http://informahealthcare.com/ijfISSN: 0963-7486 (print), 1465-3478 (electronic)

Int J Food Sci Nutr, Early Online: 1–8! 2013 Informa UK Ltd. DOI: 10.3109/09637486.2013.832171

RESEARCH ARTICLE

Resveratrol increases cerebral glycogen synthase kinasephosphorylation as well as protein levels of drebrin andtransthyretin in mice: an exploratory study

Behzad Varamini1, Angelos K. Sikalidis1,2, and Kathryn L. Bradford1

1Division of Nutritional Sciences, Cornell University, Ithaca, NY, USA and 2Dana-Farber Cancer Institute, Harvard Medical School, Harvard Institutes

of Medicine, Boston, MA, USA

Abstract

Alzheimer’s disease (AD) is characterized by intraneuronal b-amyloid plaques and hyperpho-sphorylated tau, leading to neuronal cell death and progressive memory losses. Thisexploratory work investigates if dietary resveratrol, previously shown to have broad anti-aging effects and improve AD pathology in vivo, leads to neuroprotective changes in specificprotein targets in the mouse brain. Both wild-type and APP/PS1 mice, a transgenic AD mousemodel, received control AIN-93G diet or AIN-93G supplemented with resveratrol. Pathologyparameters and AD risk were assessed via measurements on plaque burden, levels ofphosphorylated glycogen synthase kinase 3-b (GSK3-b), tau, transthyretin and drebrin. Dietaryresveratrol treatment did not decrease plaque burden in APP/PS1 mice. However, resveratrol-fed mice demonstrated increases in GSK3-b phosphorylation, a 3.8-fold increase in proteinlevels of transthyretin, and a 2.2-fold increase in drebrin. This study broadens our understand-ing of specific mechanisms and targets whereby resveratrol provides neuroprotection.

Keywords

b-amyloid, Alzheimer’s disease, drebrin,glycogen synthase kinase 3-b,neurodegeneration, resveratrol, tau,transthyretin

History

Received 2 April 2013Revised 27 July 2013Accepted 2 August 2013Published online 10 September 2013

Introduction

Alzheimer’s disease (AD) is a progressive, age-dependentneurodegenerative disorder resulting in severe cognitive impair-ment. While the decline observed during AD is poorly under-stood, it is thought that increases in amyloid beta protein (Ab), aproduct of sequential proteolysis of amyloid precursor protein(APP), leads to neurotoxic Ab 1–42 aggregates, causing down-stream oxidative damage, neuroinflammation and hyperpho-sphorylation of microtubule associated tau-proteins, resulting inneurofibrillary tangles and subsequent neuronal death. Thepresence of intraneuronal Ab plaques and neurofibrillary tanglesin the cortex and hippocampus with concomitant neuron andmemory loss are the hallmarks of AD (Bilb et al., 2012;Delacourte et al., 2002; Howlett & Richardson, 2009). AlthoughAD’s precise cause still remains unknown, several identified riskfactors are involved in AD onset, notably, ApoE4 genotype(Leduc et al., 2010; Poirier, 2003) and various dietary factors(Kawas, 2006). Currently, no treatments have been shown to stopthe progressive loss of cognitive function manifested in AD(Klafki et al., 2006).

Animal and epidemiological studies support the hypothesisthat polyphenolic constituents found in red wine and some berries,possess bioactivities that may afford protection against

cardiovascular disease and possibly, central nervous systemdisorders such as Parkinson’s, Huntington’s and potentially AD(Li et al., 2012; Rocha-Gonzalez et al., 2008). To date, somestudies have examined dietary factors against neurodegeneration,and several naturally-occurring plant compounds have been testedin treating AD (Solfrizzi et al., 2011). One of the most promisingcompounds to emerge consistently has been resveratrol, anaturally occurring polyphenol found in grape skin and redwine (Basil et al., 2012).

