Male APPSwe/PS1M146V double-Tg mice and Wt mice were randomly assigned to a sedentary group (Tg-Sed, Wt-Sed) or an aerobic exercise group (Tg-Ex, Wt-Ex). After the physical training, all mice were subjected to an eight-arm radial maze test for spatial learning and memory evaluation, then the mice were sacrificed and brain tissues of the cortex and hippocampus were extracted for further analysis.

Six-month-old male Tg mice (n = 20) and Wt male littermates (n = 20) were purchased from Beijing HFK Bioscience Co., Ltd (Beijing, China). The mice were kept under standard laboratory conditions (12:12 h light-dark cycle, at 22 $\pm$ 2 °C and 45%–55% relative humidity) and provided with food and water ad libitum. Both Tg mice and Wt mice were randomly and evenly assigned to an exercise group (Tg-Ex, n = 10; Wt-Ex, n = 10) or a sedentary group (Tg-Sed, n = 10; Wt-Sed, n = 10). Evidence has shown variations in $\beta$-amyloid pathological phenotype [22]; thus, to avoid sex-induced variations in results, the study used only male APPSwe/PS1M146V double-Tg mice.

Ethics approval was obtained from the Ethics Committee of the Beijing Sport University (2015015) and the Guiding Principles for Care and Use of Animals was followed.

Before the 12-week exercise training program, both Wt-Ex and Tg-Ex mice were familiarized with the treadmill training by running on a treadmill for 3 consecutive days at a speed of 10 m/min with a 0° inclination. After the familiarization, each training session started with a standard warm-up by running on the treadmill for 10 min at a speed of 12 m/min, followed by running for 50 min at a speed of 15 m/min, which was defined here as moderate intensity on the basis of our previous studies where mice running at a speed of 12–15 meters/min corresponded to an oxygen consumption of around 65–75% of maximal oxygen consumption [23, 24, 25]. The training was carried out once per day, 5 days per week for 12 consecutive weeks. Wt-Sed and Tg-Sed mice were placed on the treadmill for 10 min with the same time schedule as Wt-Ex and Tg-Ex mice but undertook no exercise training.

On the next day following the last training session, all mice took an eight-arm radial maze test in a quiet environment with weak light to facilitate the assessment of spatial memory. The eight-arm radial maze apparatus (JLBehv-8ARMM, Shanghai Jiliang, Shanghai, China) is comprised of eight arms, spaced equidistantly, and visual reference cues were hung 1 m above the maze apparatus. The test process has been previously described in detail [26]. Briefly, the test includes 3 days of adaption and thereafter 10 consecutive days of testing. To motivate the mice to seek the chocolate crumbs in the maze arms, the food supply to the mice was reduced 3 days before the adaption so that the body weights of the mice were reduced to 80–85% of their initial weights. Thereafter, the food supply was maintained at such a level as to keep the body weights relatively stable throughout the experiment [27, 28]. Body weights of the mice were measured every other day to avoid $>$20% reduction. During the adaptation, the mice had 3 days to familiarize themselves with the test set by freely exploring the maze for 10 min with all eight of the arms baited with chocolate crumbs (0.08 g).

Before the test, chocolate crumbs were set in four randomly selected arms, and the test began with putting the mice in the center of the platform heading towards arm number one and ended when the mice had visited all the four baited arms within a maximum time of 10 min. If the mice failed to visit all the baited arms within 10 min, the mice would be excluded from group value calculations. The test was conducted on each individual mouse for 10 consecutive days. Working memory errors, reference memory errors, and time needed to complete each trial were recorded. Evaluation of working memory error was performed by counting the number of re-entries into a baited or non-baited arm, and similarly, evaluation of reference memory error was performed by counting the number of entries into a non-baited arm. For a specific mouse, the chocolate crumbs were placed in the same four arms during the 10 trials. By the end of the tests, two mice in each group failed to finish the test within the time limit, resulting in only eight mice left in each group for statistical analysis.

