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Ubiquitin modification targets a protein for rapid degradation by the proteasome. However, polyubiquitination of proteins can result in multiple functions depending on the topology of the ubiquitin chain. Therefore, ubiquitin signaling offers a more complex and versatile biology compared with many other posttranslational modifications. One area of potential for the application of this knowledge is the field of ischemiainduced brain damage, as occurs following a stroke. The ubiquitin proteasome system may exert a dual role on neuronal outcome following ischemia. Harmful ischemia results in an overload of the ubiquitin proteasome system, and blocking the proteasome reduces brain infarction following ischemia. However, the rapid and selective degradation of proteins following brief ischemia results in endogenous protection against ischemia. Therefore, further understanding of the molecular signaling mechanisms that regulate the ubiquitin proteasome system may reveal novel therapeutic targets to reduce brain damage when ischemia is predicted or reduce the activation of the cell death mechanisms and the inflammatory response following stroke. The aim of this review is to discuss some of the recent advances in the understanding of protein ubiquitination and its implications for novel stroke therapies. [ABSTRACT FROM AUTHOR]

Osteopontin (OPN) is a secreted extracellular phosphoprotein involved in diverse biologic functions, including inflammation, cell migration, and antiapoptotic processes. Here we investigate the neuroprotective potential of OPN to reduce cell death using both in vitro and in vivo models of ischemia. We show that incubation of cortical neuron cultures with OPN protects against cell death from oxygen and glucose deprivation. The effect of OPN depends on the Arg–Gly–Asp (RGD)-containing motif as the protective effect of OPN in vitro was blocked by an RGD-containing hexapeptide, which prevents integrin receptors binding to their ligands. Osteopontin treatment of cortical neuron cultures caused an increase in Akt and p42/p44 MAPK phosphorylation, which is consistent with OPN-inducing neuroprotection via the activation of these protein kinases. Indeed, the protective effect of OPN was reduced by inhibiting the activation of Akt and p42/p44 MAPK using LY294002 and U0126, respectively. The protective effect of OPN was also blocked by the protein synthesis inhibitor cycloheximide, suggesting that the neuroprotective effect of OPN required new protein synthesis. Finally, intracerebral ventricular administration of OPN caused a marked reduction in infarct size after transient middle cerebral artery occlusion in a murine stroke model. These data suggest that OPN is a potent neuroprotectant against ischemic injury.Journal of Cerebral Blood Flow & Metabolism (2005) 25, 217–225. doi:10.1038/sj.jcbfm.9600022 [ABSTRACT FROM AUTHOR]

Bcl-2 plays a pivotal role in the control of cell death and is upregulated by ischemic tolerance. Because Bcl-2 expression is regulated by the transcription factor cyclic AMP response element-binding protein (CREB), we investigated the role of CREB activation in two models of ischemic preconditioning: focal ischemic tolerance after middle cerebral artery occlusion (MCAO) and in vitro ischemic tolerance modeled by oxygen–glucose deprivation (OGD). After preconditioning ischemia (30 minutes MCAO or 30 minutes OGD), phosphorylation of CREB was increased, and there was an increased interaction between the bcl-2 cyclic AMP-responsive element (CRE) promoter and nuclear proteins after preconditioning ischemia in vivo and in vitro. Chromatin immunoprecipitation revealed an increased interaction between CREB-binding protein and the bcl-2 CRE rather than CREB, after preconditioning ischemia. Ischemic tolerance was blocked by a CRE decoy oligonucleotide, which also blocked Bcl-2 expression. The protein kinase A inhibitor H89, the calcium/calmodulin kinase inhibitor KN62, and the MEK inhibitor U0126 blocked ischemic tolerance, but not the phosphatidylinositol 3-kinase inhibitor LY294002. H89, KN62, and U0126 reduced CREB activation and Bcl-2 expression. Taken together, these data suggest that after ischemic preconditioning CREB activation regulates the expression of the prosurvival protein Bcl-2.Journal of Cerebral Blood Flow & Metabolism (2005) 25, 234–246. doi:10.1038/sj.jcbfm.9600024 Published online 5 January 2005 [ABSTRACT FROM AUTHOR]

