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

CK2 在发育过程中 NMDAR 运输和阿尔茨海默病中的作用

基本信息

批准号：
9266717
负责人：
Antonio Sanz-Clemente
金额：
$ 24.9万
依托单位：
NORTHWESTERN UNIVERSITY AT CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-08-15 至 2018-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9266717
关键词：
Acute Alzheimer&apos s Disease Amyloid beta-Protein Award Binding Biochemical Biochemistry Calcium Cerebral cortex Complex Coupled Data Development Electrophysiology (science)Equilibrium Event Evolution Excision Functional disorder Generations Glutamate Receptor Goals Immunofluorescence Microscopy Lead Learning Lentivirus Infections Ligands Light Link Memory Molecular Molecular Genetics Mus Mutate N-Methyl-D-Aspartate Receptors NMDA receptor A1 Neurodegenerative Disorders Neurotoxins Pathologic Pharmacology Phosphorylation Phosphotransferases Play Process Property Proteins Protocols documentation Receptor Activation Recruitment Activity Regulation Reporting Research Role Scaffolding Protein Site Synapses Synaptic Receptors Synaptic plasticity Testing abeta oligomer age related calmodulin-dependent protein kinase II casein kinase II critical period density expectation genetic approach nervous system disorder neuronal circuitry postsynaptic synaptic function tool trafficking

项目摘要

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.

描述（由申请人提供）：N-甲基-D-天冬氨酸受体（NMDAR）在发育、学习、记忆和许多神经系统疾病中发挥着核心作用。在大脑皮层中，NMDAR主要由两个GluN1和两个GluN2A或GluN2B亚基组成。 NMDAR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 NMDAR）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所起的作用。因此，本 K99/R00 奖项提案的目标是确定在突触成熟过程中发生的从 GluN2B 到 GluN2A 转换过程中调节突触 GluN2 组成的精确分子机制，并确定它们是否与 AD 中的突触功能障碍有关。具体来说，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 (S1480) 的 PDZ 结合域来调节 GluN2 突触组成，并且 CK2 活性是 GluN2 亚基所必需的转变。尽管突触 CK2 已被证明很重要，但突触活性如何调节该激酶仍不清楚，因为 CK2 被认为是一种组成型活性激酶并且不受钙调节。具体目标 1 将检验以下假设：CaMKII 对 CK2 的突触募集是 GluN2B S1480 磷酸化的关键步骤，CaMKII 充当支架蛋白，在 NMDAR 激活后连接 GluN2B 和 CK2。因此，在破坏 GluN2B/CaMKII/CK2 复合物后，将使用生物化学和免疫荧光显微镜测定 GluN2B S1480 磷酸化。我的中心假设是，GluN2 亚基转换是一个具有两个连续且耦合步骤的过程，其中需要通过 CK2 磷酸化来突触去除 GluN2B，以允许突触掺入 GluN2A。这将在具体目标 2 中使用生化和电生理学方法进行测试，分析用具有缺陷 S1480 磷酸化的突变 GluN2B (GluN2B E1479Q) 替换内源性 GluN2B 后的突触 GluN2 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 的基因改变小鼠系。最近的报告支持突触外 NMDAR 过度激活在 AD 中的作用。因此，使用我之前两个目标中生成的数据和工具，我将分析 Abeta 寡聚物（AD 中的主要神经毒素）是否通过异常的 CK2 过度激活导致 GluN2B 亚基重新分布（从突触到突触外位点）（具体目标 3）。该提案的成功完成将通过阐明开发过程中调节 GluN2 亚基组成的机制并确定 AD 中潜在的新药理学靶点产生重大的积极影响。