Role of Wnt/β-catenin signaling in the protective effect of epigallocatechin-3-gallate on lead-induced impairments of spine formation in the hippocampus of rats

Abstract

The Wnt/β-catenin signaling pathway has been implicated in the development of dendritic spines, which are the structural basis for the induction of long-term potentiation. We have previously shown that exposure to Pb during development causes damage to the spines on hippocampal pyramidal neurons by decreasing the activity of the Wnt/β-catenin signaling pathway. Epigallocatechin-3-gallate (EGCG), the most abundant catechin in green tea, has been shown to recover impaired hippocampal-dependent long-term potentiation in rats exposed to Pb. We report here an investigation of whether this protective function of EGCG works by regulating the Wnt/β-catenin signaling pathway to refine the formation of spines in rats exposed to Pb during development. Sprague-Dawley rat pups were exposed to Pb from parturition to weaning and EGCG (10, 25 and 50 mg kg⁻¹) was given intraperitoneally from postnatal day 14 to postnatal day 21. We found that exposure to Pb significantly decreased the density of dendritic spines and spine head size of pyramidal neurons in the hippocampal CA1 areas; EGCG (10 and 25 mg kg⁻¹) reversed this Pb-induced spine damage. EGCG (10 and 25 mg kg⁻¹) also recovered the expression of Wnt7a and β-catenin phosphorylation after exposure to Pb. However, 50 mg kg⁻¹ of EGCG did not restore the spine morphology and the activity of the Wnt/β-catenin pathway on rats exposed to Pb. EGCG did not exert any protective effect on Pb²⁺-induced damage in cultured hippocampal neurons when Wnt7a shRNA applied. Our results show that EGCG (within a certain dose range) has a significant protective effect on spine formation and maturation through Wnt/β-catenin signaling in young rats exposed to Pb. This effect involves the up-regulation of Wnt7a expression and the attenuation of phospho-β-catenin expression. EGCG may be a potential complementary agent in the treatment of Pb poisoning.

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1. Introduction

Green tea contains polyphenols known as catechins, which have been associated with a number of benefits to health.¹ The most abundant and potent catechin in green tea is (−)-epigallocatechin-3-gallate (EGCG), which has been shown to provide protective effects in various chronic pathological conditions, including diabetes, cancer, Parkinson's disease, Alzheimer's disease, stroke and obesity.^2–4 It has been confirmed that EGCG passes easily through the blood–brain barrier and exerts protective effects on the central nervous system.^5–7 In animal models of Parkinson's disease, EGCG effectively prevents the striatal depletion of dopamine and dopaminergic neuronal loss in the substantia nigra.⁸ In vitro, it attenuates the apoptosis induced by L-dopa in rat PC12 cells.⁹ In the AβPP/PS-1 double mutant transgenic mouse model of Alzheimer's disease, EGCG reduces amyloid-induced mitochondrial dysfunction in the hippocampus, cortex and striatum.¹⁰

Lead is a well-established environmental poison and its general toxicity, particularly towards children, continues to be a major public health issue worldwide.^11–14 Exposure to Pb²⁺ during development is considered to be a high risk factor in attention-deficit hyperactivity disorder in children.^15,16 In addition, Pb²⁺ can impair the induction of hippocampal-dependent long-term potentiation (LTP), a form of synaptic plasticity.^13,17 Pb²⁺ poisoning may interfere with the release of neurotransmitters from presynaptic vesicles in addition to postsynaptic NMDAR expression and its downstream signaling.^18–20 It has been shown that the morphological alteration of spines, small protrusions in the dendrites of spiny neurons, is associated with the induction of LTP in the hippocampus.^21,22 Our previous study showed that there is a decrease in dendritic spine formation after developmental exposure to Pb²⁺ in rats;²³ this provides direct structural evidence for the Pb²⁺-induced impairment of synaptic plasticity.