The mice were anesthetized with isoflurane, then perfused with 4% paraformaldehyde (P1110, Solarbio, Beijing, China) in 0.1M sodium phosphate buffer (PBS) pH7.4 through the left ventricle of the heart. The brains were removed from the skulls and postfixed by immersion in the same fixative for 48 h at 4 °C. The cortical and hippocampal tissues were dissected and dehydrated with 20% and 30% sucrose in 0.1 M PBS at 4 °C. Subsequently, both tissues were frozen rapidly in liquid nitrogen and stored at –80 °C until use. For immunostaining, cross sections (35 µm) were cut using a cryostat (CM1850, Leica, Wetzlar, Hessian, Germany) and the sections were collected on glass slides. The sections from the left hemisphere were used for immunohistochemistry, and the right hemisphere for immunofluorescence. The immunofluorescence and immunohistochemistry staining procedures have been previously described in detail [23, 29, 30]. The primary and secondary staining antibodies are listed in Table 1.

The staining was examined under a microscope (IX71-F22PH, OLYMPUS, Tokyo, Japan) connected to a computer equipped with a digital camera (modelDP71, OLYMPUS, Tokyo, Japan). Image J software (National Institutes of Health, Bethesda, MD, USA) was used for image analysis. A$\beta$ plaque levels were quantified on six randomly selected areas of each section/mouse stained with immunofluorescence by calculating the mean fluorescence intensity, which is the ratio of integrated density to the area, using the same position on the section as a reference.

The mice were anesthetized with isoflurane (26675-46-7, Sigma-Aldrich, Saint Louis, MO, USA) then decapitated. After dissection of the cortex and hippocampus on ice, the tissues were homogenized with phosphatase inhibitors and radio immunoprecipitation assay lysis buffer (R0278, Sigma-Aldrich, St. Louis, MO, USA). The protein extraction, protein content determination, and electrophoresis procedures were the same as those described in our previous studies [23, 29, 30]. The primary and secondary antibodies are listed in Table 1.

Protein quantification was performed using Bio-Rad, ChemiDoc XRS+ System Image Lab software (version 6.0.1, Bio-Rad, Hercules, CA, USA). The protein amounts were expressed as semiquantitative ratios between the value of a specific protein and the value of the reference protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Levels of microtubule-associated protein tau (p‐tau) ser262 and p‐tau ser396 were expressed as relative values of total tau, levels of p-GSK3$\alpha$ ser21 and p-GSK3$\alpha$ tyr279 were expressed as relative values of total GSK3$\alpha$, and levels of p-GSK3$\beta$ ser9 and p-GSK3$\beta$ tyr216 were expressed as relative values of total GSK3$\beta$.

Statistical analysis was performed using SPSS software (version 22.0, SPSS Inc., Chicago, IL, USA). Group values of a specific variable were calculated based on measurements of 10 consecutive days for all mice in the group. Repeated measurement analysis of variance (ANOVA) was performed to analyze over-time changes in the eight-arm radial maze test performance among groups (i.e., Wt-Sed, Wt-Ex, Tg-Sed, and Tg-Ex), followed by Bonferroni post-hoc analysis when group, time, and group-time interaction were significant. Two-way ANOVA (with genotype [Wt or Tg] and exercise [Sed or Ex] as two factors) was used to analyze the results of western blotting, and a simple main effect (multiple comparison adjusted by the Bonferroni approach) was reported when genotype-exercise interaction was significant. The student’s t-test was applied for the analysis of immunofluorescence staining between Tg-Sed and Tg-Ex mice. A probability of less than 0.05 was used to reject the null hypothesis and deemed to indicate a statistically significant difference. All data are expressed as mean (standard deviation) (mean (SD)), except for the A$\beta$ plaque level data, which was expressed as mean $\pm$ standard error of the mean (mean $\pm$ SEM).

