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Journal of Alzheimer’s Disease 44 (2015) 561–572DOI 10.3233/JAD-140981IOS Press

561

Beneficial Effects of Dietary EGCG andVoluntary Exercise on Behavior in anAlzheimer’s Disease Mouse Model

Jennifer M. Walkera, Diana Klakotskaiaa, Deepa Ajitb,d, Gary A. Weismanb,d, W. Gibson Woodf ,Grace Y. Sunb,c,e, Peter Serfozob, Agnes Simonyib,e and Todd R. Schachtmana,∗aDepartment of Psychological Sciences, University of Missouri, Columbia, MO, USAbDepartment of Biochemistry, University of Missouri, Columbia, MO, USAcDepartment of Pathology and Anatomical Sciences, University of Missouri, Columbia, MO, USAdChristopher S. Bond Life Sciences Center, University of Missouri, Columbia, MO, USAeCenter for Translational Neuroscience, University of Missouri, Columbia, MO, USAf Department of Pharmacology, University of Minnesota, Geriatric Research Education and Clinical Center, VAMC,Minneapolis, MN, USA

Handling Associate Editor: Susan Farr

Accepted 11 September 2014

Abstract. Alzheimer’s disease (AD) is a progressive, age-dependent neurodegenerative disorder affecting specific brain regionsthat control memory and cognitive functions. Epidemiological studies suggest that exercise and dietary antioxidants are beneficialin reducing AD risk. To date, botanical flavonoids are consistently associated with the prevention of age-related diseases. Thepresent study investigated the effects of 4 months of wheel-running exercise, initiated at 2-months of age, in conjunction withthe effects of the green tea catechin (-)-epigallocatechin-3-gallate (EGCG) administered orally in the drinking water (50 mg/kgdaily) on: 1) behavioral measures: learning and memory performance in the Barnes maze, nest building, open-field, anxiety inthe light-dark box; and 2) soluble amyloid-� (A�) levels in the cortex and hippocampus in TgCRND8 (Tg) mice. UntreatedTg mice showed hyperactivity, relatively poor nest building behaviors, and deficits in spatial learning in the Barnes maze. BothEGCG and voluntary exercise, separately and in combination, were able to attenuate nest building and Barnes maze performancedeficits. Additionally, these interventions lowered soluble A�1-42 levels in the cortex and hippocampus. These results, togetherwith epidemiological and clinical studies in humans, suggest that dietary polyphenols and exercise may have beneficial effectson brain health and slow the progression of AD.

Keywords: A�PP transgenic mice, Alzheimer’s disease, behavior, exercise, EGCG, learning

INTRODUCTION

Alzheimer’s disease (AD) is a progressively degen-erative and debilitating brain disease. Its worldwideprevalence is estimated to be 35 million individuals

∗Correspondence to: Todd R. Schachtman, Department of Psy-chological Sciences, 210 McAlester Hall, University of Missouri,Columbia, MO 65211, USA. Tel.: +1 573 882 3154; Fax: +1 573882 7710; E-mail: Schachtmant@missouri.edu.

(5.5 million in the US alone), and this number isexpected to more than double in the next 40 years [1].Preventing or postponing the onset of AD and delayingor slowing its progression would lead to an improve-ment of health status and quality of life for the elderly.Since the neuropathology is likely to occur at leastdecades prior to the emergence of clinical symptoms,prevention can be effective. Epidemiological studiessuggest the benefits of dietary antioxidants, especiallybotanical polyphenols, in reducing AD risk [2, 3].

ISSN 1387-2877/15/$27.50 © 2015 – IOS Press and the authors. All rights reserved

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Population studies examining factors that maydecrease the risk of AD, such as the Kame Project,have found that drinking fruit/vegetable juices is oneof the most reliable factors inducing decreased risk forAD [4]. In addition, a higher consumption of greentea is associated with a lower prevalence of cognitiveimpairments [5].

(-)-Epigallocatechin-3-gallate (EGCG) is the majorcatechin (50% of total polyphenols) found in green teathat shows antioxidative, anti-inflammatory, and anti-amyloidogenic properties [6–8]. Studies have shownthat EGCG reduces amyloid-� (A�) levels [9–11],and in so doing can decrease A�-induced mitochon-drial dysfunction [12] and memory impairment [9].EGCG has been investigated in the Tg2576 AD mousemodel [13], where it was shown to decrease A� levelsby promoting increased amyloid-� protein precursor(A�PP) cleavage through activation of ADAM10, an�-secretase [14]. Additionally, oral administration ofEGCG (50 mg/kg/daily for 6 months) in these miceresulted in decreased levels of soluble and insolubleoligomeric A� species, as well as amelioration of spa-tial learning deficits, as shown by improved acquisitionin the radial-arm water maze [15].

