Figure 6. Neuroprotection against the TD-induced apoptosis by the activated mutant form of PDK1, PDK1A280V.
A, Cortical neurons were co-electroporated with Flag-RSK1 and either pcDNA3 (vector) or the activated PDK1 mutant PDK1A280V as indicated (3+3µg/10×106 cells). After 17 hours post-electroporation, cells were stimulated with 0 (−) or 10 (+) ng/ml BDNF for 3 hr. At 20 hr post-electroporation, the effects of PDK1A280V or BDNF were analyzed by immunocomplex kinase assay with the immunoprecipitated Flag-RSK1. While similar levels of Flag-RSK1 were detected in all immunoprecipitates (as indicated by the anti-flag western blot shown under the graph), BDNF treatment or PDK1A280V increased Flag-RSK1 NTK activity. B–C, Neurons were co-transfected with expression plasmids for β-gal (EF1αLacZ) or the activated PDK1A280V mutant (0.2 + 1 µg of plasmid DNA/5×105 neurons, respectively). The empty expression vector pcDNA3 was used as a control (Vector). After 24 hours, neurons were sham treated (control) or trophic deprived (TD) with or without 10 ng/ml BDNF, as indicated. Apoptosis was evaluated after next 24 hours. B, Representative photomicrographs of the transfected neurons, which were detected by β-gal immunoreactivity. Transfected neurons with normal chromatin morphology or its apoptotic rearrangements are pointed by arrowheads or arrows, respectively. C, As compared to BDNF, the overexpressed PDK1A280V offered partial protection against TD-induced apoptosis. In A, and C, averages of three independent experiments ±SEM are presented; *** p<0.001.