Repeated-measures ANOVA revealed that test days and groups had significant effects on the working memory errors (Fig. 1A) and reference memory errors (Fig. 1B). Both working memory errors (test days: F = 6.317, p 0.001) and reference memory errors (test days: F = 2.144, p 0.001) were significantly decreased following the progress of the test in all the four groups, and significantly different between groups (groups: working memory errors, F = 8.783, p 0.001; reference memory errors, F = 13.543, p 0.001). Interaction between groups and test days had no significant effect on either working memory errors or reference memory errors (groups $\times$ test days: working memory errors, F = 1.095, p = 0.361; reference memory errors, F = 0.489, p = 0.957). The main effect of test days and groups revealed that Tg-Sed mice showed significantly higher working memory errors (p 0.001) and reference memory errors (p = 0.003) than Wt-Sed mice, and Tg-Ex mice showed significantly lower working memory errors (p = 0.018) and reference memory errors (p = 0.042) than Tg-Sed mice (Fig. 1A,B).

Fig. 1.

Results of the eight-arm radial maze test. Both working memory errors (A) and reference memory errors (B) were significantly decreased following the progress of the test in all four groups (n = 10/group; test days: working memory errors, F = 6.317, p 0.001; reference memory errors, F = 2.144, p 0.001) and were significantly different between groups (groups: working memory errors, F = 8.783, p 0.001; reference memory errors, F = 13.543, p 0.001). Tg-Sed mice (n = 8) showed significantly higher working memory errors and reference memory errors than Wt-Sed mice (n = 8) on three test occasions. Tg-Ex mice (n = 8) had significantly lower working memory errors on two test occasions, and reference memory errors than Tg-Sed mice (n = 8) on four test occasions. Time needed to complete the test was significantly decreased following the progress of the test for all four groups (n = 10/group; test days: F = 64.724, p 0.001) and significantly different between groups (C; groups: F = 103.1, p 0.001). Tg-Sed mice needed significantly longer time to complete the test than Wt-Sed mice on seven test occasions, whereas Tg-Ex mice needed significantly shorter time to complete the test than Tg-Sed mice on five test occasions (C). Data are expressed as mean (SD). Note: Wt, wild-type; Tg, transgenic; Sed, sedentary; Ex, exercise; SD, standard deviation. ${}^{*}$p 0.05, Tg-Ex vs Tg-Sed; ${}^{**}$p 0.01, Tg-Ex vs Tg-Sed; ${}^{\mathrm{\#}}$p 0.05, Tg-Sed vs Wt-Sed; ${}^{\mathrm{\#}\mathrm{\#}}$p 0.01, Tg-Sed vs Wt-Sed.

The time needed to complete each repetition of the test is shown in Fig. 1C. The time decreased significantly following the progress of the test for all four groups (test days: F = 64.724, p 0.001), and was significantly different between groups (groups: F = 103.1, p 0.001). Significant interaction between groups and test days was also found (groups $\times$ test days: F = 14.746, p 0.001). The Bonferroni post-hoc analysis revealed that Tg-Sed mice needed significantly longer time to complete the test on day 1 (p 0.01), day 2 (p 0.01), day 3 (p 0.01), day 4 (p 0.01), day 6 (p 0.01), day 7 (p 0.01), and day 9 (p 0.01) than Wt-Sed mice on the corresponding days. In contrast, Tg-Ex mice needed significantly shorter time to complete the test on day 2 (p 0.05), day 3 (p 0.01), day 4 (p 0.05), day 6 (p 0.01), and day 7 (p 0.05) than Tg-Sed mice on the corresponding days.

A$\beta$ plaques were not observed in either the cortex or hippocampus of Wt-Sed or Wt-Ex mice, but were in both tissues of both Tg-Sed and Tg-Ex mice (Fig. 2A,B). Statistical analysis revealed significantly lower levels of A$\beta$ plaques in both tissues of Tg-Ex mice than in respective tissues of Tg-Sed mice (Fig. 2C).

Fig. 2.

