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. 2008 Nov 21;283(47):32176-87.
doi: 10.1074/jbc.M806154200. Epub 2008 Sep 24.

Effects of grape seed-derived polyphenols on amyloid beta-protein self-assembly and cytotoxicity

Kenjiro Ono  1 , Margaret M Condron, Lap Ho, Jun Wang, Wei Zhao, Giulio M Pasinetti, David B Teplow
Affiliations

Effects of grape seed-derived polyphenols on amyloid beta-protein self-assembly and cytotoxicity

Kenjiro Ono et al. J Biol Chem. .

Abstract

Epidemiological evidence suggests that moderate consumption of red wine reduces the incidence of Alzheimer disease (AD). To study the protective effects of red wine, experiments recently were executed in the Tg2576 mouse model of AD. These studies showed that a commercially available grape seed polyphenolic extract, MegaNatural-AZ (MN), significantly attenuated AD-type cognitive deterioration and reduced cerebral amyloid deposition (Wang, J., Ho, L., Zhao, W., Ono, K., Rosensweig, C., Chen, L., Humala, N., Teplow, D. B., and Pasinetti, G. M. (2008) J. Neurosci. 28, 6388-6392). To elucidate the mechanistic bases for these observations, here we used CD spectroscopy, photo-induced cross-linking of unmodified proteins, thioflavin T fluorescence, size exclusion chromatography, and electron microscopy to examine the effects of MN on the assembly of the two predominant disease-related amyloid beta-protein alloforms, Abeta40 and Abeta42. We also examined the effects of MN on Abeta-induced cytotoxicity by assaying 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide metabolism and lactate dehydrogenase activity in Abeta-treated, differentiated pheochromocytoma (PC12) cells. Initial studies revealed that MN blocked Abeta fibril formation. Subsequent evaluation of the assembly stage specificity of the effect showed that MN was able to inhibit protofibril formation, pre-protofibrillar oligomerization, and initial coil --> alpha-helix/beta-sheet secondary structure transitions. Importantly, MN had protective effects in assays of cytotoxicity in which MN was mixed with Abeta prior to peptide assembly or following assembly and just prior to peptide addition to cells. These data suggest that MN is worthy of consideration as a therapeutic agent for AD.

