Alzheimer's disease (AD) is the most common irreversible dementia involving progressive decline in memory, thinking, language, and learning capacity. AD accounts for 50%–75% of all dementia cases and disproportionally affects the elderly (7). Pathologically, AD is characterized by the accumulation of extracellular aggregates of β-amyloid peptide (Aβ) and intracellular aggregates of hyperphosphorylated tau proteins (14).
The incidence of AD is positively associated with age, smoking, female gender, low education, cardiovascular factors, and susceptibility gene mutations (20). In addition, evidence is emerging to suggest that obesity and consumption of high-fat diets (HFD) in early life are associated with an increased risk for AD in later life (2). An HFD exacerbates the neuropathology of AD and cognitive deficits in the 3xTg-AD mice (19). Delaying the onset and progression of AD is among the most pressing challenges for medical research. Yet a 5-yr delay in the onset and progression of AD could halve the disease prevalence and would have a significant effect on disease burden (28,29). The efficacy of pharmacological treatments to date has been limited to symptom control. However, none of currently available drug therapies has been consistently demonstrated to significantly alleviate AD-like disease progression and/or cognitive declines (36).
On the other hand, both clinical and epidemiological evidence suggests that interventions for delaying onset or slowing the AD-like disease progression may be crucial to AD management as an alternative or adjunct to anticholinesterase therapies (31). Physical exercise has been well known to produce beneficial effects for prevention and/or treatment of metabolic disorders such as obesity, insulin resistance, and type 2 diabetes (T2D) (4). With respect to AD neuropathology, physical exercise also improves cognitive function by promoting brain-derived neurotrophic factor (BDNF)-dependent synaptic plasticity, neurotransmission, and neurogenesis in animal studies (35), and it reduces the risk of AD and delays the onset and attenuates the AD-like disease progression with minimal cost and only a small chance of adverse effects (11). Further, exercise training inhibits β-amyloid deposition and memory deficits in conjunction with enhanced synaptic plasticity in HFD-induced amyloid precursor protein transgenic mice (23). In a recent study, we also found that treadmill running alleviated Aβ and tau pathology and synaptic instability in conjunction with cognitive deficits in the 3xTg-AD mice (6).
However, little is known regarding molecular mechanism underlying the protective role of physical exercise against the AD-like disease progression and cognitive declines due to an HFD, especially in the 3xTg-AD mice. Thus, obtaining mechanistic insights into the effects of physical exercise on AD pathology and cognitive declines associated with HFD-induced metabolic complications would certainly contribute to the development of new and improved options to treat and/or delay AD-like disease progression and its clinical consequences.
To the best of our knowledge, this is the first study to report that treadmill running attenuates exacerbated the AD-like disease progression and cognitive declines due to an HFD by alleviating impairments in brain insulin signaling, Aβ and tau pathology, and synaptic stability/plasticity, upregulating brain BDNF levels and suppressing apoptotic neuronal cell death in the 3xTg-AD mice.
The construction of 3xTg-AD mouse harboring APP Swe, PS1M146V, and tauP301L human transgenes is well documented along with the pathologic characteristics of the AD mice (26). The 3xTg-AD mice were kept in plastic cages in groups of four to five individuals depending on the age of their littermates. The mice were kept in a room maintained on 22°C ± 2°C at a constant 12-h light–12-h dark cycle with free access to tap water and chow. All experiments and procedures were reviewed and approved by the Sungkyunkwan University School of Medicine Institutional Animal Care and Use Committee in accordance with the AAALAC International Guidelines for animal experiments (protocol no. 14-51).
The overall experimental design of the study is illustrated in Figure 1A. Because of the limited availability of animals of both sexes, only male mice were used in this study. At 4 months of age, the 3xTg-AD mice (N = 30) were assigned to a chow diet (control, n = 10), an HFD (n = 10), or an HFD plus exercise (HFD + EX, n = 10) group. The chow diet consisted of 10% fat, 70% carbohydrates, and 20% protein (kcal) (Purina Mills, Seoul, Korea). The HFD consisted of 60% fat, 20% carbohydrates, and 20% protein (kcal) (D12492; Research Diet Inc., New Brunswick, NJ).