A$\beta$ plaque levels in the cortex and hippocampus. Representative images of immunofluorescence staining (upper panel) and immunohistochemical staining (lower panel) in the cortex (A) and hippocampus (B), revealing A$\beta$ plaques (white arrows) in the cortex and hippocampus, and pyramidal layer in the hippocampus (black arrows). Quantification of the fluorescence staining for A$\beta$ plaques and comparisons of the results between Tg-Sed and Tg-Ex mice in respective tissues of the cortex and hippocampus are shown in the histogram (C). In both the cortex and hippocampus, Tg-Ex mice had significantly lower levels of A$\beta$ plaques than in the respective tissues of Tg-Sed mice (n = 6/group). Data are expressed as mean $\pm$ SEM. Note: Wt, wild-type; Tg, transgenic; Sed, sedentary; Ex, exercise; SEM, standard error of the mean.${}^{**}$p 0.01 (Tg-Ex vs Tg-Sed). Scale bar = 25 µm.

Western blot analysis revealed four different protein bands with different molecular weights, representing phosphorylation of total tau, p-tau ser262, p-tau ser396, and reference protein GAPDH, respectively, in the cortex (Fig. 3 upper panel, left) and hippocampus (Fig. 3 upper panel, right; all original figures of Western Blot can be found in the Supplementary Material) of Wt-Sed, Wt-Ex, Tg-Sed, and Tg-Ex mice.

Fig. 3.

Tau phosphorylation levels in the cortex and hippocampus. Representative images of western blots for tau (A,D), p-tau ser262 (B,E), and p-tau ser396 (C,F) in the cortex (upper panel, left) and hippocampus (upper panel, right) (n = 5/group). Tau in the cortex (A) and hippocampus (D) was significantly increased in Tg-Sed mice compared with Wt-Sed mice and this increase was not blocked by the treadmill exercise. P-tau ser262/tau (B) and p-tau ser396/tau (C) in the cortex were significantly increased in Tg-Sed mice compared with Wt-Sed mice but significantly decreased in Tg-Ex mice compared with Tg-Sed mice and Wt-Ex mice compared with Wt-Sed mice. P-tau ser262/tau in the hippocampus (E) was significantly increased in Tg-Sed mice compared with Wt-Sed mice but significantly decreased in Tg-Ex mice compared with Tg-Sed mice and Wt-Ex mice compared with Wt-Sed mice. P-tau ser396/tau in the hippocampus (F) was significantly increased in Tg-Sed mice compared with Wt-Sed mice but significantly decreased in Tg-Ex mice compared with Tg-Sed mice and Wt-Ex mice compared with Wt-Sed mice. Data are expressed as mean (SD). Note: Wt, wild-type; Tg, transgenic; Sed, sedentary; Ex, exercise; SD, standard deviation. *p 0.05; **p 0.01.

Tau phosphorylation levels and their comparisons between different groups are shown in Fig. 3A–F.

Two-way ANOVA revealed that genotype had significant effects on total tau in both the cortex (genotype: F = 72.203, p 0.001) and hippocampus (genotype: F = 98.128, p 0.001), but there were no significant exercise effects or interaction between genotype and exercise on total tau in both the cortex (exercise: F = 5.012, p = 0.061; genotype $\times$ exercise: F = 0.007, p = 0.935) and hippocampus (exercise: F = 0.885, p = 0.361; genotype $\times$ exercise: F = 0.572, p = 0.460). The main effect of genotype revealed that Tg groups had significantly higher levels of total tau in both the cortex and hippocampus than in respective tissues of Wt groups (Fig. 3A,D).

Two-way ANOVA revealed that genotype and exercise had significant effects on the levels of p-tau ser262 in both the cortex (genotype: F = 8.348, p = 0.012; exercise: F = 8.820, p = 0.009) and hippocampus (genotype: F = 6.981, p = 0.021; exercise: F = 7.098, p = 0.017), but there was no significant interaction between genotype and exercise on the levels of p-tau ser262 in both the cortex (genotype $\times$ exercise: F = 0.028, p = 0.870) and hippocampus (genotype $\times$ exercise: F = 3.967, p = 0.070). The main effect of genotype and exercise revealed that Tg groups had significantly higher levels of p-tau ser262 in both the cortex and hippocampus than in respective tissues of Wt groups. Exercise groups had significantly lower levels of p-tau ser262 in both the cortex and hippocampus than in respective tissues of sedentary groups (Fig. 3B,E).

