Figure 6

Aβ oligomer treatment increases the levels of pAkt and p-mTOR in primary neurons. A. Cultured primary cortical neurons (21 DIV) plated in 96-well plates were treated with either vehicle (Veh) or increasing concentrations (0.4, 2.0, 3.0, 4.0, 10 and 20 μg/ml) of either Aβ monomer (AβM) or Aβ oligomer (AβO) for 24 h. Following fixation, In-Cell Western analysis, using primary antibodies against tubulin and phosphorylated-Akt (pAkt^S473) followed by infrared-conjugated secondary antibodies, revealed an apparent increase in pAkt levels in AβO treated cultures. C. Quantification of the pAkt/Tubulin ratio in these cultures revealed a dose-dependent statistically significant increase in AβO treated cultures at AβO concentrations above 2.0 μg/ml (2.0 μg/ml-p = 0.006; 3.0 μg/ml -p = 0.04; 4.0 μg/ml -p = 0.005; 10 μg/ml -p = 0.008; 20.0 μg/ml -p = 0.01; mean ± SD; n = 4; unpaired t test), while exposure to AβM did not reveal any alterations in the pAkt/Tubulin ratio. All comparisons were made against AβM at respective concentrations. B. In-Cell Western analysis with primary antibodies against tubulin and phosphorylated-mTOR (p-mTOR^S2448) revealed an apparent increase in the levels of p-mTOR in the middle concentration ranges (2.0 – 4.0 μg/ml). D. Quantification of the p-mTOR/tubulin ratios revealed a statistically significant (3.0 μg/ml -p = 0.04; unpaired t test; mean ± SD; n = 4) increase in AβO exposed cultures when compared to AβM exposed cultures at similar concentrations. At elevated concentrations of AβO (> 4.0 μg/ml), there was no significant increase in the p-mTOR/tubulin ratio. 'X' denotes wells with no cells (in panel A and B).