Neurochemistry International 54 (2009) 111118

Contents lists available at ScienceDirect

Neurochemistry International
journal homepage: www.elsevier.com/locate/neuint

Dietary supplementation with resveratrol reduces plaque pathology

in a transgenic model of Alzheimers disease
Saravanan S. Karuppagounder a, John T. Pinto b, Hui Xu a, Huan-Lian Chen a,
M. Flint Beal c, Gary E. Gibson a,*
a

Department of Neurology and Neurosciences, Weill Medical College of Cornell University, Burke Medical Research Institute, 785 Mamaroneck Ave.,
White Plains, NY 10605, United States
Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, NY 10595, United States
c
Department of Neurology and Neurosciences, Weill Medical College of Cornell University, 525E 68th Street, New York, NY 10021, United States
b

A R T I C L E I N F O

A B S T R A C T

Article history:
Received 9 May 2008
Received in revised form 23 October 2008
Accepted 28 October 2008
Available online 8 November 2008

Resveratrol, a polyphenol found in red wine, peanuts, soy beans, and pomegranates, possesses a wide range
of biological effects. Since resveratrols properties seem ideal for treating neurodegenerative diseases, its
ability to diminish amyloid plaques was tested. Mice were fed clinically feasible dosages of resveratrol for
forty-ve days. Neither resveratrol nor its conjugated metabolites were detectable in brain. Nevertheless,
resveratrol diminished plaque formation in a region specic manner. The largest reductions in the percent
area occupied by plaques were observed in medial cortex (48%), striatum (89%) and hypothalamus
(90%). The changes occurred without detectable activation of SIRT-1 or alterations in APP processing.
However, brain glutathione declined 21% and brain cysteine increased 54%. The increased cysteine and
decreased glutathione may be linked to the diminished plaque formation. This study supports the concept
that onset of neurodegenerative disease may be delayed or mitigated with use of dietary chemo-preventive
agents that protect against b-amyloid plaque formation and oxidative stress.
2008 Elsevier Ltd. All rights reserved.

Keywords:
Oxidative stress
Mitochondria
Alzheimers disease
Ab peptide
Stilbenes

1. Introduction
Mitochondrial dysfunction and oxidative stress have been
implicated in multiple neurodegenerative diseases including
Alzheimers disease (AD) (Balaban et al., 2005; Beal, 2005; Gibson
et al., 2005), and the changes can be plausibly linked to plaque and
tangle formation. Studies on postmortem tissues from AD patients
reveal reductions in key thiamine-dependent enzymes of the
pentose shunt (transketolase), the tricarboxylic acid cycle (TCA)
(i.e., the a-ketoglutarate dehydrogenase complex; KGDHC) and
the link of glycolysis and the TCA cycle (i.e., the pyruvate
dehydrogenase complex; PDHC) in AD (Bubber et al., 2005; Gibson
et al., 2000). Brains from AD patients also contain elevated levels
of lipid peroxidation products, as well as protein and DNA
oxidation (Nourooz-Zadeh et al., 1999; Pratico et al., 2000;
Ramassamy et al., 2000; Smith et al., 1996). In a mouse model of
plaque formation, partial knockout of manganese superoxide
dismutase (MnSOD) elevates protein carbonyl levels, Ab levels
and increased plaque burden (Li et al., 2004). On the other hand,
reducing iNOS diminishes the levels of protein tyrosine nitration

* Corresponding author. Tel.: +1 914 597 2291; fax: +1 914 597 2757.
E-mail address: ggibson@med.cornell.edu (G.E. Gibson).
0197-0186/$ see front matter 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.neuint.2008.10.008

products, lowers concentration of Ab and diminishes amyloid

plaque burden (Nathan et al., 2005). Furthermore, Ab by itself is
capable of producing free radicals and ROS (Buttereld et al.,
1994; Hensley et al., 1994; Huang et al., 1999). Plausible
mechanisms link elevated ROS to the AD-related changes in
amyloid, tau and energy metabolism (Mark et al., 1997; Pappolla
et al., 1998; Pratico, 2002; Smith et al., 1991).
The extensive data that supports an important role of oxidative
stress in neurodegenerative disorders concomitantly support the
benecial effect of antioxidants as adjunctive or supportive
therapy. Resveratrol (trans-3,5,4-trihydroxystilbene), possesses a
wide range of biological effects that include anti-oxidative, antiinammatory and anti-carcinogenic properties. Numerous reports
suggest that resveratrol acts as a potent antioxidant and induces
endogenous antioxidants levels (Miller and Rice-Evans, 1995;
Robb et al., 2008; Li et al., 2006). In vitro studies suggest that
resveratrol may protect against b-amyloid induced oxidative cell
damage in PC12 cell lines (Conte et al., 2003; Jang and Surh, 2003;
Kim et al., 2007a, 2007b), and may promote Ab clearance through
promotion of intracellular proteosomal activity in cell lines
expressing wild type or Swedish APP695 mutations (Marambaud
et al., 2005).
Several clinical studies indicate that antioxidants may delay
onset of neurodegenerative diseases (Engelhart et al., 2002; Morris

