Involvement of GSK3 and PP2A in ginsenoside Rb1's attenuation of aluminum-induced tau hyperphosphorylation

Behavioural Brain Research 241 (2013) 228–234

Contents lists available at SciVerse ScienceDirect

Behavioural Brain Research journal homepage: www.elsevier.com/locate/bbr

Research report

Involvement of GSK3 and PP2A in ginsenoside Rb1’s attenuation of aluminum-induced tau hyperphosphorylation Hai-hua Zhao a , Jing Di a , Wen-su Liu b , Hui-li Liu c , Hong Lai a , Yong-li Lü a,∗ a b c

Department of Human Anatomy, College of Basic Medicine, China Medical University, Shenyang, China Institute of Biophysics, Chinese Academy of Sciences, Beijing, China Department of Sports Medicine, College of Basic Medicine, China Medical University, Shenyang, China

h i g h l i g h t s  Ginsenoside Rb1 improves long-term aluminum exposure induced learning and memory dysfunction in mice.  Long-term aluminum exposure induced tau hyperphosphorylation in motor cortex, sensory cortex and hippocampal formation.  Ginsenoside Rb1 relieve tau phosphorylation degree by reducing active p-GSK3 and increasing PP2A protein level.

a r t i c l e

i n f o

Article history: Received 27 April 2012 Received in revised form 20 November 2012 Accepted 24 November 2012 Available online 3 December 2012 Keywords: Aluminum Ginsenoside Rb1 Tau Glycogen synthase kinase 3 Protein phosphatase-2A

a b s t r a c t Environmental agent aluminum, a well-known neurotoxin, has been proposed to play a role in the development of Alzheimer’s disease (AD), and produced clinical and pathological features which were strikingly similar to those seen in AD brain, such as neurofibrillary tangles. Ginsenoside Rb1, highly abundant active component of ginseng, has been demonstrated to be neuroprotective against various neurotoxins. In this study we investigated the effect of Rb1 on aluminum-induced tau hyperphosphorylation in ICR mice. Mice were exposed to aluminum chloride (200 mg/kg/day) for 6 months followed by a post treatment of Rb1 (20 mg/kg/day) for another 4 months. Aluminum exposure induced the cognitive ability by Morris water maze, and upregulated the tau phosphorylation level at Ser396 accompanied by increasing p-GSK and decreasing PP2A level in motor, sensory cortex and hippocampal formation. Post treatment of Rb1 significantly improved the learning and memory and reduced the tau phosphorylation by reversing the p-GSK3 and PP2A level. Our results indicate that ginsenoside Rb1 protected mice against Al-induced toxicity. The possible mechanism may be its role in preventing tau hyperphosphorylation by regulating p-GSK3 and PP2A level, which implicate Rb1 as the potential preventive drug candidate for AD and other tau pathology-related neuronal degenerative diseases. © 2013 Published by Elsevier B.V.

1. Introduction Aluminum, the third most abundant element in the earth’s crust and constituent of cooking utensils, medicines and drinking water, can easily enter human body and cross the blood–brain barrier, and thus depositing in the brain [1,2]. The definite biological function of aluminum has never been demonstrated. However, the neurotoxic effects of Al have been repeatedly demonstrated and shown to interfere with a variety of cellular and metabolic processes in the nervous system as well as several other systems [3,4]. And the relationship between aluminum and the NFTs, which was found by Klatzo et al. [5] in rabbits and Crapper et al. [6] in

∗ Corresponding author at: Department of Human Anatomy, China Medical University, Shenyang 110001, China. Tel.: +86 24 23256666 5294; fax: +86 24 23262634. E-mail addresses: [email protected], [email protected] (Y.-l. Lü). 0166-4328/$ – see front matter © 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.bbr.2012.11.037

human AD brain for the first time, led the aluminum hypothesis in Alzheimer’s disease (AD), which proposed aluminum exposure played a role in the development of this disease [7], and produced clinical and pathological features which were strikingly similar to those seen in AD brain (neuron loss, senile plaques (SP) and neurofibrillary tangles (NFTs) in selective age-related hippocampal formation and cerebral cortex, etc.). Neurofibrillary tangles contain the abnormally hyperphosphorylated forms of tau protein, a well known microtubule-associated protein [8]. Phosphorylation of certain residues on tau, specifically those of Ser396 and Ser404, has been shown to increase the fibrillogenic nature of tau and contribute to its accumulation into paired helical filaments [9–11]. The balance between the phosphorylation and dephosphorylation of tau is regulated by many kinds of proteinase, such as glycogen synthase kinase 3 (GSK3) and protein phosphatase-2A (PP2A), which were hot-studied in recent years [12–14]. In this study, we detected the phosphorylation on Ser396 of tau to further confirm the

