Nav1.7 sodium channel-induced Ca2+ influx decreases tau phosphorylation via glycogen synthase kinase-3β in adrenal chromaffin cells

Neurochemistry International 54 (2009) 497–505

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Nav1.7 sodium channel-induced Ca2+ influx decreases tau phosphorylation via glycogen synthase kinase-3b in adrenal chromaffin cells Tasuku Kanai, Takayuki Nemoto, Toshihiko Yanagita, Toyoaki Maruta, Shinya Satoh, Norie Yoshikawa, Akihiko Wada * Department of Pharmacology, Miyazaki Medical College, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki 889-1692, Japan

A R T I C L E I N F O

A B S T R A C T

Article history: Received 4 October 2008 Received in revised form 17 January 2009 Accepted 10 February 2009 Available online 24 February 2009

In cultured bovine adrenal chromaffin cells expressing Nav1.7 sodium channel isoform, veratridine increased Ser473-phosphorylation of Akt and Ser9-phosphorylation of glycogen synthase kinase-3b by 217 and 195%, while decreasing Ser396-phosphorylation of tau by 36% in a concentration (EC50 = 2.1 mM)- and time (t1/2 = 2.7 min)-dependent manner. These effects of veratridine were abolished by tetrodotoxin or extracellular Ca2+ removal. Veratridine (10 mM for 5 min) increased translocation of Ca2+-dependent conventional protein kinase C-a from cytoplasm to membranes by 47%; it was abolished by tetrodotoxin, extracellular Ca2+ removal, or Go¨6976 (an inhibitor of protein kinase Ca), and partially attenuated by LY294002 (an inhibitor of phosphatidylinositol 3-kinase). LY294002 (but not Go¨6976) abrogated veratridine-induced Akt phosphorylation. In contrast, either LY294002 or Go¨6976 alone attenuated veratridine-induced glycogen synthase kinase-3b phosphorylation by 65 or 42%; however, LY294002 plus Go¨6976 completely blocked it. Veratridine (10 mM for 5 min)-induced decrease of tau phosphorylation was partially attenuated by LY294002 or Go¨6976, but completely blocked by LY294002 plus Go¨6976; okadaic acid or cyclosporin A (inhibitors of protein phosphatases 1, 2A, and 2B) failed to alter tau phosphorylation. These results suggest that Na+ influx via Nav1.7 sodium channel and the subsequent Ca2+ influx via voltage-dependent calcium channel activated (1) Ca2+/ protein kinase C-a pathway, as well as (2) Ca2+/phosphatidylinositol 3-kinase/Akt and (3) Ca2+/ phosphatidylinositol 3-kinase/protein kinase C-a pathways; these parallel pathways converged on inhibitory phosphorylation of glycogen synthase kinase-3b, decreasing tau phosphorylation. ß 2009 Elsevier Ltd. All rights reserved.

Keywords: Nav1.7 sodium channel Calcium Protein kinase C-a Akt Glycogen synthase kinase-3b Tau

1. Introduction Voltage-dependent sodium channel activity engraves structure of neuronal circuit from early development through adulthood by regulating; e.g., differentiation of neurites into a single axon and multiple dendrites (neuronal polarity essential to unidirectional electrical signal flow); myelination and axon growth cone navigation for synapse formation; experience-driven cognition; and neuronal survival (Wada, 2006). However, the cellular mechanisms of sodium channel activity for these neuronal structural events remain elusive. Sodium channel consists of the principal a-subunit, without or with b1- to b4-subunit (Wada, 2006). The a-subunit consists of

* Corresponding author. Tel.: +81 985 85 1786; fax: +81 985 84 2776. E-mail address: [email protected] (A. Wada). Abbreviations: cPKC-a, conventional protein kinase C-a; DAG, diacylglycerol; DMSO, dimethyl sulfoxide; GSK-3, glycogen synthase kinase-3; IP3, inositol 1,4,5trisphosphate; PI3K, phosphatidylinositol 3-kinase; PIP2, phosphatidylinositol 4,5bisphosphate; PIP3, phosphatidylinositol 3,4,5-trisphosphate; PLC, phospholipase C; PYK2, proline-rich tyrosine kinase 2; SDS, sodium dodecyl sulfate. 0197-0186/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2009.02.002

four homologous domains (I–IV), each containing six transmembrane segments (S1–S6); it forms the ion-pore and the toxinbinding sites [e.g., site 1 for tetrodotoxin; site 2 for veratridine]. The nine a-subunits (Nav1.1–Nav1.9) arise from nine genes (SCN1A–SCN5A; SCN8A–SCN11A) (Wada, 2006). Nav1.7 sodium channel is encoded by SCN9A, and widely distributed in peripheral neuronal cells; besides, in PC12 cells, dorsal root ganglion neurons and NG108-15 cells, Nav1.7 sodium channel was localized predominantly in axon growth cone (D’Arcangelo et al., 1993; Toledo-Aral et al., 1997; Kawaguchi et al., 2007), a neuronal compartment sensing its environment for correct synapse formation during normal development and during regeneration following neuronal injury (Lockerbie et al., 1991; Wood et al., 1992; Wada, 2006; Farrar and Spencer, 2008). Glycogen synthase kinase-3 (GSK-3) is constitutively active in nonstimulated cells, being implicated in both the elaboration and maintenance of axon-dendrite polarity; Ser9-phosphorylation/ inactivation of GSK-3b catalyzed via multiple pathways [e.g., phosphatidylinositol 3-kinase (PI3K)] in response to insulin-like growth factor-I, leptin (anorexigenic peptide with memory enhancing activity), or Ras, or GSK-3 inhibitors (e.g., lithium)