112

S.S. Karuppagounder et al. / Neurochemistry International 54 (2009) 111118

et al., 2002). A double-blind, placebo-controlled, randomized,

multicenter trial in AD patients with moderate severity demonstrated that a-tocopherol treatment slowed the progression of the
disease (Sano et al., 1997). However, the effects were modest.
Recent studies show that resveratrol increases longevity and
delays the onset of aging in a manner similar to that of caloric
restriction (Baur et al., 2006). In a model of AD and tauopathies,
introduction of resveratrol directly into the brain ventricles
reduces neurodegeneration in the hippocampus, prevents learning
impairment, and decreases the acetylation of the known SIRT1
substrates PGC-1 a and p53 (Kim et al., 2007a). The present studies
tested whether administration of resveratrol to Tg19959 mice
alters plaque pathology.
2. Materials and methods
2.1. Animals
Tg19959 mice were produced by pronuclear microinjection of (FVB129S6F1)
embryos with a cosmid insert containing APP695 with two familial AD mutations
(KM670/671NL and V717F) under the control of the hamster PrP promoter (Chishti
et al., 2001). Male Tg19959 mice were backcrossed to (C57/B6SJL) F1 female
breeders. Genotypes of the offspring were determined by PCR analysis of tail DNA.
Animals were housed at constant temperature (22  2 8C), humidity (50  5%) and
illumination (12 h light/dark cycles) with food and water provided ad libitum. The
Institutional Animal Care and Use Committee of Weill Medical College of Cornell
University approved all procedures with the animals.
2.2. Treatment paradigm
A total of 19 Tg19959 mice at 45 days old were used. The control group included
four males and ve females, while the resveratrol group consisted of ve males and
ve females. These animals were matched for age, sex and weight. The control
group received a standard AIN-93G diet (Harlan Teklad, Madison, WI) ad libitum,
and the resveratrol group received standard AIN-93G plus 0.2% resveratrol (Harlan
Teklad, Madison, WI) ad libitum for 45 days. The calculated daily dosage in mice was
300 mg/kg (3 g food per day for a 20 g mouse). The human equivalent using a
scaling factor of 0.08 (http://www.fda.gov/cber/gdlns/dose.htm) is 24 mg/kg or
about 1.68 g per day for a 70 kg individual. Resveratrol (>98%) was purchased from
Sigma Chemical Company (St Louis, MO) and mixed to homogeneity during
manufacturing of the diets and stored at 80 8C. Food was replaced fresh in all cages
every four days. Stability of resveratrol in diet over time in cages and at room
temperature was assessed using HPLC with electrochemical detection.
2.3. Tissue preparation
Mice were sacriced by a lethal intraperitoneal dose of pentobarbitone sodium
(200 mg/kg; i.p.; Abbott Laboratories, North Chicago, IL) and perfused with 100 ml
of normal saline using a pump (Masterex, Model 7518-00, Cole-Parmer
Instrument Company, Barrington, IL) at 5 ml/min. The brains were removed and
bisected in the mid-sagittal plane. One-half was post-xed in 4% paraformaldehyde
in PBS for a minimum of 48 h and the other half was snap frozen in liquid nitrogen
and stored at 80 8C until analysis.
2.4. Analysis of resveratrol in diet and brain
The food was homogenized with 5% metaphosphoric acid (MPA) and methanol.
The homogenate was transferred to 1.5 ml Eppendorf tube and centrifuged at
13,200 rpm for 2 min. The supernatant fraction was evaporated to dryness using a
vacuum rotor vap and the residue was dissolved in 50% acetonitrile/water solution
and centrifuged at 13,200 rpm for 1 min. After centrifugation, supernatant fractions
were saved for HPLC determinations of resveratrol using electrochemical detection.
To analyze the resveratrol derivatives in the brain, about 40 mg of brain powder
was incubated with 10 ml of 10 mg/ml estriol-3-(b-glucuronidase), 0.1 ml of 0.1 M
ascorbic acid, 0.25 ml of 1.0 M ammonium acetate buffer, pH 5.0 and 1000 units of
b-glucuronidase from Helix pomatia. Glacial acetic acid (0.1 ml) was added and
hydrolysates were washed with 5.0 ml of hexane and extracted with ethyl acetate
and evaporated to dryness. The residue was dissolved in 50% methanol and
centrifuged at 14,000 rpm for 10 min.
Concentrations of resveratrol and its derivatives were measured using a
PerkinElmer Liquid Chromatograph equipped with an 8-channel coulometric array
(CoulArray) detector (ESA, Inc., Chelmsford, MA). The supernatant fractions were
injected onto a MD-150 column (3.0  150 mm; 3 mm particle size; ESA, Inc.,
Chelmsford, MA) and eluted at ambient temperature with a mobile phase consisting
of 26% acetonitrile (v/v) containing 75 mM citric acid and 25 mM ammonium
acetate (pH 2.64) at a ow rate of 0.6 ml/min. A Rheodyne injection valve with

