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Research Report

Effects of streptozotocin-induced diabetes on tau phosphorylation in the rat brain

Zhongsen Qua,b,,1 , Zongxian Jiaoc,1 , Xiaojiang Sunb , Yuwu Zhaob , Jinpeng Renb , Guogang Xua,d
Department of Pathophysiology, Key Laboratory of Neurological Diseases of Hubei Province, Tongji Medical College of Huazhong University of Science and Technology, Wuhan, Hubei 430030, PR China b Department of Neurology, The Sixth People's Hospital Affiliated to Shanghai Jiao Tong University, Shanghai 200233, PR China c Department of Pathology, Lanzhou University School of Basic Medicine, Lanzhou, Gansu 730000, PR China d Department of Neurology, The Second Hospital Affiliated to Nanchang University, Nanchang, Jiangxi 330006, PR China
a

A R T I C LE I N FO Article history: Accepted 21 January 2011 Available online 31 January 2011 Keywords: Hyperglycemia Tau hyperphosphorylation Glycogen synthase kinase-3 Protein phosphatase-2A

AB S T R A C T Brain protein kinase B (Akt) and glycogen synthase kinase-3 (GSK-3) activities are adaptable to changes of peripheral blood glucose level in vivo. GSK-3 phosphorylates microtubeassociated protein tau at multiple sites, which can be antagonized by protein phosphatase2A (PP-2A). The imbalance among these enzymes might have potential connections with diabetes mellitus (DM) and Alzheimer's disease (AD). In this study hyperglycemia rat DM model was achieved by streptozotocin (STZ) treatment. The phosphorylation of tau in the rat hippocampus was detected with specific antibodies. Insulin and Li2CO3 administration were also employed to find out the regulatory efforts of the kinases. We observed that rat hippocampus tau was hyperphosphorylated at Ser396/Ser404 (PHF-1 sites) in STZ-induced DM model, accompanied by lowered phosphorylation levels of Akt, GSK-3 and PP-2A. Lithium, a specific GSK-3 inhibitor, nearly reversed all phosphorylation of tau at above sites in 30 days. Insulin administration restored the blood glucose level in DM rats but suppressed PP-2A activity, resulting in the PHF-1 sites of tau not being dephosphorylated. These findings strongly suggest that STZ-induced hyperglycemia may cause disorder of Akt/GSK-3/PP-2A regulations in rat brain and further lead to abnormal phosphorylation of hippocampus tau. 2011 Elsevier B.V. All rights reserved.

1.

Introduction

Diabetes mellitus (DM) is a common metabolic disease in human beings with characteristic symptoms of hyperglycemia and impaired insulin secretion or insulin resistant. It has been

reported that about 6070% of DM patients also present the diabetic neuropathy manifestations in either peripheral or central nervous systems (Manschot et al., 2008; Vinik, 2004). Many clinical studies indicated an association of DM with higher risks of cognitive impairment and neurodegenerative

Corresponding author at: Department of Neurology, The Sixth People's Hospital Affiliated to Shanghai Jiao Tong University, Shanghai 200233, PR China. Fax: +86 21 64701361. E-mail address: quzs1968@yahoo.com.cn (Z. Qu). Abbreviations: AD, Alzheimer's disease; Akt, protein kinase B; DM, diabetes mellitus; GSK-3, glycogen synthase kinase-3; PHF, paired helical filament; PP-2A, protein phosphatase-2A; STZ, streptozotocin 1 These authors contributed equally to this work. 0006-8993/$ see front matter 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2011.01.084

