Abstracts

Epilepsy is one of the most common neurologic disorders, affecting approximately 50 million people worldwide and its characteristics are a predisposition to recurrent unprovoked seizures caused by abnormal hypersynchronous discharges of neurons. Through experimental modelling and clinical neuroimaging it has been shown that seizures are capable of causing neuronal death and this may contribute to epileptogenesis, impairments in cognitive function or the epilepsy phenotype. Neuronal death in this setting is, at least in part, due to the induction of the molecular machinery of apoptosis. Studies using clinical material from patients who suffer recurrent seizures and animal epilepsy models have confirmed changes in expression and activation of pro- and anti-apoptotic proteins of the caspase and Bcl-2 protein family. Another consistently up-regulated pro-apoptotic protein in seizure models is the tumour suppressor protein p53. Recently a new p53 up-regulated target gene has been identified named PUMA. PUMA, a BH3-only protein, is one of the most potent pro-apoptotic proteins of the Bcl-2 family which is also expressed in the central nervous system. In this study we examine the role of PUMA in seizure-induced neuronal death using PUMA knock-out mice., Seizures were focally evoked in adult C57Bl/6 mice by unilateral stereotaxic microinjection of kainic acid into the basolateral amygdala nucleus. The EEG was continuously monitored until lorazepam was administered to terminate seizures after 40 minutes. For Western blotting, samples were homogenized in lysis buffer and loaded on a SDS-PAGE gel. [italic]For immunohistochemical analysis,[/italic] coronal brain sections were stained with NeuN and analysis of cells exhibiting DNA fragmentation was performed using fluorescein-linked TUNEL. Data are presented as mean [plusmn] SEM. Data were analyzed using one-way ANOVA with post hoc Fisher[apos]s paired least significant difference test as appropriate. Significance was accepted at p [lt] 0.05., In C57Bl/6 wild-type mice, p53 and PUMA were up-regulated in the hippocampus shortly after seizure induction. Assessment of the phenotype of PUMA knock-out mice revealed normal numbers of hippocampal and amygdala neurons, and normal expression of the kainate receptor in the amygdala and hippocampal site of seizure elicitation. Seizures elicited by intra-amygdala kainate were of similar frequency and severity in the PUMA knock out mice. However, neuropathological analysis revealed neuron survival was significantly greater in PUMA knock-out mice than in wild-type mice and TUNEL counts were significantly lower in PUMA knock-out mice than in wild-type mice., The present data suggest that PUMA contributes to the neurodegeneration taking place during seizure-induced neuronal death and may therefore represent a therapeutic target for adjunctive therapy to prevent hippocampal damage following status epilepticus., (Supported by Health Research Board, Ireland; Science Foundation Ireland.)