Additionally, it is well known that regular physicalactivity can reduce an individual’s risk for cardiovas-cular disease, diabetes, cancer, and depression, andcan even improve cognition in healthy, older adults(see [16] for a review). Recent research suggests thatlong-term exercise may also reduce the risk of AD anddementia, and can be effective as an intervention in ADpatients (see [17] for a review). Wheel running altersthe neuropathology and behavior of AD mouse mod-els in some, but not all, studies (see [18] for a review).For example, Adlard and colleagues [19] showed that5 months of unlimited access to a home-cage run-ning wheel (beginning prior to the onset of diseasepathology; 1-month old) improved the cognitive per-formance of female TgCRND8 mice in the Morriswater maze task, compared to Tg animals experiencingstandard housing. This study also found that long-termexercise decreased extracellular amyloid burden in thecortex and hippocampus, and decreased levels of sol-uble oligomeric A� species (i.e., A�1-40 and A�1-42).In contrast, another study [20] found no cognitive ben-efit of exercise, as measured by object recognition andBarnes maze tasks, when the running wheels were pro-vided to male TgCRND8 mice at 80 days of age (micehad access up to 5-months of age; however, behav-ioral testing started after only 40 days of running wheelaccess, cf. the 150 days used by Adlard and colleagues[19]). Similarly, other studies showed differences in

the beneficial effects of early-onset versus late-onsetrunning and/or short-term versus long-term running inTg2576 and A�PP23 mice [18].

The combined effects of diet and exercise can bemore pronounced than either manipulation by itself,as shown by epidemiological as well as experimen-tal studies [21, 22]. However, studies with AD mousemodels have primarily investigated the effects of exer-cise on high-fat diet induced enhancement of ADpathology and memory impairments [23, 24]. In addi-tion, a recent study demonstrated that lipoic acidtreatment (by i.p. injections), in combination withexercise, has an extended protective effect on spatiallearning in the Morris water maze and some biochem-ical markers in NSE/A�PPswe mice [25].

The present study investigated possible preventa-tive treatment effects of voluntary exercise and EGCGdietary supplementation, alone and in combination, on:1) cognitive and behavioral impairments: spatial learn-ing and memory in the Barnes maze, nest buildingbehaviors, anxiety in the light-dark box, and locomo-tor/exploratory activity in the open-field; and 2) levelsof soluble A�1-42 in cortex and hippocampus.

MATERIALS AND METHODS

Animals

All procedures were based on a protocol approvedby the Institutional Animal Care and Use Commit-tee (IACUC) of the University of Missouri (protocol#6728). Both male and female mice were used.Forty-two TgCRND8 (Tg) mice and twelve wild-type (nTg) littermates bred by the Cell, Molecularand Animal Core Facility of the Alzheimer’s DiseaseProgram Project, University of Missouri, Columbia(http://muadppg.missouri.edu), survived to the end ofthe study and were included in the analyses. Thebreeders were obtained from Dr. David Westaway(University of Toronto), and the mice were maintainedon a hybrid genetic background (C3H/C57BL/6). Wechose this model, in part, becauseTgCRND8 micedevelop A� plaques and display behavioral deficitsby 2–3 months of age. All mice were genotyped viaDNA extraction from tail samples and PCR analysisas previously described [26, 27]. Mice were group-housed until 2-months of age and then separated intoindividual housing.

Animals were maintained on a 12-h light/darkcycle (lights on at 7:00 AM) and given unrestrictedaccess to food and water. Body weight was measuredweekly. Each mouse received a cotton pad and an

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environmental enrichment toy (small plastic house),and was observed for good health before, during, andafter testing. Behavioral procedures occurred in a sep-arate testing room.

Experimental design

Transgenic animals were assigned to one of fourgroups: 1) EGCG (n = 10, 5 females and 5 males),standard housing and EGCG (>95% purity; herbs-tech.com, Lanxi, Zhejiang, China) dissolved in water(0.2 mg EGCG/mL); 2) voluntary exercise (Ex; n = 11,6 females and 5 males), homecage running wheelaccess and normal water; 3) Combination of EGCGand exercise (Combo; n = 10, 6 females and 4 males),homecage running wheel access and EGCG; or 4)Untreated (Tg; n = 11, 7 females and 4 males), nor-mal water, standard housing. In addition, a controlgroup (normal water, standard housing) of wild-typelittermates (nTg; n = 12, 6 females and 6 males) wasused. Animals were assigned to treatment groups at 2-months of age; treatment lasted for 4-months, and micewere sacrificed at 6-months of age. Two weeks beforesacrifice, mice were tested in a variety of behavioraltasks (see Fig. 1). These tasks are extremely com-mon assessments of activity, anxiety, and learning inrodents. In sum, behavioral tests were performed inthe following order: open-field (day 1), light/dark box(day 2), Barnes maze (days 3–7), and nest building

(days 9–10). Twenty-four hours after the completionof nest building, the mice were sacrificed.

