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APAF1 is a key transcriptional target for p53 in the regulation of neuronal cell death.

Fortin A, Cregan SP, MacLaurin JG, Kushwaha N, Hickman ES, Thompson CS, Hakim A, Albert PR, Cecconi F, Helin K, Park DS, Slack RS - J. Cell Biol. (2001)

Bottom Line: Induction of neuronal cell death by camptothecin, a DNA-damaging agent that functions through a p53-dependent mechanism, resulted in increased Apaf1 mRNA in p53-positive, but not p53-deficient neurons.In transient transfections in a neuronal cell line with p53 and Apaf1 promoter-luciferase constructs, p53 directly activated the Apaf1 promoter via both p53 sites.Neurons treated with camptothecin were significantly protected in the absence of Apaf1 relative to those derived from wild-type littermates.

View Article: PubMed Central - PubMed

Affiliation: Ottawa Health Research Institute - Neuroscience, University of Ottawa, Ottawa, Ontario K1H-8M5, Canada.

ABSTRACT
p53 is a transcriptional activator which has been implicated as a key regulator of neuronal cell death after acute injury. We have shown previously that p53-mediated neuronal cell death involves a Bax-dependent activation of caspase 3; however, the transcriptional targets involved in the regulation of this process have not been identified. In the present study, we demonstrate that p53 directly upregulates Apaf1 transcription as a critical step in the induction of neuronal cell death. Using DNA microarray analysis of total RNA isolated from neurons undergoing p53-induced apoptosis a 5-6-fold upregulation of Apaf1 mRNA was detected. Induction of neuronal cell death by camptothecin, a DNA-damaging agent that functions through a p53-dependent mechanism, resulted in increased Apaf1 mRNA in p53-positive, but not p53-deficient neurons. In both in vitro and in vivo neuronal cell death processes of p53-induced cell death, Apaf1 protein levels were increased. We addressed whether p53 directly regulates Apaf1 transcription via the two p53 consensus binding sites in the Apaf1 promoter. Electrophoretic mobility shift assays demonstrated p53-DNA binding activity at both p53 consensus binding sequences in extracts obtained from neurons undergoing p53-induced cell death, but not in healthy control cultures or when p53 or the p53 binding sites were inactivated by mutation. In transient transfections in a neuronal cell line with p53 and Apaf1 promoter-luciferase constructs, p53 directly activated the Apaf1 promoter via both p53 sites. The importance of Apaf1 as a p53 target gene in neuronal cell death was evaluated by examining p53-induced apoptotic pathways in primary cultures of Apaf1-deficient neurons. Neurons treated with camptothecin were significantly protected in the absence of Apaf1 relative to those derived from wild-type littermates. Together, these results demonstrate that Apaf1 is a key transcriptional target for p53 that plays a pivotal role in the regulation of apoptosis after neuronal injury.

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Induction of Apaf1 in neurons after focal ischemia in mice. Mice were subjected to 2 h of middle cerebral artery occlusion followed by 24 h of reperfusion. (A) Sections from ipsilateral and contralateral forebrain were prepared and immunostained for Apaf1. (B) Western blot analysis of Apaf1 expression in contralateral (C) versus ipsilateral (I) cortex and striatum after focal ischemia.
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fig3: Induction of Apaf1 in neurons after focal ischemia in mice. Mice were subjected to 2 h of middle cerebral artery occlusion followed by 24 h of reperfusion. (A) Sections from ipsilateral and contralateral forebrain were prepared and immunostained for Apaf1. (B) Western blot analysis of Apaf1 expression in contralateral (C) versus ipsilateral (I) cortex and striatum after focal ischemia.

Mentions: To determine whether Apaf1 induction occurs in neuronal injury, models in which the involvement of p53 has been demonstrated previously, we examined mice subjected to ischemia via middle cerebral artery occlusion (McGahan et al., 1998; Watanabe et al., 1999). Mice were subjected to 2 h of focal ischemia followed by reperfusion for 24 h. This procedure generates an infarct in the striatum and cortex on the side of the brain ipsilateral to the occluded middle cerebral artery. Immunohistochemical staining of ischemic mice brain demonstrated increased Apaf1 immunoreactivity in the infarct region relative to the respective contralateral hemisphere (Fig. 3 A). To corroborate immunohistochemical results, Western analysis was performed to measure Apaf1 protein levels from brain tissue after ischemia. The cortex and striatum were removed at 24 h posttreatment, protein was extracted, and Apaf1 protein levels were examined. In support of our immunohistochemical results, a significant increase in the level of Apaf1 expression was evident in the ipsilateral cortex and striatum (Fig. 3 B). Consistent with our in vitro results, p53-mediated neuronal injury in vivo also results in the induction of Apaf1 protein.


