Signaling Cascades And Alcohol-Induced Neural Precursor Cell Loss

Background: Fetal alcohol syndrome(FAS) is an irreversible condition occurring in some children of mothers who have consumed alcohol during pregnancy. It is characterized mainly by craniofacial malformations, Physical and mental retardation, cardiac septal defects and minor joint abnormalities. Prenatal alcohol exposure is the leading known cause of mental retardation in the United States. Children with Fetal Alcohol Syndrome have significantly smaller brain size and thinner cerebral cortex than normal, resulting from decreases in the total number of cells. Evidence from ethanol-treated rats has shown that prenatal ethanol exposure depresses cell proliferation in the neuroepithelium of the developing cerebral cortex. The neuroepithelium is mainly composed of neural stem cells and progenitors, collectively called neural precursor cells in this proposal. Ethanol was also found to trigger excessive cell death in gastrulation and neurulation stage embryos, where neural stem cells successively form the neural plate and neural tube. Recent evidence from in vitro studies of ethanol's effects on cortical precursor cells, and cerebellar precursor cells has further confirmed that ethanol-induced cell loss is caused by two simultaneous mechanisms: cell cycle delay and excessive cell death. However, the molecular and cellular mechanisms underlying the ethanol-impaired neural precursor cell proliferation are poorly understood.Objective: To establish a method of adherent culture of neural precursor cells in vitro. Using it to study ethanol's effects on the proliferation of neural precursor cells. To research the molecular and cellular mechanisms underlying the ethanol-induced damage to neural stem cell proliferation and survival. To define molecular targets of ethanol's effects on mAChR-mediated signaling pathways which play critical roles in controlling neural precursor cell proliferation. Methods:1. Establish An in vitro model of rat neuroepithelium: Neural precursor cells were dissociated from the cerebral cortex of E13 rats and maintained in serum-free defined medium containing basic fibroblast growth factor (bFGF) and Epidermal Growth Factor (EGF), then fluorescence immunocytochemistry was applied to identify the neural precursor cells and their ability of proliferation after 5 days culture.2. To examine ethanol's effects on cell proliferation, cell viability ,apoptosis and necrosis. However, to assess the mitogenic effects of muscarinic stimulation, and the effects of pharmacological inhibitors and ethanol's effects on cell proliferation, the cultures will be deprived of bFGF for 24 h before exposure to these drugs. Then the neuroepithelial cells were exposed to different concentrations of ethanol for 24 h, and brdu incorporation , cell counting and MTT assays were employed to study the effects of ethanol on the proliferation of neural precursor cells, and PI / ho.33342 staining was used to study ethanol's effects on cell viability and apoptosis.3. To define molecular targets of ethanol's effects on mAChR-mediated signaling pathways which play critical roles in controlling neural precursor cell proliferation. To define these targets, we will evaluate ethanol's effects on muscarinic-induced kinase expression and activity using flow cytometry.Results:1. The purity of the neural stem cells which had been cultured for 5 days was 97%, and most cells were nestin-positive and possessed the ability of proliferation and self-renewal. When deprived of bFGF and EGF, neural stem cells in vitro are capable of dividing and differentiating into neurons and astrocytes.2. The concentration(25-100mM) of ethanol can inhibit the proliferation of neural precursor cells in a concentration-dependent manner.4. The concentration(0.5～2mM) of acetaldehyde have not significant effect on the proliferation of neural precursor cells, but high dose of acetaldehyde (8mM) decreased cell viability markedly. 5. Carbachol is an agonist of mAchRs, withdrawal of bFGF, cabachol could stimulate neuroepithelial-cell proliferation above the level of autonomous proliferation.6. Ethanol and MEK inhibitor PD98059 inhibit carbachol-induced neural precursor cell proliferation.7. We quantified the levels of phosphorylated forms of ERK1 and ERK2 using FCM. While 100 mM ethanol caused a modest, but significant reduction in levels of carbachol-activated phosphorylated ERKs. In contrast, carbachol-induced MAPK activity was markedly antagonized by addition of the MEK inhibitor PD98059.Conclusion:1. We have established a method of adherent culture of neural precursor cells in vitro which could be used for the further related research.2. Ethanol can inhibit the proliferation of the neural precursor cells.3. Ethanol induced cell loss studied in vitro using PI/Ho.33342 double staining.4. Acetaldehyde decreased the cell viability of neural precursor cells.5. Carbachol stimulated neural precursor cell proliferation dramatically, while ethanol and MEK inhibitor PD98059 inhibits the proliferation.6. Ethanol inhibits CCh-stimulated MAPK phosphorylation.7 MAChR-mediated signaling pathways play an important role in the proliferation of neural precursor cells, and ethanol inhibits neural precursor cell proliferation through disrupting muscarinic-activated signaling pathways.