Wheel-running

Mice in the Exercise and Combination groups hadunlimited access to a running wheel (ENV—44; MedAssociates, Inc.; 10.9 cm L × 7.62 cm W × 10.9 cmH) in their home cage until the end of the experi-ment (∼4-month duration). Data from each wheel weretransmitted wirelessly every 30 s to a HUB (DIG-804;Med Associates, Inc., St. Albans, VT), which was setup with a computer in the animal colony room. Rawdata were analyzed using Wheel Manager software(Med Associates, Inc.; Windows XP OS). Wheels werecleaned and batteries were checked regularly to ensureproper functioning.

EGCG

Mice in the EGCG and Combination treatmentgroups were provided with a drinking tube containing0.2 mg/mL EGCG in water based on previous research[15]. EGCG water was the only water made availableto these mice. Consumption of this water was mea-sured daily and fresh EGCG water was prepared everyday. Additionally, the average weekly dosage of EGCG(mg/kg) consumed was calculated based on consump-tion values and body weights. Both the EGCG and

Fig. 1. Experimental procedure.

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Combo groups had an average EGCG daily dosage of50 mg/kg across all weeks of the study.

Behavioral assessment

All mice were tested on four tasks: nest building,the open-field, the light-dark box, and the Barnes maze(see Fig. 1). With the exception of nest building, whichwas measured in each mouse’s homecage in the colonyroom just prior to lights-off, mice were acclimated tothe testing room for 30 min prior to the start of eachbehavioral test, and testing occurred between 9 : 00am–1:00 pm (first half of the light cycle). Mice werehandled for 7 days prior to the start of behavioraltesting.

Nest building

Nest building was assessed at 2 time points: 2-months old (24 h after transfer to individual housing)and 6-months old (24 h prior to sacrifice). Animalswere provided with nesting material in the form oftwo cotton pads (5 cm × 5 cm each, 5 g total weight;Ancare Wellmore, NY). Twenty-four hours later, eachanimal’s nest was video-recorded in the colony roomand later scored on a scale of 1–5 (for full details,see [28]) by two trained, independent coders. Consis-tent with the assessment by Deacon [28], nests werescored based on shape and the amount of materialused. Scores ranged from 1–5 using the following cri-teria for each score: 1 = nestlets not/barely touched,2 = nestlets partially torn, 3 = nestlets mostly shred-ded, but no identifiable nesting site (i.e., material isdistributed throughout the cage), 4 = nestlets almostentirely shredded and gathered into one-quarter of thecage, but the nest is flat, 5 = nestlets almost entirelyshredded, gathered into one-quarter of the cage; nearlyperfect nest, crater-shaped, with walls higher than themouse’s body height. Any unshredded nesting materialwas weighed and recorded for each mouse. Nest build-ing was assessed twice since an earlier experience withnest building seemed unlikely to influence later nestbuilding behavior; however, early exposure to locomo-tor or anxiety chambers or acquisition of Barnes mazelearning would likely distort the assessment of later useof these apparatuses/tasks. For example, exposure to ananxiety-inducing apparatus would not be comparablefor a mouse’s first exposure and later exposure. InitialBarnes maze acquisition would be hard to comparewith a second acquisition experience.

Open-field

The open-field is a test of exploratory and locomo-tor activity. The apparatus was made of clear acrylicplastic, and the area measured 40 cm × 40 cm. A clear,acrylic plastic wall (40 cm high) surrounded the area.Exploratory activity was measured during one 10-mintrial. The apparatus was cleaned before and after eachtrial with a 20% ethanol solution. Tests were recordedwith a Logitech digital webcam and data were analyzedusing Any-maze software (v.4.72, Stoelting Co., WoodDale, IL; Windows XP OS). Total distance traveled,percentage of time spent in the center, and entries intothe center area (which comprised 14% of the total areaof the field) were calculated.

Light-dark box

The light-dark box is a behavioral measure of bothnovelty-seeking and anxiety-like behaviors in mice;specifically, it measures the conflict between twonatural tendencies of mice: 1) exploring novel environ-ments, and 2) avoiding aversive properties of brightlylit, open areas [29].

