Cell-Free and In Vivo Characterization of the Inhibitory Activity of Lavado Cocoa Flavanols on the Amyloid Protein Ataxin-3: Toward New Approaches against Spinocerebellar Ataxia Type 3

Abstract

Spinocerebellar ataxia type 3 (SCA3) is a neurodegenerative disorder characterized by ataxia and other neurological manifestations, with a poor prognosis and a lack of effective therapies. The amyloid aggregation of the ataxin-3 protein is a hallmark of SCA3 and one of the main biochemical events prompting its onset, making it a prominent target for the development of preventive and therapeutic interventions. Here, we tested the efficacy of an aqueous Lavado cocoa extract and its polyphenolic components against ataxin-3 aggregation and neurotoxicity. The combination of biochemical assays and atomic force microscopy morphological analysis provided clear evidence of cocoa flavanols' ability to hinder ATX3 amyloid aggregation through direct physical interaction, as assessed by NMR spectroscopy. The chemical identity of the flavanols was investigated by ultraperformance liquid chromatography-high-resolution mass spectrometry. The use of the preclinical model Caenorhabditis elegans allowed us to demonstrate cocoa flavanols' ability to ameliorate ataxic phenotypes in vivo. To the best of our knowledge, Lavado cocoa is the first natural source whose extract is able to directly interfere with ATX3 aggregation, leading to the formation of off-pathway species.

Keywords: Caenorhabditis elegans; Lavado cocoa; NMR; UPLC-HR-MS; ataxin-3 protein (ATX3); flavanols; spinocerebellar ataxia type 3 (SCA3).

Figure 1

UPLC-HR-MS analysis of Lavado cocoa extracts. Base peak chromatograms obtained under the negative ionization mode (A) of total extract (1) and its related polyphenol-enriched fraction (2). Structures of major compounds identified in Lavado cocoa extract and its related polyphenol-enriched fraction (B).

Figure 2

Analysis of the Lavado cocoa extract effect on JD aggregation in cell-free system. (A) Effect of Lavado cocoa extract on JD evaluated by the ThT fluorescence assay. Different concentrations of Lavado cocoa extract (0, 0.1, 0.2, 0.5, and 1 mg/mL) were incubated with JD (50 μM) at 37 °C, and ThT fluorescence was monitored for 24 h. The data represents the mean ± standard error of the maximum fluorescence values reached at each concentration after 24 h, subtracted from the fluorescence of the related control (ThT and cocoa extract), and normalized to untreated JD (B,C). Solubility assay of JD incubated in the presence or absence of Lavado cocoa extract. Aliquots of 50 μM JD were incubated in phosphate-buffered saline (PBS) at 37 °C with different concentrations of Lavado cocoa extract (0, 0.25, 1, and 2.5 mg/mL). The soluble fractions obtained by centrifugation at different times (0, 2, 4, 6, 24, 48, and 72 h) were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) (14%), stained with EZBlue gel staining solution, and scanned at 700 nm with the Odyssey Fc System [LI-COR; (B)]. The plot represents the mean ± standard error of densitometric analysis of monomeric JD expressed as a percentage of protein amount at t = 0 h for each concentration (C). Significant differences were assessed by a 2-way factorial analysis of variance (2-way ANOVA), followed by Dunnett’s multiple comparisons test. Arrows indicate SDS-resistant aggregated species. All data were derived from at least three independent experiments. *P < 0.05; **P < 0.01; and ****P < 0.0001.

Figure 3

Cell-free effects of Lavado cocoa polyphenol-enriched fraction on JD aggregation. (A) Effect of Lavado cocoa polyphenol-enriched fraction on JD evaluated by a ThT fluorescence assay. JD (50 μM) protein was incubated with different concentrations (0, 0.01, 0.025, 0.05, and 0.1 mg/mL) of polyphenolic fraction at 37 °C. The ThT fluorescence was measured for 24 h and the graph represents the mean ± standard error of the maximum fluorescence values reached at each concentration, subtracted from the fluorescence of the related control (ThT and polyphenolic fraction), and normalized to JD. (B,C) Solubility assay of JD incubated with or without the polyphenol-enriched fraction. Different concentrations of PF (0, 0.025, 0.1, and 0.25 mg/mL) were incubated with 50 μM JD in PBS at 37 °C. At different times (0, 2, 4, 6, 24, 48, and 72 h), samples were centrifugated, and the soluble fractions were subjected to SDS-PAGE (14%), stained with EZBlue gel staining solution, and scanned at 700 nm with the Odyssey Fc System (LI-COR; B). Bars represent the mean ± standard error of densitometric analysis of monomeric JD expressed as a percentage of protein amount at t = 0 h for each concentration (C). Significant differences were assessed by a 2-way factorial analysis of variance (2-way ANOVA), followed by Dunnett’s multiple comparisons test. Arrows indicate SDS-resistant aggregated species. All data were derived from at least three independent experiments. *P < 0.05; **P < 0.01; ****P < 0.0001.