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promoted axon-dendrite polarity, increasing growth cone size and membrane expansion (Leroy et al., 2000; Jiang et al., 2005; Laurino et al., 2005; Wada et al., 2005a,b; Valerio et al., 2006; Oinuma et al., 2007). Tau, originally discovered as a cytoskeletal protein to regulate microtubule dynamics (e.g., stabilization), has been increasingly unveiled to regulate axon-dendrite polarity, axonogenesis, dendrite outgrowth, myelination, and axonal transport via as yet unidentified mechanisms in cytoskeletal and non-cytoskeletal locations (e.g., plasma membrane lipid rafts for signal transduction) (Mandell and Banker, 1996; Billingsley and Kincaid, 1997; Leroy et al., 2000; Johnson and Stoothoff, 2004; Lee, 2005). Importantly, tau associates with Src homology 3 domains of various proteins (e.g., PI3K; Src tyrosine kinase family) (Billingsley and Kincaid, 1997; Lee, 2005; Reynolds et al., 2008). These abilities of tau to execute biological functions and heteroprotein complex formation are regulated by tau phosphorylation via several protein kinases (e.g., GSK-3b). In adrenal chromaffin cells (embryologically derived from the neural crest), sodium channel a-subunit is Nav1.7 (Toledo-Aral et al., 1997; Wada, 2006; Wada et al., 2008). Veratridine-induced 22 Na+ influx via Nav1.7 sodium channel increases 45Ca2+ influx via voltage-dependent calcium channel, triggering exocytosis and synthesis of catecholamines (Wada et al., 1985, 2008; Uezono et al., 1992; Wada, 2006). In cultured bovine or frog (Rana pipiens) adrenal chromaffin cells, veratridine (50 mM for 30 min) (Shepherd and Holzwarth, 2001), or brief (1 ms) electrical stimulation or micropippete application of high K+ depolarization (Manivannan and Terakawa, 1994) immediately increased Ca2+ influx-dependent formation of growth cone; it harbored constantly exploring filopodia and lamellipodia, which finally elaborated synapse-like contacts with neighboring cells. No studies to date, however, examined whether Na+ influx via sodium channel could regulate PI3K/Akt/GSK-3/tau pathway in any given tissue. Our present study shows that veratridine-induced 22Na+ influx and the subsequent Ca2+ influx activates conventional protein kinase C-a (cPKC-a), PI3K/Akt and PI3K/cPKC-a pathways, which converge on inhibitory Ser9-phosphorylation of GSK-3b, decreasing constitutive Ser396-phosphorylation level of tau, a condition promoting growth cone development and signal transduction (Mandell and Banker, 1996; Billingsley and Kincaid, 1997; Leroy et al., 2000; Johnson and Stoothoff, 2004; Lee, 2005; Reynolds et al., 2008). 2. Materials and methods 2.1. Materials Eagle’s minimum essential medium was from Nissui Seiyaku (Tokyo, Japan). Calf serum, phenylmethylsulfonyl fluoride, leupeptin, and Tween-20 were from Nacalai Tesque (Kyoto, Japan). Cyclosporin A, cytosine arabinoside, tetrodotoxin, and veratridine were from Sigma (St. Louis, MO). LY294002, Go¨6976, and okadaic acid were from Calbiochem-Novabiochem (San Diego, CA). Horseradish peroxidaseconjugated anti-mouse or anti-rabbit antibody, ECL Plus Western Blotting Detection Reagents, and Hybond-P were from Amersham Biosciences (Piscataway, NJ). Rabbit polyclonal antibodies against Ser473-phosphorylated Akt, Ser9-phosphorylated GSK3b, or mouse monoclonal antibody against Ser396-phosphorylated tau were from Cell Signaling Technology (Beverly, MA). Mouse monoclonal GSK-3b antibody was from BD Transduction Laboratories (San Diego, CA). Mouse monoclonal Akt antibody was from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse monoclonal tau antibody was from Chemicon (Temecula, CA). Rabbit polyclonal cPKC-a antibody was from GibcoBRL (Gaithersberg, MD). Can Get signalTM immunoreaction Enhancer Solution-1 and -2 were from TOYOBO (Osaka, Japan).

