[Show abstract][Hide abstract] ABSTRACT: Rapid ischemic tolerance, induced one hour following ischemic preconditioning, is mediated via the ubiq-uitin-proteasome system and the degradation of the pro-apoptotic bcl-2 family protein Bim. Previous studies implicate adenosine A1 receptors in mediating rapid ischemic tolerance. Since the A1 adenosine receptor antagonist DPCPX (10µM) blocked rapid ischemic tolerance in our model, we investigated whether adenosine-mediated preconditioning induces rapid ischemic tolerance via the proteasomal degradation of Bim. Cultured rat cortical neurons were incubated for 60 minutes with either adenosine (1µM) or (-)-N(6)-(2-Phenyl-isopropyl) adenosine (RPIA (1µM)), prior to a harmful dose of ischemia (120min oxygen and glucose deprivation). Preconditioned cells had significantly lower levels of cell death following harmful ischemia when compared to non-preconditioned cells. The proteasome inhibitor MG132 (0.1µM) blocked the protective effect of adenosine pre-conditioning. Immunoblot analysis revealed a decrease in Bim protein levels in adenosine and RPIA preconditioned neurons. Adenosine preconditioning induced neuroprotection and Bim degradation was blocked by the MEK inhibitor UO126 (10µM). Our data suggests that pharmacological preconditioning with adenosine results in proteasomal Bim degradation mediated by p42/44 MAPK. Therefore, pharmacological approaches may be able to induce rapid ischemic tolerance via similar molecular mechanisms as ischemic preconditioning.
International Journal of Physiology, Pathophysiology and Pharmacology 01/2010; 2(1):36-44.
[Show abstract][Hide abstract] 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 Hippocampal cells, chronically treated with kynurenic acid, had kynurenic acid withdrawn to induce seizure-like activity for 40 min. 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 (10 microM) and CNQX (40 microM), 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.
Epilepsy Research 08/2006; 70(1):3-14. DOI:10.1016/j.eplepsyres.2006.02.002 · 2.02 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A previous exposure to a non-harmful ischemic insult (preconditioning) protects the brain against subsequent harmful ischemia (ischemic tolerance). In contrast to delayed gene-mediated ischemic tolerance, little is known about the molecular mechanisms that regulate rapid ischemic tolerance, which occurs within 1 h following preconditioning. Here we have investigated the degradation of the pro-apoptotic Bcl-2 family member Bim as a mechanism of rapid ischemic tolerance. Bim protein levels were reduced 1 h following preconditioning and occurred concurrent with an increase in Bim ubiquitination. Ubiquitinated proteins are degraded by the proteasome, and inhibition of the proteasome with MG132 (a proteasome inhibitor) prevented Bim degradation and blocked rapid ischemic tolerance. Inhibition of p42/p44 mitogen-activated protein kinase activation by U0126 reduced Bim ubiquitination and Bim degradation and blocked rapid ischemic tolerance. Finally, inhibition of Bim expression using antisense oligonucleotides also reduced cell death following ischemic challenge. Our results suggest that following preconditioning ischemia, Bim is rapidly degraded by the ubiquitin-proteasome system, resulting in rapid ischemic tolerance. This suggests that the rapid degradation of cell death-promoting proteins by the ubiquitin-proteasome pathway may represent a novel therapeutic strategy to reduce cell damage following neuropathological insults, e.g. stroke.