Low-intensity treadmill exercise and bright light upregulate brain-derived neurotrophic factor expression and intracellular signaling pathway in rat hippocampus and cerebral cortex

Abstract

Continuous exercise and bright light affect brain function, particularly in seasonal affective disorders. However, the underlying mechanism of action remains unclear. In this study, we examined whether low-intensity treadmill exercise and 4 weeks of bright light exposure change the expression of brain-derived neurotrophic factor (BDNF), phosphoinositide 3-kinase/AKT (PI3K/AKT) pathway, protein kinase C (PKC), extracellular signal-regulated kinases (ERK1/2), cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB), vascular endothelial growth factor (VEGF), and glycogen synthase kinase-3β (GSK-3β) in the brain of 5-week old Sprague–Dawley male rats by performing western blot analysis. We demonstrated that BDNF expression significantly increased with exercise and light exposure compared to a control group. Moreover, we found that the expression levels of PI3K, PKC, p-ERK1/2, p-CREB, p-AKT, and VEGF increased significantly in the hippocampus and cortex with exercise and light exposure compared to the levels from a control group. However, phosphorylated GSK-3β level was unchanged, and even slightly decreased with exercise and light exposure. These results suggest that low-intensity treadmill exercise and bright light exposure induce BDNF expression and activate its downstream kinase signaling pathway, which in turn activate CREB-mediated transcription of neurotrophic factors and may stimulate neurogenesis and improve neuronal functions.

Keywords:

• exercise
• bright light
• BDNF
• ERK
• CREB
• hippocampus
• cortex

Introduction

Exercise is known to increase the neurogenesis through induction of neurotrophic factors in the hippocampus, which is associated with improved cognitive function (Neeper et al. 1995; van Praag et al. 1999; Trejo et al. 2001). Bright light can relieve the symptoms of seasonal mood disorders through neurobiological changes (Kasper et al. 1990; Partonen & Lonnqvist 2000; Lau et al. 2011). It is well accepted that physical activity and phototherapy can regulate hippocampal neurogenesis and neuronal cell function. We previously showed that low-intensity treadmill exercise and/or bright light promote neurogenesis and brain-derived neurotrophic factor (BDNF) expression in an animal model (Kwon et al. 2013). However, their molecular mechanisms of action remain largely unknown.

BDNF is a nerve growth factor and critical for survival, growth, and differentiation of neurons and thus, is critical to maintain proper neuronal functions. BDNF is active in the hippocampus and cortex where it regulates neuronal excitability, synaptic plasticity, long-term potentiation (LTP), and neurogenesis (Acheson et al. 1995; Huang & Reichardt 2001; Ninan et al. 2010). BDNF is known to bind the tyrosine kinase receptor B (TrkB) receptor, modulating neurotransmitter receptors to activate various kinase signaling pathways of phosphoinositide 3-kinase/AKT (PI3K/AKT), extracellular signal-regulated kinases (ERK1/2), mitogen-activated protein kinase (MAPK), or calcium/calmodulin dependent protein kinase (CaMKII), and inducing phospho-CREB-mediated transcription (Mai et al. 2002; Garoflos et al. 2005; Luikart et al. 2008). Phosphorylation of GSK-3β mediated by PI3K/AKT inactivates GSK-3β which is known to inhibit cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB) transcriptional activity (Grimes & Jope 2001; Mai et al. 2002). The CREB target genes include neurotrophic factors such as BDNF and VEGF (Gass & Riva 2007; Lee et al. 2009) and many neuropeptides, which play important roles in neuronal plasticity and memory formation (Suzuki et al. 2011).

Here, we investigated the combined effect of exercise and light exposure on expression of BDNF and downstream signaling proteins to support their beneficial effect on brain functions.

Materials and methods

Animals

Male Sprague–Dawley rats weighing 160 ± 10 g (5-week old) were obtained from a commercial breeder (KOATECH, Gyeonggi, Korea). Experimental procedures were performed in accordance with the animal care guidelines of National Institutes of Health (NIH). Animals were housed under controlled temperature (22 ± 2°C) with an alternating 12-hour light/dark cycle (lights were turned on between 6 am and 6 pm), and provided food and water ad libitum. Rats were randomly divided into four experimental groups; control group (CG), exercise group (EG), light group (LG), and exercise plus light group (ELG; n = 5 per group).

Treadmill exercise regimen

Animals from exercise groups were subjected to treadmill (PARK TECH, Daegu, Korea) exercise for 30 min per day for 5 consecutive days, each week for 4 weeks. The exercise load consisted of a running speed of 2 m/min for the first 5 min, 5 m/min for the next 5 min, and 8 m/min for the last 20 min at 0° inclination. The exercise was performed in the morning (between 10 am and 12 pm).