glucose, and 0.5% bovine serum albumin), and treated without or with veratridine, tetrodotoxin, LY294002, Go¨6976, LY294002 plus Go¨6976, okadaic acid or cyclosporin A in Krebs-Ringer phosphate buffer, or in Ca2+-free Krebs-Ringer phosphate buffer at 37 8C for up to 10 min. Veratridine, LY294002, Go¨6976, okadaic acid, and cyclosporin A were dissolved in dimethyl sulfoxide (DMSO). When these compounds were tested, the final concentration of DMSO in the each test medium was equally adjusted to 0.2%, which did not affect phosphorylation levels of Akt and GSK-3b, or levels of Akt and GSK-3b proteins. The culture medium contained 3 mM cytosine arabinoside to suppress the proliferation of nonchromaffin cells. When chromaffin cells were further purified by differential plating (Satoh et al., 2008), phosphorylation level of GSK-3b was similar between purified and conventional chromaffin cells. Also, veratridine treatment (10 mM for 3 min) increased phosphorylation level of GSK-3b by 60 and 58% in purified and conventional chromaffin cells, compared with nontreated cells within each cell group. 2.3. Western blot analysis of Ser9-phosphorylated GSK-3b, GSK-3b, Ser473phosphorylated Akt, Akt, Ser396-phosphorylated tau, and tau Cells were washed with ice-cold Ca2+-free phosphate-buffered saline and solubilized in 500 ml of 2 sodium dodecyl sulfate (SDS) electrophoresis sample buffer (125 mM Tris–HCl [pH 6.8], 20% glycerol, 10% 2-mercaptoethanol, and 4% SDS) at 98 8C for 3 min. Total quantities of cellular proteins, as measured by the Noninterfering Protein Assay kit, were not changed between nontreated and test compound-treated cells. The same amounts of proteins (7.0–7.5 mg per lane) were separated by SDS-7.5% or -12% polyacrylamide gel electrophoresis, and transferred onto a nitrocellulose membrane (Hybond-P). The membrane was preincubated with 1% bovine serum albumin in Tween-Tris-buffered saline (10 mM Tris–HCl [pH 7.4], 150 mM NaCl, and 0.1% Tween-20), and reacted overnight at 4 8C in Can Get Signal Solution-1 with mouse or rabbit antibody (1:2000) against Ser9-phosphorylated GSK-3b, GSK-3b, Ser473-phosphorylated Akt, Akt, Ser396-phosphorylated tau, or tau (Satoh et al., 2008). After repeated washings, the immunoreactive bands were reacted in Can Get Signal Solution-2 with horseradish peroxidase-conjugated antimouse or anti-rabbit antibody, then visualized by the enhanced chemiluminescent detection system ECL Plus, and quantified by a luminoimage LAS-3000 analyzer (Fuji Film, Tokyo). 2.4. Western blot analysis of cPKC-a in soluble and particulate fractions Cells were washed with ice-cold phosphate-buffered saline, collected in 200 ml of ice-cold lysis buffer (20 mM Tris–HCl [pH 7.5], 5.5 mM EGTA, 2 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, and 5 mg/ml leupeptin), and disrupted by sonication (5 s; four times). The homogenate was centrifuged at 100,000  g for 60 min at 4 8C. An aliquot (100 ml) of the resulting supernatant was mixed with 2 SDS electrophoresis sample buffer (100 ml), and used as soluble cPKC-a. The pellet was rinsed with ice-cold lysis buffer, solubilized in 200 ml of 2 SDS electrophoresis sample buffer, and used as particulate cPKC-a. 2.5. Statistical methods All experiments were repeated five times (mean  S.E.M.). Significance (P < 0.05) was determined by one-way or two-way ANOVA with post hoc mean comparison by Newman–Keuls multiple range test. Student’s t-test was used when two means of group were compared.

3. Results 3.1. Veratridine-induced concentration- and time-dependent increase of Ser9-phosphorylated GSK-3b level in adrenal chromaffin cells Fig. 1A shows that cells were treated without or with 0.1– 100 mM veratridine for 5 min, and the cell lysates were subjected to Western blot analysis of Ser9-phosphorylated GSK-3b and GSK3b. We calculated the relative level of Ser9-phosphorylated GSK3b/GSK-3b; veratridine increased the relative level by 105% in a concentration-dependent manner (EC50 = 2.1 mM). As shown in Fig. 1B, veratridine (10 mM) increased the relative level of Ser9phosphorylated GSK-3b/GSK-3b in a time-dependent manner, attaining to 102% increase at 5 min.

2.2. Primary culture of adrenal chromaffin cells: treatment with test compounds Isolated bovine adrenal chromaffin cells were cultured (4  106 per dish, Falcon; 35 mm diameter) in Eagle’s minimum essential medium containing 10% calf serum under 5% CO2/95% air in a CO2 incubator. Three days (60–62 h) later, the cells were washed with ice-cold Krebs-Ringer phosphate buffer (154 mM NaCl, 5.6 mM KCl, 1.1 mM MgSO4, 2.2 mM CaCl2, 0.85 mM NaH2PO4, 2.15 mM Na2HPO4, 5 mM

3.2. Veratridine-induced concentration- and time-dependent increase of Ser473-phosphorylated Akt level It has been shown that Ser9-phosphorylation of GSK-3b is catalyzed by Akt, PKC, cyclic AMP-dependent protein kinase, and

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Fig. 1. Veratridine-induced increase of Ser9-phosphorylated GSK-3b level in adrenal chromaffin cells: concentration- and time-dependency. Cells were treated without (*) or with (*) (A) 0.1–100 mM veratridine for 5 min, or (B) 10 mM veratridine for up to 10 min. The cell lysates were separated by SDS-polyacrylamide gel electrophoresis and transferred to a membrane. The membrane was subjected to Western blot analysis using antibody against Ser9-phosphorylated GSK-3b (p-Ser9-GSK-3b) and GSK-3b. Blot data are typical from five independent experiments with similar results. Immunoreactivities were quantified by the luminoimage analyzer, and the relative level of p-Ser9-GSK-3b/GSK-3b was calculated. A value of 100% represents the relative level obtained in the left lane of nontreated cells at (A) 5 min and (B) 0 min. Mean  S.E.M. (n = 5). *p < 0.05, compared with left lane of nontreated cells.

p90 ribosomal S6 kinase. Multiple lines of previous studies have shown that Akt is fully activated by its sequential phosphorylation at Thr308 and Ser473 residues (Toker and Newton, 2000; Li et al., 2006b; Toker, 2008). Thr308-phosphorylation is catalyzed by PI3K/ phosphoinositide-dependent kinase-1, whereas in vitro studies proposed that Ser473-phosphorylation is mediated via several candidate protein kinases (e.g., rictor-mammalian target of rapamycin complex 2) (Toker, 2008). In our present study, Fig. 2A shows that veratridine (0.1–100 mM for 5 min) increased Ser473-phosphorylation of Akt in a concentration-dependent manner, compared to nontreated cells. Fig. 2B shows that the

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Fig. 2. Veratridine-induced increase of Ser473-phosphorylated Akt level: concentration- and time-dependency. Cells were treated without (*) or with (*) (A) 0.1–100 mM veratridine for 5 min, or (B) 10 mM veratridine for up to 10 min; the cell lysates were subjected to Western blot analysis for Ser473phosphorylated Akt (p-Ser473-Akt) and Akt. Blot data are typical from 5 independent experiments with similar results. The relative level of p-Ser473-Akt/ Akt was calculated; a value of 100% represents the relative level in the left lane of nontreated cells at (A) 5 min and (B) 0 min. Mean  S.E.M. (n = 5). *p < 0.05, compared with left lane of nontreated cells.