In view of the neuroprotective properties of EGCG, it may be able to mitigate Pb²⁺-induced neurotoxicity. It has been shown that EGCG can reverse the decrease in hippocampal LTP induction and maintenance in rats with long-term exposure to Pb²⁺.²⁴ Although EGCG exerts a protective function against synaptic plasticity deficits, there is no morphological evidence to support this phenotype. The Wnt/β-catenin signaling pathway has been reported to participate in the regulation of synapse formation and remodeling.²⁵ For example, Wnt7a (one of the Wnt ligands) has been shown to promote dendritic spine growth and synaptic strength.²⁶ Our previous study showed that down-regulation of the Wnt/β-catenin signaling activity is involved in the synaptic damage caused by exposure to Pb²⁺ during development by a decrease in the expression of Wnt7a.²³ It remains to be shown whether the protective effect of EGCG on LTP induction in rats exposed to Pb²⁺ is through the Wnt/β-catenin signaling pathway that regulates spine formation.

In the study reported here, we attempted to investigate effects of EGCG on spine formation and maturation in the hippocampus of rats after long-term exposure to Pb²⁺. We also explored whether EGCG depends on the regulation of the Wnt/β-catenin pathway to protect the morphology of spines.

2. Materials and methods

2.1 Animals and experimental design

Sprague-Dawley rats were obtained from the Laboratory Animal Center, Anhui Medical University, China and were maintained in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council 2010). The Sprague-Dawley dams were fed with standard laboratory chow and distilled water and were kept at a carefully controlled ambient temperature (20 ± 3 °C) and relative humidity (50 ± 10%). On the day of parturition, the dams were randomly divided into five groups as follows: (1) control; (2) exposed to Pb²⁺; (3) exposed to Pb²⁺ + EGCG (10 mg kg⁻¹); (4) exposed to Pb²⁺ + EGCG (25 mg kg⁻¹); and (5) exposed to Pb²⁺ + EGCG (50 mg kg⁻¹).

The protocol for exposure to Pb²⁺ has been reported previously.²⁷ The day of birth was considered as PND1. The pups exposed to Pb²⁺ and the pups exposed to Pb²⁺ + EGCG acquired Pb via the milk of their dams, whose drinking water contained 0.05% Pb²⁺ acetate (250 ppm, 30 mL per day) from parturition to weaning (PND 21). The control dams received distilled water throughout the lactation period. The pups were exposed to Pb²⁺ only via the milk of their dams. On PND 14–21, the pups in the three Pb²⁺ + EGCG groups received a daily intraperitoneal injection of EGCG (10, 25 and 50 mg kg⁻¹, respectively). This time period is considered to be a key offstage for the development of the nervous system in rodents.²⁸ The EGCG was obtained from Leshan Yujia Tea Science and Technology Development Co. Ltd and was dissolved in physiological saline.^29,30 In the same period, the offspring of the control group and the groups exposed to Pb²⁺ were injected daily with physiological saline alone. In all five groups, equal numbers of female and male pups were used. Only one pup per litter was selected for the experiment. After the last administration of EGCG (on PND 21), the animals were given 1 day of rest and then killed under deep anesthesia with CO₂. Half of the brain of each pup was immersed in the Golgi–Cox solution for morphological staining of the neurons and the other half was used to determine the concentration of Pb²⁺ and to quantitatively analyze the proteins.

2.2 Determination of the concentration of Pb²⁺ in hippocampus tissue

The concentration of Pb²⁺ in the hippocampus tissue of the pups was determined by graphite furnace atomic absorption spectrometry.³¹ The hippocampus (<0.5 g) was mixed with nitric acid (excellent pure GR, 4 mL) and 30% hydrogen peroxide (AR, 2 mL) overnight, then digested for 30 min in a microwave nitrate pyrolysis furnace (EMR Marsxpress Certificate, VB 20). The concentration of Pb²⁺ was then determined using a PerkinElmer Analyst800 spectrometer.

