Free Radical Biology & Medicine 48 (2010) 831–838

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Free Radical Biology & Medicine

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Original Contribution

Green tea averts age-dependent decline of hippocampal signaling systems related toantioxidant defenses and survival

Marco Assunção a, Maria J. Santos-Marques a, Félix Carvalho b, José P. Andrade a,⁎a Department of Anatomy, Faculty of Medicine, University of Porto, 4200-319 Porto, Portugalb REQUIMTE, Toxicology Department, Faculty of Pharmacy, University of Porto, 4099-030 Porto, Portugal

⁎ Corresponding author. Fax: +351 22 5513617.E-mail address: jandrade@med.up.pt (J.P. Andrade).

0891-5849/$ – see front matter © 2010 Elsevier Inc. Adoi:10.1016/j.freeradbiomed.2010.01.003

a b s t r a c t

a r t i c l e i n f o

Article history:Received 16 October 2009Revised 28 December 2009Accepted 1 January 2010Available online 11 January 2010

Keywords:AgingGreen teaCatechinsGlutathioneAntioxidant enzymesCREBBDNFBcl-2Hippocampal formationRatFree radicals

We previously found that prolonged consumption of green tea (GT), a rich source of antioxidantpolyphenols, protected proteins and lipids against oxidation and reduced lipofuscin deposition in the rathippocampal formation as well as improving spatial memory during aging. In this work, we sought toinvestigate whether GT treatment could interfere with age-related changes in redox status and cellularsignaling systems related to oxidative stress and survival in the same brain region. To address this issue, fivemale Wistar rats were fed with GT from 12 to 19 months of age and results were compared to those obtainedfrom controls age 19 months (C-19 M). A third group of rats was evaluated at 12 months of age to providebaseline data. At completion of the specified time points, the glutathione levels and antioxidant enzymeactivities, the activation of the transcription factors cyclic AMP response element-binding (CREB) andnuclear factor-κB (NF-κB, p50 and p65 subunits), and the levels of brain-derived neurotrophic factor (BDNF)and B-cell lymphoma-2 (Bcl-2) were measured in hippocampal formations. GT-treated rats presented higherreduced and lower oxidized glutathione levels and displayed favorable alterations in antioxidant enzymeactivities compared to C-19 M animals. In addition, GT increased CREB activation and the levels of BDNF andBcl-2, but had no effect on activation of NF-κB subunits, relative to age-matched controls. We conclude thatlong-term GT ingestion improves antioxidant systems and activates CREB in the aging rat hippocampalformation, leading to neuroprotection mediated by downstream upregulation of BDNF and Bcl-2.

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Introduction

There is currently a great research effort to understand themolecular mechanisms underlying the loss of cellular functions thataccompanies the advancement of age [1,2]. The positive evolution ofhuman longevity has increased the prevalence of several degenerativediseases, representing a potential heavy social and economical burdento society [3]. Particularly incapacitating are the neurodegenerativediseases because of the impairment of neuronal functions, which havevarying, but generally dramatic, consequences depending on theaffected brain region [4]. Neurodegeneration has been associated withmetabolic and hormonal changes resulting in deleterious alterationsin several neuronal organelles such as the mitochondria [5],endoplasmic reticulum [6], and lysosomes [7]. The accumulation ofoxidative modifications in macromolecules seems to play a pivotalrole in age-related neuronal damage, triggering a cascade of eventsthat leads to neuronal malfunctions and stimulates further increasesin inflammatory signaling that may aggravate initial damage [8]. In anattempt to cope with these undesirable cellular responses, signaling

pathways related to oxidative stress defense and survival areactivated, leading to the recruitment of molecules with antioxidant,antiapoptotic, and growth-related activities [9,10].

It has been hypothesized that dietary components such as thepolyphenols may assist in that recruitment by increasing theexpression of molecules involved in neuronal protection duringaging [11,12]. In this context, green tea (GT), a polyphenol-richbeverage largely consumed worldwide, has been associated with theprotection of cellular macromolecules from oxidative damage andmodulation of several oxidative stress markers in various experimen-tal settings [13–15]. In accordance, we have recently demonstratedthat chronic GT consumption decreases age-related protein and lipidoxidation as well as lipofuscin deposition in rat hippocampalformation [16], a brain region involved in information processingallowing acquisition and memory retention [17,18]. Therefore, it wasnot surprising that hippocampal-dependent cognitive declines ob-served during aging were also significantly prevented by GTtreatment [16]. Similar findings have been described by others whoreported favorable effects of catechins, the main polyphenoliccompounds of GT, on neurodegenerative alterations using rodentmodels of brain aging [19–22].

