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ª Federation of European Neuroscience Societies European Journal of Neuroscience, Vol. 22, pp. 1081–1089, 2005 Brain-derived neurotrophic factor induces a rapid dephosphorylation of tau protein through a PI-3Kinase signalling mechanism Evan Elliott,* Roee Atlas,* Aya Lange and Irith Ginzburg Department of Neurobiology, Weizmann Institute of Science, Rehovot 76100, Israel Keywords: Alzheimer’s disease, GSK3b, mouse, P19 cells, TrkB. Abstract The microtubule-associated protein tau is essential for microtubule stabilization in neuronal axons. Hyperphosphorylation and intracellular fibrillar formation of tau protein is a pathology found in Alzheimer’s disease (AD) brains, and in a variety of neurodegenerative disorders referred to as ‘taupathies’. In the present study, we investigated how brain-derived neurotrophic factor (BDNF), an extracellular factor that is down-regulated in AD brains, affects tau phosphorylation. BDNF stimulation of neuronally differentiated P19 mouse embryonic carcinoma cells resulted in a rapid decrease in tau phosphorylation, at phosphorylation sites recognized by Tau1, AT8, AT180 and p262-Tau antibodies. K252a, a tyrosine receptor kinase (Trk) inhibitor, attenuated this dephosphorylation event, suggesting that BNDF activation of TrkB is responsible for the tau dephosphorylation. In addition, BDNF had no affect on tau phosphorylation in the presence of wortmannin, a PI-3Kinase inhibitor, or lithium, a GSK3b inhibitor, suggesting that these two kinases are part of the signaling transduction cascade leading from TrkB receptor activation to tau dephosphorylation. These results suggest a link between a correlate of AD, decrease in BDNF levels and an AD pathology, tau hyperphosphorylation. Introduction Tau protein is a microtubule-associated protein, which is highly expressed in neurons of the central nervous system. Tau is enriched in axons, where it directly binds to, and stabilizes, microtubules, through four microtubule binding domains located at the C-terminus of the tau protein (Buee et al., 2000). In Alzheimer’s disease (AD), tau protein is found hyperphosphorylated at a variety of serine and threonine residues (Johnson & Hartigan, 1999). Hyperphosphorylation of tau protein has been shown to deter its ability to bind to, and stabilize, microtubules, which serve as the cytoskeletal backbone of the axon (Gustke et al., 1992; Cho & Johnson, 2004; Li et al., 2004). In addition, hyperphosphorylated tau selfassembles into paired helical filaments (Alonso et al., 2001), which are deposited inside the neuron (Grundke-Iqbal et al., 1986). As a result of these changes in tau structure and function, axonal length and function may be seriously reduced, promoting the neuronal degeneration that occurs in AD. An aim in AD research is to discover the mechanisms involved in regulation of tau phosphorylation. Various protein kinases, including GSK3b (Mandelkow et al., 1992) and cdk5 (Maccioni et al., 2001), have been shown to phosphorylate tau protein. Overexpression or overactivation of GSK3b in cellular and animal models induced tau hyperphosphorylation similar to that seen in AD (Lucas et al., 2001; Lee et al., 2003; Liu et al., 2003). Correspondence: Dr I. Ginzburg, as above. E-mail: [email protected] *E.E. and R.A. contributed equally to this work. Received 28 March 2005, revised 6 June 2005, accepted 28 June 2005 doi:10.1111/j.1460-9568.2005.04290.x Therefore, deregulation of GSK3b may be involved in tau hyperphosphorylation in AD patients. In addition, phosphatases, such as PP2A, have been shown to dephosphorylate tau in vivo (Gong et al., 2000; Tian & Wang, 2002). Changes in the balance of activities between tau kinases, such as GSK3b, and phosphatases, such as PP2A, may lead to tau hyperphosphorylation and neuronal degeneration. Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family of proteins, promotes neuronal survival, neurite outgrowth and synaptic plasticity through its interactions with the TrkB and p75 cellular receptors (Patapoutian & Reichardt, 2001; Poo, 2001). BDNF activation of TrkB receptor activates various signaling pathways, including Ras ⁄ MEK, PI-3K ⁄ AKT, PLCc ⁄ PKC and PKA signaling pathways (Patapoutian & Reichardt, 2001; Gallo et al., 2002). It has been shown that BDNF mRNA levels in the hippocampal formation are selectively reduced in AD patients (Phillips et al., 1991). Later research confirmed that levels of BDNF protein are reduced in the hippocampus and other cortical areas. These are areas that undergo considerable degeneration in AD brains (Murer et al., 2001). This research has raised interest in the idea of using BDNF-based therapies in AD patients to promote neuronal survival in these brain regions (Fahnestock et al., 2002). However, no previous research has shown how BDNF activity may be related to the pathologies of AD, particularly tau hyperphosphorylation. Therefore, we decided to study the possible relationship between BDNF and tau phosphorylation. Our study shows that BDNF stimulation of neuronal cells induces dephosporylation of tau protein at specific phosphorylation sites, through TrkB receptor activation, and PI-3Kinase-mediated signaling. 