Methylcobalamin increases Erk1/2 and Akt activities through the methylation cycle and promotes nerve regeneration in a rat sciatic nerve injury model

https://doi.org/10.1016/j.expneurol.2009.12.017Get rights and content

Abstract

Methylcobalamin is a vitamin B12 analog and is necessary for the maintenance of the nervous system. Although some previous studies have referred to the effects of methylcobalamin on neurons, the precise mechanism of this effect remains obscure. Here we show that methylcobalamin at concentrations above 100 nM promotes neurite outgrowth and neuronal survival and that these effects are mediated by the methylation cycle, a metabolic pathway involving methylation reactions. We also demonstrate that methylcobalamin increases Erk1/2 and Akt activities through the methylation cycle. In a rat sciatic nerve injury model, continuous administration of high doses of methylcobalamin improves nerve regeneration and functional recovery. Therefore, methylcobalamin may provide the basis for better treatments of nervous disorders through effective systemic or local delivery of high doses of methylcobalamin to target organs.

Introduction

Vitamin B12 (cobalamin) is important for normal functioning of the nervous system and its deficiency causes a systemic neuropathy called subacute combined degeneration of the spinal cord (Scalabrino et al., 1990). Vitamin B12 has analogs such as cyanocobalamin (CNCbl), methylcobalamin (MeCbl), hydroxocobalamin (OHCbl), and adenosylcobalamin (AdoCbl). In mammalian cells, CNCbl and OHCbl are inactive forms, AdoCbl acts as a coenzyme of methylmaronyl Co-A mutase in mitochondria, and MeCbl acts as a coenzyme of methionine synthase (MS), which is required for the formation of methionine from homocysteine in the methylation cycle that involves methylation of DNA or proteins (Banerjee and Ragsdale, 2003, Ghosh et al., 1991, Pfohl-Leszkowicz et al., 1991, Toohey, 2006). There are reports that vitamin B12, including MeCbl, has a beneficial effect on the nervous system. In vitro, the vitamin B complex, including vitamin B12, promotes neurite outgrowth and vitamin B12-enriched medium produces the greatest mean neurite outgrowth (Fujii et al., 1996). MeCbl protects cortical neuron and retinal cell cultures against glutamate cytotoxity (Akaike et al., 1993, Kikuchi et al., 1997). In in vivo studies, high doses of MeCbl improved nerve conduction and regeneration in a rat sciatic nerve injury model (Yamatsu et al., 1976a, Yamatsu et al., 1976b), streptozotocin-diabetic rats (Sonobe et al., 1988), and experimental acrylamide neuropathy (Watanabe et al., 1994). MeCbl promoted regeneration of motor nerve terminals that were degenerating in the anterior gracile muscle of the gracile axonal dystrophy mutant mouse (Yamazaki et al., 1994). Despite these previous reports about the effects of vitamin B12, including MeCbl, on neurons, the most effective analog and its mechanism of action on neurons remains to be clarified.

In the nervous system, the protein kinases Erk1/2 and Akt are activated by certain neurotrophins, such as nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) (Segal, 2003). Activation of Erk1/2 promotes neurite outgrowth and Akt mediates branching of neurites in dorsal root ganglion (DRG) neurons (Markus et al., 2002). The survival of cerebellar granule neurons (CGN) is promoted by the activation of Erk1/2 (Bonni et al., 1999) and Akt (Bhave et al., 1999). The activation of Erk1/2 and Akt induced by the FGL peptide (a fibroblast growth factor receptor agonist) results in neurite outgrowth and neuronal survival in primary rat neurons (Neiiendam et al., 2004).

In this study, we demonstrate that MeCbl is the most effective of several vitamin B12 analogs in promoting neurite outgrowth and neuronal survival with activation of Erk1/2 and Akt and that these effects are brought about through the methylation cycle. We also show that MeCbl promotes nerve regeneration and functional recovery in a rat model of sciatic nerve injury.

Section snippets

Cell culture

DRG neurons were cultured as described previously (Higuchi et al., 2003). DRG were obtained from Wistar rats at postnatal day (P9) and dissociated by incubation with 0.25% trypsin, 0.1% collagenase, 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

MeCbl promotes neurite outgrowth in CGN and DRG neurons

Analogs of vitamin B12 include CNCbl, MeCbl, OHCbl, and AdoCbl, with each possessing different metabolic characteristics. We performed neurite outgrowth assays with these analogs in CGN to determine the analog with the greatest effect on neurite outgrowth. Axonal length and total neurite length in the control group were 124.1 ± 6.0 μm and 191.7 ± 7.8 μm, respectively; values were higher in the MeCbl group: 206.7 ± 9.6 μm and 291.8 ± 12.5 μm, respectively (Figs. 1a, c, f, and g). Furthermore, values in

Discussion

MeCbl is an analog of vitamin B12, and vitamin B12 has various effects in the nervous system. Recent reports show that vitamin B12 is involved in some neurotrophic actions. When the central nervous system is deficient in vitamin B12, levels of epidermal growth factor, glial fibrillary acidic protein, and p75 neurotrophin receptor are decreased, and tumor necrosis factor α is increased (Magnaghi et al., 2002, Scalabrino et al., 2003, Scalabrino et al., 1999, Scalabrino et al., 2000). The

Acknowledgment

This work was supported by Grant-in-Aid for Exploratory Research.