The apparatus was the previously described open-field chamber (40 cm W × 40 cm L × 40 cm H). Half ofthe open-area was replaced with a dark, covered com-partment (20 cm W × 40 cm L × 40 cm H). A smallopening measuring 7.5 cm high and 5 cm wide facedthe center of the open field area, which allowed mice totravel freely between compartments. Testing occurredduring one trial. Each mouse was removed from itshome cage, placed into the light side of the box,and allowed to move freely for 5 min. The maze wascleaned after every trial with a 20% ethanol solution.Each trial was video-recorded and analyzed using Any-maze software. Three measures were calculated foreach mouse: 1) latency to first entry into the dark area,2) number of entries to the dark area and 3) time spentin the light area.

Barnes maze

The Barnes maze is a hippocampal-dependent spa-tial learning task where rodents learn to navigate theirway from a brightly lit, circular platform in attempt tofind a dark escape box located in one of 20 differentholes using visual cues located around the maze. Theother 19 holes did not lead to an escape box. The pro-cedure is also described elsewhere [27]. Each mousewas assigned an escape hole number; assigned holenumbers were alternated by 90◦ (i.e., the 5th hole for

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one mouse, the 10th hole for another, the 15th, the20th) to eliminate odor cues for consecutively testedmice. The escape box location remained constant forany individual mouse across test trials. Behavioral test-ing consisted initially of two shaping trials on the firstday and then 8 evaluation test trials with two trials perday over a period of four days.

A trial began by placing the mouse under a black,opaque start box positioned in the center of the plat-form. After 60 s, the box was lifted and the mouse had amaximum of 5 min (300 s) to find and enter the escapebox. If the mouse did not enter the escape box within5 min, it was gently guided there by the experimenter’shand. After 30 s, the mouse was removed from theescape box and returned to their home cage. The plat-form and escape box were cleaned after every trial witha 20% ethanol solution. Mice were allowed to rest intheir home cage for 30 min before starting their seconddaily trial. Latency (time to find the escape box) andtotal errors (nose-pokes into non-escape holes) wererecorded. Additionally, Any-maze software was usedto determine the average speed, as well as quadrantanalyses as measures of both learning and anxiety-likebehavior.

Quantification of Aβ1-42 levels

After the cessation of behavioral testing, animalswere sacrificed and their brains were removed anddissected to obtain the cerebral cortex and hippocam-pus. The hippocampus and cortex are two brain areasstrongly implicated in age- and AD-related memorydecline. Based on the known sequence of the neu-ropathological changes in the brain of the TgCRND8mice [26], the hippocampus and cortex were usedto determine whether EGCG and/or exercise wouldreduce soluble A� levels at the end point of the studywhen mice were 6 months old (a moderate state of A�pathology). Brain samples were stored at −80◦C. Sol-uble A�1-42 levels were measured as described earlier[30] using an anti-human A�1-42 sandwich ELISA kit(Invitrogen, Carlsbad, CA).

Statistical analysis

Data were analyzed by either nonparametric tests(Mann-Whitney U and Kruskal-Wallis, Open-field) orone-way (Light-dark box, A�1-42 levels) and repeated-measures two-way analysis of variance (ANOVA)(Nest building, Barnes maze). Huynh-Feldt correctionswere used for violations of sphericity. Bonferroni post-hoc tests were used for pairwise comparisons (SPSS,

V.16.0 for Mac OSX Leopard). Differences were con-sidered significant at p < 0.05 for all analyses.

RESULTS

Wheel running

Daily distance traveled (m) was calculated for eachmouse with access to a running wheel (Exercise andCombo groups). These distances were then averagedacross each week of the study, for a total of 16 weeks.Daily average running distances for each week wereanalyzed using a repeated-measures ANOVA, withweek, group, and sex as factors. No group differences(F(1,18) = 0.074, p = 0.79) or sex differences (F(1,18)= 0.555, p = 0.47) were found. Additionally, no effectof week was found (F(15,270) = 0.905, p = 0.56, Fig. 2),and no interactions were observed. No group signifi-cantly increased or decreased their running distancesacross the 16 weeks of study; males and females didnot differ in average daily running distance. Similarvalues of daily average distances have been reportedby other studies [19].

Nest building

Data were analyzed by two-way repeated measuresANOVA with Bonferroni pairwise comparisons. A sig-nificant effect of group (F(4,48) = 7.76, p < 0.0001) andage (F(1,48) = 51.94, p < 0.0001), as well as a significantinteraction (F(4,48) = 9.78, p < 0.0001) were obtained.

Fig. 2. Wheel-running activity over time in TgCRND8 mice. Mice inthe voluntary exercise (Exercise, n = 11) and Combination (Combo,n = 10) groups had unlimited access to a running wheel in their homecage from 2- to 6-months of age. EGCG at a dose of 50 mg/kg/daywas administered via drinking water to the Combo group. Dailydistance traveled (m) was calculated and then averaged across eachweek of the study. Values are means ± SEM and were analyzed byrepeated-measures ANOVA. No group differences were found.