The HFD + EX mice were made to run on a motor-driven animal treadmill with a duration of 30 min per session and a frequency of 5 d·wk−1 for 20 wk. Treadmill started with a 5-min warm-up at a speed of 5 m·min−1 followed by 20 min main exercise at a speed of 10 m·min−1 and finished with a 5-min cooldown at a speed of 5 m·min−1 in order. All animals were allowed to eat ad libitum and body weight was recorded twice a week for the duration of the study. Four weeks before the end of the 20-wk dietary with or without exercise treatments, mice underwent glucose tolerance test (GTT) and allowed a 4-wk recovery period before behavior tests.
Mice were anesthetized with Alfaxan 80 mg·kg−1 and Rompun 10 mg·kg−1 and transcardially perfused with 1× phosphate-buffered saline (PBS). For immunostaining, the whole mouse brains were excised and fixed in 4% paraformaldehyde and incubated in 30% sucrose at 4°C. The fixed brains then were cut into coronal sections at 40 μm in thickness using a microtome (CM3050S; Leica Microsystems, Nussloch, Germany) and soaked in a cold cryoprotectant solution. Serial sections containing the hippocampus and cerebral cortex were placed on glass strips. The other brains were stored at −80°C until immunoblotting and enzyme-linked immunosorbent assay (ELISA) analysis.
Morris water maze
The Morris water maze (MWM) using the method previously described (6) was used to assess spatial learning and memory. In brief, the MWM consisted of a circular pool (120 cm in diameter and 40 cm in height) made of silver metal. A translucent acrylic escape platform was placed in a fixed location away from the wall in the center of a quadrant arbitrarily labeled as the southeast quadrant. The pool was filled with water 1.5 cm above the escape platform. To prevent the use of reflected light from objects in the room as visual landmarks, all mice were tested under dim red-light illumination provided by three red bulbs in upward facing clamp lamps that were located at opposite ends of the room. The visual cues covered all four quadrants.
Although no single cue served as a beacon for the platform location, the mice had the opportunity to learn that the escape platform was located in the area with no tactile cues. A camera mounted above the pool was used to capture images of the mice swimming during behavioral testing. These images were analyzed with a computerized video tracking system (ANY-maze TM, Stoelting) using several of the available measures: (a) escape latency, i.e., time in seconds to reach the escape platform; (b) number of platform crossings; (c) total time spent in the target quadrant; (d) average swim speed (m·s−1); and (e) path lengths. All the MWM procedures began at 11:00 p.m. (±30 min). The acquisition task consisted of four trial sessions per day for three consecutive days, with each trial spaced 15 min apart. In each trial, the mouse was gently released from one random selected starting point (i.e., E, W, N, or S) and allowed to swim until escaped onto the platform (always in the southeast quadrant). Mice that failed to find the platform within 60 s were placed on it for 20 s, the same period as was allowed for the successful animals. One and a half hour after the four trial of place learning, the platform was removed from the maze, and the mice performed a probe trial test of 60 s for the 1.5-h short-term retention of memory. On the following day, the mice were tested for the cue learning of a visual platform for the 24-h long-term retention of memory. The present study was conducted in three replications with similar numbers of control, HFD, HFD + EX mice per replication.
Novel object recognition test
The novel object recognition (NOR) test was used to estimate memory capacity based on rodents' natural tendency to explore new environment. The detailed procedures are described elsewhere (36). In brief, the NOR test consisted of three sessions: habituation, familiarization, and test session. A black open field box measuring 50 cm (width) × 30 cm (height) × 50 cm (depth) was used in this test. In habituation, mice were placed to the empty NOR arena (box) and allowed to explore for 10 min·d−1 for two consecutive days. For the familiarization phase of the NOR test, two identical objects were placed near the corners on one wall in the arena. During the familiarization phase, mice were allowed to explore the two identical objects for 10 min and were returned to their home cages. During the test/recognition phase, mice were returned to the testing apparatus, presented with a third copy of the familiar object and a novel object, and were allowed to explore them for 10 min.