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diseases, such as Alzheimer's disease (AD). Furthermore, the co-occurrence of DM and AD is not rare in elderly individuals with chronic metabolic disorders (Areosa and Grimley, 2002; MacKnight et al., 2002; Janson et al., 2004; Stewart and Liolitsa, 1999). However, little is known about the underlying mechanisms of how DM and AD are coupled. Hyperphosphorylation of brain microtube-associated protein tau has been proved key in AD etiopathogenesis (Alonso et al., 2008; Gong and Iqbal, 2008). Insulin, an important signaling molecule, is capable of regulating the activity of some kinases that are responsible for tau phosphorylation (Yan et al., 2003). Therefore, both abnormal insulin signaling and pathological glucose fluctuations may contribute to the tau hyperphosphorylation, particularly in those patients with diabetes. In vivo, insulin controls the intracellular and plasma glucose level through a multi-enzyme signaling cascade including Akt and GSK-3 (Taniguchi et al., 2006). Akt is a positive regulator in this pathway, which is activated by phosphorylation of Thr308 and Ser473 by insulin signaling. GSK-3 activity is inhibited when Ser21 of its -isoform or Ser9 of its -isoform (GSK-3) is phosphorylated by Akt (Asano et al., 2007; Patel et al., 2004). Thus, down-regulation of insulin signaling leads to the dephosphorylation and consequent activation of GSK-3. GSK-3 phosphorylates tau at most sites in relation to tauopathies of AD (Mattson, 2001), which therefore could be the hinge of DM and AD. Based on these facts, insulin resistance or deficit could be a key factor that over-activates GSK-3 for tau hyperphosphorylation in DM patients. However, enhanced tau phosphorylation in rat brain was observed at conditions with either STZ-induced insulin deficiency or insulin administration-induced hyperinsulinemia in two different reports; Very interestingly, elevated blood glucose values were observed in both cases (Clodfelder-Miller et al., 2006; Freude et al., 2005). In mild cognitive impairment and AD patients, one fact that cannot neglect is the abnormal brain glucose utilization/transportation (Fellgiebel et al., 2004). Therefore, a possible casual relation between the glucose abnormality and tau phosphorylation may exist in AD. Jope group reported that Akt and GSK-3 were very sensitive to the circulating glucose level (Clodfelder-Miller et al., 2005). Moreover, in pancreatic islet -cells, glucose was found to be able to affect PP-2A activity, a powerful phosphatase that dephosphorylates many kinases and almost all tau sites reported to date (Arendt et al., 1998; Kowluru, 2005; Wang et al., 2007). Therefore, it is quite possible that abnormal alteration of glucose level in DM patients plays a role in promoting brain tau phosphorylation by its influence on the kinases/phosphatase

activities. Indeed it was reported that changes in the PP2A B regulatory subunit during insulin deficiency seemed to be related to hyperphosphorylation in STZ treated mice (Planel et al., 2007). To investigate if hyperglycemia affects brain tau phosphorylation via Akt/GSK3/PP-2A interplay, we embarked a long-term study (up to 30 days) with STZ-induced DM rat models. Phosphorylation of tau at multiple sites, accompanied by elevated GSK-3 activity and suppressed Akt/PP-2A actions, was detected in rat hippocampus in the current model. Insulin administration showed limited effects on the tau phosphorylation at PHF-1 sites in STZ-treated rats due to its simultaneous suppression of both GSK-3 and PP-2A activities. Therefore our study showed that STZ-induced diabetes interfered Akt/GSK-3/PP-2A activities and further contributed to abnormal phosphorylation of hippocampus tau.

2.
2.1.

Results
Persistent hyperglycemia in rats was induced by STZ

To determine if the hyperglycemia animal model was built successfully, the fasting blood glucose (FBG) of rats was measured. A sharp increase of FBG level to 24.563.40 mM in STZ group was observed from the third day, which is about 5 times over the control group (5.08 0.58 mM). Moreover, intra-peritoneal injection of STZ successfully induced a persistent hyperglycemia up to 30 days in rats (14.820.94 mM). As summarized in Table 1, both insulin and lithium administration showed some immediate effects of lowering blood glucose level in STZ-treated rats in 3 days, although the FBG readings were still much higher than the control group at that time. In 30 days, daily injection of 5 IU/mg insulin eventually restored FBG to normal level. Although Li2CO3 feeding helped to decrease blood glucose level, FBG still remained at relatively high level (9.240.83 mM). Meanwhile, the level of plasma insulin was quantified by radioimmunoassay in all groups. As expected, STZ caused dramatic insulin deficit in rats. High insulin concentration was detected in STZ+insulin group due to the exogenetic insulin administration.

2.2. Increased phosphorylation of tau at Tau-1 and PHF-1 sites in rats with STZ-induced hyperglycemia
After 30 days' treatments, the level of tau phosphorylation was investigated in rat hippocampus by Western-blot using

Table 1 Summary of four experimental groups. Groups Control group (Con)

Number of rats STZ injection Insulin injection Diet FBG 3 days (mM) FBG 30 days (mM) Plasma insulin (IU/L) 5 No No Normal diet 5.08 0.58 5.42 0.50 16.48 3.67

STZ group (STZ)

5 Yes No Normal diet 24.56 3.40 14.82 0.94 6.12 1.27

STZ + insulin group Ins

5 Yes Daily Normal diet 16.98 2.29# 5.60 0.50## 22.76 4.32##

STZ + Li2CO3 group (Li)

5 Yes No Li2CO3 mixed diet 21.02 2.11 9.24 0.83 7.92 1.56

STZ and FBG are abbreviations of streptozotocin and fasting blood glucose respectively. All results are presented as mean SD. **p < 0.01 vs control group; #p < 0.05 vs STZ group, ##p < 0.01 vs STZ group; p < 0.05 vs Ins group, p < 0.01 vs Ins group.

