Neuroprotection NR3A in Cultured Neurons and Ischemic Neonates

描述（由申请人提供）：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，也是脑缺血后神经元细胞死亡的主要原因。具体来说，nmdar介导的Ca2+内流和K+外排与坏死和凋亡有关。NMDAR活性受到NR3A的抑制，NR3A是一种新发现的NMDAR亚基，在新生儿神经元中大量表达，但在老年细胞中随着神经元对兴奋毒性的易感性增加而减少。NR3A是否与新生儿脑耐受性有关是一个重要但尚未确定的问题。我们假设NR3A在发育中的大脑中的表达在防止兴奋毒性损伤引起的神经元细胞死亡中起着重要的神经保护作用。我们将在野生型（WT）和NR3A缺陷型（NR3A“’”）培养的小鼠皮质神经元、转染NR亚基的HEK293细胞以及WT和NR3A“’”小鼠的新生和成年缺血性卒中模型中研究nmdar相关的神经元死亡。特异性目的1将检测NR3A表达对nmda诱导的膜电流和兴奋性毒性的抑制作用。膜片钳实验将通过NR3A的表达来检测电压依赖性降低的Mg2+阻断NMDAR，并验证NR3A选择性地减弱NMDA外向K+ (NMDA-K)电流作为NR3A诱导神经保护的新机制的假设。在表达不同水平NR3A的培养细胞中检测轻度和重度NMDA诱导的凋亡和坏死死亡。NR3A对细胞内Ca2+ ([Ca2+]i), K+ ([K+]j)， caspase激活的影响，以及它们与抑制NMDA电流和细胞死亡的关系将被评估。特异性目的2将检测NR3A对缺氧和缺血诱导的细胞死亡的神经保护作用。新生儿大脑比成人大脑更能耐受缺氧/缺血性损伤，但其潜在机制尚不清楚。脑成熟过程中NR3A表达的下降与耐受性的减弱是一致的。我们将验证NR3A的高、低表达水平对应新生儿和成人不同的缺血耐受性的假设。NR3A亚基的神经保护作用将在体外缺氧诱导的细胞死亡以及WT和NR3A“'”小鼠的缺血性卒中模型中进行研究。基于NR3A对NMDAR活性的独特抑制作用，其在发育中的大脑神经保护功能是一个合乎逻辑但未经验证的假设。这项研究将为新生儿脑缺血耐受的重要内源性自卫机制提供新的证据。这项研究的结果也可能为预防和治疗中风的受体治疗提供新的策略。

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.