increasing effect of 10 mM veratridine on Ser473-phosphorylation of Akt was time-dependent. 3.3. Veratridine-induced increases of Ser473-phosphorylated Akt and Ser9-phosphorylated GSK-3b levels: abolition by tetrodotoxin and extracellular Ca2+ removal Veratridine (10 mM for 5 min) increased Ser473-phosphorylated Akt level (Fig. 3A, lanes 1and 2) and Ser9-phosphorylated GSK-3b level (Fig. 3B, lanes 1 and 2), as shown in Figs. 1 and 2. Fig. 3 shows that tetrodotoxin (lanes 1 and 3), or extracellular Ca2+ removal (lanes 1 and 5) did not appreciably alter the basal phosphorylation levels, but completely blocked veratridine-induced increases of Ser473-phosphorylation of Akt and Ser9-phosphorylation of GSK3b (lanes 2, 4, and 6).

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Fig. 4. Veratridine-induced translocation of cPKC-a from soluble to particulate fraction: abolition by tetrodotoxin, extracellular Ca2+ removal, Go¨6976, but partial attenuation by LY294002. Cells were treated without (open column) or with (closed column) 10 mM veratridine for 5 min in normal Ca2+-containig medium supplemented with DMSO, 1 mM tetrodotoxin, 50 mM LY294002, 10 mM Go¨6976, or in Ca2+-free medium; DMSO concentration was equally adjusted to 0.2% in each medium. The cell lysates were sonicated, centrifuged at 100,000  g for 60 min at 4 8C, and saved as soluble and particulate fractions. Total cell lysate, soluble and particulate fractions were subjected to Western blot analysis. Blot data are typical from five independent experiments with similar results. Data are shown as a percentage of particulate/soluble plus particulate. Mean  S.E.M. (n = 5). * p < 0.05, compared between nontreated and veratridine-treated cells within each cell group. #p < 0.05, compared between DMSO- and LY294002-treated cell groups.

Fig. 3. Veratridine-induced increases of Ser473-phosphorylated Akt and Ser9phosphorylated GSK-3b levels: abolition by tetrodotoxin and extracellular Ca2+ removal. Cells were left nontreated at 0 min (data not shown here), or treated for 5 min without (open column) or with (closed column) 10 mM veratridine in normal Ca2+-containing medium in the absence (None) or presence of 1 mM tetrodotoxin, or in Ca2+-free medium; the cell lysates were subjected to Western blot analysis. Blot data are typical from 5 independent experiments with similar results. The relative levels of (A) p-Ser473-Akt/Akt and (B) p-Ser9-GSK-3b/GSK-3b were calculated; a value of 100% represents the relative level obtained in cells left nontreated at 0 min. Mean  S.E.M. (n = 5). *p < 0.05, compared between nontreated and veratridine-treated cells within each cell group.

3.4. Veratridine-induced translocation of cPKC-a protein from cytoplasm to membranes: abolition by tetrodotoxin, extracellular Ca2+ removal, Go¨6976, and partial attenuation by LY294002 Our previous Western blot analysis showed that, among 11 isoforms of PKC family, bovine adrenal chromaffin cells contain only three isoforms: Ca2+-dependent and diacylglycerol (DAG)dependent cPKC-a; Ca2+-independent and DAG-dependent novel PKC-e; Ca2+-independent and DAG-independent atypical PKC-z (Yanagita et al., 2000). In addition, we previously observed that, when cultured bovine adrenal chromaffin cells were treated with veratridine (1–100 mM for 1–20 min), veratridine-induced Ca2+ influx caused concentration- and time-dependent increases of both DAG level (3.0-fold) and translocation (1.7-fold) of Ca2+/

DAG/phosphatidylserine-dependent cPKC-a activity from cytoplasm to membranes (Uezono et al., 1992), a hallmark of PKC activation (Newton, 2003). As shown in Fig. 4, cells were treated without or with veratridine (100 mM for 5 min); total cell lysate (upper panel), soluble (middle panel) and particulate (lower panel) fractions were subjected to Western blot analysis for cPKC-a. When compared between nontreated (lane 1) and veratridine-treated cells (lane 2), veratridine increased particulate cPKC-a level from 42 to 88% of total cPKC-a, while dramatically decreasing soluble cPKC-a level. Veratridine-induced translocation of cPKC-a from soluble to particulate fraction was abolished by tetrodotoxin (lanes 1 and 2 vs. lanes 3 and 4) or extracellular Ca2+ removal (lanes 1 and 2 vs. lanes 5 and 6). It has been shown that intramolecular autophosphorylation of cPKC-a at its C-terminal turn motif and hydrophobic motif yields the catalytically competent mature form of cPKC-a in cytoplasm, which is the prerequisite for translocation of cPKC-a to membranes (Newton, 2003). In cultured bovine adrenal chromaffin cells, our previous Western blot analysis showed that nicotine (10 mM for 10 min)-induced Ca2+ influx via voltage-dependent calcium channel increased Ser657-phosphorylated cPKC-a level, which was prevented by Go¨6976 (Sugano et al., 2006), an inhibitor of cPKC-a (but not novel PKC-e and atypical PKC-z) (Yanagita et al., 2000). In our present study, Fig. 4 shows that Go¨6976 abolished veratridine-induced translocation of cPKC-a from soluble to particulate fraction (lanes 1 and 2 vs. lanes 9 and 10). In cultured bovine adrenal chromaffin cells, our previous finding (i.e., veratridine-induced increase of DAG level) implicates that veratridine activated phospholipase C (PLC), increasing formation of DAG and inositol 1,4,5-trisphosphate (IP3) from