1082 E. Elliott et al. Materials and methods Pharmacological treatments Cell culture Differentiated P19 cells (Day 8) were starved in serum-free Dulbecco’s modified Eagle’s medium (Life Technologies, Inc., Rockville, MD, USA) for 3 h, then pharmacological agents were used at the following concentrations for the stated times of pre-incubation before BDNF incubation: cycloheximide (Sigma; 50 lg ⁄ mL for 30 min), K252a (Alomone; 200 nm for 25 min), PD89059 (50 lm for 30 min), wortmannin (Alomone; 100 nm for 20 min), H89 (Calbiochem, La Jolla, CA, USA; 1 lm for 30 min), GF109203X (kindly provided by Prof. M. Segel; 500 nm for 15 min) and LiCl (20 mm for 10 min). P19 mouse embryonic carcinoma cells were grown in minimal essential medium (MEM, Beit-Haemek, Israel), supplemented with 5% heat-inactivated fetal calf serum in a 5% CO2 incubator at 37 C. Cells were induced toward neuronal differentiation by addition of 1 lm retinoic acid as previously described (Aronov et al., 2001). RT-PCR analysis RNA was isolated from P19 cells with Tri-Reagent (Sigma, St Louis, MO, USA), according to the manufacturer’s instructions. Then 2.5 lg RNA, for each sample, was reverse transcribed using Rtaid reverse transcriptase (MBI fermentas, Amherst, NY, USA) in a 20-lL reaction for cDNA synthesis. One microlitre from the cDNA reaction was used for PCR amplification in a 25-lL reaction with specific primers. PCR reaction with BNDF primers was carried out for: 1 min at 94 C (melting), 1 min at 61 C (annealing) and 1 min at 72 C (elongation). PCR reaction with GAPDH primers were carried out for: 1 min at 94 C (melting), 1 min at 56 C (annealing) and 1 min at 72 C (elongation). The primers used were: for TrkB, 5¢-GACCTGATCCTGACG GGTAA-3¢ and 5¢-ACAGTGAATGGAATGCACCA-3¢, which yields a fragment of 497 bp; and for GAPDH, 5¢-GCCATCAACGA CCCCTTCAT-3¢ and 5¢-TCCACACCCATCACAAACAT-3¢, which yields a fragment of 521 bp. Products were separated on a 1% agarose gel and stained with ethidium bromide. Immunoblot analysis of P19 cell extracts Differentiated P19 cells (Day 8) were starved in serum-free medium for 3 h, then incubated with or without 50 ng ⁄ mL BDNF (Alomone, Jerusalem, Israel) for 15 min (unless otherwise indicated) in 5% CO2, 37 C incubator. Cells were then washed in cold PBS buffer, and incubated for 10 min in lysis buffer [50 mm b-glycerophosphate, 1 mm EDTA, 1.5 mm EGTA, 0.1 mm orthovanadate (pH ¼ 7.3), 140 mm KCl, 1% NP40, 1% glycerol, 0.1 mm benzamidine, 10 lg ⁄ mL aprotinin, 10 lg ⁄ mL leupeptin, 5 lg ⁄ mL pepstatin, 1 mm PMSF, 1 mm DTT, 20 mm NaF]. Cleared cell extracts were obtained by centrifugation for 10 min at 14 000 g at 4 C. Protein concentration of samples was determined using Bradford Reagent. Equal amounts of protein (10–15 lg) were run on 10% SDS-PAGE, and transferred to nitrocellulose membranes. The membranes were blocked in non-fat milk (5% milk in TBS, 0.05% Tween) for 2 h, incubated with primary antibody overnight at 4 C, and washed three times for 10 min each with TBS (0.5% Tween). Membranes were then incubated with HRP-conjugated second antibody, and washed as described above. Membranes were then developed with enhanced chemiluminescence. Primary antibodies used included Tau1, Tau5, AT8 (Innogenetics, Gent, Belgium), AT180 (Innogenetics), AT270 (Innogenetics), p262 (Sigma), GAPDH (Ambion, Austin, Texas, USA), anti-cadherin antibody CH-19 (Sigma), pERK and total ERK (kindly provided by Prof. R. Seger, Weizmann Institute of Science, Israel), pGSK3b (kindly provided by Prof. G. Agam, Ben Gurion University, Israel), total GSK3b (kindly provided by Prof. H. EldarFinkelman, Tel-Aviv, University, Israel) and pAKT (kindly provided by Prof. M. Liskovitch). Immunoblots were quantitated using the NIH imager program. Results BDNF treatment induces tau dephosphorylation BDNF stimulation of neurons activates many intracellular signaling cascades, including cascades that are considered important for regulation of tau phosphorylation. To determine if BDNF stimulation can affect the state of tau phosphorylation in neurons, we utilized neuronally differentiated P19 embryonic carcinoma cells, which express tau protein and both BDNF