a 5 ml sample loop was used to manually introduce samples. The 8-channel

CoulArray detectors were set at 100, 200, 320, 380, 440, 500, 560 and 620 mV,
respectively. To assure standardization between analyses, calibration standards
were interspersed at intervals among sample runs. Peak areas were analyzed using
ESA, Inc. software (Juan et al., 1999; Sale et al., 2005).
2.5. Analysis of cysteine, ascorbate, and GSH
For determination of selected redox active compounds, brain powders were
homogenized in 10 volumes of deionized water (w/v) at 4 8C using a PotterElvehjem homogenizer tted with a Teon pestle attached to a motor. To
precipitate proteins, a 100 ml aliquot of each homogenate was added to 400 ml of
ice-cold 6.25% (w/v) MPA, and the sample was incubated for 10 min on ice. After
centrifugation at 14,000 rpm for 5 min at 4 8C, aliquots of the 5% MPA supernatant
fractions were saved for HPLC determination of redox active analytes using
electrochemical detection. Concentrations of the redox-active ascorbate and the
aminothiols, cysteine and GSH, were measured without prior derivatization using a
PerkinElmer Liquid Chromatograph equipped with an 8-channel coulometric array
(CoulArray) detector (ESA, Inc., Chelmsford, MA) (Lakritz et al., 1997; Pinto et al.,
2005). The supernatant fractions from the 5% MPA homogenates were injected
directly onto a Bio-Sil ODS-5S, 5 mm particle size, 4 mm  250 mm C18 column
(Bio-Rad) and eluted at ambient temperature with a mobile phase consisting of
50 mM NaH2PO4, 0.05 mM 1-octanesulfonic acid, 1% acetonitrile (v/v), and 0.5%
N,N-dimethylformamide (v/v) (pH 2.70) at a ow rate of 1 ml/min. PEEKTM
(polyetheretherketone) tubing was used throughout the HPLC system, and a 0.2 mm
PEEKTM lters were placed pre- and post-column to protect both column and ow
cells, respectively, from any particulate matter. A rheodyne injection valve with a
5 ml sample loop was used to manually introduce samples. The 8-channel
CoulArray detectors were set at 250, 400, 450, 500, 550, 600, 650, and 700 mV,
respectively. To assure standardization between analyses, calibration standards
were interspersed at intervals among sample runs. Peak areas were analyzed using
ESA, Inc. software. Concentrations of each redox active compound (cysteine,
ascorbic acid, and GSH) were obtained from appropriate standard curves. Protein
precipitates from 5% MPA preparations were dissolved in 500 ml of 0.1N NaOH, and
protein was quantitated by a spectrophotometric method using bicinchoninic acid
reagent (Pierce Chemical Company, Rockford, IL).
2.6. Thioavine S staining and quantitation of amyloid plaques
To demonstrate the brillar Ab deposition thioavine S staining was used.
Briey, sections were washed with distilled water for 5 min and stained in freshly
prepared and ltered aqueous 0.5% thioavine S solution for 5 min. These sections
were treated with 70% ethanol, hydrated for 5 min and mounted with cytoseal.
Sections were visualized and images were captured in Nikon Eclipse 80i microscope
with a digital camera attached to a computer (Nikon, Melville, NY) and saved as
Tagged image format les. Computer-aided quantication of Ab deposits was
performed using the MetaMorph 6.1 software (Universal Imaging Corp, Downington, PA). Three sections per mouse from lateral 0.94, 3.25, 3.72 were
analyzed. Plaque counts and percentage occupied by the thioavine S positive
plaques were quantied in the cortex, hippocampus, neostriatum, hypothalamus,
thalamus and cerebellum. The region of interest was drawn manually under 4
magnication and threshold was kept constant throughout the analysis. The
analysis was performed in a blinded fashion.
2.7. Immunohistochemistry
Serial sagittal sections (40 mm) thick were cut with a sliding microtome (Microm
Lab GmbH, Walldorf, Germany) and processed as free oating sections. The staining
protocol employed a modied ABC immunohistochemistry procedure (Karuppagounder et al., 2007; Karuppagounder et al., 2008). Briey, sections were washed
with 0.1 M potassium phosphate buffered saline (PBS, pH 7.4) and incubated in 1%
H2O2 for 30 min to quench the endogenous peroxidase. Then sections were treated
with 0.1% Triton X-100 (Sigma Chemical Co., St. Louis, MO) for 15 min and blocked
with 2% bovine serum albumin (BSA) in PBS for one hr. Sections were incubated
with mouse monoclonal 6E10 against 116 residues of human Ab, 1:1000 (Signet
laboratories, Dedham, MA) in PBS containing 1% BSA (Sigma Chemical Co., St. Louis,
MO), overnight at room temperature. After rinsing in PBS, sections were incubated
with biotinylated anti-mouse 1:200 in PBS containing 0.25% BSA (Vector
Laboratories Inc., Burlingame, CA) for one hr. Sections were then incubated in
avidinbiotinperoxidase complex for 1 h 1:100 in PBS (Vector Laboratories Inc.,
Burlingame, CA;), rinsed in PBS and developed in 0.05% 3,30 -diaminobenzidine
(DAB) (Vector Laboratories Inc., Burlingame, CA) and 0.003% H2O2 in PBS.
2.8. Immunoblot analysis
Hemi-brain powders were homogenized in cold lysate buffer (50 mM Tris-HCl
pH 7.2, 0.4% Triton X-100, 1 mM DTT, 0.2 mM EGTA, 50 m Leupeptin). Protein
concentrations of the lysates were determined by a bicinchoninic acid (BCA) assay
(Pierce Chemical Company, Rockford, IL). Forty mg of protein were electrophoresed

S.S. Karuppagounder et al. / Neurochemistry International 54 (2009) 111118

through 1020% Tristricine gels, transferred onto nitrocellulose membranes
(Pierce Chemical Company, Rockford, IL). Membrane blots were blocked with 50%
Odyssey blocking buffer, 50% Tris-buffered saline (TBS) overnight at 4 8C. After
blocking, the membranes were incubated with primary antibodies, APP C-terminal
specic polyclonal antibody G369 to epitopes 645694 of APP695 (1:1000; Dr Sam
Gandy), SIRT1 (1:1000; Santa Cruz Biotechnology, CA) or b-actin (1:10,000) (Sigma
Chemical Company, St. Louis, MO) for 120 min at room temperature. Subsequently,
blots were washed with TBS plus 0.1% Trition X-100, and incubated at room
temperature for one hour with secondary Odyssey Goat anti-Rabbit IR Dye 680
antibody and Odyssey goat anti-Mouse IRDye 800 antibody (1:10,000; LICOR
Biosciences, Lincoln, NB) and analyzed using the Odyssey infrared imaging program
(LI-COR Biotechnology, Lincoln, NB).
2.9. Statistical analysis
All values are reported as means  SEM. P < 0.05 was considered signicant.
Statistical signicance of group differences was tested by two-tailed Students t-test or
one way ANOVA analysis. SPSS (SPSS Co., Chicago, IL) software was used to perform
statistical analysis.

3. Results
3.1. Resveratrol was stable in the diet and not detectable in brain
A method to extract resveratrol from diet or brain was
developed. Linearity, characteristics plot, standard curve, and
lower limits of detection were analyzed. The retention time of
resveratrol was 5.3 min (Fig. 1A). At this time, the resveratrol
standard curve was linear over a wide range (Fig. 1D). The method
is very sensitive. The lowest point on the standard curves was
0.5 pmole/ml. Fig. 1B represents no detectable levels of resveratrol
in the control diet and Fig. 1C demonstrates the level of
reseveratrol extracted from diet containing 0.2% resveratrol. The
extraction efciency from diet was determined by adding known
quantities of resveratrol to both the control and resveratrol
containing diets. The extraction efciency was 67  3.6%. HPLC
chromatograms show Fig. 1A, a resveratrol standard (50 mM), the
control diet (Fig. 1B) and the resveratrol diet (Fig. 1C).

113

Before delivering resveratrol in this manner, its stability in food

was determined. Although several studies have reported on
resveratrol stability in various diets, the results were inconclusive.
A recent report suggested that resveratrol isolated from rhizoma
polygoni cuspidati was unstable when exposed to light, heat and
atmospheric oxygen (Chen et al., 2007). To test the stability of
resveratrol in diet, the levels of resveratrol were determined by
HPLC on diets that were kept for variable times at room
temperature. The resveratrol content did not change signicantly
between the days analyzed. The percent resveratrol content was
0.2, 0.194 and 0.216 at 0, 4 and 7 days, respectively. In agreement
with other studies (Bertelli et al., 1998; Prokop et al., 2006),
resveratrol in the experimental diet was stable for a much longer
duration than required to complete our studies.
The levels of free resveratrol or resveratrol conjugate in brain
were analyzed in 90 day old Tg19959 mice that were fed diet
containing 0.2% resveratrol for 45 days. The 0.2% resveratrol was
well tolerated without any adverse effect on body weight gain or
food intake. These results are consistent with those in rabbits fed a
hypercholesterolemic diet supplemented with resveratrol (Wilson
et al., 1996). Resveratrol is absorbed in the small intestine and
undergoes glucuronidation before entering the blood (Kuhnle
et al., 2000). The presence of glucuronide/sulfated conjugates of
resveratrol was studied by pre-incubation of tissue samples with
extracts from Helix Pomatia which contains sulfatase and bglucuronidase. Interestingly, the brain levels of resveratrol or and
its conjugated derivatives were below the detection limits
(<0.5 pmol/ml/mg tissue).
3.2. Resveratrol did not alter body weight or food intake in Tg19959
mice
The food intake of control diet or resveratrol fed animals in
addition to body weights were monitored from 7 to12 weeks.

