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（NRs）和谷氨酸回路的发育与神经系统疾病有关，在围产期谷氨酸网络发育时启动事件。自闭症谱系障碍和精神分裂症这两种常见的功能障碍就是很好的例子。对于自闭症和精神分裂症，遗传联系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在早期电路形成中的功能，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NR2B亚基的nrrs在Schaffer侧侧cal突触上产生长期突触抑制（LTD），而含有NR2A亚基的nrrs的激活在相同的接触上产生长期突触增强（LTP）。这些研究并没有揭示两个亚基之间的差异，这两个亚基负责产生二分结果的能力。我们假设NR2A和NR2B亚基尾部与NR对突触可塑性的亚基特异性作用有关。尾巴包含磷酸化和相互作用结构域，并且还具有将NR锚定到PSD-95和SAP102的差异能力，PSD-95和SAP102是主要的膜相关鸟苷酸激酶（MAGUK），将NR及其信号复合物结合到前脑和中脑区域的突触后密度。为了验证这个假设，我们制造了两个嵌合亚基。将NR2A和NR2B亚基的外膜结构域和跨膜结构域（头部）的编码区与细胞质尾部区分离，将每个亚基的头部序列与另一个亚基的尾部序列重组，从而产生两个嵌合蛋白。我们建议检测这些构建体和NR1共转染的HEK293细胞。NR功能将通过Ca++成像和全细胞贴片箝位来检查包含每个混合亚基的NR的电流和动态特性。这些反应将与表达野生型亚单位的HEK细胞的反应进行比较。我们还将在转染NR2AKO海马神经元后测试嵌合体。最后，使用慢病毒转染每种嵌合蛋白和每种野生型蛋白到NR2AKO小鼠和海马切片中贴片夹紧感染的神经元，我们将确定NR2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.