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membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) (Uezono et al., 1992). In various cells (e.g., COS-7 cells), it has been documented that translocation of PLC from cytoplasm to membrane was enhanced by phosphatidylinositol 3,4,5-trisphosphate (PIP3), a product formed from PIP2 by PI3K; thus, PI3K enhanced PLC-induced reactions, whereas PI3K inhibitors (e.g., LY294002) attenuated PLC-induced events (Bae et al., 1998; Falasca et al., 1998; Rameh et al., 1998; Maffucci and Falasca, 2007). In cultured bovine adrenal chromaffin cells, our previous and present studies showed that veratridine-induced Ca2+ influx activated Akt/GSK-3b pathway (Figs. 1 and 2), and caused DAG accumulation (Uezono et al., 1992) and cPKC-a translocation (Uezono et al., 1992; Fig. 4) (for signaling pathways, refer to Fig. 7). We, therefore, examined whether PI3K, an upstream signal of Akt/ GSK-3b, could be involved in the veratridine-induced cPKC-a translocation. As shown in Fig. 4 (lanes 1 and 2 vs. lanes 7 and 8), LY294002 attenuated veratridine-induced translocation of cPKC-a by 59%. Based on these previous and present findings, it seems that veratridine-induced Ca2+ influx activated Ca2+/PI3K/PLC/DAG/ cPKC-a pathway, in addition to the activation of classical Ca2+/ PLC/DAG/cPKC-a pathway (Fig. 7). 3.5. Veratridine-induced increase of Ser473-phosphorylated Akt level via PI3K pathway: abolition by LY294002, but not by Go¨6976 Fig. 5A shows that veratridine (10 mM for 5 min) increased Ser473-phosphorylated Akt level (lanes 1 and 2), as shown in Fig. 2. LY294002 abrogated veratridine-induced increase of Ser473phosphorylated Akt level (lanes 1 and 2 vs. lanes 3 and 4). Although Go¨6976 abolished translocation of cPKC-a from soluble to particulate fraction (Fig. 4, lanes 1 and 2 vs. lanes 9 and 10), Go¨6976 failed to block veratridine-induced increase of Ser473phosphorylated Akt level (lanes 1 and 2 vs. lanes 5 and 6); in addition, Go¨6976 did not alter abolition by LY294002 of veratridine-induced Akt phosphorylation (lanes 3 and 4 vs. lanes 7 and 8). 3.6. Veratridine-induced increase of Ser9-phosphorylated GSK-3b level via both LY294002-sensitive PI3K and Go¨6976-sensitive cPKC-a pathways (Fig. 7) Fig. 5B shows that veratridine (10 mM for 5 min) increased Ser9phosphorylated GSK-3b level (lanes 1 and 2), as shown in Fig. 1. Either LY294002 or Go¨6976 alone attenuated veratridine-induced increase of Ser9-phosphorylated GSK-3b level only by 65 (lanes 1 and 2 vs. lanes 3 and 4) or 42% (lanes 1 and 2 vs. lanes 5 and 6); however, concurrent treatment with LY294002 plus Go¨6976 abolished veratridine-induced increase of Ser9-phosphorylated GSK-3b level (lanes 1 and 2 vs. lanes 7 and 8). 3.7. Veratridine-induced decrease of Ser396-phosphorylated tau level: abolition by tetrodotoxin, extracellular Ca2+ removal, LY294002 plus Go¨6976, but not by phosphatase inhibitors It has been shown that tau associates with various signaling proteins (Lee, 2005; Reynolds et al., 2008), in addition to tubulin (Mandell and Banker, 1996; Billingsley and Kincaid, 1997; Leroy et al., 2000; Johnson and Stoothoff, 2004; Liu et al., 2007); multiple functions of tau are regulated by phosphorylation. In tau, the Cterminal Ser396 is the major phosphorylation site catalyzed by GSK3b (Billingsley and Kincaid, 1997; Johnson and Stoothoff, 2004; Li et al., 2006a; Liu et al., 2007); in tau, Ser404-phosphorylation by cyclin-dependent protein kinase 5 primes for the sequential phosphorylation at Ser400 and then Ser396 (Li et al., 2006a). In nonstimulated adrenal chromaffin cells (Fig. 6A and B, left two lanes), antibody against Ser396-phosphorylated tau detected

Fig. 5. Veratridine-induced Ser473-phosphorylation of Akt via LY294002-sensitive PI3K pathway vs. veratridine-induced Ser9-phosphorylation of GSK-3b via both LY294002-sensitive PI3K and Go¨6976-sensitive cPKC-a pathways. Cells were left nontreated at 0 min (data not shown here), or treated for 5 min without (open column) or with (closed column) 10 mM veratridine in DMSO, 50 mM LY294002, 10 mM Go¨6976, or 50 mM LY294002 plus 10 mM Go¨6976; the cell lysates were subjected to Western blot analysis. Blot data are typical from five independent experiments with similar results. The relative levels of (A) p-Ser473-Akt/Akt and (B) p-Ser9-GSK-3b/GSK-3b were calculated; a value of 100% represents the relative level in cells left nontreated at 0 min. Mean  S.E.M. (n = 5). *p < 0.05, compared between nontreated and veratridine-treated cells within each cell group; #p < 0.05, compared between DMSO- and test drug-treated cell groups.

two bands, as in mouse brain (the manufacture’s instruction); Ser396-phosphorylation of tau constitutively occurs in the absence of extracellular stimulus, as previously shown in cultured rat hippocampal (Mandell and Banker, 1996) and cortical (Lovestone et al., 1999) neurons. The Ser396-phosphorylated tau level was decreased by veratridine in a concentration- and time-dependent manner by 36%. Fig. 6C shows that veratridine-induced reduction of Ser396-phosphorylated tau level was abolished by tetrodotoxin or extracellular Ca2+ removal, as shown in Ser473-phosphorylated Akt and Ser9-phosphorylated GSK-3b levels (Fig. 3). Fig. 6D shows

