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Neurochemistry International 54 (2009) 111118

und in redwine, peanuts, soy beans, and pomegranates, possesses awide range

Contents lists availab

Neurochemistry

.e l1. Introduction

Mitochondrial dysfunction and oxidative stress have beenimplicated in multiple neurodegenerative diseases includingAlzheimers disease (AD) (Balaban et al., 2005; Beal, 2005; Gibsonet al., 2005), and the changes can be plausibly linked to plaque andtangle formation. Studies onpostmortem tissues fromADpatientsreveal reductions in key thiamine-dependent enzymes of thepentose shunt (transketolase), the tricarboxylic acid cycle (TCA)(i.e., the a-ketoglutarate dehydrogenase complex; KGDHC) andthe link of glycolysis and the TCA cycle (i.e., the pyruvatedehydrogenase complex; PDHC) inAD (Bubber et al., 2005;Gibsonet al., 2000). Brains from AD patients also contain elevated levelsof lipid peroxidation products, as well as protein and DNAoxidation (Nourooz-Zadeh et al., 1999; Pratico et al., 2000;Ramassamy et al., 2000; Smith et al., 1996). In a mouse model ofplaque formation, partial knockout of manganese superoxidedismutase (MnSOD) elevates protein carbonyl levels, Ab levelsand increased plaque burden (Li et al., 2004). On the other hand,reducing iNOS diminishes the levels of protein tyrosine nitration

products, lowers concentration of Ab and diminishes amyloidplaque burden (Nathan et al., 2005). Furthermore, Ab by itself iscapable of producing free radicals and ROS (Buttereld et al.,1994; Hensley et al., 1994; Huang et al., 1999). Plausiblemechanisms link elevated ROS to the AD-related changes inamyloid, tau and energy metabolism (Mark et al., 1997; Pappollaet al., 1998; Pratico, 2002; Smith et al., 1991).

The extensive data that supports an important role of oxidativestress in neurodegenerative disorders concomitantly support thebenecial effect of antioxidants as adjunctive or supportivetherapy. Resveratrol (trans-3,5,4-trihydroxystilbene), possesses awide range of biological effects that include anti-oxidative, anti-inammatory and anti-carcinogenic properties. Numerous reportssuggest that resveratrol acts as a potent antioxidant and inducesendogenous antioxidants levels (Miller and Rice-Evans, 1995;Robb et al., 2008; Li et al., 2006). In vitro studies suggest thatresveratrol may protect against b-amyloid induced oxidative celldamage in PC12 cell lines (Conte et al., 2003; Jang and Surh, 2003;Kim et al., 2007a, 2007b), and may promote Ab clearance throughpromotion of intracellular proteosomal activity in cell linesexpressing wild type or Swedish APP695 mutations (Marambaudet al., 2005).

Several clinical studies indicate that antioxidants may delayonset of neurodegenerative diseases (Engelhart et al., 2002; Morris

Received 9 May 2008

Received in revised form 23 October 2008

Accepted 28 October 2008

Available online 8 November 2008

Keywords:

Oxidative stress

Mitochondria

Alzheimers disease

Ab peptideStilbenes

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 glutathionemay be linked to the diminished plaque formation. This study supports the concept

that onset of neurodegenerative diseasemaybe delayed ormitigatedwithuse of dietary chemo-preventive

agents that protect against b-amyloid plaque formation and oxidative stress. 2008 Elsevier Ltd. All rights reserved.

* 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.2008.10.008Dietary supplementation with resveratin a transgenic model of Alzheimers di

Saravanan S. Karuppagounder a, John T. Pinto b, HuM. Flint Beal c, Gary E. Gibson a,*aDepartment of Neurology and Neurosciences, Weill Medical College of Cornell Univer

White Plains, NY 10605, United StatesbDepartment of Biochemistry and Molecular Biology, New York Medical College, ValhacDepartment of Neurology and Neurosciences, Weill Medical College of Cornell Univer

A R T I C L E I N F O

Article history:

A B S T R A C T

Resveratrol, a polyphenol fo

journa l homepage: wwwl reduces plaque pathologyase

u a, Huan-Lian Chen a,

Burke Medical Research Institute, 785 Mamaroneck Ave.,

Y 10595, United States

525E 68th Street, New York, NY 10021, United States

le at ScienceDirect

International

sevier .com/ locate /neuint

mistet al., 2002). A double-blind, placebo-controlled, randomized,multicenter trial in AD patients with moderate severity demon-strated that a-tocopherol treatment slowed the progression of thedisease (Sano et al., 1997). However, the effects were modest.Recent studies show that resveratrol increases longevity anddelays the onset of aging in a manner similar to that of caloricrestriction (Baur et al., 2006). In a model of AD and tauopathies,introduction of resveratrol directly into the brain ventriclesreduces neurodegeneration in the hippocampus, prevents learningimpairment, and decreases the acetylation of the known SIRT1substrates PGC-1a and p53 (Kim et al., 2007a). The present studiestested whether administration of resveratrol to Tg19959 micealters 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%) andillumination (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 inmicewas