References (59)

  • SatoY. et al.

    Amelioration by mecobalamin of subclinical carpal tunnel syndrome involving unaffected limbs in stroke patients

    J. Neurol. Sci.

    (2005)
  • ScalabrinoG. et al.

    New insights into the pathophysiology of cobalamin deficiency

    Trends Mol. Med.

    (2006)
  • TemporinK. et al.

    IL-1beta promotes neurite outgrowth by deactivating RhoA via p38 MAPK pathway

    Biochem. Biophys. Res. Commun.

    (2008)
  • TemporinK. et al.

    Interleukin-1 beta promotes sensory nerve regeneration after sciatic nerve injury

    Neurosci. Lett.

    (2008)
  • WangM. et al.

    A small molecule inhibitor of isoprenylcysteine carboxymethyltransferase induces autophagic cell death in PC3 prostate cancer cells

    J. Biol. Chem.

    (2008)
  • WatanabeT. et al.

    Ultra-high dose methylcobalamin promotes nerve regeneration in experimental acrylamide neuropathy

    J. Neurol. Sci.

    (1994)
  • WoltersM. et al.

    Cobalamin: a critical vitamin in the elderly

    Prev. Med.

    (2004)
  • YamazakiK. et al.

    Methylcobalamin (methyl-B12) promotes regeneration of motor nerve terminals degenerating in anterior gracile muscle of gracile axonal dystrophy (GAD) mutant mouse

    Neurosci. Lett.

    (1994)
  • YaqubB.A. et al.

    Effects of methylcobalamin on diabetic neuropathy

    Clin. Neurol. Neurosurg.

    (1992)
  • AisenP.S. et al.

    High-dose B vitamin supplementation and cognitive decline in Alzheimer disease: a randomized controlled trial

    JAMA

    (2008)
  • AkaikeA. et al.

    Protective effects of a vitamin B12 analog, methylcobalamin, against glutamate cytotoxicity in cultured cortical neurons

    Eur. J. Pharmacol.

    (1993)
  • AndresE. et al.

    Oral cobalamin (vitamin B(12)) treatment. An update

    Int. J. Lab. Hematol.

    (2009)
  • BanerjeeR. et al.

    The many faces of vitamin B12: catalysis by cobalamin-dependent enzymes

    Annu. Rev. Biochem.

    (2003)
  • BanksE.C. et al.

    Inhibition of cobalamin-dependent methionine synthase by substituted benzo-fused heterocycles

    FEBS J.

    (2007)
  • BergoM.O. et al.

    Inactivation of Icmt inhibits transformation by oncogenic K-Ras and B-Raf

    J. Clin. Invest.

    (2004)
  • BhaveS.V. et al.

    Brain-derived neurotrophic factor mediates the anti-apoptotic effect of NMDA in cerebellar granule neurons: signal transduction cascades and site of ethanol action

    J. Neurosci.

    (1999)
  • BoiseL.H. et al.

    Bcl-x(L) can inhibit apoptosis in cells that have undergone Fas-induced protease activation

    Proc. Natl. Acad. Sci. U. S. A.

    (1997)
  • BonniA. et al.

    Cell survival promoted by the Ras-MAPK signaling pathway by transcription-dependent and -independent mechanisms

    Science

    (1999)
  • EhrlichM.

    Expression of various genes is controlled by DNA methylation during mammalian development

    J. Cell. Biochem.

    (2003)
  • Cited by (131)

    • B vitamins on the nervous system: A focus on peripheral neuropathy

      2023, Vitamins and Minerals in Neurological Disorders
    • Upregulation of neuronal progranulin mediates the antinociceptive effect of trimetazidine in paclitaxel-induced peripheral neuropathy: Role of ERK1/2 signaling

      2022, Toxicology and Applied Pharmacology
      Citation Excerpt :

      This effect was markedly abolished by co-treatment with an ERK inhibitor (Li et al., 2017). Likewise, a vitamin B12 analog increased the number of myelinated axons and promoted sensory and motor functional recovery, in part, via ERK1/2 activation after SNI in rats (Okada et al., 2010). Additionally, PD98059 diminished numbers of proliferating Schwann cells and inhibited axonal regeneration typically boosted by treadmill training after SNI in rats (Seo et al., 2021, 2009).

    View all citing articles on Scopus

    Methylcobalamin promotes nerve regeneration.

    View full text