Two-way ANOVA revealed that genotype and exercise had significant effects on the levels of p-tau ser396 in both the cortex (genotype: F = 5.821, p = 0.031; exercise: F = 19.320, p = 0.001) and hippocampus (genotype: F = 46.984, p = 0.001; exercise: F = 15.278, p = 0.002), but there was no significant interaction between genotype and exercise on the levels of p-tau ser396 in both the cortex (genotype $\times$ exercise: F = 0.981, p = 0.335) and hippocampus (genotype $\times$ exercise: F = 3.078, p = 0.103). The main effect of genotype and exercise revealed that Tg groups had significantly higher levels of p-tau ser396 in both the cortex and hippocampus than in respective tissues of Wt groups. Exercise groups had significantly lower levels of p-tau ser396 in both the cortex and hippocampus than in respective tissues of sedentary groups (Fig. 3C,F).

Western blotting revealed three different protein bands with different molecular weights, representing total GSK3$\alpha$, p-GSK3$\alpha$ ser21/p-GSK3$\alpha$ tyr279, and reference protein GAPDH, respectively, in both tissues of each group (Fig. 4 upper panel). Quantification of the bands and comparisons of both p-GSK3$\alpha$ ser21 and p-GSK3$\alpha$ tyr279 levels in both tissues between different groups are shown in the histogram (Fig. 4A–D).

Fig. 4.

GSK3$\alpha$ kinase activity in the cortex and hippocampus. Representative images of western blots for p-GSK3$\alpha$ ser21 (A,C), p-GSK3$\alpha$ tyr279 (B,D), and GSK3$\alpha$ (A–D) in the cortex and hippocampus (n = 5/group). In the cortex of Tg-Sed mice, p-GSK3$\alpha$ ser21/GSK3$\alpha$ was significantly decreased compared with that of Wt-Sed mice but significantly increased in Tg-Ex mice compared with Tg-Sed mice and Wt-Ex mice compared with Wt-Sed mice (A). P-GSK3$\alpha$ tyr279/GSK3$\alpha$ in the cortex was significantly increased in Tg-Sed compared with Wt-Sed but significantly decreased in Tg-Ex compared with Tg-Sed mice and Wt-Ex mice compared with Wt-Sed mice (B). P-GSK3$\alpha$ ser21/GSK3$\alpha$ in the hippocampus was significantly decreased in Tg-Sed mice compared with Wt-Sed mice but significantly increased in Tg-Ex mice compared with Tg-Sed mice and Wt-Ex mice compared with Wt-Sed mice (C). P-GSK3$\alpha$ tyr279/GSK3$\alpha$ in the hippocampus was significantly increased in Tg-Sed mice compared with Wt-Sed mice but significantly decreased in Tg-Ex mice compared with Tg-Sed mice and Wt-Ex mice compared with Wt-Sed mice (D). Data are expressed as mean (SD). Note: Wt, wild-type; Tg, transgenic; Sed, sedentary; Ex, exercise; SD, standard deviation. *p 0.05; **p 0.01.

Two-way ANOVA revealed that genotype and exercise had significant effects on the levels of p-GSK3$\alpha$ ser21 (genotype: F = 7.654, p = 0.017; exercise: F = 12.397, p = 0.004) and p-GSK3$\alpha$ tyr279 (genotype: F = 17.736, p = 0.001; exercise: F = 8.980, p = 0.011) in the cortex, but there was no significant interaction between genotype and exercise on the levels of p-GSK3$\alpha$ ser21 (genotype $\times$ exercise: F = 0.713, p = 0.415) and p-GSK3$\alpha$ tyr279 (genotype $\times$ exercise: F = 0.003, p = 0.955) in the cortex. The main effect of genotype and exercise revealed that Tg groups had significantly lower levels of p-GSK3$\alpha$ ser21 and higher levels of p-GSK3$\alpha$ tyr279 in the cortex than in respective tissues of Wt groups. Exercise groups had significantly higher levels of p-GSK3$\alpha$ ser21 and lower levels of p-GSK3$\alpha$ tyr279 in the cortex than in respective tissues of sedentary groups (Fig. 4A,B).

