Moderate-Intensity Treadmill Exercise Regulates GSK3α/β Activity in the Cortex and Hippocampus of APP/PS1 Transgenic Mice

Abstract

Background: Physical exercise has been shown to be beneficial for individuals with Alzheimer's disease (AD), although the underlying mechanisms are not fully understood. Methods: Six-month-old Amyloid precursor protein/Presenilin 1 (APP/PS1) transgenic (Tg) mice and wild-type (Wt) mice were randomly assigned to either a sedentary group (Tg-Sed, Wt-Sed) or an exercise group (Tg-Ex, Wt-Ex) undertaking a 12-week, moderate-intensity treadmill running program. Consequently, all mice were tested for memory function and amyloid 6 (AB) levels and phosphorylation of tau and protein kinase В (Akt)/glycogen synthase kinase-3 (GSK3) were examined in tissues of both the cortex and hippocampus. Results: Tg-Sed mice had severely impaired memory, higher levels of A8, and increased phosphorylation of tau, GSK3a tyrosine279, and GSK36 tyrosine216, but less phosphorylation of GSK3a serine21, GSK36 serine9, and Akt serine473 in both tissues than Wt-Sed mice in respective tissues. Tg-Ex mice showed significant improvement in memory function along with lower levels of AS and less phosphorylation of tau (both tissues), GSK3a tyrosine279 (both tissues), and GSK36 tyrosine216 (hippocampus only), but increased phosphorylation of GSK3a serine21 (both tissues), GSK3 serine9 (hippocampus only), and Akt serine473 (both tissues) compared with Tg-Sed mice in respective tissues. Conclusions: Moderate-intensity aerobic exercise is highly effective in improving memory function in 9-month-old APP/PS1 mice, most likely through differential modulation of GSK3a/8 phosphorylation in the cortex and hippocampus.

Headnote

Abstract

Background: Physical exercise has been shown to be beneficial for individuals with Alzheimer's disease (AD), although the underlying mechanisms are not fully understood. Methods: Six-month-old Amyloid precursor protein/Presenilin 1 (APP/PS1) transgenic (Tg) mice and wild-type (Wt) mice were randomly assigned to either a sedentary group (Tg-Sed, Wt-Sed) or an exercise group (Tg-Ex, Wt-Ex) undertaking a 12-week, moderate-intensity treadmill running program. Consequently, all mice were tested for memory function and amyloid 6 (AB) levels and phosphorylation of tau and protein kinase В (Akt)/glycogen synthase kinase-3 (GSK3) were examined in tissues of both the cortex and hippocampus. Results: Tg-Sed mice had severely impaired memory, higher levels of A8, and increased phosphorylation of tau, GSK3a tyrosine279, and GSK36 tyrosine216, but less phosphorylation of GSK3a serine21, GSK36 serine9, and Akt serine473 in both tissues than Wt-Sed mice in respective tissues. Tg-Ex mice showed significant improvement in memory function along with lower levels of AS and less phosphorylation of tau (both tissues), GSK3a tyrosine279 (both tissues), and GSK36 tyrosine216 (hippocampus only), but increased phosphorylation of GSK3a serine21 (both tissues), GSK3 serine9 (hippocampus only), and Akt serine473 (both tissues) compared with Tg-Sed mice in respective tissues. Conclusions: Moderate-intensity aerobic exercise is highly effective in improving memory function in 9-month-old APP/PS1 mice, most likely through differential modulation of GSK3a/8 phosphorylation in the cortex and hippocampus.

Keywords: APP/PS1 mouse; aerobic exercise; memory function; amyloid-6; Akt/GSK3 pathways

1. Introduction

Alzheimer's disease (AD) is a chronic and progressive neurodegenerative disease. Extensive distribution of 8-amyloid (A 8) plaques and neurofibrillary tangles (NF Ts) in the brain are the major histopathological hallmarks of AD [1,2]. One of the hypotheses of AD pathogenesis suggests that amyloid plaques or A 6-peptides act as the direct cause and initiate cascade effects, leading to a series of reactions such as tau pathology, inflammation, synaptic dysfunction, neuronal loss, and ultimately dementia [2].

AD neuropathology initiated by AB deposition is believed to begin decades prior to the onset of clinic symptoms, and AS deposition has been observed in the cortex at 6 weeks of age and in the hippocampus at 3-4 months of age in Amyloid precursor protein/Presenilin 1 (APP/PS1) AD mouse models [2,3]. Hyperactivity of glycogen synthase kinase-3 (GSK3) is believed to be critically involved in the enhancement of A4 from 8-amyloid precursor protein (APP) and in tau hyperphosphorylation [4,5]. GSK3 is a highly conserved protein kinase for glycogen synthase and is encoded by two highly related genes: GSK3a (51 kDa) and GSK36 (47 kDa). Under physiological conditions, GSK3 is activated via phosphorylation at tyrosine residue 279 of GSK3a (p-GSK3a tyr279) and tyrosine residue 216 of GSK3/ (p-GSK3A tyr216) and inhibited via phosphorylation at serine residue 21 of GSK3a (p-GSK3a ser2 1) and serine residue 9 of GSK3 (p-GSK3 ser9) [6]. The neuronal reaction to AS and tau proteopathic stress is complex, and whether the GSK3 biochemical response 1s similar in the cortex and hippocampus 1s largely unknown.

