Figure 2

Purified Aβ oligomers induce BrdU incorporation in primary neurons. Cultured primary cortical neurons (21 DIV) were treated with vehicle (Veh) A-C, 0.4 μg/ml of Aβ monomers (AβM) D-F or 0.4 μg/ml of Aβ oligomers (AβO) G-I or 0.4 μg/ml Aβ fibrils (AβF) J-L in presence of BrdU for 24 h. Cells were fixed and immunostained with antibodies against MAP2 (A, D, G and J) and BrdU (B, E, H and K) demonstrating the induction of BrdU incorporation in MAP2 positive cells (neurons) with Aβ preparations, but not with AβM, AβF or vehicle control (merged images in C, F, I and L). Scale bar, 10 μm. M-O Quantification of the percentage of BrdU positive/MAP2 positive cells in vehicle, AβM, AβO and AβF treatment groups revealed a statistically significant (p = 0.003 for 2.0 μg/ml; p = 0.006 for 4.0 μg/ml; unpaired t test; mean ± SEM; n = 3 independent experiments) increase in the percentage of BrdU/MAP2 positive cells upon exposure to AβO, but not AβM or AβF, compared to vehicle control. P. Western blot analysis of detergent soluble cell lysates from neurons exposed to different concentrations of Aβ with an antibody against the S-phase cell cycle protein, PCNA, and an antibody against the protein loading control, GAPDH. Q. Quantification of the Westernblot for PCNA (relative to GAPDH) revealing a statistically significant (p = 0.008 for 20 μg/ml) increase in the PCNA expression in the 20 μg/ml AβO treatment group. PCNA/GAPDH ratio at 20 μg/ml AβO treatment was assigned as 100%.