- Email: [email protected]
mammalian target of rapamycin signaling pathway regulates neurite outgrowth in cerebellar granule neurons stimulated by methylcobalamin
Neuroscience Letters 495 (2011) 201–204
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Akt/mammalian target of rapamycin signaling pathway regulates neurite outgrowth in cerebellar granule neurons stimulated by methylcobalamin Kiyoshi Okada a,b,∗ , Hiroyuki Tanaka c , Ko Temporin d , Michio Okamoto a , Yusuke Kuroda a , Hisao Moritomo a , Tsuyoshi Murase a , Hideki Yoshikawa a a
Department of Orthopaedics, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan Medical Center for Translational Research, Osaka University Hospital, 2-15 Yamadaoka, Suita, Osaka 565-0871, Japan c Department of Orthopaedic Surgery, Osaka Koseinenkin Hospital, 4-2-8 Fukushima, Osaka City, Osaka 553-0003, Japan d Department of Orthopaedic Surgery, Toyonaka Municipal Hospital, 4-14-1 Shibaharatyou, Toyonaka City, Osaka 560-8566, Japan b
a r t i c l e
i n f o
Article history: Received 24 December 2010 Received in revised form 15 March 2011 Accepted 21 March 2011 Keywords: Akt Mammalian target or rapamycin Methylcobalamin Neurite outgrowth
a b s t r a c t Methylcobalamin (MeCbl), a vitamin B12 analog, promotes neurite outgrowth by activating Akt in neurons. However, Akt is involved in many cellular functions, and the downstream signal of Akt that promotes neurite outgrowth in neurons in the presence of MeCbl remains obscure. Mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase that regulates multiple cellular functions including neurite outgrowth. mTOR is regarded as important for the regeneration of injured nerves. In this study, we examined the relationship between MeCbl and mTOR activity and found that MeCbl increases mTOR activity via the activation of Akt and promotes neurite outgrowth in cerebellar granule neurons via the activation of mTOR. © 2011 Elsevier Ireland Ltd. All rights reserved.
Vitamin B12 is regarded as an important factor for maintaining the function and health of central and peripheral nerves [30]. Methylcobalamin (MeCbl), an analog of vitamin B12, has been adopted to treat diseases such as diabetic neuropathy [34] and other nervous disorders [7,8]. However, the specific effects and mechanism of MeCbl on nerves remain obscure. In a previous report, we suggested that MeCbl promotes neurite outgrowth via the activation of Akt in cerebellar granule neurons (CGNs) [19]. However, Akt is involved in many cellular functions such as neurogenesis or cell survival [24,35], and thus the specific downstream signal of Akt that promotes neurite outgrowth in neurons in the presence of MeCbl remains to be elucidated.
Abbreviations: ANOVA, one way analysis of variance; CGNs, cerebellar granule neurons; DMEM, Dulbecco’s modified Eagle’s medium; Icmt, isoprenylcysteine carboxyl methyltransferase; MeCbl, methylcobalamin; mTOR, mammalian target of rapamycin; p70S6K, p70 ribosomal S6 protein kinase; PI3K, phosphoinositide-3’ kinase; Rheb, Ras homolog enriched in brain; SEM, standard error of the mean; TSC, tuberous sclerosis complex protein; TuJ1, neural class III -tubulin. ∗ Corresponding author at: Department of Orthopaedics, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan. Tel.: +81 668793552; fax: +81 668793559. E-mail addresses: [email protected] (K. Okada), [email protected] (H. Tanaka), [email protected] (K. Temporin), [email protected] (M. Okamoto), [email protected] (Y. Kuroda), [email protected] (H. Moritomo), [email protected] (T. Murase), [email protected] (H. Yoshikawa). 0304-3940/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2011.03.065
Mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase that regulates multiple cellular functions including neurite outgrowth. Modulation of the mTOR pathway promotes axon regeneration in the central nervous system [20] and also increases the axonal growth capacity of injured peripheral nerves [1]. Thus, mTOR is known to be an important signal for the regeneration of damaged nerves in recent years. Because mTOR is a well-known Akt substrate, we examined the relationship between MeCbl and mTOR activity in this study. For CGN culture, the cerebellum was removed from Wistar rats on postnatal day (P9) as described previously [15] and dissociated by incubation with 0.25% trypsin and 200 U/ml DNase I for 30 min at 37 ◦ C. The enzymatic reaction was blocked by adding Dulbecco’s modified Eagle’s medium (DMEM, not including vitamin B12) containing 10% fetal bovine serum, and after trituration and centrifugation, the cells were resuspended in modified Sato medium (DMEM containing 5 g/ml insulin, 20 nM progesterone, 100 M putrescine, 30 nM sodium selenite, 0.1 g/ml l-thyroxine, 0.08 g/ml triiodo-l-thyronine, and 4 mg/ml bovine serum albumin). Subsequently, the cells were centrifuged and resuspended in modified Sato medium, and then plated on poly-l-lysine-coated culture dishes or four-well chamber slides. The neurons plated on the four-well chamber slides were fixed with 4% paraformaldehyde for 30 min, blocked for 1 h, and incubated overnight at 4 ◦ C with primary antibody, followed by incubation for 1 h at room temperature with secondary antibody as described previously [29], and nuclei were labeled with DAPI (Wako
202
K. Okada et al. / Neuroscience Letters 495 (2011) 201–204
Pure Chemical Industries, Osaka, Japan). The primary antibody was anti-neural class III -tubulin (TuJ1) mouse monoclonal antibody (1:1000; Covance, Berkeley, CA), and the secondary antibodies were Alexa 488-labeled goat anti-mouse IgG antibody (1:1000; Molecular Probes, Eugene, OR) or Alexa 568-labeled goat anti-mouse IgG antibody (1:1000; Molecular Probes). Neurons were cultured with MeCbl (10 M; Sigma–Aldrich, St. Louis, MO), LY294002 (50 M; Cell Signaling Technology, Beverly, MA), Akt inhibitor (5 M; Calbiochem, Darmstadt, Germany) or rapamycin (200 nM; Sigma–Aldrich) in the presence of the caspase inhibitor Z-VAD-FMK (20 M; Promega Corporation, Madison, WI), which were applied to inhibit the apoptosis of neurons caused by the addition of LY294002. The cells were cultured for 72 h and then immunostained with anti-TuJ1 antibody. The axonal length (the length of the longest neurite per TuJ1-positive neuron) was measured using an image analyzer (Lumina Vision; Mitani Co., Fukui, Japan) as described previously [28]. Only neurites that were longer than 20 m (approximately the diameter of a soma) and not in contact with other cells were measured to avoid the effect of apoptosis and cell contact [22]. The mean axonal length was calculated using at least 30 neurons in each experiment [19]. CGNs were cultured with MeCbl (10 M), LY204002 (50 M), or rapamycin (200 nM). After 10 min, cultured cells were homogenized with Kaplan buffer (50 mM Tris [pH 7.4], 150 mM NaCl, 10% glycerol, 1% NP40 and protease inhibitor cocktail) and clarified by centrifugation. Subsequently, they were separated by 12% SDS–PAGE and transferred to polyvinylidene difluoride membranes. After blocking with 5% skim milk for 1 h, the membranes were incubated with anti-Akt rabbit polyclonal antibody (1:1000; Cell Signaling Technology), anti-phospho-PKB (phospho-