receptors (Burke & Bothwell, 2003; Atlas et al., 2004). P19 cells were differentiated into neuronal cells (P19 neurons) with retinoic acid, followed by incubation with BDNF for 15 min. This length of the treatment with BDNF was chosen because it has been previously shown that BDNF-induced phosphorylation and dephosphorylation events occur within this time frame (Mai et al., 2002; Tong et al., 2004). Western blot analysis, using phosphorylation-sensitive tau antibodies, was performed on the treated cell extract. Phosphorylation-sensitive antibodies used include Tau1 (dephospho-195 ⁄ 198 ⁄ 202 tau), AT8 (phospho-202 ⁄ 205 tau), AT180 (phospho-231 tau), AT270 (phospho-181 tau) and an antibody against tau phosphorylated at Ser262. This information is summarized in Fig. 1A. Figure 1B and C shows that BDNF treatment induces dephosphorylation of tau protein at all the phosphorylation sites examined, except for the site recognized by AT270. Because the antibody Tau1 recognizes a non-phosphorylated tau epitope, Tau1 levels increased upon stimulation with BDNF. Detection of GAPDH, a commonly used housekeeping gene, was performed on the same blot to verify equal loading. To verify that tau phosphorylation, and not synthesis, was affected by BDNF stimulation, blots were analysed with Tau5, a phosphorylation-independent antibody that recognizes total tau. The results demonstrate that tau protein levels were unchanged following BDNF treatment, as tested by the Tau5 antibody. To verify further that the observed increase in Tau1 reactivity is not a result of increased synthesis of tau protein, we pretreated P19 neurons with cycloheximide, a translation inhibitor, followed by treatment with BDNF. As shown in Fig. 1D, Tau1 reactivity increased in response to BDNF treatment, further indicating that BDNF stimulation is affecting the phosphorylation levels of tau protein, and not translation. To test the kinetics of the effect of BDNF on tau phosphorylation, cells were treated with BDNF for variable time periods. Figure 1E shows that the maximal effect is observed after 15 min of treatment. Therefore, we continued performing our experiments at this time point. These results demonstrate that BDNF stimulation of P19 neurons induces an immediate, transient dephosphorylation of tau protein. We concentrated the rest of our studies on the affect of BDNF on the phosphorylation sites recognized by Tau1 and AT8 because we observed the strongest effect with these antibodies. It has been previously demonstrated that P19 cells develop a neuronal morphology during the differentiation process, and differentiate into mature neurons in culture (Bain et al., 1994). To differentiate ª 2005 Federation of European Neuroscience Societies, European Journal of Neuroscience, 22, 1081–1089 BDNF stimulation induces tau dephosphorylation 1083 Fig. 1. Stimulation of P19 neurons with BDNF induces dephosphorylation of tau protein. (A) Schematic representation of tau protein and the epitopes that the phosphorylation-dependent tau antibodies recognize. (B) P19 neurons were stimulated for 15 min with BDNF (50 ng ⁄ mL). 10–15 lg Cell extract was separated by SDS-PAGE and analysed by Western blot with Tau1 (dephospho-195 ⁄ 198 ⁄ 202 tau), AT8 (p202 ⁄ p205 tau), AT180 (p231 tau), AT270 (p181 tau), p262, Tau5 (total tau protein) and GAPDH antibodies. BDNF stimulation induced a decrease in band intensities of the AT8, AT180 and p262 antibodies, correlating with a decrease in phosphorylation at these epitopes. Tau1 recognizes unphosphorylated tau, and therefore the increase in Tau1 band intensity represents a decrease in tau phosphorylation at the Tau1 epitope. Two lanes are shown for each stimulus, representing two experiments. (C) Quantitative analysis of A, using NIH imager. Results from each antibody were normalized against level of GAPDH signal detected. For Tau1, n ¼ 9; for AT8, n ¼ 7; for Tau5, n ¼ 4 (*P < 0.05, unpaired Student’s t-test). (D) P19 neurons were pretreated with cycloheximide (50 lg), stimulated with BDNF and then lysed. Extract was separated by SDS-PAGE, and analysed by Western blot with Tau1 and GAPDH antibodies. Bands were quantitated using NIH imager (n ¼ 4). Tau1 immunoreactivity was normalized against level of GAPDH signal detected. BDNF induces a rise in Tau1 levels, even in the presence of cycloheximide (*P < 0.05, unpaired Student’s t-test). (E) P19 neurons were stimulated with BDNF for differing time periods. Extract was examined by Western blot with Tau1, AT8, and Tau5 antibodies, and band intensities were quantified using NIH imager (n ¼ 3). Maximal Tau1 immunoreactivity was observed following stimulation for 15 min. P19 cells into neurons, the cells are initially