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Fig. 6. Veratridine-induced concentration- and time-dependent decrease of Ser396-phosphorylated tau level: abolition by tetrodotoxin, extracellular Ca2+ removal, and LY294002 plus Go¨6976, but not by okadaic acid and cyclosporin A. Cells were left nontreated at 0 min (data not shown here), or treated without (*, open column) or with (*, closed column) (A) 0.1–100 mM veratridine for 5 min; (B) 10 mM veratridine for up to 10 min; (C) for 5 min in normal Ca2+-containing medium without (None) or with 1 mM tetrodotoxin, or in Ca2+-free medium; (D) in DMSO, 50 mM LY294002, 10 mM Go¨6976, or 50 mM LY294002 plus 10 mM Go¨6976; (E) in DMSO, 30 nM okadaic acid, or 10 mM cyclosporin A. The cell lysates were subjected to Western blot analysis for Ser396-phosphorylated tau (p-Ser396-Tau) and tau; arrows indicate two bands in both p-Ser396-Tau and tau, as shown in the manufacture’s instruction and LoPresti et al. (1995). Blot data are typical from five independent experiments with similar results. The relative level of p-Ser396-Tau/Tau was calculated; a value of 100% represents the relative level in cells left nontreated at 0 min. Mean  S.E.M. (n = 5). *p < 0.05, compared between nontreated and veratridine-treated cells within each cell group; #p < 0.05, compared between DMSO- and test drug-treated cell groups.

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that veratridine-induced reduction of Ser396-phosphorylated tau level was partially attenuated by either LY294002 or Go¨6976 alone, but abolished by LY294002 plus Go¨6976; also, the concurrent treatment with LY294002 plus Go¨6976 was required to abolish veratridine-induced Ser9-phosphorylation of GSK-3b (Fig. 5B). It has been shown that Ser396-phosphorylation site of tau was subjected to the dephosphorylation by protein phosphatases 1, 2A, and 2B (calcineurin) (Billingsley and Kincaid, 1997). In cultured bovine adrenal chromaffin cells, we previously showed that cyclosporin A inhibited calcineurin activity (IC50 = 500 nM) (Satoh et al., 2008). In our present study, okadaic acid (an inhibitor for protein phosphatases 1 and 2A), or cyclosporin A failed to prevent veratridine-induced reduction of Ser396-phosphorylated tau level (Fig. 6E). 4. Discussion In our present study, veratridine increased Ser473-phosphorylation of Akt and Ser9-phosphorylation of GSK-3b in a concentration- and time-dependent manner, which were abolished by tetrodotoxin or extracellular Ca2+ removal. In bovine adrenal chromaffin cells, veratridine-induced 22Na+ influx occurred in Ca2+-free medium to the same extent as in Ca2+-containing medium (Wada et al., 1985). Therefore, veratridine-induced Ca2+ influx led to Akt phosphorylation and GSK-3b phosphorylation. Veratridine caused translocation of cPKC-a from cytoplasm to membranes, which was abrogated by tetrodotoxion, extracellular Ca2+ removal, or Go¨6976. Distinctively, veratridine-induced membrane translocation of cPKC-a was attenuated partially by LY294002, suggesting that PI3K pathway partly contributes to cPKC-a translocation (Fig. 7). Previous studies showed that extracellular stimuli (e.g., serum) caused rapid translocation of PLC-g from cytoplasm to plasma membrane; PLC-g was directly tethered to PIP3 formed by PI3K in membrane (Falasca et al., 1998;

Fig. 7. Nav1.7 sodium channel and decrease of Ser396-phosphorylation of tau. Na+ influx via Nav1.7 increases Ca2+ influx via calcium channel. Intracellular Ca2+ activates (1) PLC pathway; (2) PI3K/Akt pathway; (3) PI3K/PLC pathway. They converge on GSK-3b Ser9-phosphorylation, decreasing tau Ser396-phosphorylation.