Although GT polyphenols possess intrinsic antioxidant capacity,very little is known about the mechanisms underlying the beneficial

832 M. Assunção et al. / Free Radical Biology & Medicine 48 (2010) 831–838

effects of this beverage on specific cerebral regions [23]. Therefore, inthis study, we intended to evaluate whether prolonged GT ingestionmight interfere with cellular defensive functions in the aging rathippocampal formation. Measurements of antioxidant defensivesystems such as glutathione levels and antioxidant enzyme activities[24] were performed in hippocampal homogenates. We also evalu-ated the activation of transcription factors involved in neuronalsignaling systems related to oxidative stress and survival [25], namelycyclic AMP response element-binding (CREB) and nuclear factor-κB(NF-κB, p50 and p65 subunits). These transcription factors were usedto evaluate the balance between prosurvival and proinflammatorystates as seen in other experimental settings such as after dietaryenergy restriction [25,26], ethanol consumption [27], or ingestion offlavonoids [28]. Finally, the levels of brain-derived neurotrophic factor(BDNF) and the antiapoptotic B-cell lymphoma-2 (Bcl-2) protein,known to be regulated by the above-mentioned transcription factorsand both associated with neuronal plasticity and implicated inneurogenesis [29–31], were also quantified in the hippocampalformation.

Materials and methods

Chemicals and reagents

All chemicals used were of analytical grade and the solutions wereprepared using distilled water. Reduced glutathione (GSH), oxidizedglutathione (GSSG), glutathione reductase (GR; EC 1.8.1.7), reducednicotinamide adenine dinucleotide phosphate (NADPH), 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB), 2-vinylpyridine, Hepes, KCl, EDTA,EGTA, protease inhibitors (aprotinin, leupeptin, and pepstatin),Igepal, DTT, PMSF, glycerol, and all other reagents necessary forenzymatic determinations were obtained from Sigma–Aldrich (St.Louis, MO, USA). All other chemicals were purchased from Merck(Darmstadt, Germany) unless otherwise stated.

Animals and treatments

Fifteen male Wistar rats were obtained from Charles River(Barcelona, Spain) and maintained individually under controlledlight (12-h light/dark cycle) and temperature (21–22°C) conditions.At 12 months of age, animals weighing 614 ± 12 g were randomlydivided into three groups of five rats each. Control rats had access totap water and were evaluated at 12 (C-12 M) or 19 (C-19 M) monthsof age. GT-treated rats (GT-19 M) drank an infusion of GT as the soleavailable fluid from 12 to 19 months of age. All groups had access tostandard pellet food throughout the experiment. Ad libitumingestion of both food and fluid was monitored every other dayand rat weight was recorded weekly. To prepare GT, 1 L of boilingwater (100°C) was dispensed over three green tea bags (1.3 g perbag) and infused for 5 min. The infusion was allowed to cool to roomtemperature and transferred to dark bottles to avoid oxidation oflight-sensitive components and renewed every other day. Thecatechin composition of the GT, in a representative sample, wasdetermined by high-performance liquid chromatography [32] andwas as follows: (−)-epigallocatechin-3-gallate (EGCG), 556.7 mg/L;(−)-epicatechin, 243.4mg/L; (−)-epicatechin-3-gallate, 126.4mg/L;(+)-gallocatechin-3-gallate, 32.5 mg/L. All experimental procedureswere conducted following the European Community guidelines(86/609/EEC) and the Portuguese Act (129/92) for the use ofexperimental animals. Every possible effort was made to minimizethe number of animals used and their suffering.

Tissue collection and preparation

All rats were deeply anesthetized by intraperitoneal injection ofsodium pentobarbital [80 mg/kg body weight (bw)] and transcar-

