Effects of ethanol on transforming growth factor B1-dependent and -independent mechanisms of neural stem cell apoptosis

Stem cell vitality is critical for the growth of the developing brain. Growth factors can define the survival of neural stem cells (NSCs) and ethanol can affect growth factor-mediated activities. The present study tested two hypotheses: (a) ethanol causes the apoptotic death of NSCs and (b) this effect is influenced by the ambient growth factor. Monolayer cultures of non-immortalized NS-5 cells were exposed to fibroblast growth factor (FGF) 2 or transforming growth factor (TGF) β1 in the absence or presence of ethanol for 48. h. Ethanol killed NSCs as measured by increases in the numbers of ethidium bromide+ and annexin V+ cells and decreases in the number of calcein AM+ (viable) cells. These toxic effects were promoted by TGFβ1. A quantitative polymerase chain reaction array of apoptosis-related mRNAs revealed an ethanol-induced increase (≥ 2-fold change; p< 0.05) in transcripts involved in Fas ligand (FasL) and tumor necrosis factor (TNF) signaling. These effects, particularly the FasL pathway, were potentiated by TGFβ1. Immunocytochemical analyses of NS-5 cells showed that transcriptional alterations translated into consistent up-regulation of protein expression. Experiments with the neocortical proliferative zones harvested from fetal mice exposed to ethanol showed that ethanol activated similar molecular systems in vivo. Thus, ethanol induces NSC death through two distinct molecular mechanisms, one is initiated by TGFβ1 (FasL) and another (through TNF) which is TGFβ1-independent.