Cyanidin-3-Glucoside Reverses Ethanol-Induced Inhibition of Neurite Outgrowth: Role of Glycogen Synthase Kinase 3 Beta

Ethanol is a potent teratogen for the developing central nervous system (CNS), and fetal alcohol syndrome (FAS) is the most common nonhereditary cause of mental retardation. Ethanol disrupts neuronal differentiation and maturation. It is important to identify agents that provide neuroprotection against ethanol neurotoxicity. Using an in vitro neuronal model, mouse Neuro2a (N2a) neuroblastoma cells, we demonstrated that ethanol inhibited neurite outgrowth and the expression of neurofilament (NF) proteins. Glycogen synthase kinase 3β (GSK3β), a multifunctional serine/threonine kinase negatively regulated neurite outgrowth of N2a cells; inhibiting GSK3β activity by retinoic acid (RA) and lithium induced neurite outgrowth, while over-expression of a constitutively active S9A GSK3β mutant prevented neurite outgrowth. Ethanol inhibited neurite outgrowth by activating GSK3β through the dephosphorylation of GSK3β at serine 9. Cyanidin-3-glucoside (C3G), a member of the anthocyanin family rich in many edible berries and other pigmented fruits, enhanced neurite outgrowth by promoting p-GSK3β(Ser9). More importantly, C3G reversed ethanol-mediated activation of GSK3β and inhibition of neurite outgrowth as well as the expression of NF proteins. C3G also blocked ethanol-induced intracellular accumulation of reactive oxygen species (ROS). However, the antioxidant effect of C3G appeared minimally involved in its protection. Our study provides a potential avenue for preventing or ameliorating ethanol-induced damage to the developing CNS.