2.3 Golgi–Cox staining

To analyze the changes in the morphology of the dendritic spines, rat brains were stained using the Golgi–Cox procedure.³² The brains were first stored in the dark for 2 days (37 °C) in a Golgi–Cox solution and were then sectioned at a thickness of 200 μm in the coronal plane with a vibratome (VT1000S, Leica, Germany). All the sections with hippocampus tissue were collected and stained with ammonia for 60 min, followed by Kodak Film Fix for 20 min, and then dehydrated, cleaned and mounted using a resinous medium. The pyramidal neurons in the hippocampus CA1 area were then imaged with a Nikon wide-field microscope (Eclipse 80i) using a 40× objective. The images were analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Neurons that had a pyramidal-shaped cell body and a clear secondary dendrite were chosen for analysis. The total dendritic protrusion density was counted according to a previously reported method³³ and the spine density was estimated as the number of spines on each terminal dendrite per 10 μm. The head size of the mushroom-like spines on pyramidal and granule neurons was measured by a plug-in for the ImageJ software.³⁴

2.4 Western blotting

The hippocampus proteins in the CA1 areas were dissolved in lysis buffer (with PMSF) for 1 h on ice. The protein concentration was determined using the bicinchoninic acid method. Equal amounts of protein were loaded onto an SDS-PAGE gel. The antibodies used were: rabbit anti-Wnt7a (Abcam), rabbit anti-β-catenin (Cell Signaling Technology), rabbit anti-phospho-β-catenin (Cell Signaling Technology) and rabbit anti-β-actin (Abcam). Sample bands developed on the films were quantified by the ImageJ software. All the results were normalized against β-actin.

2.5 Semi-quantitative RT-PCR

Using an RNA kit (Axygen, USA), total RNA was extracted from the hippocampus of the rats with or without exposure to Pb²⁺ and with exposure to Pb²⁺ and 10, 25 and 50 mg kg⁻¹ EGCG. The Oligo dT primer was used to complete the reverse transcription reaction into cDNA according to the manufacturer's instructions (Trans Gene, China).

The 20 μL reaction pool of RT-PCR was composed of: 2 μL of 10× buffer; 0.4 μL of Taq DNA polymerase; 1.6 μL of dNTP; 0.8 μL of forward and reverse primer; 1 μL of cDNA template; and 13.4 μL of deionized water. The primers used in this protocol were: CCAGTTCAAACCTCGCCATTAG-AAGGAATCAGCCATACAGTCGTG for Wnt7a and CCTGAAGTACCCCATTGA AC-GAGGTCTTTACGGATGTCAAC for β-actin. The reaction procedure was set as one cycle of 94 °C for 5 min, 30 cycles of 94 °C for 30 s, 57 °C for 30 s, then 72 °C for 1 min 40 s followed by the dissociation stage at 72 °C for 10 min. The Wnt7a mRNA levels were normalized to β-actin under the same conditions.

2.6 Neuronal cultures and small interfering Wnt7a RNA transfection

To determine whether the protective effect of EGCG on spine formation was via Wnt7a, the cultured hippocampal neurons exposed to Pb²⁺ were transfected with Wnt7a shRNA (Biomics Biotechnologies, Guangzhou, China) using Lipofectamine 2000 at DIV 9.²³ The neurons were then treated with EGCG (50 μM) for 48 h and fixed with 4% paraformaldehyde at DIV 12.²⁴ Neuronal images were acquired with an Olympus confocal microscope (FV1000, BX61WI). For analysis, three to five dendritic segments were examined in each neuron with Golgi–Cox staining.

2.7 Data analysis

All data are given as mean ± SEM values. The statistical significance of the differences among groups was examined using one-way ANOVA analysis followed by the Bonferroni test as post hoc analysis; p < 0.05 indicated a significant difference.

3. Results

3.1 Concentrations of Pb²⁺ in the rat hippocampus

The Pb²⁺ concentration in the hippocampus was 0.025 ± 0.002 μg g⁻¹ in the control group and 0.407 ± 0.10 μg g⁻¹ in the group exposed to Pb²⁺. The Pb²⁺ concentrations in the group exposed to Pb²⁺ were significantly higher than those in the control group (p < 0.001) (Fig. 1). The Pb²⁺ concentration in all the EGCG groups did not show a significant difference from the group exposed to Pb²⁺ (p > 0.05).

Fig. 1 Concentration of Pb in the hippocampus of rats in the control group, the group exposed to Pb²⁺ and the groups exposed to Pb²⁺ + EGCG. Data are presented as mean ± SEM values. ***p < 0.001.