Akt) (pSer473) rabbit polyclonal antibody (1:1000; Sigma–Aldrich), anti-mTOR rabbit monoclonal antibody (1:1000; Cell Signaling Technology), anti-phospho-mTOR (Ser2448) rabbit monoclonal antibody (1:1000; Cell Signaling Technology), anti-p70 ribosomal S6 protein kinase (p70S6K) rabbit monoclonal antibody (1:1000; Cell Signaling Technology) or anti-phospho-p70S6K (Thr389) rabbit monoclonal antibody (1:1000; Cell Signaling Technology) at 4 ◦ C overnight, followed by incubation with horseradish peroxidaseconjugated secondary antibody (1:1000; GE Healthcare, Little Chalfont, UK) and ECL reagents (GE Healthcare). The densities of Akt, phospho-Akt, mTOR, phospho-mTOR, p70S6K and phopho-p70S6K were measured using Scion Image software (Scion Corporation, Frederick, MD). To calculate the normalized density, we divided the density of phospho-Akt, phospho-mTOR or phophop70S6K based on the density of Akt, mTOR, or p70S6K, respectively, in the same membrane. Data are expressed as mean ± standard error of the mean (SEM). Statistical evaluation was performed by one-way ANOVA and post hoc Scheffe’s test using StatView software, version 5.0 (SAS Institute, Cary, NC). To determine whether mTOR activity is related with the neurite outgrowth facilitated by MeCbl, we performed neurite outgrowth assays with LY294002 [26], which is an inhibitor of phosphoinositide-3’ kinase (PI3K) and blocks PI3K-dependent Akt activation, Akt inhibitor [5], and rapamycin, which is an inhibitor of mTOR [27] (Fig. 1). MeCbl increased axonal length from 183.9 ± 11.1 m (mean ± SEM) to 320.8 ± 16.0 m in CGNs (Fig. 1A and B). LY294002 did not decrease the axonal length of neurons (158.8 ± 7.0 m) but inhibited the effect of MeCbl on neurite outgrowth (158.6 ± 8.7 m), which was determined from the
Fig. 1. MeCbl promotes neurite outgrowth via the activation of Akt and mTOR. (A) CGNs from P9 rats were cultured for 72 h with MeCbl, LY294002, Akt inhibitor and/or rapamycin. Fluorescence micrographs from each group are shown. White arrows indicate the longest axon measured in this experiment. (B) The axonal length after culturing with MeCbl, LY294002, Akt inhibitor and/or rapamycin was measured from fluorescence micrographs. Significance was determined by one-way ANOVA and the post hoc Scheffe’s test. Error bars represent SEM. Results are representative of three independent experiments. * p < 0.05 compared with control group.
K. Okada et al. / Neuroscience Letters 495 (2011) 201–204
203
Fig. 2. MeCbl increases the activity of Akt and mTOR. CGNs from P9 rats were cultured for 10 min in the presence or absence of MeCbl, LY294002, or rapamycin. The activity of Akt (A), mTOR (B), and p70S6K (C) was detected by western blotting. Quantification of Akt (A), mTOR (B), and p70S6K (C) activity as the normalized density is shown below each figure of Western blots. Significance was determined by one-way ANOVA and the post hoc Scheffe’s test. Error bars represent SEM. Results are representative of three independent experiments. * p < 0.05 compared with control group.
axonal length in the LY + MeCbl group. Akt inhibitor and rapamycin decreased axonal length to 116.7 ± 4.8 m and 121.8 ± 5.3 m, which were not affected by the addition of MeCbl (110.6 ± 4.0 m and 133.9 ± 5.9 m). Based on these results, it is suggested that MeCbl promotes neurite outgrowth via the activation of Akt and mTOR. According to the result of the neurite outgrowth assay, MeCbl facilitated neurite outgrowth in CGNs, and this effect could not have occurred under the inactivation of Akt and mTOR. Thus, we attempted to detect the activities of Akt and mTOR in CGNs in the presence of MeCbl by Western blotting (Fig. 2). Based on the evaluation of Akt and mTOR activities, MeCbl produced a 3.3 ± 0.5-fold stronger activation of Akt (Fig. 2A) and a 3.0 ± 0.5-fold stronger activation of mTOR (Fig. 2B) than that observed in the control group. LY294002 decreased the activities of Akt and mTOR, and MeCbl did not increase the activities of Akt and mTOR in the presence of LY294002. The addition of rapamycin, which did not influence the Akt activity, inhibited the MeCbl-induced activation of mTOR. In addition, to reveal whether MeCbl exactly increased the activity of mTOR or not, we examined the phosphorylation of p70S6K, which was directly phosphorylated by activated mTOR [17]. MeCbl promoted a 2.0 ± 0.1-fold stronger activation of p70S6K than that observed in control group, which was inhibited by LY294002 and rapamycin (Fig. 2C). These results suggest that MeCbl activates Akt and mTOR, and Akt is an upstream signal of mTOR in CGNs stimulated by MeCbl. In this study, we investigated the mechanism of neurite outgrowth in neurons stimulated by MeCbl, a vitamin B12 analog. Vitamin B12 (cobalamin) is important for the maintenance of nerves, and its deficiency causes a systemic neuropathy called subacute combined degeneration of the spinal cord [23]. Vitamin B12 has analogs such as cyanocobalamin, MeCbl, hydroxocobalamin, and adenosylcobalamin. In mammalian cells, cyanocobalamin and hydroxocobalamin are inactive forms, adenosylcobalamin acts as a coenzyme of methylmalonyl CoA mutase, and MeCbl acts as a coenzyme of methionine synthase in a specific metabolic cycle called the methylation cycle. Adenosylcobalamin is mainly distributed in the liver, and MeCbl is mainly distributed in the serum and spinal fluid. Thus, MeCbl is considered the most effective analog of vitamin B12 pertaining to the nervous system. In previous reports, MeCbl protected cortical neuron and retinal cell cultures against glutamate cytotoxicity [2,10]. In in vivo studies, high doses of MeCbl improved nerve conduction in streptozotocin-diabetic rats [25] and in experimental acrylamide-induced neuropathy [32]. MeCbl promoted the regeneration of motor nerve terminals that were degenerating in
