Arctigenin Attenuates Learning and Memory Deficits through PI3k/Akt/GSK-3β Pathway Reducing Tau Hyperphosphorylation in Aβ-Induced AD Mice

Abstract

Arctigenin is a phenylpropanoid dibenzylbutyrolactone lignan compound possessing antitumor, anti-inflammatory, anti-influenza, antioxidant, antibacterial, and hypoglycaemic activities. Our previous study demonstrated that arctigenin exerts neuroprotective effects both in vitro and in vivo in a Parkinsonʼs disease model. However, the exact mechanism through which arctigenin improves amyloid beta-induced memory impairment by inhibiting the production of the hyperphosphorylated tau protein is unknown. Amyloid β _1–42 was slowly administered via the intracerebroventricular route in a volume of 3 µL (≈ 410 pmmol/mouse) to mice. The mice were administered arctigenin (10, 40, or 150 mg/kg) or vehicle starting from the second day after amyloid β _1–42 injection to the end of the experiment. Behavioural tests were performed from days 9 to 15. On day 16 after the intracerebroventricular administration of amyloid β _1–42, the mice were sacrificed for biochemical analysis. Arctigenin (10–150 mg/kg) significantly attenuated the impairment of spontaneous alternation behaviours in the Y-maze task, decreased the escape latency in the Morris water maze test, and increased the swimming times and swimming distances to the platform located in the probe test. Arctigenin attenuated the level of phosphorylated tau at the Thr-181, Thr-231, and Ser-404 sites in the hippocampus, and increased the phosphorylation levels of phosphatidylinositol-3-kinase, threonine/serine protein kinase B, and glycogen synthase kinase-3β. Arctigenin effectively provides protection against learning and memory deficits and in inhibits hyperphosphorylated tau protein expression in the hippocampus. The possible mechanism may occur via the phosphatidylinositol-3-kinase/protein kinase B-dependent glycogen synthase kinase-3β signalling pathway.

Key words

Alzheimerʼs disease tau - glycogen synthase kinase-3β - phosphatidylinositol-3-kinase - threonineserine protein kinase B

Introduction

Alzheimerʼs disease (AD) is a neurological condition characterised by progressive cognitive deterioration and loss of memory. Extracellular β-amyloid (Aβ) deposits are proposed to serve as initial pathological hallmarks of AD [1], [2]. The deposition of Aβ in AD may lead to the formation of senile plaques (SPs) followed by intraneuronal neurofibrillary tangles (NFTs) composed of aggregates of hyperphosphorylated tau [3]. Tau proteins are microtubule-associated proteins mainly expressed in neurons. Physiologically, their primary function is to stabilise the neuronal cytoskeleton [4]. However, tau phosphorylation may affect the ability of the tau protein to bind microtubules and promote their assembly, resulting in cognitive impairments [5], [6]. Based on these observations, inhibiting Aβ-induced tau phosphorylation is of great importance for the development of therapeutic strategies in AD.

Various kinases and phosphatases are involved in the regulation of tau phosphorylation [7]. Glycogen synthase kinase-3β (GSK-3β), a major tau kinase, is a downstream target of the phosphatidylinositol-3-kinase (PI3K)/threonine/serine protein kinase B (Akt) signalling pathway. Substantial evidence shows that PI3K/Akt activation prevents Aβ-induced neurotoxicity in cells [8] and in mouse models of AD [9]. GSK-3β regulates both tau phosphorylation and Aβ production [10], [11] through inactivation of the PI3K/Akt pathway. Therefore, the PI3K/Akt/GSK-3β signalling pathway is an important target for the diagnosis and treatment of AD.

Arctigenin is a phenylpropanoid dibenzylbutyrolactone lignan compound that occurs naturally in Arctium lappa L. (Asteraceae), Bardanae Fructus, Ipomoea cairica (L.) Sweet (Convolvulaceae), Saussurea medusa Maxim. (Asteraceae), and Torreya nucifera (L.) Siebold & Zucc. (Taxaceae) [12], [13]. Previous studies have shown that arctigenin possesses antitumour, anti-inflammatory, anti-influenza, antioxidant, antibacterial, and hypoglycaemic activities [14], [15]. Our previous study demonstrated that arctigenin exerts neuroprotective effects both in vitro and in vivo in a Parkinsonʼs disease model [16]. This present study examines the effect of arctigenin on learning and memory impairments induced by an injection of amyloid beta (Aβ _1–42) in a mouse model. Furthermore, its effects on hyperphosphorylated tau proteins and the possible mechanisms were investigated.