Fig. 1. Standardization of the resveratrol assay to test the presence in brain and stability in diet. Levels of resveratrol were determined by HPLC using electrochemical
detection. Representative HPLC chromatograms from resveratrol standard show that the retention time of resveratrol peak was 5.3 min (A). (B) represents no detectable
levels of resveratrol in the control diet and (C) demonstrates the level of reseveratrol extracted from diet containing 0.2% resveratrol. The standard curves for resveratrol
measurements reveal a large linear range and a limit of detection of 0.5 picomole/mL (D). The inset is an expansion of the lower range of the curve.

114

S.S. Karuppagounder et al. / Neurochemistry International 54 (2009) 111118

Fig. 2. Resveratrol, body weight and food intake. Forty-ve days old Tg19959 mice were fed AIN-93G diet with or without 0.2% resveratrol. The body weight (A) and the food
intake (B) were determined in mice from 712 weeks of age. Data represent means  SEM of control (n = 8) and 0.2% resveratrol (n = 9) groups from 23 independent
determinations.

Before treatment (day 45), the control or resveratrol group mice

weighed 18.2  0.6 or 18.3  0.7 g, respectively, and after treatment
(90 days) animals weighed 22.0  0.3 or 22.4  0.43 g, respectively.
Body weight did not vary between the control and resveratrol groups
(Fig. 2A). The food intake in control and resveratrol fed groups were
3.11  0.11 g and 3.15  0.12 g, respectively at 45 days and 3.23
 0.13 g and 3.31  0.06 g, respectively at 90 days. Food intake did
not vary between groups (Fig. 2B).

3.3. Resveratrol treatment reduced plaque pathology in Tg19959 mice

To test the effect of resveratrol on amyloid plaque pathology, 45
day old Tg19959 mice were fed a control AIN-93G diet with or
without 0.2% resveratrol for 45 days. Resveratrol markedly reduced
thioavine S positive compact plaques and 6E10 positive diffuse
plaques compared to mice on control diet (Fig. 3A and B). Quantication of thioavine S positive plaques revealed signicant

Fig. 3. Resveratrol and plaque pathology. Forty ve days old Tg19959 mice were fed AIN-93G diet with or without 0.2% resveratrol. Animals were sacriced at 90 days old, and
the brains were processed for thioavine S staining. (A) Representative saggital brain sections from lateral levels -0.94, -3.25, -3.72, respectively, showed that resveratrol
decreased thioavin S immunoreactivity (scale bar, 500 mm). (B) Resveratrol also increased decreased 6E10 immunoreactivity stained with 6E10 antibody directed against 1
17 amino acids of Ab in representative cortex sections (Scale bar, 200 mm). (C) Graphs shows the plaque counts and percentage area occupied by plaques quantied from the
cortex, hippocampus, striatum, thalamus and hypothalamus region. Data represent means  SEM of control (n = 8) and 0.2% resveratrol (n = 9) groups from 23 independent
experiments. * Denotes control differs from resveratrol group (P < 0.05).

S.S. Karuppagounder et al. / Neurochemistry International 54 (2009) 111118

115

Fig. 4. Resveratrol and APP CTF levels. Forty-ve day old Tg19959 mice were fed AIN-93G diet with or without 0.2% resveratrol. Animals were sacriced at 90 days. The whole
hemi-brain extracts from control and resveratrol fed mice were analyzed by Western blot using G369 antibody. (A) Representative Western blots probed with G369 antibody,
an antibody specic against both APP holo protein and C-terminal fragments for control and resveratrol groups are shown. (B) The densitometric analysis of APP holo protein
and C-terminal fragments band intensity do not reveal any differences. Data are means  SEM of control (n = 5) and resveratrol (n = 5) groups.

reduction in plaque counts and plaque burden in medial cortex

(P < 0.05), striatum (P < 0.05), hypothalamus (P < 0.05) of resveratrol-fed mice compared to mice fed the control diet (Fig. 3C).
Although, plaque counts and plaque burden declined in hippocampus, the change was statistically insignicant (Fig. 3C).
3.4. Resveratrol treatment did not alter amyloid precursor protein
(APP) carboxyterminal fragments in Tg19959 mice
To investigate the mechanism responsible for the resveratrolinduced reduction in plaque counts, the APP cleavage products
full length APP, b CTF (C99) and a CTF (C83) levels were
determined by Western blot using APP G369 antibody (against
the APP cytoplasmic tail). Resveratrol did not alter the levels
of high molecular weight APP species (holo APP), b CTF (C99)
or a CTF (C83) levels (Fig. 4A and B).

3.5. Resveratrol treatment increased the cysteine levels, but reduced

the GSH levels in Tg19959 mice
To test the effect of resveratrol on redox status, cysteine,
ascorbate and GSH levels were determined in control mice and in
mice fed the resveratrol diet. Resveratrol feeding signicantly
increased the cysteine levels by 54% (Fig. 5A; P < 0.05) compared to
control. By contrast, resveratrol diminished GSH levels by 21%
(Fig. 5B; P < 0.05), and did not alter ascorbate levels (Fig. 5C).
3.6. Resveratrol treatment did not alter SIRT 1 levels in Tg19959 mice
Since previous studies demonstrated that resveratrol activates
the NAD-dependent protein deactylase SIRT1, a discovery that
provided a molecular link to neurite stability and neuronal
protection, we tested whether SIRT1 expression was upregulated

Fig. 5. Resveratrol and brain redox status. Forty-ve day old Tg19959 mice were fed AIN-93G diet with or without 0.2% resveratrol for 45 days and sacriced at 90 day. Values
are expressed as nmol/mg protein. Data represent means  SEM of control (n = 8) and 0.2% Resveratrol (n = 9) groups from 23 independent experiments. * Denotes control and
resveratrol groups vary (P < 0.05).