the anterior gracile muscle of the gracile axonal dystrophy mutant mouse [33]. In a previous study, we demonstrated that MeCbl was the most effective of all vitamin B12 analogs tested for promoting neurite outgrowth and neuronal survival in CGNs and DRG neurons [19]. MeCbl is necessary for the formation of methionine from homocysteine, and this metabolic process is involved in the methylation cycle, which is related to certain methylation reactions [30]. In a previous report, we suggested that MeCbl at concentration ≥100 nM promoted neurite outgrowth and neuronal survival through this cycle including S-adenosylmethionine, which was a downstream metabolite of MeCbl and also promoted neurite outgrowth and neuronal survival [19]. In addition, we suggested that MeCbl activated Erk1/2 and Akt through the methylation cycle, and these signals were necessary for neurite outgrowth and neuronal survival. The specific signal for neurite outgrowth in neurons stimulated by MeCbl was previously obscure. To address this issue, we revealed that MeCbl promoted neurite outgrowth in CGNs via the Akt/mTOR signaling pathway in this study. mTOR is the master regulator of several cell functions and is crucial for neuronal development and long-term modification of synaptic strength [9]. The pathway of mTOR activation leads from some receptor tyrosine kinases such as tyrosine receptor kinase B through the induction of PI3K and Akt kinases to the phosphorylation and inhibition of tuberous sclerosis complex proteins (TSC1 and TSC2). TSC1/TSC2 complex acts as a GTPase-activating protein for Ras homolog enriched in brain (Rheb). The inhibition of TSC1/TSC2 complex elevates GTP-bound Rheb, which stimulates the phophorylation of mTOR. Rapamycin is a bacterial natural product, which binds directly to the binding domain of mTOR and inhibits its phosphorylation [6]. mTOR is believed to act primarily by phophorylating eIF-4E binding protein and p70S6K, which are important regulators of protein translation. mTOR regulates neuronal polarity through Rap1B and the local translation of CRMP2 and tau [11,16]. In recent reports, mTOR promoted axonal regeneration in the adult central nervous system and increased the axonal growth of injured peripheral nerves [1,20]. In our experiments, MeCbl increased the activation of mTOR and p70S6K (Fig. 2), while rapamycin, an inhibitor of mTOR, decreased the effect of MeCbl on neurite outgrowth (Fig. 1). These results implicated mTOR as the main signal of neurite outgrowth in neurons stimulated by MeCbl. Akt is the main upstream regulator of mTOR in many cellular functions [9], and in our experiments, the inhibition of PI3K-dependent Akt activation by LY294002 decreased the activation of mTOR (Fig. 2) and both LY294002 and Akt inhibitor blocked
204
K. Okada et al. / Neuroscience Letters 495 (2011) 201–204
neurite outgrowth facilitated by MeCbl (Fig. 1). Based on these results, we believe that MeCbl increases the phosphorylation of mTOR and neurite outgrowth through PI3K and Akt. In addition, mTOR regulates not only neurite outgrowth but also myelination of the central nervous system [17]. In previous in vivo experiments, MeCbl increased remyelination in rat sciatic nerve injury models [19]. In clinical studies, decreased levels of vitamin B12 were found in patients with multiple sclerosis, which causes the demyelination of nerves [14]. Thus, we considered the possibility that MeCbl also controls the myelination of nerve tissues through mTOR. The major question for the signaling pathways in neurons affected by MeCbl is how MeCbl, which is involved in some methylation reactions, activates specific protein kinases such as Akt and mTOR. The methylation reactions include two major pathways: DNA methylation and protein methylation. DNA methylation is an important cellular phenomenon that promotes genetic stability in various species [18]. However, if MeCbl regulates the activities of Akt or mTOR by DNA methylation, the regulatory mechanism must require at least 1 h because of the time required to methylate DNA and produce the specific protein [21]. In our study, MeCbl activated mTOR 10 min after its addition (Fig. 2). Thus, it is unlikely that MeCbl increased the activity of mTOR via DNA methylation. Protein methylation is known to stabilize certain protein functions. One carboxyl methyltransferase related to the methylation of certain proteins, isoprenylcysteine carboxyl methyltransferase (Icmt), acts on proteins formed via precursors and synthesized with a Cterminal CAAX tetrapeptide motif (C is cysteine; A is generally an aliphatic residue; X is a variable residue) [4]. Methylation of the cysteine residue of the C-terminal CAAX motif of the protein has been shown to be important for the localization, stability, and functions of proteins [12]. Ras, which is upstream of Akt in its signaling pathway [13], is a CAAX protein, and its methylation by Icmt is very important for its function [3]. Furthermore, methylation of Ras regulates the Akt and mTOR signaling pathways [31]. Therefore, based on these previous reports, we propose the hypothesis that MeCbl increases the methylation of Ras and promotes its function, and Ras activates mTOR through activation of Akt, which facilitates neurite outgrowth. There is little evidence to support such a hypothesis, but we suggest the possibility that MeCbl promotes neurite outgrowth via the methylation of upstream proteins of Akt such as Ras. In this study, we found that the Akt/mTOR signaling pathway controls neurite outgrowth facilitated by MeCbl. We believe that MeCbl has a great potential to treat several nervous disorders, and further investigation of the effects of MeCbl may reveal new insights into nerve regeneration and the treatment of several nervous diseases and injuries.
[6] [7] [8] [9] [10]
[11] [12] [13] [14] [15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24] [25]
[26]
[27]
Acknowledgments This work was supported by a Grant-in-Aid for Exploratory Research, a Grant-in-Aid for Research Activity Start-up, and the General Insurance Association of Japan.
