Postsynaptic Signaling by Norepinephrine and cAMP

去甲肾上腺素和 cAMP 的突触后信号传导

• 批准号: 10445917
• 负责人: JOHANNES W HELL
• 金额: $ 47.14万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-02-01 至 2026-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10445917
• 关键词: Acute; Adenylate Cyclase; Adrenergic Receptor; Affect; Agonist; Alzheimer' s Disease; Arousal; Attention; Attention deficit hyperactivity disorder; Binding; Binding Sites; Biological Assay; Brain; Cell surface; Cells; Complex; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cytosol; Data; Dendritic Spines; Diffuse; Disease; Drug Targeting; Endoplasmic Reticulum; Endosomes; Ensure; Fostering; Glutamate Receptor; Glutamates; Goals; Hippocampus (Brain); Image; Individual; Injections; Isoproterenol; Link; Mediating; Mediator of activation protein; Membrane; Memory impairment; Microscopy; Modeling; Molecular; Monitor; Mus; Mutation; Neurologic; Neurons; Norepinephrine; Organic Cation Transporter; Peptides; Phosphorylation; Play; Point Mutation; Post-Traumatic Stress Disorders; Probability; Proteins; Psyche structure; Recycling; Regulation; Reporting; Research; Role; Schizophrenia; Science; Signal Transduction; Site; Sleep; Sleep Deprivation; Slice; Sodium Chloride; Stimulus; Surface; Synapses; Synaptic Transmission; Testing; Tetanus; Theta Rhythm; Time; Up-Regulation; Vertebral column; Western Blotting; Work; alertness; cognitive function; density; endosome lumen; inhibitor; knock-down; monoamine; nervous system disorder; new therapeutic target; noradrenaline transporter; phosphoric diester hydrolase; polypeptide; postsynaptic; protein kinase A kinase; response; targeted treatment; trafficking; uptake

Postsynaptic Signaling by Norepinephrine and cAMP Abstract Norepinephrine (NE) regulates attention and alertness. The β2AR adrenergic receptor (β2AR) is the prevalent postsynaptic NE effector at glutamatergic synapses, where it interacts with AMPARs and the postsynaptic L- type Ca2+ channel (LTCC) CaV1.2. These complexes also contain all downstream effectors of β2AR (Gs, adenylyl cyclases (ACs) and the cAMP-dependent protein kinase PKA) for highly localized signaling by cAMP (see our pioneering work in Science 293, 98; Science 293, 2205; EMBO J 29, 482; EMBO J 35, 1330; Science Signaling10, eaaf9659 and eaaf9647). Our exciting preliminary data indicate that 1) NE enters neurons and endosomes via the NE transporters OCT3 and PMAT to stimulate intracellular β2AR signaling and drive AMPARs to the cell surface; 2) the cAMP degrading phosphodiesterase PDE4A5, which is increased during sleep deprivation and mediates the resulting memory impairment, is linked to the β2AR - AMPAR complex via PSD- 95; 3) CaV1.2 forms a supercomplex with AMPARs, which allows efficient activation of CaV1.2 during synaptic transmission at postsynaptic sites with NE present. Aim 1 is to test whether activation of intracellular β2ARs upon NE uptake by OCT3 & PMAT triggers AMPAR phosphorylation and surface delivery, thereby promoting LTP as PKA is known to stimulate AMPAR surface insertion. We will test whether NE-induced phosphorylation (immunoblotting; IB) and surface insertion of GluA1 (immunofluorescent microscopy; IF) and NE-dependent LTP are abrogated by inhibitors and KO of OCT3 &PMAT. This will provide for the first time lacking but important evidence for intracellular signaling by NE in neurons. Aim 2 is to test whether the cAMP-hydrolyzing PDE4A5 curbs upregulation of AMPARs by β2AR - cAMP - PKA signaling. We will test whether displacing PDE4A5 from GluA1 with polypeptides elevates GluA1 phosphorylation by PKA and surface expression of GluA1 in HCs (IF) and postsynaptic AMPARs responses (mEPSC, EPSC). Aim 3 is to determine the AMPAR - CaV1.2 interaction sites and test the functional relevance of the AMPAR - CaV1.2 supercomplex formation. We will disrupt the interaction by mutations and acute peptide application to determine its role in Ca2+ influx into dendritic spines and GluA1 surface insertion by live imaging. This work will define unexplored fundamental molecular mechanisms of how NE regulates postsynaptic functions. It will elucidate new molecular details that might be affected in neurological diseases such as Alzheimer’s disease, which is at least in part due to dysregulation of CaV1.2 and glutamate receptors by β2AR signaling. NE signaling is also relevant for ADHD and PTSD. The postsynaptic assembly of specific signaling components that control PKA-mediated phosphorylation of AMPARs and CaV1.2 and their functional interactions constitute potentially effective and specific targets for drugs that disrupt some of these interactions while not affecting others (e.g., our peptides DSPL and Pep2 that specifically displace the β2AR from AMPAR but not CaV1.2 and vice versa, respectively EMBO J 35,1330). Page 1