Given that GSK3 is relevant to the pathophysiological processes of AD, inhibition of GSK3 activity has been deemed to be an optional strategy in AD prevention and treatment. Physical inactivity is generally believed to be one of the risk factors associated with AD [7,8], whereas physical exercise has been proven to reduce both the progression of AD-like neuropathology in the TgCRND8 mouse model [9,10] and the risk of AD [11], and improve cognitive function in AD patients [12]. Many studies have been performed in both AD animal models and AD patients to explore the molecular mechanisms underlying exercise-induced improvement in memory or cognition. In several studies on AD animal models, exercise induced significant reductions in AB levels and tau phosphorylation at multiple sites in both the cortex and hippocampus [13-17], which was believed to be associated with inhibition of GSK3 activity via enhancement in levels of p-GSK3a/5 ser21/9 [17-19]. However, in a study on 6month-old APP/PS1 transgenic (Tg) mice, housing mice in an enriched environment for 4 weeks was unable to modulate GSK38 (ser9) activity in either the hippocampus or the cortex [20]. The discrepancies in these results might be due to variations in the forms of stimulation/exercise, intervention times in the AD animals/patients (AD stage), and duration of the interventions. In a previous study, 3week voluntary wheel running in 15-19-month-old mice did not significantly effect A 6 levels [21]. However, in another study, 5-month treadmill exercise in 3- month and 12month-old mice [15] significantly reduced soluble A 840/42 in mice of both ages and significantly reduced AS plaque burden in the 3-month-old mice but not the 12-month-old mice. Therefore, intervention type and duration as well as AD stage seem to play important roles in the outcomes.

To our knowledge, only a few studies have investigated the underlying molecular mechanisms of memory function improvement in AD mouse models induced by aerobic exercise with respect to regulation in protein kinase B (Akt)/GSK3 activity in both the cortex and hippocampus. More importantly, no study has yet investigated the mechanisms in term of GSK3a/6 activity in both the cortex and hippocampus of moderate AD stage APP/PS1 mice. To fill these knowledge gaps, 6-month-old APPSwe/PS1M146V double Tg AD mice and wild-type (Wt) mice were subjected to 12-week, moderate-intensity treadmill running. After the training, spatial learning and memory were tested, and levels of AB, phosphorylation of tau, phosphorylation of Akt at ser473 (a major upstream modulator of GSK3), and phosphorylation of GSK3a and GSK36 at tyr279/216 and ser21/9 were examined in tissues of both the cortex and hippocampus from both Tg mice and Wt mice. By examining the effects of aerobic exercise on memory functionin Tg mice, and levels of A 3, phosphorylation of tau, phosphorylation of Akt at ser473, and phosphorylation of GSK3a and GSK3 at tyr279/216 and ser21/9 in both the cortex and hippocampus, we aimed to explore the molecular mechanisms underlying the beneficial effects of physical exercise on AD development.

2. Materials and Methods

2.1 Experimental Design

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 eightarm 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.

2.2 Animals and Ethic Approval

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 + 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, п = 10; Wt-Ex, n = 10) or a sedentary group (Tg-Sed, n = 10; Wt-Sed, n = 10). Evidence has shown variations in 8-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.

2.3 Aerobic Exercise Training

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 [23-25]. 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.

2.4 Eight-Arm Radial Maze Test

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 (JLBehvSARMM, 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 nonbaited 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.

2.5 Brain Tissue Processing and Immunostaining

The mice were anesthetized with isoflurane, then perfused with 4% paraformaldehyde (P1110, Solarbio, Beijing, China) in 0. IM 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 um) 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 (IX71F22PH, 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. АВ 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.

2.6 Western Blotting

The mice were anesthetized with isoflurane (2667546-7, 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-GSK3a ser21 and p-GSK3a tyr279 were expressed as relative values of total GSK3a, and levels of p-GSK3 ser9 and p-GSK3 3 tyr216 were expressed as relative values of total GSK3 5.

2.7 Statistical Analysis

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 posthoc 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 f-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 AB plaque level data, which was expressed as mean + standard error of the mean (mean + SEM).

3. Results

3.1 Eight-Arm Radial Maze Test

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, р < 0.001; reference memory errors, F = 13.543, р < 0.001). Interaction between groups and test days had no significant effect on either working memory errors or reference memory errors (groups х test days: working memory errors, F = 1.095, р = 0.361; reference memory errors, F = 0.489, р = 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. 1 A,B).

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, р < 0.001), and was significantly different between groups (groups: F= 103.1, р < 0.001). Significant interaction between groups and test days was also found (groups x test days: F = 14.746, р < 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 (р < 0.01), day 3 (р < 0.01), day 4 (р < 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.

3.2 AB Plaques

AB 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 AB plaques in both tissues of Tg-Ex mice than in respective tissues of Tg-Sed mice (Fig. 2C).