aggregated on nonadherent plates for 4 days in the presence of retinoic acid (1 lm), followed by disaggregation, and plated on adherent plates in medium lacking retinoic acid. It has previously been reported that tau protein is expressed from Day 4 (day of plating), and continues to be expressed throughout differentiation, peaking at Day 8 (Atlas et al., 2004). Our original experiments, shown in Fig. 1, were carried out on Day 8 of neuronal differentiation. We followed these experiments by examining the effect of BDNF stimulation on tau phosphorylation at different time points of P19 neuronal differentiation. P19 neurons were stimulated with BDNF at Days 5, 6 or 8 of differentiation. As can be seen in Fig. 2, there is a significant increase in the BDNF effect on tau phosphorylation between Days 5 and 6 of differentiation. The effect on tau phosphorylation remains the same between Days 6 and 8. Therefore, all the cellular machinery necessary for BDNF stimulation to dephosphorylate tau is fully functional by Day 6 of P19 neuronal differentiation, when P19 neurons acquire extended neurites. BDNF activation of TrkB receptor dephosphorylates tau protein BDNF-dependent signaling is mediated by the TrkB and p75 receptors. Both receptors have been shown to be present in P19 neurons (Burke & Bothwell, 2003). To determine TrkB expression in differentiated P19 neurons, RT-PCR analysis was performed on RNA isolated from differentiated P19 cells. TrkB expression increased continuously from Day 2 to Day 8 of differentiation (Fig. 3C). In order to determine if tau phosphorylation state is mediated through the TrkB receptor, K252a, a selective inhibitor of Trk receptors (Koizumi et al., ª 2005 Federation of European Neuroscience Societies, European Journal of Neuroscience, 22, 1081–1089 1084 E. Elliott et al. Fig. 2. BDNF has differential effects on tau phosphorylation levels during P19 neuronal differentiation. (A) Neuronally differentiated P19 cells from Day 5, 6 or 8 of differentiation were stimulated with BDNF for 15 min. Cell extracts were separated on 10% SDS-PAGE and Western blot analysis was performed using Tau1 and Tau5 antibodies. (B) Quantitative analysis, from four experiments (n ¼ 4), represented as mean ± SEM. Tau1 immunoreactivity was normalized against level of Tau5 signal detected (*P < 0.05, unpaired Student’s t-test). 1988; Tapley et al., 1992), was employed. After incubation with K252a, P19 neurons were stimulated with BDNF, and tau phosphorylation was verified with the appropriate antibodies. As shown in Fig. 3A and B, BDNF stimulation, in the presence of K252a, had no significant effect on levels of tau phosphorylation, as detected by AT8 and Tau1 antibodies. Therefore, we conclude that the effect of BDNF on tau phosphorylation is dependent on functional TrkB receptor signaling. Roles of PI-3Kinase, AKT and GSK3b in tau dephosphorylation BDNF stimulation of TrkB has previously been shown to activate various signal transduction cascades, including MEK ⁄ ERK, PI-3K ⁄ AKT, PKC and PKA pathways (Patapoutian & Reichardt, 2001; Gallo et al., 2002). Each of these pathways has been independently shown to affect tau phosphorylation levels. In order to distinguish which signaling pathway is responsible for the influence of TrkB receptor on tau phosphorylation, we treated the cells with inhibitors and ⁄ or activators that specifically affect the tested pathways. Figure 4A shows that BDNF stimulation induces phosphorylation of the ERK kinase in P19 neurons, and that this phosphorylation is inhibited by pre-incubation with the MEK inhibitor PD98059 (Dudley et al., 1995). The MEK inhibitor was subsequently used to test if the influence of BDNF on tau phosphorylation is reliant on the MEK ⁄ ERK pathway. P19 neurons were pretreated with PD89059, followed by incubation with, or without, BDNF. PD98059 treatment alone caused a small decrease in tau phosphorylation (Fig. 4B and C), which suggests that the MEK ⁄ ERK pathway is involved in promoting tau protein phosphorylation. Cell extracts of P19 neurons treated with PD98059 and BDNF exhibited a decrease in immunoreactivity to AT8 antibody, and an increase in reactivity to Tau1 antibody, as compared with those that were not treated with BDNF (Fig. 4B and C). As can be seen, the ERK inhibitor did not inhibit the effect of BDNF Fig. 3. BDNF induces tau dephosphorylation through the activation of the TrkB receptor. (A) P19 neurons were incubated with or without 200 nm K252a (TrkB inhibitor), followed by incubation with or without BDNF. Cells were lysed and Western blot analysis was performed with Tau1, AT8 and Tau5 antibodies. Two lanes are shown for each stimulus. (B) Quantitative analysis, from