Studies performed during the previous grant period established that the IRS-1 mediated signal transduction pathway was important in promoting cell growth and survival particularly in the context of insulin and IGF-1 stimulation, and that ethanol exposure profoundly inhibits growth factor signaling through this pathway. In addition, we delineated some of the differential effects of ethanol on insulin/IGF-1 stimulated signaling as they pertain to different cell types and cellular functions. In this regard, we observed that ethanol inhibits insulin-stimulated survival but has little adverse effects on DNA synthesis in neuronal cells. This phenomenon was linked to the prominent inhibition of insulin-stimulated IRS-1-PI3K-Akt pathway and relative preservation of downstream signaling through Erk MAPK. In preliminary studies, we found that the IRS-1-PI3K pathway is also an important mediator of neuronal migration, and that the effects are mediated through the aspartyl (asparaginyl)-beta-hydroxylase (AAH) gene, which we isolated. We have demonstrated that expression of the AAH gene is regulated by insulin/IGF-1 stimulated signaling through IRS-1-PI3K, and that ethanol inhibition of neuronal migration may be mediated by its inhibitory effects on insulin/IGF-1 stimulated cyclin-dependent kinase 5 (cdk-5) activity and PI3K-Akt-Gsk-3 phosphorylation. In addition, we have obtained preliminary evidence that the effects of AAH in promoting neuronal migration are mediated through downstream Notch signaling pathways. These observations are particularly exciting because cdk-5 and Notch have known roles in neuronal migration during development. In this application, we propose to utilize chronic gestational and in vitro exposure models in hypothesis-driven experiments that are organized under the following 3 specific aims:
Specific Aim #1 : Evaluate the effects of ethanol on insulin/IGF-1 stimulated signaling in relation to neuronal migration;
Specific Aim #2 : Characterize the regulation of insulin/IGF-1 stimulated AAH expression and AAH-mediated neuronal migration;
Specific Aim #3 : Define the upstream and downstream signaling pathways that mediate neuronal migration and that are inhibited by ethanol. Importantly, the third Specific Aim will link AAH gene expression to upstream signaling through cdk-5, and downstream signaling through Notch, and evaluate the effects of ethanol on these mechanisms. We expect that the results from these investigations will demonstrate the mechanisms by which chronic gestational exposure to ethanol inhibits insulin/IGF-1 stimulated signaling through IRS-1 and its associated downstream effector molecules, leading to impaired neuronal migration during development.
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