3.3 Tau Phosphorylation

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.

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, р < 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, р = 0.061; genotype x exercise: F = 0.007, р = 0.935) and hippocampus (exercise: F - 0.885, р= 0.361; genotype x exercise: 7 = 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, р = 0.009) and hippocampus (genotype: F = 6.981, р = 0.021; exercise: F = 7.098, р = 0.017), but there was no significant interaction between genotype and exercise on the levels of p-tau ser262 in both the cortex (genotype x exercise: F = 0.028, р = 0.870) and hippocampus (genotype x exercise: F = 3.967, р = 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, р = 0.031; exercise: F = 19.320, р = 0.001) and hippocampus (genotype: F = 46.984, р = 0.001; exercise: F = 15.278, р = 0.002), but there was no significant interaction between genotype and exercise on the levels of p-tau ser396 in both the cortex (genotype x exercise: F = 0.981, р = 0.335) and hippocampus (genotype x 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).

3.4 GSK 3 Kinase Activity in the Cortex and Hippocampus

Western blotting revealed three different protein bands with different molecular weights, representing total GSK3a, p-GSK3a ser21/p-GSK3a 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-GSK3a ser21 and p-GSK3a tyr279 levels in both tissues between different groups are shown in the histogram (Fig. 4A-D).

Two-way ANOVA revealed that genotype and exercise had significant effects on the levels of p-GSK3a ser21 (genotype: F = 7.654, p = 0.017; exercise: F = 12.397, ? = 0.004) and p-GSK3a tyr279 (genotype: F = 17.736, p = 0.001; exercise: F = 8.980, ? = 0.011) in the cortex, but there was no significant interaction between genotype and exercise on the levels of p-GSK3a ser21 (genotype x exercise: F = 0.713, ? = 0.415) and p-GSK3a tyr279 (genotype x 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-GSK3a ser21 and higher levels of p-GSK3a tyr279 in the cortex than in respective tissues of Wt groups. Exercise groups had significantly higher levels of p-GSK3a ser21 and lower levels of p-GSK3a 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-GSK3a ser21 (genotype: F = 9.573, р = 0.009; exercise: F = 6.217, р = 0.028) and p-GSK3a tyr279 (genotype: F = 5.397, р = 0.035; exercise: = 33.209, р < 0.001) in the hippocampus, but there was no significant interaction between genotype and exercise on the levels of p-GSK3a ser21 (genotype x exercise: F = 0.684, р = 0.424) and p-GSK3a tyr279 (genotype x exercise: F = 0.156, р = 0.699) in the hippocampus. The main effect of genotype and exercise revealed that Tg groups had significantly lower levels of pGSK3a ser21 and higher levels of p-GSK3a tyr279 in the hippocampus than in respective tissues of Wt groups. Exercise groups had significantly higher levels of p-GSK3a ser21 and lower levels of p-GSK3a tyr279 in the hippocampus than in respective tissues of sedentary groups (Fig. 4C,D).

P-GSK35 ser9 and p-GSK38 tyr216 levels in both tissues are shown in Fig. 5. Western blotting revealed three different protein bands with different molecular weights, representing total GSK3p3, p-GSK38 ser9/pGSK35 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. SA-D).

Two-way ANOVA revealed that genotype had significant effects on the levels of p-GSK3 ser9 (genotype: F = 34.192, р = 0.001) and p-GSK3 В 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-GSK35 ser9 (exercise: F = 11.851, р = 0.003; genotype x exercise: F = 5.349, р = 0.034) but not on p-GSK36 tyr216 (exercise: F = 3.952, р = 0.064; genotype x exercise: F = 0.095, р = 0.762) in the cortex. The main effect of genotype revealed that Tg groups had significantly lower levels of p-GSK3 8 ser9 and higher levels of p-GSK36 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-GSK38 ser9 (р < 0.01) than Wt-Sed mice (Fig. SA).

Two-way ANOVA revealed that genotype and exercise had significant effects on the levels of p-GSK3 5 ser9 (genotype: F = 5.821, р = 0.034; exercise: F = 6.871, р = 0.024) and p-GSK3 а tyr216 (genotype: F = 39.914, р = 0.001; exercise: 7 = 8.074, р = 0.019; genotype x exercise: F = 10.467, р = 0.010) in the hippocampus, but there was no significant interaction between genotype and exercise on the levels of p-GSK3 6 ser9 (genotype x exercise: F = 0.393, р = 0.637) in the hippocampus. The main effect of genotype and exercise revealed that Tg groups had significantly lower levels of p-GSK38 ser9 in the hippocampus than in respective tissues of Wt groups. Exercise groups had significantly higher levels of p-GSK3 5 ser9 in the hippocampus than in respective tissues of (Fig. 5C). The multiple comparison test revealed that TgSed mice had significantly higher levels of p-GSK3Atyr216 (p < 0.01) than Wt-Sed mice (Fig. 5D). In contrast, Tg-Ex mice (р = 0.011) had significantly lower levels of p-GSK33 tyr216 than Tg-Sed mice, and similar levels of p-GSK36 tyr216 in Wt-Ex and Wt-Sed mice (p = 0.785) (Fig. 5D).