3.2 Effect of EGCG on the spine density in the hippocampal CA1 areas of rats exposed to Pb²⁺

To explore the protective effect of EGCG on spine formation, the spine density in the hippocampal CA1 areas was measured. Compared with the control group, the group exposed to Pb²⁺ showed a decrease in the dendritic spine density in hippocampal CA1 pyramidal neurons (27%, p < 0.001, Fig. 2). After the administration of 10 and 25 mg kg⁻¹ EGCG, the dendritic spine density increased by about 10.68 and 21.98%, respectively, compared with the group exposed to Pb²⁺ (p < 0.01, p < 0.001). However, 50 mg kg⁻¹ EGCG did not affect the spine density.

Fig. 2 Effect of EGCG on the dendritic spine density of pyramidal neurons (CA1 areas) in the hippocampus of rats exposed to Pb²⁺. Left: dendritic branch and spines stained with the Golgi–Cox solution; scale bar 10 μm. Right: histogram showing changes in dendritic spine density (spines/10 μm). Data are presented as mean ± SEM values. **p < 0.01, ***p < 0.001.

3.3 Effect of EGCG on spine width in hippocampal CA1 areas in rats exposed to Pb²⁺

The head width of mushroom-like spines was measured to explore the effects of EGCG on spine maturation. Fig. 3 shows that the group exposed to Pb²⁺ exhibited a decrease in the head width of spines in the hippocampal CA1 areas (control, 0.59 ± 0.018 μm; Pb²⁺, 0.41 ± 0.013 μm; p < 0.001) and that EGCG (10 and 25 mg kg⁻¹) significantly reversed the alteration in the spine head width (10 mg kg⁻¹, 0.52 ± 0.019 μm; Pb²⁺ + EGCG 25 mg kg⁻¹, 0.53 ± 0.027 μm; p < 0.01; Fig. 3). However, 50 mg kg⁻¹ EGCG did not affect spine maturity after exposure to Pb²⁺.

Fig. 3 Effect of EGCG on the width of the mushroom-type spines of pyramidal neurons (CA1 areas) in the hippocampus of rats exposed to Pb²⁺. The histogram shows the change of width of the dendritic spines after long-term exposure to Pb²⁺ with or without EGCG. Data are presented as mean ± SEM values. **p < 0.01 and ***p < 0.001.

3.4 Effect of EGCG on Wnt7a expression in the hippocampal CA1 areas in rats exposed to Pb²⁺

As Wnt proteins are involved in synapse formation and synaptic strength, the expression of Wnt7a on hippocampal CA1 areas was analyzed by western blotting. Fig. 4 shows that, after exposure to Pb²⁺, the relative expression of Wnt7a decreased by about 10.89% in the CA1 areas (p < 0.001). After treatment with 10 and 25 mg kg⁻¹ EGCG, it significantly increased by about 6.36 and 7.15% (p < 0.01), whereas 50 mg kg⁻¹ EGCG did not cause any significant change.

Fig. 4 Effect of EGCG on the expression of Wnt7a in the hippocampal CA1 areas exposed to Pb²⁺. The representative immunoblot and histograms show the expression of Wnt7a in CA1 areas normalized to β-actin. Data are presented as mean ± SEM values. **p < 0.01, ***p < 0.001.

To investigate effect of EGCG on Wnt7a gene expression, total RNA was extracted from the rat hippocampus for semi-quantitative RT-PCR analysis. Fig. 5 shows that the expression of Wnt7a mRNA significantly decreased by 54.98% on long-term exposure to Pb²⁺ (p < 0.05). After treatment with 10 and 25 mg kg⁻¹ EGCG, the level of Wnt7a mRNA increased by 56.23 and 55.42% compared with the group exposed to Pb²⁺ (p < 0.05). The results showed a change in the level of transcription of Wnt7a agents after treatment with EGCG, which is consistent with the translation effect.

