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

• 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
• 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' 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

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核心作用。在大脑皮层中，NMDAR主要由两个GluN 1和两个GluN 2A或GluN 2B亚基组成。NMDAR的许多功能特性由GluN 2亚基决定，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）和其他年龄相关神经退行性疾病发展中的作用。因此，本K99/R 00奖项提案的目标是定义在突触成熟期间发生的从GluN 2B到GluN 2A的转换期间调节突触GluN 2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，酪蛋白激酶2（CK 2）在这些过程中的作用将进行分析，因为我以前已经证明，CK 2调节GluN 2突触的组成，磷酸化的PDZ结合域的GluN 2B（S1480）和CK 2的活性是必需的GluN 2亚基开关。虽然突触CK 2已被证明是重要的，但突触活动如何调节这种激酶仍然不清楚，因为CK 2被认为是一种组成型活性激酶，它不受钙调节。具体目标1将检验以下假设：CaMKII对CK 2的突触募集是GluN 2B S1480磷酸化的关键步骤，CaMKII在NMDAR激活后充当连接GluN 2B和CK 2的支架蛋白。因此，GluN 2B S1480磷酸化将在GluN 2B/CaMK II/CK 2复合物破坏后使用生物化学和免疫荧光显微镜测定。我的中心假设是，GluN 2亚基开关是一个过程，有两个顺序和耦合的步骤，其中需要通过CK 2磷酸化的GluN 2B的突触清除，以允许GluN 2A的突触掺入。这将在特定目标2中使用生物化学和电生理学方法进行测试，分析S1480磷酸化缺陷的突变型GluN 2B（GluN 2B E1479 Q）替代内源性GluN 2B后的突触GluN 2组成。几种分子遗传学方法将用于这种替换，包括慢病毒感染和产生表达GluN 2B E1479 Q的遗传改变的小鼠系。最近的报道支持突触外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AD中的主要神经毒素Abeta寡聚体是否通过异常CK 2过度激活导致GluN 2B亚基的重新分布（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GluN 2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积极影响。

项目成果

An Antibody Feeding Approach to Study Glutamate Receptor Trafficking in Dissociated Primary Hippocampal Cultures.

研究分离的原代海马培养物中谷氨酸受体贩运的抗体喂养方法。

• DOI: 10.3791/59982
• 发表时间: 2019
• 期刊: Journal of visualized experiments : JoVE
• 作者: Chiu,AndrewM;Barse,Levi;Hubalkova,Pavla;Sanz-Clemente,Antonio
• 通讯作者: Sanz-Clemente,Antonio

Diversity in NMDA receptor composition: many regulators, many consequences.

• DOI: 10.1177/1073858411435129
• 发表时间: 2013-02
• 期刊: The Neuroscientist : a review journal bringing neurobiology, neurology and psychiatry
• 作者: Sanz-Clemente A;Nicoll RA;Roche KW
• 通讯作者: Roche KW