four experiments (n ¼ 4), represented as mean ± SEM. Tau1 and AT8 immunoreactivity was normalized against level of Tau5 signal detected. BDNF stimulation of P19 neurons pretreated with K252a, a TrkB inhibitor, did not significantly affect tau phosphorylation (*P < 0.05, unpaired Student’s t-test). (C) RT-PCR analysis of RNA extract from P19 cells in different stages of neuronal differentiation. TrkB-specific and GAPDH-specific primers were used. TrkB mRNA levels increased significantly throughout neuronal differentiation. stimulation on tau protein phosphorylation. Therefore, the observed change in tau phosphorylation, upon BDNF stimulation, is independent of the MEK ⁄ ERK pathway. We next investigated the role of the PI-3K ⁄ AKT pathway in the effect of the TrkB receptor on tau phosphorylation. TrkB receptor is known to activate PI-3Kinase, which results in the phosphorylation, and activation, of the kinase AKT (Patapoutian & Reichardt, 2001). Independent studies have already demonstrated that PI-3 Kinase ⁄ AKT signaling can regulate tau phosphorylation (Liu et al., 2003; Baki et al., 2004). In the current study, we show that BDNF treatment of P19 neurons induces phosphorylation of AKT at its site of activation, as would be expected (Fig. 4D). This phosphorylation was prevented when neurons were pretreated with the PI-3Kinase inhibitor wortmannin. Cell extracts prepared from P19 neurons pretreated with wortmannin and stimulated with BDNF were analysed with antibodies for tau. Both Tau1 and AT8 immunoreactivities were not significantly changed by stimulation with BDNF in comparison with control, in cells that were pretreated with wortmannin (Fig. 4E and F). Therefore, ª 2005 Federation of European Neuroscience Societies, European Journal of Neuroscience, 22, 1081–1089 BDNF stimulation induces tau dephosphorylation 1085 Fig. 4. PI-3K ⁄ AKT signaling, and not MEK signaling, is essential for BDNF to affect tau phosphorylation. (A) P19 neurons were pre-incubated with 50 lm PD98059 (MEK inhibitor), then incubated with or without BDNF. Cell extract was separated on SDS-PAGE and analysed by Western blot with antibodies against pERK and total ERK. (B) Cell extract from A was analysed with Tau1 and AT8 antibodies. BDNF stimulation decreased tau phosphorylation even in the presence of MEK inhibitor. (C) Quantitative analysis, from four experiments (n ¼ 4), represented as mean ± SEM. Tau1 and AT8 immunoreactivity was normalized against level of Tau5 signal detected (*P < 0.05, unpaired Student’s t-test). (D) P19 neurons were incubated with or without 100 nm wortmannin (PI-3K ⁄ AKT inhibitor), followed by incubation with or without BDNF. Cell extract was separated by SDS-PAGE and analysed by Western blot with pAKT and GAPDH antibodies. BDNF stimulation increased pAKT immunoreactivity in cells not treated with wortmannin. Treatment with Wortmannin inhibited increase in pAKT immunoreactivity upon BDNF treatment. Two lanes are shown per stimili with BDNF. (E) Cell extract from experiment in C was analysed by Western blot with Tau1, AT8 and Tau5 antibodies. BDNF stimulation did not affect tau phosphorylation in those cells that were pretreated with wortmannin. (F) Quantitative analysis, from four experiments (n ¼ 4), represented as mean ± SEM. Tau1 and AT8 immunoreactivity was normalized against level of Tau5 signal detected (*P < 0.05, unpaired Student’s t-test). the influence of BDNF stimulation on tau phosphorylation levels is dependent on the PI-3K ⁄ AKT signal transduction pathway. To support further our conclusion that the PI3-K ⁄ AKT signal transduction pathway is responsible for the influence of TrkB on tau phosphorylation, we examined the possible effects of two major signal transduction pathways, namely the PKA and PKC pathways. To determine if PKA can be part of the signal transduction pathway leading to tau dephosphorylation through TrkB activation by BDNF, P19 neurons were incubated with H89, a PKA inhibitor (Chijiwa et al., 1990). PKA inhibition had no significant effect on tau phosphorylation at Tau1 and AT8 epitopes (Fig. 5A and B). In addition, we examined the effects of PKA inhibition on the Ser262 phospho-epitope, as this is a preferred site for PKA phosphorylation. PKA inhibition induced a small dephosphorylation at Ser262, which was much less pronounced than the dephosphorylation induced by BDNF signaling. Moreover, BDNF induced a strong dephosphorylation of tau protein at all epitopes tested even when the cells were preincubated with PKA inhibitor (Fig. 5A and B). Therefore, neither PKA activation nor inactivation is responsible for tau dephosphorylation of AT8, Tau1 and p262 phosphorylation epitopes upon BDNF stimulation. Extensive