Results

The effect of arctigenin on spontaneous alternation behaviour was evaluated using a Y-maze test. As shown in [Fig. 1 B], the model group exhibited significantly reduced spontaneous alternation behaviour compared to the control group [F(5,42) = 6.986, p < 0.001]. Arctigenin (40 and 150 mg/kg) attenuated the impairment of spontaneous alternation behaviour. LSD post hoc analysis showed that the 40 and 150 mg/kg groups treated with arctigenin presented significantly alleviated effects (p < 0.05; [Fig. 1 B]). The group administered donepezil (1.3 mg/kg) was used as a positive control. The total number of arm entries was not significantly different among the groups [F(5,42) = 0.114, p > 0.05; [Fig. 1 C]].

Fig. 1 Effects of arctigenin on learning and memory deficits induced by ICV Aβ in the Y-maze test. A Chemical structure of arctigrenin. B Alternation (%) from Y-maze test. C The total number of arm entries from the Y-maze test. All of the results are expressed as the means ± SD; n = 8 animals; ^###p < 0.001 vs. control; *p < 0.05, **p < 0.01 vs. Aβ.

To test whether Aβ _1–42 induces spatial memory deficits in mice, we assessed the behavioural performance of the mice using the Morris water maze test. During the training period, a significant difference in the performance of six different mouse groups was observed [F_group = 5.596, p < 0.001; F_day = 37.349, p < 0.001; F_{group × day} = 1.178, p = 0.276; [Fig. 2 A]]. The mice in the Aβ group consistently took longer to find the platform than those in the control group ([Fig. 2 A]). Post hoc analyses indicated a significant decrease in the escape latency of mice treated with 150 mg/kg and 40 mg/kg arctigenin (p < 0.05). In the probe test, the exploration time spent and the distance travelled by the Aβ mice in searching for the target quadrant were significantly lower compared with the control mice, and the mice treated with arctigenin (10, 40, and 150 mg/kg) spent more time [F(5,42) = 3.361, p < 0.05; [Fig. 2 B]] and travelled a longer distance [F(5,42) = 2.846, p < 0.05; [Fig. 2 C]] in their attempt to search for the target quadrant compared with the Aβ-treated mice. LSD post hoc analyses showed that the arctigenin-treated mice in all three dose groups demonstrated a significant improvement (p < 0.05). No significant differences in swim speed were observed among the animal groups [F(5,42) = 0.471, p = 0.796; [Fig. 2 D]]. Donepezil also significantly increased the time and distance spent searching for the target quadrant. Taken together, these data suggest that arctigenin ameliorates learning and memory deficits induced by intracerebroventricular (ICV) Aβ _1–42 in mice.

Fig. 2 Effects of arctigenin on learning and memory deficits induced by ICV Aβ in the Morris water maze test. A Changes in the latency to reach the platform during the training period. Changes in the swimming time (B) and swimming distance (C) in the target quadrant in the probe trial. Aβ-treated mice exhibited a decrease in the swimming time and swimming distance in the target quadrant compared to the control mice. These effects were reversed by treatment with arctigenin. D No significant differences in swim speed were observed among the groups. All of the results are expressed as the means ± SD; n = 8 animals; ^#p < 0.05, ^##p < 0.01 vs. control; *p < 0.05, **p < 0.01 vs. Aβ.

We found that arctigenin inhibits the production of phosphorylated tau and measured the expression of phosphorylated tau through Western blot analysis. The results indicate that the level of phosphorylated tau at the Thr-181, Thr-231, and Ser-404 sites in the hippocampus was significantly higher in the Aβ group than in the control group [Thr-231: F(5,12) = 74.542, p < 0.01; Ser-231: F(5,12) = 35.584, p < 0.01; Ser-404: F(5,12) = 62.515, p < 0.01; [Fig. 3]], but was significantly lower in the group treated with arctigenin than in the Aβ group (p < 0.01, [Fig. 3]).