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Figures

FIGURE 1.
FIGURE 1.
Structures of MN and NGA. MN is water-soluble polyphenolic extract from Vitis vinifera grape seeds (17). MN comprises catechin and epicatechin in monomeric (∼8%), oligomeric (∼75%), and polymeric (∼17%) forms. The structure of a typical MN component composed of catechin and epicatechin base units and their derivatives is shown.
FIGURE 2.
FIGURE 2.
ThT binding. Aβ40 (A and C) or Aβ42 (B and D) were incubated for 7 days at 37 °C in 10 mm phosphate, pH 7.4, in the presence of 0 (○), 5 (•), or 25 (▵) μm NGA (A and B) or MN (C and D). Periodically, aliquots were removed, and ThT binding levels were determined. Binding is expressed as mean fluorescence (in arbitrary fluorescence units (FU)) ± S.E (error bars). Each figure comprises data obtained in three independent experiments.
FIGURE 3.
FIGURE 3.
Aβ assembly morphology. EM was used to determine the morphologies of assemblies of Aβ40 (A, C, E, and G) or Aβ42 (B, D, F, and H) incubated at 37 °C for 7 days in 10 mm sodium phosphate, pH 7.4. Peptides were incubated in buffer alone (A and B) or in the presence of 25 μm NGA (C and D), 5 μm MN (E and F), or 25 μm MN (G and H). White, quasicircular structures of diameter ∼10–30 nm were seen in some samples. These do not appear to be proteinaceous. Scale bars indicate 100 nm.
FIGURE 4.
FIGURE 4.
Protofibril formation. Aβ peptides were incubated alone (○) or in the presence of 25 μm NGA (•) or 25 μm MN (▵). Periodically during incubation, aliquots were analyzed by SEC to quantify protofibril formation. A, SEC of Aβ42 incubated alone at 37 °C for 10 days reveals a prominent protofibril peak eluting at ∼12 min, along with a LMW Aβ peak at ∼24 min. mAU, milliabsorbance units. Similar chromatograms were obtained for Aβ40. The areas under the protofibril peaks (see A) were integrated to determine temporal changes in protofibril amounts in samples containing Aβ40 (B) or Aβ42 (C). Areas are expressed as mean area ± S.E (error bars). Each figure comprises data obtained in three independent experiments.
FIGURE 5.
FIGURE 5.
Protofibril morphology. EM was used to determine the morphologies of protofibrils obtained by SEC following incubation of Aβ40 (A, C, and E) or Aβ42 (B, D, and F) at 37 °C for 10 days in 10 mm sodium phosphate, pH 7.4. Peptides were incubated in buffer alone (A and B) or in the presence of 25 μm NGA (C and D) or 25 μm MN (E and F). Scale bars indicate 100 nm.
FIGURE 6.
FIGURE 6.
Aβ oligomerization. PICUP, followed by SDS-PAGE and silver staining, was used to determine the effects of MN or NGA on oligomerization of Aβ40 (A), Aβ42 (B), or GST (C). Lanes 1, molecular weight markers; lanes 2, proteins alone (no cross-linking); lanes 3, proteins alone; lanes 4, proteins plus NGA (25 μm); lanes 5, proteins plus NGA (250 μm); lanes 6, proteins plus MN (25 μm); and lanes 7, protein plus MN (250 μm). Each gel is representative of each of three independent experiments.
FIGURE 7.
FIGURE 7.
Aβ secondary structure dynamics. Aβ40 (A, C, and E) or Aβ42 (B, D, and F) were incubated at 37 °C for 7 days in 10 mm phosphate, pH 7.4, in buffer alone (A and B) or in the presence of 25 μm NGA (C and D) or 25 μm MN (E and F). Spectra were acquired immediately at the start of the incubation period, day zero (○), and after days 2 (▾), 3(□), 6 (▪), and 7 (⋄). The spectra presented at each time are representative of those obtained during each of three independent experiments.
FIGURE 8.
FIGURE 8.
MTT metabolism. Aβ40 (A and C) or Aβ42 (B and D) were incubated in 10 mm sodium phosphate, pH 7.4, at 37 °C for 0, 2 (Aβ42), or 3 (Aβ40), and 7 days prior to addition to differentiated PC12 cells. In one set of experiments (A and B), the peptides were co-incubated with NGA or MN prior to addition to cells (pre-incubation). In a second set of experiments (C and D), NGA or MN were mixed with the peptides following the incubations (post-incubation) and immediately prior to addition to cells. Effects of untreated (closed bars), NGA-treated (cross-hatched bars), and MN-treated (open bars) Aβ on cell metabolism were determined fluorometrically using MTT (see “Experimental Procedures”) 24 h after peptide addition. Aβ40 or Aβ42 fibrils were used as a positive control and to provide a means for normalization of data among experiments. Data are presented as toxicity relative to fibrils, Tr, according to the formula Tr = Tsample/Tfibrils, where Tsample and Tfibrils are the percentage of toxicity in samples and the fibril controls, respectively (see “Experimental Procedures”). Tr is presented as mean ± S.E. (error bars). Statistical significance among groups was determined using one-way fractional analysis of variance and multiple comparison tests. Differences reaching statistical significance are noted by line segments between samples, along with their associated p values, where * signifies p < 0.05 and ** signifies p < 0.01. Multiple comparisons show differences between buffer and MN and between NGA and MN. Unless noted, no significant differences were observed between buffer and NGA.
FIGURE 9.
FIGURE 9.
LDH activity. Cell death was assessed by measurement of extracellular LDH activity in samples prepared and analyzed as specified in the legend for Fig. 8 and under “Experimental Procedures.” Data are presented as specified in the legend for Fig. 8.
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