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）处，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连接，所述支架蛋白在学习和记忆的突触可塑性中发挥重要作用。AMPAR通过NMDAR激活被募集到树突棘，在海马神经元中通过也增加棘大小和肌动蛋白聚合的途径诱导长时程增强（LTP）期间。AMPAR-GluR 1亚基被cAMP依赖性蛋白激酶（PKA）磷酸化可能促进LTP期间招募的AMPARs的表面表达。相反，长期抑制（LTD）的诱导导致钙调神经磷酸酶蛋白磷酸酶2B（CaN）介导的PKA磷酸化GluR 1的去磷酸化，从突触中去除AMPAR，以及棘肌动蛋白解聚，随后是棘收缩。然而，协调调节AMPAR定位，磷酸化和脊柱结构可塑性的机制还没有得到很好的理解。A-激酶锚定蛋白（AKAP）79/150（人79/啮齿动物150）是一种PKA和CaN锚定蛋白，通过PSD-95和SAP 97膜相关鸟苷酸激酶（MAGUK）支架与NMDAR和AMPAR连接。AKAP 79/150通过结合磷脂酰肌醇-4，5-二磷酸（PIP 2）、F-肌动蛋白和钙粘蛋白粘附分子的N-末端碱性区靶向于棘。重要的是，上一个资助期和最近的初步研究结果表明，AKAP 79通过其靶向结构域的棕榈酰化被招募到LTP中的棘，AKAP 79过表达通过MAGUK结合增强树突棘大小和AMPAR活性。相反，在LTD中涉及AMPAR抑制和脊柱收缩的NMDAR-CaN信号通路破坏AKAP 79/150与肌动蛋白、MAGUKs和钙粘蛋白的相互作用，并导致AKAP和锚定的PKA从突触丢失。这种来自棘的AKAP 79/150易位依赖于肌动蛋白重组和磷脂酶C（PLC）对PIP 2的切割，初步研究表明棕榈酰化的额外调节。因此，AKAP 79/150可能在可塑性中发挥重要的结构和信号作用。由于PKA和CaN信号在神经元中的复杂性以及支架蛋白如AKAP 79/150的多功能性，使用简单的药理学、敲除或RNAi方法来理解这些蛋白所提供的特定突触后功能是相当大的挑战，因为这些方法一次性消除了所有功能。因此，在本项目中，我们将在培养的大鼠神经元中将RNAi敲除与突变体替代方法配对，此外还使用新型AKAP 150敲入突变小鼠来探测特定AKAP 79/150膜靶向基序和蛋白质-蛋白质相互作用在LTD和LTP诱导期间控制突触后结构和功能的功能。我们将测试的假设是，通过棕榈酰化（Aim 1）、MAGUK支架相互作用（Aim 1）和CaN锚定（Aim 2和3）调节AKAP 79/150突触后靶向和信号传导协调调节树突棘结构和AMPAR可塑性功能。 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AKAP 79/150组织的神经元兴奋性突触后信号传导过程被认为与神经系统疾病（如阿尔茨海默氏症和癫痫）和精神健康疾病（如唐氏综合征和精神分裂症）中突触可塑性和认知改变的机制有关。这些相同的途径也与理解过度的谷氨酸受体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有关，因此了解AKAP 79/150通过其结构相互作用和信号功能控制这些事件的作用对于了解人类疾病中改变的基本突触过程非常重要。

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.