Puerarin Ameliorates D-Galactose Induced Enhanced Hippocampal Neurogenesis and Tau Hyperphosphorylation in Rat Brain

Abstract

Enhanced neurogenesis has been reported in the hippocampus of patients with Alzheimer’s disease (AD), the most common neurodegenerative disorder characterized with amyloid-β (Aβ) aggregation, tau hyperphosphorylation, and progressive neuronal loss. Previously we reported that tau phosphorylation played an essential role in adult hippocampal neurogenesis, and activation of glycogen synthase kinase (GSK-3), a crucial tau kinase, could induce increased hippocampal neurogenesis. In the present study, we found that treatment of D-galactose rats with Puerarin could significantly improve behavioral performance and ameliorate the enhanced neurogenesis and microtubule-associated protein tau hyperphosphorylation in the hippocampus ofD-galactose rat brains. FGF-2/GSK-3 signaling pathway might be involved in the effects of Puerarin on hippocampal neurogenesis and tau hyperphosphorylation. Our finding provides primary in vivo evidence that Puerarin can attenuate AD-like enhanced hippocampal neurogenesis and tau hyperphosphorylation. Our finding also suggests Puerarin can be served as a treatment for age-related neurodegenerative disorders, such as AD.

Keywords

Alzheimer’s disease glycogen synthase kinase hippocampus neurogenesis Puerarin spatial memory tau hyperphosphorylation

INTRODUCTION

Alzheimer’s disease (AD) is the most common type of dementia in the aging population, which accounts for over 20 million cases worldwide. Brain aging is one of the strongest risk factors for this disease. Patients with AD experience symptoms which includecognitive alterations and memory loss. The dementia in AD is characterized by progressive neuronal loss, formation of amyloid-β (Aβ)-containing plaques, and the presence of neurofibrillary tangles (NFT) composed of hyperphosphorylated tau [1]. More recent studies have provided evidence that altered neurogenesis might also participate in the neurodegenerative process in AD. Some available data from AD patients reported increased expression of neurogenesis markers in hippocampus not only during the onset but also during the middle and advanced stages of AD [2, 3]. Nevertheless, investigations from animal models of AD are more complicated: impaired hippocampal neurogenesis has been reported in many transgenic mouse models of AD [4, 5], while increased neurogenesis was also observed in certain AD animal models [6 –8]. Many studies of transgenic mouse models which express mutant forms of amyloid-β protein precursor (AβPP) or presenilin-1 and of triple transgenic mice which show Aβ overproduction indicate that the decrease in neurogenesis is associated with the presence of toxic amyloid-β peptides (Aβ₄₂) [4 , 10].However, Yetman and Jankowsky reported that amyloid plaques were not localized in the proliferative region of the neurogenesis niche; furthermore, overexpression of mutated human amyloid-β protein precursor (hAβPP) in the granule cell layer of the dentate gyrus has no impact on hippocampal neurogenesis [11]. These studies indicate the increase in hippocampal neurogenesis observed in AD patients may be due to other factors than Aβ.

Neurogenesis persists in the normal adult mammalian brain in the olfactory bulb, the subventricular zone (SVZ) and the subgranular zone (SGZ) of the hippocampus. It is thought that the hippocampus plays an important role in learning and memory that helps animals adapt to everyday life, and much evidence accumulated over the past decade supports the hypothesis that hippocampal neurogenesis is required for hippocampus-dependent learning and memory recall. However, an increase in neurogenesis may not always result in improved function, and more recent work reports no relation between neurogenesis decay from age and memory retention and retrieval [12 –14]. Some studies demonstrate that dentate gyrus neurogenesis influences some hippocampus-dependent behaviors, such as trace and contextual fear conditioning, but not spatial learning [15]. The most recent work by Akers et al. even demonstrates that adult hippocampal neurogenesis may promote forgetting [16]. Excessive neurogenesis in pathological circumstances could even lead to cognitive impairment by altering hippocampal circuits [14]. Until now, the underlying mechanism for the enhanced hippocampal neurogenesis in the brain of patients with AD remains intricate.