Bright light exposure

Light therapy was administered at an intensity of 10,000 lx (Gagné et al. 2007). A lamp for artificial light of 10,000 lx illumination was obtained from a commercial company (Danbee, Gyeonggi, Korea), and placed at a distance of 80 cm from animals during the experiments. For the LG, the bright light was exposed for 30 min, which is the matched time period with the EG. ELG was treated at the same time for 30 min.

Tissue preparation

Animals were sacrificed 24 hours after 4 weeks of treatment. Rats were anesthetized with an intraperitoneal injection of 10 mg/kg of Zoletil 50 (Virbac, Carros, France). The brains were quickly removed and hippocampus and cortex were dissected and frozen. The frozen samples were transferred to sterile 1.5 mL microcentrifuge tubes containing 550 µL of lysis buffer (T-buffer; Themo Scientific) with protease inhibitors. Homogenized tissues were incubated for 10 min on ice and sonicated. Samples were centrifuged at 4°C for 30 min at 12,000 rpm and supernatants were collected. Protein concentration was determined using a bicinchoninic acid protein assay (Bio-Rad, Hercules, CA, USA), and samples were stored at –80°C.

Western blot analysis

Protein extracts were separated by SDS-polyacrylamide gel and transferred to nitrocellulose membranes. The membranes were incubated with the following primary antibodies (diluted at 1:1000); BDNF (Santa Cruz), PI3K (Santa Cruz), PKCα (Santa Cruz), PKCδ (Santa Cruz), ERK1/2 (Cell Signaling Technology), AKT (Cell Signaling Technology), p-AKT (Cell Signaling Technology), CREB (Cell Signaling Technology), p-CREB (Cell Signaling Technology), GSK3β (Cell Signaling Technology), p-GSK3β (Cell Signaling Technology), and VEGF (Santa Cruz). After washing in Tween 20 in Tris-buffered saline (0.5% TBST), the membranes were incubated with their corresponding secondary antibodies. The proteins were detected with the enhanced chemiluminescence (ECL) solution (Pierce, Rockford, IL) and images were captured by the LAS-4000 system (Fujifilm, Tokyo, Japan). The protein levels were normalized to those of α-tubulin (Sigma).

Statistical analysis

Differences between control and experimental groups were determined by using unpaired Student's t-test, and one-way repeated measures analysis of variance were used to analyze the differences among all the groups. Multiple comparisons were performed with Duncan's method. Values are expressed as mean ± standard error of the mean (SEM) and a p value <0.05 was considered as statistically significant.

Results

Exercise and light exposure increased BDNF protein levels

We examined whether BDNF expression in the hippocampus and cortex is altered by low-intensity treadmill exercise and bright light by performing western blot analysis. BDNF expression was increased in the EG, LG, and ELG compared to the CG in both hippocampus and cortex (Figure 1). Moreover, there was an additive effect on BDNF expression in the ELG in cortex (Figure 1B). The statistical significance was indicated.

Figure 1. Changes in BDNF protein expression in the hippocampus and cortex after low-intensity treadmill exercise and bright light exposure. Western blots of BDNF proteins from lysates of rat hippocampus (A) and cortex (B) and their quantification. a, p < 0.05, b, p < 0.001 vs. control group and ^#p < 0.001, vs. exercise and light group. CG, control group; EG, exercise group; LG, light group; ELG, exercise plus light group.

Exercise and light exposure increased the protein levels of PI3K, PKC, and p-ERK

Once BDNF binds to its receptor, TrkB, it activates downstream targets leading to various neuronal processes. The signaling cascade is mediated through PLC/PKC, PI3K/AKT, and ERK1/2 (p44/p42 MAPK) and regulates synaptic plasticity, proliferation, differentiation, and neuronal survival. Thus, we examined the expression of PI3K, PKCα, PKCδ (Figure 2), and phosphorylated Erk1/2 (p44/p42; Figure 3) in the hippocampus and cortex by western blot analysis. The expression of PI3K, PKCα, PKCδ, and p-ERK was significantly increased in the EG, LG, and ELG compared to the CG. Our results suggest that exercise and light exposure activate various kinase signaling molecules, downstream of BDNF.

Figure 2. Changes in PI3K, PKCα, and PKCδ protein expression in the hippocampus and cortex after low-intensity treadmill exercise and bright light exposure. Western blots of PI3K, PKCα, and PKCδ proteins from lysates of rat hippocampus (A) and cortex (B) and their quantification. a, p < 0.05, b, p < 0.01, c, p < 0.001 vs. control group, ^#p < 0.01 vs. light group, and ^##p < 0.001 vs. exercise group and light group. CG, control group; EG, exercise group; LG, light group; ELG, exercise plus light group.