3.5 Akt Phosphorylation in the Cortex and Hippocampus

Western blotting revealed three different protein bands with different molecular weights, representing p-Akt serd73, 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.

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, р = 0.001; exercise: Е = 12.950, р = 0.004) and hippocampus (genotype: F = 5.484, р = 0.044; exercise: F = 5.202, р = 0.048), but there was no significant interaction between genotype and exercise on the levels of p-Akt ser473 in the cortex (genotype x exercise: F = 0.221, р = 0.648) and hippocampus (genotype x exercise: F = 1.433, р = 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. GA,B).

4. Discussion

The study revealed that moderate-stage AD APP/PS1 mice had significantly impaired memory function, along with significantly higher levels of AB plaques, total tau, p-tau ser262, and p-tau ser396 in both the cortex and hippocampus than Wt mice, whereas 12-week, moderateintensity treadmill exercise in AD mice induced significant improvements in memory function together with a significant reduction in AS plaques and all the three tau molecules in both tissues. Importantly, the moderate-stage AD APP/PS1 mice had significantly lower levels of both pGSK3a ser21 and p-GSK3 6 ser9, but higher levels of both p-GSK3a tyr279 and p-GSK36 tyr216 in both tissues than Wt sedentary mice. However, after the 12-week exercise training, the hippocampus had significantly higher levels of both p-GSK3a ser21 and p-GSK36 ser9 but lower levels of both p-GSK3a tyr279 and p-GSK3 tyr216, whereas the cortex had significantly higher levels of p-GSK3a ser21 but only lower levels of p-GSK3a tyr279, in comparison with sedentary AD mice. The results strongly suggest the effectiveness of the training in improving memory function in the AD mice, which was believe occurred most likely through differential regulation of the Akt/GSK3 signaling pathways in the cortex and hippocampus.

The double-Tg APPSwe/PS1M146V mouse strain 1s currently widely used in studying the pathogenesis of AD, and impaired memory in these mice has been reported previously [31-33]. In our study, we observed significantly impaired memory function in the moderate-stage AD APP/PS1 mice. However, the impaired memory was rescued mice after the 12-week aerobic exercise training program. This is consistent with previous studies where regular exercise led to cognitive improvement/reservation, as reflected by lower working memory errors and reference memory errors [34,35], and shorter time to complete the Maze test in other AD mouse models such as the tripleTg and TgCRNDS lines. Regular exercise also decreased completion time of the Floor Maze test in AD patients [36,37]. However, contradictory results showing no improvement in cognitive performance after regular exercise have also been observed in 10-month-old APP/PS1 mice, 9-month-old triple-Tg AD female mice, and AD patients [38-41]. The reasons for this are attributed to many different factors such as exercise type and intensity, health status, and wide range of Tg animal strains and AD stages [34,36,37,42]. The present observation of significant improvement in memory function in the moderate-stage AD stage Tg-Ex mice suggests both the efficiency of the training protocol and the increased susceptibility of the mice to the training, i.e., memory function can be improved in moderate-stage AD APP/PS1 mice.

The double-Tg APPSwe/PS1M146V mouse strain 15 characterized by initial and progressive enhancement of AB plaques [31-33]. Indeed, clusters of AS plaques were clearly observed in both the cortex and hippocampus of the 6-month-old AD mice, similar to the observations in double-Tg APPSwe/PS1M146V mice [31-33]. However, the 12-week moderate-intensity exercise training program induced a significant reduction in A plaques in both tissues, suggesting that moderate-intensity aerobic exercise is effective in regulating A 8 plaque production in moderatestage AD mice. Similar results have been observed in other AD mouse models where exercise training in Tg2576 AD mice resulted in lower АВ plaques in both the cortex and hippocampus [43], and enriched housing of TgCRND8 AD mice induced the reduction of AB plaque burden [44].

Intracellular accumulation of phosphorylated tau is another hallmark lesion in AD [45]. The present study extensively studied tau by examining total tau, p-tau ser262, and p-tau ser396 in both the cortex and hippocampus. The results revealed that while the AD mice had significantly higher levels of total of tau, p-tau ser262, and p-tau ser396 in both the cortex and hippocampus than in that of Wt mice, moderate-intensity exercise training induced significant reductions in all three tau molecules in both tissues (see Fig. 3). The results are consistent with the observations on AB plaques in AD-exercised mice, confirming the effectiveness of the exercise in preventing or delaying deterioration of AD development in moderate-stage AD mice. Interestingly, the study revealed that the exercise also induced a significant reduction in p-tau ser262 and ptau ser396 in both the cortex and hippocampus of Wt mice compared with Wt sedentary mice, indicating the significance of physical exercise in preventing the occurrence of AD. Similar to the present observations, exercise-induced reduction in A 5 plaques and tau phosphorylation have been observed previously [13,16], along with improvement in cognitive function and behaviors [16,17,46]. Considering the observations in the improvement in memory function, and the reductions in A6 plaques and tau molecules in the AD-exercised mice, we assume that the reductions are closely associated with the improvement in memory function. Nonetheless, it is worth noting that a previous study on another AD mouse model revealed that exercise had no significant effect on 12-month TgCRNDS8 mice in abnormal APP metabolism, tau pathology, or angiogenesis [12].