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phosphorylation-dependent and site-specific tau antibodies, Tau-1 and PHF-1. The immunoreactivity of Tau-1 epitopes (Ser198, Ser199 and Ser202) significantly decreased in rat hippocampus of STZ group, and that of PHF-1 sites (Ser396 and Ser404) increased accordingly (Fig. 1). Also shown in Fig. 1, under the conditions of STZ-induced DM, the phosphorylation level of tau at Ser396 and Ser404 remained constant despite of daily insulin administration. But significant de-phosphorylation at Tau-1 site was observed in this group. In contrast, both Tau-1 and PHF-1 sites of tau phosphorylation were reversed by lithium, a specific GSK-3 inhibitor. No significant change in the level of total tau (probed by 111e polyclonal antibody) was seen in all groups. These results suggest that high blood glucose may contribute to the hyperphosphorylation of tau in vivo at both circumstances of insulin deficiency or sufficiency and GSK-3 may play a role in this pathophysicological process.

2.3. Brain GSK-3 was up-regulated by dephosphorylation at Ser9; down-regulation of PP-2A also involved in tau phosphorylation in STZ-induced DM
To reveal the effects of abnormal changes of circulation glucose and insulin on GSK-3 and Akt, the two key kinases in insulin signaling pathway, enzyme activity assays for both were performed. Our results showed that brain GSK-3 activity was evaluated by 1.5 times over control, with a decrease of Akt activity (p < 0.01 vs control) in STZ group (Fig. 2a, c), which were in accord with the level of dephosphorylation of GSK-3 at Ser9 and Akt at Ser473 (Fig. 2b, d). Both lithium and insulin suppressed the GSK-3 activity in STZ-induced DM rats. However, they might have achieved this by adopting different mechanisms: lithium directly inhibited GSK-3, while insulin activated insulin-Akt signaling cascade to down-regulate GSK-3 (Fig. 2). The expression of total GSK-3 and Akt remained constant compared to the control group. Taken together, these results raised a big question why GSK-3 suppression nearly restored all tau hyperphosphorylation in lithium group but not in the insulin group. Therefore, GSK-3 could not be the sole enzyme that regulates tau phosphorylation in diabetic rats. And the actions of PP-2A, an important protein phosphatase, were thereby monitored in the current study. PP-2A could dephosphorylate almost all tau phosphorylation sites and decreased activity of PP-2A has been related to tauopathy in AD. In the present study, PP-2A activities were shown lower than the control in all other three groups, only differed in degree (Fig. 3).

3.

Discussion

The association between diabetes and high risks of developing AD is increasingly recognized, although the detailed mechanisms remained unclear. Mice models with either insulin deficit or high peripheral insulin level were reported to exhibit tau hyperphosphorylation at multiple sites in the brain. Hence, it is quite possible that other factors may also contribute to tauopathy of AD in DM patients. In the study, we showed that pathological increase of blood glucose could up-regulate tau phosphorylation at PHF-1 sites in rat brain. And imbalanced regulations of GSK-3 and PP-2A in pathological hyperglycemia could directly affect tau hyperphosphorylation.

Fig. 1 Changes of hippocampus tau phosphorylation at Tau-1 and PHF-1 sites. 30 days after treatments, tau phosphorylation at various sites was determined. The phosphorylation level of tau shown in the plots was standardized by total tau detected with 111e. (a) Tau phosphorylation at Tau-1 epitopes, **P < 0.01 vs control group, ## P < 0.01 vs STZ group. (b) Tau phosphorylation at PHF-1 epitopes, **p < 0.01 vs con group, p < 0.01 vs Ins group.

