Role of Rac1 GTPase in JNK signaling and delayed neuronal cell death following global cerebral ischemia

The overall goal of this study was to determine the role of Rac1 in POSH/MLK/JNK signaling and delayed neuronal cell death following cerebral ischemia. Temporal studies revealed that Rac1 GTPase activation was significantly elevated in hippocampus CA1 at 10 min to 72 h after cerebral ischemia reperfusion, with peak levels 30 min to 6 h after reperfusion. Total Rac1 protein levels were not significantly changed following cerebral ischemia. Rac1 has been shown to interact with POSH (plenty of SH3s), a scaffold protein that binds to and regulates MLK3 and JNK activation. Co-immunoprecipitation (Co-IP) studies revealed that POSH-Rac1-MLK3 complex formation displayed a significant and prolonged elevation after reperfusion, with a correlative increase in phosphorylation/activation of MLK3 as compared to sham controls. Intracerebroventricular administration of Racl antisense oligonucleotides (AS-ODNs) significantly attenuated Rac1 levels and Racl activation at 30 min after reperfusion, with a correlated significant attenuation of POSH-MLK3-Rac1 complex formation and MLK3 activation in hippocampus CA1. Infusion of Racl AS-ODNs also significantly attenuated post-ischemic activation of JNK, downstream of MLK3, and strongly protected the hippocampus CA1 from ischemic damage. Missense oligos had no effect on any of the parameters measured. The Racl AS-ODNs results were further confirmed by administration of a Rac1 inhibitor (NSC23766), which markedly attenuated activation of Racl and JNK, and significantly attenuated apoptotic delayed neuronal cell death following cerebral ischemia. As a whole, these studies demonstrate an important role for Racl in activation of the prodeath MLK3-JNK kinase signaling pathway and delayed neuronal cell death following cerebral ischemia. (C) 2009 Elsevier B.V. All rights reserved.