S1). == Pharmacologic inhibition of p53 prevents Puma induction after SE == To determine which of the three potential pathways were responsible for inducing Puma, we focused on p53, for which specific pharmacological and genetic disruption was possible. proapoptotic protein acting acutely to influence neuronal death subsequently alters the phenotype of epilepsy in the long-term, supporting the concept that apoptotic pathway activation is a trigger of epileptogenesis.Engel, T., Murphy, B. M., Hatazaki, Rabbit polyclonal to ZNF439 S., Jimenez-Mateos, E. M., Concannon, C. G., Woods, I., Prehn, J. H. M., Henshall, D. C. Reduced hippocampal damage and epileptic seizures afterstatus epilepticusin mice lacking proapoptotic Puma. Keywords:apoptosis, Bcl-2, BH3-only protein, epileptogenesis, kainate, p53 Prolonged seizures[status epilepticus(SE)] in adult rodents and humans can cause hippocampal damage and precipitate temporal lobe epilepsy (TLE)(1,2,3,4). Although neuronal death has been implicated as a pathogenic substrate of TLE, 20-HETE this remains largely unproven. However, recent animal studies suggest that hippocampal hilar and CA3 lesions are particularly critical for precipitating post-SE epilepsy(5, 6). Consistent with this, CA3 neuroprotection has been associated with reduced incidence of spontaneous seizures after experimental SE(6,7,8). The p53 transcription factor is a critical regulator of apoptosis, which has long been implicated as an effector of seizure-induced neuronal death(9). Seizure-like activityin vitroand prolonged seizuresin vivocause p53 accumulation and increased transcriptional activity(9,10,11,12). Genetic ablation or pharmacologic inhibition of p53 reduces neuronal damage following glutamate receptor overactivationin vitro(13, 14)and neuronal loss after seizuresin vivo(14,15,16). However, the mechanism by which p53 induces neuronal death after seizures is unknown. Among the target genes under 20-HETE p53 control is Puma (p53-up-regulated modulator of apoptosis), a member of the Bcl-2 homology domain 3 (BH3)-only subgroup of Bcl-2 family proteins(17,18,19). Puma is one of the most potent proapoptotic proteins, which derives from its avid binding to all prosurvival Bcl-2 family proteins(20)and possibly an ability to directly activate Bax at mitochondria(21). The proapoptotic actions of p53 20-HETE in neurons have been proposed to derive from transcriptional activation of Puma(22, 23). Presently, we investigated whether Puma is the effector of p53-induced neuronal death after SE. We also tested whether the absence ofpumainfluenced the subsequent development of recurrent spontaneous seizures. == MATERIALS AND METHODS == == Mouse model of focal-onset SE == All animal procedures were performed in accordance with the principals of the European Communities Council Directive (86/609/EEC) and were approved by the Research Ethics Committee of the Royal College of Surgeons in Ireland. Adult (2025 g) male mice were used: C57BL/6 (Harlan, Bicester, UK),p53+/+,/on a C57BL/6 background (Jackson Laboratory, Bar Harbor, ME, USA), andpuma+/+,+/,/on a C57BL/6 background (Andreas Strasser, Molecular Genetics of Cancer Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia). Food and water were availablead libitum. Mice underwent seizures induced by unilateral stereotactic microinjection of kainic acid (KA) into the amygdala, as described previously(24, 25). Briefly, mice were anesthetized using isoflurane (35% induction, 12% maintenance) and kept normothermic in a stereotactic frame. Mice were affixed with skull-mounted electrodes (Bilaney Consultants, Sevenoaks, UK) above dorsal hippocampus and frontal cortex to record surface electroencephalogram (EEG) using a Grass Comet digital EEG (Medivent, Lucan, Ireland). A guide cannula was affixed over the dura (coordinates from bregma: AP=0.94; L=2.85 mm), and the entire skull assembly was fixed in place with dental cement. 20-HETE Anesthesia was discontinued, EEG recordings were commenced, and then a 31-gauge internal cannula was inserted into the lumen of the guide to inject KA (Ocean Produce International, Dartmouth, NS, Canada; 0.3 g in 0.2 l vehicle; PBS, pH adjusted to 7.4) into the amygdala. Nonseizure control mice received 0.2 l intra-amygdala vehicle. Lorazepam (6 mg/kg, intraperitoneal or intravenous) was administered 40 min after KA, and the EEG was recorded for up to 1 h thereafter. Mice were euthanized at different time points after anticonvulsant for biochemical analysis and histopathology, or following completion of spontaneous seizure monitoring (see below). Brains were microdissected on ice and processed for mRNA and protein analysis, or flash-frozen whole in 2-methylbutane at 30C for immunohistochemistry. Brains from additional naive (noninstrumented) wild-type andpuma+/,/mice were used to examine hippocampal and amygdala neuroanatomy and gene expression. == Epilepsy monitoring == Long-term EEG recording to define the emergence of epilepsy was performed using implantable EEG telemetry units [Data Sciences International (DSI), St. Paul, MN, USA], as described previously(8, 24). Mice underwent the same initial surgical procedure for affixing the injection cannula but were equipped with EEG transmitters (F20-EET, DSI) to record bilateral EEG from skull overlying the dorsal ipsilateral and contralateral hippocampi. EEG data were acquired using the Dataquest A.R.T. system (DSI). Following surgery, mice received.