H.-h. Zhao et al. / Behavioural Brain Research 241 (2013) 228–234

aluminum neurotoxicity on tau and the role of GSK3 and PP2A in this process. Ginseng, a traditional anti-aging medicine, has been used as a tonic remedy in China for more than 2000 years [15]. Its dominant active components, called ginsenosides (GSS), now contain about 30 different forms. Our previous studies have found that total-ginsenosides (t-GSS) could upregulate the BDNF and its high affinity receptor TrkB level in aging rat brain [16,17]. We also found t-GSS could promote the regeneration of senescent cholinergic nerve fibers [18,19]. However the total protective functions of GSS on the central nerve system have not been clearly understood. Ginsenoside Rb1 (Rb1) is an ethanol extract from ginseng and is highly abundant in t-GSS, with an established safety record. Studies have demonstrated the Rb1 anti-neuroinflammation effect in A␤induced loss of learning and memory of rats [20]. And the results above led us to explore the exact roles of Rb1 on aluminum-induced tau hyperphosphorylation. 2. Materials and methods 2.1. Animals A total 32 female ICR mice (8-week-old) were purchased from the Experimental Animal Center, China Medical University. These mice were maintained on a standard 12-h light/dark cycle with free access to a standard diet and water available ad libitum. Animals were treated humanely and with regard to alleviation of suffering according to the care and use of medical laboratory animals (Ministry of Health PR China, 1998) and the guidelines of the laboratory animal ethical standards of China Medical University. 2.2. Animal groups At the beginning, 32 female ICR mice were divided into 2 groups: normal control group (NC) (n = 8) drinking distilled water and aluminum (Al)-exposed group (n = 24) treated with AlCl3 (200 mg/kg body weight) in drinking water. After 6 months, the Al-exposed group was randomly divided into three groups (n = 8 each) with different disposal for the next 4 months: All-time-Al-exposed group (Al-1) was continually exposed to Al; Stop Al exposure group (Al-2) started to drink distilled water as NC; Rb1-treated group(Rb1) was fed with ginsenoside Rb1 (20 mg/kg/day, purchased from Victory, WKQ0240, Sichuan, China) in drinking water without AlCl3 .

229

retrieval. Then the sections were placed in PBS containing 3% hydrogen peroxide (H2 O2 ) for 10 min. After rinsing with PBS, the sections were treated with 5% bovine serum albumin (BSA) for 10 min, and then incubated overnight with rabbit anti-phospho-tau396 (1:1000; SAB, #11102), rabbit anti-phospho-GSK3 ␣/␤ (Tyr279 + Tyr216) (1:1000; Bioss, bs-2073R), and rabbit anti-PP2A (1:1000, Bioss, bs-0029R) at 4 ◦ C in a humidified chamber. Control sections were treated with identical solutions but without primary antibody. After rinsing several times with PBS, sections were incubated with biotinylated goat anti-rabbit immunoglobulin G (IgG) for 30 min at 37 ◦ C, followed by amplification with streptavidin peroxidase for 20 min at 37 ◦ C. The sections were then rinsed in PBS and incubated with 0.025% 3,3diaminobenzidine (DAB) plus 0.0033% H2 O2 in PBS for 5 min. The stained sections were dehydrated through graded alcohol solutions, cleared in xylene, and covered with neutral. 2.6. Western blot analyses The preparation of the western blots was performed as described previously [23]. Briefly, tissues of motor cortex, sensory cortex, and hippocampal formation were homogenized at 1:5 (wt:vol) in an ice-cold lysis buffer. The resulting homogenate was centrifuged at 12,000 rpm for 30 min at 4 ◦ C. The supernatant was collected, and total protein levels were measured using a BCA protein assay kit (Pierce Biotechnology, Rockford, IL). Proteins (30 ␮g) were separated on 10% sodium dodecyl sulfate (SDS) polyacrylamide gels and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, CA). The membranes were blocked with 5% skim milk in TBS containing 0.1% Tween-20 for 3 h and then incubated with a primary antibody overnight at 4 ◦ C. The primary antibodies used were rabbit anti-phosphotau396 (1:1000; SAB, #11102), rabbit anti-tau396 (1:1000; SAB, #21093), rabbit anti-phospho-GSK3 ␣/␤ (Tyr279 + Tyr216) (1:1000; Bioss, bs-2073R), rabbit antiGSK3␤ (1:1000, Bioss, bs-0028R), rabbit anti-PP2A (1:1000, Bioss, bs-0029R), mouse anti ␤-actin (1:2000; Santa Cruz, sc-47778), and mouse anti-glyceraldehyde 3phosphate dehydrogenase (GAPDH, 1:1000, Bioss, bsm-0978 M). Bound secondary antibodies were visualized using an enhanced chemiluminescence (ECL) kit (Pierce Biotechnology) using ChemDoc XRS with Quantity One software (BioRad, Hercules, CA). Blots were repeated at least three times for every condition. After development, the band intensities were quantified using Image-pro Plus 6.0 analysis software. 2.7. Statistical analysis All values are expressed as mean (M) ± standard error (S.E.). Statistically significant differences were determined by the analysis of variance in SPSS statistical software (SPSS, Inc., Chicago, IL). Differences were considered significant at p < 0.05.

