TRPV1-mediated induction of a protective heat shock response after stroke

描述(申请人提供)：在美国，缺血性中风是导致死亡和残疾的主要原因之一；然而，目前的治疗选择非常有限。我们最近通过对清醒小鼠的TRPV1通道的选择性药理兴奋来促进治疗性低温(TC=33�C)，从而证明了中风后显著的神经保护作用。我们在目前的提案中扩展了这些发现，证明了来自TRPV1激动剂的神经保护的很大一部分实际上是独立于低温效应的。在探索这种新的低温非依赖效应的机制时，我们发现TRPV1激动剂促进了大脑中的保护性热休克反应(HSR)。TRPV1是一种钙离子渗透性非选择性阳离子通道，存在于体温调节系统的热敏感觉纤维以及大脑中的多种其他类型的细胞中。HSR是一种内源性细胞反应，它涉及热休克蛋白(HSPs)的快速转录激活，热休克蛋白有助于防止或逆转细胞损伤。这种细胞保护机制有助于身体对包括中风在内的各种侮辱和伤害做出生存反应。在大脑中，Hsp27和Hsp70似乎在中风后的神经恢复中发挥关键作用。该项目的总体目标是证明TRPV1通道激动剂是一种有效的治疗策略，可以促进HSR的激活和缺血性卒中后的持续神经保护。具体地说，我们将1)确定TRPV1介导的HSR在未损伤和卒中脑中的诱导机制和细胞特异性，以及2)确定TRPV1介导的HSR诱导是否在中风后提供持续的神经保护。将对培养的脑星形胶质细胞和神经元进行初步研究，以测试TRPV1介导的Hsp27和Hsp70的诱导机制，使用药物调节剂和从机制特异性基因敲除(KO)小鼠建立的培养。这些研究之后将进行整体动物研究，以确定在野生型和机制特异的KO小鼠中，TRPV1激动剂在未受损伤的脑和局灶性脑缺血后诱导HSR的体内剂量-反应、时间进程和细胞特异性。最后，将对中风小鼠进行研究，以确定TRPV1介导的HSR诱导是否在再灌注28天内提供持续的神经保护。神经保护将通过组织学/免疫荧光方法来评估，以确定细胞的特异性存活、血脑屏障完整性和细胞凋亡，并通过行为测试来证明最终的功能恢复。Hsp27和Hsp70在神经保护中的具体作用将在各自的KO小鼠身上确定。预计这些研究将证明TRPV1激动剂在大脑中的新的神经保护机制，并为改进中风和其他神经退行性疾病的治疗策略奠定基础。

DESCRIPTION (provided by applicant): Ischemic stroke is among the leading causes of death and disability in the United States; however, current treatment options are very limited. We recently demonstrated significant neuroprotection following stroke by promoting therapeutic hypothermia (Tc=33� C) through selective pharmacological agonism of TRPV1 channels in conscious mice. We extend these findings in the present proposal by demonstrating that a significant portion of the neuroprotection derived from TRPV1 agonism is actually independent of the hypothermic effect. While seeking the mechanism of this novel hypothermia- independent effect, we discovered that TRPV1 agonists promote a protective heat shock response (HSR) in the brain. TRPV1 is a Ca2+-permeable non-selective cation channel found within heat- sensitive sensory fibers of the thermoregulatory system as well as multiple other cell types in the brain. The HSR is an endogenous cellular response that involves rapid transcriptional activation of heat shock proteins (Hsps) which contribute to the prevention or reversal of cellular damage. This cytoprotective mechanism contributes to the body's survival response to a variety of insults and injuries, including stroke. In the brain, Hsp27 and Hsp70 appear to play critical roles in neurorecovery from stroke. The overall aim of this project is to demonstrate TRPV1 channel agonism as an effective therapeutic strategy to promote HSR activation and sustained neuroprotection following ischemic stroke. Specifically, we will 1) determine the mechanism and cell specificity of TRPV1-mediated HSR induction in the uninjured and stroke brain and 2) determine if TRPV1-mediated HSR induction provides sustained neuroprotection following stroke. Initial studies will be performed with cultured brain astrocytes and neurons to test proposed mechanisms of TRPV1-mediated induction of Hsp27 and Hsp70, using pharmacological modulators and cultures established from mechanism-specific knockout (KO) mice. These studies will be followed by whole animal studies to determine the in vivo dose- response, time course, and cell specificity of HSR induction by TRPV1 agonism in the uninjured brain and the brain following focal ischemic stroke in wild type and mechanism-specific KO mice. Lastly, studies will be performed with stroke mice to determine if TRPV1-mediated HSR induction provides sustained neuroprotection through 28 days of reperfusion. Neuroprotection will be evaluated by histological/immunofluorescence methods to determine cell specific survival, BBB integrity, and apoptosis and by behavior testing to demonstrate ultimate functional recovery. The specific roles of Hsp27 and Hsp70 in neuroprotection will be determined with respective KO mice. It is expected that these studies will demonstrate novel neuroprotective mechanisms of TRPV1 agonism in the brain and lay the groundwork for improved treatment strategies for stroke and other neurodegenerative diseases.