[28]
[29]
[30]
References [1] N. Abe, S.H. Borson, M.J. Gambello, F. Wang, V. Cavalli, Mammalian target of rapamycin (mTOR) activation increases axonal growth capacity of injured peripheral nerves, J. Biol. Chem. 285 (2010) 28034–28043. [2] A. Akaike, Y. Tamura, Y. Sato, T. Yokota, Protective effects of a vitamin B12 analog, methylcobalamin, against glutamate cytotoxicity in cultured cortical neurons, Eur. J. Pharmacol. 241 (1993) 1–6. [3] M.O. Bergo, B.J. Gavino, C. Hong, A.P. Beigneux, M. McMahon, P.J. Casey, S.G. Young, Inactivation of Icmt inhibits transformation by oncogenic K-Ras and B-Raf, J. Clin. Invest. 113 (2004) 539–550. [4] M.A. Grillo, S. Colombatto, S-adenosylmethionine and protein methylation, Amino Acids 28 (2005) 357–362. [5] Y. Hu, L. Qiao, S. Wang, S.B. Rong, E.J. Meuillet, M. Berggren, A. Gallegos, G. Powis, A.P. Kozikowski, 3-(Hydroxymethyl)-bearing phosphatidylinositol ether lipid
[31]
[32]
[33]
[34] [35]
analogues and carbonate surrogates block PI3-K, Akt, and cancer cell growth, J. Med. Chem. 43 (2000) 3045–3051. S. Huang, M.A. Bjornsti, P.J. Houghton, Rapamycins: mechanism of action and cellular resistance, Cancer Biol. Ther. 2 (2003) 222–232. Y. Izumi, R. Kaji, Clinical trials of ultra-high-dose methylcobalamin in ALS, Brain Nerve 59 (2007) 1141–1147. M.A. Jalaludin, Methylcobalamin treatment of Bell’s palsy, Method Find. Exp. Clin. Pharmacol. 17 (1995) 539–544. J. Jaworski, M. Sheng, The growing role of mTOR in neuronal development and plasticity, Mol. Neurobiol. 34 (2006) 205–219. M. Kikuchi, S. Kashii, Y. Honda, Y. Tamura, K. Kaneda, A. Akaike, Protective effects of methylcobalamin, a vitamin B12 analog, against glutamate-induced neurotoxicity in retinal cell culture, Invest. Ophthalmol. Vis. Sci. 38 (1997) 848–854. Y.H. Li, H. Werner, A.W. Puschel, Rheb and mTOR regulate neuronal polarity through Rap1B, J. Biol. Chem. 283 (2008) 33784–33792. T. Magee, M.C. Seabra, Fatty acylation and prenylation of proteins: what’s hot in fat, Curr. Opin. Cell Biol. 17 (2005) 190–196. A. Markus, J. Zhong, W.D. Snider, Raf and akt mediate distinct aspects of sensory axon growth, Neuron 35 (2002) 65–76. A. Miller, M. Korem, R. Almog, Y. Galboiz, Vitamin B12, demyelination, remyelination and repair in multiple sclerosis, J. Neurol. Sci. 233 (2005) 93–97. F Mimura, S. Yamagishi, N. Arimura, M. Fujitani, T. Kubo, K. Kaibuchi, T. Yamashita, Myelin-associated glycoprotein inhibits microtubule assembly by a Rho-kinase-dependent mechanism, J. Biol. Chem. 281 (2006) 15970–15979. T. Morita, K. Sobue, Specification of neuronal polarity regulated by local translation of CRMP2 and Tau via the mTOR-p70S6K pathway, J. Biol. Chem. 284 (2009) 27734–27745. S.P. Narayanan, A.I. Flores, F. Wang, W.B. Macklin, Akt signals through the mammalian target of rapamycin pathway to regulate CNS myelination, J. Neurosci. 29 (2009) 6860–6870. E.D. Nelson, E.T. Kavalali, L.M. Monteggia, Activity-dependent suppression of miniature neurotransmission through the regulation of DNA methylation, J. Neurosci. 28 (2008) 395–406. K. Okada, H. Tanaka, K. Temporin, M. Okamoto, Y. Kuroda, H. Moritomo, T. Murase, H. Yoshikawa, Methylcobalamin increases Erk1/2 and Akt activities through the methylation cycle and promotes nerve regeneration in a rat sciatic nerve injury model, Exp. Neurol. 222 (2010) 191–203. K.K. Park, K. Liu, Y. Hu, P.D. Smith, C. Wang, B. Cai, B. Xu, L. Connolly, I. Kramvis, M. Sahin, Z. He, Promoting axon regeneration in the adult CNS by modulation of the PTEN/mTOR pathway, Science 322 (2008) 963–966. A. Pfohl-Leszkowicz, G. Keith, G. Dirheimer, Effect of cobalamin derivatives on in vitro enzymatic DNA methylation: methylcobalamin can act as a methyl donor, Biochemistry 30 (1991) 8045–8051. S.L. Rankin, C.S. Guy, K.M. Mearow, Neurite outgrowth is enhanced by lamininmediated down-regulation of the low affinity neurotrophin receptor, p75NTR, J. Neurochem. 107 (2008) 799–813. G Scalabrino, B. Monzio-Compagnoni, M.E. Ferioli, E.C. Lorenzini, E. Chiodini, R. Candiani, Subacute combined degeneration and induction of ornithine decarboxylase in spinal cords of totally gastrectomized rats, Lab. Invest. 62 (1990) 297–304. N. Shioda, F. Han, K. Fukunaga, Role of Akt and ERK signaling in the neurogenesis following brain ischemia, Int. Rev. Neurobiol. 85 (2009) 375–387. M. Sonobe, H. Yasuda, I. Hatanaka, M. Terada, M. Yamashita, R. Kikkawa, Y. Shigeta, Methylcobalamin improves nerve conduction in streptozotocindiabetic rats without affecting sorbitol and myo-inositol contents of sciatic nerve, Horm. Metab. Res. 20 (1988) 717–718. S. Subramaniam, N. Shahani, J. Strelau, C. Laliberte, R. Brandt, D. Kaplan, K. Unsicker, Insulin-like growth factor 1 inhibits extracellular signalregulated kinase to promote neuronal survival via the phosphatidylinositol 3-kinase/protein kinase A/c-Raf pathway, J. Neurosci. 25 (2005) 2838–2852. L. Swiech, M. Perycz, A. Malik, J. Jaworski, Role of mTOR in physiology and pathology of the nervous system, Biochim. Biophys. Acta 1784 (2008) 116–132. H. Tanaka, T. Yamashita, M. Asada, S. Mizutani, H. Yoshikawa, M. Tohyama, Cytoplasmic p21(Cip1/WAF1) regulates neurite remodeling by inhibiting Rhokinase activity, J. Cell Biol. 158 (2002) 321–329. K. Temporin, H. Tanaka, Y. Kuroda, K. Okada, K. Yachi, H. Moritomo, T. Murase, H. Yoshikawa, IL-1beta promotes neurite outgrowth by deactivating RhoA via p38 MAPK pathway, Biochem. Biophys. Res. Commun. 365 (2008) 375–380. J.I. Toohey, Vitamin B12 and methionine synthesis: a critical review. Is nature’s most beautiful cofactor misunderstood? Biofactors 26 (2006) 45–57. M. Wang, W. Tan, J. Zhou, J. Leow, M. Go, H.S. Lee, P.J. Casey, A small molecule inhibitor of isoprenylcysteine carboxymethyltransferase induces autophagic cell death in PC3 prostate cancer cells, J. Biol. Chem. 283 (2008) 18678–18684. T. Watanabe, R. Kaji, N. Oka, W. Bara, J. Kimura, Ultra-high dose methylcobalamin promotes nerve regeneration in experimental acrylamide neuropathy, J. Neurol. Sci. 122 (1994) 140–143. K. Yamazaki, K. Oda, C. Endo, T. Kikuchi, T. Wakabayashi, Methylcobalamin (methyl-B12) promotes regeneration of motor nerve terminals degenerating in anterior gracile muscle of gracile axonal dystrophy (GAD) mutant mouse, Neurosci. Lett. 170 (1994) 195–197. B.A. Yaqub, A. Siddique, R. Sulimani, Effects of methylcobalamin on diabetic neuropathy, Clin. Neurol. Neurosurg. 94 (1992) 105–111. H. Zhao, R.M. Sapolsky, G.K. Steinberg, Phosphoinositide-3-kinase/akt survival signal pathways are implicated in neuronal survival after stroke, Mol. Neurobiol. 34 (2006) 249–270.