Tau is a neuronal microtubule-associated protein, which plays a key role in regulating microtubuledynamics, axonal transport, and neurite outgrowth [17]. The biological functions of tau protein are predominately modulated by site-specific phosphorylation, and the level of tau phosphorylation is developmentally regulated, which is higher in fetal neurons and decreases with age during development [17, 18]. Fetal-tau phosphorylation can also be observed in the adult brain [19]. It has been demonstrated that the presenceof tau phosphorylated in fetal epitopes is related with adult neurogenesis in the SGZ; and phosphorylated tau is coexpressed temporally and spatially with immature neuronal markers DCX and NeuroD [20, 21]; and tau phosphorylation plays an essential role in adult hippocampal neurogenesis [20]. However, inappropriate phosphorylation of tau could lead to the development of tau pathology and result in cognitive impairment [18]. Accumulating evidence has demonstrated that hyperphosphorylated tau sequestered normal microtubule-associated proteins (MAPs), disrupts microtubule dynamics, blocks intracellular trafficking of the neurons and increases tau aggregation [18]. Furthermore, the amount of tau-related neurofibrillary pathologies is closely correlated with the degree of cognitive decline in AD patients [18]. Thus regulating the adult hippocampal neurogenesis via reducing tau phosphorylation level might be a potential therapeutic strategy for treatment of aging-associated neurodegenerative disorders, such as AD.

Puerarin [7-hydroxy-3-(4-hydroxyphenyl)-1-ben-zopyran-4-one 8-(b-D-glucopyranoside)] is an isoflavone component extracted from a Chinese herb, namely Kudzu (Radix puerariae). It has been widely used as a traditional medicine for treating various diseases including coronary artery disease, hypertension, diabetes mellitus, and ischemic stroke [22]. The neuroprotective effects of Puerarin in AD animals have also been reported in several studies: Zhou et al. demonstrated that treatment with Puerarin could ameliorate learning and memory deficits in APP/PS1 transgenic mice [23]; it could also attenuate glutamate-induced neurofilament axonal transport impairment [24]. However, little is known about the effects of Puerarin on hippocampal neurogenesis and the underlying mechanisms.

To elucidate the function and underlying mechanism of Puerarin in age-associated neurodegenerative disorders, we employed the D-galactose to induce rat aging model. D-galactose is a naturally occurring substance in the body. However, at high levels it can accelerate the process of aging by inducing oxidative stress in various organ systems [25 –28]. Previous studies with D-galactose have demonstrated that continuous subcutaneous injection of D-galactose in mice could induce forebrain cholinergic neuronal loss [29], decrease density of synapses [26], and create cognitive impairment [26 , 30]. Therefore, injection with D-galactose in rodents is a well-known model for brain aging to develop neurodegenerative disorders, such as AD. In the present study, we investigated the effects of Puerarin on the level of neurogenesis and tauphosphorylation in the hippocampus, as well as the behavioral performance in D-galactose-treated rats. We found that administration with Puerarin could remarkably improve spatial learning and memory impairment, suppress enhanced hippocampal neurogenesis and tau hyperphosphorylation induced by intraperitoneal injection of D-galactose. We also found that the level of immature neuron markers was positively correlated with that of tau phosphorylation, and FGF-2/GSK-3β pathway seems to play an important role in the efficacy of Puerarin.

MATERIALS AND METHODS

Antibodies and Chemicals

Puerarin (solubilized in 1, 2-propanediol) was purchased from KAMP Pharmaceuticals Co. Ltd (Hunan, China). Goat polyclonal antibody anti-DCX waspurchased from Santa Cruz Biotechnology Inc. (Delaware, CA, USA). Rabbit pAb pS396 against tau phosphorylated at Ser396 site were from Biosource International Inc. (Camarillo, CA, USA). Mouse mAb Tau-1 against tau unphosphorylated at Ser198/199/202 was from Chemicon International Inc. (Temecula, CA, USA). Mouse mAb Tau-5 against total tau was from Thermo Fisher scientific Inc. (Waltham, MA, USA). Rabbit monoclonal antibody against total GSK-3β and polyclonal antibody against GSK-3β phosphorylated at Ser9/21 were obtained from Cell Signaling Technology Inc. (Beverly, MA, USA). HistostainTM-SP kits were from Zymed (South San Francisco, CA, USA). Mouse mAb against b-actin were from Boster Bio-engineering Limited Company (Wuhan, China). Peroxidase-conjugated goat anti-rabbit, and goat anti-mouse secondary antibodies, chemiluminescent substrate kit were from Boster Bio-engineering Limited Company (Wuhan, China). D-galactose and diaminobenzidine (DAB) were from Sigma (St Louis, MO, USA).

