Role of NR2A and NR2B Intracellular Tails in Hippocampal LTP and LTD

描述(申请人提供)：N-甲基-D-天冬氨酸谷氨酸受体(NRs)和谷氨酸回路的发育与神经系统疾病有关，在谷氨酸网络发育的围产期启动事件。自闭症谱系障碍和精神分裂症这两种常见的这类功能障碍就是很好的例子。对于自闭症和精神分裂症，遗传连锁分析已经确定了参与正常NR功能的分子。我们的目标是了解NR在早期回路形成中的作用，并提供有助于减轻或消除这种破坏性疾病的信息。最近，含有NR2B亚单位的NRs在Schaffer侧支-CA1突触产生长期突触抑制(LTD)，而含有NR2A亚单位的NRs激活在相同接触处产生长期突触增强(LTP)。这些研究没有揭示两个亚基之间的差异，这两个亚基负责产生二分性结果的能力。我们推测NR2A型和NR2B型亚基尾巴参与了NR对突触可塑性的亚基特异性效应。尾巴含有磷酸化和相互作用结构域，还具有将NR锚定到PSD-95和SAP102的不同能力，PSD-95和SAP102是主要的膜相关鸟苷晚期激酶(MAGUK)，它将NR及其信号复合体与前脑和中脑区域的突触后密度结合在一起。为了验证这一假设，我们制作了两个嵌合亚单位。NR2A和NR2B亚基的外区和跨膜区的编码区与胞质尾区分离，每个亚基的头序列与另一个亚基的尾序列重组，从而产生两个嵌合蛋白。我们建议检测与这些构建物和NR1共转染的HEK293细胞。NR功能将通过钙离子成像和全细胞膜片钳来检测，以检测包含每个杂交亚基的NRs的电流和动力学特性。这些反应将与表达野生型亚基的HEK细胞的反应进行比较。我们还将在将嵌合体导入NR2AKO海马神经元后进行测试。最后，通过慢病毒将每个嵌合体和每个野生型蛋白导入NR2AKO小鼠，并在海马片上膜片钳住受感染的神经元，我们将确定NR2亚单位尾部是否决定LTP和LTD。

DESCRIPTION (provided by applicant): N-methyl-D-aspartate glutamate receptors (NRs) and glutamate circuit development have been implicated in neurological diseases with initiating events in perinatal periods when glutamate networks are developing. Two prevalent dysfunctions of this type, autism spectrum disorders and schizophrenia, are good examples. For both autism and schizophrenia, genetic linkage analyses have identified molecules involved in normal NR function. Our goal is to understand NR function in early circuit formation and provide information that would help mitigate or eliminate such devastating diseases. Recently, NRs containing NR2B subunits were shown to produce long-term synaptic depression (LTD) at Schaffer collateral-CAl synapses, and activation of NRs containing NR2A subunits produced long-term synaptic potentiation (LTP) at the same contacts. These studies did not reveal the differences between the two subunits responsible for their abilities to produce dichotomous outcomes. We hypothesize that the NR2A and NR2B subunit tails are responsible for the subunit-specific effect of the NR on synaptic plasticity. The tails contain phosphorylation and interaction domains and also have a differential ability to anchor the NR to PSD-95 and SAP102, the predominant membrane-associated guanylate kinases (MAGUK) that bind the NR and its signaling complex to post- synaptic densities in fore-and mid-brain regions. To test this hypothesis, we made two chimeric subunits. The coding regions of the external and transmembrane domains ( heads) of the NR2A and NR2B subunits were separated from the cytoplasmic tail regions, and the head sequences for each subunit were recombined with the tail sequences of the other, thereby producing two chimeric proteins. We propose to examine HEK293 cells co-transfected with each of these constructs and NR1. NR function will be examined with Ca++ imaging and with whole-cell patch-clamping to examine the currents and dynamic properties of the NRs containing each hybrid subunit. These responses will be compared with those from HEK cells expressing wild-type subunits. We will also test the chimeras after transfection into NR2AKO hippocampal neurons. Finally, using lentiviral transfection of each chimeric and each wild-type protein into NR2AKO mice and patch-clamping infected neurons in hippocampal slices, we will determine if the NR2 subunit tails dictate LTP versus LTD.