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Fig. 3. Effects of EGCG, wheel running activity and their combina-tion on nest building behavior in TgCRND8 mice. Nest buildingscores at 2-months and 6-months of age for Tg (n = 11) mice,their nTg littermates (n = 12), EGCG (n = 10), Exercise (Ex, n = 11),and Combo (n = 10) groups. Data are expressed as mean ± SEM.Significant group and age effects were revealed by two-wayrepeated-measures ANOVA (p < 0.0001). Letters above the bars indi-cate group differences. See text for details on group comparisons.

At 2-months of age, Tg mice showed poor nest con-struction compared to wild-type littermates, as shownby lower nest building scores (Fig. 3). At 6-months ofage, untreated Tg mice still showed poor nest construc-tion compared to nTg mice (p < 0.001), while micein all three intervention groups showed nest construc-tion comparable to nTg mice (p > 0.05, ns). Pairwisecomparisons indicated significant differences betweenuntreated Tg mice and EGCG (p < 0.05), Exercise(p < 0.01), as well as Combo (p < 0.001) groups. Theseintervention groups did not differ from each otheralthough the mean value was the lowest for the EGCGgroup (see Fig. 3).

Open-field

Due to unequal variances between groups, data wereanalyzed using nonparametric Kruskal-Wallis tests andMann-Whitney U tests were used for pairwise com-parisons. Between-group differences were found fortotal distance traveled (H = 18.05, df = 4, p = 0.001)and number of center area entries (H = 12.04, df = 4,p = 0.017). No differences were found for percentagetime spent in the center (p = 0.141, data not shown).Consistent with the results of earlier studies, pair-wise comparisons showed that untreated Tg mice werehyperactive compared to nTg controls (p < 0.01). TheEGCG condition was also hyperactive (p = 0.001) rela-tive to the nTg control, but no difference in locomotionwas observed between the EGCG and untreated Tggroups. Mice in the Exercise group and Combina-tion group showed reduced locomotion compared toconditions that did not receive exercise opportunities(Fig. 4A); it may not be surprising that these groups

Fig. 4. Effects of EGCG, wheel running activity and their combi-nation on open-field behavior in TgCRND8 mice. Total distancetraveled (A) and number of center area entries (B) in the open-field for untreated TgCRND8 mice (Tg, n = 11), their wild-typelittermates (nTg, n = 12), EGCG (n = 10), Exercise (Ex, n = 11), andCombo groups (n = 10). Resulted represented as box plots. A) Sig-nificant between-groups differences were found for total distancetraveled during a single, 10-min trial in the open-field (p = 0.01,Kruskal-Wallis test). Letters above the bars indicate group differ-ences. See text for details on group comparisons. B) Significantgroup differences were found for the number of center area entries(p = 0.017, Kruskal-Wallis test). ∗ indicates significant differenceas compared to all the other groups. See text for details on groupcomparisons.

would show less locomotion since they had substantialwheel running experience. Nonetheless, the differencebetween the nTg and untreated Tg group confirms ear-lier findings.

Additionally, mice in the Exercise group (p = 0.033),EGCG group (p = 0.02), and Combination group(p = 0.014) made more entries into the center area com-pared to nTg mice (Fig. 4B). The untreated Tg groupalso made more entries to the center area compared tonTg mice (p = 0.04) and did not differ from any of theother Tg groups. Therefore, all Tg groups made morecenter entries compared to nTg littermates and did notdiffer from each other.

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Light-dark box

Data were analyzed using one-way ANOVAs, withgroup as a factor and the following as dependent mea-sures (as mentioned earlier): 1) latency to first entryinto the dark area, 2) number of entries to the darkarea, and 3) percentage of time spent in the light zone.Bonferonni post-hoc tests were used for pairwise com-parisons. Two mice that never entered the dark zonewere excluded from the analyses.

No between-group differences were found for per-centage time spent in the light zone (F(4,46) = 0.81,p = 0.543, data not shown). Overall, there was a slighttrend toward group differences in latency to firstenter into darkness (F(4,46) = 2.00, p = 0.110, Fig. 5A).Pairwise comparisons showed that the nTg group laten-cies did not differ from the Tg group. However, the

Fig. 5. Effects of EGCG, wheel running activity and their combina-tion on behavior in the light-dark box in TgCRND8 mice. Latencyto first entry into the dark area (A) and number of entries to thedark area (B) in the light-dark box during a single 5-min trial foruntreated TgCRND8 mice (Tg, n = 11), their wild-type littermates(nTg, n = 12), EGCG (n = 10), Exercise (Ex, n = 10), and Combogroups (n = 9). Data are expressed as mean ± SEM. Significant groupeffects were revealed by one-way ANOVA for the number of entries(p = 0.041). ∗ indicates significant difference as compared to the nTggroup. See text for details of the analyses.