去甲肾上腺素和cAMP的突触后信号传导 摘要 去甲肾上腺素（NE）调节注意力和警觉性。β 2 AR肾上腺素能受体（β 2 AR）是最常见的 突触后NE效应器在神经元能突触，在那里它与AMPAR和突触后L- 型钙通道（LTCC）CaV1.2.这些复合物还含有β 2 AR的所有下游效应物（Gs，腺苷酰 环化酶（AC）和cAMP依赖性蛋白激酶PKA）用于通过cAMP的高度定位的信号传导（参见我们的 科学的开创性工作293，98;科学293，2205; EMBO J 29，482; EMBO J 35，1330;科学 Signaling 10、eaaf 9659和eaaf 9647）。我们令人兴奋的初步数据表明：1）NE进入神经元， 通过NE转运蛋白OCT 3和PMAT刺激细胞内β2AR信号传导并驱动AMPAR 2）cAMP降解磷酸二酯酶PDE 4A 5，其在睡眠期间增加 剥夺并介导导致的记忆障碍，通过PSD与β 2AR-AMPAR复合物相关。 95; 3）CaV1.2与AMPAR形成超复合物，其允许在突触形成期间有效激活CaV1.2。 在NE存在的突触后位点的传递。目的1是测试细胞内β 2 AR的激活是否在 通过OCT 3和PMAT的NE摄取触发AMPAR磷酸化和表面递送，从而促进LTP， 已知PKA刺激AMPAR表面插入。我们将测试NE诱导的磷酸化是否 （免疫印迹; IB）和GluA 1的表面插入（免疫荧光显微镜; IF）以及NE依赖性LTP 被OCT 3和PMAT的抑制剂和KO废除。这将首次提供缺乏但重要的 神经元中NE的细胞内信号传导的证据。目的2是测试cAMP水解PDE 4A 5是否 通过β 2 AR- cAMP - PKA信号转导抑制AMPAR的上调。我们将测试是否将PDE 4A 5从 GluA 1与多肽通过PKA和HC中GluA 1的表面表达提高GluA 1磷酸化（IF） 和突触后AMPAR反应（mEPSC、EPSC）。目的3是确定AMPAR -CaV 1.2相互作用 位点，并测试AMPAR-CaV 1.2超复合物形成的功能相关性。我们会破坏 突变和急性肽应用的相互作用，以确定其在Ca 2+流入树突棘中的作用 和GluA 1表面插入。这项工作将定义未探索的基本分子 NE如何调节突触后功能的机制。它将阐明新的分子细节 影响神经系统疾病，如阿尔茨海默氏病，这是至少部分由于失调， CaV1.2和谷氨酸受体通过β2AR信号传导。NE信号也与ADHD和PTSD有关。的 控制PKA介导的AMPAR磷酸化的特异性信号成分的突触后组装 和CaV1.2及其功能性相互作用构成了潜在有效和特异性的药物靶点， 破坏这些相互作用中的一些而不影响其他相互作用（例如，我们的肽DSPL和Pep 2， 从AMPAR置换β2AR，但不置换CaV 1.2，反之亦然，分别参见EMBO J 35，1330）。 第1页