TRPM2 channels and synaptic dysfunction following ischemic injury in the developing brain.

项目摘要 心脏骤停引起的全脑缺血会导致许多神经后遗症，包括 学习和记忆。这些缺陷在儿童身上和成年人身上都很明显。由此产生的神经学 儿童心脏骤停(CA)的后遗症可能是由神经元死亡和生理改变引起的 幸存的神经元。TRPM2通道是被高氧化应激激活的非选择性离子通道 在预防神经毒性和细胞功能障碍方面是引人注目的靶点。我们的实验室最近设计了一种 一种新的TRPM2通道抑制剂，称为talM2NX，以更好地了解TRPM2在神经元中的作用 死亡和功能障碍。评估神经元功能障碍的一个有用的方法是调查突触的水平 功能。神经元在海马区经历突触可塑性(LTP)的能力 被认为是一种与生俱来的功能衡量标准，是一种被广泛接受的学习和 记忆。该建议利用一种新的幼年小鼠心脏骤停模型(p21-25)来研究 TRPM2通道激活导致突触功能和认知功能损害的假说 全球脑缺血后的缺陷。我们发现在幼年CA引导后不久抑制TRPM2 保护突触功能，尽管在神经元死亡方面没有显著变化。我们将进一步测试 通过在延迟时间点(PCA后7-14天)抑制TRPM2来进行假说。将对TRPM2进行抑制 有药理作用(体内tATM2NX)或基因调控(TRPM2-/-，慢病毒shRNA TRPM2敲除) 功能将通过电生理学和行为进行评估。初步数据显示，推迟 抑制TRPM2可逆转CA后突触损伤。我们将利用信号转导技术 确定缺血诱导的TRPM2激活后突触损伤的机制。我们将重点关注 假设激活TRPM2信号钙调神经磷酸酶，导致突触功能下降。最后，我们 研究人员发现，虽然幼年动物的缺血会导致长达14天的突触功能受损 CA，有显著的内生恢复到对照水平。该项目的最终目标将调查 发育变化在神经元TRPM2表达中的作用从青春期到成年期可能 解释了内源性突触功能受损的恢复。总体而言，这个项目具有很高的转化率 通过重新定义儿童全脑缺血后治疗窗口的机会具有潜力。 该提案中概述的实验将为独立的研究生涯提供重要的培训。在这 建议，我将学习海马慢病毒shRNA的转染，行为测试，以及信号转导 评估蛋白质表达和功能的技术。组建的导师团队具有丰富的经验和 确保完成这一项目的专业知识。在完成这项建议后，我将把这些技能与 我已经熟悉并申请R01的电生理技术，以进一步表征 成年幼鼠心脏骤停后TRPM2活性的变化这项工作的影响将在 建立一种新的治疗策略来改善年轻大脑中缺血的神经学后果。

Project Summary Global cerebral ischemia caused by cardiac arrest results in many neurological sequelae, including deficits in learning and memory. These deficits are as evident in children as they are in adults. The resulting neurological sequelae from cardiac arrest (CA) in children likely arise from both neuronal death and altered physiology in surviving neurons. TRPM2 channels are non-selective ion channels that are activated by hyperoxidative stress and are compelling targets in preventing neurotoxicity and cellular dysfunction. Our lab has recently designed a novel inhibitor of TRPM2 channels, known as tatM2NX, to better understand the role of TRPM2 in neuronal death and dysfunction. A useful measure to assess neuronal dysfunction is to investigate the level of synaptic function. The ability for neurons to undergo synaptic plasticity (long-term potentiation; LTP) in the hippocampus is recognized as an innate measure of function and is a widely accepted cellular model for learning and memory. This proposal makes use of a novel cardiac arrest model in juvenile mice (p21-25) to investigate the hypothesis that activation of TRPM2 channels contributes to impairment of synaptic function and cognitive deficits following global cerebral ischemia. We have found that inhibiting TRPM2 soon after juvenile CA leads to preservation of synaptic function, despite no significant change in neuronal death. We will further test the hypothesis by inhibiting TRPM2 at delayed time points (7-14 days after PCA). Inhibition of TRPM2 will be done with pharmacology (in vivo tatM2NX) or genetic modulation (TRPM2-/-, lentiviral shRNA TRPM2 knockdown) and function will be assessed by electrophysiology and behavior. Preliminary data suggest that delayed inhibition of TRPM2 reverses synaptic impairments after CA. We will use signal transduction techniques to identify the mechanism for synaptic impairment after ischemia-induced TRPM2 activation. We will focus on the hypothesis that activation of TRPM2 signals calcineurin, leading to decreased synaptic function. Finally, we have found that while ischemia in young animals results in impairment of synaptic function up to 14 days after CA, there is remarkable endogenous recovery to control levels. The final aim of the project will investigate the role of developmental changes in TRPM2 expression in neurons through puberty into adulthood that may account for endogenous recovery of impaired synaptic function. Overall, this project has high translational potential through the opportunity of redefining therapeutic windows after global cerebral ischemia in children. Experiments outlined in this proposal will provide important training for an independent research career. In this proposal, I will learn hippocampal lentivirus shRNA transfection, behavior testing, and signal transduction techniques to assess protein expression and function. The mentor team assembled has experience and expertise to assure completion of this project. Upon completion of this proposal, I will combine these skills with electrophysiology techniques that I am already familiar with and apply for an R01 to further characterize TRPM2 activity in the maturing juvenile mouse after cardiac arrest. The impact of this work will be in establishing a novel therapeutic strategy to improve neurological consequences of ischemia in the young brain.