Neuroprotection NR3A in Cultured Neurons and Ischemic Neonates

描述(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，并在很大程度上导致脑缺血后神经细胞的死亡。具体地说，NMDAR介导的钙内流和钾流出与坏死和细胞凋亡有关。NMDAR活性被NR3A抑制，NR3A是一种新发现的NMDAR亚单位，在新生神经元中大量表达，但在衰老细胞中表达减弱，神经元对兴奋毒性的易感性增加。NR3A是否参与新生儿脑耐受是一个重要但尚未确定的问题。我们推测，NR3A在发育中的脑中的表达在防止兴奋性毒性损伤诱导的神经细胞死亡中起着重要的神经保护作用。将在野生型(WT)和NR3A缺失(NR3A“‘”)培养的小鼠皮质神经元中，在转NR亚单位的HEK293细胞中，以及在WT和NR3A“’”小鼠的新生和成年缺血性卒中模型中，研究NMDAR相关神经元的死亡。特定目的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。膜片钳实验将检测NR3A表达减少的NMDAR的电压依赖的MG2阻断，并验证NR3A选择性减弱NMDA外向钾电流(NMDA-K)作为NR3A诱导的神经保护的新机制的假设。在表达不同水平的NR3A的培养细胞中，将检测到轻度和严重的NMDA损伤诱导的凋亡和坏死性死亡。我们将评估NR3A对细胞内钙([钙]i)、钾([K]j)、半胱氨酸天冬氨酸氨基转移酶(Caspase)激活的影响，以及它们与被抑制的NMDA电流和细胞死亡的关系。特定目的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。新生儿脑比成人脑对缺氧/缺血损伤有更强的耐受性，但其潜在机制尚不清楚。NR3A在大脑成熟过程中表达的下降与耐受性的减弱是平行的。我们将检验这一假设，即NR3A高水平和低水平的表达对应于新生儿和成年人不同的缺血耐受性。NR3A亚单位的神经保护作用将在体外低氧诱导的细胞死亡以及WT和NR3A“‘”小鼠的缺血性中风模型中进行检验。基于NR3A对NMDAR活性的独特抑制作用，它在发育中的大脑中的神经保护功能是一个合乎逻辑但未经检验的假说。这项研究将为一种重要的内源性自卫机制提供新的证据，该机制可能是新生儿对脑缺血耐受的基础。这项研究的发现也可能为卒中的预防和治疗提供一种新的受体治疗策略。

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.