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 at80 8C. Foodwas replaced fresh in all cagesevery 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 themid-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 1min. 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 10ml of 10mg/ml estriol-3-(b-glucuronidase), 0.1 ml of 0.1 Mascorbic 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 andhydrolysates 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; 3mm particle size; ESA, Inc.,Chelmsford,MA) and eluted at ambient temperaturewith amobile phase consisting

of 26% acetonitrile (v/v) containing 75 mM citric acid and 25 mM ammonium

S.S. Karuppagounder et al. / Neuroche112acetate (pH 2.64) at a ow rate of 0.6 ml/min. A Rheodyne injection valve witha 5ml sample loop was used to manually introduce samples. The 8-channelCoulArray 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 Potter-Elvehjem 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 ofice-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 supernatantfractions 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, 5mm 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) tubingwas used throughout theHPLC system, and a 0.2 mmPEEKTM 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

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

Sectionswere visualized and imageswere captured in Nikon Eclipse 80imicroscope

with a digital camera attached to a computer (Nikon, Melville, NY) and saved as

Tagged image format les. Computer-aided quantication of Ab deposits wasperformed using the MetaMorph 6.1 software (Universal Imaging Corp, Down-

ington, PA). Three sections per mouse from lateral 0.94, 3.25, 3.72 wereanalyzed. 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 4magnication and threshold was kept constant throughout the analysis. The

analysis was performed in a blinded fashion.

2.7. Immunohistochemistry

Serial sagittal sections (40 mm) thickwere cut with a slidingmicrotome (MicromLab GmbH,Walldorf, Germany) and processed as free oating sections. The staining

protocol employed a modied ABC immunohistochemistry procedure (Karuppa-

gounder 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 (Signetlaboratories, 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). Proteinconcentrations of the lysates were determined by a bicinchoninic acid (BCA) assay

ry International 54 (2009) 111118(Pierce Chemical Company, Rockford, IL). Fortymg of protein were electrophoresed

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. Afterblocking, 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) (SigmaChemical 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 wasdeveloped. Linearity, characteristics plot, standard curve, andlower limits of detection were analyzed. The retention time ofresveratrol was 5.3 min (Fig. 1A). At this time, the resveratrolstandard curve was linear over a wide range (Fig. 1D). The methodis very sensitive. The lowest point on the standard curves was0.5 pmole/ml. Fig. 1B represents no detectable levels of resveratrolin the control diet and Fig. 1C demonstrates the level of

Before delivering resveratrol in this manner, its stability in foodwas determined. Although several studies have reported onresveratrol stability in various diets, the results were inconclusive.A recent report suggested that resveratrol isolated from rhizomapolygoni cuspidati was unstable when exposed to light, heat andatmospheric oxygen (Chen et al., 2007). To test the stability ofresveratrol in diet, the levels of resveratrol were determined byHPLC on diets that were kept for variable times at roomtemperature. The resveratrol content did not change signicantlybetween the days analyzed. The percent resveratrol content was0.2, 0.194 and 0.216 at 0, 4 and 7 days, respectively. In agreementwith other studies (Bertelli et al., 1998; Prokop et al., 2006),resveratrol in the experimental diet was stable for a much longerduration than required to complete our studies.

The levels of free resveratrol or resveratrol conjugate in brainwere analyzed in 90 day old Tg19959 mice that were fed dietcontaining 0.2% resveratrol for 45 days. The 0.2% resveratrol waswell tolerated without any adverse effect on body weight gain orfood intake. These results are consistent with those in rabbits fed ahypercholesterolemic diet supplemented with resveratrol (Wilsonet al., 1996). Resveratrol is absorbed in the small intestine andundergoes glucuronidation before entering the blood (Kuhnleet al., 2000). The presence of glucuronide/sulfated conjugates ofresveratrol was studied by pre-incubation of tissue samples withextracts from Helix Pomatia which contains sulfatase and b-glucuronidase. Interestingly, the brain levels of resveratrol or andits conjugated derivatives were below the detection limits(

Before treatment (day 45), the control or resveratrol group miceweighed 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 were3.11 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 didnot vary between groups (Fig. 2B).