Fig. 5 Effect of EGCG on the relative levels of Wnt7a mRNA in the hippocampus of rats after long-term exposure to Pb²⁺. The expression of Wnt7a mRNA was evaluated by RT-PCR; quantification shows the expression of Wnt7a mRNA in the hippocampus normalized to β-actin. Data are presented as mean ± SEM values. *p < 0.05.

3.5 Effect of EGCG on the expression of Wnt pathway components in the hippocampal CA1 areas of rats exposed to Pb²⁺

β-Catenin plays a key part in the canonical Wnt pathway.³⁴ In the absence of Wnt signaling, the level of β-catenin phosphorylation and ubiquitination will be increased. To further determine the activity of the Wnt pathway, the level of phosphorylation of β-catenin in the hippocampus was examined. Exposure to Pb²⁺ significantly increased the expression of phosphorylated β-catenin by 36.50% in the CA1 areas (p < 0.01, Fig. 6). After treatment with 10 and 25 mg kg⁻¹ EGCG, the phosphorylation level of β-catenin was significantly decreased by about 22.71 and 33.32% (p < 0.05, p < 0.01, Fig. 6), but 50 mg kg⁻¹ EGCG did not have any effect.

Fig. 6 Effect of EGCG on the expression of phosphorylated β-catenin in hippocampal CA1 areas exposed to Pb²⁺. Representative immunoblot and quantification show the ratio of expression of phosphorylated β-catenin to the total β-catenin in CA1 areas. Data are presented as mean ± SEM values. *p < 0.05, **p < 0.01, ***p < 0.001.

3.6 Loss of Wnt7a expression: EGCG could not invert the impairment of spine formation after exposure to Pb²⁺

To examine the involvement of Wnt7a in the protection by EGCG, we applied Wnt7a shRNA to the cultured hippocampal neurons. Compared with the control group, exposure to 1 μM Pb²⁺ reduced the density of the spines (23.6%, p < 0.05, Fig. 7). After treatment with 50 μM EGCG, the dendritic spine density increased by about 27% compared with the group exposed to Pb²⁺ (p < 0.05). However, compared with the group treated with EGCG, the amount of spines decreased by 29.6% when Wnt7a shRNA was added (p < 0.01, Fig. 7). These results show that Wnt7a shRNA prohibited the EGCG-induced spine density from increasing. These data show that Wnt7a plays an essential role in spine formation.

Fig. 7 Effect of EGCG on spine formation in cultured hippocampal neurons with Wnt7a shRNA after exposure to Pb²⁺. The histograms show the change in dendritic spine density in hippocampus cells after stable transfection with Wnt7a shRNA and EGFP (spines/10 μm). Data are presented as mean ± SEM values. *p < 0.05, **p < 0.01.

4. Discussion

Environmental exposure to Pb has been implicated in the impairment of synaptic plasticity and in the formation of spines in the hippocampus.²³ This study showed that treatment with EGCG could inhibit the decrease in spine density and maturity in hippocampal CA1 areas after exposure to Pb²⁺ during development. The effect of EGCG occurred within a certain dose range and a dose of 25 mg kg⁻¹ had a higher potential to recover the Pb²⁺-induced morphological damage in the spines. We also found that EGCG up-regulates the activity of the Wnt/β-catenin signaling pathway to reverse the impairment in spine morphology. Once the expression of Wnt7a had been blocked by Wnt7a shRNA, EGCG did not have any protective effect on the Pb²⁺-induced damage to the hippocampal neurons.

The developing nervous system is sensitive to environmental insults.³⁵ Pb²⁺ exposure may reduce the overall cognitive function of children. It is well known that exposure to Pb²⁺ during development significantly reduces hippocampal-dependent LTP,^36,16 which is viewed as a key model for neuronal plasticity and memory. LTP induction elicits the formation of additional synapses between the activated axon terminals and dendritic spines and LTP is associated with the growth and stabilization of newly emerging dendritic spines.³⁷ Our previous study showed that developmental exposure to Pb²⁺ significantly decreases spine formation in hippocampal CA1 areas, which explains the Pb²⁺-related impairment of LTP induction. The mature spine is characterized as a “mushroom-like” shape, with thin necks and bulbous heads, which make contact with the presynaptic terminal for the transmission of signals.^38,39 Changes in the shape of the spines, such as shortening and/or widening of the neck, reduce their electrical resistance and thus increase the strength of the synapses. This study has indicated that EGCG reverses the decrease in spine density and the width of the mushroom heads of dendritic spines after exposure to Pb²⁺. It implies that EGCG could improve spine formation and maturity to refine synaptic efficacy in rats exposed to Pb²⁺. To some extent, this trend is consistent with the protective effects of EGCG on LTP induction in the Pb²⁺-treated hippocampus.