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Akt/mammalian target of rapamycin signaling pathway regulates neurite outgrowth in cerebellar granule neurons stimulated by methylcobalamin Kiyoshi Okada a,b,∗ , Hiroyuki Tanaka c , Ko Temporin d , Michio Okamoto a , Yusuke Kuroda a , Hisao Moritomo a , Tsuyoshi Murase a , Hideki Yoshikawa a a
Department of Orthopaedics, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan Medical Center for Translational Research, Osaka University Hospital, 2-15 Yamadaoka, Suita, Osaka 565-0871, Japan c Department of Orthopaedic Surgery, Osaka Koseinenkin Hospital, 4-2-8 Fukushima, Osaka City, Osaka 553-0003, Japan d Department of Orthopaedic Surgery, Toyonaka Municipal Hospital, 4-14-1 Shibaharatyou, Toyonaka City, Osaka 560-8566, Japan b
a r t i c l e
i n f o
Article history: Received 24 December 2010 Received in revised form 15 March 2011 Accepted 21 March 2011 Keywords: Akt Mammalian target or rapamycin Methylcobalamin Neurite outgrowth
a b s t r a c t Methylcobalamin (MeCbl), a vitamin B12 analog, promotes neurite outgrowth by activating Akt in neurons. However, Akt is involved in many cellular functions, and the downstream signal of Akt that promotes neurite outgrowth in neurons in the presence of MeCbl remains obscure. Mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase that regulates multiple cellular functions including neurite outgrowth. mTOR is regarded as important for the regeneration of injured nerves. In this study, we examined the relationship between MeCbl and mTOR activity and found that MeCbl increases mTOR activity via the activation of Akt and promotes neurite outgrowth in cerebellar granule neurons via the activation of mTOR. © 2011 Elsevier Ireland Ltd. All rights reserved.
Vitamin B12 is regarded as an important factor for maintaining the function and health of central and peripheral nerves [30]. Methylcobalamin (MeCbl), an analog of vitamin B12, has been adopted to treat diseases such as diabetic neuropathy [34] and other nervous disorders [7,8]. However, the specific effects and mechanism of MeCbl on nerves remain obscure. In a previous report, we suggested that MeCbl promotes neurite outgrowth via the activation of Akt in cerebellar granule neurons (CGNs) [19]. However, Akt is involved in many cellular functions such as neurogenesis or cell survival [24,35], and thus the specific downstream signal of Akt that promotes neurite outgrowth in neurons in the presence of MeCbl remains to be elucidated.
Abbreviations: ANOVA, one way analysis of variance; CGNs, cerebellar granule neurons; DMEM, Dulbecco’s modified Eagle’s medium; Icmt, isoprenylcysteine carboxyl methyltransferase; MeCbl, methylcobalamin; mTOR, mammalian target of rapamycin; p70S6K, p70 ribosomal S6 protein kinase; PI3K, phosphoinositide-3’ kinase; Rheb, Ras homolog enriched in brain; SEM, standard error of the mean; TSC, tuberous sclerosis complex protein; TuJ1, neural class III -tubulin. ∗ Corresponding author at: Department of Orthopaedics, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan. Tel.: +81 668793552; fax: +81 668793559. E-mail addresses: [email protected] (K. Okada), [email protected] (H. Tanaka), [email protected] (K. Temporin), [email protected] (M. Okamoto), [email protected] (Y. Kuroda), [email protected] (H. Moritomo), [email protected] (T. Murase), [email protected] (H. Yoshikawa). 0304-3940/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2011.03.065
Mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase that regulates multiple cellular functions including neurite outgrowth. Modulation of the mTOR pathway promotes axon regeneration in the central nervous system [20] and also increases the axonal growth capacity of injured peripheral nerves [1]. Thus, mTOR is known to be an important signal for the regeneration of damaged nerves in recent years. Because mTOR is a well-known Akt substrate, we examined the relationship between MeCbl and mTOR activity in this study. For CGN culture, the cerebellum was removed from Wistar rats on postnatal day (P9) as described previously [15] and dissociated by incubation with 0.25% trypsin and 200 U/ml DNase I for 30 min at 37 ◦ C. The enzymatic reaction was blocked by adding Dulbecco’s modified Eagle’s medium (DMEM, not including vitamin B12) containing 10% fetal bovine serum, and after trituration and centrifugation, the cells were resuspended in modified Sato medium (DMEM containing 5 g/ml insulin, 20 nM progesterone, 100 M putrescine, 30 nM sodium selenite, 0.1 g/ml l-thyroxine, 0.08 g/ml triiodo-l-thyronine, and 4 mg/ml bovine serum albumin). Subsequently, the cells were centrifuged and resuspended in modified Sato medium, and then plated on poly-l-lysine-coated culture dishes or four-well chamber slides. The neurons plated on the four-well chamber slides were fixed with 4% paraformaldehyde for 30 min, blocked for 1 h, and incubated overnight at 4 ◦ C with primary antibody, followed by incubation for 1 h at room temperature with secondary antibody as described previously [29], and nuclei were labeled with DAPI (Wako
202
K. Okada et al. / Neuroscience Letters 495 (2011) 201–204
Pure Chemical Industries, Osaka, Japan). The primary antibody was anti-neural class III -tubulin (TuJ1) mouse monoclonal antibody (1:1000; Covance, Berkeley, CA), and the secondary antibodies were Alexa 488-labeled goat anti-mouse IgG antibody (1:1000; Molecular Probes, Eugene, OR) or Alexa 568-labeled goat anti-mouse IgG antibody (1:1000; Molecular Probes). Neurons were cultured with MeCbl (10 M; Sigma–Aldrich, St. Louis, MO), LY294002 (50 M; Cell Signaling Technology, Beverly, MA), Akt inhibitor (5 M; Calbiochem, Darmstadt, Germany) or rapamycin (200 nM; Sigma–Aldrich) in the presence of the caspase inhibitor Z-VAD-FMK (20 M; Promega Corporation, Madison, WI), which were applied to inhibit the apoptosis of neurons caused by the addition of LY294002. The cells were cultured for 72 h and then immunostained with anti-TuJ1 antibody. The axonal length (the length of the longest neurite per TuJ1-positive neuron) was measured using an image analyzer (Lumina Vision; Mitani Co., Fukui, Japan) as described previously [28]. Only neurites that were longer than 20 m (approximately the diameter of a soma) and not in contact with other cells were measured to avoid the effect of apoptosis and cell contact [22]. The mean axonal length was calculated using at least 30 neurons in each experiment [19]. CGNs were cultured with MeCbl (10 M), LY204002 (50 M), or rapamycin (200 nM). After 10 min, cultured cells were homogenized with Kaplan buffer (50 mM Tris [pH 7.4], 150 mM NaCl, 10% glycerol, 1% NP40 and protease inhibitor cocktail) and clarified by centrifugation. Subsequently, they were separated by 12% SDS–PAGE and transferred to polyvinylidene difluoride membranes. After blocking with 5% skim milk for 1 h, the membranes were incubated with anti-Akt rabbit polyclonal antibody (1:1000; Cell Signaling Technology), anti-phospho-PKB (phospho-
