Ethanol decreases Glial Derived Neurotrophic Factor (GDNF) protein releasebut not mRNA expression and increases GDNF-stimulated Shc phosphorylation in the developing cerebellum

Background: Ethanol exposure during development leads to substantial neuron al loss in multiple regions of the brain. Although differentiating Purkinje cells of the cerebellum are particularly vulnerable to ethanol exposure, t he mechanisms underlying ethanol-induced Purkinje cell loss have not been w ell defined. Our previous research indicated that exogenous Glial-Derived N eurotrophic Factor (GDNF) attenuated ethanol-induced Purkinje cell loss in cerebellar explant cultures, which suggests that ethanol, in turn, may decr ease endogenous trophic factor-mediated survival mechanisms. Methods: The present experiments used an explant culture model of the devel oping rat cerebellum to test the hypothesis that ethanol decreases endogeno us trophic support by limiting the availability of trophic factors, such as GDNF, or by altering the activation of key adapter proteins such as Shc (S rc homology domain carboxy-terminal) that couple GDNF binding to multiple i ntracellular signaling pathways. GDNF mRNA and protein levels were measured by reverse northern blot analysis and sandwich enzyme-linked immunosorbent assay respectively, whereas Shc phosphorylation was measured by immunoprec ipitation/western immunoblot analysis. Results: The developing cerebellum expresses both GDNF mRNA and protein in vitro. Ethanol exposure (68, 103, or 137 mM) had no effect on cerebellar le vels of GDNF mRNA. However, ethanol (68 and 137 mM) decreased levels of GDN F protein released into culture medium. In addition, ethanol itself had no effect on Shc phosphorylation. However, in the presence of the highest dose of ethanol (137 mM) GDNF did stimulate Shc phosphorylation. Conclusions: Together, these results suggest that ethanol decreases GDNF-me diated trophic support of Purkinje cells in the developing cerebellum. Howe ver, GDNF in turn activates intracellular signaling pathways throughout the developing cerebellum as part of its Purkinje cell-selective neuroprotecti ve response to ethanol exposure.