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

描述（由申请人提供）：我们的长期目标是确定神经递质受体如何促进新生儿缺氧缺血性（HI）脑损伤的机制，并寻找新生儿HI的新的特异性治疗靶点。我们对这个项目的假设是：Fyn通过调节突触和突触外NR 2B信号通路来促进新生儿HI脑损伤目的1：确定Fyn如何调节新生儿HI后不同突触后位置的NR 2B复合物组成。假设：在正常脑和HI后，突触和突触外NMDAR组装不同的蛋白质复合物，这些蛋白质复合物被Fyn修饰。1A）从假手术和HI损伤的WT、Fyn过表达小鼠、PP 2处理的小鼠和Y1472敲入小鼠中纯化突触和突触外膜。1B）使用IP从两个隔室中拉下NR 2B复合物。1C）使用蛋白质组学分析来鉴定突触和突触外NR 2B结合配偶体。1D）突触内和突触外NR 2B结合配偶体。目标二：评估新生儿HI后Y1472处NR 2B的Fyn磷酸化的功能意义假设：Y1472处NR 2B的Fyn磷酸化通过超氧化物和一氧化氮（NO）形成的上调介导HI脑损伤2A）评估HI后Y1472敲入小鼠的脑损伤。2B）研究在HI后Y1472处的NR 2B磷酸化调节超氧化物和NO形成的机制。2C）确定Y1472是否影响NR 2B表达和突触/突触外定位。2D）确定Y1472是否影响NR 2B与下游信号传导蛋白的缔合。目的3：体外研究Fyn介导的NR 2B磷酸化位点的功能及HI后三个位点的协同作用。假设：NR 2B上特定残基的Fyn磷酸化激活存活或细胞死亡信号通路。3A）。使用LEMPRA（慢病毒介导的蛋白质替代测定）构建体敲除内源性NR 2B，并同时在原代皮质神经元中表达外源性NR 2B与单磷酸化突变体（Y1472、Y1252或Y1336）、双磷酸化突变体或三磷酸化突变体。3B）。在氧葡萄糖剥夺或NMDA暴露后，用LEMPRA构建体测量神经元中的细胞死亡和NMDAR表面表达。3C）。用LEMPRA构建体对细胞进行电生理学和钙成像，作为NMDAR活性的功能读数。脑缺氧缺血是新生儿死亡和儿童残疾的主要原因。这一建议代表了第一次系统和深入的检查如何Fyn介导的NR 2B磷酸化参与新生儿HI脑损伤的发病机制。此外，位置依赖性NR 2B结合伴侣的鉴定尚未在发育中的大脑中表征，或者它如何响应HI而变化。这项工作将提供新的治疗方法，以选择性地将NMDAR或Fyn从有害后果中解偶联，同时保留其在新生儿中的重要有益功能。

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.