Akt) (pSer473) rabbit polyclonal antibody (1:1000; Sigma–Aldrich), anti-mTOR rabbit monoclonal antibody (1:1000; Cell Signaling Technology), anti-phospho-mTOR (Ser2448) rabbit monoclonal antibody (1:1000; Cell Signaling Technology), anti-p70 ribosomal S6 protein kinase (p70S6K) rabbit monoclonal antibody (1:1000; Cell Signaling Technology) or anti-phospho-p70S6K (Thr389) rabbit monoclonal antibody (1:1000; Cell Signaling Technology) at 4 ◦ C overnight, followed by incubation with horseradish peroxidaseconjugated secondary antibody (1:1000; GE Healthcare, Little Chalfont, UK) and ECL reagents (GE Healthcare). The densities of Akt, phospho-Akt, mTOR, phospho-mTOR, p70S6K and phopho-p70S6K were measured using Scion Image software (Scion Corporation, Frederick, MD). To calculate the normalized density, we divided the density of phospho-Akt, phospho-mTOR or phophop70S6K based on the density of Akt, mTOR, or p70S6K, respectively, in the same membrane. Data are expressed as mean ± standard error of the mean (SEM). Statistical evaluation was performed by one-way ANOVA and post hoc Scheffe’s test using StatView software, version 5.0 (SAS Institute, Cary, NC). To determine whether mTOR activity is related with the neurite outgrowth facilitated by MeCbl, we performed neurite outgrowth assays with LY294002 [26], which is an inhibitor of phosphoinositide-3’ kinase (PI3K) and blocks PI3K-dependent Akt activation, Akt inhibitor [5], and rapamycin, which is an inhibitor of mTOR [27] (Fig. 1). MeCbl increased axonal length from 183.9 ± 11.1 m (mean ± SEM) to 320.8 ± 16.0 m in CGNs (Fig. 1A and B). LY294002 did not decrease the axonal length of neurons (158.8 ± 7.0 m) but inhibited the effect of MeCbl on neurite outgrowth (158.6 ± 8.7 m), which was determined from the
Fig. 1. MeCbl promotes neurite outgrowth via the activation of Akt and mTOR. (A) CGNs from P9 rats were cultured for 72 h with MeCbl, LY294002, Akt inhibitor and/or rapamycin. Fluorescence micrographs from each group are shown. White arrows indicate the longest axon measured in this experiment. (B) The axonal length after culturing with MeCbl, LY294002, Akt inhibitor and/or rapamycin was measured from fluorescence micrographs. Significance was determined by one-way ANOVA and the post hoc Scheffe’s test. Error bars represent SEM. Results are representative of three independent experiments. * p < 0.05 compared with control group.
K. Okada et al. / Neuroscience Letters 495 (2011) 201–204
203
Fig. 2. MeCbl increases the activity of Akt and mTOR. CGNs from P9 rats were cultured for 10 min in the presence or absence of MeCbl, LY294002, or rapamycin. The activity of Akt (A), mTOR (B), and p70S6K (C) was detected by western blotting. Quantification of Akt (A), mTOR (B), and p70S6K (C) activity as the normalized density is shown below each figure of Western blots. Significance was determined by one-way ANOVA and the post hoc Scheffe’s test. Error bars represent SEM. Results are representative of three independent experiments. * p < 0.05 compared with control group.
axonal length in the LY + MeCbl group. Akt inhibitor and rapamycin decreased axonal length to 116.7 ± 4.8 m and 121.8 ± 5.3 m, which were not affected by the addition of MeCbl (110.6 ± 4.0 m and 133.9 ± 5.9 m). Based on these results, it is suggested that MeCbl promotes neurite outgrowth via the activation of Akt and mTOR. According to the result of the neurite outgrowth assay, MeCbl facilitated neurite outgrowth in CGNs, and this effect could not have occurred under the inactivation of Akt and mTOR. Thus, we attempted to detect the activities of Akt and mTOR in CGNs in the presence of MeCbl by Western blotting (Fig. 2). Based on the evaluation of Akt and mTOR activities, MeCbl produced a 3.3 ± 0.5-fold stronger activation of Akt (Fig. 2A) and a 3.0 ± 0.5-fold stronger activation of mTOR (Fig. 2B) than that observed in the control group. LY294002 decreased the activities of Akt and mTOR, and MeCbl did not increase the activities of Akt and mTOR in the presence of LY294002. The addition of rapamycin, which did not influence the Akt activity, inhibited the MeCbl-induced activation of mTOR. In addition, to reveal whether MeCbl exactly increased the activity of mTOR or not, we examined the phosphorylation of p70S6K, which was directly phosphorylated by activated mTOR [17]. MeCbl promoted a 2.0 ± 0.1-fold stronger activation of p70S6K than that observed in control group, which was inhibited by LY294002 and rapamycin (Fig. 2C). These results suggest that MeCbl activates Akt and mTOR, and Akt is an upstream signal of mTOR in CGNs stimulated by MeCbl. In this study, we investigated the mechanism of neurite outgrowth in neurons stimulated by MeCbl, a vitamin B12 analog. Vitamin B12 (cobalamin) is important for the maintenance of nerves, and its deficiency causes a systemic neuropathy called subacute combined degeneration of the spinal cord [23]. Vitamin B12 has analogs such as cyanocobalamin, MeCbl, hydroxocobalamin, and adenosylcobalamin. In mammalian cells, cyanocobalamin and hydroxocobalamin are inactive forms, adenosylcobalamin acts as a coenzyme of methylmalonyl CoA mutase, and MeCbl acts as a coenzyme of methionine synthase in a specific metabolic cycle called the methylation cycle. Adenosylcobalamin is mainly distributed in the liver, and MeCbl is mainly distributed in the serum and spinal fluid. Thus, MeCbl is considered the most effective analog of vitamin B12 pertaining to the nervous system. In previous reports, MeCbl protected cortical neuron and retinal cell cultures against glutamate cytotoxicity [2,10]. In in vivo studies, high doses of MeCbl improved nerve conduction in streptozotocin-diabetic rats [25] and in experimental acrylamide-induced neuropathy [32]. MeCbl promoted the regeneration of motor nerve terminals that were degenerating in