3.3. Resveratrol treatment reduced plaque pathology in Tg19959mice

To test the effect of resveratrol on amyloid plaque pathology, 45day old Tg19959 mice were fed a control AIN-93G diet with orwithout 0.2% resveratrol for 45 days. Resveratrolmarkedly reducedthioavine S positive compact plaques and 6E10 positive diffuseplaques compared to mice on control diet (Fig. 3A and B). Quan-tication of thioavine S positive plaques revealed signicant

Fig. 2. Resveratrol, body weight and food intake. Forty-ve days old Tg19959mice were fed AIN-93G diet with or without 0.2% resveratrol. The body weight (A) and the foodintake (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 independentdeterminations.

S.S. Karuppagounder et al. / Neurochemistry International 54 (2009) 111118114Fig. 3. Resveratrol and plaque pathology. Forty ve days old Tg19959micewere fed AIN-9the brains were processed for thioavine S staining. (A) Representative saggital brain s

decreased thioavin S immunoreactivity (scale bar, 500 mm). (B) Resveratrol also increase17 amino acids of Ab in representative cortex sections (Scale bar, 200 mm). (C) Graphs shcortex, hippocampus, striatum, thalamus and hypothalamus region. Data represent mea

experiments. * Denotes control differs from resveratrol group (P < 0.05).3G diet with orwithout 0.2% resveratrol. Animals were sacriced at 90 days old, and

ections from lateral levels -0.94, -3.25, -3.72, respectively, showed that resveratrol

d decreased 6E10 immunoreactivity stainedwith 6E10 antibody directed against 1

ows the plaque counts and percentage area occupied by plaques quantied from the

ns SEM of control (n = 8) and 0.2% resveratrol (n = 9) groups from 23 independent

93G

blot

rol a

ean

S.S. Karuppagounder et al. / Neurochemistry International 54 (2009) 111118 115Fig. 4. Resveratrol and APP CTF levels. Forty-ve day old Tg19959mice were fed AIN-hemi-brain extracts from control and resveratrol fedmicewere analyzed byWestern

an antibody specic against both APP holo protein and C-terminal fragments for cont

and C-terminal fragments band intensity do not reveal any differences. Data are mreduction in plaque counts and plaque burden in medial cortex(P < 0.05), striatum (P < 0.05), hypothalamus (P < 0.05) of resver-atrol-fed mice compared to mice fed the control diet (Fig. 3C).Although, plaque counts and plaque burden declined in hippo-campus, 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 resveratrol-induced reduction in plaque counts, the APP cleavage productsfull length APP, b CTF (C99) and a CTF (C83) levels weredetermined by Western blot using APP G369 antibody (againstthe APP cytoplasmic tail). Resveratrol did not alter the levelsof high molecular weight APP species (holo APP), b CTF (C99)or a CTF (C83) levels (Fig. 4A and B).

Fig. 5. Resveratrol and brain redox status. Forty-ve day old Tg19959micewere fed AIN-9are expressed as nmol/mg protein. Data represent means SEM of control (n = 8) and 0.2resveratrol groups vary (P < 0.05).diet with or without 0.2% resveratrol. Animals were sacriced at 90 days. The whole

using G369 antibody. (A) RepresentativeWestern blots probedwith G369 antibody,

nd resveratrol groups are shown. (B) The densitometric analysis of APP holo protein

s SEM of control (n = 5) and resveratrol (n = 5) groups.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 inmice fed the resveratrol diet. Resveratrol feeding signicantlyincreased the cysteine levels by 54% (Fig. 5A; P < 0.05) compared tocontrol. 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 activatesthe NAD-dependent protein deactylase SIRT1, a discovery thatprovided a molecular link to neurite stability and neuronalprotection, we tested whether SIRT1 expression was upregulated

3G diet with or without 0.2% resveratrol for 45 days and sacriced at 90 day. Values

% Resveratrol (n = 9) groups from 23 independent experiments. * Denotes control and

93G

as q

mistin 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 studiessuggest that oxidative stress and mitochondrial dysfunction areimportant causative factors in the development and progression ofseveral neurodegenerative diseases including AD (Gibson et al.,2000; Ojaimi et al., 1999; Balaban et al., 2005). Induction of mildimpairment of oxidative metabolism, oxidative stress and inam-mation induced by thiamine deciency alters BACE1 levels andmetabolism of APP and/or Ab and promotes accumulation ofplaques in Tg 19959 mice (Karuppagounder et al., 2008). Ifoxidative stress is critical in the alteration of APP processing andthe pathogenesis of AD, treatment with the appropriate antiox-idants 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 aclinically relevant manner. Dietary resveratrol at the leveladministered in these studies had a striking effect on brain eventhough resveratrol was not measurable. Although resveratrol andits derivatives have been detected following gastric gavage, orintraperitoneal administration (Wang et al., 2002), they have notbeen detected following dietary administration (current results)(Asensi et al., 2002; Sale et al., 2005). This is true even though thedaily dosage was higher in our experiments (300 mg/kg) thanbolus injections in other experiments. This same dosage protectedthe vasculature of mice fed a high calorie diet (Baur et al., 2006).Thus, the data suggests that the striking biological effects ofresveratrol in the diet result from trace levels entering the brain, aneffect on brain vasculature or a peripheral change that affects