Two-way ANOVA revealed that genotype and exercise had significant effects on the levels of p-GSK3$\alpha$ ser21 (genotype: F = 9.573, p = 0.009; exercise: F = 6.217, p = 0.028) and p-GSK3$\alpha$ tyr279 (genotype: F = 5.397, p = 0.035; exercise: F = 33.209, p 0.001) in the hippocampus, but there was no significant interaction between genotype and exercise on the levels of p-GSK3$\alpha$ ser21 (genotype $\times$ exercise: F = 0.684, p = 0.424) and p-GSK3$\alpha$ tyr279 (genotype $\times$ exercise: F = 0.156, p = 0.699) in the hippocampus. The main effect of genotype and exercise revealed that Tg groups had significantly lower levels of p-GSK3$\alpha$ ser21 and higher levels of p-GSK3$\alpha$ tyr279 in the hippocampus than in respective tissues of Wt groups. Exercise groups had significantly higher levels of p-GSK3$\alpha$ ser21 and lower levels of p-GSK3$\alpha$ tyr279 in the hippocampus than in respective tissues of sedentary groups (Fig. 4C,D).

P-GSK3$\beta$ ser9 and p-GSK3$\beta$ tyr216 levels in both tissues are shown in Fig. 5. Western blotting revealed three different protein bands with different molecular weights, representing total GSK3$\beta$, p-GSK3$\beta$ser9/p-GSK3$\beta$ tyr216, and reference protein GAPDH, respectively, in both tissues of each individual group (upper panel in Fig. 5A–D). Quantification of the bands and comparisons between different groups are shown in the histograms (Fig. 5A–D).

Fig. 5.

GSK3$\beta$ kinase activity in the cortex and hippocampus. Representative images of western blots for p-GSK3$\beta$ ser9 (A,C), p-GSK3$\beta$ tyr216 (B,D), and GSK3$\beta$ (A–D) in the cortex and hippocampus (n = 5/group). P-GSK3$\beta$ ser9/GSK3$\beta$ in the cortex was significantly decreased in Tg-Sed mice compared with Wt-Sed mice but only increased in Wt-Ex mice compared with Wt-Sed (A). P-GSK3$\beta$ tyr216/GSK3$\beta$ in the cortex was significantly increased in Tg-Sed mice compared with Wt-Sed mice but similar between Tg-Ex and Tg-Sed mice, and between Wt-Ex and Wt-Sed mice (B). P-GSK3$\beta$ ser9/GSK3$\beta$ in the hippocampus was significantly decreased in Tg-Sed mice compared with Wt-Sed mice but significantly increased in Tg-Ex mice compared with Tg-Sed mice and Wt-Ex mice compared with Wt-Sed mice (C). P-GSK3$\beta$ tyr216/GSK3$\beta$ in the hippocampus was significantly increased in Tg-Sed mice compared with Wt-Sed mice but significantly decreased in Tg-Ex mice compared with Tg-Sed mice, and similar between Wt-Ex and Wt-Sed mice (D). Data are expressed as mean (SD). Note: Wt, wild-type; Tg, transgenic; Sed, sedentary; Ex, exercise; SD, standard deviation. *p 0.05; **p 0.01.