dially perfused with saline solution (4°C). The brains were rapidlyremoved from the skulls into a petri dish placed on ice. Eachhippocampal formation was immediately dissected, weighed, codedto allow blind estimations, frozen in liquid nitrogen, and stored at−80°C until further processing. Randomly chosen left or righthippocampal formations were homogenized in ice-cold 50 mMphosphate buffer (pH 7.4) with 0.1% (v/v) Triton X-100 using aglass–Teflon homogenizer. Homogenates were centrifuged at16,000 g for 10 min at 4°C. Aliquots of the resulting supernatantswere collected and stored at −80°C for quantification of glutathioneredox status and antioxidant enzyme activities. Quantification of theprotein concentration in supernatants was performed according tothe method of Lowry et al. [33], using bovine serum albumin asstandard. The contralateral hippocampal formations were used for thepreparation of cytoplasmic and nuclear extracts. Briefly, samples werehomogenized in 1ml of cell lysis buffer, pH 7.9 (10mMHepes, 10 mMKCl, 1.5 mMMgCl2, 0.5 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.25 mMPMSF, and 30 μl/ml Igepal), and after vortexing were incubated on icefor 15 to 30min. The lysates were centrifuged at 2000 g for 10min andthe cytoplasmic extracts were collected and stored at −80°C untilquantification of Bcl-2 and BDNF by enzyme-linked immunosorbentassay (ELISA). Pellets were then resuspended in 60 μl of nucleus lysisbuffer, pH 7.9 (20 mM Hepes, 420 mM NaCl, 1.5 mM MgCl2, 0.5 mMEDTA, 20% glycerol, 1 mM DTT, 0.25 mM PMSF, and 5 μg/ml proteaseinhibitor cocktail containing 1 mg/ml aprotinin, 1 mg/ml pepstatin,and 1mg/ml leupeptin), vigorously vortexed, and incubated on ice for30 min. Lysates were centrifuged at 16,000 g for 20 min at 4°C and thenuclear extracts collected, aliquotted, and stored at −80°C forposterior semiquantification of NF-κB (p50 and p65 subunits) andCREB by oligonucleotide-based ELISA. The protein content of bothextracts was determined by the Bradford method [34], using bovineserum albumin as standard. All the biochemical assays were adaptedto a 96-well plate reader (Infinite M200; Tecan, Switzerland).

Measurement of glutathione redox status

Total glutathione (GSX) and GSSG levels were evaluated kineti-cally at 415 nm by the DTNB–GR recycling assay and measured fromstandard curves as previously described [35]. Both GSX and GSSGcontents were expressed as nmol/mg protein. GSH content wascalculated by subtracting GSSG (in GSH equivalents) from the GSXconcentration.

Measurement of antioxidant enzyme activities

Enzyme activities were determined as described earlier [36]. Totalsuperoxide dismutase (SOD) (combined copper and zinc (Cu/Zn) andmanganese (Mn) SOD) activity was assessed based on the inhibitionof nitroblue tetrazolium (NBT) reduction upon superoxide generationby the xanthine–xanthine oxidase system at 560 nm and expressedas units/mg protein (1 unit is the amount of SOD required to inhibitthe rate of NBT reduction by 50%). Catalase (CAT) activity wasevaluated by monitoring the rate of H2O2 decomposition at 240 nmusing an extinction coefficient (ɛ) of 0.0394 M−1 cm−1 and expressedas units/mg protein, 1 unit being defined as the amount of enzymerequired to hydrolyze 1 μmol of H2O2 per minute [37]. GR activity wasassayed spectrophotometrically by following NADPH oxidation at340 nm and expressed as nmol of NADPH/min/mg protein (ɛ=6.2 mM–1 cm–1). Selenium-dependent glutathione peroxidase (Se-GPX) activity was measured by monitoring NADPH oxidation at340 nm when GSSG is reduced back by GR and expressed as nmolof NADPH/min/mg protein (ɛ= 6.2 mM−1 cm−1). GlutathioneS-transferase (GST) activity was assayed through the formation ofGSH conjugates with 1-chloro-2,4-dinitrobenzene at 340 nm andexpressed as nmol of S-2,4-dinitrophenylglutathione/min/mg pro-tein (ɛ= 9.6 mM−1 cm−1).

Fig. 1. Hippocampal glutathione levels in 12-month-old (C-12 M), 19-month-old(C-19 M), and 19-month-old GT-treated (GT-19 M) rats. (A) Reduced form, GSH; (B)oxidized form, GSSG. Columns represent means and vertical bars correspond to 1 SEM.⁎⁎Pb 0.01 vs C-12M; ⁎⁎⁎Pb 0.0001 vs C-12M;+Pb0.05 vs C-19M;++Pb0.01 vs C-19M;n= 5 in each group.

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Measurement of activation of transcription factors