A number of in vitro studies have demonstrated resveratrol’sability to protect against aging and age-associated neuronaldegradation (Parker et al., 2005) through reduced levels ofsecreted and intracellular Ab peptides (Marambaud et al., 2005;Sgarbossa, 2012). It is well established that much of resveratrol’sneuroprotective activity is partially due to its action as a calorierestriction mimetic, (Baur et al., 2006; Jiang, 2008; Kaeberlein,2013; Lagouge et al., 2006) thereby inducing the sirtuin family ofproteins whose upregulation is strongly associated with neuro-protection in several AD models (Albani et al., 2010; Anekonda &Reddy, 2006; de Oliveira et al., 2010). It has been demonstratedin vivo that, resveratrol reduced neurodegeneration and cognitivedecline in mice expressing a coactivator of cyclin-dependentkinase 5 and displaying massive forebrain degeneration with ADfeatures (Kim et al., 2007). In another study, resveratrol reducedoverall plaque pathology in a APP/PS1 AD transgenic mousemodel (Karuppagounder et al., 2009). Further, AD transgenicmice consuming a Cabernet Sauvignon red wine containingresveratrol and other polyphenols for seven months, demonstratedimproved spatial-memory functions and decreased Ab peptides(Wang et al., 2006).

Correspondence: Dr Behzad Varamini, Division of Nutritional Sciences,Cornell University, B40 Savage Hall, Ithaca, NY 14853, USA. Tel: (607)255-1031. Fax: (607) 255-1033. E-mail: bv29@cornell.edu

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While several mechanisms for resveratrol’s protectiveeffects in AD have been proposed, further identification andelucidation of targets is needed. Several studies suggest thatresveratrol could also act on sirtuin-independent targets that leadto neuroprotection (Albani et al., 2010; Barger et al., 2008;Dasgupta & Milbrandt, 2007). Additionally, resveratrol wasshown to modulate glycogen synthase kinase-3 (GSK3), a proteincentral to a variety of biological processes including neurode-generation, in vitro (Cecchinato et al., 2007; Klinge et al., 2005;Lin et al., 2011). Further, resveratrol’s interaction with post-synaptic receptors suggests that its neuroprotective benefits couldalso be exerted at the synapse environment (Basil et al., 2012;Zhang et al., 2008). Developmentally regulated brain protein(drebrin) is a key post-synaptic protein critical to maintainingproper synaptic function, losses of which have been reported inAD (Dun et al., 2012; Shim & Lubec, 2002). Finally, in vitro,resveratrol has been postulated to affect transthyretin (TTR), anAb scavenger (Klinge et al., 2005). Here we report our resultsfrom testing the hypothesis that resveratrol modulates theaforementioned pivotal proteins in vivo both in AD transgenicand wild-type mice.

Materials and methods

Animals

All procedures involving animals were approved by CornellUniversity’s Institutional Animal Care and Use Committee priorto commencing experiments. Aged male (41–44-weeks-old)B6.Cg-Tg (APPswe, PSEN1DE9)85Dbo/J and standard wild-type (wt) genotypic age-matched controls were purchased fromJackson Laboratories (Bar Harbor, ME). These transgenic miceexpress two mutations associated with early-onset AD: a chimericmouse/human amyloid precursor protein (Mo/HuAPP695swe)and a mutant human presenilin 1 (PS1-dE9). Phenotypically, themice spontaneously develop Ab plaque by 6–7 months of age withprogressive increases in plaque up to 19 months (Garcia-Allozaet al., 2006; Savonenko et al., 2005). Experimentally, all micewere housed in vented pathogen-free cages at a constanttemperature (22� 1 �C), humidity (44� 4%) and illumination(12-h light/dark repeated cycles) with fresh food and waterprovided ad libitum daily.

Treatments

Resveratrol (498% purity) was purchased from OrchidPharmaceuticals (Aurangabad, India) tested and mixed to homo-geneity in the dark during manufacturing of the diets. Two dietswere used: Conventional AIN-93G (control) and AIN-93Gsupplemented with 0.19% w/w resveratrol.

A total of 18 male mice were used, nine wild-type and ninetransgenic. During a nine-week acclimation period where all micewere singly housed in holding cages at Cornell University’sfacility and fed AIN-93G diet ad libitum, the experimental dietaryregimes began at 50–53 weeks of age. Of the wild-type mice, sixmice were assigned to receive control AIN-93G diet (Dyets Inc,Bethlehem, PA) while three mice were assigned to receive AIN-93G supplemented with resveratrol. Of the transgenic mice, threemice received the control diet and six mice received theresveratrol-supplemented AIN-93G (r-AIN-93G). The dailyresveratrol consumption is calculated to be 174 mg/kg/d (3.3 gfood per day for a 36 g mouse) for 16 weeks. Diets were stored at4 �C and replaced with fresh in all cages daily. All animals wereinspected daily while body weight and feed intake measurementswere performed at standard times on a weekly basis throughoutthe experimental period.