Fig. 3 Effects of arctigenin on the expression of Thr-181, Thr-231 Ser-404, and Tau-5 in the hippocampus (A). Arctigenin (40 mg/kg and 150 mg/kg) significantly decrease Thr-181 (B), Thr-231 (C), and Ser-404 (D), as determined by Western blotting. All of the results are expressed as the means ± SD; n = 3 animals; ^###p < 0.001 vs. control; *p < 0.05, **p < 0.01 vs. Aβ.

We examined the phosphorylation levels of PI3K, Akt, and GSK-3β to investigate the potential involvement of the PI3K/Akt pathway in the arctigenin-induced protection against phosphorylated tau. Aβ injection significantly decreased the phosphorylation levels of PI3K [F(5,12) = 60.413, p < 0.001; [Fig. 4]], Akt [F(5,12) = 92.591, p < 0.001; [Fig. 4]], and GSK-3β [F(5,12) = 4.328, p < 0.05; [Fig. 4]]. Treatment with arctigenin at doses of 150 mg/kg and 40 mg/kg significantly increased the phosphorylation levels of PI3K (p < 0.01; [Fig. 4]), Akt (p < 0.01; [Fig. 4]), and GSK-3β (p < 0.05; [Fig. 4]).

Fig. 4 Effects of arctigenin on the expression of p-PI3K, p-AKT, and p-GSK-3β in the hippocampus (A). Arctigenin (40 mg/kg and 150 mg/kg) significantly increase p-PI3K (B), p-AKT (C), and p-GSK-3β (D) protein expression levels, as determined by Western blotting. All of the results are expressed as the means ± SD; n = 3 animals; ^###p < 0.001 vs. control; **p < 0.01, ***p < 0.01 vs. Aβ.

Discussion

Extracellular Aβ deposits and intracellular neurofibrillary tangles in the brain parenchyma involved in cognitive functions are assumed to be pathological hallmarks of AD. Abnormal fibres composed of hyperphosphorylated tau protein can form NFTs in neuronal cell bodies or other cell types of the brain [17]. Arctigenin treatment was reported to be effective in reversing the deficits in memory and learning by using APP/PS1 transgenic AD mice models. These improvements are hypothesised to be regulated by both the inhibition of Aβ production and promoting Aβ clearance through the AKT/mTOR and AMPK/Raptor signalling pathways [18], but the mechanism through which arctigenin improves Aβ-induced memory impairment by inhibiting tau hyperphosphorylation in vivo has not been elucidated. In this study, we demonstrated that arctigenin effectively provides protection against learning and memory deficits and in inhibiting tau hyperphosphorylation in the hippocampus. The possible mechanism involves inhibition of the PI3K/Akt-dependent GSK-3β signalling pathway.

Because AD patients display learning and memory disabilities, in this study, we investigated the behavioural changes in Aβ _1–42-treated mice administered arctigenin using the Y-maze test and the Morris water maze test. The results showed that the ICV injection of Aβ _1–42 caused impairments in learning and memory performance of the mice and that treatment with arctigenin significantly attenuated the Aβ _1–42-induced spatial learning and memory impairments in a dose-dependent manner.

Tau is a major microtubule-associated protein that plays a key role in the outgrowth of neuronal processes and the development of neuronal polarity. Abnormal levels and hyperphosphorylation of the tau protein are the underlying cause of a group of neurodegenerative diseases, including AD. Among all of the phosphorylation sites on the tau protein, the majority are serines (53 % of phosphorylation sites of tau) and threonines (41 %) [19]. On the basis that Thr-231, Thr-181, and Ser-404 were reported to be identified in AD patients [20], the role of tau phosphorylation sites in Aβ _1–42-injected mice were investigated by using Western blot analysis. As shown in [Fig. 3], compared with the control group, Aβ _1–42 increased the levels of tau protein phosphorylated at the three sites. However, arctigenin attenuated the above-described level of the tau protein. The result indicated that arctigenin effectively protects against tau phosphorylation.

