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

• 批准号: 7321490
• 负责人: Shan P. Yu
• 金额: $ 25.55万
• 依托单位: MEDICAL UNIVERSITY OF SOUTH CAROLINA
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-01 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7321490
• 关键词: Adult; Affect; Apoptosis; Apoptotic; Attenuated; Birth; Brain; Brain Hypoxia-Ischemia; Brain Ischemia; Caspase; Cell Death; Cells; Cessation of life; Cultured Cells; Defense Mechanisms; Event; Glucose; Glutamate Receptor; Glutamates; Human; Hypoxia; In Vitro; Infant; Infarction; Injury; Investigation; Ischemia; Ischemic Brain Injury; Ischemic Stroke; Knock-out; Lead; Link; Mediating; Membrane; Modeling; Modification; Mus; N-Methyl-D-Aspartate Receptors; N-Methylaspartate; Necrosis; Neonatal; Neurons; Play; Predisposition; Prevention; Receptor Activation; Regulation; Role; Stimulus; Stroke; Testing; Week; age related; base; critical developmental period; deprivation; excitotoxicity; in vivo; insight; neonate; neuron apoptosis; neuron loss; neuroprotection; novel; patch clamp; prevent; receptor; research study; voltage

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，并且在很大程度上负责脑缺血后神经元细胞死亡。具体而言，NMDAR介导的Ca2+流入和K+外排与坏死和凋亡有关。 NR3A抑制了NMDAR活性，NR3A是一种新发现的NMDAR亚基，在新生儿神经元中大量表达，但在较老的细胞中减少了神经元脆弱性对兴奋性毒性的脆弱性。 NR3A是否有助于新生儿脑耐受性是一个重要但不确定的问题。我们假设发育中的大脑中的NR3A表达在预防兴奋性毒性损伤引起的神经元细胞死亡方面起着重要的神经保护作用。 NMDAR相关的神经元死亡将在野生型（WT）和NR3A缺陷型（NR3A“'”）中培养的小鼠皮质神经元，在用NR亚基转染的HEK293细胞中，以及新生儿和成人和成人缺血性的wt和nr3a”小鼠。具体目标1将检查NR3A表达对NMDA诱导的膜电流和兴奋性毒性的抑制作用。斑块夹实验将通过NR3A表达检查NR3A表达的NMDAR电压依赖性MG2+块的降低，并检验NR3A选择性地将NMDA向外降低了NMDA向外K+（NMDA-K）作为NR3A诱导的神经保护的新机制。在表达不同水平的NR3A的培养细胞中，将检查轻度和严重的NMDA侮辱性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抑制NMDA电流和细胞死亡的关系。具体目标2将检查NR3A对低氧和缺血诱导的细胞死亡的神经保护作用。与成人大脑相比，新生儿大脑对低氧/缺血性损伤的耐受性更大，但基本机制的定义不当。脑成熟过程中NR3A表达的下降与耐受性的降低相似。我们将检验以下假设：NR3A表达的高水平和低水平与新生儿和成年人的缺血性公差相对应。 NR3A亚基的神经保护作用将在缺氧引起的体外细胞死亡以及WT和NR3A的缺血性卒中模型中检查。基于NR3A对NMDAR活性的独特抑制作用，其在发育中的大脑中的神经保护功能是逻辑但未测试的假设。该研究将为重要的内源性自卫机制提供新的证据，该机制可能是对脑缺血的新生儿耐受性的基础。这项调查的发现还可能表明一种在预防和治疗中风的受体疗法的新策略。