Tissue preparation

After 16 weeks on the dietary intervention, mice were terminatedby CO2 inhalation and rapidly dissected. Brains were quicklyremoved and a thin coronal section containing cerebrum andhippocampus was excised using a rodent brain matrix and fixed in10% neutral buffered formalin. The rest of the brain was separatedinto several regions, flash frozen in liquid nitrogen, and stored at�80 �C until analysis.

Immunoblot analysis

Right cerebrum was homogenized in ice-cold lysis buffer(150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 50 mM TrispH 7.5) with protease (Protease inhibitor cocktail, SigmaAldrich, St. Louis, MO) and phosphatase (PhosSTOP Roche,Indianapolis, IN) inhibitors as indicated by manufacturer.Samples for immunoblot analyses were processed as describedpreviously (Sikalidis & Stipanuk, 2010). Briefly, samples werecentrifuged for 4 minutes at 4 �C at 13 000� g to obtain thesoluble protein fraction. Protein concentrations were determinedby a bicinchoninic acid (BCA) assay (Pierce ChemicalCompany, Rockford, IL). The amount of 25 mg of protein wasloaded and analyzed by one-dimensional SDS-PAGE (12% w/vacrylamide), then electroblotted at 4 �C overnight onto 0.45 mmImmobilin-P PVDF membranes (Millipore, Medford, MA) andimmunoblotted for drebrin (1:1000, Abcam, Cambridge, MA),insulin degrading enzyme (1:500, Abcam, Cambridge, MA),transthyretin (1:5000, Abcam, Cambridge, MA), total glycogensynthase kinase 3-b (1:1000, Cell Signaling Technology,Danvers, MA), and phospho-glycogen synthase kinase 3-b(Ser-9) (1:1000, Cell Signaling Technology, Danvers, MA).Visualization of bands was accomplished using horseradishperoxidase-coupled (HRP) secondary antibodies and chemilu-minescent substrates (West Dura, Pierce) with exposure toautoradiography film. Film images were digitized and analyzedusing NIH ImageJ 1.63 software (National Institutes of Health,Bethesda, MD). Band intensities were normalized againstcorresponding bands for b-actin, tau and glycogen synthasekinase 3-b for loading and transfer controls.

Immunohistochemistry

Coronal sections of 5mm thickness were cut with a slidingmicrotome and processed as free floating sections at CornellUniversity’s Histology Laboratory. Briefly, sections were washedwith TBS pH 7.6 and incubated in 3% hydrogen peroxide forfive minutes to block endogenous peroxidase activity. Sectionswere blocked using rabbit serum and incubated with monoclonalmouse anti-human beta-amyloid 6F/3D primary antibody, 1:50(DakoCytomation, Glostrup, Denmark) in antibody diluent for90 minutes. The secondary antibody, a biotinylated goat anti-mouse, was applied and the slides were incubated for 15 minutesat room temperature. Sections were then incubated in strepta-vidin-peroxidase conjugate for 10 minutes at room temperature.The chromogen, 3,3-diaminobenzidine-tetra hydrochloride (DABfrom DakoCytomation) was applied to the slides for one minuteat room temperature and slides were counterstained usinghematoxylin for two minutes and rinsed with distilled water.Slides were dehydrated using ethyl alcohol and cleared withxylene before applying cover-slips using Permount mountingmedia (Fisher Scientific, Pittsburgh, PA).

Plaque counts and percentage occupied by the 6F/3D antibody,were quantified in the cerebrum and hippocampus. The region ofinterest was drawn manually under 4� magnification, the imageswere set to threshold, and plaques were analyzed for major

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changes using Metamorph 7.1 software (Molecular Devices,Sunnyvale, CA). Multiple trained assessors confirmed the resultsof the analysis in a blinded fashion.