3. Results 2.3. Morris water maze After 4-months ginsenoside Rb1 treatment, the Morris water maze test (MWM) [21] was selected as a method of evaluation of spatial learning and memory. Briefly, the maze consisted of a circular stainless steel tank (120 cm in diameter and 35 cm in height), which was divided by 4 fixed points on its perimeter to 4 quadrants. It contained an escape platform of 10 × 10 × 10 cm of the same color as the rest of the basin (to eliminate any false positive results due to vision) was placed in a constant quadrant of the basin throughout the trials and kept 1.5 cm below the water surface. Mice were placed gently at a start point in the middle of the rim of a quadrant not containing the escape area with their face to the wall. If the mouse failed to find the escape platform within 90 s it was gently guided to the platform and allowed to stay on it for 30 s. Animals had 4 trials per day separated by 10 min for 8 successive days (acquisition trials) during which the time latency to reach the platform (escapes latency) was evaluated. On the 9th day, 24 h after the previous training, probe trials were started in which the escape platform was removed and the animals were allowed to swim freely for 120 s before the end of the session. In probe trials the times of crossing the platform (passing times) were calculated. 2.4. Tissue preparation After behavioral tests, mice (12 months old) were anesthetized with sodium pentobarbital (50 mg/kg, i.p.) and decapitated. The brains were immediately removed and split into halves. Half of the brain was paraffin-embedded, and 7 mm-thick sections encompassing the entire midbrain were prepared for immunohistochemistry. The other half of the brain tissue of motor cortex, sensory cortex and hippocampal formation was prepared as the mouse brain in stereotaxic coordinates [22] referred and directly frozen in liquid nitrogen before storage at −80 ◦ C for western blot analyses.

3.1. Ginsenoside Rb1’ function on Al-induced learning and memory impairment After being treated with Rb1 for 4 months, we tested the spatial learning and memory ability of the 4 groups by Morris water maze. Fig. 1A showed the track on the last day in place navigation test of the 4 groups. We found the trajectory of NC and Rb1 group were mostly in a towards-type, spending shorter distance and time to find the platform than Al-1 and -2 group, which swam by a random-type to reach the platform. Fig. 1B showed the trajectory of 4 groups in spatial probe test. Most of the mice of NC and Rb1 group swam in the quadrant of platform. However, Al-1 and -2 group swam mostly around the wall. Statistical data (Table 1 and Fig. 1C) showed that the escape latency to find the submerged platform in place navigation test gradually declined as training times increased in 4 groups. And significant difference among groups began to appear on the 5th day of the test. In the 120 s probe test, with the platform removed, the crossing times of Al-1 and -2 group was significantly less than NC group (NC: 5.20 ± 1.14, Al-1: 2.60 ± 0.58, Al-2: 2.60 ± 0.27, p < 0.05;). The crossing times in Rb1 group (4.20 ± 0.80) were similar as NC, but not significantly greater than Al-1 and -2 group (Fig. 1D). 3.2. Ginsenoside Rb1’ function on p- tau Ser396 level