116

S.S. Karuppagounder et al. / Neurochemistry International 54 (2009) 111118

Fig. 6. Resveratrol and SIRT1 levels. Forty-ve day old Tg19959 mice were fed AIN-93G diet with or without 0.2% resveratrol and sacriced at 90 days. Whole hemi-brain
extracts were analyzed by Western blot using SIRT1 antibody (A) and the band was quantied by densitometry (B) Values represent mean  SEM of control (n = 5) and
resveratrol (n = 5) groups.

in Tg19959 mice fed resveratrol. As demonstrated in Fig. 6A and B,

resveratrol treatment did not increase SIRT1 expression.
4. Discussion
Accumulating evidences from experimental and human studies
suggest that oxidative stress and mitochondrial dysfunction are
important causative factors in the development and progression of
several neurodegenerative diseases including AD (Gibson et al.,
2000; Ojaimi et al., 1999; Balaban et al., 2005). Induction of mild
impairment of oxidative metabolism, oxidative stress and inammation induced by thiamine deciency alters BACE1 levels and
metabolism of APP and/or Ab and promotes accumulation of
plaques in Tg 19959 mice (Karuppagounder et al., 2008). If
oxidative stress is critical in the alteration of APP processing and
the pathogenesis of AD, treatment with the appropriate antioxidants should be benecial and diminish plaque formation.
The dosage of resveratrol that was used in the current studies
(0.2% in diet) is a practical amount and was administered in a
clinically relevant manner. Dietary resveratrol at the level
administered in these studies had a striking effect on brain even
though resveratrol was not measurable. Although resveratrol and
its derivatives have been detected following gastric gavage, or
intraperitoneal administration (Wang et al., 2002), they have not
been detected following dietary administration (current results)
(Asensi et al., 2002; Sale et al., 2005). This is true even though the
daily dosage was higher in our experiments (300 mg/kg) than
bolus injections in other experiments. This same dosage protected
the vasculature of mice fed a high calorie diet (Baur et al., 2006).
Thus, the data suggests that the striking biological effects of
resveratrol in the diet result from trace levels entering the brain, an
effect on brain vasculature or a peripheral change that affects
brain.
The data demonstrate that resveratrol reduced plaque counts
and plaque burden most effectively in medial cortex, striatum and
hypothalamus. The causative factors or thresholds may differ in
various brain regions. For example, thiamine deciency (i.e., mild
impairment of oxidative metabolism) exacerbates plaque formation in regions that will eventually get plaques and induces plaque
formation in brain regions that do not normally contain plaques
(Karuppagounder et al., 2008). The current results demonstrate
that resveratrol can reduce plaques, but only in select regions.
Thus, a more effective paradigm is required to reduce plaque
burden throughout the brain. The relation of these restricted
changes in plaque formation to learning and memory is critical.
However, additional studies are required to optimize the response
of the resveratrol or its derivatives before examining the
interaction of learning and memory in multiple mouse models
of plaque formation.

The exact mechanism(s) underlying reduction of plaque

pathology by resveratrol is (are) unknown. The reduction in
plaque pathology did not appear to be due to altered APP
processing towards the non-amyloidgenic pathway. Resveratrol
did not alter levels of a CTF or b CTF (C99) or high molecular
weight APP species (holo APP). In vitro, resveratrol promotes Ab
clearance by increasing the intracellular proteosomal activity. To
demonstrate the role of proteasomes in vitro, Marambaud et al.,
used a variety of selective proteasomal inhibitors and siRNA. These
approaches are far more equivocal for in vivo studies. The effective
concentrations (about 40 mM; Marambaud et al., 2005) exceed
those in brain in the current study (<0.5 nM) by orders of
magnitude. Resveratrol has been postulated to have its benecial
effects on life span, neurodegeneration and to prevent impaired
memory by activation of SIRT1 (Kim et al., 2007a; Lagouge et al.,
2006). However, we did not see activation of SIRT-1. Other reports
suggest that resveratrol has other SIRT 1 independent actions
(Dasgupta and Milbrandt, 2007).
In the current study, the surprising resveratrol-induced
decline in glutathione and increase in cysteine is consistent with
a modest oxidant effect in brain. The decline in GSH could reect a
decreased synthesis, increased degradation or altered redox
status. GSH consists of glycine, cysteine, and glutamate. Thus, GSH
serves as a storage form of cysteine. Addition of cysteinyl
analogues to cells (e.g., N-acetylcysteine, 2-oxo-4-thiazolidine
carboxylic acid) would normally increase glutathione. Thus, the
decline in glutathione with increased cysteine suggests that brain
glutathione is providing additional cysteine residues. Since the
endogenous concentration of glutathione is much higher than
that of cysteine in brain, a small decline in glutathione can lead to a
large increase in cysteine. Further, an age-related decrease in
plasma cysteine suggests that high cysteine may be important in
diminishing plaque formation (Chen et al., 1989). Increasing
intracellular cysteine protects against multiple oxidant injuries.
In vitro and in vivo experiments have used N-acetylcysteine (NAC)
to increase cellular cysteine. NAC protects SHSY-5Y neuroblastoma cells from oxidative stress and cell toxicity due to H2O2, UV
light and amyloid-b142. Also, NAC promotes release of amyloidb142 from cells and diminishes oxidant induced tau phosphorylation (Olivieri et al., 2001). NAC down regulates transcription of
the amyloid precursor protein in human neuroblastoma cells
(Studer et al., 2001). Thus, the resveratrol induced increase in
cysteine may be protective and prevent plaque formation in the
same way as NAC.
An alternative tantalizing speculation is that the resveratrolinduced reduction in plaques may be through cysteine or
resveratrol chelation of copper or zinc. Cysteine chelates copper
(Baker and Czarnecki-Maulden, 1987; Cakir et al., 2001; Hallman
et al., 1971; Li and Manning, 1955) and zinc (Baker and Czarnecki-

