Figure 7. RSK1/2 is required for PDK1-mediated neuroprotection.

A, Freshly isolated cortical neurons were co-electroporated with indicated shRNA constructs, together with expression plasmids for β-gal (EF1αLacZ) and either FLAG-RSK1 or FLAG-RSK2 as indicated (2.5 + 0.2 + 0.3 µg of each plasmid DNA/5×10⁶ neurons, respectively). If mixed, shRNA plasmids were combined eqimolarly and applied at 2.5 µg of total plasmid DNA/5×10⁶ neurons. The controls included the shRNA vector pSuper and the shRSK2- or shRSK1 mixes for shRSK1 or shRSK2, respectively. After 72 hours, the levels of FLAG-RSK1 or FLAG-RSK2 but not β-gal decreased in response to isoform-specific shRNAs indicating efficient and selective knockdown. B, Neurons were co-transfected with the β-gal plasmid (pON260) and the indicated shRNAs (0.2 + 0.5 µg of plasmid DNA/0.5×10⁶ neurons, respectively). After 48 hours, neurons were sham- or TD-treated with or without 10 ng/ml BDNF for additional 24 hours. In neurons receiving control plasmids (pSuper or shGFP), BDNF reduced the apoptotic responses to TD. The protection against TD was removed by knocking down either RSK1 or RSK2 indicating that each of these RSK isoforms was required for BDNF to suppress apoptosis. C, Neurons were co-transfected with pON260, the indicated shRNAs, and the activated PDK1A280V mutant (0.2 + 0.2 + 0.8 µg of plasmid DNA/0.5×10⁶ neurons, respectively). After 24 hours, neurons were trophic deprived (TD) for the next 24 hours. PDK1A280V reduced TD-induced apoptosis in neurons receiving shGFP while it failed to protect the recipients of the equimolar mix of shRSK1-1, shRSK1-2, shRSK2-1, and shRSK2-2 (shRSKs). In contrast, the equimolar mix of shERK1-1, shERK1-2, shERK2-1, and shERK2-2 (shERKs) did not affect the PDK1A280V-mediated neuroprotection. Results shown in A, were replicated in two independent experiments. In B–C, averages of triplicate determinations from 3 independent experiments ±SEM are shown; **, p<0.01; ***, p<0.001; NS, p>0.05.