Postsynaptic Signaling by Norepinephrine and cAMP

去甲肾上腺素和 cAMP 的突触后信号传导

基本信息

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
项目类别：
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&apos 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

项目摘要

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的突触后信号传导抽象的去甲肾上腺素（NE）调节注意力和机敏性。 β2AR肾上腺素受体（β2AR）是普遍的谷氨酸能突触的突触后NE效应子与AMPAR相互作用和突触后L- 类型Ca2+通道（LTCC）CAV1.2。这些复合物还包含β2AR的所有下游效应（gs，adenylyl CAMP高度局部信号传导的周期（ACS）和cAMP依赖性蛋白激酶PKA（请参阅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;科学信号10，EAAF9659和EAAF9647）。我们令人兴奋的初步数据表明1）NE进入神经元和内体通过NE转运蛋白OCT3和PMAT刺激细胞内β2AR信号传导并驱动AMPARS 到细胞表面； 2）营地降解磷酸二酯酶PDE4A5，在睡眠期间增加剥夺并介导了由此产生的记忆障碍，与pSD-与β2AR -AMPAR复合物有关 95; 3）CAV1.2形成具有AMPAR的超复合物，它允许在突触中有效激活Cav1.2 与NE存在的突触后部位的传播。 AIM 1是测试是否激活细胞内β2AR OCT3和PMAT触发AMPAR磷酸化和表面递送的NE吸收，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GLUA1（免疫荧光显微镜；如果）和NE依赖性LTP的表面插入被Oct3＆PMAT的抑制剂和KO废除。这将提供第一次缺乏但很重要的 NE在神经元中NE的细胞内信号传导的证据。 AIM 2是测试营地 - 水解PDE4A5是否通过β2AR -CAMP -PKA信号传导对AMPAR的遏制上调。我们将测试是否从中取代PDE4A5 glua1与多肽的GLUA1通过PKA升高GLUA1磷酸化，而HCS中GLUA1的表面表达（if）和突触后AMPARS响应（MEPSC，EPSC）。 AIM 3是确定AMPAR -CAV1.2相互作用位点并测试AMPAR -CAV1.2超复合形成的功能相关性。我们会破坏通过突变和急性胡椒的相互作用，以确定其在Ca2+影响树突状刺中的作用通过实时成像插入GLUA1表面。这项工作将定义意外的基本分子 NE如何调节突触后功能的机制。它将阐明可能是受到阿尔茨海默氏病等神经系统疾病的影响，至少部分是由于失调 CAV1.2和β2AR信号传导的谷氨酸受体。 NE信号传导也与ADHD和PTSD有关。这控制PKA介导的AMPAR磷酸化的特定信号成分的突触后组装和CAV1.2及其功能相互作用构成了对药物的潜在有效和特定靶标的破坏其中一些相互作用的同时不影响其他相互作用（例如，我们的肽dspl和pep2具体从AMPAR中取代β2AR，而不是CAV1.2，反之亦然，EMBO J 35,1330）。第1页