The possible mechanisms of Pb-related impairment of the central nervous system have been recognized as an imbalance of the pro-oxidant/antioxidant ratio and the disturbance of the calcium ion balance.^17,18 It has been reported that EGCG can be used in the treatment of neurodegenerative diseases, such as Parkinson's disease and Alzheimer's disease, and reduces focal ischemia/reperfusion-induced injury in the central nervous system by its antioxidative and ion-chelating properties.^8,9,40 We found here that EGCG could slightly decrease the accumulation of Pb in the developing brain of rats. This suggests that EGCG may reverse the Pb²⁺-related impairment in the hippocampus through its antioxidative activity rather than its chelating properties.²⁴ It has been shown that natural compounds with antioxidant properties have anti-inflammatory and neuroprotective effects in the prevention of seizure-induced pathology.^41,42 Among these compounds, vitamin E (as α-tocopherol) has been confirmed to reduce spine loss in patients with seizures.⁴³ This study has shown that EGCG, an antioxidant, also has the potential to improve the morphological stability of dendritic spines in hippocampal CA1 areas after exposure to Pb²⁺. It provides structural evidence for our previous physiological study, which showed that EGCG significantly reduced LTP impairment in the hippocampal CA1 area in developing rats exposed to Pb²⁺.²⁴ Interestingly, our results showed that 25 mg kg⁻¹ EGCG seems to have a better protective effect than 10 and 50 mg kg⁻¹ EGCG in improving the spine density and size stability of mature spines in damaged hippocampal CA1 areas. These results showed that EGCG exerts a protective function within a certain dose range in rats with long-term exposure to Pb²⁺, which is consistent with our previous study.²⁴ The underlying mechanism of the dose-dependent effect should be explored in future work.

It has been well-established that Wnt signaling plays an important part in the formation, growth and function of synapses⁴⁴ and that disorders in the Wnt/β-catenin pathway have been implicated in neurodegenerative and psychiatric diseases.⁴⁵ We found that exposure to Pb²⁺ during development significantly decreased the expression of Wnt7a and increased the phosphorylation levels of β-catenin, suggesting a decrease in the activity of the Wnt/β-catenin pathway. One week of administration of EGCG (10 and 25 mg kg⁻¹) recovered the alteration in the activity of the Wnt/β-catenin pathway. After the expression of Wnt7a had been blocked to decrease the activity of the pathway, EGCG was unable to reverse the impairment of spine formation in rats exposed to Pb²⁺. These results suggest that EGCG could protect the synaptic plasticity of spiny neurons in the hippocampus with involvement of the activation of the Wnt/β-catenin pathway. However, the exact mechanism underlying this effect needs to be investigated further.

This study has demonstrated that developmental exposure to Pb²⁺ resulted in a significant decrease in spine density and spine maturation in the hippocampal CA1 areas of Sprague-Dawley rat pups. We also provided evidence of the neuroprotective action of EGCG against Pb²⁺-induced morphological alteration of the spines. This study suggests that Wnt7a is an important target in spine formation through the modulating expression of key molecules in the Wnt/β-catenin pathway.

Conflict of interest

The authors declare no conflict of interest.

Acknowledgements

This work was supported by the National Key Basic Research Program of China (973 Program, no. 2012CB525003), the National Science Foundation of China (nos 21477031, 31200851, and 31401671), the Program for New Century Excellent Talents in University (NCET-12-0835), the Specialized Research Fund for the Doctoral Program of Higher Education (no. 20130111110024) and the Huangshan Young Scholar Fund of Hefei University of Technology (407-037030).

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Footnote

† These authors contributed equally to this work.

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