Brain Ab ELISA analysis

Left cerebrum was homogenized in carbonate buffer (100 mMNa2CO3, 50 mM NaCl, pH 11) containing protease inhibitors(Protease inhibitor cocktail, Sigma Aldrich, St. Louis, MO).The homogenate was centrifuged at 14 000� g for 20 minutes at4 �C. The carbonate soluble supernatant fraction was transferredto a new tube and stored at �80 �C until analysis. The pellet wasfurther homogenized in guanidine solution (5 M guanidine HCl in50 mM Tris-HCl, pH 8.0). The homogenate was mixed by rockingfor four hours at room temperature and centrifuged at 14 000� gfor 20 minutes at 4 �C. After centrifugation, the supernatant(insoluble fraction) was transferred to a new tube and stored at�80 �C until analysis. Soluble and insoluble Ab40 and Ab42levels were determined using the Human b-Amyloid 1-40 and1-42 ELISA kits (Invitrogen, Camarillo, CA) according to themanufacturer’s protocols.

Statistics

Values reported are means� SEM and significance was acceptedat p50.05. Statistical significance was tested by two tailedStudent’s t-test and ANOVA using JMP 7 (SAS Institute Inc,Cary, NC).

Results

Resveratrol treatment increased levels of phosphorylatedGSK-3 b, drebrin and transthyretin in cerebrum of wildtype and transgenic mice

Neither body weight nor total food intake varied between dietarygroups (Table 1). Western blot analysis demonstrated that dietaryresveratrol led to significantly increased levels of phosphylatedGSK3-b in both the wild-type and AD transgenic groups (1.9-foldphosphorylation increase, Figure 1A, p50.05, pooled data/resveratrol effect), while genetic background did not change themeasured outcome (Figure 1B). For GSK3 measurements, levelsof phosphorylated GSK3-b (associated with inhibition of GSK3-bactivity) were normalized to total levels of GSK-b. Resveratrolfeeding also significantly increased drebrin protein levels in bothwild-type and transgenic animals (2.2-fold increase, Figure 2A;p50.05, pooled data/resveratrol effect), while genetic back-ground did not change the measured outcome (Figure 2B). Inaddition, resveratrol significantly increased levels of TTRcompared to control diet fed mice (3.8-fold increase, Figure 3A;p50.05, pooled data/resveratrol effect), while genetic back-ground did not change the measured outcome (Figure 3B).However, resveratrol did not increase TTR in the resveratrol-fedtransgenics compared to resveratrol-fed wild-type mice(p¼ 0.11).

Resveratrol did not alter plaque load and levels of Abprotein secreted or tau phosphorylation in APP/PS1transgenic AD mice

Quantification of plaque areas in stained brain tissue sectionsrevealed no obvious differences in plaque burden in the

Figure 1. GSK3-beta phosphorylation levels in the brain of wild-type and transgenic mice fed control versus resveratrol supplemented diet. (A) TotalGSK3-beta and phospho-GSK3-beta (Ser-9) from brain were measured and levels of phosphorylated GSK3-beta were normalized to total levels ofGSK3-beta. Data represent means� SEM of wild-type control diet treated (N¼ 6), wild-type resveratrol treated (N¼ 3), transgenic control diet-treated(N¼ 3) and transgenic resveratrol-treated (N¼ 6) groups, (p50.05). (B) Total GSK3-beta and phospho-GSK3-beta (Ser-9) from brain were measuredand levels of phosphorylated GSK3-beta were normalized to total levels of GSK3-beta. In both wild-type and transgenic groups, resveratrolsupplementation significantly decreased GSK3 activity (1.9-fold phosphorylation increase; p50.05, pooled data shown). Data represent means� SEMof control (N¼ 9) and resveratrol (N¼ 9) groups. Representative Western blot images shown.

Table 1. Body weights and feed intake for APP/PS1 transgenic AD mice.

Prior to treatment* Post treatment

Dietary groupAIN93G(control) Resv-AIN93G

AIN93G(control) Resv-AIN93G

Body weight (g) 33.0� 0.8 33.8� 1.0 37.5� 0.7 39.0� 0.9Food intake

(g/day)3.0� 0.1 3.1� 0.1 3.1� 0.1 3.2� 0.2

Body weight and feed intake measurements were recorded on a weeklybasis throughout the experimental period. Changes in body weight andtotal food intake did not vary between dietary groups. Data representmeans� SEM for control (N¼ 9) and 0.19% wt/wt resveratrol (N¼ 9)groups.

*Treatment was 16 weeks dietary resveratrol supplementation at 0.19%wt/wt. Before treatment, all mice consumed standard control diet,AIN93G.