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Maffucci and Falasca, 2007). In COS-1 cells, wortmannin (an inhibitor of PI3K) blocked serum-induced membrane translocation of PLC-g (Falasca et al., 1998). In various cells (e.g., NIH 3T3 cells; HepG2 cells), wortmannin or LY294002 inhibited PLC activity, decreasing PLC-catalyzed IP3 formation and IP3-induced intracellular Ca2+ release (Bae et al., 1998; Rameh et al., 1998; Maffucci and Falasca, 2007). Thus, our present and previous (Uezono et al., 1992) studies implicate that veratridine-induced Ca2+ influx activated PI3K/PIP3/PLC pathway, increasing DAG production and translocation of cPKC-a (Fig. 7). In our present study, veratridine-induced Ca2+ influx-dependent Akt phosphorylation was abrogated by LY294002, as shown in the attenuation of Ca2+-dependent cPKC-a translocation by LY294002 (see above). These results raise the question of how Ca2+ influx can transduce the signal to PI3K (Fig. 7). PI3K is a heterodimer composed of p85 regulatory subunit and p110 catalytic subunit. Our knowledge about PI3K activation, however, is primitive; when cells are activated by receptor tyrosine kinases and Ras, Src homology 2 domain of p85 is recruited to Tyrphosporylation residues of target molecules, relieving the constitutive inhibition of p110 by p85 in nonstimulated cells (Geering et al., 2007). In PC12 cells, previous studies showed that Ca2+ influx via voltage-dependent calcium channel led to activation of Shc/ Grb2/Sos1/Ras pathway, promoting neurite outgrowth (Rosen et al., 1994; Rusanescu et al., 1995; Rosen and Greenberg, 1996); direct interaction between Ras and PI3K was essential to growth factor-induced activation of PI3K (Rodriguez-Viciana et al., 1994). In neuronal cells (e.g., hippocampus; PC12 cells), depolarizationinduced Ca2+ influx activated proline-rich tyrosine kinase 2 (PYK2); also, PYK2 activated Ras (Lev et al., 1995; Corvol et al., 2005). Based on these previous studies, it may be possible that Ca2+ influx activates PI3K in adrenal chromaffin cells via (1) Ca2+/Shc/ Grb2/Sos1/Ras/PI3K, (2) Ca2+/PYK2/Ras/PI3K, and/or (3) Ca2+/ PYK2-catalyzed Tyr-phosphorylation of as yet undefined molecule(s), which created the binding site(s) for PI3K activation. In striking contrast to the abolition by LY294002, Go¨6976 did not prevent veratridine-induced Akt phosphorylation, suggesting that cPKC-a is not an upstream signal of Akt. On the other hand, veratridine-induced GSK-3b phosphorylation was partially blocked by either LY294002 or Go¨6976, while being completely prevented by LY294002 plus Go¨6976. These findings show that Akt and cPKC-a pathways function as independent parallel signaling branches, which converged on Ser9-phosphorylation of GSK-3b (Fig. 7). In our present study, veratridine decreased constitutive Ser396phosphorylation level of tau in a concentration- and timedependent manner, which was abolished by tetrodotoxin or extracellular Ca2+ removal. Reduction of tau phosphorylation was partially blocked by LY294002 or Go¨6976, while being completely prevented by LY294002 plus Go¨6976; in contrast, phosphatase inhibitors failed to attenuate it. As shown in Fig. 7, these results suggest that veratridine-induced Na+ influx via Nav1.7 sodium channel and the subsequent Ca2+ influx via calcium channel decreased tau phosphorylation level via (1) Ca2+/PLC/DAG/cPKC-a/ GSK-3b; (2) Ca2+/PI3K/Akt/GSK-3b; (3) Ca2+/PI3K/PLC/DAG/cPKCa/GSK-3b. It has been shown that Nav1.7 sodium channel is predominantly localized at axon growth cone in dorsal root ganglion neurons, PC12 cells (Toledo-Aral et al., 1997) and NG108-15 cells (Kawaguchi et al., 2007); in addition, cell surface number of Nav1.7 sodium channel was up-regulated by neuronal differentiation of PC12 cells (D’Arcangelo et al., 1993) and NG108-15 cells (Kawaguchi et al., 2007). In sealed axon growth cone particles isolated from fetal rat brain, Lockerbie et al. (1991) demonstrated that veratridine-induced gating of sodium channel caused plasmalemmal expansion of growth cone in a Ca2+-dependent

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manner; surprisingly, depolarization per se promoted insertion of sodium channel from cytoplasm into the growth cone plasmalemma (Wood et al., 1992). In cultured frog or bovine adrenal chromaffin cells, veratridine (50 mM for 30 min) (Shepherd and Holzwarth, 2001), or brief electrical stimulation or high K+ depolarization (Manivannan and Terakawa, 1994) immediately increased Ca2+ influx-dependent formation of growth cone; at the growth cone, its constantly exploratory sprouting filopodia and lammelipodia culminated in synapse-like contacts with neighboring cells. Little is known, however, about the mechanisms whereby neuronal activity integrates architecture and function of growth cone. Previous studies documented that growth cone outgrowth was specifically promoted by insulin-like growth factor-I via PI3K pathway (but not brain-derived neurotrophic factor) (Pfenninger ¨ zdinler and Macklis, 2006; Sosa et al., 2006). Among et al., 2003; O PI3K pathway, GSK-3b has been noted; insulin-like growth factorI-, leptin-, or Ras-induced PI3K-mediated Ser9-phosphorylation/ inactivation of GSK-3b, or GSK-3 inhibitors (e.g., lithium) promoted axon-dendrite polarity by increasing growth cone size and membrane expansion (Leroy et al., 2000; Jiang et al., 2005; Laurino et al., 2005; Wada et al., 2005a,b; Valerio et al., 2006; Oinuma et al., 2007). In PC12 cells, nerve growth factor rapidly (<3 min) increased lamellipodia formation via PI3K pathway (Posern et al., 2000), when activation of nerve growth factor receptor p140trk and p75NGF rapidly caused Na+ influx-dependent membrane depolarization (Shimazu et al., 2005). Taken together, these results implicate that sodium channel activity contributes to PI3K/GSK-3b-dependent outgrowth of growth cone. In neuronal cells, tau was predominantly distributed in axon, with its level progressively increasing toward growth cone (Black et al., 1996), where tau was indispensable to the establishment and maintenance of axon-dendrite neuronal polarity (Billingsley and Kincaid, 1997; Johnson and Stoothoff, 2004). In addition to the well-documented interaction between tau’s C-terminus and tubulin, tau’s N-terminus has been unveiled to interact with Src homology 3 domains of various proteins (e.g., PI3K; PLC; Src; Fyn); tau phosphorylation regulated the tau’s ability to form heteroprotein complex (Lee, 2005; Reynolds et al., 2008), where the catalytic activity of Src or Fyn was enhanced (Sharma et al., 2007). In PC12 cells, Brandt et al. (1995) demonstrated that tau’s Nterminus bound to growth cone membrane in a microtubuleindependent manner, which was indispensable to nerve growth factor-induced neuronal process formation. In Chinese hamster ovary (CHO) cells, expression study by Leroy et al. (2000) showed that tau increased outgrowth of neuronal processes; the outgrowth was inhibited by GSK-3b-catalyzed phosphorylation of tau; in contrast, graded inhibition of GSK-3b activity by LiCl (0.1–25 mM) gradually restored the tau-induced outgrowth, depending on the concentration of LiCl. In developing rat cultured hippocampal neurons, Mandell and Banker (1996) showed that tau phosphorylation level decreased in proximo-distal gradient; 80% phosphorylated at proximal axon, but only 20% phosphorylated at growth cone. Reynolds et al. (2008) showed that phosphorylation level of tau regulated tau’s function as a scaffolding protein; when tau was phosphorylated by GSK-3b, binding of tau to Src homology 3 domains of various proteins (e.g., PI3K; PLC; Fyn) was decreased, such that no binding occurred with hyperphosphorylated tau isolated from post-mortem human brains of Alzheimer’s disease, compared to those of healthy individuals. Sodium channel activity engraves neuronal architecture in normal development and repairs remyelination after neuronal injury (Wada, 2006). In cultured sympathetic neuron of newborn rat, local depolarization of growing neurite promoted its differentiation into axon (Singh and Miller, 2005). In zebrafish retinal ganglion neuron, sodium channel activity increased arbor length

