Phospho-regulation of NMDA receptors in neonatal brain hypoxia-ischemia

描述(申请人提供)：我们的长期目标是明确神经递质受体在新生儿缺氧缺血性脑损伤中的作用机制，并寻找治疗新生儿缺氧缺血性脑损伤的新的和特定的治疗靶点。我们的假设是：Fyn通过调节突触和突触外的NR2B信号通路参与了新生儿HI脑损伤的发生。目的1：确定Fyn在新生儿HI后如何调节不同突触后NR2B复合体的组成。假设：突触和突触外NMDAR在正常大脑和缺氧缺血后组装不同的蛋白质复合体，这些复合体被Fyn修饰。1)纯化WT、Fyn高表达小鼠、PP2处理小鼠和Y1472敲入小鼠的突触膜和突触外膜。1b)使用IP从两个隔间中拉下NR2B复合体。1c)使用蛋白质组学分析确定突触和突触外NR2B结合伙伴。1d)验证突触和突触外NR2B结合伙伴。目的：评价新生儿缺氧缺血后Y1472位NR2B的Fyn磷酸化的功能意义假说：Y1472位NR2B的Fyn磷酸化通过上调超氧化物和一氧化氮(NO)的形成来介导缺氧缺氧性脑损伤，评估缺氧缺血后Y1472敲入鼠的脑损伤。2B)研究Y1472位NR2B磷酸化调控HI后超氧化物歧化和NO生成的机制。2C)确定Y1472是否影响NR2B的表达和突触/突触外的定位。2D)确定Y1472是否影响NR2B与下游信号蛋白的关联。目的3：确定Fyn介导的NR2B磷酸化位点在体外缺氧缺血后的个体功能及3个位点的协同作用。假设：NR2B上特定残基的Fyn磷酸化激活了生存或细胞死亡信号通路。3A)。利用慢病毒介导的蛋白替换实验(LEMPRA)构建敲除内源性NR2B的方法，同时利用单个(Y1472、Y1252或Y1336)、双或三个磷酸化突变体在原代皮质神经元中表达外源NR2B。3B)。在缺氧、葡萄糖剥夺或NMDA暴露后，测量带有LEMPRA结构的神经元的细胞死亡和NMDAR表面表达。3C)。对具有LEMPRA结构的细胞进行电生理和钙成像，作为NMDAR活性的功能读数。脑缺氧缺血是导致儿童新生儿死亡和残疾的主要原因。这一提议代表了第一次系统和深入地研究Fyn介导的NR2B磷酸化如何参与新生儿HI脑损伤的发病机制。此外，在发育中的大脑中，依赖于位置的NR2B结合伙伴的识别还没有被表征，或者它如何改变对HI的反应。这项工作将为新的治疗方法提供信息，以选择性地将NMDAR或FYN从有害后果中分离出来，同时保留它们对新生儿的重要有益功能。

DESCRIPTION (provided by applicant): Our long-term objectives are to define mechanisms how neurotransmitter receptors contribute to neonatal hypoxic-ischemic (HI) brain injury, and to search for novel and specific therapeutic targets for neonatal HI. Our hypothesis for this project is: Fyn contributes to neonatal HI brain injury through regulation of both synaptic and extrasynaptic NR2B signaling pathways Aim 1: Determine how Fyn regulates NR2B complex composition at different postsynaptic locations after neonatal HI. Hypothesis: Synaptic and extrasynaptic NMDAR assemble different protein complexes in normal brain and after HI, which are modified by Fyn. 1A) Purify synaptic and extrasynaptic membranes from sham and HI-injured WT, Fyn overexpressing mice, PP2-treated mice and Y1472 knock-in mice. 1B) Use IP to pull down NR2B complexes from the two compartments. 1C) Use proteomic analysis to identify synaptic and extrasynaptic NR2B binding partners. 1D) Validate synaptic and extrasynaptic NR2B binding partners. Aim 2: Assess the functional significance of Fyn phosphorylation of NR2B at Y1472 after neonatal HI Hypothesis: Fyn phosphorylation of NR2B at Y1472 mediates HI brain injury through upregulation of superoxide and nitric oxide (NO) formation 2A) Assess brain injury in Y1472 knock-in mice following HI. 2B) Investigate the mechanisms by which NR2B phosphorylation at Y1472 regulates superoxide and NO formation after HI. 2C) Determine if Y1472 affects NR2B expression and synaptic/extrasynaptic localization. 2D) Determine if Y1472 affects NR2B association with downstream signaling proteins. Aim 3: Determine the individual function of Fyn-mediated NR2B phosphorylation site and the coordination of three sites following HI in vitro. Hypothesis: Fyn phosphorylation of specific residues on the NR2B activates either survival or cell death signaling pathways. 3A). Use the LEMPRA (lentiviral-mediated protein replacement assay) constructs to knockdown endogenous NR2B and simultaneously express exogenous NR2B with single (Y1472, Y1252 or Y1336), double or triple phosphorylation mutants in primary cortical neurons. 3B). Measure cell death and NMDAR surface expression in neurons with LEMPRA constructs following oxygen glucose deprivation or NMDA exposure. 3C). Perform electrophysiology and calcium imaging for cells with LEMPRA constructs as functional readouts of NMDAR activity. Brain hypoxia-ischemia is a leading cause of neonatal mortality and disabilities in children. This proposal represents the first systematic and in-depth examination of how Fyn-mediated NR2B phosphorylation is involved in the pathogenesis of HI brain injury in the newborn. Moreover, identification of location-dependent NR2B binding partners has not been characterized in the developing brain, or how it changes in response to HI. This work will inform novel therapeutic approaches to selectively uncouple the NMDAR or Fyn from harmful consequences while preserving their important beneficial functions in neonates.