Each time, objects and open field arena were repeatedly cleaned with alcohol (70%, v/v) to avoid the olfactory cues. Discrimination index normalized the ratio of time spent with the novel object divided by time spent with the familiar object plus novel object.
Glucose tolerance test
After fasting overnight, mice were given a bolus intraperitoneal injection (1.5 g·kg−1 body weight) of 20% glucose (Sigma-Aldrich, St. Louis, MO). Then blood was taken from the tail vein before and 15, 30, 45, 60, and 120 min after intraperitoneal glucose injection. Blood glucose levels were measured with a One Touch II glucose meter (Lifescan; Johnson & Johnson, New Brunswick, NJ). Areas under the curve (AUC) for the GTT were calculated using a linear trapezoid method.
Serum insulin levels were measured with a commercially available ELISA (ALPCO, Salem, NH). Serum total cholesterol (TC) and triglyceride (TG) were measured with commercially available enzymatic kits (Wako Chemicals USA, Inc., Richmond, VA).
Soluble Aβ ELISA
For soluble Aβ ELISA analysis, the whole hippocampus and cerebral cortex were collected from the brains and homogenized in 50 mM Tris and/or HCl buffer pH 7.6 with 150 mM NaCl. The homogenized tissues were then centrifuged at 75,000g and 4°C for 30 min. The supernatants were collected. Soluble Aβ1–40 and Aβ1–42 levels in hippocampal and cortical tissues were measured with commercial ELISA kits (IBL, Minneapolis, MN) according to the manufacturer's instructions.
The immunohistochemistry of the mouse hippocampus and cerebral cortex was performed as described previously (34). Briefly, brain sections were treated with 0.6% H2O2 in PBS (pH 7.5) to block endogenous peroxidase. The sections were washed with PBS and blocked using 0.3% Triton X-100 and 3% normal goat serum in PBS for 1 h. The sections then were incubated with mouse antiamyloid (Millipore, Billerica, MA) at 4°C overnight and washed in PBS. Subsequently, the sections were incubated at room temperature for 1 h with biotinylated secondary goat antimouse IgG antibodies (Vector Laboratories, Burlingame, CA) and washed with PBS. In addition, the sections were incubated at room temperature for 1 h with biotinylated secondary goat antimouse IgG antibodies (Vector Laboratories) in ABC solution (Vector Laboratories) and in diaminobenzidine solution for 2–5 min. After three washes with PBS, the sections were mounted onto gelatinized glass slides and air-dried overnight. The sections were dehydrated in ethanol, cleared in xylene, and mounted with resinous mounting medium (EukittTM, Sigma-Aldrich) under the coverslip.
Immunofluorescence was performed as described previously with minor modifications (42). Brain sections were blocked with 3% normal goat serum (Gibco, Grand Island, NY) and incubated overnight with the following primary antibodies at 4°C: mouse anti-NeuN (Millipore), rabbit anticleaved caspase-3 (Cell Signaling, Beverly, MA), rabbit anti-GFAP (Millipore), and mouse anti-IBA1 (Millipore). The brain sections were then washed with PBS and incubated in the appropriate secondary antibodies for 2 h. The secondary antibodies used were antimouse IgG Alexa Fluor-488 and antirabbit IgG Alexa Fluor-594. DNA was stained with DAPI (Santa Cruz Biotechnology, Paso Robles, CA) and mounted on a glass slide.
TUNEL assay was performed as described previously with minor modifications. (21). Cells undergoing apoptosis generate free DNA ends that can be labeled in situ using terminal deoxynucleotidyltransferase by incorporating an exogenously added, labeled nucleotide into DNA strands. In situ Death Detection Kits (Roche, Mannheim, Germany) were used to detect apoptotic cells by specific staining. Briefly, brain sections were rinsed three times in PBS for 5 min and then treated with 3% H2O2 in PBS for 10 min to inactivate endogenous peroxidase. After three washes in PBS at room temperature, the sections were then permeabilized with Triton X-100 at 4°C for 2 min and flooded with deoxynucleotidyltransferase enzyme and digoxigenin–dUTP reaction buffer (TUNEL) reagent for 37°C for 1 h. The sections were washed with PBS and visualized using Converter-POD with 0.03% diaminobenzidine. Methyl green was used as a counterstain, and the samples were cover slipped.