and branch number of growth cone (Hua et al., 2005). In extending growth cone of cultured Xenopus spinal neuron, electrical stimulation converted netrin-1-induced repulsion into attraction in young culture, while enhancing netrin-1-induced attraction in older culture (Ming et al., 2001). In growth cone of older culture, electrical stimulation converted myelin-associated glycoproteininduced repulsion into attraction (Ming et al., 2001). In growth cone of Xenopus laevis spinal neuron, Nishiyama et al. (2008) documented that diffusible guidance molecules (e.g., semaphorin 3A; brain-derived neurotrophic factor) caused attraction via sodium channel activation, while causing repulsion via chloride channel activation; exquisitely, clamping to depolarization converted semaphorin 3A-induced repulsion into attraction. Among multiple steps elaborating neuronal circuit, formation of axondendrite neuronal polarity and growth cone navigation are the initial and most crucial events. Our present finding that Nav1.7 sodium channel activity decreased tau phosphorylation may provide a new avenue for understanding neuronal activitydependent neuronal development and reparation. Acknowledgements We thank Keiko Kawabata and Shoko Tokashiki for technical and secretarial assistance. This study was supported in part by a Grant-in-Aid for The 21st Century COE (Centers of Excellence) Program (Life science), Scientific Research (B) (to AW 30131949); and Scientific Research (C), Young Scientists (A) (to TY 60295227); from the Ministry of Education, Culture, Sports, Science and Technology, Japan. References Bae, Y.S., Cantley, L.G., Chen, C.-S., Kim, S.-R., Kwon, K.-S., Rhee, S.G., 1998. Activation of phospholipase C-g by phosphatidylinositol 3,4,5-trisphposphate. J. Biol. Chem. 273, 4465–4469. Billingsley, M.L., Kincaid, R.L., 1997. Regulated phosphorylation and dephosphorylation of tau protein: effects on microtubule interaction, intracellular trafficking and neurodegeneration. Biochem. J. 323, 577–591. Black, M.M., Slagher, T., Moshiach, S., Obrocka, M., Fischer, I., 1996. Tau is enriched on dynamic microtubules in the distal region of growing axons. J. Neurosci. 16, 3601–3619. Brandt, R., Le´ger, J., Lee, G., 1995. Interaction of tau with the neural plasma membrane mediated by tau’s amino-terminal projection domain. J. Cell Biol. 131, 1327–1340. Corvol, J.-C., Valjent, E., Toutant, M., Enslen, H., Irinopoulou, T., Lev, S., Herve´, D., Girault, J.-A., 2005. Depolarization activates ERK and proline-rich tyrosine kinase 2 (PYK2) independently in different cellular compartments in hippocampal slices. J. Biol. Chem. 280, 660–668. D’Arcangelo, G., Paradiso, K., Shepherd, D., Brehm, P., Halegoua, S., Mandel, G., 1993. Neuronal growth factor regulation of two different sodium channel types through distinct signal transduction pathways. J. Cell Biol. 122, 915–921. Falasca, M., Logan, S.K., Lehto, V.P., Baccante, G., Lemmon, M.A., Schlessinger, J., 1998. Activation of phospholipase Cg by PI 3-kinase-induced PH domainmediated membrane targeting. EMBO J. 17, 414–422. Farrar, N.R., Spencer, G.E., 2008. Pursuing a ‘‘turning point’’ in growth cone research. Dev. Biol. 318, 102–111. Geering, B., Cutillas, P.R., Vanhaesebroeck, B., 2007. Regulation of class IA PI3Ks: is there a role for monomeric PI3K subunits? Biochem. Soc. Trans. 35 (Pt 2), 199– 203. Hua, J.Y., Smear, M.C., Baier, H., Smith, S.J., 2005. Regulation of axon growth in vivo by activity-based competition. Nature 434, 1022–1026. Jiang, H., Guo, W., Liang, X., Rao, Y., 2005. Both the establishment and the maintenance of neuronal polarity require active mechanisms: critical roles of GSK3b and its upstream regulators. Cell 120, 123–135. Johnson, G.V.W., Stoothoff, W.H., 2004. Tau phosphorylation in neuronal cell function and dysfunction. J. Cell Sci. 117, 5721–5729. Kawaguchi, A., Asano, H., Matsushita, K., Wada, T., Yoshida, S., Ichida, S., 2007. Enhancement of sodium current in NG108-15 cells during neural differentiation is mainly due to an increase in Nav1.7 expression. Neurochem. Res. 32, 1469–1475. Laurino, L., Wang, X.X., de la Houssaye, B.A., Sosa, L., Dupraz, S., Ca´ceres, A., Pfenninger, K.H., Quiroga, S., 2005. PI3K activation by IGF-1 is essential for the regulation of membrane expansion at the nerve growth cone. J. Cell Sci. 118, 3653–3663. Lee, G., 2005. Tau and src family tyrosine kinases. Biochim. Biophys. Acta 1739, 323– 330.

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