EGCG remodels mature alpha-synuclein and amyloid-beta fibrils and reduces cellular toxicity

Abstract

Protein misfolding and formation of beta-sheet-rich amyloid fibrils or aggregates is related to cellular toxicity and decay in various human disorders including Alzheimer's and Parkinson's disease. Recently, we demonstrated that the polyphenol (-)-epi-gallocatechine gallate (EGCG) inhibits alpha-synuclein and amyloid-beta fibrillogenesis. It associates with natively unfolded polypeptides and promotes the self-assembly of unstructured oligomers of a new type. Whether EGCG disassembles preformed amyloid fibrils, however, remained unclear. Here, we show that EGCG has the ability to convert large, mature alpha-synuclein and amyloid-beta fibrils into smaller, amorphous protein aggregates that are nontoxic to mammalian cells. Mechanistic studies revealed that the compound directly binds to beta-sheet-rich aggregates and mediates the conformational change without their disassembly into monomers or small diffusible oligomers. These findings suggest that EGCG is a potent remodeling agent of mature amyloid fibrils.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

EGCG remodels αS fibrils into benign amorphous protein assemblies. (A) EGCG (50 μM) induced remodeling of fibrillar αS-His (50 μM) into spherical assemblies monitored by negative-stain EM and intermittent contact mode AFM. Scale bars = 200 nm (EM), 500 nm (AFM). 1st derivative (dz/dt) scans are shown for AFM to enhance contrast. (B) Loss of β-sheet structure of αS fibrils (50 μM) after incubation with EGCG. Fibrils were sonicated for 1 h before CD measurement. (C) Loss of ThT fluorescence after incubation of αS fibrils (50 μM) with EGCG (50 μM) under constant agitation at 37 °C was monitored for 72 h; error bars = SD, n = 3. (D) Time-dependent loss of seeding capacity of αS fibrils after incubation with EGCG. Fibrils (10% wt/wt) were preincubated with or without EGCG for the indicated times, sonicated for 1 h, and added to fresh monomeric αS (50 μM) in phosphate buffer. Seeded fibril formation was monitored by ThT fluorescence for 90 h. (E) Nondenaturing FRA of NP-40 resistant (total) aggregates after incubation of αS fibrils (50 μM) with EGCG (50 μM) for 10 min. EGCG-specific staining by NBT, and αS staining by anti-αS antibody shows rapid binding of EGCG to αS fibrils.

Fig. 2.

Direct remodeling of αS fibrils. (A) Red and green fluorescently labeled fibrils of αS formed for 7 d (total monomer concentration 50 μM, 2.5% αS-A488 or 2.5% αS-Cy5) were mixed and incubated with equimolar EGCG for 24 h. Fluorescence microscopy after incubation shows aggregates predominantly labeled by either the green or the red fluorophores, scale bar = 1 μm. (B) Pearson coefficients from colocalization studies of mixed green/red fibril remodeling and of green/red monomer coaggregation for 24 h (30 images, 10–50 aggregates/image). (C) EGCG binding precedes remodeling. Fibrillar αS (50 μM) was incubated with EGCG (50 μM, 250 μM) for 10 min in TBS. Aggregates were pelleted for 20 min at 200,000 × g, washed, resuspended in TBS and incubated at 37 °C for 10 min–6 h. SDS insoluble αS was quantified by anti-αS antibody (11) staining, aggregate-bound EGCG was detected by NBT staining after FRA. (D) Quantitative analysis of EGCG binding affinity. Fibrillar αS (100 nM) was incubated with EGCG (20 nM–10 μM) overnight at 37 °C in PBS. Aggregate-bound EGCG was detected by NBT staining after FRA and quantified densitometrically to determine EGCG binding affinities. Unspecific NBT membrane staining was subtracted and EGCG staining was normalized and fitted by a single binding site model; Kd = 100 ± 20 nM, mean ± SD, n = 4.

Fig. 3.

EGCG remodels and detoxifies cellular αS aggregates. (A) Fibrillar αS (1 μM) aggregates were introduced into HEK-293 cells expressing wild-type αS (HEK-293 wt αS). After 6 h cells were washed and incubated for 1–3 d in the absence or presence of 20 μM EGCG. NP-40 resistant (total) aggregates as well as SDS-resistant aggregates were quantified by FRA after cell lysis using anti-αS antibody. (B) Cytotoxicity of αS aggregates to HEK-293 wt αS cells was assayed by LDH-release. LDH signals were normalized to cells neither treated with αS aggregates nor treated with EGCG and incubated for 3 d. Values represent means ± SD, n = 8; *** P < 0.0005. (C) Reduction in metabolic activity of PC12 cells after addition of αS fibrils. Fibrils (100 μM) were incubated with different amounts of EGCG for 24 h at 37 °C and diluted into the cell culture media at the indicated concentrations. MTT reduction was normalized to cells neither treated with αS aggregates nor treated with EGCG. Values represent means ± SD (n = 3); * P < 0.01, ** P < 0.001.

Fig. 4.

EGCG remodels Aβ42 fibrils and oligomers into benign SDS-resistant structures. (A) Fibrillar Aβ42 (15 μM) was incubated with EGCG (15 μM) for up to 24 h in PBS at 37 °C. EGCG-induced remodeling of Aβ42 fibrils into spherical assemblies was monitored by atomic force microscopy. (B) Fibrillar Aβ42 (equivalent 15 μM monomer) was incubated in the absence (Black) or presence of EGCG (15 μM, Blue; 75 μM Red) in PBS at 37 °C. Loss of ThT fluorescence was monitored at time intervals of 10 min, preceded by 5 s shaking each. (C) Immunofluorescence microscopy shows loss of intracellular Aβ aggregates (Green) in 7PA2 cells after ECGG (10 μM) treatment for 3 d. (phalloidin, Red; DAPI, Blue). (D) LDH release in 7PA2 cells after introduction of Aβ42 aggregates. The assay was performed as described in Fig. 3B. Values represent means ± SD, n = 8; *** P < 0.0005. (E) Aβ42 aggregates (15 μM) were incubated with the indicated amounts of EGCG for 24 h at 37 °C. Aggregates were then were diluted into the cell culture media at the indicated concentrations and added to PC12 cells. Metabolic activity was monitored after 3 d by MTT reduction. Values represent means ± SD (n = 3) normalized to cells neither treated with Aβ nor with EGCG; * P < 0.01, ** P < 0.001.