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

描述（由申请人提供）：N-甲基-D-天冬氨酸谷氨酸受体（NR）和谷氨酸回路发育与神经系统疾病有关，在谷氨酸网络发育的围产期发生起始事件。两种常见的功能障碍，自闭症谱系障碍和精神分裂症，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功能，并提供有助于减轻或消除此类破坏性疾病的信息。最近，含有NR 2B亚基的NR显示在Schaffer侧支-CA 1突触处产生长时程突触抑制（LTD），并且含有NR 2A亚基的NR的激活在相同接触处产生长时程突触增强（LTP）。这些研究并没有揭示负责产生二分结果的能力的两个亚基之间的差异。我们假设NR 2A和NR 2B亚基尾部负责NR对突触可塑性的亚基特异性作用。尾部含有磷酸化和相互作用结构域，并且还具有将NR锚至PSD-95和SAP 102的不同能力，PSD-95和SAP 102是将NR及其信号传导复合物结合至前脑和中脑区域中的突触后密度的主要膜相关鸟苷酸激酶（MAGUK）。为了验证这一假设，我们制作了两个嵌合亚基。将NR 2A和NR 2B亚基的外部和跨膜结构域（头部）的编码区与胞质尾区分离，并且将每个亚基的头部序列与另一个亚基的尾部序列重组，从而产生两种嵌合蛋白。我们建议检查用这些构建体和NR 1中的每一个共转染的HEK 293细胞。将用Ca++成像和全细胞膜片钳检查NR功能，以检查含有每个杂合亚基的NR的电流和动态特性。将这些反应与来自表达野生型亚基的HEK细胞的那些反应进行比较。我们还将在转染到NR 2AKO海马神经元中后测试嵌合体。最后，使用慢病毒将每种嵌合蛋白和每种野生型蛋白转染到NR 2 AKO小鼠中，并在海马切片中对感染的神经元进行膜片钳，我们将确定NR 2亚基尾部是否决定LTP与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.