Animals’ treatment and morris water maze test

Male Sprague-Dawley rats (weight 300±30 g, 4 months old) were obtained from the Experiment Animal Center of Tongji Medical College. All animals were kept at 24±2°C on daily 12 h light–dark cycles with ad libitum access to food and water. The animal experiments were carried out according to the ‘Policies on the Use of Animals and Humans in Neuroscience Research’ approved by the Society for Neuroscience in 1995. The rats were housed in groups of 4 rats per cage for at least 2 weeks before the day of experimentation. Then the rats received water maze training, carried out from 12:00 to 17:00 every day for 7 consecutive days (4 trials per day). The swimming pathway and escape latency of the rats to find the hidden platform were recorded each day. After 7 days of Morris water maze training, rats were randomly divided into 3 groups (n = 10 for every group): (1) control rats treated with saline (NS) as a vehicle for 8 weeks; (2) rats treated with D-galactose ((160 mg/kg per day) for 8 weeks; (iii) rats treated with D-galactose ((160 mg/kg per day) for 8 weeks and Puerarin (80 mg/kg per day) for 4 weeks. After 8 weeks of treatment, spatial learning and memory was measured again by Morris water maze.

To study different dose or time effects of Puerarin on the level of hippcoampal neurogenesis, another 40 rats were randomly divided into 8 groups: each group received D-galactose (160 mg/kg per day) for 4 weeks first and then the therapy group rats treated withD-galactose ((160 mg/kg per day) and Puerarin at40, 80, or 160 mg/kg per day for 4 weeks, or withD-galactose ((160 mg/kg per day) for 4 weeks and with Puerarin at 80 mg/kg per day for 4, 8, or 12 weeks (n = 5 for every group).

To compare the effect of Puerarin pretreatment with post-treatment on hippocampal neurogenesis, 20 rats were randomly divided into 4 groups (n = 5 for every group): (1) control rats treated with NS for 8 weeks; (2) rats treated with D-galactose (160 mg/kg per day) for 8 weeks; (3) rats first injected with D-galactose (160 mg/kg per day) for 4 w, then injected with D-galactose ((160 mg/kg per day) and Puerarin at 80 mg/kg per day for another 4 weeks; (4) rats pretreated with Puerarin at 80 mg/kg per day for 4 weeks, then injected with D-galactose for 8 weeks. The dose effect of Puerarin pretreatment was also studied by pre-injected with Puerarin at 40 mg/kg or 80 mg/kg per day for 4 weeks before the D-galactose injection.

Immunohistochemistry

Rats were deeply anesthetized and transcardially perfused with 200 ml 0.9% NaCl rapidly, followed by 400 ml 4% paraformaldehyde solution (pH 7.2–7.6). After perfusion, the brain was removed from the skull and postfixed in the same fixation solution for another 12 h at 4°C. Coronal brain sections of hippocampal tissue were cut at 30μm with a freezing microtome (CM1900, Leica Microsystems, Wetzlar, Germany) and were collected in multi-well dishes in series representatives of the hippocampus.

The sections were permeabilized with with 0.3% H₂O₂–PBS–1% Triton X-100 for 20 min to block endogenous peroxidase, and were blocked in PBS-0.3% Triton X-100-3% bovine serum albumin (BSA) for 60 min at room temperature and then incubated with DCX (1:400) for 48 h at 4°C. The immunoreaction was detected using horseradish peroxidase-labeled antibodies for 1 h at 37°C and visualized with the DAB tetrachloride system for brown color (Olympus Optical, Tokyo, Japan). For all immunostaining studies, incubation of the braintissue without the primary antibodies served as negative controls for immunohistochemistry.

Western blotting

After the rats were sacrificed, the hippocampi were immediately homogenized in ice-cold 50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 10 mM NaF, 1 mM Na₃VO₄, 5 mM EDTA, 2 mM benzamidine, 1.0 mM phenylmethylsulfonyl fluoride (PMSF), 10μg/ml each of leupeptin, aprotinin, and pepstatin A. The tissue homogenate was added to one-third volume of sample buffer containing 200 mM Tris–HCl, pH 7.6, 8% SDS, 40% glycerol, 40 mM DTT and boiled for 10 min in a water bath, then centrifuged at 12,000×g for 15 min at 25°C. The supernatant was stored at –80°C for western blotting analysis. The protein concentration in the supernatant was measured by BCA kit according to manufacturer’s instruction.