The caspase family of cell death proteases has been implicated in the mechanism of neuronal death following seizures. We investigated the expression and processing of caspases 6 and 7, putative executioner caspases. Brief limbic seizures were evoked by intraamygdala kainic acid to elicit unilateral death of target hippocampal CA3 neurons in the rat. Seizures rapidly induced cleavage of constitutively expressed caspase-6, followed by elevated VEIDase activity and the proteolysis of lamin A. Neuronal caspase-6 immunoreactivity was markedly upregulated within cortex and hippocampus in relation to bursts of polyspike paroxysmal discharges. In contrast, while caspase-7 expression also increased within cortical and hippocampal neuronal populations in response to the same seizure patterns, caspase-7 was not proteolytically activated. These data highlight differences in expression and activation of caspases 6 and 7 in response to identifiable seizure patterns, focusing potential therapeutic targets for neuroprotection in epilepsy. [Copyright &y& Elsevier]

We have previously shown that the cell death-promoting protein Bcl-2-interacting mediator of cell death (Bim) is ubiquitinated and degraded following a neuroprotection-conferring episode of brief ischernia (preconditioning). Here, we identify the E3 ligase that ubiquitinates Bim in this model, using a proteomics approach. Using phosphorylated GST-Bim as bait, we precipitated and identified by mass spectrometry tripartite motif protein 2 (TRIM2), a RING (really interesting new gene) domain-containing protein. The reaction between TRIM2 and Bim was confirmed using co-immunoprecipitation followed by immunoblotting. We show that TRIM 2 binds to Bim when it is phosphorylated by p42/p44 MAPK but does not interact with a nonphosphorylatable Bim mutant (3ABim). 12-O-tetradecanoylphorbol13-acetate activation of p42/p44 MAPK drives Bim ubiquitination in mouse embryonic fibroblast cells and is associated with an increased interaction between TRIM2 and Bim. One hour following preconditioning ischemia, the binding of Bim to TRIM2 increased, consistent with the time window of enhanced Bim degradation. Blocking p42/p44 MAPK activation following preconditioning ischemia with U0126 or using the nonphosphorylatable 3ABim reduced the binding between Bim and TRIM2, Immunodepletion of TRIM2 from cell lysates prepared from preconditioned cells reduced Bim ubiquitination. Finally, suppression of TRIM2 expression, using lentivirus transduction of shRNAmir, stabilized Bim protein levels and blocked neuroprotection observed in rapid ischemic tolerance. Taken together, these data support a role for TRIM2 in mediating the p42/p44 MAPK-dependent ubiquitination of Bim in rapid ischemic tolerance. [ABSTRACT FROM AUTHOR]

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-κB and HIF-1α, 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. [Copyright &y& Elsevier]

Programmed cell death pathways have been implicated in the mechanism by which neurons die following brief and prolonged seizures, but the significance of proapoptotic Bcl-2 family proteins in the process remains poorly defined. Expression of the death agonist Bcl-2-interacting mediator of cell death (Bim) is under the control of the forkhead in rhabdomyosarcoma (FKHR) transcription factors. This prompted us to examine the response of this pathway to experimental seizures and in hippocampi from patients with intractable temporal lobe epilepsy. A short period of status epilepticus in rats that damaged the hippocampus activated FKHR/FKHRL-1 and induced a significant increase in expression of Bim. Blocking of FKHR/FKHRL-1 dephosphorylation after seizures improved hippocampal neuronal survival in vivo, and Bim antisense oligonucleotides were neuroprotective against seizures in vitro. Inhibition of Akt increased the FKHR/Bim response and DNA fragmentation within the normally resistant cortex. Analysis of hippocampi from patients with intractable epilepsy revealed that Bim levels were significantly lower than in controls and FKHR was inhibited; we were able to reproduce these results experimentally in rats by evoking multiple brief, noninjurious electroshock seizures. We conclude that Bim expression may be a critical determinant of whether seizures damage the brain, and that its control may be neuroprotective in status epilepticus and epilepsy. [ABSTRACT FROM AUTHOR]