APAF1 is a key transcriptional target for p53 in the regulation of neuronal cell death.

Fortin A, Cregan SP, MacLaurin JG, Kushwaha N, Hickman ES, Thompson CS, Hakim A, Albert PR, Cecconi F, Helin K, Park DS, Slack RS - J. Cell Biol. (2001)

Induction of Apaf1 in neurons after focal ischemia in mice. Mice were subjected to 2 h of middle cerebral artery occlusion followed by 24 h of reperfusion. (A) Sections from ipsilateral and contralateral forebrain were prepared and immunostained for Apaf1. (B) Western blot analysis of Apaf1 expression in contralateral (C) versus ipsilateral (I) cortex and striatum after focal ischemia.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2198828&req=5

fig3: Induction of Apaf1 in neurons after focal ischemia in mice. Mice were subjected to 2 h of middle cerebral artery occlusion followed by 24 h of reperfusion. (A) Sections from ipsilateral and contralateral forebrain were prepared and immunostained for Apaf1. (B) Western blot analysis of Apaf1 expression in contralateral (C) versus ipsilateral (I) cortex and striatum after focal ischemia.
Mentions: To determine whether Apaf1 induction occurs in neuronal injury, models in which the involvement of p53 has been demonstrated previously, we examined mice subjected to ischemia via middle cerebral artery occlusion (McGahan et al., 1998; Watanabe et al., 1999). Mice were subjected to 2 h of focal ischemia followed by reperfusion for 24 h. This procedure generates an infarct in the striatum and cortex on the side of the brain ipsilateral to the occluded middle cerebral artery. Immunohistochemical staining of ischemic mice brain demonstrated increased Apaf1 immunoreactivity in the infarct region relative to the respective contralateral hemisphere (Fig. 3 A). To corroborate immunohistochemical results, Western analysis was performed to measure Apaf1 protein levels from brain tissue after ischemia. The cortex and striatum were removed at 24 h posttreatment, protein was extracted, and Apaf1 protein levels were examined. In support of our immunohistochemical results, a significant increase in the level of Apaf1 expression was evident in the ipsilateral cortex and striatum (Fig. 3 B). Consistent with our in vitro results, p53-mediated neuronal injury in vivo also results in the induction of Apaf1 protein.

Bottom Line: Induction of neuronal cell death by camptothecin, a DNA-damaging agent that functions through a p53-dependent mechanism, resulted in increased Apaf1 mRNA in p53-positive, but not p53-deficient neurons.In transient transfections in a neuronal cell line with p53 and Apaf1 promoter-luciferase constructs, p53 directly activated the Apaf1 promoter via both p53 sites.Neurons treated with camptothecin were significantly protected in the absence of Apaf1 relative to those derived from wild-type littermates.

View Article: PubMed Central - PubMed

Affiliation: Ottawa Health Research Institute - Neuroscience, University of Ottawa, Ottawa, Ontario K1H-8M5, Canada.

ABSTRACT
p53 is a transcriptional activator which has been implicated as a key regulator of neuronal cell death after acute injury. We have shown previously that p53-mediated neuronal cell death involves a Bax-dependent activation of caspase 3; however, the transcriptional targets involved in the regulation of this process have not been identified. In the present study, we demonstrate that p53 directly upregulates Apaf1 transcription as a critical step in the induction of neuronal cell death. Using DNA microarray analysis of total RNA isolated from neurons undergoing p53-induced apoptosis a 5-6-fold upregulation of Apaf1 mRNA was detected. Induction of neuronal cell death by camptothecin, a DNA-damaging agent that functions through a p53-dependent mechanism, resulted in increased Apaf1 mRNA in p53-positive, but not p53-deficient neurons. In both in vitro and in vivo neuronal cell death processes of p53-induced cell death, Apaf1 protein levels were increased. We addressed whether p53 directly regulates Apaf1 transcription via the two p53 consensus binding sites in the Apaf1 promoter. Electrophoretic mobility shift assays demonstrated p53-DNA binding activity at both p53 consensus binding sequences in extracts obtained from neurons undergoing p53-induced cell death, but not in healthy control cultures or when p53 or the p53 binding sites were inactivated by mutation. In transient transfections in a neuronal cell line with p53 and Apaf1 promoter-luciferase constructs, p53 directly activated the Apaf1 promoter via both p53 sites. The importance of Apaf1 as a p53 target gene in neuronal cell death was evaluated by examining p53-induced apoptotic pathways in primary cultures of Apaf1-deficient neurons. Neurons treated with camptothecin were significantly protected in the absence of Apaf1 relative to those derived from wild-type littermates. Together, these results demonstrate that Apaf1 is a key transcriptional target for p53 that plays a pivotal role in the regulation of apoptosis after neuronal injury.

Show MeSH
Related in: MedlinePlus