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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.
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Robert Meller,
Simon John Thompson,
Theresa Ann Lusardi, Andrea Nicole Ordonez,
Michelle Dawn Ashley,
Veronica Jessick,
Weihzen Wang,
Daniel John Torrey,
David Clifford Henshall,
Philip R Gafken,
Julie Anne Saugstad,
Zhi-Gang Xiong,
Roger Pancoast Simon
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ABSTRACT: Ischemic tolerance is an endogenous neuroprotective mechanism in brain and other organs, whereby prior exposure to brief ischemia produces resilience to subsequent normally injurious ischemia. Although many molecular mechanisms mediate delayed (gene-mediated) ischemic tolerance, the mechanisms underlying rapid (protein synthesis-independent) ischemic tolerance are relatively unknown. Here we describe a novel mechanism for the induction of rapid ischemic tolerance mediated by the ubiquitin-proteasome system. Rapid ischemic tolerance is blocked by multiple proteasome inhibitors [carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG132), MG115 (carbobenzoxy-L-leucyl-L-leucyl-L-norvalinal), and clasto-lactacystin-beta-lactone]. A proteomics strategy was used to identify ubiquitinated proteins after preconditioning ischemia. We focused our studies on two actin-binding proteins of the postsynaptic density that were ubiquitinated after rapid preconditioning: myristoylated, alanine-rich C-kinase substrate (MARCKS) and fascin. Immunoblots confirm the degradation of MARCKS and fascin after preconditioning ischemia. The loss of actin-binding proteins promoted actin reorganization in the postsynaptic density and transient retraction of dendritic spines. This rapid and reversible synaptic remodeling reduced NMDA-mediated electrophysiological responses and renders the cells refractory to NMDA receptor-mediated toxicity. The dendritic spine retraction and NMDA neuroprotection after preconditioning ischemia are blocked by actin stabilization with jasplakinolide, as well as proteasome inhibition with MG132. Together these data suggest that rapid tolerance results from changes to the postsynaptic density mediated by the ubiquitin-proteasome system, rendering neurons resistant to excitotoxicity.
Journal of Neuroscience 02/2008; 28(1):50-9. · 7.11 Impact Factor
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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.
Journal of Biological Chemistry 04/2006; 281(11):7429-36. · 4.77 Impact Factor