Figure 3. Changes in ERK1/2 protein expression in the hippocampus and cortex after low-intensity treadmill exercise and bright light exposure. Western blots of p-ERK 1/2 proteins from lysates of rat hippocampus (A) and cortex (B) and their quantification. a, p < 0.01, b, p < 0.001 vs. control group and ^†p < 0.001 vs. light group, ^#p < 0.001 vs. exercise group and light group. CG, control group; EG, exercise group; LG, light group; ELG, exercise plus light group.

Exercise and light exposure increased the level of p-CREB

The activated protein kinases are translocated to nucleus and phosphorylate CREB to activate p-CREB-mediated transcription (Gonzalez & Montminy 1989; Karin & Hunter 1995; Mayr & Montminy 2001). CREB in the brain has well-known role in neuronal plasticity and LTP. Thus, we examined p-CREB level in the hippocampus and cortex by western blot analysis. We found that p-CREB protein levels were increased in the EG, LG, and ELG compared to the CG (Figure 4). Notably, the significance of increase was higher in the combined stimulus group (ELG) than in EG or LG with a single stimulus.

Figure 4. Changes in CREB protein expression in the hippocampus and cortex after low-intensity treadmill exercise and bright light exposure. Western blots of p (phospho)-CREB and T (total)-CREB proteins from lysates of rat hippocampus (A) and cortex (B) and their quantification. a, p < 0.01, b, p < 0.001 vs. control group and ^#p < 0.01, ^##p < 0.001 vs. exercise group and light group. CG, control group; EG, exercise group; LG, light group; ELG, exercise plus light group.

Exercise and light exposure increased p-AKT levels, but not p-GSK-3β levels

GSK-3β is a critical downstream target of the PI3K/AKT pathway and its activity is inhibited by AKT-mediated phosphorylation. GSK-3β is known to inhibit CREB binding activity. Thus, we examined whether exercise or light exposure affects the levels of p-AKT, activated AKT, by western blot analysis. We found p-AKT levels were significantly increased in the EG, LG, and ELG compared to the CG in the hippocampus (Figure 5A), and further, we observed an additive effect by exercise and light exposure in the cortex (Figure 5B). Subsequently, we examined expression of p-GSK-3, which is phosphorylated by AKT. While p-AKT level was enhanced by exercise and light exposure, p-GSK-3 level was not changed, and even slightly decreased in the hippocampus of EG, LG, and ELG and in the cortex of ELG (Figure 6), indicating that GSK-3β may be not the direct target of BDNF-PI3K/AKT-mediated signaling pathway in our regimen of exercise and light exposure.

Figure 5. Changes in AKT protein expression in the hippocampus and cortex after low-intensity treadmill exercise and bright light exposure. Western blots of p (phospho)-AKT and T (total)-AKT proteins from lysates of rat hippocampus (A) and cortex (B) and their quantification. a, p < 0.05, b, p < 0.01, c, p < 0.001 vs. control group and ^#p < 0.01 vs. exercise group and light group. CG, control group; EG, exercise group; LG, light group; ELG, exercise plus light group.

Figure 6. Changes in GSK-3β protein expression in the hippocampus and cortex after low-intensity treadmill exercise and bright light exposure. Western blots of p (phospho)-GSK-3β and T (total)-GSK-3β proteins from lysates of rat hippocampus (A) and cortex (B) and their quantification. a, p < 0.05, b, p < 0.01, c, p < 0.001 vs. control group and ^#p < 0.001 vs. exercise group and light group. CG, control group; EG, exercise group; LG, light group; ELG, exercise plus light group.

Exercise and light exposure increased VEGF protein levels

CREB activation increases expression of neurotrophic factors, such as BDNF and VEGF. Therefore, we examined whether exercise or light exposure affects VEGF level by western blot analysis. We found that VEGF levels were significantly increased in the EG, LG, and ELG compared to the CG in the hippocampus and cortex (Figure 7). We propose a model that exercise and light exposure stimulate BDNF receptors and the downstream kinase signaling pathways. The following activated CREB increases expression of various neurotrophic factors, possibly leading to neurogenesis.

Figure 7. Changes in VEGF protein expression in the hippocampus and cortex after low-intensity treadmill exercise and bright light exposure. Western blots of VEGF proteins from lysates of rat hippocampus (A) and cortex (B) and their quantification. a, p < 0.05, b, p < 0.01, c, p < 0.001 vs. control group and ^#p <0.001 vs. exercise group and light group. CG, control group; EG, exercise group; LG, light group; ELG, exercise plus light group.

