Regulation of AKAP79 Postsynaptic Membrane Targeting

描述（由申请人提供）：在神经元兴奋性突触的突触后密度（PSD）， AMPA （AMPAR）和NMDA （NMDAR）谷氨酸受体通过支架蛋白网络与树突棘中的信号蛋白和肌动蛋白细胞骨架相连，这些蛋白在学习和记忆的突触可塑性中起重要作用。ampar在海马神经元长时程增强（LTP）诱导过程中通过NMDAR激活被募集到树突棘，其途径也增加了棘的大小和肌动蛋白聚合。camp依赖性蛋白激酶（PKA）对AMPAR-GluR1亚基的磷酸化可能促进LTP过程中募集的ampar的表面表达。相反，长期抑制（LTD）的诱导导致钙调磷酸酶2B （CaN）介导的PKA磷酸化GluR1的去磷酸化，突触中ampar的去除和脊柱肌动蛋白的解聚，随后脊柱收缩。然而，协调调节AMPAR定位、磷酸化和脊柱结构可塑性的机制尚不清楚。a -激酶锚定蛋白（AKAP） 79/150 （human79/rodent150）是一种PKA和CaN锚定蛋白，通过PSD-95和SAP97膜相关鸟苷酸激酶（MAGUK）支架与NMDARs和AMPARs连接。AKAP79/150通过结合磷脂酰肌醇-4,5-二磷酸（PIP2）、F-actin和cadherin粘附分子的n端碱性区靶向脊柱。重要的是，上一个资助期的研究结果和最近的初步研究表明，AKAP79通过其靶向结构域的棕榈酰化被招募到LTP的脊柱中，并且AKAP79过表达通过MAGUK结合增加树突脊柱的大小和AMPAR活性。相反，与AMPAR抑制和脊柱萎缩相关的NMDAR-CaN信号通路破坏了AKAP79/150与肌动蛋白、MAGUKs和钙粘蛋白的相互作用，导致AKAP和锚定的PKA从突触中丢失。这种来自脊柱的AKAP79/150易位依赖于肌动蛋白重组和PIP2的磷脂酶C （PLC）裂解，初步研究表明棕榈酰化是额外的调节作用。因此，AKAP79/150可能在可塑性中发挥重要的结构和信号作用。由于神经元中PKA和CaN信号的复杂性以及支架蛋白（如AKAP79/150）的多功能性，使用简单的药理学、敲除或RNAi方法来了解这些蛋白所服务的特定突触后功能是一个相当大的挑战，因为这些方法可以一次消除所有功能。因此，在本项目中，除了使用一种新的AKAP150敲入突变小鼠外，我们还将在培养的大鼠神经元中配对RNAi敲入和突变体替代方法，以探索在LTD和LTP诱导过程中，特定AKAP79/150膜靶向基元的功能和蛋白质-蛋白质相互作用在控制突触后结构和功能中的作用。我们将测试的假设是，通过棕榈酰化（Aim 1）、MAGUK脚手架相互作用（Aim 1）和CaN锚定（Aim 2和3）调节AKAP79/150突触后靶向和信号传导，协调调节树突棘结构和AMPAR功能。公共卫生相关性：我们正在研究的控制树突棘结构和谷氨酸受体功能的akap79 /150组织的神经元兴奋性突触后信号传导过程被认为与阿尔茨海默氏症和癫痫等神经系统疾病以及唐氏综合症和精神分裂症等精神健康障碍中突触可塑性和认知改变的机制有关。这些相同的通路也与理解谷氨酸受体过度激活如何导致神经退行性疾病、脑损伤和中风中的兴奋性毒性神经元死亡有关。特别是，谷氨酸受体活性和树突棘结构变化的调节与可塑性和兴奋性都有关系，因此了解AKAP79/150通过其结构相互作用和信号功能在控制这些事件中的作用，对于理解人类疾病中改变的基本突触过程是重要的。

DESCRIPTION (provided by applicant): At the postsynaptic density (PSD) of neuronal excitatory synapses, AMPA (AMPAR) and NMDA (NMDAR) glutamate receptors are linked to signaling proteins and the actin cytoskeleton in dendritic spines through a network of scaffolding proteins that play important roles during synaptic plasticity underlying learning and memory. AMPARs are recruited to dendritic spines through NMDAR activation during induction of long term potentiation (LTP) in hippocampal neurons through pathways that also increase spine size and actin polymerization. Phosphorylation of AMPAR-GluR1 subunits by the cAMP-dependent protein kinase (PKA) may promote surface expression of AMPARs recruited during LTP. In contrast, induction of long-term depression (LTD) leads to calcineurin-protein phosphatase 2B (CaN) mediated dephosphorylation of PKA phosphorylated GluR1, removal of AMPARs from synapses and depolymerization of spine actin followed by spine shrinkage. However, mechanisms for coordinately regulating AMPAR localization, phosphorylation, and spine structural plasticity are not well understood. A-kinase-anchoring protein (AKAP) 79/150 (human79/rodent150) is a PKA and CaN anchoring protein linked to NMDARs and AMPARs through PSD-95 and SAP97 membrane-associated guanylate kinase (MAGUK) scaffolds. AKAP79/150 is targeted to spines by an N-terminal basic region that