4, pH 5, pH 6, and pH 7. The three replicates per condition were combined before centrifugation (except for the control at pH 6, where each replicate sample was analyzed separately; see (A). (B) Amount of protein measurable with the Fluidity One (F1) MDS instrument (supernatant + pellet) in μg in the end-product of $\alpha$-synuclein at pH 7 in a non-binding surface plate without additional glass beads (left). The dotted line indicates the used amount of protein. AFM height images of the control (black frame), $\alpha$-synuclein with EGCG (1:1) (cyan frame) and of $\alpha$-synuclein with EGCG_ox (1:5) (magenta frame). The image scale is 5 × 5 μM. The color range represents the height from −2 to 10 nm (left and middle) and −10 to 25 nm (right).

Figure 6

Time-resolved AFM height images of $\alpha$-synuclein aggregation at pH 4 in a non-binding surface plate without glass beads. The colors of the frame correspond to the conditions (Figure 3): control (black frame), EGCG (1:1) (cyan frame), EGCG (1:5) (green frame), EGCG_ox (1:1) (purple frame), and EGCG_ox (1:5) (magenta frame). The image scale is 5 × 5 μM. The color range of the image represents the height range from −5 to 20 nm.

Figure 7

Aggregation kinetics of $\alpha$-synuclein at pH 4 in a non-binding surface plate under quiescent conditions in the absence of glass beads. The fibril formation was monitored in the presence and absence of EGCG or EGCG_ox and in wells that were pre-treated with EGCG-solutions (A) and the corresponding concentration measurement by Fluidity One after 160 h (B) with AFM height images (D) of the aggregation products of $\alpha$-synuclein (black frame) in the presence of EGCG_ox (1:1) (purple frame) and (1:5) (magenta frame), in the pre-treated wells with EGCG_ox (1:1) (light purple frame) and (1:5) (light magenta frame), and the overview of the three replicates per condition (C). The image scale is 5 × 5 μM. The color range represents the height from −3 to 12 nm.

Figure 8

(A) The effects of EGCG and EGCG_ox on the aggregation kinetics of $\alpha$-synuclein, in particular the growth of fibrils, at different pH values (pH 3 to pH 7) in the presence of 5% seeds monitored in a non-binding surface plate under quiescent conditions and a AFM height image of the sample at pH 7 in presence of EGCG (1:5) and (B) AFM height images of $\alpha$-synuclein in the presence of EGCG_ox (1:5) at different pH values after the aggregation experiment. The image scale is 5 × 5 μM.

Figure 9

(A) The seeding efficiency, expressed in seeding units (s.u., [49]), determined by fitting the kinetics of the 5% seeding experiments with y = 1 − e^−kt after normalization between zero and one. Only the kinetics that showed the shape expected for a strongly seeded aggregation curve [49] were analyzed. (B) The effects of EGCG and EGCG_ox on the aggregation kinetics of $\alpha$-synuclein at different pH values (pH 3 to pH 7) in the presence of 0.5% seeds monitored in a non-binding surface plate under quiescent conditions. (C) The t₅₀ of $\alpha$-synuclein at different pH values (pH 3 to pH 7) in a non-binding surface plate without additional seeds with shaking (black bar), with 0.5% seeds (dark grey bar) and 5% seeds (light grey bar) under quiescent conditions and in the presence of 1:5 EGCG_ox (violet).

Figure 10

(A) The effects of EGCG and EGCG_ox on the seeded aggregation, when the seeds were pre-incubated with stoichiometric amounts of the compound for 2 h at RT before adding them to a 25 μM monomer-solution at pH 5, pH 6, and pH 7 to a final concentration of 5% (in monomer equivalents). The samples, where the fibrils were pre-incubated with the compound, contained still 1.25 μM EGCG or EGCG_ox. (B) 10 μM fibrils at pH 4, pH 6, and pH 7 were incubated in the presence of 10 μM EGCG or EGCG_ox in a non-binding surface plate at 37 °C for over 100 h (left), and then, 50 μM fresh monomer was added (right).

Figure 11

Schematic depiction of the effects of the compound EGCG on $\alpha$-synuclein amyloid fibril formation. (A) EGCG oxidizes at pH 7, whereas it is stable at pH 6 and below. (B) illustrates that $\alpha$-synuclein cannot bind to the non-binding surface of the multi-well plate, while in particular, EGCG_ox can bind to the surface and facilitate the formation of amyloid fibrils. (C) EGCG displayed almost no effect on a strongly seeded aggregation reaction (5% seeds), whereas a weakly seeded aggregation reaction (0.5% seeds) is inhibited more strongly. (D) The compound seems to interact with amyloid fibrils, but was not found to be able to remodel the fibrils into amorphous, seeding-incompetent aggregates. When fresh monomer was added, the fibrils had the same seeding efficiency as the control fibrils.