Effect of treadmill exercise on PI3K/AKT/mTOR, autophagy, and Tau hyperphosphorylation in the cerebral cortex of NSE/htau23 transgenic mice.

PURPOSE: Neurofibrillary tangles, one of pathological features of Alzheimer's disease, are produced by the hyperphosphorylation and aggregation of tau protein. This study aimed to investigate the effects of treadmill exercise on PI3K/AKT/mTOR signal transmission, autophagy, and cognitive ability that are involved in the hyperphosphorylation and aggregation of tau protein.
METHODS: Experimental animals (NSE/htau23 mice) were divided into non-transgenic control group (Non-Tg-Control; CON; n = 7), transgenic control group (Tg-CON; n = 7), and transgenic exercise group (Tg-Treadmill Exercise; TE; n = 7). The Tg-TE group was subjected to treadmill exercise for 12 weeks. After the treadmill exercise was completed, the cognitive ability was determined by conducting underwater maze tests. Western blot was conducted to determine the phosphorylation status of PI3K/AKT/mTOR proteins and autophagy-related proteins (Beclin-1, p62, LC3-B); hyperphosphorylation and aggregation of tau protein (Ser199/202, Ser404, Thr231, PHF-1); and phosphorylation of GSK-3β, which is involved in the phosphorylation of tau protein in the cerebral cortex of experimental animals.
RESULTS: In the Tg-TE group that was subjected to treadmill exercise for 12 weeks, abnormal mTOR phosphorylation of PI3K/AKT proteins was improved via increased phosphorylation and its activity was inhibited by increased GSK-3β phosphorylation compared with those in the Tg-CON group, which was used as the control group. In addition, the expression of Beclin-1 protein involved in autophagosome formation was increased in the Tg-TE group compared with that in the Tg-CON group, whereas that of p62 protein was reduced in the Tg-TE group compared with that in the Tg-CON group. Autophagy was activated owing to the increased expression of LC3-B that controls the completion of autophagosome formation. The hyperphosphorylation and aggregation (Ser199/202, Ser404, Thr231, PHF-1) of tau protein was found to be reduced in the Tg-TE group compared with that in the Tg-CON group. Furthermore, in the underwater maze test, the Tg-TE group showed a reduced escape time and distance compared with those of the Tg-CON group, suggesting that learning and cognitive ability were improved.
CONCLUSION: These findings suggest that aerobic exercise such as treadmill exercise might be an effective approach to ameliorate the pathological features (or neurofibrillary tangles) of Alzheimer's disease.

INTRODUCTION

Alzheimer’s disease is the most common form of dementia, exhibiting memory deterioration and cognitive impairment. This disease is characterized by the presence of amyloid plaque (AP) and neurofibrillary tangles (NFT) [1]. AP are intracellular inclusion bodies composed of amyloid β peptides generated by the degradation of amyloid precursor proteins, while NFTs are aggregates of hyperphosphorylated tau proteins [2].

Tau is a microtubule-binding protein, which functions to facilitate assembly and stabilization of microtubules. Under normal conditions, it maintains homeostasis of phosphorylation and dephosphorylation. It also regulates cytoskeletal stability, which in turn modulates the maintenance of axon morphology [3]. However, hyperphosphorylation of serine and threonine residues of tau protein decreases the binding affinity of tau to microtubules causing aggregation of microtubules, which leads to the formation of paired helical filament (PHF) [4,5]. Intracellular hyperphosphorylation of tau protein is morphologically manifested by the formation of NFT, which is known to induce apoptosis in neuronal cells. This condition is referred to as tauopathy and its clinical characteristics include deterioration of brain function and apoptosis in neuronal cells [6]. Although the exact mechanism, involved in intercellular signaling pathways, that influences the formation of NFTs are not completely clear, mTOR has been reported to be deeply involved in this process [7-9].