Equal amounts of protein were separated by a 10% SDS-polyacrylamide gel, and then transferred electrically onto PVDF membranes. The membranes were blocked with 5% defatted milk dissolved in TBS-Tween-20 (TBS-T) (50 mM Tris–HCl, pH 7.6, 150 mM NaCl, 0.2% Tween-20) for 1 h at 37°C, and incubated respectively with DCX (1:100), NeuroD (1:100), pS396 (1:1000), Tau-1 (1:30,000), GSK-3β (1:1000), phospho-GSK-3β (Ser-9/21) (1:1000), or β-actin (1:1000) overnight at 4°C. Immunostaining was visualized with a chemiluminescent substrate kit and Kodak Film and quantitatively analyzed by Kodak Digital Science 1D software (Eastman Kodak Co., New Haven, CT, USA). The band intensity was measured as the sum optical density and the relative level to each control was expressed.

RNA extraction and real-time quantitative RT-PCR

Total RNA was isolated from frozen hippocampi using Trizol (Takara, Japan), according to the manufacturer’s protocol. RNA (2.95μg) was reverse transcribed to cDNA using the cDNA Reverse Transcription Kit (Takara, Japan). Real-time quantitative PCR (Rt-qPCR) was performed using an Applied Bio-systems ViiA 7 Real-Time PCR system (Applied Biosystems, Foster City, CA, USA). The qPCR ampli-fication procedure was as follows: 50°C for 2 min and95°C for 10 min, followed by 40 cycles of 95°C for 5 s and 60°C for 30 s. Primers are provided as follows: FGF-2 forward: GGAGAAGAGCGACCCACA;FGF2 reverse: CGTTTCAGTGCCACATACC; β-actinforward: 5^′-CACGATGGAGGGGCCGGACTCATC-3^′; β-actin reverse: 5^′-TAAAGACCTCTATGCCA-ACACAGT-3^′. All primers for qPCR were designed by using Primer Premier 5.0 software. The level of FGF-2 mRNA expression was normalized against β-actin using the 2 ^–^ΔΔCT method.

Statistic analysis

Data were expressed as means±SEM and analyzed with SPSS 12.0 statistical software (SPSS Inc., Chicago, IL). The results were analyzed by one-way factorial ANOVA followed by the Fisher’s PLSD post hoc test. Differences were considered significant atp < 0.05.

RESULTS

Puerarin improved D-galactose induced spatial memory deficits in rats

Previous studies had demonstrated that D-galactose could induce spatial memory deficits [26 , 30, 31]. In the present study, we investigated the effect of Puerarin on the D-galactose induced behavioral impairment by Morris water maze. After 7 days of training, all rats were able to find the hidden platform within 10 s. Then the rats were subgrouped randomly and received intraperitoneal injection with NS, D-galactose, or Puerarin, separately or in combination. After 4 weeks of Puerarin treatment, spatial memory retention was detected by Morris water maze. On the first day after the treatment, all rats spent more time to find the hidden platform than the day before the injection. However, it was shown that D-galactose injection significantly prolonged the latency compared with NS-treated group, while supplement of Puerarin remarkably shortened the D-galactose-induced increase of the latency (Fig. 1B). Moreover, the Puerarin-treated rats significantly improved the searching strategy in 2 days (Fig. 1A). Instead of taking a tortuous swimming path to the hidden platform as seen in D-galactose treated group, the Puerarin-treated rats were able to find the platform in less than 10 s (Fig. 1A, B). These results suggest that Puerarin can efficiently improve the D-galactose induced spatial memory deficits in the rats.

