MegaNatural GSPE is a highly purified 100% water-soluble polyphenolic extract from Vitis vinifera grape seeds (Polyphenolics). The geographic source of the grape seeds used to generate MegaNatural GSPE is California, where grapes are usually harvested between August and October. The GSPE extraction procedure is based on standard procedures at Polyphenolics. Specifically, the solvent used for the extraction of the raw material in the form of dried seeds is water for 2 h at ~200–210°F, and the ratio of fresh grape/seeds to finished extract is 30 –50:1. No excipients are used in the extraction procedure, and the native extract is 100% from seeds. Finished product is spray dried into a powder and contains >90% total polyphenols as gallic acid equivalents and moisture content of <10%. GSPE is stable in the factory-sealed container at room temperature for at least 3 years.
HPLC analysis shows that MegaNatural GSPE
is comprised of catechin and epicatechin in monomeric, oligomeric, and polymeric forms (supplemental Fig. 1A
, available at www.jneurosci.org
as supplemental material). Typically, MegaNatural GSPE
contains ~8% monomers, 75% oligomers, and ~17% polymers. We arbitrarily used the molecular weight of catechin and epicatechin dimer (which is the most abundant form of oligomer in GSPE
) to calculate the molarity for GSPE
in this study. A unique feature of this extract is that it is readily absorbed through the intestinal mucosa because of the modification of the constituent polyphenols (Siva et al., 2006
). Moreover, MegaNatural GSPE
extract is well tolerated in both humans (Dr. A. Shrikhande, personal communication) and rodents (Bentivegna and Whitney, 2002
Given the difficulty of reproducing the nutraceuticals consistently, we tried two different lots (lot #25952501-30 and lot #05642505-1), and both lots showed similar chemical profiles by normal-phase HPLC (data not shown) and reproducibly prevented AD-type Aβ aggregation in vitro (data not shown). For the in vivo study, we used lot #25952501-30.
In vitro Aβ1–40 and Aβ1–42 aggregation assay
peptides were purchased from American Peptide or synthesized chemically. For Western blotting, peptides were solubilized in hexafluoroisopropanol (Sigma) and dried overnight (Klein, 2002
stock was dissolved in phosphate buffer, pH 7.4 (Invitrogen), at 200 µm
(100 µg/ml in PBS) were mixed with different concentrations of GSPE
at 1:1 volume and incubated at 37°C for 24 h. The effect of GSPE
on Aβ aggregation was analyzed by Western blot analysis using 6E10 antibody.
Photo-induced cross-linking of unmodified proteins assay
Freshly isolated low-molecular-weight Aβ1–42
) or Aβ1–40
) peptide was mixed with 1 µl of 1 (1×), 2 (2×), 5 (5×), or 10 (10×) mm
tris(2,2′-bipyridyl)dichlororuthenium(II) [Ru(Bpy)] and 1 µl of 20 (1×), 40 (2×), 100 (5×), or 200 (10×) mm
ammonium persulfate (APS) in the presence or absence of 50 µm GSPE
in 10 mm
phosphate, pH 7.4. The mixture was irradiated for 1 s and quenched immediately with 10 µl of Tricine sample buffer (Invitrogen) containing 5% β-mercaptoethanol (Bitan et al., 2001
). The reaction was subjected to SDS-PAGE and visualized by silver staining (SilverXpress; Invitrogen).
Tg2576 mice and GSPE treatment
In this study, we used Tg2576 AD transgenic mice (catalog #001349; Taconic). In this Tg2576 AD mouse model, Aβ peptide content in the brain accumulates exponentially between 7 and 15 months of age (Hsiao et al., 1996
; Kawarabayashi et al., 2001
). Therefore, we treated animals for 5 months, starting at 6 (for behavioral testing) and 10 (for neuropathology and mechanistic study) months of age.