Activated p50 and p65 NF-κB subunits and phospho-Ser133 CREBwere assessed by oligonucleotide-based ELISA (Upstate, Lake Placid, NY,USA) in nuclear extracts. During the assay, a double-stranded biotiny-lated oligonucleotide containing the flanked DNA-binding consensussequence (capture probe) for NF-κB (5′-GGGACTTTCC-3′) or CREB (5′-TGACGTCA-3′) was incubated for 2 h at room temperature, on a shakingplatform, simultaneous with the nuclear extract in a 96-well streptavi-din-coated microtiter plate. The active NF-κB subunits or CREB wereimmobilized on the biotinylated double-stranded capture probe boundto the streptavidin-coated well. After the incubation period any inactiveunbound material was washed away with the appropriate buffer. Thebound NF-κB subunits and CREB were detected independently withspecific monoclonal primary antibodies incubated for 1 h at room tem-perature. The plateswere thenwashedwith buffer and incubatedwith ahorseradish peroxidase-conjugated secondary antibody for 30 min atroom temperature. Color was developed after the addition of thehydrogen peroxide/tetramethylbenzidine (TMB) chromogenic sub-strate and was proportional to the quantity of each of the transcriptionfactors analyzed in each sample. The reaction was stopped by adding0.5 M HCl. Specificity of the binding to the nucleotide sequence wasdetermined by the incubation of specific competitor double-strandedoligonucleotide, nonspecific double-stranded oligonucleotide, andpositive cell extract alongwith the samples. Absorbancewasmeasuredat 450 nm using a 96-well plate reader. Background values weresubtracted and results are shownas optical density (OD) of the samplesdivided by the protein content of the extract expressed in milligrams.

Measurement of BDNF and Bcl-2 levels

BDNF was assayed in the cytoplasmic extracts using the ChemiKineBDNF sandwich ELISA kit (Upstate) according to the manufacturer'sinstructions. Briefly, the cytoplasmic extracts and BDNF standards wereincubated overnight on a shaking platform at 4°C, followed by washingwith the appropriate buffer. Plates were incubated with biotinylatedmouse anti-human BDNF monoclonal antibody at room temperaturefor 2 h on a shaking platform,washed, and incubatedwith streptavidin–enzymeconjugate for 1hat roomtemperature. Subsequently, theplateswere incubatedwith peroxidase substrate and TMB solution to achievea colorimetric reaction. The reaction was stopped by adding 1 M HCl.Absorbancewasmeasured at 450 nmusing a plate reader. The amountof BDNF in each well was determined by interpolation to the linearrange of a curve obtained in the same assay with known concentra-tions of BDNF. Results were expressed as picograms of BDNF detectedper milligram of protein. For the quantitative detection of Bcl-2, 100 μlof cytoplasmic extract was used for the ELISA, performed according tothe manufacturer's instructions (Bender MedSystems, Vienna, Aus-tria). The experimental protocolwas similar to that described for BDNFexcept that the incubation period lasted 2 h. Absorbance wasmeasured at 450 nm. Background values were subtracted and resultsare shown as nanograms of Bcl-2 per milligram of protein.

Statistical analysis

The unpaired Student t test was performed to assess differencesbetween groups. All results are presented as means ± standard errorof the mean (SEM). A P valueb0.05 was considered to indicate astatistically significant difference.

Results

Animals and treatments

Rats from the C-12 M group ingested a mean amount of 42.68 ±0.70 g of food and drank 63.73 ± 1.67ml/kg bw per day at 12months

of age. These amounts were similar to those consumed by C-19 M andGT-19 M animals at that same age. After 7 months of treatment, C-19 M and GT-19 M rats ingested 36.95 ± 0.34 and 36.04 ± 0.27 g offood/kg bw per day, respectively. Also, no differences were found inthe volume of liquid drunk by C-19 M (52.61 ± 1.00 ml/day/kg bw)and GT-19 M (54.55 ± 0.94 ml/day/kg bw) rats. The mean dailyamount of catechins ingested by GT-treated rats was 52.32 ±0.90 mg/kg bw, calculated according to the average GT intake bythese animals.

The mean body weight of the rats was similar among all groups atthe beginning of the experiment and there was a gradual incrementthroughout the period of treatment. At 19 months of age, groupspresented at 905 ± 41 (C-19M) and 909 ± 32 g (GT-19 M), whereasthe C-12 M animals weighed 774 ± 12 g. No differences were foundamong the mean brain weight of C-12 M (1.60 ± 0.02 g), C-19 M(1.63 ± 0.04 g), and GT-19 M (1.61 ± 0.02 g) rats. Likewise,hippocampal formation weights were similar among groups andranged between 110 and 130 mg (120.0 ± 2.4 mg).

Glutathione redox status

Glutathione levels measured in hippocampal tissue homogenatesof rats from all groups are shown in Fig. 1. GSH concentration in the C-19 M group was reduced 50% compared to C-12 M animals (Pb0.0001). C-19M rats also showed an increase in the GSSG levels (86%)relative to the younger controls (Pb 0.01). Conversely, markedlyenhanced GSH (77%) and decreased GSSG (53%) levels were found inGT-treated rats versus their age-matched group (Pb 0.01 and Pb 0.05,respectively).