Puerarin inhibits D-galactose-induced enhanced hippocampal neurogenesis

Increased hippocampal neurogenesis has been observed in patients of AD [2, 3] and some animal models [6 –8]. In the present study, we examined the expression of neurogenesis marker proteins doublecortin (DCX) and neuroD in rat hippocampus to explore the possible mechanisms underlying the effect of Puerarin on D-galactose-induced cognitive impairment. We found that the immunoreactivity of DCX and neuroD increased in D-galactose-treated rat hippocampus, while Puerarin suppressed the enhanced neurogenesis level induced by D-galactose (Fig. 2A-C).We further investigated the influence of Puerarin on hippocampal neurogenesis at different dosages and time. We found that treatment of Puerarin at any of the three dosages (40, 80, and 160 mg/kg per day) could significantly inhibit the D-galactose-induced enhanced neurogenesis in rat hippocampus (Fig. 2D, E). The effect was most remarkable at 40 mg/kg per day, but not dosage-dependent as expected (Fig. 2D, E). Then, we prolonged the injection time from 4 weeks to 12 weeks to explore the time effect of Puerarin on hippocampal neurogenesis. In the time-course study, we only injected the rats at 80 mg/kg per day and observed that the attenuation of increased hippocampal neurogenesis was still distinct after 12 weeks of Puerarin treatment, but no obvious difference was observed among the three time points (Fig. 2F-I).

We also tested the effect of pretreatment with Puerarin on the level of hippocampal neurogenesis in D-galactose-treated rat brain. It was shown that either pre-injection or post-injection with Puerarin could significantly suppress the enhanced hippocampal neurogenesis induced by D-galactose, while the effect of post-injection with Puerarin seems more remarkable (Fig. 3A, B). We also discovered that the effect of pre-injection with Puerarin at lower dosage (40 mg/kg per day) seemed more obvious (Fig. 3C, D). These data indicate that supplementation with Puerarin at any time could significantly inhibit the increased hippocampal neurogenesis induced by chronic D-galactose treatment, and the effect was most remarkable at 40 mg/kg per day.

Puerarin attenuates D-galactose-induced tau hyperphosphorylation in rat hippocampus

Hyperphosphorylation of tau plays an important role in neurodegeneration and memory deficits in AD patients [18]. Interestingly, some sites in tau protein are hyperphosphorylated both during embryonic and early postnatal periods and in the degenerating neurons of the AD brain. Previous studies have demonstrated that tau protein in adult hippocampal progenitor cells are phosphorylated at Ser 195/198/199/202(the Tau-1 site) [32]; phosphorylated tau probed by pS396 was observed as being essential in adult hippocampal neurogenesis [20], and these sites are also hyperphosphorylated in AD brain [18]. Therefore, we further measured the level of tau phosphorylation at Ser 195/198/199/202 and Ser 396 in rat hippocampus in the current study. The effect of Puerarin on D-galactose-induced tau hyperphosphorylation was shown by western blotting that the immunoreactivity of pS396 was enhanced and Tau-1 staining was decreased in D-galactose-injected rats (Fig. 4A, B), suggesting that D-galactose induces tau hyperphosphorylation at Ser396 (pS396) and Ser195/198/199/202 (Tau-1 reacts with non-phosphorylated tau). Treatment with Puerarin attenuated the D-galactose-induced tau hyperphosphorylation at both pS396 and Tau-1 epitopes, but total tau probed by Tau-5 was not changed by the treatment (Fig. 4A, B).

We also examined the effect of Puerarin on tau phosphorylation at different dosages and times, and the effect was similar to that of neurogenesis in rat hippocampus: the effect was most noticeable at 40 mg/kg per day, but not dosage-dependent (Fig. 4 C, D); and the attenuation of tau hyperphosphorylation was still obvious after 12 weeks of Puerarin treatment (Fig. 4E, F). By Pearson analysis, positive correlation of the levels of p-tau and DCX/neuroD was shown (Fig. 5).

These data together indicate Puerarin could arrest the D-galactose-induced tau hyperphosphorylation and enhanced neurogenesis parallel, and that maycontribute to the improved effect Puerarin has on learning and memory in the rats.

Puerarin prevents D-galactose-induced GSK-3β overactivation and FGF-2 transcription in rat hippocampus

GSK-3β is one of the most crucial tau kinases involved in AD-like tau hyperphosphorylation. Therefore, we measured the total GSK-3β (tGSK-3β) and the Serine-9 phosphorylated (pSGSK-3β, the inactive form) in rat hippocampus by western blotting. We found that the total level of tGSK-3β was unchanged in D-galactose-injected or Puerarin-treated rats(Fig. 6A, B). However, we observed that the level of pSGSK-3β was obviously decreased in D-galactose-injected rat hippocampus compared to the control, and supplement of Puerarin for 4 weeks could significantly increase the level of pSGSK-3β (Fig. 6A, B).