mTOR is a sub-signaling protein of Phosphoinositide 3-kinase (PI3K) and Protein kinase B (PKB; also known as AKT) and is expressed ubiquitously in the living body, and two multi-protein complexes named TOR Complex 1 and TOR Complex 2, which are functionally distinct in composition and temperament, form the core of the catalytic domain [10]. These mTOR protein complexes are localized in the center of complex signaling pathways that are activated by intracellular stress or growth factor signals. Moreover, they are involved in regulation of various cellular functions, such as protein synthesis, cell growth, biosynthesis of ribosome and mitochondria, cytoskeletal formation, and autophagy [11,12]. mTOR can undergo self-phosphorylation via its own serine/threonine kinase activities, and regulates the synthesis of other proteins by inhibiting the activation of 4E-BP, an inhibitor protein of mRNA translation process, and through the activation of p70-S6K phosphorylation [13]. Autophagy is a process that sequesters intra-cellular waste by-products, degenerative protein aggregates, and deteriorated organelles in double-membraned vesicle, referred to as autophagosomes, and fuses autophagosomes with lysosome, followed by the degradation by lysosomal proteases [14,15]. The by-products of degradation can be reused for generating energy required for the survival of cells or for building new organelles; thus, autophagy is called a cellular recycling factory [16].

According to recent reports, mTOR was hyperactivated in an Alzheimer’s animal model. This hyperactivity of mTOR was deeply involved in hyperphosphorylation of Aβ and tau [17-20]. In other words, it was reported that hyperactivation of mTOR reduced autophagy and directly contributed to hyperphosphorylation and aggregation of tau proteins [21,22]. Caccamo et al. (2013) reported that, treatment of Alzheimer’s transgenic mice (P301S mice) with rapamycin, an mTOR inhibitor, activated autophagy and decreased hyperphosphorylation of tau proteins. This suggests that inhibition of mTOR expression can increase autophagy and decrease hyperphosphorylation, thus, preventing aggregation of tau proteins. However, mTOR is also engaged in the synthesis of proteins related to learning and memory in the brain [23,24]; hence, if its activity is completely inhibited by treatment with rapamycin, an mTOR inhibitor, it is likely to inhibit the synthesis of proteins related to learning and memory. Thus, appropriately controlling mTOR or avoiding its over-expression is important [22].

No effective treatment of Alzheimer’s disease is yet available. Although many researchers have attempted to develop pharmacological treatments of Alzheimer’s disease, their efficacy was reported to be temporary or radical treatment was reported to be not feasible. Physical activity, or continued exercise has been gaining much attention as preventive and therapeutic approaches to Alzheimer’s disease. According to the studies that analyzed effects of exercise on degenerative neurological diseases, such as Alzheimer’s disease, exercise contributed to the improvement of cognitive functions and prevention of degenerative neurological disease. These were achieved due to positive changes in brain health, such as increased brain volume, generating neuronal cells and growth factors, and promoting cerebral vasculogenesis [25-27]. Moreover, another study reported that exercise can reduce the expressions of Aβ-42 and hyperphosphorylated tau, which are pathological markers of Alzheimer’s disease [28-30]. Many studies have shown that exercise might reduce AP and NTF, which are the pathological features of Alzheimer’s disease, but studies investigating the effect of exercise on molecular signaling pathways that might reduce the hyperphosphorylation and aggregation of tau protein, or determining whether exercise might control mTOR signaling pathway and autophagy-related proteins are lacking.

Therefore, the present study investigated the effects of long-term treadmill exercise on hyperphosphorylation of tau protein, mTOR signaling pathway, autophagy and cognitive function. For this, we used Alzheimer’s transgenic mice with overexpression of NSE/htau23 fusion gene, generated by recombining human tau23 gene under the regulation of NSE (neuron-specific enolase) promoter.

Methods

Experimental animals

In the present study, tau disease model mice that over-express fusion genes (NSE/htau23 fusion gene) because of the presence of htau23 gene under the control of NSE-gene promoter were used; they were provided by the Korea Food and Drug Administration Laboratory Animal Resources and bred for 18 months at H University Animal Laboratory. During the experimental period, the mice were provided food (Purina rat food-5057), which was produced in accordance with AIN-76A dietary adjustment for experimental animals as recommended by the American Dietetic Association, and food and water were provided ad libitum. The mice were divided into non-transgenic control group (Non-Tg-Control; CON; n = 7), transgenic control group (Tg-CON; n = 7), and transgenic exercise group (Tg-Treadmill Exercise; TE; n = 7). The study protocol was submitted to and approved by the H University Animal Experiment Ethics Committee.

Treadmill exercise

After a prior adaptive training (20 min/day, 15cm/sec, 5 days/week) by using a rodent treadmill (8 Lanes, Daemyung Scientific Co, Ltd, Korea) was completed, NSE/htau23 mice were subjected to exercise of 5 days per week for 12 weeks by modifying the exercise program suggested by Cho et al. [29] in consideration of the exercise performance ability of NSE/htau23 mice. The exercise protocol is shown in .