Considering that GSK-3β plays a central role in the pathogenesis of AD and regulates tau phosphorylation, we studied the effect of GSK-3β on AD [21], [22], [23], [24], [25]. Phosphorylation of Ser-9 (inhibitory) and Tyr-216 (activating) for GSK-3β is regulated by multiple mechanisms, including the PI3K/Akt pathway [26], [27]. Hyperactivation of the PI3K/AKT/GSK-3β pathway is linked to the disrupted clearance of Aβ and tau, synaptic loss, and cognitive decline in AD. Nevertheless, the PI3K/AKT pathway is downregulated in AD transgenic mice and AD patients [28], [29]. Our data showed that arctigenin increases the GSK-3β (Ser-9) phosphorylation level, suppresses PI3K/Akt activation, and increases the PI3K p85 and Akt Ser 473 phosphorylation levels. These results suggest that the Aβ-induced tau hyperphosphorylation is attenuated by arctigenin via the PI3K/Akt/GSK-3β cascade. Furthermore, the point that Aβ promotes tau phosphorylation, and arctigenin reduces the accumulation of Aβ [18], may be in correlation with the reduction in the levels of tau phosphorylation.

In this study, we provide the first demonstration that arctigenin exerts protective effects against ICV Aβ _1–42-induced learning and memory deficits by the reduction of tau hyperphosphorylation through the PI3K/Akt-dependent GSK-3β signalling pathway. These data demonstrate that arctigenin possesses anti-tau hyperphosphorylation activity, which may be a potential therapeutic target in AD treatment.

Materials and Methods

Reagents and instruments

Amyloid_1–42 and phenylmethylsulfonyl fluoride (PMSF) were purchased from Sigma-Aldrich. The total protein extraction kit was purchased from Nanjing KeyGen Biotech. Co., Ltd., and a Page-Ruler prestained protein ladder (10–170 kDa) was purchased from Thermo Fisher Scientific Chemical Reagent Co., Ltd. The BCA protein assay kit, SDS-PAGE gel preparation kit, SDS-PAGE sample loading buffer (5 ×), HRP-labelled goat anti-rabbit IgG (H+L), HRP-labelled goat anti-mouse IgG (H+L), and BeyoECL Plus (ECL-like Western reagent) were obtained from the Beyotime Institute of Biotechnology. Antibodies against Ser-404, Thr-231, Thr-181, PI3K, p-PI3K (P85), AKT, p-AKT (Ser-473), GSK-3β, p- GSK-3β (Ser-9), and β-actin were purchased from Santa Cruz Biotechnology, Ltd. Antibodies against Tau-5 were obtained from Invitrogen Biotechnology, Ltd. Arctigenin, with a purity higher than 98 %, was prepared by our laboratory, and its chemical structure is shown in [Fig. 1 A] [30]. Donepezil, with a purity higher than 98 %, was obtained from Eisai, Ltd. All other chemicals were of analytical grade.

Experimental animals

Equal numbers of adult male and female ICR mice (n = 48; 8 weeks of age) weighing 20–22 g were obtained from the Beijing HFK Bioscience Co., Ltd., and used in the study. The animals were housed in a polyacrylic cage with four mice per cage and maintained under standard housing conditions (22 °C ± 2 °C and humidity 50 % ± 5 %) with a 12-h light/12-h dark cycle. Food and water were available ad libitum. All efforts were made to minimise the number of animals and their suffering throughout the experiments. The food was withdrawn 16–18 h prior to the surgical procedure. All animal studies were performed in strict accordance with the P. R. China legislation on the use and care of laboratory animals and the guidelines established by the Institute for Experimental Animals at Liaoning University of Traditional Chinese Medicine (131/2010; approved on November 1, 2011).

Animal model and amyloid beta administration

Aβ _1–42 was dissolved in sterile physiological saline and aggregated by incubation at 37 °C for 5 days prior to injection, according to a previous report [31].

The mice were anaesthetised with chloral hydrate (300 mg/kg i. p.) and placed on a stereotaxic apparatus (David Kopf Instruments) using a mouse adaptor (Kopf 921), and the skull was then exposed. Aβ _1–42 was injected via the ICV route slowly in a volume of 3 µL (≈ 410 pmol/mouse) on day 1, as described by Chi et al. [32]. The coordinates from the bregma were the following: AP − 0.5 mm, ML − 1.0 mm, and DV − 3 mm [33]. The control animals were injected with equivalent volumes of sterile physiological saline. The mice were orally administered arctigenin (10, 40, or 150 mg/kg), donepezil (1.3 mg/kg), or vehicle by gavage immediately after the second Aβ _1–42 injection. The mice were then treated once daily or 1 h before the behavioural tests.