2.5. Immunohistochemical staining Paraffin sections were dewaxed in xylene and rehydrated through graded alcohol solutions and then rinsed in 0.1 M PBS (pH 7.2–7.4). Sections were processed in 0.05 M citrate buffer (pH 6.0) and heated in 95 ◦ C water for 10 min for antigen

According to the results of MWM experiments, we stained and observed the brain area of motor, sensory cortex and the hippocampal formation by immunohistochemistry. We chose the most

230

H.-h. Zhao et al. / Behavioural Brain Research 241 (2013) 228–234

Fig. 1. The Mirros water maze test. A: The trajectory of each group on the last day of place navigation test. B: The trajectory of each group in 120s probe test. C: The escape latency of 4 groups in place navigation test. D: The crossing times of 4 groups in 120s probe test. * p < 0.05 compared to NC group. # p < 0.05 compared to Rb1 group.

typical staining of hippocampal CA3, the brain area closely associated with learning and memory [24], to show the distribution of p-tau, p-GSK3 and PP2A expression in four groups (Fig. 2). The positive staining of p-tau396 was mainly expressed in nerve fibers and the cytoplasm around the nucleus, especially in the nerve fibers

of 4 groups (Fig. 2A1–D1).The p-tau396 staining in the nerve fiber layer of CA3 area of Al-1 and -2 group were deeper than the control group(Fig. 2B1). Although the Al-2 group returned to normal drinking water after 6-months Al-exposure, the p-tau396 staining was still deeper than NC group (Fig. 2C1), suggesting that Al can increase

H.-h. Zhao et al. / Behavioural Brain Research 241 (2013) 228–234

231

Table 1 The escape latency in place navigation test of 4 groups (unit: second).

*

Test day

Control

1 2 3 4 5 6 7 8

74.10 74.30 65.90 51.45 44.65 49.25 44.15 35.65

± ± ± ± ± ± ± ±

Al-1 10.83 11.81 7.77 9.59 8.30 7.37 7.55 7.51

82.17 85.25 81.17 72.67 76.17 79.08 73.75 70.08

Al-2 ± ± ± ± ± ± ± ±

13.66 14.12 12.49 14.32 11.80* # 8.87* # 11.92* # 11.95* #

79.29 80.57 72.94 68.82 65.64 75.07 70.29 60.00

Rb1-treated ± ± ± ± ± ± ± ±

11.53 11.87 10.82 12.85 9.66 7.84* # 9.82* # 10.87

75.71 69.57 60.57 59.36 47.43 56.43 43.07 39.71

± ± ± ± ± ± ± ±

12.05 12.19 14.34 9.75 7.40 4.34 8.62 10.18

p < 0.05 vs. NC group and # p < 0.05 vs. Rb1 group, respectively.

the tau phosphorylation level in the hippocampal CA3. On the other hand, p-tau positive staining in Rb1 group was much lighter than Al-1 and -2 group, close to NC (Fig. 2D1). In our western blot test, the p-tau levels in hippocampal formation of the Al-1 and -2 group were higher than NC and Rb group (p < 0.05 compared to NC and Rb1), conforming with the results of immunohistochemistry. We also tested the p-tau396 protein level in motor and sensory cortex, the learning and memory associated brain area [25], by western blot (Fig. 3A). The results in motor cortex were similar to those in the hippocampal formation (increased p-tau396 level in Al-1 and -2 group, p < 0.05). We found slightly change but not statistically significant difference in sensory cortex among 4 groups (Fig. 3A–S).

Our results suggested that ginsenoside Rb1 could effectively reduce the p-tau level in the three brain regions associated closely with learning and memory. 3.3. Ginsenoside Rb1’ function on p-GSK3 and PP2A Molecular mechanisms that regulate Tau phosphorylation are complex and currently incompletely understood. A specific phospho-pattern will result from the balance between kinases and phosphatases. A large number of studies proved that glycogen synthase kinase-3 (Glycogen synthase kinase, GSK3) and protein phosphatase 2A (Protein phosphatase 2A, PP2A) were two of the

Fig. 2. The expression of p-tau Ser396 (A1-D1), p-GSK3␣/␤ (Tyr279 + Tyr216) (A2-D2) and PP2A (A3-D3) in hippocampal CA3 of 4 groups: A(1–3): NC group; B(1–3): Al-1 group; C(1–3): Al-2 group; D(1–3): Rb1 group. bar = 100 ␮m.

