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