DOI: 10.3109/09637486.2013.832171 Resveratrol provides neuroprotection in mice 3

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hippocampus or cerebrum between dietary groups, although thepossibility of subtle differences cannot be entirely ruled out(Figure 4). Quantification, by ELISA, of Ab contents in thecarbonate soluble and insoluble (guanidine-soluble) fractions inthe cerebrum was conducted to determine the effect of dietaryresveratrol on cerebral Ab protein levels. No significant differ-ence in either soluble or insoluble Ab40 and Ab42 was revealedbetween groups (Figure 5). These results are consistent with the

immunohistochemical analyses that showed no significant differ-ence between Ab plaque deposition in hippocampus andcerebrum between dietary groups. Resveratrol administrationalso did not result in differences in tau phosphorylation at Ser-396and Ser-404, sites whose hyperphosphorylation is associated withAD (Figure 6).

In summary, compared to control diet feeding, mice fedresveratrol-supplemented diet for 16 weeks exhibited higher

Figure 2. Resveratrol treatment increased levels of drebrin in the brain. (A) Protein levels of drebrin in the brain of wild-type and transgenic mice fedcontrol diet and resveratrol supplemented. Data represent means� SEM of wild-type control diet treated (N¼ 6), wild-type resveratrol treated (N¼ 3),transgenic control diet-treated (N¼ 3) and transgenic resveratrol-treated (N¼ 6) groups (p50.05). (B) Resveratrol feeding significantly increaseddrebrin levels in both wild-type and transgenic mice (2.2-fold increase; p50.05, pooled data shown). Data represent means� SEM of control (N¼ 9)and resveratrol (N¼ 9) groups. Representative Western blot images shown.

Figure 3. Cerebrum protein levels of TTR in response to resveratrol treatment. (A) Protein levels of TTR in the brain of wild-type and transgenic micefed control diet and resveratrol supplemented. Data represent means� SEM of wild-type control diet-treated (N¼ 6), wild-type resveratrol-treated(N¼ 3), transgenic control diet-treated (N¼ 3) and transgenic resveratrol-treated (N¼ 6) groups, (p50.05). (B) Resveratrol significantly increasedlevels of TTR in wild-type and transgenic animals compared to control diet (3.8-fold increase; p50.05, pooled data shown). Data representmeans� SEM of control diet (N¼ 9) and resveratrol diet (N¼ 9) groups. Representative Western blot images shown.

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cerebrum levels of phospho-GSK-3b, drebrin and TTR protein,regardless of genotype. Resveratrol-fed mice did not demonstratedifferent Ab plaque loads in the hippocampus or cerebrum, orsecreted Ab levels and tau phosphorylation in cerebrum. Takentogether our data suggest that resveratrol daily dietary intake at alevel of 0.19% w/w, exerts a protective effect for AD in the brainof mice especially at the early signaling level.

Discussion

The serine/threonine protein kinase GSK3, was originallyidentified as an enzyme which regulates glycogen synthesis butis now known to affect numerous physiological events byinteracting with a number of substrates in insulin resistance,tumorigenesis, inflammation, cardiac/cardiovascular function andneurodegeneration (Wada, 2009). Further, GSK3 is intimatelyinvolved with memory formation, inflammation, as well as tauphosphorylation and other pathological hallmarks of AD, thusleading a number of researchers to classify it as a highlypromising pharmaceutical target and formulate the so-termedGSK3 hypothesis of AD (Avila et al., 2010; Hooper et al., 2008).Previous studies have shown that GSK co-localizes with neuro-fibrillary tangles (Ly et al., 2013; Pei et al., 1999) and its activityis increased in the frontal cerebrum and hippocampus in AD(Blalock et al., 2004; Leroy et al., 2007; Wang et al., 2011). In thecontext of studies examining apoptosis, cell cycle regulation andischemia, in vitro data have confirmed resveratrol’s interactionwith the phosphatidylinositol-3-kinase/Akt pathway leading to aninactivation of GSK3-b by phosphorylation of Ser-9 (Benitezet al., 2007; Cecchinato et al., 2007; Filippi-Chiela et al., 2011).However, to date, no studies have reported the effect of dietaryresveratrol in a mammalian system particularly on the regulationof brain GSK3. In our study, mice fed resveratrol demonstrated1.9-fold increase in GSK3-b Ser-9 phosphorylation compared tocontrol diet (Figure 1). Phosphorylation at Ser-9 inhibits GSK3-bactivity; namely, aberrant phosphorylation of tau on a number ofdisease-associated epitopes such as Ser-396 and Ser-404 (Bilbet al., 2012). Surprisingly, we detected no differences in total tauor phospho-tau Ser-396 and Ser-404 (Figure 6). No change in tauphosphorylation could be explained by several factors. First, whenacting alone, GSK3-b phosphorylation of tau occurs at very slowrates and increases rapidly when tau is pre-phosphorylated atseparate sites by other protein kinases, such as PKA on Ser-214thus initiating a rapid site-sequential phosphorylation cascade(Singh et al., 1995). Since resveratrol’s inhibition of GSK3-b didnot lead to differences in phospho-tau Ser-396 and Ser-404, it ispossible that tau was not sufficiently primed via pre-phosphor-ylation by other kinases. Second, our measurement was on thesoluble fraction of the brain lysate, and that insoluble tau couldrepresent a different phosphorylation pattern on Ser-396 and Ser-404. Third, other post-translational modifications of tau such asglycosylation have been shown to play a significant role in itsregulation of phosphorylation. For example, deglycosylation oftau suppresses subsequent phosphorylation at Ser-404 (Liu et al.,2002). Regardless, clearly resveratrol is able to phosphorylate andinactivate the critical target GSK3-b and that could well representan important mechanism underlying resveratrol’s neuroprotectiveeffects.