Exercise and Combo groups showed increased laten-cies compared to nTg mice (p = 0.026 and p = 0.012,respectively). The Tg group did not differ from theExercise group, but there was a trend toward shorterlatencies for the Tg group than the Combo group(p = 0.091). Nonetheless, these findings are limitedgiven the absence of a difference between the nTgand untreated Tg group, and the fact that groups givenexercise opportunities showed longer latencies to exit.

A significant between-group difference wasobserved for the number of entries into the darkzone (F(4,46) = 2.70, p = 0.041, Fig. 5B). There wasno difference between the nTg and untreated Tggroups on the number of dark zone entries. Althoughanalyses revealed that Exercise (p = 0.011) and Combo(p = 0.068) groups showed fewer dark zone entries, theabsence of a difference between nTg and Tg groupsand the fact that these former groups had exerciseopportunities limits conclusions stemming from theseresults.

Barnes maze

Mice that jumped from the platform were excludedfrom the analyses. Results were analyzed using arepeated-measures ANOVA with day and group as fac-tors, and latency and errors as dependent measures.Huynh-Feldt corrections were used for violations ofsphericity and Bonferonni post-hoc tests were used forpairwise comparisons.

Within-subjects contrasts showed a significant effectof day, such that all mice decreased their escape laten-cies (F(1,46) = 68.89, p < 0.001) and errors (F(1,46)= 34.01, p < 0.001) across test days. Additionally, therewas a significant effect of group for both latency(F(4,46) = 4.19, p = 0.006) and errors (F(4,46) = 4.49,p = 0.004, Fig. 6A and B). No group × day interactionswere found.

Pairwise comparisons between groups showed thatuntreated Tg mice demonstrated poor spatial learn-ing, as measured by increased latencies (p = 0.041)and errors (p = 0.003) compared to nTg mice. Thisconfirms earlier results showing maze performancedeficiencies in TgCRND8 mice [27, 31]. Addition-ally, EGCG, Exercise, and Combo groups performedcomparably to nTg mice; EGCG and Combo groupsshowed improved performance (decreased latencies)compared to untreated Tg mice (p < 0.05). An ANOVAconducted on the latency data on Day 4 (the finalday) of testing obtained a main effect of groups(p = 0.0253). The untreated Tg group showed poorerperformance than the nTg group (p < 0.01); and both

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Fig. 6. Effects of EGCG, wheel running activity and their combina-tion on Barnes maze acquisition. Latency (sec) by day (A) and errors(number) by day (B) for untreated TgCRND8 mice (Tg, n = 10),their wild-type littermates (nTg, n = 12), EGCG (n = 9), Exercise(Ex, n = 10), and Combo groups (n = 10). Testing consisted of 8training trials (2 trials/day) over four days. Data are expressed asmean ± SEM. Significant group effects were revealed by two-wayrepeated-measures ANOVA for both latency (p = 0.006) and error(p = 0.004). See text for details of the analyses. C) Barnes mazeacquisition (area under the curve as calculated from Days 1–4).Data are expressed as mean ± SEM. A significant effect of groups(p = 0.0103) was revealed. ∗ indicates significant difference as com-pared to all the other groups.

the EGCG and the Exercise groups showed shorterlatencies than the untreated Tg group (p < 0.01), andthe Combo group also produced shorter latencies thanthe untreated Tg group (p < 0.05). These results con-firm earlier work showing group differences involvingTg mice on maze learning emerging on later test trials[15, 32]. Similarly, error data on Day 4 revealed a maineffect of group (p = 0.0084) with significant differencesbetween untreated Tg as compared to the other groups

Fig. 7. Effects of EGCG, wheel running activity and their combina-tion on soluble A�1-42 levels in cortex (A) and hippocampus (B) foruntreated TgCRND8 mice (Tg, n = 11), their wild-type littermates(nTg, n = 12), EGCG (n = 10), Exercise (Ex, n = 11), and Combogroups (n = 10). A�1-42 levels were quantified by ELISA. Data areexpressed as mean ± SEM. Significant group effects were revealedby one-way ANOVA for both cortex and hippocampus (p < 0.0001).Letters above the bars indicate group differences. See text for detailsof the analyses.

(p < 0.05). Additional analyses revealed no significantgroup differences in average speed, percentage of timespent in the center of the maze or in the target quadrant(data not shown).