To explore the molecular mechanisms underlying the improvement in memory function and the significant reduction in A8 plaques in both tissues of AD mice following a 12-week exercise training program, the study further examined Akt/GSK3 signaling pathways. GSK3 deregulation has been proven to account for memory impairment, increased A4 production, hyper-phosphorylation of tau, and inflammatory responses in AD [47]. Indeed, our study revealed that the AD sedentary mice had significantly lower levels of both p-GSK3a ser21 and p-GSK38 ser9, but higher levels of both p-GSK3a tyr279 and p-GSK36 tyr216 (see Figs. 4,5) in both tissues, along with significantly lower memory function than Wt sedentary mice. As p-GSK3a tyr279 and p-GSK3 3 tyr216 function to activate GSK3, whereas p-GSK3a ser21 and p-GSK3/ ser9 inhibit GSK3 [48], the reduced levels of p-GSK3a ser21 and p-GSK36 ser9 together with the enhanced levels of pGSK3a tyr279 and p-GSK3/ tyr216 in the AD sedentary mice are believed to work coordinately to activate GSK3a and GSK36, leading to AS plaque formation and resulting in enhanced А В plaque levels (see Fig. 3). These findings suggest that GSK3a and GSK3 5 phosphorylation were similarin both tissues, although A 8 pathology occurred earlier in the cortex than the hippocampus.

After the 12-week exercise training program in the AD mice, the hippocampus had significantly higher levels of both p-GSK3a ser21 and p-GSK3A ser9 but lower levels of both p-GSK3a tyr279 and p-GSK3A tyr216, whereas the cortex had significantly higher levels of p-GSK3a ser21 but lower levels of p-GSK3a tyr279 in comparison to respective tissues of AD sedentary mice (see Figs. 4,5). The activity of GSK3a/ß is believed to be maintained through phosphorylation at both tyr279/216 sites (activation) and ser21/9 sites (inhibition) [49], though the activity of GSK3 has also been suggested to be regulated through phosphorylation at Ser9/21, rather than Tyr 216/279 [50]. Thus, in both tissues of the exercised-AD mice, the enhanced levels of both p-GSK3a ser21 and p-GSK36 ser9 together with the reduced levels of both p-GSK3a tyr279 and p-GSK3 5 tyr216 are believed to work coordinately to result in the reduction in GSK3 activity, which subsequently led to the decrease in AA plaques and tau phosphorylation. However, the exercise training had significant impact on both GSK36 and GSK3a in the hippocampus, but not on GSK3 in the cortex of the AD mice, indicating that the aerobic exercise is sufficient to suppress the GSK3 signal in the hippocampus, but not the cortex of moderate-stage AD mice.

To further explore the molecular signaling pathways of GSK3 activation, we examined the activity of the GSK3 upstream regulator Akt. The results revealed that both tissues of AD sedentary mice had significantly lower p-Akt serd73 levels than respective tissues of Wt sedentary mice (see Fig. 6). However, after the 12-week exercise training program, both tissues of the AD mice showed significantly higher levels of p-Akt ser473 than respective tissues of AD sedentary mice. As GSK3 could be inactivated by Akt ser473 phosphorylation [51,52], the increased p-Akt ser473 levels indicated enhanced inhibition of GSK3 activity in both tissues of AD exercised-mice.

Considering all the results, we propose a scenario of molecular reactions in the two regions of the brain of the APP/PS1 mice in response to the exercise training as shown in Fig. 7: the exercise induced enhancement in p-Akt ser473 in both the cortex and hippocampus, which subsequently led to enhancement in inhibition of both GSK3a and GSK3A by increasing both p-GSK3a ser21 and pGSK3 6 ser9 but decreasing both p-GSK3a tyr279 and pGSK36 tyr216 in the hippocampus, as well as by increasing p-GSK3a ser21 but decreasing p-GSK3a tyr279 in the cortex. The inhibition of GSK3a and GSK3 activity in the cortex and hippocampus resulted in the reduction 1n both ptau ser396 and p-tau ser262, as well as in AB plaques in both regions, and eventually improved memory function or delayed memory deterioration in the AD-exercised mice.

5. Conclusions

In conclusion, moderate-intensity aerobic training regulates GSK3a/A activity in the cortex and hippocampus of 9-month-old APP/PS1 mice. The effects of exercise are more prominent in the hippocampus, but similar in the cortex. As such, it is never too late to initiate an exercise program to delay the progression of AD, which serves as a promising preventive intervention.

6. Limitations

The present study used APP/PS1 Tg mice, Which overexpress a chimeric mouse/human APP (Mo/HuAPP695swe) and a mutant human presenilin 1 (PS1-dE9), both of which exist in the central nervous system and are associated with early-onset familial AD. Because this mouse model displays very similar biochemical aspects to that of human AD, particularly regarding amyloid pathology, it is widely used in studies of AD. However, it has inherent limitations with respect to the human AD condition, as it represents only a small percentage of all AD patients. In most AD patients, the disease is sporadic with a multifactorial etiology, making it challenging to generalize findings obtained from this model to all AD patients. Sporadic AD, the most common form, has not yet been faithfully modeled. Nevertheless, the APP/PS1 model is a powerful tool in AD research.