232

H.-h. Zhao et al. / Behavioural Brain Research 241 (2013) 228–234

Fig. 3. The relative content of p-tau ser396 (A), p-GSK3␣/␤ (Tyr279 + Tyr216) (B) and PP2A (C) in motor cortex (M), sensory cortex (S) and hippocampal formation (H) (C) of each group. * p < 0.05 compared to NC group. # p < 0.05 compared to Rb1 group.

most important proteinase balancing the tau phos- or dephosphorylation state, respectively. The positive reactions of p-GSK3 and PP2A were mainly distributed in the cytoplasm around the nucleus in our immunohistochemical results (Fig. 2). Fig. 2B2–3 showed more positive p-GSK3 in Al-1 and -2 group than in NC, while PP2A level decreased in these two groups (Fig. 2C2–3). The expression patterns of p-GSK3 and PP2A in Rb1-treated mice were close to NC, suggesting that ginsenoside Rb1 could reduce the active p-GSK3 and increase the protein expression of PP2A, which

could directly and indirectly regulate the phosphorylation level of tau. Our western blot study confirmed the results in immumohistochemistry. P-GSK3 of Al-1 and -2 group was increased (p < 0.05, Fig. 3B) and PP2A was reduced (p < 0.05, Fig. 3C) in the motor, sensory cortex and hippocampus, which were related with learning and memory. Ginsenoside Rb1 could effectively reduce and increase the level of p-GSK3 and PP2A, respectively (p < 0.05, Fig. 3B, C), indicating the role of Rb1 in reducing the p-tau level.

H.-h. Zhao et al. / Behavioural Brain Research 241 (2013) 228–234

4. Discussion 4.1. Ginsenoside Rb1 improves long-term aluminum exposure induced learning and memory dysfunction in mice Studies showed aluminum exposure could significantly increase cognitive dysfunction in the MWM and other behavioral tests, such as radial arm maze, passive avoidance test and rota-rod test [26,27]. However the mechanism underlining Al-induced loss of learning and memory has not been exactly well understood. In this study, we found the mice with 6-month-Al-exposure (Al2 group) or more Al-exposure (Al-1 group) have longer escape latency and reduced crossing times, and its swimming trajectory distributed along the wall in MWM test. It was known that tau protein became hyperphosphorylated, detached from microtubules, abnormally localized to the soma and dendrites, was cleaved by caspases and aggregated into neurofibrillary lesions. There are at least thirty phosohorylation site of tau playing a role in the pathogenesis of AD [28]. However, studies have showed the phosphorylation of tau at Ser396 reduces its affinity for MTs and demonstrate that the abnormal phosphorylation of tau in AD involves Ser396 [29]. Immunohistochemistry and western blot results in Al-1 and -2 group showed there was higher tau phosphorylation level in major brain regions than NC. These processes disrupt cellular transport and cause synapse loss and, ultimately, death of plenty of neurons lead to disrupted neural circuits and cognitive decline [30]. Our results suggested that long-term Al-exposure caused elevated levels of tau phosphorylation, which could lead to learning and memory damage. Studies had shown that ginsenosides could improve the A␤- [20], scopolamine- [31] and chronic morphine injection-induced learning and memory damage [32]. In this study, the MWM behavioral test indicated that the mice of 4 months ginsenoside Rb1 treatment spent much shorter time to find the platform than the Al-exposed group, and their sport modes were similar with normal control group. Interestingly, stopping Al exposure for 4 months (the results of Al-2 group) could not improve the ability of finding the platform, suggesting that ginsenoside Rb1 did reverse the Al-induced spatial learning ablility defect, rather than the results of stopping Al exposure. In addition, results of immunohistochemical staining and western blot also confirmed that tau phosphorylation level in the motor, sensory cortex and hippocampal formation of Rb1 group was lower than that of Al-1 and -2 group, but similar to NC group, suggesting that ginsenoside Rb1 could improve the Al-induced learning and memory defect by reducing the level of tau phosphorylation in mice. 4.2. Ginsenoside Rb1 relieve tau phosphorylation degree by reducing active p-GSK3 and increasing PP2A protein level Studies showed that aluminum might participate in the hyperphosphorylation of tau in Alzheimer’s disease [33–35]. In this study, we detected the tau phosphorylation in motor, sensory cortex and hippocampal formation, and found that p-tau level was significantly increased in motor cortex and hippocampal formation, suggesting aluminum could induce the hyperphosphorylation of tau in these learning and memory related brain areas, which might be a potential factor leading dementia. A large body of biochemical, genetic, and cell biological evidences implicate GSK-3 and PP2A as major enzymes responsible for regulation of tau phosphorylation in vivo [12–14]. GSK-3 exists as two isoforms, alfa and beta, which both phosphorylate tau in vitro and appear as granules in pyramidal cells of hippocampal neurons [36]. Phosphorylation of GSK-3␣/␤ at Tyr216 and Tyr279 is believed to maintain the constitutive activity of GSK-3 in neurons [37]. PP2A is the major tau phosphatase that regulates its phosphorylation at multiple sites in human brain [38]. Previous studies had showed the reduced PP2A