research has shown that BDNF activates the PKC signal transduction pathway (Zirrgiebel et al., 1995; Patapoutian & Reichardt, 2001). To examine the role of PKC activation in tau phosphorylation, GF109203X, a potent inhibitor of PKC, was used. BDNF stimulation of P19 neurons induced dephosphorylation of tau at the AT8 and Tau1 epitopes in cells that were incubated with, or without, the PKC inhibitor (Fig. 5C and D). Total tau levels, as measured by the Tau5 antibody, were not affected by this treatment. Therefore, the BDNFinduced tau dephosphorylation is independent of PKC activity. From these experiments, we conclude that stimulation of P19 neurons with BDNF dephosphorylates tau at the AT8 and Tau1 epitopes solely through the activation of the PI-3K ⁄ AKT pathway by TrkB receptor activation. The PI3-K ⁄ AKT signal transduction pathway has known downstream effectors that regulate cell survival and proliferation (Brazil et al., 2004). One of these effectors is the GSK3b serine ⁄ threonine kinase, which can be inhibited by AKT-mediated phosphorylation on Ser9 (Cross et al., 1995). GSK3b is a major tau kinase, which phosphorylates tau protein at various sites, including at the Tau1, AT8, AT180 and p262 epitopes (Munoz-Montano et al., 1997; Wang et al., 1998; Lee et al., 2003). Previous work has also shown that BDNF stimulation of cells results in the inhibition of GSK3b through phosphorylation on Ser9 (Mai et al., 2002). In light of these data, we tested whether tau dephosphorylation caused by BDNF treatment may be mediated by GSK3b inhibition. We demonstrate, in Fig. 6A, that ª 2005 Federation of European Neuroscience Societies, European Journal of Neuroscience, 22, 1081–1089 1086 E. Elliott et al. Fig. 5. PKA and PKC signaling are not essential for BDNF to affect tau phosphorylation. (A) P19 neurons were incubated with 1 lm H89 (PKA inhibitor), and then stimulated with BDNF. Cell extract was separated by SDS-PAGE and analysed by Western blot with Tau1, AT8, p262 and Tau5 antibodies. BDNF stimulation decreased tau phosphorylation even in the presence in PKA inhibitor. (B) Quantitative analysis, from four experiments (n ¼ 4), represented as mean ± SEM. Tau1 and AT8 immunoreactivity was normalized against level of Tau5 signal detected (*P < 0.05, unpaired Student’s t-test). (C) P19 neurons were incubated with GF109203X (PKC inhibitor), and then stimulated with BDNF. Cell extract was separated by SDS-PAGE and analysed by Western blot with Tau1, AT8 and Tau5 antibodies. BDNF dephosphorylates tau even in the presence of PKC inhibitor. (D) Quantitative analysis, from three experiments (n ¼ 3), represented as mean ± SEM. Tau1 and AT8 immunoreactivity was normalized against level of Tau5 signal detected (*P < 0.05, unpaired Student’s t-test). Fig. 6. GSK3b is an important regulator of Tau phosphorylation in P19 neurons. (A) P19 neurons were stimulated with BDNF in the presence, or absence, of PI-3Kinase inhibitor wortmannin. Cell extract was separated by SDS-PAGE and analysed with Western blot with antibodies against pGSK3b and total GSK3b. (B) P19 neurons were incubated with LiCl (GSK3b inhibitor) for a variety of times and concentrations, and then lysed. Cell extract was separated with SDS-PAGE, and analysed by Western blot with Tau1 and Tau5 antibodies. GSK3b inhibition induced tau dephosphorylation in a time-dependent manner. Two lanes are shown per stimili with lithium. (C) P19 neurons were incubated with 10 mm LiCl for 5 min, and then stimulated with BDNF for 15 min. Cell extract was separated with SDS-PAGE, and analysed by Western blot with Tau1, AT8 and Tau5 antibodies. BDNF stimulation did not affect tau phosphorylation in those cells where GSK3b was previously inhibited. (D and E) Quantitative analysis, from four experiments (n ¼ 4), represented as mean ± SEM. Tau1 and AT8 immunoreactivity was normalized against the level of Tau5 signal detected (*P < 0.05, unpaired Student’s t-test). ª 2005 Federation of European Neuroscience Societies, European Journal of Neuroscience, 22, 1081–1089 BDNF stimulation induces tau dephosphorylation 1087 Fig. 7. BDNF does not up-regulate presenilin function. P19 neurons were incubated with BDNF for 15 min, and then lysed. Extract was separated by SDS-PAGE and analysed by Western blot with antibody against N-cadherin. Levels of CTF-N-cadherin are not affected by BDNF stimulation. Two independent experiments are shown. BDNF stimulation induces phosphorylation at Ser9, and therefore inhibition, of GSK3b in P19 neurons. In addition, pretreatment of P19 neurons with wortmannin decreased the basal level of GSK3b phosphorylation, and prevented the BDNF-induced phosphorylation of GSK3b. Therefore, PI-3Kinase