Small molecule inhibitors of glycogen synthase kinase-3 beta modulate radioprotection in developing hippocampus    Free

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Neurocognitive deficits are devastating consequences in cancer patients undergoing cranial irradiation due to the damage of hippocampus. In our recent studies we have shown that lithium prevents radiation-induced apoptosis in hippocampal neurons leading to the improved cognitive performance of irradiated mice. One of the molecular mechanisms of lithium neuroprotection involves inhibition of Glycogen Synthase Kinase-3 beta(GSK-3β). In the present study we demonstrate that small molecule inhibitors of GSK-3β could be used to protect hippocampal neurons from the damaging effects of radiation. We utilized GSK-3β down-stream targets β-catenin and cyclin D1 to monitor GSK-3β activity. Based on the immunoblot analysis of β-catenin accumulation in the cytosol of mouse hippocampal neurons HT-22, the optimal effective concentration of GSK-3β inhibitors SB415286, SB216763, AR-AO14418 and BIO was 25µM, 10µM, 1µM and 1µM respectively. The optimal time of treatment and was 16 hours for all four inhibitors tested. In case of combined treatment, HT-22 cells pretreated with the inhibitors and irradiated with 3 Gy also demonstrated increased accumulation of β-catenin. However, only pretreatment with SB415286 showed increased accumulation of both β-catenin and cyclin D1, which was similar to the protective effect of lithium. This protective effect was confirmed in clonogenic studies. Pretreatment of HT-22 with SB415286 or AR-AO14418 for 16 hours before irradiation showed significantly increased cell survival as compared to cells treated with radiation alone. This phenomenon was not observed in glioblastoma cell lines D54 or GL-261. To elucidate the mechanism of this selective increased survival in normal hippocampal cells we monitored for program cell death by Annexin V-FITC or DAPI staining. Irradiated HT-22 cells which were pretreated with SB415286 demonstrated 2 fold decrease in apoptosis than irradiated cells. Similar results were obtained using irradiated mice. Seven-days old C57BL6J pups were either irradiated with the single dose of 7 Gy or treated with SB415286 (1mg/Kg) followed by irradiation. SB415286 pretreatment protected hippocampal neurons from radiation-induced apoptosis as demonstrated by TUNEL staining. We confirmed the specificity of the protective effects of the small molecule GSK-3β inhibitors with genetic inhibition of the endogenous GSK-3β activity by expressing the kinase inactive GSK-3β in a bicistronic construct functioning as a dominant negative. In conclusion, small molecule inhibitors of GSK-3β protect normal hippocampal neurons but not the glioblastoma cells from radiation damage by preventing radiation-induced apoptosis in cell culture and in vivo. The GSK-3β inhibitors could be valuable pharmacological compound in protecting from radiation-induced neurological defects.