In a series of studies supported by this grant, we have demonstrated that the anti-apoptotic Bcl-2 family member Bcl-XL plays a particularly important role in promoting neuron survival. Massive apoptotic death of immature neurons is seen in the Bcl-XL-deficient embryonic nervous system and this effect is prevented by concomitant genetic disruption of bax, apaf-1, caspase-9, or caspase-3. Although the downstream effectors of Bcl-XL and Bax-dependent neuronal death have been largely defined, the critical upstream regulators of this pathway have not. We hypothesize that two members of the pro-apoptotic BH3 domain-only Bcl-2 subfamily, Bim and DPS, act in concert to regulate Bcl-XL:Bax-dependent neuron apoptosis. To critically test this hypothesis, we generated mice carrying various combinations of genetic disruptions in bcl-x, bim, and dp5. Our preliminary studies have demonstrated that both Bim-deficient and DPS-deficient embryos exhibit slightly decreased neuronal programmed cell death and that this effect is markedly enhanced by dual deficiency of Bim and DPS. Interestingly, DPS deficiency significantly reduced immature neuron death throughout the Bcl-XL-deficient embryonic nervous system while Bim deficiency selectively rescued neurons in the trigeminal ganglion. Preliminary studies of telencephalic neurons in vitro suggest that bim and dp5 mRNA expression is regulated by neurotrophic factor signaling.
In specific aim one, we will define the neurodevelopmental significance of the Bim/DPS death pathway and characterize the molecular mechanism(s) controlling its activation.
In specific aim two, we will determine the in vivo relevance of Bim and DPS in acute ethanol-induced neuron apoptosis in the neonatal mouse brain, a model of human fetal alcohol syndrome, and define the signaling events regulating bim and dp5 expression following ethanol exposure. We have previously shown that acute ethanol neurotoxicity is regulated by the Bcl-XL:Bax- dependent apoptotic pathway and is, at least in part, due to ethanol's NMDA receptor antagonist properties. We now have preliminary in vitro data showing that Bim deficiency inhibits the neuronal death inducing action of MK801, a potent NMDA receptor antagonist. In total, these studies will yield new insights into the molecular pathways regulating both physiological and pathological neuron death.
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