Hyperglycemia was reported to contribute to dementia of the Alzheimer type (DAT) (Craft et al., 1993; Hoyer et al., 1991). In hyperglycemia, excessive cerebral glucose could be converted to sorbitol and fructose. Increase of such metabolites in the brain alters the activities of some protein kinases, which in turn affects the phosphorylation of their target proteins including tau (Yagihashi et al., 2001). Tau protein is a substrate

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Fig. 2 Changes in activity and site-specific phosphorylation of GSK-3 and Akt. The activities of Akt (a) and GSK3 (c) in rat hippocampus extracts were measured by using specific peptide substrates. (b) and (d) showed the phosphorylation levels of specific sites of Akt and GSK3 respectively. No significant change in total Akt and GSK-3 was seen. **p < 0.01 vs control group; # p < 0.05 vs STZ group, ##p < 0.01 vs STZ group; p < 0.05 vs Ins group, p < 0.01 vs Ins group.

for some kinases, such as GSK-3. Abnormally phosphorylated tau is prone to aggregate as paired helical filaments (PHFs), which may chronically form neurofibrillary tangles that are the characteristics of AD (Benzing and Mufson, 1995). To investigate if hyperglycemia is related with tau hyperphosphorylation in vivo, here we employed STZ, a selective pancreatic -cells toxin, to generate diabetic hyperglycemia model. In our 30-day experiments, persistent high blood glucose was achieved, accompanied with elevated tau phosphorylation at multiple sites in rat hippocampus. Since STZ induced both hyperglycemia and low insulin in rat (Table 1), a further experiment with insulin administration was conducted to maintain high insulin level in

STZ-treated rats. It is observed that high tau phosphorylation at Ser396 and Ser404 still existed in this case, although no significant phosphorylation of tau at Ser198, Ser199 and Ser202. Therefore, these findings strongly suggest that hyperglycemia affects brain tau phosphorylation in STZ-induced diabetic rats. Important kinases such as GSK-3 and Akt were further investigated. GSK-3 is known to be the key enzyme that can phosphorylate tau at most sites that found in AD brain. Here, upregulated GSK-3 activity along with inhibition of Akt were detected in STZ-treated rats. Lithium specifically inhibits GSK-3 (Engel et al., 2006), according to our result, which might play its role directly instead of phosphorylation, and it

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Relative PP-2A activity(% of Con)
1.2 1 0.8 0.6

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**

was shown to have such regulatory functions that link insulin and PP-2A (Inoki et al., 2006; Meske et al., 2008). Further investigations are to be done to confirm the involvement of TSC/mTOR under these conditions.

4.
0.4 0.2 0 Con STZ Ins Li

Conclusion

Fig. 3 Changes of hippocampus PP-2A activity in different groups. The activity of PP-2A was assayed by using the rat hippocampus extracts among the four experimental groups. **p < 0.01 vs control group; p < 0.05 vs Ins group.

Chronic hyperglycemia in DM may break the equilibrium between GSK-3 and PP-2A and put them at high risk of developing AD due to the misled tau phosphorylation. Insulin regulations via insulin/Akt (and possibly TSC/mTOR) signaling cascade only generates partial protective effects of tau under the pathological conditions in the current rat model.

5.
5.1.

Experimental procedures
Antibodies and chemicals

showed dramatic effects of reversing tau phosphorylation at both Tau-1 and PHF-1 sites (Fig. 1). This provides a clue of the involvement of GSK-3 in this diabetic tau hyperphosphorylation rat model. Akt/GSK-3 are two essential kinases in insulin signaling cascade. Insulin deficiency leads to inactivation of Akt. Suppression of Akt results in up-regulation of GSK-3. Insulin administration, vice versa, activates Akt and thereby inhibits GSK-3 activity, as shown in Fig. 2. However, insulin administration did not prevent tau from phosphorylation at PHF-1 sites in STZ-induced hyperglycemia rats. Thus, GSK-3 could not be the only factor that affects tau phosphorylation in this case. A recent study reported that in normal neuron, the coupling of GSK-3 and PP-2A equilibrates the phosphorylation/dephosphorylation of tau and thereby helps prevent tau from aggregating to PHFs under some transient stress conditions (Meske et al., 2008). Since PP-2A dephosphorylates all tau sites reported to date, it could be of great importance to detect the activity of PP-2A in our rat models. In fact, GSK-3 alone could not phosphorylate tau at Ser262, a residue essential in inducing the self-assembly of tau to PHFs. Accordingly, only some specific kinase combinations, or GSK-3 with inhibited protein phosphatase-2A (PP-2A) could convert normal tau protein to the AD-like hyperphosphorylated status. Although we have not managed to get the right antibodies to detect tau phosphorylation at Ser262, down regulation of PP-2A was identified in both STZ group and STZ+ insulin group compared to the control, which was related to the phosphorylation level of tau in the insulin administration group (Fig. 3). Lithium did not show inhibitory effects on PP-2A in our experiments. Therefore, both up-regulated GSK-3 and downregulated PP-2A in STZ-induce hyperglycemia contribute to the brain tau hyperphosphorylation. On the other hand, the dual suppression of GSK-3 and PP-2A by insulin administration could be the reason why PHF-1 sites of tau remained at high level of phosphorylation. Insulin suppresses GSK-3 activity via insulin/ Akt signaling cascade. But how does it affect PP-2A, as observed in the current study? There might be a pathway of regulating PP-2A activity that also could be initiated by insulin. According to some recent studies, tuberous sclerosis complex (TSC)/mTOR pathway