S.S. Karuppagounder et al. / Neurochemistry International 54 (2009) 111118

Maulden, 1987; Cakir et al., 2001; Chen and Liao, 2003; Hallman
et al., 1971; Li and Manning, 1955). The chelation appears
pharmacologically important. Resveratrol inhibits human low
density lipoproteins (LDL) oxidation by chelating copper (Frankel
et al., 1993; Belguendouz et al., 1997; Fremont et al., 1999). Cu and
Zn are enriched in Ab deposits in AD, which are solubilized by Cu/
Zn-selective chelators in vitro. A bioavailable Cu/Zn chelator
decreases brain Ab deposition by 49% (Cherny et al., 2001). Thus,
the resveratrol induced increase in cysteine may help to reduce
plaques by chelation of copper in a manner shown for studies of
Cu/Zn chelators.
In conclusion, our results demonstrate that resveratrol treatment for 45 days reduced the plaque pathology in a region specic
manner, decreased brain glutathione and increased its precursor
product cysteine. Further studies need to be conducted to elucidate
the precise mechanism(s) by which resveratrol reduces Ab
pathology and alters brain glutathione status. This study supports
an important concept that onset of neurodegenerative disease may
be delayed or mitigated with use of dietary chemo-preventive
agents that protect against Ab plaque formation and oxidative
stress.
Acknowledgements
These studies were supported by Alzheimers Drug Discovery
Foundation & Institute for the Study of Aging and NIH grants
AG14600 and P01 AG14930.
References
Asensi, M., Medina, I., Ortega, A., Carretero, J., Bano, M.C., Obrador, E., Estrela, J.M.,
2002. Inhibition of cancer growth by resveratrol is related to its low bioavailability. Free Rad. Biol. Med. 33, 387398.
Baker, D.H., Czarnecki-Maulden, G.L., 1987. Pharmacologic role of cysteine in
ameliorating or exacerbating mineral toxicities. J. Nutr. 117, 10031110.
Balaban, R.S., Nemoto, S., Finkel, T., 2005. Mitochondria, oxidants, and aging. Cell
120, 483495.
Baur, J.A., Pearson, K.J., Price, N.L., Jamieson, H.A., Lerin, C., Kalra, A., Prabhu, V.V.,
Allard, J.S., Lopez-Lluch, G., Lewis, K., Pistell, P.J., Poosala, S., Becker, K.G., Boss, O.,
Gwinn, D., Wang, M., Ramaswamy, S., Fishbein, K.W., Spencer, R.G., Lakatta, E.G.,
Le Couteur, D., Shaw, R.J., Navas, P., Puigserver, P., Ingram, D.K., de Cabo, R.,
Sinclair, D.A., 2006. Resveratrol improves health and survival of mice on a highcalorie diet. Nature 444, 337342.
Beal, M.F., 2005. Mitochondria take center stage in aging and neurodegeneration.
Ann. Neurol. 58, 495505.
Belguendouz, L., Fremont, L., Linard, A., 1997. Resveratrol inhibits metal iondependent and independent peroxidation of porcine low-density lipoproteins.
Biochem. Pharmacol. 53, 13471355.
Bertelli, A.A., Gozzini, A., Stradi, R., Stella, S., Bertelli, A., 1998. Stability of resveratrol
over time and in the various stages of grape transformation. Drugs Under Exp.
Clin. Res. 24, 207211.
Bubber, P., Haroutunian, V., Fisch, G., Blass, J.P., Gibson, G.E., 2005. Mitochondrial
abnormalities in Alzheimer brain: mechanistic implications. Ann. Neurol. 57,
695703.
Buttereld, D.A., Hensley, K., Harris, M., Mattson, M., Carney, J., 1994. Beta-Amyloid
peptide free radical fragments initiate synaptosomal lipoperoxidation in a
sequence-specic fashion: implications to Alzheimers disease. Biochem. Biophys. Res. Commun. 200, 710715.
Cakir, S., Bicer, E., Eleman, A., 2001. Synthesis, spectroscopic and voltammetric
studies of mixed-ligand copper(II) complexes of amino acids. Trans. Met. Chem.
26, 8995.
Chen, C.J., Liao, S.L., 2003. Zinc toxicity on neonatal cortical neurons: involvement of
glutathione chelation. J. Neurochem. 85, 443453.
Chen, T.S., Richie Jr., J.P., Lang, C.A., 1989. The effect of aging on glutathione and
cysteine levels in different regions of the mouse brain. Proc. Soc. Exp. Biol. Med.
190, 399402.
Chen, Y.B., Sun, B.X., Chen, J.X., 2007. Study on the stability of resveratrol in rhizoma
polygoni cuspidati. Zhong Yao Cai 30, 805807.
Cherny, R.A., Atwood, C.S., Xilinas, M.E., Gray, D.N., Jones, W.D., McLean, C.A.,
Barnham, K.J., Volitakis, I., Fraser, F.W., Kim, Y., Huang, X., Goldstein, L.E., Moir,
R.D., Lim, J.T., Beyreuther, K., Zheng, H., Tanzi, R.E., Masters, C.L., Bush, A.I., 2001.
Treatment with a copper-zinc chelator markedly and rapidly inhibits betaamyloid accumulation in Alzheimers disease transgenic mice. Neuron 30, 665
676.
Chishti, M.A., Yang, D.S., Janus, C., Phinney, A.L., Horne, P., Pearson, J., Strome, R.,
Zuker, N., Loukides, J., French, J., Turner, S., Lozza, G., Grilli, M., Kunicki, S.,