233

activity and GSK-3 level in al-treated animals [39]. In this study, we used immunohistochemistry and western blotting to show the pGSK3 at Tyr216 or Tyr279 and PP2A protein level in motor, sensory cortex and hippocampal formation of 4 groups. We found p-GSK3 was significantly increased with a declined PP2A protein level in all three brain regions, which indicated that these two factors might be involved in Al-induced tau hyperphosphorylation in this model. Ginsenoside Rb1 had been proved to increased pp2a activity in OA-induced dementia rats [40] and down-regulated GSK-3␤ activity by PI3K activation [41]. Thus we compared the actived pGSK3 and PP2A level among the four groups to investigate the role of ginsenoside Rb1 on the Al-induced tau phosphorylation. The immunohistochemical staining, as well as the results in western blot detection, showed that the two indicators above of Rb1-treated mice were significantly reversed when compared with Al-1 and -2 group, yet similar to the NC. Our results suggested ginsenoside Rb1 could reduce the Al-induced phosphorylation of tau by decreasing activated p-GSK3 and increasing the PP2A level, which might explain how ginsenoside Rb1 worked on improving learning and memory of Al-poisoned mice. 5. Conclusions In conclusion, our results show the protective function of ginsenoside Rb1 on Al-induced cognitive deficits by Morris water maze. Moreover, in this study, the role of GSK3 and PP2A on phosand dephosphorylation of tau might be involved in this protective process. Conflict of interest The author declares that there are no conflicts of interest. Acknowledgments This study was supported by the Science and Technology Plan Projects, Liaoning Province, China (No. 2010225034) and the National Natural Science Foundation of China (No. NSFC81171248). We are also grateful to Prof. Zhan-You Wang, Dr. Na Xin and Dr. Song Yu from Laboratory of Cell Engineering and Cell Therapy at China Medical University, for their experiment equipments and technical assistance. References [1] Yokel RA, Allen DD, Ackley DC. The distribution of aluminum into and out of the brain. Journal of Inorganic Biochemistry 1999;76(2):127–32. [2] Yokel RA, Wilson M, Harris WR, Halestrap AP. Aluminum citrate uptake by immortalized brain endothelial cells: implications for its blood-brain barrier transport. Brain Research 2002;930(1–2):101–10. [3] Kaizer RR, Corrêa MC, Gris LR, da Rosa CS, Bohrer D, Morsch VM, et al. Effect of long-term exposure to aluminum on the acetylcholinesterase activity in the central nervous system and erythrocytes. Neurochemical Research 2008;33(11):2294–301. [4] Abu-Taweel GM, Ajarem JS, Ahmad M. Neurobebavioral toxic effects of perinatal oral exposure to aluminum on the developmental motor reflexes, learning, memory and brain neurotransmitters of mice offspring. Pharmacology Biochemistry and Behavior 2012;101(1):49–56. [5] Klatzo I, Wisniewski H, Streicher E. Experimental production of neurofibrillary degeneration. Journal of Neuropathology and Experimental Neurology 1965;24:187–99. [6] Crapper DR, Krishnan SS, Dalton AJ. Brain aluminum distribution in Alzheimer’s disease and experimental neurofibrillary degeneration. Science 1973;180:511–3. [7] Gupta VB, Anitha S, Hegde ML, Zeccal L, Garruto RM, Shankar SK, et al. Aluminum in Alzheimer’s disease: are we still at a crossroad? Cellular and Molecular Life Sciences 2005;62(2):143–58. [8] Grundke-Iqbal I, Iqbal K, Quinlan M, Tung YC, Zaidi MS, Wisniewski HM. Microtubule-associated protein tau. A component of Alzheimer paired helical filaments. Journal of Biological Chemistry 1986;261(13): 6084–9.