binds phosphatidylinositol-4,5-bisphosphate (PIP2), F-actin, and cadherin adhesion molecules. Importantly, findings from the last funding period and recent preliminary studies indicate that AKAP79 is recruited to spines in LTP through palmitoylation of its targeting domain and that AKAP79 overexpression enhances dendritic spine size and AMPAR activity through MAGUK binding. In contrast, NMDAR-CaN signaling pathways implicated in AMPAR depression and spine shrinkage in LTD disrupt AKAP79/150 interactions with actin, MAGUKs and cadherins and lead to loss of the AKAP and anchored PKA from synapses. This AKAP79/150 translocation from spines depends on actin reorganization and phospholipase C (PLC) cleavage of PIP2, and preliminary studies suggest additional modulation by palmitoylation. Thus, AKAP79/150 is likely to play important structural and signaling roles in plasticity. Due to the complexity of PKA and CaN signaling in neurons and the multi-functionality of scaffold proteins such as AKAP79/150, it is a considerable challenge to understand the specific postsynaptic functions served by these proteins using simple pharmacologic, knock-out or RNAi approaches because these methods eliminate all functions at once. Thus, in this project we will pair RNAi knockdown with a mutant replacement approach in cultured rat neurons in addition to using a novel AKAP150 knock-in mutant mouse to probe the functions of specific AKAP79/150 membrane targeting motifs and protein-protein interactions in control of postsynaptic structure and function during induction of LTD and LTP. The hypotheses that we will be testing are that regulation of AKAP79/150 postsynaptic targeting and signaling by palmitoylation (Aim 1), MAGUK scaffolding interactions (Aim 1), and CaN anchoring (Aims 2 & 3) coordinately regulate dendritic spine structure and AMPAR function in plasticity. PUBLIC HEALTH RELEVANCE: The AKAP79/150-organized neuronal excitatory postsynaptic signaling processes we are studying that control dendritic spine structure and glutamate receptor function are believed to be relevant for mechanisms of altered synaptic plasticity and cognition in neurological disorders such as Alzheimer's and epilepsy and mental health disorders such as Down syndrome and schizophrenia. These same pathways also have relevance for understanding how excessive glutamate receptor activation leads to excitotoxic neuronal death in neurodegenerative diseases, brain injury and stroke. In particular, regulation of glutamate receptor activity and dendritic spine structural changes have been implicated in both plasticity and excitoxicity, thus understanding the role of AKAP79/150 in controlling these events through both its structural interactions and signaling functions is important for understanding basic synaptic processes that are altered in human disease.