Espresso Coffee Mitigates the Aggregation and Condensation of Alzheimer's Associated Tau Protein

Abstract

Espresso coffee is among the most consumed beverages in the world. Recent studies report a protective activity of the coffee beverage against neurodegenerative disorders such as Alzheimer's disease. Alzheimer's disease belongs to a group of disorders, called tauopathies, which are characterized by the intraneuronal accumulation of the microtubule-associated protein tau in fibrillar aggregates. In this work, we characterized by NMR the molecular composition of the espresso coffee extract and identified its main components. We then demonstrated with in vitro and in cell experiments that the whole coffee extract, caffeine, and genistein have biological properties in preventing aggregation, condensation, and seeding activity of the repeat region of tau. We also identified a set of coffee compounds capable of binding to preformed tau fibrils. These results add insights into the neuroprotective potential of espresso coffee and suggest candidate molecular scaffolds for designing therapies targeting monomeric or fibrillized forms of tau.

Keywords: Alzheimer′s disease; NMR; bioactive molecules; coffee; liquid−liquid phase separation; protein aggregation; tau protein.

Figure 1

Domain organization of full-length tau, tau^FL, and of the shorter construct comprising the four-repeat region, tau^4RD. Red bars indicate the position of hexapeptide motifs known as aggregation nuclei.

Figure 2

(A) ¹H NMR profile of 5 mg/mL lyophilized espresso coffee extract. The downfield spectral region (5–10 ppm) is displayed with 4-fold higher intensity than the highfield region for better visualization. The spectrum has been recorded at 600 MHz and 25 °C. Peak assignments are indicated. Caff: caffeine, Trigo: trigonelline, CGAs: chlorogenic acids, Lac: lactate, and Cho: choline. (B) Molecular structures of coffee-derived molecules analyzed in this study.

Figure 3

Time course of tau conformational transitions. (A–C) ThT fluorescence-based aggregation kinetics curves measured on 50 μM tau^4RD in the presence of coffee extract (A), caffeine (B), or genistein (C). Compound concentrations were 0 (black), 50 (orange), or 400 (red) μg/mL. Measurements were carried out on four replicates and data are reported as mean ± s.d. Solid lines correspond to the best-fit curves determined using an empirical sigmoid function. (D–F) Far-UV CD spectra recorded on 6 μM tau^4RD in the absence or presence of coffee compounds. Measurements were performed immediately after sample preparation (continuous curves) and after 72 h (dotted curves) incubation of a concentrated stock (50 μM protein and 50 or 400 μg/mL compounds) in static conditions at 37 °C. Molar concentrations of compounds were 0.26 mM (50 μg/mL) and 2 mM (400 μg/mL) caffeine and 0.18 mM (50 μg/mL) and 1.5 mM (400 μg/mL) genistein.

Figure 4

Transmission electron microscopy of tau^4RD aggregates. Representative TEM images of tau^4RD aggregates in the presence of different concentrations of coffee extract or single compounds. Samples contained tau^4RD in buffer (A) with 50 μg/mL (B) or 400 μg/mL (C) coffee extract, 50 μg/mL (D) or 400 μg/mL (E) caffeine, 50 μg/mL (F) or 400 μg/mL (G) genistein. The protein was 50 μM. Samples were incubated for 48 h at 37 °C in static conditions. Scale bars are 500 nm (red) and 200 nm (light blue).

Figure 5

Influence of coffee compounds on tau condensation. Representative fluorescence microscopy images displaying condensates of tau^4RD/heparin in simple buffer (A) or in the presence of 280 μg/mL coffee extract (B), caffeine (1.4 mM) (C), and genistein (1 mM) (D). Images were acquired at 0, 5, and 30 min after mixing components. Protein was 35 μM and heparin was 8.75 μM. The scale bar is 10 μm. (E–H) Distribution plots of droplet diameters corresponding to conditions of panels (A–D) at 5 min; orange lines are best-fit log-normal curves.

Figure 6

Interaction of coffee compounds with tau aggregates. Saturation transfer difference (A, B) and WaterLOGSY (C, D) NMR spectra acquired on a 5 mg/mL espresso coffee mixture (A, C) or 0.8 mg/mL (4 mM) caffeine (B, D) in the absence (red) and presence (black) of tau^4RD filaments (80 μM monomer) in 20 mM deuterated phosphate buffer, pH 7.4 at 25 °C. The downfield regions in (A, C) are displayed with four-fold higher intensity compared to the highfield regions on the right. (E, F) ¹H NMR spectrum of 5 mg/mL coffee (E) or 0.8 mg/mL caffeine (F) in 20 mM deuterated phosphate buffer, pH 7.4 (1), STD (2), and WaterLOGSY (3) spectra acquired on 5 mg/mL coffee (E) or 0.8 mg/mL caffeine (F) in the presence of tau^4RD filaments.

Figure 7

(A) Schematic depiction of sample preparation for cell viability and seeding-based aggregation assays in cellular models. A first centrifugation step was performed for the isolation of the fibrils (pellets in blue) from soluble tau (aggregates in dark red and monomers as blue spheres) obtained after the fibrillization reaction of tau^4RD in buffer (gray frame), in the presence of 50 μg/mL coffee extract (orange frame) and in the presence of 400 μg/mL coffee extract (red frame). The latter sample did not contain any aggregate and therefore the first centrifugation did not produce pellets. The solution obtained after centrifugation of tau^4RD aggregated in the presence of 400 μg/mL coffee extract was further treated: a filtration step (through 100 kDa MWCO) was performed to separate monomers (filtrate) from soluble aggregates (retentate) used for seeding-based aggregation assays. The figure was created with BioRender.com. (B) Cell viability assay performed on H4-APPswe neuroglioma cells nontreated (NT) or treated for 48 h with pellets or retentate obtained as depicted in A (the color code is maintained). One-way statistical analysis ANOVA followed by Dunnett′s multiple comparison test was performed, ns = nonsignificant, p = *0.01–0.05, **0.001–0.01, ***0.0001–0.001. (C) Immunoblot analysis of the cellular insoluble fraction after treatment with samples of tau^4RD aggregated in buffer and in the presence of 400 μg/mL coffee extract (prepared as depicted in A, red frame). HEK293 cells overexpressing tau^FL/P310L-GFP were treated with the samples for 48 h and the Triton-insoluble fractions were blotted with the TAU-5 antibody. The last lane represents cells without the treatment (NT).