Behavioural analysis

The Y-maze test was performed 9 days after Aβ injection. The maze had three arms, and the dimensions of each arm were 40 × 12 × 10 cm (length × height × width). Each mouse was placed at the terminus of one arm and allowed to move freely through the maze for 5 min. The total number of arm entries (N) and the sequence of entries were recorded. Alternation behaviour was defined as entries into all three arms on consecutive occasions. The alternation rate (%) was calculated as follows: alternation behaviour (%) = number of alternations/(N − 2) × 100.

The Morris water maze test was performed 10 days after Aβ injection. The Morris water maze consisted of a circular pool (100 cm in diameter and 50 cm in height) that was filled with water (25 °C ± 1 °C and 30 cm in depth). A platform (10 cm in diameter) was placed 1 cm below the water surface in one of four identical quadrants. The mice were trained twice a day for 5 consecutive days with an inter-trial interval of 3 h. The mice were allowed to escape by swimming to the platform, and the escape latency was recorded for 60 s. After 60 s, if a mouse failed to locate the platform, it was then placed on the platform for 20 s. On the sixth day, the mice were administered a probe test. In this test, the platform was removed, and each mouse was allowed to explore the pool for 60 s. The escape latency (time spent in the target quadrant), swimming distance, and swimming speed were measured using a computer system with a video camera. All trials were completed between 08 : 00 and 18 : 00 h (on days 1, 2, 3, 4, and 5 of the test).

Western blotting analysis

Sixteen days after the ICV Aβ _1–42 injection, the rats were sacrificed. Brain tissues were rinsed twice with cold PBS, disrupted with 400 µL of cold hypotonic buffer (10 mM HEPES pH 7.9, 10 mM KCL, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, and 0.5 M PMSF) for 15 min on ice, and incubated for an additional 5 min after the addition of 25 µL of 10 % NP-40. The samples were then sonicated for 15 s and centrifuged at 12 000 × g and 4 °C for 10 min. The protein levels were determined using the Bradford assay (Beyotime). Protein extracts (50 µg) were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and then electrophoretically transferred to polyvinylidene difluoride membranes (Millipore). After incubation at room temperature with 5 % nonfat milk in PBS for 2 h, the membranes were incubated overnight at 4 °C with the indicated primary antibodies against Tau-5 (Invitrogen), Ser-404, Thr-231, Thr-181, PI3K, p-PI3K (P85), AKT, p-AKT (Ser-473), GSK-3β, p- GSK-3β (Ser-9), and β-actin (Santa Cruz). The membranes were then incubated with secondary anti-mouse IgG or anti-rabbit IgG antibodies (Santa Cruz) for 2 h at room temperature. Protein bands were developed using electrochemiluminescent Western blot reagents (Cwbiotech). The intensity was quantified by densitometry using Quantity One 4.6.2 software (Bio-Rad) and corrected with the corresponding β-actin level. The results are expressed as percentages of the control or Aβ levels.

Statistical analysis

The data are expressed as the means ± SD. The statistical significance was determined through one-way or two-way ANOVA followed by Fisherʼs LSD multiple comparisons test. A p value less than 0.05 was considered to indicate statistical significance. The data were analysed using SPSS 17.0.

Acknowledgements

The authors acknowledge the funding provided by the National Basic Research 973 Program of China (2013CB531803), the National Eleventh-Five-Year Plan in R&D of Important New Medicines (2009ZX09103–423), the 2013 Program for Liaoning Innovative Research Team in University (LT LT2013020), and NSFC (81373939).

Conflict of Interest

The authors declare no conflicts of interest.

