β-Amyloid (Aβ_1-40), a component of the mature senile plaque in Alzheimer’s disease, is proposed to contribute to the disease pathology by influencing neuronal signalling events. The aim of this study was to examine the role of the cell cycle regulatory protein, p53, in Aβ_1-40-mediated induction of the apoptotic pathway in cultured cortical neurons.

Cultured rat cortical neurons were prepared from humanely killed rats as previously described (Fogarty et al. 2003). Cells were exposed to aggregated Aβ_1-40 (2 µM, 5 min) and p53 activity was assessed by Western immunoblot using an anti-phospho-p53^ser15 or anti-phospho-p53^ser392 antibody. Bax expression was assessed by Western immunoblot using an anti-bax antibody. Caspase-3 activity and cleavage of the DNA repair enzyme, poly-ADP ribose polymerase (PARP), were assessed by immunocytochemistry using anti-active caspase-3 and anti-cleavage site-specific PARP antibodies, respectively. To assess the role of p53 in mediating the modulatory effects of Aβ_1-40 on caspase-3 activity and PARP cleavage, the cells were pretreated with the p53 inhibitor, pifithrin-α (100 nM) for 3 h. All results are expressed as means ± S.E.M. and P < 0.05 was considered significant.

Aβ_1-40 significantly increased mean phospho-p53^ser15 expression from 0.99 ± 0.01 to 1.75 ± 0.19 arbitrary units (a.u.) at 5 min (P < 0.01, Student’s paired t test, n = 6) and from 1.01 ± 0.26 to 1.49 ± 0.13 a.u. at 1 h (P < 0.01, Student’s paired t test, n = 6). No change in expression of phospho-p53^ser392 was mediated by Aβ_1-40; thus phospho-p53^ser392 expression was 1.00 ± 0.03 a.u. in control and 0.96 ± 0.09 a.u. in Aβ-treated cells at 5 min, and 0.99 ± 0.08 a.u. in control and 0.98 ± 0.10 a.u. in Aβ-treated cells at 1 h (n = 6). Aβ_1-40 significantly increased mean bax expression from 1.00 ± 0.03 to 1.35 ± 0.09 a.u. at 6 h (P < 0.05, ANOVA, n = 6) and this was abolished by pifithrin-α (0.95 ± 0.04 a.u., n = 6). Aβ significantly increased the percentage of cells displaying anti-active caspase-3 immunoreactivity from 24 ± 2 % to 48 ± 2 % at 24 h (P < 0.01, ANOVA, n = 6 coverslips) and this increase was abolished by pifithrin-α (24 ± 1 %, n = 6 coverslips). Similarly, Aβ significantly increased the percentage of cells displaying cleaved PARP (85 kDa) immunoreactivity from 38 ± 3 % to 67 ± 3 % at 72 h (P < 0.01, ANOVA, n = 6 coverslips) and this increase was abolished by pifithrin-α (31 ± 3 %, n = 6 coverslips).

These results demonstrate that Aβ_1-40 induces phosphorylation of p53 in cultured cortical neurons, leading to increased bax expression, caspase-3 activation and PARP cleavage. This pathway is likely to be involved in Aβ_1-40-mediated neuronal cell death and may be of importance in Alzheimer’s disease.

This work was supported by the Health Research Board, Ireland.