234

H.-h. Zhao et al. / Behavioural Brain Research 241 (2013) 228–234

[9] Eidenmuller J, Fath T, Mass T, Pool M, Sontag E, Brandt R. Phosphorylationmimicking glutamate clusters in the proline-rich region are sufficient to simulate the functional deficiencies of hyperphosphorylated tau protein. Biochemical Journal 2001;357(Pt):759–67. [10] Fath T, Eidenmüller J, Brandt R. Tau-mediated cytotoxicity in a pseudohyperphosphorylation model of Alzheimer’s disease. Journal of Neuroscience 2002;22(22):9733–41. [11] Léger J, Kempf M, Lee G, Brandt R. Conversion of serine to aspartate imitates phosphorylation-induced changes in the structure and function of microtubule-associated protein tau. Journal of Biological Chemistry 1997;272(13):8441–6. [12] Guadaqna S, Esiri MM, Williams RJ, Francis PT. Tau phosphorylation in human brain: relationship to behavioral disturbance in dementia. Neurobiology of Aging 2012 [Epub ahead of print]. [13] Martin L, Page G, Terro F. Tau phosphorylation and neuronal apoptosis induced by the blockade of PP2A preferentially involve GSK3␤. Neurochemistry International 2011;59(2):235–50. [14] Obeid R, Schlundt J, Umanskaya N, Herrmann W, Herrmann M. Folate is related to phosphorylated neurofilament-H and P-tau(Ser396) rat brain. Journal of Neurochemistry 2011;117(6):1047–54, in http://dx.doi.org/10.1111/j. 1471-4159.2011.07280.x. [15] Attele AS, Wu JA, Yuan CS. Ginseng pharmacology: multiple constituents and multiple actions. Biochemical Pharmacology 1999;58(11): 1685–93. [16] Zhao HH, Lai H, Zeng L, Lü YL. Effects of ginsenosides on BDNF in nucleus basalis of Meynert and cerebral cortex of aged rats. Acta Anatomica Sinica 2006;37(2):223–5. [17] Lai H, Zhao HH, Zeng L, Yang JP, Fang X, Lü YL. Effects of ginsenosides on BDNF and TrkB protein expression in hippocampal formation of aged rats. Chinese Journal of Histochemistry & Cytochemistry 2006;15(1):60–5. [18] Lai H, Wang TM, Zhao HH, Zeng L, Gao J, Lü YL. Effects of ginsenosides on cholinergic fibers in hippocampal formation of aged rats. Chinese Journal of Anatomy 2006;29(2):249–51. [19] Gao J, Zhao HH, Lai H, Gao XH, Li ZS, Lü YL. Effect of ginseng total saponins on the expression of Acetylcholinesterase in cerebral cortex of aged rats. Progress of Anatomical Sciences 2009;15(1):97–9. [20] Wang Y, Liu J, Zhang Z, Bi P, Qi Z, Zhang C. Anti-neuroinflammation effect of ginsenoside Rb1 in a rat model of Alzheimer disease. Neuroscience Letters 2011;487(1):70–2. [21] Liu HL, Zhao G, Cai K, Zhao HH, Shi LD. Treadmill exercise prevents decline in spatial learning and memory in APP/PS1 transgenic mice through improvement of hippocampal long-term potentiation. Behavioural Brain Research 2011;218(2):308–14. [22] George P, Keith BJF. The mouse brain in stereotaxic coordinates. 2nd ed. San Diego: Academic Press; 2001. [23] Yu S, Zheng W, Xin N, Chi ZH, Wang NQ, Nie YX, et al. Curcumin prevents dopaminergic neuronal death through inhibition of the c-Jun N-terminal kinase pathway. Rejuvenation Research 2010;13(1):55–64. [24] Kesner RP. Behavioral functions of the CA3 subregion of the hippocampus. Learning and Memory 2007;14(11):771–81. [25] Ungerleider LG. Functional brain imaging studies of cortical mechanisms for memory. Science 1995;270(5237):769–75.