Two-way ANOVA revealed that genotype had significant effects on the levels of p-GSK3$\beta$ ser9 (genotype: F = 34.192, p = 0.001) and p-GSK3$\beta$ tyr216 (genotype: F = 15.190, p = 0.001) in the cortex. Significant exercise effects and interaction between genotype and exercise were observed on the levels of p-GSK3$\beta$ ser9 (exercise: F = 11.851, p = 0.003; genotype $\times$ exercise: F = 5.349, p = 0.034) but not on p-GSK3$\beta$ tyr216 (exercise: F = 3.952, p = 0.064; genotype $\times$ exercise: F = 0.095, p = 0.762) in the cortex. The main effect of genotype revealed that Tg groups had significantly lower levels of p-GSK3$\beta$ ser9 and higher levels of p-GSK3$\beta$ tyr216 in the cortex than in respective tissues of Wt groups. The multiple comparison test revealed that Wt-Ex mice had significantly higher levels of p-GSK3$\beta$ ser9 (p 0.01) than Wt-Sed mice (Fig. 5A).

Two-way ANOVA revealed that genotype and exercise had significant effects on the levels of p-GSK3$\beta$ ser9 (genotype: F = 5.821, p = 0.034; exercise: F = 6.871, p = 0.024) and p-GSK3$\beta$ tyr216 (genotype: F = 39.914, p = 0.001; exercise: F = 8.074, p = 0.019; genotype $\times$ exercise: F = 10.467, p = 0.010) in the hippocampus, but there was no significant interaction between genotype and exercise on the levels of p-GSK3$\beta$ ser9 (genotype $\times$ exercise: F = 0.393, p = 0.637) in the hippocampus. The main effect of genotype and exercise revealed that Tg groups had significantly lower levels of p-GSK3$\beta$ ser9 in the hippocampus than in respective tissues of Wt groups. Exercise groups had significantly higher levels of p-GSK3$\beta$ ser9 in the hippocampus than in respective tissues of sedentary groups (Fig. 5C). The multiple comparison test revealed that Tg-Sed mice had significantly higher levels of p-GSK3$\beta$ tyr216 (p 0.01) than Wt-Sed mice (Fig. 5D). In contrast, Tg-Ex mice (p = 0.011) had significantly lower levels of p-GSK3$\beta$ tyr216 than Tg-Sed mice, and similar levels of p-GSK3$\beta$ tyr216 in Wt-Ex and Wt-Sed mice (p = 0.785) (Fig. 5D).

Western blotting revealed three different protein bands with different molecular weights, representing p-Akt ser473, total Akt, and reference protein GAPDH, respectively, in both tissues of each group (Fig. 6 upper panel). Quantification of the bands and comparisons between different groups are shown in Fig. 6.

Fig. 6.

Akt kinase activity in the cortex and hippocampus. Representative images of western blots of p-Akt ser473, total Akt, and reference protein GAPDH in the cortex (upper panel, left) and hippocampus (upper panel, right; n = 5/group). P-Akt ser473/Akt in the cortex was significantly decreased in Tg-Sed mice compared with Wt-Sed mice but significantly increased in Tg-Ex mice compared with Tg-Sed mice and Wt-Ex mice compared with Wt-Sed mice (A). P-Akt ser473/Akt in the hippocampus was significantly decreased in Tg-Sed mice compared with Wt-Sed mice, and both significantly increased in Tg-Ex and Wt-Ex mice (B). Data are expressed as mean (SD). Note: Wt, wild-type; Tg, transgenic; Sed, sedentary; Ex, exercise; SD, standard deviation. *p 0.05; **p 0.01.

Two-way ANOVA revealed that genotype and exercise had significant effects on the levels of p-Akt ser473 in the cortex (genotype: F = 38.969, p = 0.001; exercise: F = 12.950, p = 0.004) and hippocampus (genotype: F = 5.484, p = 0.044; exercise: F = 5.202, p = 0.048), but there was no significant interaction between genotype and exercise on the levels of p-Akt ser473 in the cortex (genotype $\times$ exercise: F = 0.221, p = 0.648) and hippocampus (genotype $\times$ exercise: F = 1.433, p = 0.262). The main effect of genotype and exercise revealed that Tg groups had significantly lower levels of p-Akt ser473 in both the cortex and hippocampus than in respective tissues of Wt groups. Exercise groups had significantly higher levels of p-Akt ser473 in both the cortex and hippocampus than in respective tissues of sedentary groups (Fig. 6A,B).