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Neuroprotection NR3A in Cultured Neurons and Ischemic Neonates

NR3A 对培养神经元和缺血新生儿的神经保护作用

基本信息

批准号：
7999235
负责人：
Shan P. Yu
金额：
$ 26.58万
依托单位：
EMORY UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-09-01 至 2013-11-30
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): Excessive activation of the N-methyl-D-aspartate receptor (NMDAR) is a key event in excitotoxicity and largely responsible for neuronal cell death after brain ischemia. Specifically, NMDAR-mediated Ca2+ influx and K+ efflux have been linked to necrosis and apoptosis. NMDAR activities are inhibited by NR3A, a newly identified NMDAR subunit that is abundantly expressed in neonatal neurons but diminished in older cells with increased neuronal vulnerability to excitotoxicity. Whether NR3A contributes to the neonatal brain tolerance is an important but undetermined question. We hypothesize that NR3A expression in the developing brain plays an important neuroprotective role in preventing neuronal cell death induced by excitotoxic insults. NMDAR-associated neuronal death will be studied in wild type (WT) and NR3A-deficient (NR3A"'") cultured mouse cortical neurons, in HEK293 cells transfected with NR subunits, and in neonatal and adult ischemic stroke models of WT and NR3A"'" mice. Specific Aim 1 will examine the inhibitory effects of NR3A expression on NMDA-induced membrane currents and excitotoxicity. Patch clamp experiments will examine a reduced voltage-dependent Mg2+ block of NMDAR by NR3A expression, and test the hypothesis that NR3A selectively attenuates the NMDA outward K+ (NMDA-K) current as a novel mechanism of NR3A-induced neuroprotection. Mild and severe NMDA insult-induced apoptotic and necrotic death will be examined in cultured cells expressing different levels of NR3A. Effects of NR3A on intracellular Ca2+ ([Ca2+]i), K+ ([K+]j), caspase activation, and their relationships to suppressed NMDA currents and cell death will be assessed. Specific Aim 2 will examine the neuroprotective effect of NR3A against hypoxia- and ischemia-induced cell death. The neonatal brain is more tolerant of hypoxic/ischemic injury than the adult brain, but the underlying mechanism is not well defined. The decline of NR3A expression during brain maturation parallels the weakening of tolerance. We will test the hypothesis that the high and low levels of NR3A expression correspond to the different ischemic tolerances of neonates and adults. The neuroprotective role of the NR3A subunit will be examined in hypoxia-induced cell death in vitro as well as in ischemic stroke models of WT and NR3A"'" mice. Based on the unique inhibitory effect of NR3A on NMDAR activities, its neuroprotective function in the developing brain is a logical but untested hypothesis. The investigation will provide novel evidence for an important endogenous self-defense mechanism which may underlie neonatal tolerance to brain ischemia. The findings from this investigation may also suggest a new strategy of receptor therapy in prevention and treatment of stroke.

描述（由申请方提供）：N-甲基-D-天冬氨酸受体（NMDAR）的过度激活是兴奋性毒性的关键事件，并在很大程度上导致脑缺血后神经元细胞死亡。具体而言，NMDAR介导的Ca 2+内流和K+外流与坏死和凋亡有关。NMDAR活性被NR 3A抑制，NR 3A是一种新鉴定的NMDAR亚基，在新生神经元中大量表达，但在老年细胞中减少，神经元对兴奋性毒性的脆弱性增加。NR 3A是否有助于新生儿脑耐受是一个重要但尚未确定的问题。我们推测NR 3A在发育中的大脑中的表达在防止兴奋性毒性损伤诱导的神经元细胞死亡中起重要的神经保护作用。将在野生型（WT）和NR 3A缺陷型（NR 3A“”）培养的小鼠皮质神经元中、在用NR亚基转染的HEK 293细胞中以及在WT和NR 3A“”小鼠的新生和成年缺血性中风模型中研究NMDAR相关的神经元死亡。具体目标1将检查NR 3A表达对NMDA诱导的膜电流和兴奋性毒性的抑制作用。膜片钳实验将检查NR 3A表达对NMDAR的降低的电压依赖性Mg 2+阻断，并测试NR 3A选择性地减弱NMDA外向K+（NMDA-K）电流作为NR 3A诱导的神经保护的新机制的假设。将在表达不同水平NR 3A的培养细胞中检查轻度和重度NMDA损伤诱导的凋亡和坏死性死亡。将评估NR 3A对细胞内Ca 2+（[Ca 2 +]i）、K+（[K+]j）、半胱天冬酶活化的影响及其与抑制的NMDA电流和细胞死亡的关系。具体目标2将检查NR 3A对缺氧和缺血诱导的细胞死亡的神经保护作用。新生儿脑比成人脑更耐受缺氧/缺血损伤，但其潜在机制尚未明确。NR 3A在脑成熟过程中表达的下降与耐受性的减弱相平行。我们将检验NR 3A表达的高水平和低水平对应于新生儿和成人的不同缺血耐受性的假设。NR 3A亚基的神经保护作用将在体外缺氧诱导的细胞死亡中以及在WT和NR 3A+小鼠的缺血性中风模型中进行检查。基于NR 3A对NMDAR活性的独特抑制作用，其在发育中的脑中的神经保护功能是一个合乎逻辑但未经检验的假设。这项研究将为新生儿脑缺血耐受的重要内源性自我防御机制提供新的证据。本研究的结果也可能为脑卒中的预防和治疗提供一种新的受体治疗策略。