Antioxidant enzyme activities

Total SOD and CAT activities, evaluated in hippocampal tissuehomogenates of C-12 M, C-19 M, and GT-19 M rats, are presented inTable 1. SOD activity was similar between the C-12 M and the C-19 Mgroups but increased 49% after chronic GT ingestion in comparison

Table 1Superoxide dismutase and catalase activities in the hippocampal formation

Group Total SOD CAT

C-12M 0.140 ± 0.004 131.4 ± 9.73C-19M 0.127 ± 0.008 492.3 ± 100.51⁎⁎GT-19M 0.189 ± 0.020+ 227.5 ± 27.91+

Values are expressed as means ± SEM (n= 5 in each group). Units: total SOD, U/mgprotein (1 U is the concentration of enzyme that inhibits the reduction of NBT by 50%);CAT, U/mg protein (1 U is the amount of enzyme required to hydrolyze 1 μmol of H2O2

per minute). C-12 M, 12-month-old controls; C-19 M, 19-month-old controls; GT-19 M,19-month-old GT-treated rats; SOD, superoxide dismutase; CAT, catalase.⁎⁎Pb 0.01 vs C-12 M.+Pb 0.05 vs C-19 M.

Fig. 2. (A) Cyclic AMP response element-binding (CREB) and (B) nuclear factor-κB(NF-κB; p50 and p65 subunits) activation in hippocampal nuclear extracts of12-month-old (C-12 M), 19-month-old (C-19 M), and 19-month-old GT-treated(GT-19 M) rats. Columns represent means and vertical bars correspond to 1 SEM.⁎⁎Pb 0.01 vs C-12M;++Pb0.0001 vs C-19M; n= 5 in each group.

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with C-19 M animals (Pb 0.05). Concerning CAT activity, there was anincrease of 275% in C-19 M rats relative to C-12 M animals (Pb 0.01).In addition, a significant reduction of 54% of CAT activity was observedin GT-treated rats compared to age-matched controls (Pb 0.05).

Table 2 summarizes the activities of glutathione-related enzymesin hippocampal tissue homogenates from all groups of rats. GTanimals displayed a reduction of 55% in GR activity compared toC-19 M rats (Pb 0.0001). However, no differences regarding theactivity of this enzyme were found between the C-12 M and theC-19 M groups. Regarding Se-GPX activity, there was an age-dependent decrease of 43% (Pb 0.001) that was not changed by GTintake. Finally, the activity of GST in C-19 M rats was augmented byapproximately 31% compared to the C-12 M group (Pb 0.001),whereas treatment with GT led to a decrease of 36% in GST activitycompared with age-matched controls (Pb 0.01).

Activation of transcription factors

The activation of CREB and NF-κB (p50 and p65 subunits)evaluated in hippocampal nuclear extracts of C-12 M, C-19 M, andGT-19M rats is shown in Fig. 2. The data revealed that CREB activationdiffered among groups. In particular, the activation of this transcrip-tion factor in the C-19 M group was 72% higher than in C-12 M rats(Pb 0.01). Furthermore, GT treatment induced a marked increase inCREB activation (93%) in comparison with age-matched animals (Pb0.0001; Fig. 2A). Compared with C-12 M controls, C-19 M rats showeda 50% decrease in the p50 subunit (Pb 0.01). However, no significantdifferences were found between these two groups regarding theactivation of the p65 subunit. GT administration did not result in anysignificant change concerning the activation of both subunits of NF-κBcompared with the C-19 M group of animals (Fig. 2B).

BDNF and Bcl-2 levels

The results obtained for BDNF and Bcl-2 levels in hippocampalcytoplasmic extracts from C-12 M, C-19 M, and GT-19 M rats can beseen in Fig. 3. A significant decrease of 30% in the levels of BDNF wasobserved in C-19 M animals compared to C-12 M rats (Pb 0.05). In

Table 2Glutathione-related enzyme activities in the hippocampal formation

Group GR Se-GPX GST

C-12M 7.22 ± 0.17 10.04 ± 0.50 42.86 ± 1.22C-19M 7.10 ± 0.17 5.75 ± 0.33⁎⁎⁎ 55.96 ± 2.25⁎⁎⁎GT-19M 3.20 ± 0.25+++ 6.70 ± 0.46 35.68 ± 3.89++

Values are expressed as means ± SEM (n= 5 in each group). Units: GR, Se-GPX, andGST, pmol/min/mg protein. C-12 M, 12-month-old controls; C-19 M, 19-month-oldcontrols; GT-19 M, 19-month-old GT-treated rats; GR, glutathione reductase; Se-GPX,selenium-dependent glutathione peroxidase; GST, glutathione S-transferase.⁎⁎⁎Pb 0.001 vs C-12 M.+++Pb 0.0001 vs C-19 M.++Pb 0.01 vs C-19 M.