GSK-3β can be activated by fibroblast growth factor-2 (FGF-2) in hippocampal neural precursors [32]. To investigate whether Puerarin suppressed the GSK-3 overactivation by regulating the level of FGF-2, we examined the mRNA level of FGF-2 by QRT-PCR. It was observed that the mRNA level of FGF-2 was significantly enhanced in D-galactose-injected rat hippocampus, while Puerarin treatment suppressed the increased mRNA level of FGF-2 in rat hippocampus effectively (Fig. 6C, D).

DISCUSSION

AD is the most common cause of dementia in the aging population, and there is still no effective cure for this disease. Progressive neuronal loss is a hallmark pathology observed in the AD brains [33], therefore some researchers speculate that increased hippocampal neurogenesis might be compensatory for the neuronal loss in the neurodegenerative disorder. However, some studies revealed that an increase in adult hippocampal neurogenesis may not result in improved function and may even promote forgetting [15, 16]. Our previous studies have demonstrated that tau phosphorylation plays an essential role in adult hippocampal neurogenesis, and that inducing increased tau phosphorylation can lead to enhanced hippocampal neurogenesis [20]. Hyperphosphorylated tau is the major protein subunit of NFT, which is believed to be closely correlated with the cognitive deficits in AD [18]. Therefore, we postulated that reducing tau phosphorylation level and ameliorating hippocampal neurogenesis might befeasible therapeutic strategies for AD.

In the present study we tested this idea in the D-galactose rat model, which is a well-recognized model for studying aging-related memory disorders. Data from previous studies have demonstrated that chronic administration with D-galactose treatment could induce memory and synaptic impairment, oxidative and mitochondrial dysfunction, glial activation reduction in immune responses, thus leading to neurodegeneration [31]. In the current study, we demonstrated that chronic treatment with D-galactose significantly induced cognitive dysfunction, which is consistent with previous evidence. By intraperitoneal injection for four continuous weeks, we found that Puerarin could effectively reverse D-galactose-induced memory deficits, reducing the escape latency time and improving the searching strategy to find the hidden platform in the Morris water maze test.

Puerarin is the major isoflavonoid derived from Radix puerariae (kudzu root) that has been widely used in China as a traditional medicine for treating various diseases for thousands of years. It has been reported that Puerarin increases cerebral blood flow in dogs [34] and reduces cerebral and spinal cord injury in ischemia-reperfusion animals [35 –37]. Besides, studies have demonstrated that Puerarin has neuroprotective effects against beta-amyloid-induced neurotoxicity in PC12 cells [38] and cultured hippocampal neurons [39, 40], and can improve cognitive performance in AβPP/PS1 mice through activation of the Akt/GSK-3β signaling pathway [23]. In our present study, we found that Puerarin could also significantly decrease D-galactose-induced enhanced hippocampal neurogenesis. The specific function of the neurogenesis remains controversial. Some evidence supports the hypothesis that neurogenesis is required for hippocampus-dependent learning and memory recall [41 –43], while some other studies show surprising limitations in the ways new neurons in the adult brain can improve function [15 , 44–46]. Specifically, Akers et al. demonstrated that hippocampal neurogenesis will lead to competitive circuit modification and thus contribute to forgetting [16]. This may partly explain why increased hippocampal neurogenesis cannot improve brain function in AD patients.

Neurogenesis is a complex process characterized by several progressive steps, including proliferation, migration, differentiation and maturation. Each of these phases may be regulated by distinct molecular mechanisms and could be susceptible to different pathological conditions [47]. In the current study, we choose DCX and NeuroD, which are mostly expressed during migration and differentiation stages, to explore the effect of Puerarin on hippocampal neurogenesis in the aging animal model. Besides, upregulated expression of DCX and NeuroD has also been reported in the brains of patients with AD [2]. Our results in this study revealed that the expression of DCX and NeuroD were enhanced in the hippocampus of D-galactose rat brain, while Puerarin could significantly suppress the enhanced hippocampal neurogenesis induced by D-galactose. We also found that supplements of Puerarin at different concentrations and different time lengths could effectively inhibit the increased hippocampal neurogenesis induced by D-galactose, and the effects were long lasting.