Discussion

The main finding of this study was that low-intensity treadmill exercise and bright light activate signaling pathways leading to VEGF and BDNF induction, which are closely related to neurogenesis. We showed increased p-ERK1/2, p-AKT and activation of PI3K and PKC pathway, as well as increased p-CREB and VEGF in the hippocampus and cortex of rats after exercise and light exposure.

Exercise stimulates the sympathetic nervous system causing a neuronal and hormonal stress response which induces calcium influx and cAMP signaling cascades resulting in activation of BDNF transcription (Zheng et al. 2012). A recent study showed that exercise affects epigenetic regulation of BDNF, inducing promoter demethylation and activation of methyl-CpG-binding protein 2 in rat hippocampus (Gomez-Pinilla et al. 2011).

Light received by the retina stimulates hypothalamic orexinergic neurons in the hypothalamic suprachiasmatic nucleus (SCN) and in the serotonergic nucleus in the dorsal raphe (DRN; Adidharma et al. 2012). The SCN is a principal circadian clock and orexin is a neuropeptide regulating the sleep/wake cycle. Serotonin production directly correlates with bright sunlight exposure and is often decreased in people with depression. These neuronal and hormonal changes stimulate neurotransmission, inducing calcium influx and cAMP signaling cascades and resulting in activation of BDNF transcription (Zheng et al. 2012).

BDNF promotes neuronal growth, development, and survival, and is also associated with learning and memory through regulation of synaptic plasticity (Bibel & Barde 2000; Mizuno et al. 2000). A previous study has shown that learning in water maze increases BDNF mRNA expression in the rat hippocampus (Kesslak et al. 1998). In agreement with our study, exercise such as treadmill running increased neurogenesis in hippocampus and enhanced spatial learning and LTP (Neeper et al. 1995; van Praag et al. 1999; Kwon et al. 2013). A recent study has found that the improved spatial learning and memory by exercise was correlated with an increase in the number of cholinergic neurons (Ang et al. 2006). Thus, our study supports that behaviors such as physical exercise and learning protect neurons at risk in aging and neurodegenerative diseases by BDNF signaling (Binder & Scharfman 2004). Additionally, BDNF was shown to produce an antidepressant effect in animal behavioral models of depression through MAPK kinase signaling (MAPK/ERK). BDNF heterozygote mice exhibited a depressive phenotype when combined with a low-dose MEK inhibitor or stress exposure (Shirayama et al. 2002; Duman et al. 2007). Thus, environmental challenge such as light deprivation might be treated with BDNF expression or by activating its downstream signaling molecules. All of these support that BDNF induced by exercise and light exposure stimulates neuronal functions and alleviates neuropathological symptoms.

Activation of CREB is known to increase the proliferation and survival of newborn neurons, and enhance the differentiation and maturation of neurons in the adult mouse hippocampus (Bender et al. 2001; Nakagawa et al. 2002; Fujioka et al. 2004). Previous studies demonstrated that BDNF induced by exercise and antidepressant treatment activates PI3K/AKT, GSK-3β, and CREB leading to neuronal survival (Mai et al. 2002; Chen & Russo-Neustadt 2005). In this study, we exposed rats to bright light along with treadmill exercise, expecting antidepressant and further synergistic effects. We propose here that PI3K/AKT signaling mediates these effects via CREB transcriptional activation. Although GSK-3β was not directly associated with the signaling pathway in our study, other downstream targets may involve CREB activation and remain to be further studied.

VEGF has well-known roles in angiogenesis during development and in various diseases, and moreover, recent reports showed its neurotrophic and neuroprotective effects in neurons and glial cells. Thus, VEGF has therapeutic potentials for treating ischemia, traumatic injury as well as chronic neurodegenerative diseases perhaps through an increase in angiogenesis and stimulation of neuronal functions (Greenberg & Jin 2004; Rosenstein & Krum 2004; Zachary 2005). In our study, we found that VEGF was significantly increased after exercise and light exposure, supporting its promising roles in treating cognitive diseases, depression, or mood disorders through its neurotrophic and neuroprotective effects.

In summary, low-intensity treadmill exercise and bright light exposure activate BDNF and its downstream biochemical signaling leading to induction of neurotrophic factors. We also found that the combined treatment of exercise and light therapy more effectively stimulates BDNF-mediated neuronal functions.

Acknowledgments

We would like to thank Lauren Shields at the Gladstone Institutes affiliated with the University of California, San Francisco (UCSF) for editing our manuscript. This research was supported by the Basic Science Research Program through the National Research Foundation (NRF) of Korea [No. 2005-0049415].