the anterior gracile muscle of the gracile axonal dystrophy mutant mouse [33]. In a previous study, we demonstrated that MeCbl was the most effective of all vitamin B12 analogs tested for promoting neurite outgrowth and neuronal survival in CGNs and DRG neurons [19]. MeCbl is necessary for the formation of methionine from homocysteine, and this metabolic process is involved in the methylation cycle, which is related to certain methylation reactions [30]. In a previous report, we suggested that MeCbl at concentration ≥100 nM promoted neurite outgrowth and neuronal survival through this cycle including S-adenosylmethionine, which was a downstream metabolite of MeCbl and also promoted neurite outgrowth and neuronal survival [19]. In addition, we suggested that MeCbl activated Erk1/2 and Akt through the methylation cycle, and these signals were necessary for neurite outgrowth and neuronal survival. The specific signal for neurite outgrowth in neurons stimulated by MeCbl was previously obscure. To address this issue, we revealed that MeCbl promoted neurite outgrowth in CGNs via the Akt/mTOR signaling pathway in this study. mTOR is the master regulator of several cell functions and is crucial for neuronal development and long-term modification of synaptic strength [9]. The pathway of mTOR activation leads from some receptor tyrosine kinases such as tyrosine receptor kinase B through the induction of PI3K and Akt kinases to the phosphorylation and inhibition of tuberous sclerosis complex proteins (TSC1 and TSC2). TSC1/TSC2 complex acts as a GTPase-activating protein for Ras homolog enriched in brain (Rheb). The inhibition of TSC1/TSC2 complex elevates GTP-bound Rheb, which stimulates the phophorylation of mTOR. Rapamycin is a bacterial natural product, which binds directly to the binding domain of mTOR and inhibits its phosphorylation [6]. mTOR is believed to act primarily by phophorylating eIF-4E binding protein and p70S6K, which are important regulators of protein translation. mTOR regulates neuronal polarity through Rap1B and the local translation of CRMP2 and tau [11,16]. In recent reports, mTOR promoted axonal regeneration in the adult central nervous system and increased the axonal growth of injured peripheral nerves [1,20]. In our experiments, MeCbl increased the activation of mTOR and p70S6K (Fig. 2), while rapamycin, an inhibitor of mTOR, decreased the effect of MeCbl on neurite outgrowth (Fig. 1). These results implicated mTOR as the main signal of neurite outgrowth in neurons stimulated by MeCbl. Akt is the main upstream regulator of mTOR in many cellular functions [9], and in our experiments, the inhibition of PI3K-dependent Akt activation by LY294002 decreased the activation of mTOR (Fig. 2) and both LY294002 and Akt inhibitor blocked
204
K. Okada et al. / Neuroscience Letters 495 (2011) 201–204
neurite outgrowth facilitated by MeCbl (Fig. 1). Based on these results, we believe that MeCbl increases the phosphorylation of mTOR and neurite outgrowth through PI3K and Akt. In addition, mTOR regulates not only neurite outgrowth but also myelination of the central nervous system [17]. In previous in vivo experiments, MeCbl increased remyelination in rat sciatic nerve injury models [19]. In clinical studies, decreased levels of vitamin B12 were found in patients with multiple sclerosis, which causes the demyelination of nerves [14]. Thus, we considered the possibility that MeCbl also controls the myelination of nerve tissues through mTOR. The major question for the signaling pathways in neurons affected by MeCbl is how MeCbl, which is involved in some methylation reactions, activates specific protein kinases such as Akt and mTOR. The methylation reactions include two major pathways: DNA methylation and protein methylation. DNA methylation is an important cellular phenomenon that promotes genetic stability in various species [18]. However, if MeCbl regulates the activities of Akt or mTOR by DNA methylation, the regulatory mechanism must require at least 1 h because of the time required to methylate DNA and produce the specific protein [21]. In our study, MeCbl activated mTOR 10 min after its addition (Fig. 2). Thus, it is unlikely that MeCbl increased the activity of mTOR via DNA methylation. Protein methylation is known to stabilize certain protein functions. One carboxyl methyltransferase related to the methylation of certain proteins, isoprenylcysteine carboxyl methyltransferase (Icmt), acts on proteins formed via precursors and synthesized with a Cterminal CAAX tetrapeptide motif (C is cysteine; A is generally an aliphatic residue; X is a variable residue) [4]. Methylation of the cysteine residue of the C-terminal CAAX motif of the protein has been shown to be important for the localization, stability, and functions of proteins [12]. Ras, which is upstream of Akt in its signaling pathway [13], is a CAAX protein, and its methylation by Icmt is very important for its function [3]. Furthermore, methylation of Ras regulates the Akt and mTOR signaling pathways [31]. Therefore, based on these previous reports, we propose the hypothesis that MeCbl increases the methylation of Ras and promotes its function, and Ras activates mTOR through activation of Akt, which facilitates neurite outgrowth. There is little evidence to support such a hypothesis, but we suggest the possibility that MeCbl promotes neurite outgrowth via the methylation of upstream proteins of Akt such as Ras. In this study, we found that the Akt/mTOR signaling pathway controls neurite outgrowth facilitated by MeCbl. We believe that MeCbl has a great potential to treat several nervous disorders, and further investigation of the effects of MeCbl may reveal new insights into nerve regeneration and the treatment of several nervous diseases and injuries.
[6] [7] [8] [9] [10]
[11] [12] [13] [14] [15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24] [25]
[26]
[27]
Acknowledgments This work was supported by a Grant-in-Aid for Exploratory Research, a Grant-in-Aid for Research Activity Start-up, and the General Insurance Association of Japan.