Monoclonal antibodies to PHF-1 (labels phosphorylated tau at Ser396 and Ser404), and to Tau-1 (recognizes non-phosphorylated tau at Ser198, Ser199, or Ser202), were gifts from Dr. P. Davies (Albert Einstein College of Medicine, Bronx, NY) and Dr. L. Binder (Northwestern University, Chicago, IL) respectively, and 111e (recognizes total tau) was provided by Dr. Iqbal K and Dr. GrundkeIqbal I (New York State Institute for Basic Research, Staten Island, NY). Antibodies to phospho-Ser9-GSK/total GSK and phosphoSer473-Akt/total Akt were from Cell Signaling Technology (Beverly, MA, USA). Goat anti-rabbit peroxidase-conjugated secondary antibodies and phosphocellulose paper were from Pierce Chemical Co. (Rockford, IL). Alkaline phosphatase-labeled IgG and polyvinylidene difluoride membranes were from Amersham Pharmacia (NJ, USA). Phospho-GS peptide 2 (GSK-3 substrate) was obtained from Upstate Biotechnology Inc. (Lake Placid, NY). Bicinchoninic acid (BCA) protein detection kit and TMB were obtained from Pierce (Rockford, IL, USA). [-32P] ATP was bought from Yahui Biologic & Medicinal Engineering Co. (Beijing, China). Streptozotocin (STZ), diaminobenzidine, insulin and all other chemicals not specified were purchased from Sigma (St Louis, MO, USA).

5.2.

Animal and treatments

All the rats used in the current studies were treated in compliance with the Animal Experiments Guidelines and Animal Care of Chinese Academy of Sciences. 4-month-old SpragueDawley rats (Grade II, male, weight 200250 g) were supplied by Experimental Animal Center, Chinese Academy of Sciences, Shanghai. The rats were randomly divided into 4 groups: control group, streptozotocin (STZ) group, STZ plus insulin group, STZ plus Li2CO3 group. Except for the control group, all the other rats received intra-peritoneal injection with 55 mg/kg STZ. For STZ plus insulin group, 24 h after STZ injection, the rats were injected with zinc insulin (5 IU/kg) daily. STZ plus Li2CO3 group: the rats were fed with food with 0.4% Li2CO3 when STZ was injected. Blood glucose was monitored with sample from caudal vein every morning before feeding. All rats were sacrificed 30 days after treatments. Blood samples from rat left ventricle were kept for plasma insulin

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assays using radioimmunoassay kit from Beijing Nuclear Institute (China).

5.3.

Preparation of rat hippocampal extracts

The hippocampus was immediately removed and homogenized at 4 C using a Teflon glass homogenizer in 50 mM TrisHCl pH 7.4, 150 mM NaCl, 10 mM NaF, 1 mM Na3VO4, 10 mM -mercaptoethanol (-ME), 5 mM ethylenediaminetetraacetic acid (EDTA), 2 mM benzamidine, 1.0 mM phenylmethylsulfonyl fluoride (PMSF), 5 g/ml leupeptin, 5 g/ml aprotinin and 2 g/ml pepstatin. The tissue homogenates were then divided into two portions. One was centrifuged at 12,000 g for 20 min at 4 C, and the supernatant was stored at 80 C for enzyme activity assays. The other portion was mixed in 2:1 (v/v) ratio with lysis buffer containing 200 mM TrisHCl pH 7.6, 8% SDS, and 40% glycerol and boiled for 10 min in a water bath, then centrifuged at 12,000 g for 30 min. The supernatant was stored at 80 C for Western blot analysis. The concentration of protein in the hippocampal extracts was measured by BCA kit according to the manufacturer's instruction (Pierce, Cheshire, UK).