[26] Kumar A, Prakash A, Dogra S. Neuroprotective effect of carvedilol against aluminum induced toxicity: possible behavioral and biochemical alterations in rats. Pharmacological Report 2011;63(4):915–23. [27] Abdel-Aal RA, Assi AA, Kostandy BB. Rivastigmine reverses aluminum-induced behavioral changes in rats. European Journal of Pharmacology 2011;659(23):169–76. [28] Billingsley ML, Kincaid RL. Regulated phosphorylation and dephosphorylation of tau protein: effects on microtubule interaction, intracellular trafficking and neurodegeneration. Biochemical Journal 1997;323(Pt 3):577–91. [29] Bramblett GT, Goedert M, Jakes R, Merrick SE, Trojanowski JQ, Lee VM. Abnormal tau phosphorylation at Ser396 in Alzheimer’s disease recapitulates development and contributes to reduced microtubule binding. Neuron 1993;10(6):1089–99. [30] Buée L, Bussière T, Buée-Scherrer V, Delacourte A, Hof PR. Tau protein isoforms, phosphorylation and role in neurodegenerative disorders. Brain Research Brain Research Reviews 2000;33(1):95–130. [31] Wang Q, Sun LH, Jia W, Liu XM, Dang HX, Mai WL, et al. Comparison of ginsenosides Rg1 and Rb1 for their effects on improving scopolamineinduced learning and memory impairment in mice. Phytotherapy Research 2010;24(12):1748–54. [32] Qi D, Zhu Y, Wen L, Liu Q, Qiao H. Ginsenoside Rg1 restores the impairment of learning induced by chronic morphine administration in rats. Journal of Psychopharmacology 2009;23(1):74–83. [33] Li W, Ma KK, Sun W, Paudel HK. Phosphorylation sensitizes microtubuleassociated protein tau to Al(3+)-induced aggregation. Neurochemical Research 1998;23(12):1467–76. [34] Murayama H, Shin RW, Higuchi J, Shibuya S, Muramoto T, Kitamoto T. Interaction of aluminum with PHFtau in Alzheimer’s disease neurofibrillary degeneration evidenced by desferrioxamine-assisted chelating autoclave method. American Journal of Pathology 1999;155(3):877–85. [35] Walton JR. Evidence for participation of aluminum in neurofibrillary tangle formation and growth in Alzheimer’s disease. Journal of Alzheimer’s Disease 2010;22(1):65–72. [36] Hanger DP, Hughes K, Woodgett R, Brion JP, Anderton BH. Glycogen synthase kinase-3 induces Alzheimer’s disease-like phosphorylation of tau: generation of paired helical filament epitopes and neuronal localization of the kinase. Neuroscience Letters 1992;147(1):58–62. [37] Forde JE, Dale TC. Glycogen synthase kinase 3: a key regulator of cellular fate. Cellular and Molecular Life Sciences 2007;64(15):1930–44. [38] Liu F, Grundke-lqbal I, Iqbak K, Gong CX. Contributions of protein phosphatases PP1, PP2A, PP2B and PP5 to the regulation of tau phosphorylation. European Journal of Neuroscience 2005;22(8):1942–50. [39] Singla N, Dhawan DK. Regulatory role of zinc during aluminum-induced altered carbohydrate metabolism in rat brain. Journal of Neuroscience Research 2012;90(3):698–705. [40] Li YK, Chen XC, Zhu YG, Peng XS, Zeng YQ, Sheng J, et al. Ginsenoside Rb1 attenuates okadaic acid-induced Tau protein hyperphosphorylation in rat hippocampal neurons. Sheng Li Xue Bao 2005;57(2):154–60. [41] Zhao R, Zhang Z, Song Y, Wang D, Qi J, Wen S. Implication of phosphatidylinositol-3 kinase/Akt/glycogen synthase kinase-3␤ pathway in ginsenoside Rb1’s attenuation of beta-amyloid-induced neurotoxicity and tau phosphorylation. Journal of Ethnopharmacology 2011;133(3):1109–16.