Quantification of histology
Quantitative immunohistochemistry and immunofluorescence for the hippocampus and cerebral cortex were performed on fields located in sections of 2.5 to 2.7 mm posterior to the bregma. Images were captured using a Nikon Eclipse TE 2000-U microscope (Nikon, Tokyo, Japan) and an FV10i FLUOVIEW confocal microscope (Olympus, Tokyo, Japan) for immunohistochemistry and immunofluorescence, respectively. For cell count, images were photographed and quantified in three hippocampal and cortical fields (160 × 160 μm) randomly chosen from each brain section. A total of three fields were manually analyzed per mouse by one blinded experimenter. An average cell count per field was obtained and reported for each mouse.
Western blotting was performed as described previously (33). Ten to fifteen micrograms of total proteins was loaded on 7.5%–15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Membranes were blocked in 5% nonfat dry milk/0.05% Tris-buffered saline with Tween 20 (TBST) for 1 h and incubated overnight at 4°C with the appropriate primary antibodies. The primary antibodies used were rabbit anti-p-Tau S404 (ThermoScientific, Rockford, IL), rabbit anti-pAKT S473, rabbit anti-AKT, rabbit anti-p-GSK3β S9, rabbit anti-GSK3β, rabbit anti-PSD95, rabbit anti-BACE1 (Cell Signaling), rabbit anti-ADAM10, mouse anti-Bcl-2, mouse anti-Bax (Santa Cruz Biotechnology), rabbit anti-IDE (Abcam, Cambridge, UK), rabbit anti-BDNF (Alomone Labs, Jerusalem, Israel), mouse anti-synaptophysin (Millipore), and rabbit anti-β-actin (Bethyl Laboratories, Montgomery, TX). The membrane was subsequently incubated with horseradish peroxidase conjugated secondary antibodies. Blots were developed with a chemiluminescent horseradish peroxidase substrate kit (Millipore). Band density was determined by ImageJ (National Institutes of Health, Bethesda, MD).
Data are presented as means ± SD. Except for the MWM outcomes, a one-way ANOVA followed by the Tukey post hoc multiple comparison tests, if necessary, were used to compare any significant differences in the outcome variables among the groups. Two-way ANOVA analyses were used to test the main effects of time (i.e., D1, D2, D3, and D4) and group (i.e., control, HFD, and HFD + EX) and time–group interaction on the escape latency to the hidden platform in the MWM test. Statistical significances were tested at P = 0.05 by using the SPSS-PC software (version 20.0).
Treadmill running alleviates an HFD-induced metabolic complications and brain insulin signaling defects in the 3xTg-AD mice
With respect to metabolic parameters, there were significant group differences in final weight (F = 18.904, P < 0.001), TC (F = 470.823 P < 0.001), TG (F = 4.870, P = 0.028), glucose (F = 3.539, P = 0.045), insulin (F = 4.573, P = 0.043), homeostasis model assessment of insulin resistance (HOMA-IR) (F = 15.444, P < 0.001), and AUC of GTT (F = 3.539, P = 0.045) among the three groups of AD mice. Tukey post hoc tests showed that the HFD mice had significantly higher mean values in final weight (P < 0.001), TC (P < 0.001), TG (P = 0.023), insulin (P = 0.036), HOMA-IR (P < 0.001), and AUC of GTT (P < 0.001) compared with the control mice. These findings suggest that chronic exposure to an HFD resulted in obesity and metabolic complications in the 3xTg-AD mice (Table 1).