Abbreviations

AD, Alzheimer's disease; AB, B-amyloid; APP, 6-amyloid precursor protein, GSK3, glycogen synthase kinase-3; NF Ts, neurofibrillary tangles.

Availability of Data and Materials

The datasets generated for this study are available on request to the corresponding authors.

Author Contributions

PH and BG have contributed equally to this work. LZ and JY designed the research study. PH, BG and LM performed the research. PH, BG and LM analyzed the data. PH and BG wrote the manuscript. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.

Ethics Approval and Consent to Participate

The study was approved by the local ethics committee of Beijing Sport University (2015015) and carried out according to the requirements of The Guiding Principles for Care and Use of Animals.

Acknowledgment

The authors would like to gratefully thank the ethics committee of the Beijing Sport University and Key Laboratory of Physical Fitness and Exercise of the Beijing Sport University for supporting in this study.

Funding

This study was supported by National Natural Science Foundation of China (31571229, 31271278) to LZ.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Material

Supplementary material associated with this article can be found, in the online version, at https://doi.org/10. 31083/).jin2307136.

Sidebar

Submitted: 2 February 2024 Revised: 1 April 2024 Accepted: 7 April 2024 Published: 23 July 2024

References

References

[1] De Strooper B, Karran E. The Cellular Phase of Alzheimer's Disease. Cell. 2016; 164: 603-615.

[2] Güell-Bosch J, Lope-Piedrafita $, Esquerda-Canals С, Montoliu-Gaya L, Villegas S. Progression of Alzheimer's disease and effect of scFv-h3D6 immunotherapy in the 3xTg-AD mouse model: An in vivo longitudinal study using Magnetic Resonance Imaging and Spectroscopy. NMR in Biomedicine. 2020; 33: e4263.

[3] Radde В, Bolmont T, Kaeser SA, Coomaraswamy J, Lindau D, Stoltze L, et al. Abeta42-driven cerebral amyloidosis in trans- genic mice reveals early and robust pathology. EMBO Reports. 2006; 7: 940-946.

Benilova I, Karran Е, De Strooper В. The toxic АВ oligomer and Alzheimer's disease: an emperor in need of clothes. Nature Neuroscience. 2012; 15: 349-357.

De-Paula VJ, Radanovic M, Diniz BS, Forlenza OV. Alzheimer's disease. Sub-cellular Biochemistry. 2012; 65: 329-352.

Cohen P, Goedert M. GSK3 inhibitors: development and therapeutic potential. Nature Reviews. Drug Discovery. 2004; 3: 479-487.

Reitz C, Brayne C, Mayeux R. Epidemiology of Alzheimer disease. Nature Reviews. Neurology. 2011; 7: 137-152.

Norton S, Matthews FE, Barnes DE, Yaffe K, Brayne C. Potential for primary prevention of Alzheimer's disease: an analysis of population-based data. The Lancet. Neurology. 2014; 13: 788- 794.

Adlard PA, Perreau VM, Pop V, Cotman CW. Voluntary exercise decreases amyloid load in a transgenic model of Alzheimer's disease. The Journal of Neuroscience: the Official Journal of the Society for Neuroscience. 2005; 25: 4217-4221.

Cho JY, Um HS, Kang EB, Cho IH, Kim CH, Cho JS, et al. The combination of exercise training and alpha-lipoic acid treatment has therapeutic effects on the pathogenic phenotypes of Alzheimer's disease in NSE/APPsw-transgenic mice. International Journal of Molecular Medicine. 2010; 25: 337-346.

Scarmeas N, Luchsinger JA, Schupf N, Brickman AM, Cosentino S, Tang MX, et al. Physical activity, diet, and risk of Alzheimer disease. JAMA. 2009; 302: 627-637.

Meng Q, Lin MS, Tzeng IS. Relationship Between Exercise and Alzheimer's Disease: A Narrative Literature Review. Frontiers in Neuroscience. 2020; 14: 131.

Ohia-Nwoko O, Montazari $, Lau YS, Eriksen JL. Long-term treadmill exercise attenuates tau pathology in P301S tau transgenic mice. Molecular Neurodegeneration. 2014; 9: 54.

Bo H, Kang W, Jiang N, Wang X, Zhang Y, Ji LL. Exerciseinduced neuroprotection of hippocampus in APP/PS1 transgenic mice via upregulation of mitochondrial 8-oxoguanine DNA glycosylase. Oxidative Medicine and Cellular Longevity. 2014; 2014: 834502.

Zhao G, Liu HL, Zhang H, Tong XJ. Treadmill exercise enhances synaptic plasticity, but does not alter 8-amyloid deposition in hippocampi of aged APP/PS1 transgenic mice. Neuroscience. 2015; 298: 357-366.

