Figure 7.

STEP increases neuronal vulnerability to excitotoxic cell death. A, Hippocampal neurons isolated from embryonic rat pups were transfected with EGFP expression vector and either a STEP expression vector or an empty expression vector (pcDNA). Two days after transfection, cells were stimulated with NMDA (50 μm) for 15 min, and cell viability was quantified 8 h later using Hoechst staining. A total of 326 pcDNA- and 366 STEP-transfected cells were examined. Arrows identify representative transfected cells that are dying (condensed nuclei); arrowheads denote healthy cells. Scale bar, 50 μm. B, Quantitation of the percentage of transfected neurons undergoing cell death. Relative to control vector transfection, transfection with STEP increased the toxic effects of NMDA (***p < 0.001, significantly different from control vector transfection). C, STEP attenuates MAPK-dependent gene expression. Neurons were transfected with Gal4-ELK and E1B-luciferase reporter and either the STEP expression vector or empty expression vector (pcDNA). Cells were stimulated (15 min) with NMDA (20 μm) or TPA (1 μm) and lysed 6 h later. Both NMDA and TPA stimulated a significant increase in MAPK-dependent reporter gene expression. STEP overexpression blocked both NMDA- and TPA-mediated gene expression. Pretreatment (30 min) with FK506 (1 μm) blocked the repressive effects of STEP. The values are means ± SEM of quadruplicate determinations and are expressed as fold stimulation normalized to the unstimulated pcDNA control, which was set equal to one. ***p < 0.001, significant difference relative to control buffer treatment.