Ethanol, a teratogen, results in central nervous system (CNS) dysfunction when developmental exposure occurs. One consequence of early ethanol exposure is irreversible depletion of neurons. However, the molecular mechanisms underlying ethanol teratogenecity remain unknown. In searching for target genes of ethanol, we identified that a gene encoding the ribosomal large subunit protein (rpL7a) is ethanol-responsive. At physiologically relevant concentrations, ethanol up-regulates the expression of rpL7a in cultured neuronal cells as well as in the developing cerebellum. The expression of rpL7a is dynamically regulated during neuronal development and ectopic over-expression of rpL7a activates p53 and induces neuronal apoptosis. Ribosomal proteins participate in various extra-ribosomal activities; rpL7a is clearly one of the ribosomal proteins involved in multiple extra-ribosomal functions. Our hypothesis is that ethanol-induced ectopic over-expression of rpL7a causes neuronal death and disruption of cell cycle kinetics. Three sets of in vitro experiments (Specific Aims 1-3) will be performed to test this hypothesis.
In Specific Aim 1, we will determine whether ectopic over-expression of rpL7a alone affects neuronal survival and proliferation. Inducible rpL7a expression will be precisely controlled to a level similar to ethanol-induced alteration. Subsequently, the effect of ectopic over-expression of rpL7a on cell survival and cell cycle kinetics will be examined.
In Specific Aim 2, we will determine whether ethanol-induced damages are mediated by rpL7a, rpL7a will be knocked down using RNA interference. The effect of ethanol on survival and cell cycle kinetics will be examined in the cells with suppressed expression of rpL7a.
In Specific Aim 3, we will investigate the mechanisms by which rpL7a mediates ethanol-induced damages. Ectopic over-expression of rpL7a activates p53. We will determine the role of rpL7a in ethanol modulation of p53 activation. As a unit, the series of experiments will explore the novel function of rpL7a in ethanol-induced neuronal damages. The favorable outcome of proposed studies will help to identify a therapeutic target to ameliorate alcohol neurotoxicity. Furthermore, the studies will provide an important insight into the mechanisms of extra-ribosomal function of ribosomal proteins.
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| Luo, Jia (2005) The role of matrix metalloproteinases in the morphogenesis of the cerebellar cortex. Cerebellum 4:239-45 |
