HSP27 is a member of the small heat shock protein family, a group of ubiquitous stress proteins that are expressed in virtually all organisms. The expression of HSP27 is markedly induced in the brain after cerebral ischemia, and experimental evidence suggests that HSP27 is a promising endogenous neuroprotectant against injury-induced neuronal cell death. The studies outlined in this proposal attempt to investigate the neuroprotective effect of HSP27 and the underlying molecular mechanism in models of cerebral ischemia. The overall hypothesis to be tested is that enhanced expression and phosphorylation- dependent activation of HSP27 protects against ischemic brain injury via novel anti-apoptotic mechanisms. We have recently created transgenic mice overexpressing either the wild-type HSP27 or a non- phosphorylatable HSP27 mutant. Using both transgenic and gene-transfection approaches, we have obtained exciting preliminary results suggesting that overexpression of HSP27 protects against ischemic cell death in both in vivo and in vitro settings, that the neuroprotective effect of HSP27 is dependent on phosphorylation-mediated activation of the protein, and that HSP27 may achieve the neuroprotective effect by directly inhibiting ASK1 and ASK1-dependent apoptosis signaling pathways. The proposed studies outlined in this application will further explore HSP27 as a neuroprotective molecule in cerebral ischemia, and results from this research may have future therapeutic implications for stroke. The following specific objectives are proposed:
Aim 1. Test the hypothesis that transgenic overexpression and phosphorylation-dependent activation of HSP27 protects against focal ischemic brain injury.
Aim 2. Test the hypothesis that the neuroprotective effect of HSP27 against ischemic neuronal injury is mediated via novel anti-apoptotic mechanisms involving the disruption of ASK1-dependent apoptosis signaling pathways.
Aim 3. Test the hypothesis that TAT protein transduction domain-mediated delivery of HSP27 into the brain protects against focal ischemic brain injury. Studies in both in vivo and in vitro models are proposed. The in vivo animal model of focal cerebral ischemia mimics many aspects of pathophysiological changes in the brain after clinical ischemia. The in vitro models will complement the in vivo studies by allowing for precise mechanistic studies to be performed.
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