117

Morissette, C., Paquette, J., Gervais, F., Bergeron, C., Fraser, P.E., Carlson, G.A.,
George-Hyslop, P.S., Westaway, D., 2001. Early-onset amyloid deposition and
cognitive decits in transgenic mice expressing a double mutant form of
amyloid precursor protein 695. J. Biol. Chem. 276, 2156221570.
Conte, A., Pellegrini, S., Tagliazucchi, D., 2003. Synergistic protection of PC12
cells from beta-amyloid toxicity by resveratrol and catechin. Brain Res. Bull.
62, 2938.
Dasgupta, B., Milbrandt, J., 2007. Resveratrol stimulates AMP kinase activity in
neurons. Proc. Natl. Acad. Sci. U.S.A. 104, 72177222.
Engelhart, M.J., Geerlings, M.I., Ruitenberg, A., van Swieten, J.C., Hofman, A., Witteman, J.C., Breteler, M.M., 2002. Dietary intake of antioxidants and risk of
Alzheimer disease. J. Am. Med. Assoc. 287, 32233229.
Frankel, E.N., Waterhouse, A.L., Kinsella, J.E., 1993. Inhibition of human LDL oxidation by resveratrol. Lancet 341, 11031104.
Fremont, L., Belguendouz, L., Delpal, S., 1999. Antioxidant activity of resveratrol and
alcohol-free wine polyphenols related to LDL oxidation and polyunsaturated
fatty acids. Life Sci. 64, 25112521.
Gibson, G.E., Haroutunian, V., Zhang, H., Park, L.C., Shi, Q., Lesser, M., Mohs, R.C.,
Sheu, R.K., Blass, J.P., 2000. Mitochondrial damage in Alzheimers disease varies
with apolipoprotein E genotype. Ann. Neurol. 48, 297303.
Gibson, G.E., Blass, J.P., Beal, M.F., Bunik, V., 2005. The alpha-ketoglutarate-dehydrogenase complex: a mediator between mitochondria and oxidative stress in
neurodegeneration. Mol. Neurobiol. 31, 4363.
Hallman, P.S., Perrin, D.D., Watt, A.E., 1971. The computed distribution of copper(II)
and zinc(II) ions among seventeen amino acids present in human blood plasma.
Biochem. J. 121, 549555.
Hensley, K., Carney, J.M., Mattson, M.P., Aksenova, M., Harris, M., Wu, J.F., Floyd, R.A.,
Buttereld, D.A., 1994. A model for beta-amyloid aggregation and neurotoxicity
based on free radical generation by the peptide: relevance to Alzheimer disease.
Proc. Natl. Acad. Sci. U.S.A. 91, 32703274.
Huang, X., Cuajungco, M.P., Atwood, C.S., Hartshorn, M.A., Tyndall, J.D., Hanson, G.R.,
Stokes, K.C., Leopold, M., Multhaup, G., Goldstein, L.E., Scarpa, R.C., Saunders,
A.J., Lim, J., Moir, R.D., Glabe, C., Bowden, E.F., Masters, C.L., Fairlie, D.P., Tanzi,
R.E., Bush, A.I., 1999. Cu(II) potentiation of alzheimer abeta neurotoxicity.
Correlation with cell-free hydrogen peroxide production and metal reduction.
J. Biol. Chem. 274, 3711137116.
Jang, J.H., Surh, Y.J., 2003. Protective effect of resveratrol on beta-amyloid-induced
oxidative PC12 cell death. Free Radical Biology & Medicine 34, 11001110.
Juan, M.E., Lamuela-Raventos, R.M., de la Torre-Boronat, M.C., Planas, J.M., 1999.
Determination of trans-resveratrol in plasma by HPLC. Anal. Chem. 71, 747
750.
Karuppagounder, S.S., Shi, Q., Xu, H., Gibson, G.E., 2007. Changes in inammatory
processes associated with selective vulnerability following mild impairment of
oxidative metabolism. Neurobiol. Dis. 26, 353362.
Karuppagounder, S.S., Xu, H., Shi, Q., Chen, L.H., Pedrini, S., Pechman, D.P., Baker,
H.A., Beal, M.F., Gandy, S.E., Gibson, G.E., 2008. Thiamine deciency induces
oxidative stress and exacerbates the plaque pathology in Alzheimers mouse
model. Neurobiol. Aging, doi: 10.1016/j.neurobiolaging.2007.12.013.
Kim, D., Nguyen, M.D., Dobbin, M.M., Fischer, A., Sananbenesi, F., Rodgers, J.T.,
Delalle, I., Baur, J.A., Sui, G., Armour, S.M., Puigserver, P., Sinclair, D.A., Tsai, L.H.,
2007a. SIRT1 deacetylase protects against neurodegeneration in models for
Alzheimers disease and amyotrophic lateral sclerosis. EMBO J. 26, 31693179.
Kim, H.J., Lee, K.W., Lee, H.J., 2007b. Protective effects of piceatannol against betaamyloid-induced neuronal cell death. Ann. N. Y. Acad. Sci. 1095, 473482.
Kuhnle, G., Spencer, J.P., Chowrimootoo, G., Schroeter, H., Debnam, E.S., Srai, S.K.,
Rice-Evans, C., Hahn, U., 2000. Resveratrol is absorbed in the small intestine as
resveratrol glucuronide. Biochem. Biophys. Res. Commun. 272, 212217.
Lagouge, M., Argmann, C., Gerhart-Hines, Z., Meziane, H., Lerin, C., Daussin, F.,
Messadeq, N., Milne, J., Lambert, P., Elliott, P., Geny, B., Laakso, M., Puigserver, P.,
Auwerx, J., 2006. Resveratrol improves mitochondrial function and protects
against metabolic disease by activating SIRT1 and PGC-1alpha. Cell 127, 1109
1122.
Lakritz, J., Plopper, C.G., Buckpitt, A.R., 1997. Validated high-performance liquid
chromatographyelectrochemical method for determination of glutathione and
glutathione disulde in small tissue samples. Anal. Biochem. 247, 6368.
Li, N.C., Manning, R.A., 1955. Some metal complexes of sulfur containing amino
acids. J. Am. Chem. Soc. 77, 52255228.
Li, F., Calingasan, N.Y., Yu, F., Mauck, W.M., Toidze, M., Almeida, C.G., Takahashi, R.H.,
Carlson, G.A., Flint Beal, M., Lin, M.T., Gouras, G.K., 2004. Increased plaque
burden in brains of APP mutant MnSOD heterozygous knockout mice. J.
Neurochem. 89, 13081312.
Li, Y., Cao, Z., Zhu, H., 2006. Upregulation of endogenous antioxidants and phase 2
enzymes by the red wine polyphenol, resveratrol in cultured aortic smooth
muscle cells leads to cytoprotection against oxidative and electrophilic stress.
Pharmacol. Res. 53, 615.
Marambaud, P., Zhao, H., Davies, P., 2005. Resveratrol promotes clearance of
Alzheimers disease amyloid-beta peptides. J. Biol. Chem. 280, 3737737382.
Mark, R.J., Lovell, M.A., Markesbery, W.R., Uchida, K., Mattson, M.P., 1997. A role for
4-hydroxynonenal, an aldehydic product of lipid peroxidation, in disruption of
ion homeostasis and neuronal death induced by amyloid beta-peptide. J.
Neurochem. 68, 255264.
Miller, N.J., Rice-Evans, C.A., 1995. Antioxidant activity of resveratrol in red wine.
Clin. Chem. 41, 1789.
Morris, M.C., Evans, D.A., Bienias, J.L., Tangney, C.C., Bennett, D.A., Aggarwal, N.,
Wilson, R.S., Scherr, P.A., 2002. Dietary intake of antioxidant nutrients and the

118

S.S. Karuppagounder et al. / Neurochemistry International 54 (2009) 111118

risk of incident Alzheimer disease in a biracial community study. J. Am. Med.

