Figure 5

TUDCA prevents Aβ peptide-induced apoptosis through a PI3K-dependent pathway. Cultured neurons were incubated with either vehicle (control), 25 µM Aβ fragment 25–35, 100 µM TUDCA, or a combination of Aβ plus TUDCA, as described in Materials and Methods. Cells were pretreated with 200 nM wortmannin for 1 h prior to adding TUDCA, and the PI3K inhibitor was left in the culture medium during incubation with both Aβ and TUDCA. A: Wortmannin slightly increased Aβ toxicity at 24 h, but almost completely abolished the inhibitory effect of TUDCA in Aβ-induced apoptosis. Histograms show mean ± SEM values of nuclear fragmentation for at least 5 different experiments. *P < 0.01 from controls treated with wortmannin. B: PI3K inhibition was associated with reduced Akt phosphorylation by Aβ at 5 min, TUDCA alone, or a combination of Aβ plus TUDCA. After incubation with wortmannin, total proteins were processed for Western blot analysis. Following SDS-PAGE and transfer, the nitrocellulose membranes were incubated with polyclonal antibodies for p-Akt1 and total Akt1/2. Representative Western blots are shown for Akt phosphorylation. C: Wortmannin almost completely abolished the inhibitory effect of TUDCA on Aβ-mediated Bax translocation from cytosol to mitochondria at 24 h. After incubation with wortmannin, total, cytosolic, and mitochondrial proteins were processed for Western blot analysis. Following SDS-PAGE and transfer, the nitrocellulose membranes were incubated with antibodies for p-Bad and Bax. Histograms are mean ± SEM for at least 3 independent experiments. §P < 0.05 and *P < 0.01 from controls treated with wortmannin.