is a key regulator of GSK3b phosphorylation, and mediates the BDNF-induced phosphorylation of GSK3b. To determine if GSK3b inhibition can induce tau phosphorylation, we inhibited GSK3b with lithium. Lithium is extensively used as a GSK3b inhibitor in both animals and cell culture (MunozMontano et al., 1997; Stambolic et al., 1997). We incubated P19 neurons with lithium for various periods of time, and found that GSK3b inhibition results in a strong increase in Tau1 immunoreactivity, and decrease in AT8 reactivity, corresponding to tau dephosphorylation (Fig. 6). Therefore, GSK3b inhibition is an effective method to induce tau dephosphorylation in P19 neurons. When we incubated P19 neurons with lithium for 5 min, and with ⁄ without BDNF for an additional 15 min, we found no difference in Tau1 or AT8 reactivity between those neurons that were given BDNF and those that were not treated (Fig. 6B and C). Therefore, BDNF stimulation is unable to decrease tau phosphorylation levels when GSK3b activity is previ- ously inhibited. These data suggest that tau dephosphorylation observed after BDNF stimulation may be due to AKT-mediated inhibition of GSK3b kinase activity. Recent research has shown a role for presenilin, a transmembrane protein that is mutated in many cases of familiar AD, in tau phosphorylation. Presenilin can induce tau dephosphorylation through GSK3b inhibition by PI-3Kinase signaling (Baki et al., 2004). As this is the same mechanism through which BDNF may dephosphorylate tau, we examined whether BDNF-induced tau dephosphorylation may be mediated through an up-regulation of presenilin. An established method to detect up-regulation of presenilin is to test for changes in the level of cytoplasmic fragment N-cadherin (CTF-N-cad) (Baki et al., 2004). Presenilin, a constituent of the c-Secretase, is necessary for the proteolysis and degradation of CTF-N-cad, which is a 40-kDa fragment of N-cadherin. Therefore, if BDNF up-regulates presenilin, we would expect to detect lower CTF-N-cad levels in the cell. As seen in Fig. 7, there was no change in CTF-N-cad levels in P19 neurons after stimulation with BDNF. We therefore conclude that BDNF does not induce an immediate up-regulation of presenilin, and BDNF-mediated tau dephosphorylation is independent of presenilin activity. Discussion In the present study, we examined the effects of BDNF stimulation on tau phosphorylation in P19 neuronal cells. P19 neurons are an appropriate cellular model for the investigation of these effects because they express both BNDF receptors, TrkB and p75 (Burke & Bothwell, 2003), and they develop distinct axons, where tau protein is enriched (Aronov et al., 2001). BDNF stimulation of P19 neurons induced an immediate dephosphorylation of tau protein at sites recognized by the phosphorylation-dependent antibodies Tau1, AT8, AT180 and p262 (Fig. 1). Phosphorylation at the epitope recognized Fig. 8. Proposed model of the effect of BDNF on tau phosphorylation levels. BDNF activation of TrkB receptor leads to PI-3Kinase activation, and subsequent activation of AKT. Activated AKT phosphorylates, and inhibits, GSK3b. Inhibition of GSK3b induces dephosphorylation of tau due to the loss of activity of this tau kinase. This mechanism is a very rapid form of tau protein regulation at critical phases of neuronal differentiation. ª 2005 Federation of European Neuroscience Societies, European Journal of Neuroscience, 22, 1081–1089 1088 E. Elliott et al. by AT270 (Thr181), was not affected by BDNF treatment. This phoshorylation site is differentially regulated from the other sites tested (Wang et al., 1998), which may explain the difference in the BDNF effect. The intensity of dephosphorylation at the Tau1 epitope increased upon maturation of the differentiating neurons, from Day 5 to Day 6 of differentiation (Fig. 2). This most likely reflects the increase in the neuronal phenotype of P19 neurons during this time period (Atlas et al., 2004). A Trk receptor inhibitor, K252a, attenuated the effect of BDNF stimulation on tau phosphorylation (Fig. 3). This indicates that BDNF induces tau dephosphorylation through activating the TrkB receptor. Wortmannin, a PI-3Kinase inhibitor, also attenuated the effect of BDNF stimulation on tau phosphorylation, indicating that BDNFinduced tau dephosphorylation at the AT8 and Tau1 epitopes is dependent on PI-3Kinase signaling (Fig. 4). Inhibitors of other signaling pathways that TrkB receptor kinase influences, including the MEK ⁄ ERK, PKA and PLC ⁄ PKC pathways, did not attenuate the ability of BDNF to affect tau phosphorylation (Figs 4 and 5). An additional observation from these experiments is that inhibition of MEK by PD98059 