Kang EB, Cho JY. Effect of treadmill exercise on PI3K/AKT/mTOR, autophagy, and Tau hyperphosphorylation in the cerebral cortex of NSE/htau23 transgenic mice. Journal of Exercise Nutrition & Biochemistry. 2015; 19: 199-209.

Herring A, Münster Y, Metzdorf J, Bolczek В, Krüssel $, Krieter D, ef al. Late running is not too late against Alzheimer's pathology. Neurobiology of Disease. 2016; 94: 44-54.

Liu HL, Zhao G, Zhang H, Shi LD. Long-term treadmill exercise inhibits the progression of Alzheimer's disease-like neuropathology in the hippocampus of APP/PS1 transgenic mice. Behavioural Brain Research. 2013; 256: 261-272.

Isla AG, Vazquez-Cuevas FG, Peña-Ortega Е. Exercise Prevents Amyloid-6-Induced Hippocampal Network Disruption by Inhibiting GSK36 Activation. Journal of Alzheimer's Disease: JAD. 2016; 52: 333-343.

Hu YS, Long N, Pigino G, Brady ST, Lazarov O. Molecular mechanisms of environmental enrichment: impairments in Akt/GSK3B, neurotrophin-3 and CREB signaling. PLoS ONE. 2013; 8: 64460.

Parachikova A, Nichol KE, Cotman CW. Short-term exercise in aged Tg2576 mice alters neuroinflammation and improves cognition. Neurobiology of Disease. 2008; 30: 121-129.

McGurran H, Glenn JM, Madero EN, Bott NT. Prevention and Treatment of Alzheimer's Disease: Biological Mechanisms of Exercise. Journal of Alzheimer's Disease. 2019; 69: 311-338.

Mu L, Са: J, Си В, Yu L, Li C, Liu QS, et al. Treadmill Exercise Prevents Decline in Spatial Learning and Memory in 3xTg-AD Mice through Enhancement of Structural Synaptic Plasticity of the Hippocampus and Prefrontal Cortex. Cells. 2022; 11: 244.

Xu L, Li M, Wei A, Yang M, Li С, Liu R, et al. Treadmill exercise promotes E3 ubiquitin ligase to remove amyloid 3 and Ptau and improve cognitive ability in APP/PS1 transgenic mice. Journal of Neuroinflammation. 2022; 19: 243.

Hoydal MA, Wisloff U, Kemi OJ, Ellingsen O. Running speed and maximal oxygen uptake in rats and mice: practical implications for exercise training. European Journal of Cardiovascular Prevention and Rehabilitation: Official Journal of the European Society of Cardiology, Working Groups on Epidemiology & Prevention and Cardiac Rehabilitation and Exercise Physiology. 2007; 14: 753-760.

Huang FL, Huang KP, Wu J, Boucheron C. Environmental enrichment enhances neurogranin expression and hippocampal learning and memory but fails to rescue the impairments of neurogranin null mutant mice. The Journal of Neuroscience: the Official Journal of the Society for Neuroscience. 2006; 26: 6230- 6237.

Ren WJ, Liu Y, Zhou LJ, Li W, Zhong Y, Pang RP, et al. Peripheral nerve injury leads to working memory deficits and dysfunction of the hippocampus by upregulation of TNF-a in rodents. Neuropsychopharmacology: Official Publication of the American College of Neuropsychopharmacology. 2011; 36: 979-992.

Xu H, Baracskay P, O'Neill J, Csicsvari J. Assembly Responses of Hippocampal CAI Place Cells Predict Learned Behavior in Goal-Directed Spatial Tasks on the Radial Eight-Arm Maze. Neuron. 2019; 101: 119-132.e4.

Li C, Li Y, Zhao Z, Lv Y, Gu В, Zhao L. Aerobic exercise regulates synaptic transmission and reactive oxygen species production in the paraventricular nucleus of spontaneously hypertensive rats. Brain Research. 2019; 1712: 82-92.

Qiu D, Zhou $, Donnelly J, Xia D, Zhao L. Aerobic exercise attenuates abnormal myelination and oligodendrocyte differentiation in 3xTg-AD mice. Experimental Gerontology. 2023; 182: 112293.

Borchelt DR, Thinakaran G, Eckman CB, Lee MK, Davenport F, Ratovitsky T, ef al. Familial Alzheimer's disease-linked presenilin 1 variants elevate Abetal-42/1-40 ratio in vitro and in vivo. Neuron. 1996; 17: 1005-1013.

Puoliväli J, Wang J, Heikkinen T, Heikkilá M, Tapiola T, van Groen T, et al. Hippocampal A beta 42 levels correlate with spatial memory deficit in APP and PS1 double transgenic mice. Neurobiology of Disease. 2002; 9: 339-347.

Pedrós I, Petrov D, Allgaier M, Sureda Е, Barroso E, BeasZarate C, et al. Early alterations in energy metabolism in the hippocampus of APPswe/PS1dE9 mouse model of Alzheimer's disease. Biochimica et Biophysica Acta. 2014; 1842: 1556-1566.

