Neuroprotective effect of HSP27 in Cerebral Ischemia

描述(申请人提供)：HSP27是小热休克蛋白家族的成员，这是一组普遍存在的应激蛋白，几乎在所有生物体中都有表达。热休克蛋白27在脑缺血后明显诱导表达，实验证据表明，热休克蛋白27是一种很有前途的内源性神经保护剂，可对抗损伤诱导的神经细胞死亡。本研究试图探讨HSP27在脑缺血模型中的神经保护作用及其分子机制。有待检验的总体假设是，HSP27的增强表达和磷酸化依赖的激活通过新的抗细胞凋亡机制保护缺血性脑损伤。我们最近创造了过度表达野生型HSP27或非磷酸化HSP27突变体的转基因小鼠。通过转基因和基因转染法，我们已经获得了令人振奋的初步结果，表明HSP27的过表达在体内和体外都能保护缺血细胞的死亡，HSP27的神经保护作用依赖于磷酸化介导的蛋白激活，HSP27可能通过直接抑制ASK1和ASK1依赖的凋亡信号通路来实现神经保护作用。本申请中提出的研究将进一步探索HSP27在脑缺血中作为神经保护分子的作用，这项研究的结果可能对未来中风的治疗具有指导意义。目的1.验证HSP27的转基因过表达和磷酸化依赖的激活对局灶性脑缺血损伤具有保护作用的假说。目的2.验证HSP27对缺血性神经元损伤的神经保护作用是通过阻断ASK1依赖的细胞凋亡信号通路等新的抗细胞凋亡机制来实现的。目的3.验证TAT蛋白转导结构域介导的热休克蛋白27脑内转导对局灶性脑缺血损伤的保护作用。提出了体内和体外两种模型的研究。在体局灶性脑缺血动物模型模拟了临床脑缺血后脑内许多方面的病理生理变化。体外模型将通过允许进行精确的机制研究来补充体内研究。

DESCRIPTION (provided by applicant): 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.