[28]
[29]
[30]
References [1] N. Abe, S.H. Borson, M.J. Gambello, F. Wang, V. Cavalli, Mammalian target of rapamycin (mTOR) activation increases axonal growth capacity of injured peripheral nerves, J. Biol. Chem. 285 (2010) 28034–28043. [2] A. Akaike, Y. Tamura, Y. Sato, T. Yokota, Protective effects of a vitamin B12 analog, methylcobalamin, against glutamate cytotoxicity in cultured cortical neurons, Eur. J. Pharmacol. 241 (1993) 1–6. [3] M.O. Bergo, B.J. Gavino, C. Hong, A.P. Beigneux, M. McMahon, P.J. Casey, S.G. Young, Inactivation of Icmt inhibits transformation by oncogenic K-Ras and B-Raf, J. Clin. Invest. 113 (2004) 539–550. [4] M.A. Grillo, S. Colombatto, S-adenosylmethionine and protein methylation, Amino Acids 28 (2005) 357–362. [5] Y. Hu, L. Qiao, S. Wang, S.B. Rong, E.J. Meuillet, M. Berggren, A. Gallegos, G. Powis, A.P. Kozikowski, 3-(Hydroxymethyl)-bearing phosphatidylinositol ether lipid
[31]
[32]
[33]
[34] [35]
analogues and carbonate surrogates block PI3-K, Akt, and cancer cell growth, J. Med. Chem. 43 (2000) 3045–3051. S. Huang, M.A. Bjornsti, P.J. Houghton, Rapamycins: mechanism of action and cellular resistance, Cancer Biol. Ther. 2 (2003) 222–232. Y. Izumi, R. Kaji, Clinical trials of ultra-high-dose methylcobalamin in ALS, Brain Nerve 59 (2007) 1141–1147. M.A. Jalaludin, Methylcobalamin treatment of Bell’s palsy, Method Find. Exp. Clin. Pharmacol. 17 (1995) 539–544. J. Jaworski, M. Sheng, The growing role of mTOR in neuronal development and plasticity, Mol. Neurobiol. 34 (2006) 205–219. M. Kikuchi, S. Kashii, Y. Honda, Y. Tamura, K. Kaneda, A. Akaike, Protective effects of methylcobalamin, a vitamin B12 analog, against glutamate-induced neurotoxicity in retinal cell culture, Invest. Ophthalmol. Vis. Sci. 38 (1997) 848–854. Y.H. Li, H. Werner, A.W. Puschel, Rheb and mTOR regulate neuronal polarity through Rap1B, J. Biol. Chem. 283 (2008) 33784–33792. T. Magee, M.C. Seabra, Fatty acylation and prenylation of proteins: what’s hot in fat, Curr. Opin. Cell Biol. 17 (2005) 190–196. A. Markus, J. Zhong, W.D. Snider, Raf and akt mediate distinct aspects of sensory axon growth, Neuron 35 (2002) 65–76. A. Miller, M. Korem, R. Almog, Y. Galboiz, Vitamin B12, demyelination, remyelination and repair in multiple sclerosis, J. Neurol. Sci. 233 (2005) 93–97. F Mimura, S. Yamagishi, N. Arimura, M. Fujitani, T. Kubo, K. Kaibuchi, T. Yamashita, Myelin-associated glycoprotein inhibits microtubule assembly by a Rho-kinase-dependent mechanism, J. Biol. Chem. 281 (2006) 15970–15979. T. Morita, K. Sobue, Specification of neuronal polarity regulated by local translation of CRMP2 and Tau via the mTOR-p70S6K pathway, J. Biol. Chem. 284 (2009) 27734–27745. S.P. Narayanan, A.I. Flores, F. Wang, W.B. Macklin, Akt signals through the mammalian target of rapamycin pathway to regulate CNS myelination, J. Neurosci. 29 (2009) 6860–6870. E.D. Nelson, E.T. Kavalali, L.M. Monteggia, Activity-dependent suppression of miniature neurotransmission through the regulation of DNA methylation, J. Neurosci. 28 (2008) 395–406. K. Okada, H. Tanaka, K. Temporin, M. Okamoto, Y. Kuroda, H. Moritomo, T. Murase, H. Yoshikawa, Methylcobalamin increases Erk1/2 and Akt activities through the methylation cycle and promotes nerve regeneration in a rat sciatic nerve injury model, Exp. Neurol. 222 (2010) 191–203. K.K. Park, K. Liu, Y. Hu, P.D. Smith, C. Wang, B. Cai, B. Xu, L. Connolly, I. Kramvis, M. Sahin, Z. He, Promoting axon regeneration in the adult CNS by modulation of the PTEN/mTOR pathway, Science 322 (2008) 963–966. A. Pfohl-Leszkowicz, G. Keith, G. Dirheimer, Effect of cobalamin derivatives on in vitro enzymatic DNA methylation: methylcobalamin can act as a methyl donor, Biochemistry 30 (1991) 8045–8051. S.L. Rankin, C.S. Guy, K.M. Mearow, Neurite outgrowth is enhanced by lamininmediated down-regulation of the low affinity neurotrophin receptor, p75NTR, J. Neurochem. 107 (2008) 799–813. G Scalabrino, B. Monzio-Compagnoni, M.E. Ferioli, E.C. Lorenzini, E. Chiodini, R. Candiani, Subacute combined degeneration and induction of ornithine decarboxylase in spinal cords of totally gastrectomized rats, Lab. Invest. 62 (1990) 297–304. N. Shioda, F. Han, K. Fukunaga, Role of Akt and ERK signaling in the neurogenesis following brain ischemia, Int. Rev. Neurobiol. 85 (2009) 375–387. M. Sonobe, H. Yasuda, I. Hatanaka, M. Terada, M. Yamashita, R. Kikkawa, Y. Shigeta, Methylcobalamin improves nerve conduction in streptozotocindiabetic rats without affecting sorbitol and myo-inositol contents of sciatic nerve, Horm. Metab. Res. 20 (1988) 717–718. S. Subramaniam, N. Shahani, J. Strelau, C. Laliberte, R. Brandt, D. Kaplan, K. Unsicker, Insulin-like growth factor 1 inhibits extracellular signalregulated kinase to promote neuronal survival via the phosphatidylinositol 3-kinase/protein kinase A/c-Raf pathway, J. Neurosci. 25 (2005) 2838–2852. L. Swiech, M. Perycz, A. Malik, J. Jaworski, Role of mTOR in physiology and pathology of the nervous system, Biochim. Biophys. Acta 1784 (2008) 116–132. H. Tanaka, T. Yamashita, M. Asada, S. Mizutani, H. Yoshikawa, M. Tohyama, Cytoplasmic p21(Cip1/WAF1) regulates neurite remodeling by inhibiting Rhokinase activity, J. Cell Biol. 158 (2002) 321–329. K. Temporin, H. Tanaka, Y. Kuroda, K. Okada, K. Yachi, H. Moritomo, T. Murase, H. Yoshikawa, IL-1beta promotes neurite outgrowth by deactivating RhoA via p38 MAPK pathway, Biochem. Biophys. Res. Commun. 365 (2008) 375–380. J.I. Toohey, Vitamin B12 and methionine synthesis: a critical review. Is nature’s most beautiful cofactor misunderstood? Biofactors 26 (2006) 45–57. M. Wang, W. Tan, J. Zhou, J. Leow, M. Go, H.S. Lee, P.J. Casey, A small molecule inhibitor of isoprenylcysteine carboxymethyltransferase induces autophagic cell death in PC3 prostate cancer cells, J. Biol. Chem. 283 (2008) 18678–18684. T. Watanabe, R. Kaji, N. Oka, W. Bara, J. Kimura, Ultra-high dose methylcobalamin promotes nerve regeneration in experimental acrylamide neuropathy, J. Neurol. Sci. 122 (1994) 140–143. K. Yamazaki, K. Oda, C. Endo, T. Kikuchi, T. Wakabayashi, Methylcobalamin (methyl-B12) promotes regeneration of motor nerve terminals degenerating in anterior gracile muscle of gracile axonal dystrophy (GAD) mutant mouse, Neurosci. Lett. 170 (1994) 195–197. B.A. Yaqub, A. Siddique, R. Sulimani, Effects of methylcobalamin on diabetic neuropathy, Clin. Neurol. Neurosurg. 94 (1992) 105–111. H. Zhao, R.M. Sapolsky, G.K. Steinberg, Phosphoinositide-3-kinase/akt survival signal pathways are implicated in neuronal survival after stroke, Mol. Neurobiol. 34 (2006) 249–270.