Mechanisms of CaMKII Binding to GluN2B and its Role in Synaptic Plasticity and Me

描述(由申请人提供)： 长时程增强(LTP)是一个重要的细胞过程，在调节突触强度方面发挥重要作用，而突触强度被认为是学习和记忆的基础。钙/钙调蛋白依赖的蛋白激酶II(CaMKII)和NMDA型谷氨酸受体2B(GluN2B)是LTP中两个重要的蛋白质。蛋白质之间的相互作用是正常诱导LTP所必需的。这些蛋白质的功能与许多神经疾病有关，如癫痫、中风和成瘾。特别令人感兴趣的是CaMKII在LTP维持阶段的作用。虽然该蛋白在LTP诱导中具有明确的作用，但其在维持中的功能尚不清楚。许多研究表明，CaMKII的酶活性不是必需的；然而，最近有研究表明，CaMKII与NMDAR-复合体之间的结构相互作用可能具有重要作用。进一步推测，由于这种相互作用的几个关键方面，CaMKII-GluN2B结合特异性地介导了这一效应。也就是说，CaMKII到GluN2B的易位是由LTP刺激诱导的，刺激消退后相互作用保持活跃状态，刺激后结合也持续。因此，GluN2B在LTP诱导后保持了CaMKII的活性和定位，为CaMKII在LTP维持中的结构作用提供了理论解释。本研究将从三个方面进一步探讨CaMKII-GluN2B结合在LTP维持中的作用，特别是假设这种相互作用是维持所需的，以及该过程的行为相关，即记忆存储。目的1：CaMKII的酶活性如何影响GluN2B结合？假设：CaMKII与GluN2B的相互作用不需要酶活性。目的利用两种ATP竞争性抑制剂CaMKII，H7和星形孢子素(ST)，使用以下方法：(I)使用纯化蛋白在体外实验中测量CaMKII与GluN2B的结合，以及(Ii)在异源细胞表达系统和原代神经元培养中刺激诱导CaMKII到GluN2B的转位。目的2：LTP的维持是由CaMKII-GluN2B的持续关联介导的吗？假设：LTP刺激后CaMKII与GluN2B的持续结合是LTP维持所必需的。目的通过比较野生型(WT)和CaMKII-GluN2B结合的无能敲门小鼠(KI)在CaMKII-NMDAR复合体和CaMKII-NMDAR复合体在用CaMKII抑制剂talCN21处理下的LTP维持减少方面进行比较。目标3：记忆巩固需要CaMKII-NMDAR复合体吗？假设：记忆存储依赖于CaMKII-NMDAR-复合体的持久联系。目的是通过在Morris水迷宫(MWM)上训练WT小鼠，然后测试它们在服用talCN21后的记忆保持，期望该抑制剂将逆转记忆巩固。

DESCRIPTION (provided by applicant): Long-term potentiation (LTP) is a cellular process important in the mediation of synaptic strength, which is thought to underlie learning and memory. Ca2+/Calmodulin-dependent protein kinase II (CaMKII) and the NMDA-type glutamate receptor subunit 2B (GluN2B) are two important proteins in LTP. The interaction between the proteins is additionally required for normal induction of LTP. The functions of these proteins have been associated with a number of neurological disorders such as epilepsy, stroke, and addiction. Of particular interest is the role f CaMKII in the maintenance phase of LTP. While this protein has a well- defined role in LTP induction, its function in maintenance is unclear. Many studies have demonstrated that enzymatic activity of CaMKII is not required; however, it has recently been suggested that a structural interaction between CaMKII and the NMDAR-complex may have an important role. It is further speculated that CaMKII-GluN2B binding specifically mediates this effect due to a few key aspects of that interaction. Namely, translocation of CaMKII to GluN2B is induced by LTP stimuli, the interaction maintains CaMKII in an active state after stimuli have subsided, and binding also persists post-stimuli. Therefore, GluN2B maintains CaMKII activity and localization after LTP induction, providing a theoretical explanation for the structural role of CaMKII in LTP maintenance. The current study will further investigate the role of CaMKII-GluN2B binding in LTP maintenance in three aims, specifically hypothesizing that the interaction is required for maintenance as well as the behavioral correlate of that process, i.e. memory storage. Aim 1: How does enzymatic activity of CaMKII effect GluN2B binding? Hypothesis: Enzymatic activity of CaMKII is not required for its interaction with GluN2B. Aim will be addressed using two ATP competitive inhibitors of CaMKII, H7 and staurosporine (ST), using the following approaches: (i) measurement of CaMKII-GluN2B binding in an in vitro assay using purified proteins, and (ii) stimulus-induced translocation of CaMKII to GluN2B in heterologous cellular expression systems and in primary neuron cultures. Aim 2: Is LTP maintenance mediated by the persistent association of CaMKII-GluN2B? Hypothesis: Sustained binding of CaMKII to GluN2B after LTP stimuli is required for LTP maintenance. Aim will be addressed by comparing wild type (WT) and CaMKII-GluN2B binding incompetent knockin mice (KI) with respect to (i) reduction in LTP maintenance measured under the treatment with the CaMKII inhibitor tatCN21 and (ii) reduction in CaMKII-NMDAR complexes under tatCN21 treatment. Aim 3: Are CaMKII-NMDAR complexes required for memory consolidation? Hypothesis: Memory storage is dependent on the persistent association of CaMKII-NMDAR-complexes. Aim will be addressed by training WT mice on the Morris Water Maze (MWM) and then testing their memory retention after administration of tatCN21, expecting that the inhibitor will reverse memory consolidation.