Role of CK2 in NMDAR trafficking during development and in Alzheimer's disease

描述（由申请人提供）：n -甲基- d -天冬氨酸受体（NMDARs）在发育、学习、记忆和许多神经系统疾病中发挥核心作用。在大脑皮层中，NMDARs主要由两个GluN1和两个GluN2A或GluN2B亚基组成。NMDARs的许多功能特性是由GluN2亚基决定的，因此它们受到严格的控制机制。我的长期研究目标是了解谷氨酸受体失调（特别是NMDARs）在阿尔茨海默病（AD）和其他与年龄相关的神经退行性疾病的发展中的作用。因此，这项K99/R00奖励提案的目标是确定在突触成熟过程中从GluN2B到GluN2A转换过程中调节突触GluN2组成的精确分子机制，并确定它们是否参与AD的突触功能障碍。具体来说，我们将分析酪蛋白激酶2 （CK2）在这些过程中的作用，因为我之前已经证明CK2通过磷酸化GluN2B的PDZ结合域（S1480）来调节GluN2突触组成，并且CK2活性是GluN2亚基开关所必需的。尽管突触CK2已被证明是重要的，但突触活性如何调节这种激酶仍然不清楚，因为CK2被认为是一种组成活性激酶，它不受钙的调节。特异性Aim 1将验证CaMKII在突触募集CK2是GluN2B S1480磷酸化的关键步骤的假设，CaMKII在NMDAR激活后作为支架蛋白连接GluN2B和CK2。因此，GluN2B S1480磷酸化将在GluN2B/CaMKII/CK2复合物被破坏后，通过生物化学和免疫荧光显微镜来确定。我的中心假设是GluN2亚基转换是一个具有两个连续和耦合步骤的过程，其中需要通过CK2磷酸化突触去除GluN2B以允许GluN2A的突触结合。这将在Specific Aim 2中使用生化和电生理方法进行测试，分析内源性GluN2B被S1480磷酸化缺陷的突变GluN2B （GluN2B E1479Q）取代后突触GluN2的组成。几种分子遗传学方法将用于这种替代，包括慢病毒感染和产生表达GluN2B E1479Q的转基因小鼠系。最近的报告支持突触外NMDARs过度激活在AD中的作用。因此，我将使用我之前两个目标中生成的数据和工具，分析阿尔茨海默病的主要神经毒素Abeta寡聚物是否通过CK2异常过度激活导致GluN2B亚基（从突触到突触外位点）的重新分布（Specific Aim 3）。该提案的成功完成将对阐明GluN2亚基组成在发育过程中的调节机制和确定AD中潜在的新药理靶点具有重大的积极影响。

DESCRIPTION (provided by applicant): N-methyl-D-aspartate receptors (NMDARs) play a central role in development, learning, memory, and in many neurological disorders. In cerebral cortex NMDARs are mainly composed of two GluN1 and two GluN2A or GluN2B subunits. Many functional properties of NMDARs are determined by GluN2 subunits, so they are subjected to strict control mechanisms. My long-term research objective is to understand the role that glutamate receptors dysregulation (in particular, NMDARs) plays in the development of Alzheimer's disease (AD) and other age-related neurodegenerative diseases. Therefore, the goal of this K99/R00 award proposal is to define the precise molecular mechanisms that regulate synaptic GluN2 composition during the switch from GluN2B to GluN2A that occurs during synaptic maturation and to determine if they are involved in synaptic dysfunction in AD. Specifically, the role of casein kinase 2 (CK2) in these processes will be analyzed, since I have previously demonstrated that CK2 regulates GluN2 synaptic composition by phosphorylating the PDZ binding domain of GluN2B (S1480) and that CK2 activity is required for the GluN2 subunit switch. Although synaptic CK2 has been shown to be important, how synaptic activity regulates this kinase remains obscure, since CK2 is considered a constitutively active kinase and it is not regulated by calcium. Specific Aim 1 will test the hypothesis that synaptic recruitment of CK2 by CaMKII is a key step for GluN2B S1480 phosphorylation, with CaMKII acting as a scaffolding protein to link GluN2B and CK2 after NMDAR activation. Therefore, GluN2B S1480 phosphorylation will be determined after disruption of the GluN2B/CaMKII/CK2 complex, using biochemistry and immunofluorescence microscopy. My central hypothesis is that the GluN2 subunit switch is a process with two sequential and coupled steps, in which the synaptic removal of GluN2B by CK2 phosphorylation is required to allow synaptic incorporation of GluN2A. This will be tested using biochemical and electrophysiological approaches in the Specific Aim 2, analyzing the synaptic GluN2 composition after the replacement of endogenous GluN2B by mutated GluN2B with defective S1480 phosphorylation (GluN2B E1479Q). Several molecular genetic approaches will be used for this replacement including lentivirus infection and the generation of a genetically-altered mouse line expressing GluN2B E1479Q. Recent reports support a role for extrasynaptic NMDARs overactivation in AD. Therefore, using the data and tools generated in my two previous Aims I will analyze if Abeta oligomers, main neurotoxins in AD, leads to a redistribution in GluN2B subunit (from synaptic to extrasynaptic sites) via aberrant CK2 overactivation (Specific Aim 3). The successful completion of this proposal will have a significant positive impact by elucidating the mechanisms regulating GluN2 subunit composition during development and identifying a potential new pharmacological target in AD.