On the other hand, the HFD + EX mice had significantly lower TC (P < 0.001), HOMA-IR (P = 0.001), and AUC of GTT (P = 0.002) compared with the HFD mice. In addition, the HFD + EX mice had similar concentrations in TC (P = 0.275), glucose (P = 0.998), and HOMA-IR (P = 0.685) but significantly higher final weight (P < 0.001) and AUC of GTT (P = 0.004) compared with the control mice. Together, the current findings suggested that treadmill running significantly attenuated T2D-like metabolic complications caused by an HFD in the 3xTg-AD mice (Table 1).
In conjunction with the metabolic complications, the HFD mice had significantly lower levels of pAKT/AKT and p-GSK3β/GSK3β proteins in the hippocampus and cerebral cortex compared with the control mice (Fig. 1B–D), suggesting HFD-induced impairment of brain insulin signaling. On the other hand, the HFD + EX mice had significantly higher levels of pAKT/AKT and p-GSK3β/GSK3β proteins in the hippocampus and cerebral cortex compared with the HFD mice (Fig. 1B–D), suggesting a protective effect of treadmill running against HFD-induced impairment of brain insulin signaling.
Treadmill running alleviates an HFD-induced exacerbation of amyloid and tau pathology in the 3xTg-AD mice
With respect to Aβ pathology, the HFD mice had significantly higher BACE1 and lower IDE proteins in the hippocampus and cerebral cortex compared with the control mice (Fig. 2A and B). Furthermore, the HFD mice had significantly higher Aβ plaque and Aβ1–40/42 proteins in the hippocampus and cerebral cortex (Fig. 2C–E), collectively suggesting HFD-induced exacerbation of Aβ plaque pathology via the upregulation of the exacerbated amyloidogenic pathway.
On the other hand, the HFD + EX mice had significantly higher IDE proteins and lower BACE1 in the hippocampus and cerebral cortex compared with the HFD mice (Fig. 2A and B). In addition, the HFD + EX mice had significantly lower Aβ plaque and Aβ1–40/42 proteins in the hippocampus and cerebral cortex compared with the HFD mice (Fig. 2C–E), collectively suggesting a protective effect of treadmill running against HFD-induced exacerbation of amyloid pathology (Fig. 2).
In addition, the HFD mice had significantly higher p-tau/tau proteins in the hippocampus and cerebral cortex compared with the control mice, suggesting HFD-induced exacerbation of tau neuropathology. On the other hand, the HFD + EX mice had significantly lower p-tau/tau proteins in the hippocampus and cerebral cortex compared with the HFD mice, suggesting a protective effect of treadmill running against HFD-induced exacerbation of tau pathology (Fig. 2A and B).
Treadmill running alleviates an HFD-induced deterioration of synaptic stability/plasticity and downregulation of BDNF in the 3xTg-AD mice
With respect to molecular markers of synaptic function, the HFD mice had significantly lower hippocampal PSD95 and BDNF proteins and cortical PSD95, synaptophysin, and BDNF proteins compared with the control mice (Fig. 2F and G), suggesting an HFD-induced deterioration of synaptic plasticity/stability and downregulation of BDNF in the hippocampus and cerebral cortex.
On the other hand, the HFD + EX mice had significantly higher hippocampal PSD95 and BDNF proteins and significantly higher cortical PSD95, synaptophysin, and BDNF proteins compared with the HFD mice (Fig. 2F and G). In addition, the HFD + EX mice had significantly higher hippocampal PSD95 and BDNF proteins and significantly higher cortical synaptophysin and BDNF proteins compared with the control mice (Fig. 2F and G). Taken together, the findings of this study suggest that treadmill running reverses the HFD-induced deterioration of synaptic stability/plasticity and the downregulation of BDNF in the hippocampus and cerebral cortex of the 3xTg-AD mice.
Treadmill running alleviates an HFD-induced exacerbation of apoptotic neuronal cell death in the 3xTg-AD mice
The HFD mice had significantly higher numbers of microglia and astrocyte in the hippocampus compared with the control mice, with similar but nonsignificant differences in the number of microglia and astrocyte in the cerebral cortex between the control and the HFD mice (Fig. 3). In addition, the HFD mice had significantly lower Bcl-2 proteins and NeuN-positive cells and higher Bax and cleaved caspase-3 proteins in conjunction with significantly higher levels of hippocampal and cortical DNA fragmentation compared with the control mice, collectively suggesting HFD-induced exacerbation of apoptotic neuronal cell death (Fig. 4).
