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The ubiquitin-proteasome system is the major non-lysosymal system for degrading proteins in the cell; the work leading to its discovery was awarded the Nobel Prize in Chemistry in 2004. In addition to small ubiquitin-like modifiers (e.g. Sumo and Nedd8), ubiquitin is involved in the complex regulation of the levels and function of many proteins and signaling pathways involved in determining cell fate. The cell death regulatory proteins, such as Bcl-2 family proteins and caspases are targeted for degradation by the ubiquitin proteasome system (UPS). In addition to mediating the degradation of proteins, the UPS regulates function and translocation of proteins, many of which play a role in the determination of cell fate. For example the UPS can regulate the activity of transcription factors, such as P53, NF-kappaB and HIF-1 alpha, which control the expression of protein mediators of cell death. Aberrant UPS function has been reported in multiple neuropathologies including Parkinson's diseases and ischemia. With the number of ubiquitin conjugating and de-conjugating enzymes reaching close to the levels of protein kinases and phosphatases, it is clear that ubiquitination is an important biological regulatory step for proteins.

Seizure-induced neuronal death may involve engagement of the BCL-2 family of apoptosis-regulating proteins. In the present study we examined the activation of proapoptotic BAD in cultured hippocampal neurons following seizures induced by removal of chronic glutamatergic transmission blockade. Kynurenic acid withdrawal elicited an increase in seizure-like electrical activity, which was inhibited by blockers of AMPA (CNQX) and NMDA (MK801 and AP5) receptor function. However, only NMDA receptor antagonists inhibited calcium entry as assessed by fura-2, and cell death of hippocampal neurons. Seizures increased proteolysis of caspase-3 and terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) of cells. Seizure-like activity induced dephosphorylation of BAD and the disruption of its constitutive interaction with 14-3-3 proteins. In turn, BAD dimerized with antiapoptotic BCL-Xl after seizures. However, the absence of neuroprotective effects of pathway intervention suggests that BAD may perform a reinforcement rather than instigator role in cell death following seizures in vitro.

The consequences of activation of tumour necrosis factor receptor 1 (TNFR1) during neuronal injury remain controversial. The apoptosis signal-regulating kinase 1 (ASK1), a mitogen-activated protein kinase kinase kinase, can mediate cell death downstream of TNFR1. Presently, we examined the formation of the TNFR1 signalling cascade and response of ASK1 during seizure-induced neuronal death. Brief (40 min) seizures were induced in rats by intra-amygdala microinjection of kainic acid, which elicited unilateral hippocampal CA3 neuronal death. Seizures caused a rapid decline in the expression of the silencer of death domains protein within injured CA3. Co-immunoprecipitation analysis revealed a commensurate assembly of a TNFR1 scaffold complex containing TNFR-associated death domain protein, receptor interacting protein and TNFR-activating factor 2. In addition, recruitment of TNFR-activating factor 2 was likely promoted by Bcl10-mediated sequestering of cellular inhibitor of apoptosis protein 2. Apoptosis signal-regulating kinase 1 was sequestered in a complex that contained the molecular chaperone 14-3-3beta and protein phosphatase 5. Seizures triggered its dissociation, and the phosphorylation of the ASK1 substrates, mitogen-activated protein kinase kinase 3/6 and 4. Subsequently, protein phosphatase 5 translocated into the nuclei of degenerating CA3 neurons, while ASK1 colocalized with the adaptor proteins Daxx and TNFR-activating factor 2 at the outer membrane of injured CA3 neurons. Neutralizing antibodies to TNFalpha reduced the numbers of DNA damaged cells within the injured hippocampus. These data suggest ASK1 may be involved in the mechanism of seizure-induced neuronal death downstream of a TNFR1 death-signalling complex.

Activation of cysteinyl aspartate-specific proteases (caspases) may underlie apoptotic cell death in brain. Terminal, executioner caspases 3, 6 and 7 likely contribute to such cell death in a stimulus- and cell type-specific manner. Here we investigate the processing and activation of caspases 3, 6 and 7 in rat C6 glioma cells induced to undergo apoptosis by staurosporine (STS) treatment as a model of apoptosis in glia. Proteolysis and activation of caspases 3 and 7 as determined by immunoblotting and substrate-specific cleavage assay (DEVDase) preceded caspase-6 proteolysis and increased VEIDase activity following STS treatment. Activation of caspase-6 was paralleled by cleavage of the nuclear envelope protein lamin-A. These results highlight temporal differences in the activation of the triad of executioner caspases 3, 6 and 7 during glial cell apoptosis.

The mechanism by which seizures induce neuronal death is not completely understood. Caspase-8 is a key initiator of apoptosis via extrinsic, death receptor-mediated pathways; we therefore investigated its role in mediating seizure-induced neuronal death evoked by unilateral kainic acid injection into the amygdala of the rat, terminated after 40 min by diazepam. We demonstrate that cleaved (p18) caspase-8 was detectable immediately following seizure termination coincident with an increase in cleavage of the substrate Ile-Glu-Thr-Asp (IETD)-p-nitroanilide and the appearance of cleaved (p15) Bid. Expression of Fas and FADD, components of death receptor signaling, was increased following seizures. In vivo intracerebroventricular z-IETD-fluoromethyl ketone administration significantly reduced seizure-induced activities of caspases 8, 9, and 3 as well as reducing Bid and caspase-9 cleavage, cytochrome c release, DNA fragmentation, and neuronal death. These data suggest that intervention in caspase-8 and/or death receptor signaling may confer protection on the brain from the injurious effects of seizures., (Copyright 2001 Academic Press.)

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. [ABSTRACT FROM AUTHOR]

Abstract: In response to harmful stresses, cells induce programmed cell death (PCD) or apoptosis. Seizures can induce neural damage and activate biochemical pathways associated with PCD. Since seizures trigger intracellular calcium overload, it has been presumed that the intrinsic cell death pathway mediated by mitochondrial dysfunction would modulate cell death following seizures. However, previous work suggests that the extrinsic cell death pathway may initiate the damage program. Here we investigate intrinsic versus extrinsic cell death pathway activation using caspase cleavage as a marker for activation of these pathways in a rat in vitro model of seizures. Hippocampal cells, chronically treated with kynurenic acid, had kynurenic acid withdrawn to induce seizure-like activity for 40min. Subjecting rat hippocampal cultures to seizures increased cell death and apoptosis-like DNA fragmentation using TUNEL staining. Seizure-induced cell death was blocked by both MK801 (10μM) and CNQX (40μM), which suggests multiple glutamate receptors regulate seizure-induced cell death. Cleavage of the initiator caspases, caspase 8 and 12 were increased 4h following seizure, and cleavage of the quintessential executioner caspase, caspase 3 was increased 4h following seizure. In contrast, caspase 9 cleavage only increased 24h following seizure. Using an affinity labeling approach to trap activated caspases in situ, we show that caspase 8 is the apical caspase activated following seizures. Finally, we show that the caspase 8 inhibitor Ac-IETD-CHO was more effective at blocking seizure-induced cell death than the caspase 9 inhibitor Ac-LEHD-CHO. Taken together, our data suggests the extrinsic cell death pathway-associated caspase 8 is activated following seizures in vitro. [Copyright &y& Elsevier]

Abstract: 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 ∼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. [Copyright &y& Elsevier]