contrast, prolonged GT consumption increased BDNF levels 122%relative to age-matched controls (Pb 0.0001; Fig. 3A). Rats from theC-19 M group had significantly lower levels of the antiapoptoticprotein Bcl-2 than C-12 M animals (Pb 0.001), corresponding to adecrease of approximately 67%. However, in GT-treated rats there was

Fig. 3. (A) Brain-derived neurotrophic factor (BDNF) and (B) antiapoptotic Bcl-2levels in hippocampal cytoplasmic extracts of 12-month-old (C-12 M), 19-month-old (C-19 M), and 19-month-old GT-treated (GT-19 M) rats. Columns representmeans and vertical bars correspond to 1 SEM. ⁎Pb 0.05 vs C-12M; ⁎⁎⁎Pb 0.001 vsC-12M;++Pb0.01 vs C-19M;++Pb0.0001 vs C-19M; n= 5 in each group.

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an increase in Bcl-2 levels of 159% compared with the C-19 M group(Pb 0.01; Fig. 3B).

Discussion

The hippocampal formation is one of the most affected brainregions during normal aging; striking biochemical and morphologicalchanges that take place there are implicated in age-dependentlearning and memory declines [38,39]. Redox imbalances areconsidered critical to the aging process in this limbic area, asaccumulation of oxidative damage to macromolecules triggers acascade of cellular events that usually culminate in impairment ofneuronal function [40,41]. In this study, we demonstrate the existenceof important improvements in redox status and cellular signalingsystems related to oxidative stress, survival, and neuronal functions inthe aging rat hippocampal formation after 7 months of GT intake.Indeed, deterioration of the oxidative status in this brain regionduring aging was confirmed by the finding that C-19 M rats presentedlower GSH and higher GSSG levels compared to C-12 M animals.Conversely, none of these age-related alterations were apparent afterchronic GT consumption.

Although there seems to be at least three distinct mechanisms bywhich polyphenolic compounds may protect cells, i.e., directlylowering levels of redox reactive species, preventing the influx ofcalcium, and increasing intracellular GSH [42], the latter seems to beresponsible for most of the neuroprotective activity of catechins [43].The endogenous antioxidant GSH system has a major biologicalimportance, as it can protect neurons against the damage inflicted byfree radicals, maintaining intracellular redox homeostasis, which iscritical to normal physiological processes [44,45]. Thus, the preserva-tion of GSH levels in GT-treated animals compared to the youngercontrols deserves to be emphasized as it may translate into higherprotection of neurons against redox reactive species and othernoxious compounds [45]. In accordance, other studies have demon-strated that EGCG increased GSH content, providing neuroprotection[43], and prevented β-amyloid cytotoxicity, by upregulating GSH,through induction of γ-glutamylcysteine ligase (GCL), the rate-limiting enzyme in GSH biosynthesis [46]. Additionally, certainpolyphenols can modulate GSH levels in astrocytes and neurons byincreasing mRNA levels of the GCL modifier subunit [47].

Decreased hippocampal GR activity was present in GT-treated rats,apparently in contrast to the increased GSH/GSSG ratio, given thatthis enzyme is responsible for maintaining glutathione in the reducedform [45]. However, the lowered activity of this enzyme in thepresence of polyphenol-rich beverages has also been reported alongwith the increase in GSH levels [36]. Although the mechanismunderlying this effect is yet poorly understood [48,49], it can berelated to a direct inhibition of GR by polyphenols or, more likely, to alesser expenditure of GSH in scavenging reactions and a consequentdecrease in the need of GSSG recycling, resulting from an externalsource of antioxidants. Another antioxidant enzyme, SOD, detoxifiessuperoxide radicals and the resulting hydrogen peroxide is eliminatedmainly by CAT and GPX [24]. The isoenzymes Cu/Zn-and Mn-SOD areboth involved in antioxidant actions and neuronal protection [50,51]and considered to be the first and most important line of enzymaticdefense against oxygen radicals. Total SOD activity was similarbetween the C-12 M and the C-19 M groups, it was increased afterGT ingestion. Actually, it has been demonstrated that catechintreatment increased both the activity and the mRNA level of Cu/Zn-and Mn-SOD subtypes in cultured rat astrocytes [52]. In line withthese findings, administration of EGCG, the most abundant GTcatechin, resulted in the increment of total SOD activity in aged ratbrain [44]. Regarding CAT activity, it was markedly increased inC-19 M rats compared to C-12 M animals. Although reports on age-related changes in rat brain CAT activity are not unequivocal [53–56],it seems to vary inversely with that of Se-GPX in older controls,