On the other hand, the HFD + EX mice had a significantly lower number of microglia in the hippocampus and only a trend toward the decrease in the number of astrocyte in the hippocampus compared with the HFD mice, with similar but nonsignificant differences in the number of microglia and astrocyte in the cerebral cortex between the HFD and the HFD + EX mice (Fig. 3). In addition, the HFD + EX mice had a significantly higher hippocampal Bcl-2 proteins and NeuN-positive cells and significantly lower hippocampal Bax and cleaved caspase-3 proteins in conjunction with significantly lower DNA fragmentation in the hippocampus and cerebral cortex compared with the HFD mice (Fig. 4). Together, the current findings of the study suggest that treadmill running alleviates HFD-induced exacerbation of apoptotic neuronal cell death in the 3xTg-AD mice.
Treadmill running alleviates an HFD-induced exacerbation of cognitive deficits in the 3xTg-AD mice
With respect to the MWM test, a two-way ANOVA analysis showed significant time (F = 41.506 and P < 0.001), group (F = 30.194 and P < 0.001), and time–group interaction (F = 2.190 and P = 0.044) in the escape latency to the hidden platform. Tukey post hoc analyses showed that the HFD mice had a longer escape latency (P = 0.001) at the 2-d time point over the 4-d trial course as compared with the control mice, with no such significant difference in the escape latency between the control and the HFD + EX mice or between the HFD and the HFD + EX mice. In addition, the HFD mice had a longer escape latency at the 3-d (P < 0.001 and P < 0.001, respectively) and 4-d (P < 0.001 and P < 0.001, respectively) time points as compared with either the control mice or the HFD + EX mice. Overall, the findings of the study suggested that the 3xTg-AD mice had a significantly reduced MWM performance due to an HFD, which was substantially improved by treadmill running (Fig. 5A).
With respect to memory retention, the AD mice had a comparable 4-h short-term memory formation in the probe test (i.e., time spent in target quadrant, F = 0.166, P = 0.848, and the number of platform crossings, F = 0.855, P = 0.437), independent of the dietary treatments. However, the control mice and the HFD + EX mice had significantly greater 24-h long-term memory formation compared with the HFD mice, with no statistically significant difference in the 24-h long-term memory between the former groups. In addition, the control and the HFD + EX mice had a longer time spent in target quadrant (F = 6.757, P = 0.004) compared with the HFD mice, with no significant difference between the control and the HFD + EX mice (Fig. 5B and C). The control mice had a greater the number of platform crossings (F = 5.325, P = 0.012) compared with the HFD mice, with no significant difference between the control and the HFD + EX mice (Fig. 5B and C).
In the NOR test, AD mice spent a similar amount of time exploring a familiar object in the training phase, independent of the dietary treatments. In the test phase, performed 2 h after habituation, however, only the HFD + EX mice spent significantly more time spent exploring a new object, with no group differences in discrimination index (Fig. 5D–G). Overall, the current findings of the study suggest that exacerbated cognitive deficits caused by an HFD in the 3xTg-AD mice were recovered after treadmill running.
The health benefits of exercise training on the progression of metabolic diseases have been well established (22). Interestingly enough, metabolic diseases and AD share common abnormalities, including impaired glucose tolerance, insulin resistance, inflammation, and amyloidogenesis (8). Therefore, we hypothesized that an intervention addressing the risk factors for metabolic disorders would be also effective in treating AD pathology and AD-like cognitive deficits. To the best of our knowledge, this is the first study to report that treadmill running attenuates AD-like disease progression and cognitive deficits caused by an HFD in the 3xTg-AD mice.