Drebrin is a dendritic spine protein which plays an importantrole in neurogenesis and synaptic function, losses of which havebeen reported beyond 70% in a number of separate independentstudies and in subjects with only mild cognitive impairment(Geraldo et al., 2008). Levels of drebrin are significantlydecreased in AD and correlate inversely with tau pathology andBrack scores (Counts et al., 2012; Dun & Chilton, 2010;Dun et al., 2012; Julien et al., 2008; Kojima & Shirao, 2007)

Figure 5. Cerebrum protein levels of secreted Ab proteins in mice.(A) ELISA analyses of Ab40 and Ab42 levels in the carbonate solublefraction of cerebrum homogenate show no significant differences betweendietary groups. (B) ELISA analyses of Ab40 and Ab42 levels in thecarbonate insoluble (guanidine-soluble) fraction of cerebrum homogenateshow no significant differences between dietary groups. Data representmeans� SEM of control (N¼ 3) and 0.19% resveratrol (N¼ 6) groups.

Figure 4. Resveratrol did not alter plaque load in APP/PS1 transgenic ADmice. (A) Representative coronal brain sections from AD transgenic miceshowing hippocampus and cortex stained with antibody specific forextracellular beta-amyloid. (B) Graph describes percentage of areaoccupied by plaques from the hippocampus and cortex. Data representmeans� SEM of control (N¼ 3) and 0.19% resveratrol (N¼ 6) groups.

DOI: 10.3109/09637486.2013.832171 Resveratrol provides neuroprotection in mice 5

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while positively correlate with Mini-Mental State Examinationscores (Counts et al., 2012). In our study, resveratrol-fed miceexhibited 2.2-fold increases in cerebrum drebrin (Figure 2).Increases were observed regardless of wild-type or transgenicstate, with both groups of animals exhibiting increased drebrinwhen fed resveratrol. Differences in drebrin levels did not existbetween wild-type and AD mice on control diet, therefore thistransgenic model does not mimic the common loss of drebrin inAD. Since drebrin has been demonstrated to constitute animportant predictor in memory function in even mildly impairedsubjects, increases in drebrin induced by diet could lead toimproved neuronal health in populations that are otherwisedementia-free (Counts et al., 2006).

As evidence has indicated an association between altered APPprocessing and increased amyloid production and the develop-ment of AD (Bertram & Tanzi, 2008), a concerted effort hasbeen made to develop drugs and treatments that specificallydecrease the production or increase the clearance of Ab(van Marum, 2008).