For analysis of memory acquisition in the Barnesmaze, the area under the latency curve (AUC) from Day1 to Day 4 was calculated for each animal and groupcomparisons were performed by one-way ANOVA(Fig. 6C). Significant differences between groups forarea under the learning curve were found (p = 0.0103).Pairwise comparisons indicated that untreated Tg miceshowed much higher AUC values compared to eithernTg mice or all three Tg groups (ps < 0.05).

Amyloid-β levels

Data were analyzed using a one-way ANOVAwith group as a factor and Bonferroni post-hoc

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tests for pairwise comparisons. In the cortex, resultsshowed a significant effect of group (F(4,48) = 104.19,p < 0.0001). Pairwise comparisons indicated thatuntreated Tg mice showed much higher levels of sol-uble A� compared to all three Tg groups (ps < 0.05),and none of these groups differed significantly fromeach other (see Fig. 7A).

In the hippocampus, results also showed a signifi-cant effect of group (F(4,48) = 37.82, p < 0.0001) withsignificant differences between Tg and EGCG, Exer-cise, as well as Combo groups (Fig. 7B). Although theCombo group showed the numerically lowest A�1-42level, there was no significant difference when it wascompared to either the EGCG or Exercise group.

DISCUSSION

The present study showed that 4 months ofEGCG dietary supplementation (∼50 mg/kg daily) andhomecage access to a running wheel (both alone andin combination), beginning at the onset of diseasepathology, improved nest building behaviors (previ-ously unexamined in the TgCRND8 model of AD)and spatial learning, and reduced levels of solubleA�1-42 in the cortex and hippocampus of TgCRND8mice (∼25–35% decrease). These findings are con-sistent with previous research using this dosage ofEGCG in the Tg2576 strain [15], as well as researchusing voluntary exercise treatment in the TgCRND8strain [19]. However, the current study started theseinterventions when animals were already experiencingcognitive loss and pathology. Brain tissue A�1-42 lev-els double in TgCRND8 mice from 4 until 8 weeksof age, and a sharp increase in both A�1-42 levelsand A�1-42/A�1-40 ratios can be detected by 10 weeksof age [26]. The earliest cognitive deficits previouslyreported in TgCRND8 mice were demonstrated at 8–9weeks of age using an object recognition task [33].This is the first study to explore the potential interac-tion of the effects of EGCG and exercise in these mice;it is valuable to know that, given the constraints of thepresent procedure, these manipulations have signifi-cant effects but do not appear to interact.

Nest building

Nest construction by mice is important for sur-vival in three ways: 1) conservation of body heat, 2)shelter from predators/environmental conditions, and3) reproduction and social interaction. This naturalbehavior of mice has been used to test changes inemotional states, such as apathy and social withdrawal

[34]. Murine models of AD (A�PPswe/PS1: [34, 35];Tg2676: [36]; 3xTg-AD: [37]), as well as mice withhippocampal lesions, show poorer nest constructioncompared to wild-type littermates. It has also beenargued that nest-building behaviors may be indicativeof executive functioning and behavioral organizationin mice [35]. This task has not been evaluated in theTgCNRD8 model of AD. In accordance with previousfindings using both single- and double-mutant A�PPmouse models of AD [35, 36], we found that untreatedTg mice showed poor nest construction compared towild-type littermates, at both 2-months and 6-monthsof age. It is important to mention that the deficit in nest-ing behavior was observed early in life, so it could beviewed as an indicator of cognitive dysfunction duringprodromal AD. Additionally, poor nest construction at6-months of age was associated with increased levelsof soluble A�1-42 in the cortex of Tg mice, indicat-ing that this behavioral task may indeed be sensitiveto changes in executive functioning in mice, as otherauthors [35] have argued. Our laboratory has testedmore than eighty mice for nest building at 2-monthsof age, and consequently found a significant effect ofgenotype, but no significant differences between malesand females in nesting behavior (data not shown).This observation is in agreement with the findings forTg2576 mice [38].

Four months of unlimited access to a running wheelreversed nest building deficits in TgCRND8 mice.Mice supplemented with EGCG (but without accessto a running wheel) also showed some improvement innest building behavior, but the effect was not as robustin this group compared to Tg mice who had accessto running wheels. These findings indicate that dailyintake of EGCG (at least 50 mg/kg) and daily physi-cal activity can help ameliorate dysfunction in dailyliving activities (i.e., nest building) in the TgCRND8strain; however, regular physical activity may be moreeffective, compared to daily EGCG intake alone.

Exploratory/locomotor activity

Consistent with our previous findings, untreatedTgCRND8 mice showed hyperactivity compared tonTg littermates in the open-field. Four months ofEGCG treatment failed to reduce this behavioral symp-tom; additionally, circling or “looping” was observedin some of these animals in the open-field. Four monthsof running wheel access, however, did reduce hyper-activity in these mice (see Fig. 4). There was a trendtoward higher daily distances traveled on the homecagerunning wheels for mice in the Combination group

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compared to exercise-only mice, but only at the verybeginning of the treatment period (see Fig. 2).