suggesting that peroxide detoxification occurs mainly via CAT inC-19 M rats, in which reduced Se-GPX activity was also found.Supporting our data, recent studies have shown a decline in GPXactivity in the hippocampus and cerebral cortex of rodents duringaging [55,57]. GT treatment reduced CAT activity by more than one-half, which may be understood in light of the powerful hydrogen-donating antioxidant and free radical scavenger actions of GTpolyphenols [14,15], limiting the formation of peroxides and avoidingCAT upregulation. In contrast, Se-GPX activity was not significantlyaltered after GT drinking. On the other hand, C-19 M animalspresented increased activity of GST, a glutathione-consuming en-zyme, in conjugation reactions [24], which may partially account forthe lower GSH content observed in these rats, further contributing to afavorable redox status in the old animals.

In a different experimental setting, EGCG increased the activity ofSOD and protected against glycation endproduct-induced neurotox-icity by decreasing the accumulation of radical oxygen species,inhibiting the formation of malondialdehydes and protein carbonyls[58]. Reductions in the hippocampal levels of these last two oxidativemarkerswere already described in 12-month-old rats fed for 7monthswith GT [16]. We have also shown, in these same animals, thataccumulation of lipofuscin, a marker of senescence correlated withincreasing age and impairment of brain functions [59,60], wasdecreased by GT [16]. Although redox changes provide a solidexplanation for these ameliorations caused by GT treatment, thereare other cellular mechanisms underpinning such alterations. Forexample, it is recognized that GT polyphenols can be involved in themodulation of antioxidant response elements in the promoter regionof genes encoding proteins related to antioxidant protection [61] andalso implicated in the regulation of multiple and crucial neuronalsignaling pathways [62,63]. Among these are the NF-κB and CREBpathways, which control cellular responses involved in the regulationof antioxidant enzymes, antiapoptotic proteins, and neurotrophicfactors critical for the proper functioning of neurons [25].

NF-κB was selected to be quantified in these experiments as itslevels are affected by redox balance because of the numerous bindingproteins and large number of gene-specific sequences recognizing NF-κB [64,65]. NF-κB seems to fortify cellular protection against oxidativestress through increase in GSH levels by inducing biosynthesis of theGCL heavy subunit [65,66]. Although the human GCLmodifier (GCLM)subunit does not contain a κB element, and the role of NF-κB in ratGCLM activation is still a matter of debate, it was recently shown thatNF-κB can modulate GCLM and glutathione synthetase because of itsability to interfere with c-Jun phosphorylation for activator protein-1(AP-1)-mediated transcription [67]. The general mechanisms of GCLregulation are primarily through 12-O-tetradecanoylphorbol-13-acetate-responsive element (TRE) and the electrophile-responsiveelement (EpRE) [68,69]. As the transcription factor Nrf2 is a principalplayer in the activation of the EpRE, and c-Jun is a binding partner ofNrf2 in the activation of EpRE-dependent transcription, the modula-tion and phosphorylation of c-Jun are required in the activation of AP-1/TRE-mediated transcription [68]. This ability of NF-κB to regulateGCL expression through interplay with other transcription factorpathways illustrates the complexity of redox balance responses, theirdetailed mapping representing an enormous challenge [67]. Inneurons and glia, NF-κB proteins are ubiquitously expressed mostlyas the p50–p65 heterodimer and p50–p50 homodimer, which, inaddition to regulating physiological processes [64,70], participate inpathological events after, for example, trauma or ischemia [71,72].However, the functional role of NF-κB in neuronal signaling pathwaysclosely related to cell survival and death is complex. It seems that theinduction of certain NF-κB-regulated genes in neurons is activateddifferentially, depending on subunit composition and strength andduration of stimuli [73]. This may explain the age-dependent decreasein NF-κB p50 subunit activation and the absence of alterations in thep65 subunit found in the current experiments. Moreover, although

Fig. 4. Model of the possible mechanisms whereby long-term green tea ingestionprevents hippocampal-dependent spatial memory decline associated with aging.Important modifications in redox status biomarkers were found in GT-treated ratsalong with decreased accumulation of lipofuscin. Green tea treatment also activatedsignaling pathways that induced the expression of genes encoding growth factors andantiapoptotic proteins. Antioxidant defense enzymes may also be under the control ofCREB. Increased neuronal survival and plasticity may arise from those alterationssupporting the prevention of age-related spatial memory impairment observed in ratstreated for 7 months with green tea.

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dietary polyphenols have been described as modulating the activationof NF-κB [74], GT-treated rats presented the same hippocampal NF-κBp50 and p65 subunit activation as age-matched controls. Contrastingwith this, the antioxidant effect of a GT extract on β-amyloid peptide-induced neuronal death was also associated with the attenuation ofNF-κB activation in a concentration-dependent manner [75].