Cass SP. Alzheimer's Disease and Exercise: A Literature Review. Current Sports Medicine Reports. 2017; 16: 19-22.

Silva MVF, Loures CDMG, Alves LCV, de Souza LC, Borges KBG, Carvalho MDG. Alzheimer's disease: risk factors and potentially protective measures. Journal of Biomedical Science. 2019; 26: 33.

Baker LD, Frank LL, Foster-Schubert K, Green PS, Wilkinson CW, McTiernan A, et al. Effects of aerobic exercise on mild cognitive impairment: a controlled trial. Archives of Neurology. 2010; 67: 71-79.

Stern Y. Cognitive reserve in ageing and Alzheimer's disease. The Lancet. Neurology. 2012; 11: 1006-1012.

Steen Jensen C, Portelius E, Siersma V, Hogh P, Wermuth L, Blennow К, ef al. Cerebrospinal Fluid Amyloid Beta and Tau Concentrations Are Not Modulated by 16 Weeks of Moderateto High-Intensity Physical Exercise in Patients with Alzheimer Disease. Dementia and Geriatric Cognitive Disorders. 2016; 42: 146-158.

Marlatt MW, Potter MC, Bayer TA, van Praag H, Lucassen PJ. Prolonged running, not fluoxetine treatment, increases neurogenesis, but does not alter neuropathology, in the 3xTg mouse model of Alzheimer's disease. Current Topics in Behavioral Neurosciences. 2013; 15: 313-340.

Xu ZQ, Zhang LQ, Wang О, Marshall С, Xiao N, Gao JY, et al. Aerobic exercise combined with antioxidative treatment does not counteract moderate- or mid-stage Alzheimer-like pathophysiology of APP/PS1 mice. CNS Neuroscience & Therapeutics. 2013; 19: 795-803.

Ruiz JR, Gil-Bea F, Bustamante-Ara N, Rodriguez-Romo G, Fiuza-Luces C, Serra-Rexach JA, ef al. Resistance training does not have an effect on cognition or related serum biomarkers in nonagenarians: a randomized controlled trial. International Journal of Sports Medicine. 2015; 36: 54-60.

Cui MY, Lin Y, Sheng JY, Zhang X, Cui RJ. Exercise Intervention Associated with Cognitive Improvement in Alzheimer's Disease. Neural Plasticity. 2018; 2018: 9234105.

Thomas R, Zimmerman SD, Yuede KM, Cirrito JR, Tai LM, Timson BF, ef al. Exercise Training Results in Lower Amyloid Plaque Load and Greater Cognitive Function in an Intensity Dependent Manner in the Tg2576 Mouse Model of Alzheimer's Disease. Brain Sciences. 2020; 10: 88.

Ambrée O, Leimer U, Herring A, Gortz N, Sachser N, Heneka MT, et al. Reduction of amyloid angiopathy and Abeta plaque burden after enriched housing in TgCRNDS8 mice: involvement of multiple pathways. The American Journal of Pathology. 2006; 169: 544-552.

Neddens J, Temmel M, Flunkert S, Kerschbaumer B, Hoeller С, Loeffler T, et al. Phosphorylation of different tau sites during progression of Alzheimer's disease. Acta Neuropathologica Communications. 2018; 6: 52.

Mankhong $, Kim $, Moon $, Lee KH, Jeon HE, Hwang BH, et al. Effects of Aerobic Exercise on Tau and Related Proteins in Rats with the Middle Cerebral Artery Occlusion. International Journal of Molecular Sciences. 2020; 21: 5842.

Bhat RV, Andersson U, Andersson $, Knerr L, Bauer U, Sundgren-Andersson AK. The Conundrum of GSK3 Inhibitors: Is it the Dawn of a New Beginning? Journal of Alzheimer's Disease: JAD. 2018; 64: S547-S554.

Luo J. Glycogen synthase kinase 3beta (GSK3beta) in tumorigenesis and cancer chemotherapy. Cancer Letters. 2009; 273: 194-200.

Pei JJ, Gong CX, An WL, Winblad В, Cowburn RF, GrundkeТаба! I, et al. Okadaic-acid-induced inhibition of protein phosphatase 2A produces activation of mitogen-activated protein kinases ERK1/2, MEK1/2, and p70 $6, similar to that in Alzheimer's disease. The American Journal of Pathology. 2003; 163: 845-858.

Shaw M, Cohen P, Alessi DR. Further evidence that the inhibition of glycogen synthase kinase-3beta by IGF-1 is mediated by PDK 1/PKB-induced phosphorylation of Ser-9 and not by dephosphorylation of Tyr-216. FEBS Letters. 1997; 416: 307-311.

Sakamoto К, Arnolds DEW, Ekberg I, Thorell A, Goodyear LJ. Exercise regulates Akt and glycogen synthase kinase-3 activities in human skeletal muscle. Biochemical and Biophysical Research Communications. 2004; 319: 419-425.

McGurran H, Glenn JM, Madero EN, Bott NT. Prevention and Treatment of Alzheimer's Disease: Biological Mechanisms of Exercise. Journal of Alzheimer's Disease: JAD. 2019; 69: 311- 338.