Although we expected to see EGCG reduce hyper-activity, as has been found for other nutraceuticals[39–41] in AD animals, only wheel running activ-ity was effective in influencing open-field behaviorin TgCRND8 mice. Furthermore, exercise has beenshown to not influence or slightly improve spe-cific AD-related changes in open-field behavior, suchas hypoactivity [42]. Running decreases anxiety-likebehavior, but increases exploration, in normal mice[43].

Anxiety

There are discrepancies in the literature regard-ing whether there are anxiety differences betweenuntreated TgCRND8 and wild-type mice (see [27]for a discussion). There is some evidence that EGCGhas anxiolytic effects on behavior in the elevated-plus maze [44], but that study used wild-type CD1mice. Exercise has been shown to decrease measuresof anxiety in different transgenic AD mouse mod-els (A�PP/PS1: [45]; 3x-Tg-AD: [42]). We did notfind any significant differences in anxiety between Tggroups in the open-field test, as measured by percent-age of time spent in the center area. Mice with access torunning wheels showed reduced anxiety-like behaviorcompared to untreated Tg mice, as measured by fewerentries to the dark zone in the light-dark box test, butthis effect was not found for EGCG-only mice.

Spatial learning and memory

In line with previous work [27], untreatedTgCRND8 mice demonstrated poor spatial learningperformance in the Barnes maze task compared towild-type littermates, as shown by longer escape laten-cies and more errors. We expected the EGCG andExercise groups to show some cognitive improve-ments (e.g., shorter latencies, fewer errors) based onprevious research [15, 19]; however, these publishedstudies used longer treatment periods for exercise (5–6months), and in the case of EGCG administration, adifferent transgenic AD mouse model (i.e., Tg2576:[15]). In line with previous research using longer wheelrunning exercise durations, we found that a shorter run-ning duration (4 months) still improved spatial learninglatencies in Tg mice, compared to untreated Tg con-trols. However, in contrast to other research usinga combination antioxidant/exercise intervention [25,42], no additive effects were found in our Combina-

tion group. In general, our Combination group did notshow improved cognitive performance compared toeither the exercise-only or EGCG-only groups. Never-theless, our studies together with others provide strongsupport for a complex multifaceted effect of EGCGand exercise on AD pathology. The time course ofthe efficacy of these effects in the improvement ofcognitive functions and reduction of soluble A�1-42levels may differ between animal models of AD. Thereduction of cognitive deficits can be specific to spa-tial memory performance and it may not generalize toother learning tasks. These beneficial changes may ormay not be related to alterations in soluble A�1-42 lev-els, A�PP processing, and other physiological markersof the disease. In addition, there may be differencesbetween males and females. Important sex-specificdifferences in adrenocortical hyperactivity have beendemonstrated in TgCRND8 mice between the age of30 and 120 days [46]. However, no sex-specific differ-ences have been reported in behavioral tests, althoughseveral studies like the present one used both femaleand male mice in different maze learning tests, novelobject recognition test and the open-field [33, 47–51].Our study used both sexes, but the animal numberswere not sufficient to observe sex differences so sex-specific effects cannot be ruled out completely. Futurestudies with larger sample sizes would allow us to testgroups separately in order to examine the influence ofsex in behavior and determine sex-specific effects ofdietary interventions and exercise.

CONCLUSION

Oral administration of EGCG, as well as a mod-erate duration of voluntary exercise, can amelioratesome of the behavioral manifestations and cognitiveimpairments of AD as modeled by the TgCRND8strain. Previous research using the TgCRND8 strain[19] found that voluntary exercise reduced solubleA�1-42 levels in the cortex and improved spatial learn-ing in the Morris water maze, when wheel runningbegan before the development of disease pathology.The present study shows that regular physical activ-ity, as well as daily EGCG dietary supplementation,can exert beneficial effects on cognition and lower A�burden in the brain of TgCRND8 mice, when it isstarted after the onset of disease pathology. Exercisedid seem to be more effective than EGCG-alone inimproving nest building behaviors, and both exerciseand EGCG were effective in reducing the cognitivedeficit observed in Barnes maze performance.

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ACKNOWLEDGMENTS

This work was supported by a University of Mis-souri Research Council grant and 2P01 AG018357(NIA), 1P50 AT006273 (NCCAM, ODS, NCI) fromNIH. We thank Dr. David Westaway for providing theTgCRND8 mice and Jennifer Hamilton for maintain-ing the colony and genotyping.

Authors’ disclosures available online (http://www.j-alz.com/disclosures/view.php?id=2547).

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