Assoc. 287, 32303237.
Nathan, C., Calingasan, N., Nezezon, J., Ding, A., Lucia, M.S., La Perle, K., Fuortes, M.,
Lin, M., Ehrt, S., Kwon, N.S., Chen, J., Vodovotz, Y., Kipiani, K., Beal, M.F., 2005.
Protection from Alzheimers-like disease in the mouse by genetic ablation of
inducible nitric oxide synthase. J. Exp. Med. 202, 11631169.
Nourooz-Zadeh, J., Liu, E.H., Yhlen, B., Anggard, E.E., Halliwell, B., 1999. F4-isoprostanes as specic marker of docosahexaenoic acid peroxidation in Alzheimers disease. J. Neurochem. 72, 734740.
Ojaimi, J., Masters, C.L., Opeskin, K., McKelvie, P., Byrne, E., 1999. Mitochondrial
respiratory chain activity in the human brain as a function of age. Mech. Ageing
Dev. 111, 3947.
Olivieri, G., Baysang, G., Meier, F., Muller-Spahn, F., Stahelin, H.B., Brockhaus, M.,
Brack, C., 2001. N-acetyl-L-cysteine protects SHSY5Y neuroblastoma cells from
oxidative stress and cell cytotoxicity: effects on beta-amyloid secretion and tau
phosphorylation. J. Neurochem. 76, 224233.
Pappolla, M.A., Chyan, Y.J., Omar, R.A., Hsiao, K., Perry, G., Smith, M.A., Bozner, P.,
1998. Evidence of oxidative stress and in vivo neurotoxicity of beta-amyloid in a
transgenic mouse model of Alzheimers disease: a chronic oxidative paradigm
for testing antioxidant therapies in vivo. Am. J. Pathol. 152, 871877.
Pinto, J.T., Van Raamsdonk, J.M., Leavitt, B.R., Hayden, M.R., Jeitner, T.M., Thaler, H.T.,
Krasnikov, B.F., Cooper, A.J., 2005. Treatment of YAC128 mice and their wildtype littermates with cystamine does not lead to its accumulation in plasma or
brain: implications for the treatment of Huntington disease. J. Neurochem. 94,
10871101.
Pratico, D., 2002. Alzheimers disease and oxygen radicals: new insights. Biochem.
Pharmacol. 63, 563567.
Pratico, D., Clark, C.M., Lee, V.M., Trojanowski, J.Q., Rokach, J., FitzGerald, G.A., 2000.
Increased 8,12-iso-iPF2alpha-VI in Alzheimers disease: correlation of a noninvasive index of lipid peroxidation with disease severity. Ann. Neurol. 48, 809812.
Prokop, J., Abrman, P., Seligson, A.L., Sovak, M., 2006. Resveratrol and its glycon
piceid are stable polyphenols. J. Med. Food 9, 1114.

Ramassamy, C., Averill, D., Beffert, U., Theroux, L., Lussier-Cacan, S., Cohn, J.S.,
Christen, Y., Schoofs, A., Davignon, J., Poirier, J., 2000. Oxidative insults are
associated with apolipoprotein E genotype in Alzheimers disease brain. Neurobiol. Dis. 7, 2337.
Robb, E.L., Page, M.M., Wiens, B.E., Stuart, J.A., 2008. Molecular mechanisms
of oxidative stress resistance induced by resveratrol: Specic and progressive induction of MnSOD. Biochem. Biophys. Res. Commun. 367, 406
412.
Sale, S., Tunstall, R.G., Ruparelia, K.C., Potter, G.A., Steward, W.P., Gescher, A.J., 2005.
Comparison of the effects of the chemopreventive agent resveratrol and its
synthetic analog trans 3,4,5,40 -tetramethoxystilbene (DMU-212) on adenoma
development in the Apc(Min+) mouse and cyclooxygenase-2 in human-derived
colon cancer cells. Int. J. Cancer 115, 194201.
Sano, M., Ernesto, C., Thomas, R.G., Klauber, M.R., Schafer, K., Grundman, M.,
Woodbury, P., Growdon, J., Cotman, C.W., Pfeiffer, E., Schneider, L.S., Thal,
L.J., 1997. A controlled trial of selegiline, alpha-tocopherol, or both as treatment
for Alzheimers disease. The Alzheimers Disease Cooperative Study. N. Engl. J.
Med. 336, 12161222.
Smith, C.D., Carney, J.M., Starke-Reed, P.E., Oliver, C.N., Stadtman, E.R., Floyd, R.A.,
Markesbery, W.R., 1991. Excess brain protein oxidation and enzyme dysfunction in normal aging and in Alzheimer disease. Proc. Natl. Acad. Sci. U.S.A. 88,
1054010543.
Smith, M.A., Perry, G., Richey, P.L., Sayre, L.M., Anderson, V.E., Beal, M.F., Kowall, N.,
1996. Oxidative damage in Alzheimers. Nature 382, 120121.
Studer, R., Baysang, G., Brack, C., 2001. N-Acetyl-L-cystein downregulates betaamyloid precursor protein gene transcription in human neuroblastoma cells.
Biogerontology 2, 5560.
Wang, Q., Xu, J., Rottinghaus, G.E., Simonyi, A., Lubahn, D., Sun, G.Y., Sun, A.Y., 2002.
Resveratrol protects against global cerebral ischemic injury in gerbils. Brain Res.
958, 439447.
Wilson, T., Knight, T.J., Beitz, D.C., Lewis, D.S., Engen, R.L., 1996. Resveratrol
promotes atherosclerosis in hypercholesterolemic rabbits. Life Sci. 59, 1521.