Fig. 6. Resveratrol and SIRT1 levels. Forty-ve day old Tg19959 mice were fed AIN-extracts were analyzed by Western blot using SIRT1 antibody (A) and the band w

resveratrol (n = 5) groups.

S.S. Karuppagounder et al. / Neuroche116brain.The data demonstrate that resveratrol reduced plaque counts

and plaque burden most effectively in medial cortex, striatum andhypothalamus. The causative factors or thresholds may differ invarious brain regions. For example, thiamine deciency (i.e., mildimpairment of oxidative metabolism) exacerbates plaque forma-tion in regions that will eventually get plaques and induces plaqueformation in brain regions that do not normally contain plaques(Karuppagounder et al., 2008). The current results demonstratethat resveratrol can reduce plaques, but only in select regions.Thus, a more effective paradigm is required to reduce plaqueburden throughout the brain. The relation of these restrictedchanges in plaque formation to learning and memory is critical.However, additional studies are required to optimize the responseof the resveratrol or its derivatives before examining theinteraction of learning and memory in multiple mouse modelsof plaque formation.The exact mechanism(s) underlying reduction of plaquepathology by resveratrol is (are) unknown. The reduction inplaque pathology did not appear to be due to altered APPprocessing towards the non-amyloidgenic pathway. Resveratroldid not alter levels of a CTF or b CTF (C99) or high molecularweight APP species (holo APP). In vitro, resveratrol promotes Abclearance by increasing the intracellular proteosomal activity. Todemonstrate the role of proteasomes in vitro, Marambaud et al.,used a variety of selective proteasomal inhibitors and siRNA. Theseapproaches are far more equivocal for in vivo studies. The effectiveconcentrations (about 40 mM; Marambaud et al., 2005) exceedthose in brain in the current study (

mistMaulden, 1987; Cakir et al., 2001; Chen and Liao, 2003; Hallmanet al., 1971; Li and Manning, 1955). The chelation appearspharmacologically important. Resveratrol inhibits human lowdensity lipoproteins (LDL) oxidation by chelating copper (Frankelet al., 1993; Belguendouz et al., 1997; Fremont et al., 1999). Cu andZn are enriched in Ab deposits in AD, which are solubilized by Cu/Zn-selective chelators in vitro. A bioavailable Cu/Zn chelatordecreases brain Ab deposition by 49% (Cherny et al., 2001). Thus,the resveratrol induced increase in cysteine may help to reduceplaques by chelation of copper in a manner shown for studies ofCu/Zn chelators.

In conclusion, our results demonstrate that resveratrol treat-ment for 45 days reduced the plaque pathology in a region specicmanner, decreased brain glutathione and increased its precursorproduct cysteine. Further studies need to be conducted to elucidatethe precise mechanism(s) by which resveratrol reduces Abpathology and alters brain glutathione status. This study supportsan important concept that onset of neurodegenerative diseasemaybe delayed or mitigated with use of dietary chemo-preventiveagents that protect against Ab plaque formation and oxidativestress.

Acknowledgements

These studies were supported by Alzheimers Drug DiscoveryFoundation & Institute for the Study of Aging and NIH grantsAG14600 and P01 AG14930.

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S.S. Karuppagounder et al. / Neurochemistry International 54 (2009) 111118118

Dietary supplementation with resveratrol reduces plaque pathology in a transgenic model of Alzheimer's diseaseIntroductionMaterials and methodsAnimalsTreatment paradigmTissue preparationAnalysis of resveratrol in diet and brainAnalysis of cysteine, ascorbate, and GSHThioflavine S staining and quantitation of amyloid plaquesImmunohistochemistryImmunoblot analysisStatistical analysis

ResultsResveratrol was stable in the diet and not detectable in brainResveratrol did not alter body weight or food intake in Tg19959 miceResveratrol treatment reduced plaque pathology in Tg19959 miceResveratrol treatment did not alter amyloid precursor protein (APP) carboxyterminal fragments in Tg19959 miceResveratrol treatment increased the cysteine levels, but reduced the GSH levels in Tg19959 miceResveratrol treatment did not alter SIRT 1 levels in Tg19959 mice

DiscussionAcknowledgementsReferences