T2D-like metabolic complications, brain insulin resistance, and AD neuropathology
In this study, we found that chronic exposure to an HFD resulted in T2D-like metabolic complications and impaired brain insulin signaling in conjunction with exacerbated Aβ and tau pathology in the 3xTg-AD mice, implying insulin resistance as a common factor linking T2D and AD pathogenesis. In support of the current findings, brain insulin resistance, which is characterized by decreased brain levels of insulin and insulin receptor (IR) and impaired brain insulin signaling via stimulation of IR serine kinase-dependent pathway, was reported in both postmortem analysis and in animal models of AD (3,9). On the other hand, we found that treadmill running attenuated HFD-induced exacerbations of T2D-like metabolic complications, brain insulin resistance, and Aβ and tau pathology in the 3xTg-AD mice. Together, the findings of the current study suggest that treadmill running-induced stimulation of brain insulin signaling, perhaps via the IR tyrosine kinase-dependent pathway, may play an important role in suppressing HFD-induced exacerbation of Aβ and tau pathology in the 3xTg-AD mice.
In support of the above interpretation of the results, Muller et al. (25) found that voluntary wheeling running increased IR contents, pTyrIR, and pAkt in alien strain (CF1) mice. Likewise, Park et al. (27) found that forced treadmill running increased IR contents, pTyrIR, and pAkt in the hypothalamus of diabetic rats. Yang et al. (40) demonstrated that intranasal insulin treatment normalized hyperphosphorylated tau, decreased AKT activation, and GSK-3β overactivation in an animal model of T2D. Vandal et al. (38) showed that a single insulin injection eliminated the deleterious effects of HFD on memory and soluble Aβ levels via decreased Aβ production and/or increased Aβ clearance in the 3xTg-AD mice. Together, we assume that treadmill running-induced stimulation of the IR tyrosine kinase-dependent pathway and subsequent improvement of brain insulin sensitivity plays an important role in attenuating HFD-induced exacerbation of Aβ production and tau phosphorylation by increasing IDE activity and decreasing GSK3 activity in the 3xTg-AD mice.
In contrast to the current findings, however, HFD-induced cognitive impairments were found in 3xTg-AD male mice (19) and other AD mice (30) accompanying no changes in β-amyloid and tau pathology, suggesting that the HFD-induced cognitive impairments are independent of changes in AD neuropathology. Therefore, caution should be taken in the interpretation of the current findings regarding responses in AD neuropathology to HFD. A further study is required to resolve the contrasting findings regarding changes in AD neuropathology to HFD and the underlying mechanism(s).
Apoptotic neuronal cell death and synaptic instability
In addition to exacerbated AD pathology, we also found that chronic exposure to an HFD increased defects in synaptic stability/plasticity, neuronal inflammation, and apoptotic neuronal cell death in the 3xTg-AD mice. The findings of our study are in line with those of previous studies reporting HFD-induced increases in neuronal inflammation and apoptotic neuronal cell death in conjunction with brain insulin resistance and exacerbated AD pathology in experimental mouse models of AD (17,39) and wild-type mice (5,18). Similarly, AD neuropathology-mediated apoptotic cell death is a characteristic of the affected brain regions of patients with AD (32).
On the other hand, we found that treadmill running alleviated HFD-induced increases in neuronal inflammation and apoptotic cell death in the affected brain regions of the 3xTg-AD mice. In support of our findings, Ghasemi et al. (12) showed that insulin treatment prevented Aβ pathology-mediated hippocampal apoptosis and oxidative stress in male SD rats. Wu et al. (39) showed that treadmill running alleviated LPS-induced disturbance of hippocampal neurogenesis in wild-type mice. In age Tg-NSE/PS2m mice, Um et al. (37) found that treadmill running suppressed Aβ-mediated neuronal cell death and increased molecular markers of hippocampal anti-inflammation, neurogenesis, and synaptic plasticity.