At least 20% of the total protein in the cerebrospinal fluid isrepresented by TTR, which is synthesized and secreted by thechoroids plexus (Schreiber, 2002) and has been shown to bind andsequester Ab protein and prevent Ab aggregation (Tsuzuki et al.,2000). As shown in vitro, purified TTR inhibits Ab fibrils(Guerreiro et al., 2012) and in Caenorhabditis Elegans leads tosignificant reductions in Ab plaques (Diomede et al., 2013).Further, an inverse relationship has been found between levels ofTTR in human cerebrospinal fluid and severity of AD (Ribeiroet al., 2012). Previous studies have shown that resveratrol inhibitsamyloidogenic variant M-TTR-induced cytotoxicity (Reixachet al., 2006). Additionally, resveratrol-binding sites have beendiscovered broadly in the rodent brain and appear mostconcentrated in the choroids plexus where TTR is produced(Han et al., 2006). In our study, dietary resveratrol increasedlevels of TTR protein by 3.8-fold (Figure 3). However, increasedTTR levels did not translate into decreases in Ab plaque, and TTR

levels were not significantly higher in resveratrol-fed AD miceversus control diet AD mice, though a notable trend in the meanvalues suggested an increase (p¼ 0.11), (Figure 4). Resveratrolhas been shown to be a potent mitogen-activated protein kinase(MAPK) activator, leading to activation of protein kinase A(PKA) or protein kinase C (PKC) (Klinge et al., 2005). KinasesPKA and PKC regulate cAMP response element binding protein(CREB), which is suggested to activate downstream genes such asTTR and others involved in memory (Miyamoto, 2006;Taubenfeld et al., 2001). Our data, along with previous studies,suggest that resveratrol could increase TTR expression throughbinding at the choroids plexus, potentially leading to activationand initiation of MAPK, PKA, PKC or CREB signaling cascades.In this study, it is possible that increases in TTR were notsignificant enough to translate into measurable differences in Ab,or that TTR has a limited window of opportunity during Abaccumulation to sequester and prevent intraneuronal Ab aggre-gation. In spite of this, dietary modulation of TTR by resveratrolprovides a new putative mechanism whereby resveratrol couldexert anti-amyloidogenic effects.

Similar to the findings of Karuppagounder and colleagues, ourstudy did not find differences in levels of hippocampal plaquebetween control diet and resveratrol-fed AD mice(Karuppagounder et al., 2009). Interestingly they also do notsee a difference in the amyloid precursor protein. Some differ-ences in the plaque load at the cortex region may be attributed tothe somewhat different AD model used (especially in the responseof the cortex) and the oxidation status as implied by the markedlydifferent levels of glutathione. Despite no obvious changes inamyloid biology, our study reveals putative mechanisms wherebyresveratrol may mediate neuroprotection, especially at the initial/early signaling level. Similarly, Joseph and colleagues reportedthat dietary supplementation with blueberries (known to containresveratrol) prevents cognitive impairment by promoting signal-ing processes associated with learning and memory withoutdetectable changes in amyloid deposition (Joseph et al., 2003).

Figure 6. Levels of phosphorylated tau at Ser-396 or Ser-404 in the cerebrum region of mouse brain. Total tau and P-tau (Ser-396 and Ser-404) frombrain cerebrum was measured and levels of phosphorylated tau were normalized to total levels of tau. Resveratrol did not significantly alter levels of tauphosphorylation at sites Ser-396 or Ser-404. Representative Western blot images shown.

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In humans, a number of studies show that progressive neurode-generation may occur in AD patients despite removal of plaques,and many cognitively normal humans display Ab plaques inequivalent densities as Alzheimer’s diseased individuals (Daviset al., 1999; Holmes et al., 2008). Our findings confirm andextend the role of resveratrol as a plausibly neuroprotectivenutrient and open the door to a variety of new mechanisms andprotein targets whereby resveratrol exerts its beneficial action,such as degradation of Ab (TTR) positive structural and post-synaptic changes (drebrin), and inhibition of tau pathology(GSK). It will be worthwhile to investigate whether these broadpleiotropic effects are SIRT1-dependent or could also be foundunder conditions of calorie restriction.

Acknowledgements

The authors are gratefully indebted to the guidance, advice, and use oflaboratory resources provided by Dr J. Thomas Brenna at CornellUniversity. In addition, the authors acknowledge the kind assistance ofSylvia Allen at the Biotechnology Mouse Facility, Marlene Nardi atCornell University’s Histology laboratory, Carol Bayles at Cornell’sIntegrated Microscopy Center, and the Soloway laboratory at CornellUniversity for providing equipment for brain dissections.

Declaration of interest

The authors declare no conflicts of interest.BV was supported by NIH grant DK07158.

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