Deletion of Puma protects hippocampal neurons in a model of severe status epilepticus.

Prolonged seizures (status epilepticus) can activate apoptosis-associated signaling pathways. The extent to which such pathways contribute to cell death might depend on the insult intensity, whereby the programmed or apoptotic cell death component is reduced when seizures are more severe or protracted. We recently showed that mice lacking the pro-apoptotic Bcl-2 homology domain 3-only protein Puma (Bbc3) were potently protected against damage caused by status epilepticus. In the present study we examined whether Puma deficiency was protective when the seizure episode was more severe. Intra-amygdala microinjection of 1 microg kainic acid (KA) into C57BL/6 mice triggered status epilepticus that lasted about twice as long as with 0.3 microg KA prior to lorazepam termination. Hippocampal damage was also significantly greater in the higher-dose group. Over 80% of degenerating neurons after seizures were positive for DNA fragmentation assessed by terminal deoxynucleotidyl dUTP nick end labeling (TUNEL). Microscopic analysis of neuronal nuclear morphology in TUNEL-positive cells revealed the proportion displaying large rounded clumps of condensed chromatin was approximately 50% lower in the high-dose versus low-dose KA group. Nevertheless, compared to heterozygous and wild-type mice subject to status epilepticus by high-dose KA, neuronal death was reduced by approximately 50% in the hippocampus of Puma-deficient mice. These data suggest aspects of the apoptotic component of seizure-induced neuronal death are insult duration- or severity-dependent. Moreover, they provide further genetic evidence that seizure-induced neuronal death is preventable by targeting so-called apoptosis-associated signaling pathways and Puma loss likely disrupts caspase-independent or non-apoptotic seizure-induced neuronal death.

Contrasting patterns of Bim induction and neuroprotection in Bim-deficient mice between hippocampus and neocortex after status epilepticus.

Prolonged seizures (status epilepticus) are associated with brain region-specific regulation of apoptosis-associated signaling pathways. Bcl-2 homology domain 3-only (BH3) members of the Bcl-2 gene family are of interest as possible initiators of mitochondrial dysfunction and release of apoptogenic molecules after seizures. Previously, we showed that expression of the BH3-only protein, Bcl-2 interacting mediator of cell death (Bim), increased in the rat hippocampus but not in the neocortex after focal-onset status epilepticus. In this study, we examined Bim expression in mice and compared seizure damage between wild-type and Bim-deficient animals. Status epilepticus induced by intra-amygdala kainic acid (KA) caused extensive neuronal death within the ipsilateral hippocampal CA3 region. Hippocampal activation of factors associated with transcriptional and posttranslational activation of Bim, such as CHOP and c-Jun NH(2)-terminal kinases, was significant within 1 h. Upregulation of bim mRNA was evident after 2 h and Bim protein increased between 4 and 24 h. Hippocampal CA3 neurodegeneration was reduced in Bim-deficient mice compared with wild-type animals after seizures in vivo, and short interfering RNA molecules targeting bim reduced cell death after KA treatment of hippocampal organotypic cultures. In contrast, neocortical Bim expression declined after status epilepticus, and neocortex damage in Bim-deficient mice was comparable with that in wild-type animals. These results show region-specific differential contributions of Bim to seizure-induced neuronal death.

Microarray profile of seizure damage-refractory hippocampal CA3 in a mouse model of epileptic preconditioning.

A neuroprotected state can be acquired by preconditioning brain with a stimulus that is subthreshold for damage (tolerance). Acquisition of tolerance involves coordinate, bi-directional changes to gene expression levels and the re-programmed phenotype is determined by the preconditioning stimulus. While best studied in ischemic brain there is evidence brief seizures can confer tolerance against prolonged seizures (status epilepticus). Presently, we developed a model of epileptic preconditioning in mice and used microarrays to gain insight into the transcriptional phenotype within the target hippocampus at the time tolerance had been acquired. Epileptic tolerance was induced by an episode of non-damaging seizures in adult C57Bl/6 mice using a systemic injection of kainic acid. Neuron and DNA damage-positive cell counts 24 h after status epilepticus induced by intraamygdala microinjection of kainic acid revealed preconditioning given 24 h prior reduced CA3 neuronal death by approximately 45% compared with non-tolerant seizure mice. Microarray analysis of over 39,000 transcripts (Affymetrix 430 2.0 chip) from microdissected CA3 subfields was undertaken at the point at which tolerance was acquired. Results revealed a unique profile of small numbers of equivalently up- and down-regulated genes with biological functions that included transport and localization, ubiquitin metabolism, apoptosis and cell cycle control. Select microarray findings were validated post hoc by real-time polymerase chain reaction and Western blotting. The present study defines a paradigm for inducing epileptic preconditioning in mice and first insight into the global transcriptome of the seizure-damage refractory brain.