Adult female Tg2576 mice, which have a more robust plaque neuropathology (Callahan et al., 2001
) and low mortality rate compared with the male Tg2576 mice, were assigned to two different groups: the GSPE
treatment group and the water control group. The same kind of Tg2576 female mice were also used in our previous study on cabernet sauvignon (Wang et al., 2006
). Mice were fed with 200 mg/kg/d GSPE
delivered in their drinking water, which is equivalent to a human dose of 1 g/d using Food and Drug Administration criteria for converting drug equivalent dosages across species (http://www.fda.gov/cber/gdlns/dose.htm
). Animals had ad libitum
access to the liquid and standard chow. Drinking solutions were changed every 3 d. After 5 months of treatment, mice were anesthetized with the general anesthetic ketamine HCl and xylazine (Fort Dodge Animal Health) and killed by decapitation. Brains were harvested as described previously (Wang et al., 2005
). All animal studies were approved by the Institutional Animal Care and Use Committee of Mount Sinai School of Medicine.
Behavioral assessment of cognitive functions by Morris water maze test
Spatial learning memory was assessed by the Morris water maze behavioral test, as described previously (Morris, 1984
). In this assay, mice were tested in a 1.25 m circular pool filled with water mixed with nontoxic white paint (Dick Blick Art Materials). Mice were trained to mount a hidden/submerged (1.5 cm below water surface) escape platform (14 × 14 cm) in a restricted region of the pool. Spatial memory is assessed by recording the latency time for the animal to escape from the water onto a submerged escape platform as a function of the number of learning trials during the learning phase. Twenty-four hours after the learning phase, mice were subjected to a 45 s probe trial wherein the escape platform was removed. The water maze activity was monitored with the San Diego Instrument Poly-Track video tracking system.
Assessment of AD-type amyloid neuropathology
in the brain were quantified by sandwich ELISA (BioSource), as described previously (Wang et al., 2005
). Stereological assessment of AD-type amyloid burden in Tg2576 mice was analyzed as described previously (Wang et al., 2005
). Briefly, 4% paraformaldehyde-fixed brain was sectioned, and every 15th section was selected from a random start position and processed for thioflavin-S staining. The amyloid burden was estimated using the Cavalieri principle with a small-size grid (50 × 50 µm) for point counting. Estimates of plaque volume were obtained using a systematic random sampling procedure at 40× magnification.
Brain soluble Aβ oligomer analysis
The level of soluble Aβ oligomers was measured both by dot blot assay and Western blot analysis (McLaurin et al., 2006
) and was also quantified by ELISA. Briefly, soluble amyloid peptide was extracted in PBS supplemented with protease inhibitor mixture stock (25× aqueous solution; Roche Applied Science). After centrifugation at 78,500 × g
for 1 h at 4°C, the supernatant was analyzed. Five micrograms of total protein were spotted on nitrocellulose membrane and probed with antibody A11 (Biosource), specific for oligomeric forms of Aβ. The immunoreactive signals were visualized using enhanced chemiluminescence detection (Pierce) and quantified densitometrically (Quantity One; Bio-Rad). The same sample was also used for Western analysis. Seventy-five micrograms of total proteins were separated by 10–20% Tris-Tricine gel, transferred to a nitrocellulose membrane, and probed with either antibody 6E10 (Signet) or A11. Immunoreactive signals were visualized and quantified. For quantitative oligomeric Aβ analysis, the same sample was applied to a commercially available ELISA kit that specifically detects aggregated β amyloid using protocols provided by the manufacturer (Invitrogen).
APP processing and α-, β-, and γ-secretase activity
Expression of holoamyloid precursor protein (APP) was examined by Western blot analysis with the C8 antibody. Immunoprecipitation was performed for detection of soluble APP (sAPP)-α or sAPP-β as described previously (Wang et al., 2005
). α-, β-, and γ-secretase activities were assessed using commercially available kits (R&D Systems) (Ho et al., 2004
; Wang et al., 2005
). The expression of neprilysin and insulin-degrading enzyme (IDE) were analyzed by Western blot using commercially available antibody (IDE antibody from Abcam and neprilysin antibody from Alpha Diagnostic International).
All values are expressed as mean and SEM. Differences between means were analyzed using either two-way repeated-measures ANOVA or two-tailed Student’s t test. In all analyses, the null hypothesis was rejected at the 0.05 level. All statistical analyses were performed using the Prism Stat program (GraphPad Software).