0.2 ng/l inhibitor 1 (PP-1 specific inhibitor), and 0.06 mg/ml hippocampal exacts. The reaction was started by addition of -32P phosphorylase-a. After incubation for 30 min at 30 C, the reaction mixture (5 l) was spotted on a chromatography paper already spotted with 10 l stop solution (4 mM cold ATP in 20% trichloroacetic acid, TCA). The released -32P was separated from the substrate by ascending chromatography in 5% TCA in 200 mM NaCl, and the radioactivity was counted by liquid scintillation counter.

5.5.

Western blot

5.4.

Enzyme activity assays

The GSK-3 activity in rat hippocampal exacts was measured using phospho-GS peptide 2 as described previously (Pei et al., 1997; Tsujio et al., 2000). Briefly, 7.5 g tissue extracts were incubated for 30 min at 30 C with 20 M peptide substrate and 200 M [-32P] ATP (1500 cpm/pmol ATP) in 30 mM Tris, pH 7.4, 10 mM MgCl2, 10 mM NaF, 1 mM Na3VO4, 2 mM ethylene glycol tetraacetic acid (EGTA), and 10 mM -ME in a total volume of 25 l. The reaction was stopped by addition of 25 l of 300 mM O-phosphoric acid. The reaction mixture was applied in triplicates on phosphocellulose paper (Pierce). The filter papers were washed three times with 75 mM O-phosphoric acid, dried, and counted by liquid scintillation counter (A-5082, TECAN, Austria). GSK-3 activities were calculated with picomoles of phosphate incorporated/mg protein/min at 30 C and expressed as relative activity against control. The Akt activity was measured using histone2B as a substrate as described previously (Jacob et al., 1999). Briefly, after the immunoprecipitates were washed with lysis buffer and kinase buffer, 40 l of kinase buffer contains 200 M [-32P] ATP (5 Ci), 100 M ATP, and 1 g/l histone2B, the samples were incubated at 30 C for 15 min, and spotted onto P81 filter papers, and the filter papers were washed by 75 mM O-phosphoric acid, dried, and counted by liquid scintillation counter. Akt activity was also expressed as relative activity against control. PP-2A activity assay was conducted according to Gong et al. (1993) and Mawal-Dewan et al. (1994). Phosphorylase-b (2 mg/ml) was phosphorylated into phosphorylase-a by incubating for 10 min at 30 C in 40 mM TrisHCl (pH 8.5), 20 mM -ME, 0.2 mM CaCl2, 15 mM MgCl2, and 0.5 mM -32P-ATP with 10 mg/ml phosphorylase kinase. The product of -32P-phosphorylase was separated from free ATP on a Sephadex G-50 column, and the protein-containing fractions were collected. The activity of PP-2A toward -32P-phosphorylase-a was assayed by the release of -32P as described previously. The reaction was carried out in a 20 l reaction mixture containing 50 mM Tris pH 7.0, 10 mM -ME, 0.1 mM EDTA, 7.5 mM caffeine, 7.5 ng/ml -32P phosphorylase-a,

The phosphorylation of tau was detected by Western blot. 20 g proteins were loaded in each lane. Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes. After a 1 h incubation in a solution of 5% nonfat dry milk in 1Tris-buffered saline (TBS), the membranes were immunoblotted using primary antibodies to PHF-1 (1:500), Tau-1 (1:30,000) and 111e (1:3000) at 4 C overnight. Afterwards, the reactions were developed with 0.5 g/ml secondary antibody alkaline phosphatase-labeled IgG. 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium (BCIP/NBT) was used as the substrate to visualize the reactions. The Phospho-GSK-3 and total GSK-3 were determined with antibodies to p-GSK-3 (1:1000) and total GSK-3 (1:1000) respectively. Detection of Akt was performed with antibodies to phospho-Ser473Akt and total Akt (dilution 1:800). The immunoblots were developed using horseradish peroxidase (HRP)-conjugated goat anti-rabbit or goat anti-rat IgG (1:1000) followed by detection with enhanced chemiluminescence. The intensity of the protein bands from Western blot were analyzed by 1D Image Analysis software (KODAK, USA).

5.6.

Statistical analysis

All experiments in the current study were repeated five times. The data were presented as mean SD and analyzed with SPSS 13.0 (Illinois, USA). One-way analysis of variance (ANOVA) and least significant differences (LSDs) post hoc tests were selected to determine the different means among groups.

Acknowledgments
Zhongsen Qu was supported by grants from China Postdoctoral Science Foundation (20060390635), and Zongxian Jiao was supported by the Fundamental Research Funds for the Central Universities (lzujbky-2009-92). We also thank Dr. Yanqiu Deng of Tianjin Medical University for useful discussion.

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