• References

• 1 Qi Y, Zou LB, Wang LH, Jin G, Pan JJ, Chi TY, Ji XF. Xanthoceraside inhibits pro-inflammatory cytokine expression in Aβ _25–35/IFN-γ-stimulated microglia through the TLR2 receptor, MyD88, nuclear factor-κB, and mitogen-activated protein kinase signaling pathways. J Pharmacol Sci 2013; 122: 305-317
• 2 Krause DL, Müller N. Neuroinflammation, microglia and implications for anti-inflammatory treatment in Alzheimerʼs disease. Int J Alzheimers Dis 2010; 2010: 732806
• 3 Zumkehr J, Rodriguez-Ortiz CJ, Cheng D, Kieu Z, Wai T, Hawkins C, Kilian J, Lim SL, Medeiros R, Kitazaw M. Ceftriaxone ameliorates tau pathology and cognitive decline via restoration of glial glutamate transporter in a mouse model of Alzheimerʼs disease. Neurobiol Aging 2015; 36: 2260-2271
• 4 Reddy PH. Abnormal tau, mitochondrial dysfunction, impaired axonal transport of mitochondria, and synaptic deprivation in Alzheimerʼs disease. Brain Res 2011; 1415: 136-148
• 5 Dugger BN, Tu M, Murray ME, Dickson DW. Disease specificity and pathologic progression of tau pathology in brainstem nuclei of Alzheimerʼs disease and progressive supranuclear palsy. Neurosci Lett 2011; 491: 122-126
• 6 Tang Z, Ioja E, Bereczki E, Hultenby K, Li C, Guan Z, Winblad B, Pei JJ. mTor mediates tau localization and secretion: Implication for Alzheimerʼs disease. Biochim Biophys Acta 2015; 1853: 1646-1657
• 7 Wang JZ, Liu F. Microtubule-associated protein tau in development, degeneration and protection of neurons. Prog Neurobiol 2008; 85: 148-175
• 8 Wei W, Wang X, Kusiak JW. Signaling events in amyloid beta-peptide-induced neuronal death and insulin-like growth factor I protection. J Biol Chem 2002; 277: 17649-17656
• 9 Stein TD, Johnson JA. Lack of neurodegeneration in transgenic mice overexpressing mutant amyloid precursor protein is associated with increased levels of transthyretin and the activation of cell survival pathways. J Neurosci 2002; 22: 7380-7388
• 10 Hoppe JB, Coradini K, Frozza R, Oliveira CM, Meneghetti AB, Bernardi A, Pires ES, Beck RCR, Salbego CG. Free and nanoencapsulated curcumin suppress β-amyloid-induced cognitive impairments in rats: involvement of BDNF and Akt/GSK-3β signaling pathway. Neurobiol Learn Mem 2013; 106: 134-144
• 11 Koh SH, Kim SH, Kwon H, Park Y, Kim KS, Song CW, Kim J, Kim MH, Yu HJ, Henkel JS, Jung HK. Epigallocatechin gallate protects nerve growth factor differentiated PC12 cells from oxidative-radical-stress-induced apoptosis through its effect on phosphoinositide 3-kinase/Akt and glycogen synthase kinase-3. Brain Res Mol Brain Res 2003; 118: 72-81
• 12 Wang W, Pan Q, Han XY, Wang J, Tan RQ, He F, Dou DQ, Kang TG. Simultaneous determination of arctiin and its metabolites in rat urine and feces by HPLC. Fitoterapia 2013; 86: 6-12
• 13 Swarup V, Ghosh J, Mishra M, Basu A. Novel strategy for treatment of Japanese encephalitis using arctigenin, a plant lignan. J Antimicrob Chemother 2008; 3: 679-688
• 14 Zhao F, Wang L, Liu K. In vitro anti-inflammatory effects of arctigenin, a lignan from Arctium lappa L., through inhibition on iNOS pathway. J Ethnopharmacol 2009; 122: 457-462
• 15 Kang HS, Lee JY, Kim CJ. Anti-inflammatory activity of arctigenin from Forsythiae Fructus. J Ethnopharmacol 2008; 116: 305-312
• 16 Li DW, Liu QP, Jia D, Dou DQ, Wang XF, Kang TG. Protective effect of arctigenin against MPP+ and MPTP-induced neurotoxicity. Planta Med 2014; 80: 48-55
• 17 Avila J. Tau phosphorylation and aggregation in Alzheimerʼs disease pathology. FEBS Lett 2006; 580: 2922-2927