Figure 4

Cell-free effects of the Lavado cocoa polyphenolic-enriched fraction on ATX3Q55 aggregation. (A) Effect of the polyphenol-enriched fraction on ATX3Q55 evaluated by a ThT fluorescence assay. Different concentrations of polyphenol-enriched fraction (0, 0.025, 0.1, and 0.25 mg/mL) were incubated with ATX3Q55 (25 μM) at 37 °C, and ThT fluorescence was measured for 24 h. The means ± standard error of the maximum fluorescence values reached for each concentration at 24 h subtracted from the fluorescence value of the related control (ThT and polyphenolic fraction) are normalized to ATX3Q55. (B,C) Solubility assay of ATX3Q55 incubated in the presence of a polyphenol-enriched fraction. Aliquots of 25 μM ATX3Q55 were incubated in PBS with different concentrations of polyphenol-enriched fraction (0, 0.025, 0.1, 0.25, and 0.5 mg/mL) at 37 °C. After centrifugation at different time points (0, 2, 4, 6, 24, 48, and 72 h), the soluble fractions were obtained and analyzed by SDS-PAGE (12%). Gels were stained with EZBlue gel staining solution and scanned at 700 nm with an Odyssey Fc System (LI-COR; B). The densitometric analysis of monomeric ATX3Q55 protein was plotted and represents the mean ± standard error as a percentage of the protein amount at t = 0 h for each concentration (C). For the statistical analysis, significant differences were assessed by a 2-way factorial analysis of variance (2-way ANOVA), followed by Dunnett’s multiple comparisons test. Arrows indicate SDS-resistant aggregated species. All data were derived from at least three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. (D) Atomic force microscopy (AFM) morphological analysis of the ATX3Q55 aggregates. AFM images were acquired on a sample containing 25 μM ATX3Q55 incubated in the absence (1–4) or the presence (5–8) of the polyphenol-enriched fraction (0.5 mg/mL) for different times (0, 2, 4, 6, and 24 h).

Figure 5

STD NMR characterization of Lavado cocoa polyphenols’ interaction with ATX3Q55 protein. ¹H NMR (A) and STDD (B) spectra of a mixture of Lavado cocoa polyphenol-enriched fraction (10 mg/mL) and ATX3Q55 protein (7 μM) in PBS (10% D₂O), pH 7.2. STD spectra were acquired with 1024 scans and a 2 s saturation time at 600 MHz, 25 °C. An amplification factor of 5 (5×) of the aromatic regions was used to optimize the visualization of the signals of interest in each spectrum.

Figure 6

Polyphenol fraction effect on ATX3 transgenic C. elegans lifespan and motility. (A1-2-3) Effect of Lavado cocoa polyphenol-enriched fraction on wild-type and ataxic C. elegans strains survival. One-day synchronized adult worms were placed on a plate seeded with heat-killed OP50 in the presence or absence of 0.5 mg/mL of a polyphenol-enriched fraction at 25 °C. Nematodes were transferred daily on a new plate, and the number of alive was reported until all were dead. Representative Kaplan–Meier survival curves of AT3Q17-GFP (1) and AT3Q130-GFP (2) treated and not treated animals were reported. A statistical analysis was reported in panel 3. (B1-2) Effect of the polyphenol-enriched fraction on wild-type (1) and ataxic C. elegans strains (2) motility. One-day synchronized adult worms were placed on a plate seeded with heat-killed OP50 in the presence or absence of 0.5 mg/mL of the polyphenol-enriched fraction at 25 °C. Worms were transferred daily on a new plate, and body bends were counted for 20 s after 24, 48, 72, and 96 h of treatment. Data are expressed as the percentage of motility increase at time 0 h. Error bars represent standard errors. Each test was repeated at least four times, and for each treatment, at least 30 worms were used. Significant differences were assessed by a 2-way factorial analysis of variance (2-way ANOVA), followed by Bonferroni’s multiple comparisons test *P < 0.05; **P < 0.01; ****P < 0.0001; and #P < 0.00001.