Effects of Homocysteine on ERK Signaling and Cell Proliferation in Fetal Neural Stem Cells In Vitro
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ORIGINAL PAPER
Effects of Homocysteine on ERK Signaling and Cell Proliferation in Fetal Neural Stem Cells In Vitro Hai Yan • Xumei Zhang • Suhui Luo • Huan Liu • Xuan Wang • Yuxia Gao • John X. Wilson • Guowei Huang
Published online: 30 October 2012 Ó Springer Science+Business Media New York 2012
Abstract The aim of the present study was to determine if the excitatory amino acid homocysteine (Hcy) alters ERK signaling and cell proliferation in fetal neural stem cells (NSCs) in vitro. NSCs were isolated from fetal rats and grown in serum-free suspension medium. The cells were identified as NSCs by their expression of immunoreactive Sox2. NSCs were assigned to one of four treatment groups: vehicle control, low-dose Hcy group (Hcy-L, medium contained 30 lmol/L Hcy), middle-dose Hcy group (Hcy-M, 100 lmol/L Hcy) and high-dose Hcy group (Hcy-H, 300 lmol/L Hcy). Cell proliferation was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Protein expression levels of ERK1/2 and phosphorylated ERK1/2 were detected by Western blot. The effects of Hcy on NSC death, including apoptosis, were assessed by using flow cytometry and trypan blue exclusion. The results showed that NSCs grew as neurospheres in the serum-free medium. Hcy decreased ERK1/2 protein phosphorylation and NSC proliferation, but it did not induce cell death or apoptosis within the concentration from 30 to 300 lmol/L. The above results are consistent with the hypothesis that Hcy decreases fetal NSC proliferation by inhibiting ERK signaling. H. Yan X. Zhang S. Luo H. Liu X. Wang G. Huang (&) Department of Nutrition and Food Hygiene, School of Public Health, Tianjin Medical University, Tianjin 300070, China e-mail: [email protected] Y. Gao Department of Cardiology, General Hospital of Tianjin Medical University, Tianjin 300152, China J. X. Wilson Department of Exercise and Nutrition Sciences, School of Public Health and Health Professions, University at Buffalo, Buffalo, NY 14214-8028, USA
Keywords Neural stem cells Homocysteine Proliferation ERK signaling In vitro
Introduction Abnormally high concentration of the excitatory amino acid homocysteine (Hcy) is a risk factor for Alzheimer’s disease, neurodegenerative dementias, memory deficit and cognitive impairment without dementia [1]. Hcy can induce neural injury through overstimulation of N-methylD-aspartate receptors [2]. Hcy also sensitizes neurons to excitotoxicity by multiple mechanisms, including DNA damage, poly (ADP-ribose) polymerase activation and p53 induction [3]. Further, experiments with chick embryo have shown that Hcy increases neural crest cell migration from the neural tube and inhibits neural crest cell differentiation [4], while elevated serum Hcy is correlated with embryonic malformations related to neural crest cell development [5]. One cause of elevated Hcy concentration is folate deficiency, because folate is a cofactor for the enzyme (5,10methyenetetralhydrofolate reductase) that converts Hcy to methionine. For example, folate defici
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