P53 target genes in the regulation of neuronal cell death

The role of the tumour suppressor p53 in neuronal cell death following acute injury is well documented. However, the transcriptional targets upregulated by this transcription factor and required to mediate its apoptotic function have not been identified. Although Bax has previously been reported as a direct target for p53, its expression levels are not elevated in neurons. I set out to find key p53 targets involved in injury-induced apoptosis in neurons. Using data generated from DNA microarray analysis of total RNA isolated from neurons undergoing p53-induced apoptosis as the basis for my search, I proceeded to identify two novel p53 target genes. The first of these was Apaf1, a gene crucial for the activation of caspases via the mitochondrial apoptotic pathway. Apaf1 was upregulated in several models of p53-mediated neuronal cell death including the in vivo model of middle cerebral artery occlusion. Electrophoretic mobility shift assay revealed that p53 could directly bind to two p53 consensus binding sites located within the Apaf1 promoter, and luciferase reporter assays using Apaf1 promoter-luciferase constructs indicated that p53 could directly activate the Apaf1 promoter via both p53 sites. In the absence of Apaf1, neurons were significantly more resistant to apoptosis induced by DNA damage as compared to their wildtype counterparts. These results established Apaf1 as an important transcriptional target for p53 in the regulation of neuronal apoptosis. The second gene of interest was the proapoptotic gene SIVA. SIVA mRNA was induced in models of p53-dependent apoptosis in cultured neurons as well as in stroke injury in vivo. Its expression was sufficient to induce apoptosis in cultured neurons. Analysis of the SIVA gene identified two p53 consensus binding sites located within intron 1 along with two E2F sites within the SIVA promoter. Electrophoretic mobility shift assay demonstrated that both transcription factors could bind to their respective consensus binding sites, and luciferase reporter assays indicated that E217 and p53 could induce activity from the SIVA promoter. These findings indicated that the proapoptotic gene, SIVA, was a direct transcriptional target for both p53 and E2F1. Altogether, my research identified two novel transcriptional targets for p53 in the modulation of neuronal apoptosis