The activation of CREBwas also quantified in this study, as it is welllinked to neuronal survival and synaptic plasticity [76–78]. Also, it isrecognized as a very solid molecular marker of memory processing inthe hippocampal formation for spatial learning, and the activation ofthe CREB signaling pathway plays an important role in spatialmemory formation [76,79]. CREB activation controls the inductionof many downstream genes, including antioxidant enzymes such asSOD and key prosurvival proteins such as BDNF and Bcl-2, known tobe unambiguously related to the above-mentioned effects [30,80]. Thetranscriptional activation related to phosphorylation at Ser133induces translocation of the cytoplasmic CREB to the nucleus andwe have quantified phosphorylated CREB in nuclear extracts ofhippocampal tissue. However, reports on CREB activation in thehippocampal formation of aged rodents have yielded conflictingresults [20,81,82]. Here, we have found an augmentation of CREBactivation in the hippocampal formation during aging. In agreement,Monti and colleagues [82] showed that CREB activation is higher inthe hippocampus of memory-impaired aged rats compared withyoung adults, although this activationwas not affected by the learningexperience. More interestingly, we have demonstrated that prolongedGT treatment increased hippocampal phosphorylated CREB to almosttwice the concentration observed in the age-matched animals. Thisresult comes in agreement with a previous study showing that (−)-epicatechin rapidly stimulates CREB phosphorylation and subse-quently increases CREB-regulated gene expression in cortical neurons[83].

As CREB phosphorylation may be involved in neuroprotectionthrough the modulation of genes related to prosurvival factors[28,30,31], the levels of the antiapoptotic protein Bcl-2 and theneurotrophin BDNF were quantified in the hippocampal tissueextracts. Of significance, it seems that Bcl-2 and BDNF proteins actin concert to mediate protective functions in neurons and ultimatelyyield synapse-specific structural changes relative to long-termmemory formation [29,84]. It is also important to note that Bcl-2can increase intracellular GSH capacity through NF-κB activation [65].In accordance with previous studies [20,85], we found that hippo-campal levels of both Bcl-2 and BDNF were decreased in 19-month-old rats comparedwith 12-month-old baseline controls, suggesting anattenuation of prosurvival pathways with age. On the other hand, GTwas able not only to prevent the age-related reduction in hippocam-pal BDNF levels but also to stimulate its increase relative to the age-matched control group. These variations are in parallel with thoseobserved in Bcl-2 content and support the conjoint action of bothmediators in the neuroprotective effect of GT intake. Supporting ourobservations are recent results showing that prolonged administra-tion of GT catechins increased CREB phosphorylation and theexpression of BDNF and Bcl-2 in the aging mouse hippocampus andsimultaneously prevented age-related spatial learning and memorydecline [20]. CREB activation enhances BDNF and Bcl-2 expression,which can further activate CREB, a feed-forward mechanism involvedin the regulation of synaptic protein synthesis and axonal anddendritic growth in the hippocampal formation [86–88]. In thisregard, Li and colleagues [20] also found that GT catechin-treatedmice dose-dependently presented higher levels of phosphorylatedCREB and BDNF associated with an increase in the hippocampalpostsynaptic density proteins PSD95 and Ca2+/calmodulin-depen-dent kinase II compared to age-matched controls. These interestingresults corroborate our prior observations that chronic GT consump-tion prevented the age-associated loss of ability to perform a spatialwater maze task [16]. Altogether, the increased neuronal survival and

plasticity (neurogenesis, neurite outgrowth, and synaptogenesis)unequivocally related to enhanced levels of Bcl-2 and BDNF andother prosurvival factors may lead to the prevention of age-relatedspatial memory decline previously observed in rats treated for7 months with GT (Fig. 4) [16,26].

GT contains strong bioactive compounds that act on the centralnervous system [89,90], and an understanding of some theirmechanisms of action is crucial for the evaluation of the effects ofthis beverage in the aging brain. Here, we show that long-term GTingestion improves antioxidant protection systems, increases activa-tion of the transcription factor CREB, and upregulates BDNF and Bcl-2in the hippocampal formation during aging. The fact that thesemolecular targets are unambiguously involved in neuroprotectiveactions [31,78] highlights the possibility that their modulation mayvery well constitute plausible molecular mechanisms through whichGT relates to increased resistance to age-associated impairments.

Acknowledgments

We are grateful to Professor Victor de Freitas for quantification ofGT catechins. This workwas supported by the Fundação para a Ciênciae a Tecnologia—Centro de Morfologia Experimental, Unit 121/94 andSFRH/BD/19497/2004.

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