• 18 Zhu ZY, Yan JM, Jiang W, Yao XG, Chen J, Chen LL, Li CJ, Hu LH, Jiang HL, Shen X. Arctigenin effectively ameliorates memory impairment in Alzheimerʼs disease model mice targeting both β-amyloid production and clearance. J Neurosci 2013; 33: 13138-13149
• 19 Martin L, Page G, Terro F. Tau phosphorylation and neuronal apoptosis induced by the blockade of PP2A, preferentially involve GSK3β . Neurochem Int 2011; 59: 235-250
• 20 Fujimura M, Usuki F, Sawada M, Takashima A. Methylmercury induces neuropathological changes with tau hyperphosphorylation mainly through the activation of the c-jun-N-terminal kinase pathway in the cerebral cortex, but not in the hippocampus of the mouse brain. Neurotoxicology 2009; 30: 1000-1007
• 21 Peng CX, Hu J, Liu D, Hong XP, Wu YY, Zhu LQ, Wang JZ. Disease-modified glycogen synthase kinase-3β intervention by melatonin arrests the pathology and memory deficits in an Alzheimerʼs animal model. Neurobiol Aging 2013; 34: 1555-1563
• 22 Jin G, Wang LH, Ji XF, Chi TY, Qi Y, Jiao Q, Xu Q, Zhou XY, Zhang R, Zou LB. Xanthoceraside rescues learning and memory deficits through attenuating beta-amyloid deposition and tau hyperphosphorylation in APP mice. Neurosci Lett 2014; 573: 58-63
• 23 Xiong YS, Wang DL, Tan L, Wang X, Chen LM, Gong CX, Wang JZ, Zhu LQ. Inhibition of glycogen synthase kinase-3 reverses tau hyperphosphorylation induced by Pin1 down-regulation. CNS Neurol Disord Drug Targets 2013; 12: 436-443
• 24 Dewachter I, Ris L, Jaworski T, Seymour CM, Kremer A, Borghgraef P, Vijver HD, Godaux E, Van Leuven F. GSK3β, a centre-staged kinase in neuropsychiatric disorders, modulates long term memory by inhibitory phosphorylation at serine-9. Neurobiol Dis 2009; 35: 193-200
• 25 Noh MY, Chun K, Kang BY, Kim H, Park JS, Lee HC, Kim YH, Ku S, Kim SH. Newly developed glycogen synthase kinase-3 (GSK-3) inhibitors protect neuronal cells death in amyloid-beta induced cell model and in a transgenic mouse model of Alzheimerʼs disease. Biochem Biophys Res Commun 2013; 435: 274-281
• 26 Liu P, Zou LB, Wang LH, Jiao Q, Chi TY, Ji XF, Jin G. Xanthoceraside attenuates tau hyperphosphorylation and cognitive deficits in intracerebroventricular-streptozotocin injected rats. Psychopharmacology (Berl) 2014; 231: 345-356
• 27 Maqbool M, Mobashir M, Hoda N. Pivotal role of glycogen synthase kinase-3: A therapeutic target for Alzheimerʼs disease. Eur J Med Chem 2016; 107: 63-81
• 28 Heras-Sandoval D, Pérez-Rojas JM, Hernández-Damián J, Pedraza-Chaverri J. The role of PI3K/AKT/mTOR pathway in the modulation of autophagy and the clearance of protein aggregates in neurodegeneration. Cell Signal 2014; 26: 2694-2701
• 29 Lou HY, Fan PH, Perez RG. Neuroprotective effects of linarin through activation of the PI3K/Akt pathway in amyloid-β-induced neuronal cell death. Bioorg Med Chem 2011; 19: 4021-4027
• 30 Hou Q, He F, Dou DQ, Kang TG, Li HF. [New preparation method of arctigenin from Fructus Arctii]. Chin J New Drugs 2011; 20: 871-873
• 31 Jana M, Palencia CA, Pahan K. Fibrillar amyloid-β peptides activate microglia via TLR2: implications for Alzheimerʼs disease. J Immunol 2008; 181: 7254-7262
• 32 Chi TY, Wang LH, Ji XF, Li W, Wang Y, Xia M, Zou LB. [Protective effects of xanthoceraside on learning and memory impairment induced by intracerebroventricular injections of Aβ _1–42 in mice]. J Shenyang Pharm Univ 2010; 27: 314-320
• 33 Laursen SE, Belknap JK. Intranerebroventricular injections in mice. Some methodological refinements. J Pharmacol Methods 1986; 16: 335-357