The Hsp90-binding benzoquinoid ansamycin, geldanamycin (GA) protects Ht22 cells and immature primary rat cortical neuron cultures from glutamate induced oxidative toxicity. Furthermore, preliminary results suggest that GA given during resuscitation improves neurological outcome in rats subjected to global ischemia induced by asphyxial cardiac arrest. GA binding to HSP90 disrupts various intracellular signaling pathways and leads to induction of Hsp70, depletion of the Raf-1 protooconcogene, and reduced activation of downstream targets of Raf-1, ERK-1 and ERK-2. The downregulation of ERK activation caused by GA treatment might be an important component of its neuroprotective activity since inhibition of an ERK activating kinases (i.e. MEK-1) also protects against oxidative toxicity in Ht22 cells and primary rat cortical neuron cultures. We hypothesize that manipulation of Hsp90 function may be a useful strategy to impact various signal transduction pathways in vivo that trigger neuronal cell death. The identification of the molecular mechanisms involved in neuroprotection associated with pharmacological manipulation of Hsp90 function is the major goal of this application.
In specific aim 1 we will determine the impact of Hsp90 regulated signaling pathway on glutamate-induced oxidative toxicity in the Ht22 mouse hippocampal cell line and address the following questions. Does Hsp70 induction contribute to the protective effects of Hsp90 binding drugs in vitro? Is persistent ERK activation necessary and sufficient for glutamate-induced oxidative toxicity in Ht22 cells? Does glutamate-induced oxidative toxicity affect other members of the MAPK family (i.e. JNK/SAPK and P38MAPK)? In specific aim 2 we will determine the impact of Hsp90-regulated signaling pathways on glutamate-induced oxidative toxicity in immature primary rat cortical neuron cell cultures. Are GA and U1026 protective against oxidative toxicity in immature primary rat cortical neuron cell cultures? What biochemical events are associated with neuroprotective effects of Hsp90-binding drugs in immature primary rat cortical neuron cultures? Finally in Specific Aim 3 we will determine whether Hsp90 binding drugs are effective post-treatment neuroprotective agents in rat models of global ischemia. Do Hsp90-binding drugs improve neurological outcome following asphyxial cardiac arrest or middle cerebral artery occlusion? Do Hsp90-binding drugs induce Hsp70 in vivo? Do Hsp90-binding drugs affect MAPK activation in vivo?