In addition, BDNF is important in neuronal growth and neuronal survival (1). Along with nerve growth factor and glial cell-derived neurotrophic factor, BDNF is important in neuronal growth and survival, thereby contributing to synaptic stability/plasticity as well as memory formation (1). Exercise training increases serum (13) and brain BDNF levels (1,16), thereby contributing to synaptic stability/plasticity and neuronal cell survival. In this study, we found that treadmill running alleviated HFD-induced downregulation of pre- and postsynaptic markers and brain BDNF levels in conjunction with alleviating impaired brain insulin signaling and exacerbated Aβ and tau pathology. Together, the current findings of the study suggest that treadmill running-induced upregulation of brain BDNF levels in conjunction with treadmill running-induced alleviation of brain insulin resistance and AD neuropathology plays an important role in suppressing defects in pre- and postsynaptic stability/plasticity and apoptotic neuronal cell death caused by an HFD in the 3xTg-AD mice.
In support of our findings, García-Mesa et al. (10) showed that voluntary wheel running increased antioxidant and neuroplasticity markers, including catalase, p-CREB, and BDNF, which contributed to protecting aging-related body frailty and behavior and cognitive deficits in ovariectomized 3xTg-AD mice. Zhao et al. (41) found that treadmill running enhanced hippocampal synaptic plasticity and reduced soluble Aβ levels in conjunction with increased consolidation of long-term potentiation in age APP/PS1 mice. Molteni et al. (24) showed that voluntary wheel running reversed HFD-mediated decreases of synaptic plasticity markers in female Fisher rats.
Cognitive deficits and progression of AD pathology
Finally, we found that chronic exposure to an HFD resulted in a significantly reduced long-term memory formation in the MWM test in the 3xTg-AD mice. On the other hand, treadmill running attenuated HFD-induced long-term memory deficit and resulted in a longer time spent exploring novel object in the 3xTg-AD mice. Along with those cognitive benefits, we also found that treadmill running attenuated defects in brain insulin signaling, exacerbation of Aβ and tau pathology, defects in pre- and postsynaptic stability/plasticity, and apoptotic neuronal cell death caused by an HFD in the 3xTg-AD mice. Together, we assume that treadmill running-induced alleviating effects on brain insulin resistance, AD neuropathology, and apoptotic neuronal cell death in conjunction with the upregulation of brain BDNF levels collectively contribute to the alleviation of cognitive deficits in the 3xTg-AD mice fed an HFD.
In support of our findings, HFD-induced cognitive deficits were found in the 3xTg-AD mice accompanying exacerbated Aβ and tau pathology (3) and accompanying no changes in Aβ and tau pathology (19). Similarly, HFD-induced cognitive deficits were also reported in other AD mice accompanying exacerbated β-amyloid and tau pathology (15,18) and accompanying no changes in β-amyloid and tau pathology (30). In fact, several factors including brain insulin resistance, synaptotoxicity, oxidative stress, and apoptotic neuronal cell death are blamed for increases in cognitive impairments caused by an HFD in the AD mice. Therefore, a further study is required to elucidate the exact mechanism(s) by which exercise training attenuates HFD-induced exacerbation of cognitive deficits in the AD mice.
In summary, we investigated the protective role of treadmill running against progression of AD pathology and cognitive deficits caused by an HFD in the 3xTg-AD mice. This is the first study to report that treadmill running attenuates AD-like disease progression and cognitive declines due to an HFD in the 3xTg-AD mice. The findings of this study provide experimental evidence to support exercise training as a promising strategy against AD-like disease progression and cognitive declines associated with HFD-induced metabolic complications. Our work adds to the growing body of evidence that exercise training reduces the risk of metabolic disorders in AD and attenuates cognitive declines, at least in this transgenic animal model of AD. Yet a randomized controlled trial involving AD patients is necessary to confirm the current findings and to assess the efficacy of lifestyle modification as a nonpharmacological strategy to lower the risk of metabolic disorders and cognitive declines in AD patients.
The National Research Foundation Grant funded by the Korean Government (grant no. NRF-2013S1A2A2034953) supported this work.
The authors have no conflicts of interest to disclose.
The results of the study are presented clearly, honestly, and without fabrication, falsification, or inappropriate data manipulation and do not constitute endorsement of the American College of Sports Medicine.
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Keywords:© 2017 American College of Sports Medicine
INSULIN RESISTANCE; EXERCISE TRAINING; ALZHEIMER'S DISEASE; COGNITIVE DECLINE