Until now, mechanisms regulating neurogenesis in the adult hippocampus and its role in neurodegenerative disorders are not fully understood. Our previous studies have demonstrated that tau phosphorylation plays an essential role in adult hippocampal neurogenesis [20]. Tau is a phosphoprotein containing 79 putative Ser/Thr phosphorylation sites [18]. The expression and phosphorylation of tau is developmentally regulated: it is higher in fetal neurons and decreases with age during development [19 , 49]. However, fetal-tau phosphorylation can also be found in the adult brain and tau becomes hyperphosphorylated during neurodegeneration, suggesting a relationship between neuronal development and neurodegeneration. It is known that phosphorylated tau has a reduced affinity for microtubules, leading to decreased microtubule stability. The developmentally and functionally regulated phosphorylation of tau may aid newborn neurons to control microtubule dynamics during normal neurite outgrowth and maturation [18]. The similarity between tau phosphorylation in newborn neurons and AD degenerating neuronal cells indicate the brain exposed to toxic signals may respond to activate the machinery for regeneration, which may be involved in the etiology of neurodegeneration [18]. In the current study, we examined the level of tau phosphorylation at Ser 396 and Ser 195/198/199/202 sites, which are hyperphosphorylated both in developing neuronal cells and AD degenerating neurons. Our results revealed that the expression of p-tau was increased in D-galactose rat hippocampus, while Puerarin could remarkably inhibit the increased tau phosphorylation induced by D-galactose. Furthermore, we observed in the present study that the expression of p-tau was positively correlated with that of neurogenesis markers in the hippocampus of rat brain. These results are consistent with our previous studies, which have confirmed that the expression and distribution of the p-tau was synchronous to that of the immature neuron markers [20]. Other research also demonstrated that elevated levels of phosphorylated tau correlate with the presence of dynamic microtubules during periods of high plasticity in the developing brain [48], and tau hyperphosphorylation may induce neuronal cell cycle [50, 51]. Although the mechanism which underlies the tau-involved neurogenesis is intricate, we can speculate from these data that Puerarin treatment may reverse the D-galactose-induced enhanced hippocampal neurogenesis by regulating tau phosphorylation.

Tau phosphorylation is regulated by multiple protein kinases and protein phosphatases, among them, GSK-3β is the most implicated in the hyperphosphorylation of tau in AD brains [17, 18]. GSK-3β is expressed at high levels in brain, and the changing profile of GSK-3 activity during brain development was similar to that of tau phosphorylation and neurogenesis [52, 53]. Our previous studies have demonstrated that tau hyperphosphorylation induced by activating GSK-3β could promote hippocampal neurogenesis [20]. Here we found that the activity of GSK-3 was increased in D-galactose-treated rat brain, while Puerarin could effectively inhibit the enhanced GSK-3 activity. These results suggested that GSK-3 was involved in the effect of Puerarin on regulating hippocampal neurogenesis. GSK-3β can be activated by fibroblast growth factor-2(FGF-2) in hippocampal progenitor cells [32], and increased level of FGF-2 and GSK-3 has been observed in the brains of patients with AD [54 –56]. Therefore, we further investigated whether FGF-2 was involved in the influence of Puerarin on hippocampal neurogenesis. Interestingly, we observed the mRNA level of FGF-2 was increased in D-galactose rat brain, while Puerarin could significantly prevent the increased level of FGF-2 in rat hippocampus. These data together indicate that FGF-2/GSK-3β signaling pathway may play an important role in the effect of Puerarin ameliorating D-galactose-induced spatial memory deficit, enhanced hippocampal neurogenesis and tau hyperphosphorylation. However, it is still unknown how Puerarin regulates the level of FGF-2 in adult hippocampus, and further study may be needed to clarify the underlying mechanisms.

Taken together, we have found in the present study that Puerarin could efficiently improve D-galactose-induced cognitive impairments and ameliorate the enhanced hippocampal neurogenesis and tau hyperphosphorylation. FGF-2/GSK-3β signaling pathway might partially underlie the effects of Puerarin. Our data provide primary evidence for a new therapeutic strategy to treat chronic neurodegenerative disorders, such as AD.

Footnotes

ACKNOWLEDGMENTS

This work was supported by National Natural Science Foundation of China grant (81100245).

Authors’ disclosures available online ().

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