induced dephosphorylation of tau protein. This finding is consistent with earlier data, which suggest that ERK may influence tau phosphorylation. ERK has been shown to phosphorylate tau in vitro at the site recognized by Tau1 and AT8 (Drewes et al., 1992; Illenberger et al., 1998). Our results suggest that endogenous ERK may have an effect on tau phosphorylation. These findings shed light on the mechanisms of tau phosphorylation in vivo. The relatively flexible secondary structure of tau protein makes it an easy target for many cellular kinases. In-vitro studies have demonstrated that at least 17 different kinases are involved in tau phosphorylation (Johnson & Hartigan, 1999). Despite the mass of data on possible tau kinases, there is still much ambiguity with regard to which of these kinases is directly affecting tau phosphorylation in physiological processes. Some reports have worked on implicated PI-3Kinase, MAP Kinase and PKC as regulators of tau phosphorylation in physiological processes (Ferreira et al., 1997; Baki et al., 2004). Although all of these kinases may have roles in regulation of tau phosphorylation, our data highlight the role of PI-3Kinase by showing that a physiologically relevant ligand, BDNF, can regulate tau dephosphorylation through PI-3Kinase signaling. In this study, we demonstrate that inhibition of GSK3b, a major tau kinase, by BDNF is induced by PI-3Kinase signaling (Fig. 6A). This is the same signaling pathway that is responsible for the effects of BDNF on tau phosphorylation. In vivo inhibition of GSK3b, with lithium, resulted in tau dephosphorylation (Fig. 6). Therefore, inhibition of GSK3b is an adequate mechanism to induce dephosphorylation of tau protein. Subsequent treatment with BDNF did not induce further tau dephosphorylation. This series of data suggests that the BDNF-induced dephosphorylation of tau protein is due to inhibition of GSK3b by PI-3Kinase signaling. In addition, our results demonstrated that BDNF has no affect on presenilin activity, thus ruling out a role for presenilin in BDNF-induced tau dephosphorylation (Fig. 7). Figure 8 represents a model suggesting how BDNF simulation of P19 cells may lead to tau dephosphorylation, based on the data presented here. GSK3b has gained much attention as a kinase responsible for tau phosphorylation. It has also been shown to induce neuronal apoptosis in cell culture (Pap & Cooper, 1998; Hetman et al., 2000) and is a mandatory element of b-amyloid-induced neurotoxicity (Takashima et al., 1993). Therefore, GSK3b inhibitors have been proposed as therapeutic agents against central nervous system injury and toxicity (Bhat et al., 2004). Based on the fact that GSK3b inhibitors induce dephosphorylation of tau, they may also be useful as AD treatments. The transient dephosphorylation of tau by BDNF-induced signaling is both physiologically and pathologically significant. BDNF stimulation of neurons induces a chemoattractant response of the growth cone in growing axons (Ming et al., 2002). Dephosphorylation of tau may help to stabilize microtubules in the advancing axonal growth cone. Earlier research has also shown that BDNFinduced lamellipodia formation in Xenopus embryonic nerves is dependent on microtubule dynamics (Gibney & Zheng, 2003). Dephosphorylation of tau protein may be a mechanism through which BDNF regulates microtubule dynamics of emerging axonal processes formed at the growth cones and lamellipodia. The transient nature of the dephosphorylation would be necessary to preserve the neuron’s ability to respond to new signals, and to respond to changes in the levels of extracellular BDNF. The transient dephosphorylation of tau by BDNF is very significant for AD research. The finding that there is a decrease in BDNF mRNA and protein levels in AD brains (Phillips et al., 1991; Murer et al., 2001) led to the hypothesis that lack of BDNF signaling could play a part in AD progression. Our results suggest a link between BDNF signaling and an AD molecular pathology, tau phosphorylation. Although the dephosphorylation of tau by BDNF signaling is transient, the cumulative lack of BDNF signaling may promote hyperphosphorylation. Further research, utilizing mice that lack BDNF signaling, should determine if a lack in BDNF signaling can indeed contribute to tau hyperphosphorylation. In addition, recent work has demonstrated that b-amyloid peptides, at sublethal concentrations, can down-regulate BDNF function (Tong et al., 2004). Extracellular plaques containing b-amyloid is the second major pathology observed in AD, other than formation of tau filaments (Masters et al., 1985; Hardy & Selkoe, 2002). 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