Ethanol Decreases Glial Derived Neurotrophic Factor (GDNF) Protein Release but Not mRNA Expression and Increases GDNF-Stimulated Shc Phosphorylation in the Developing Cerebellum

Background: Ethanol exposure during development leads to substantial neuronal loss in multiple regions of the brain. Although differentiating Purkinje cells of the cerebellum are particularly vulnerable to ethanol exposure, the mechanisms underlying ethanol-induced Purkinje cell loss have not been well defined. Our previous research indicated that exogenous Glial-Derived Neurotrophic Factor (GDNF) attenuated ethanol-induced Purkinje cell loss in cerebellar explant cultures, which suggests that ethanol, in turn, may decrease endogenous trophic factor-mediated survival mechanisms. Methods: The present experiments used an explant culture model of the developing rat cerebellum to test the hypothesis that ethanol decreases endogenous trophic support by limiting the availability of trophic factors, such as GDNF, or by altering the activation of key adapter proteins such as Shc (Src homology domain carboxy-terminal) that couple GDNF binding to multiple intracellular 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 immunoprecipitation/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 levels of GDNF mRNA. However, ethanol (68 and 137 mM) decreased levels of GDNF 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-mediated trophic support of Purkinje cells in the developing cerebellum. However, GDNF in turn activates intracellular signaling pathways throughout the developing cerebellum as part of its Purkinje cell-selective neuroprotective response to ethanol exposure.