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：
10557151
负责人：
JOHANNES W HELL
金额：
$ 47.59万
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/10557151
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 membrane Cell surface Cells Co-Immunoprecipitations Complex Cyclic AMP Cyclic AMP Receptors Cyclic AMP-Dependent Protein Kinases Cytosol DLG4 gene Data Dendritic Spines Diffuse Disease Drug Targeting Endoplasmic Reticulum Endosomes Ensure Fostering Glutamate Receptor Glutamates Goals Hippocampus Image Individual Injections Isoproterenol Learning Link Mediating Mediator 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 Deprivation Slice Sodium Chloride Stimulus Surface Synapses Synaptic Membranes 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 synaptic function 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) 调节注意力和警觉性 β2AR 肾上腺素受体 (β2AR) 是最常见的。谷氨酸能突触的突触后 NE 效应器，与 AMPAR 和突触后 L-相互作用 Ca2+ 型通道 (LTCC) CaV1.2 这些复合物还包含 β2AR 的所有下游效应子（Gs、腺苷酸）。环化酶 (AC) 和 cAMP 依赖性蛋白激酶 PKA）用于 cAMP 的高度局部化信号传导（请参阅我们的科学 293, 98；科学 293, 2205；EMBO J 35, 1330；信号传导 10、eaaf9659 和 eaaf9647)。我们令人兴奋的初步数据表明 1) NE 进入神经元并内体通过 NE 转运蛋白 OCT3 和 PMAT 刺激细胞内 β2AR 信号传导并驱动 AMPAR 2) cAMP 降解磷酸二酯酶 PDE4A5，该酶在睡眠期间增加剥夺并介导由此产生的记忆障碍，通过 PSD- 与 β2AR - AMPAR 复合体相连 95; 3) CaV1.2 与 AMPAR 形成超级复合物，从而可以在突触期间有效激活 CaV1.2 存在 NE 的突触后位点的传递目的 1 是测试细胞内 β2AR 是否被激活。 OCT3 和 PMAT 摄取 NE 会触发 AMPAR 磷酸化和表面递送，从而促进 LTP 已知 PKA 会刺激 AMPAR 表面插入，我们将测试 NE 是否诱导磷酸化。（免疫印迹；IB）和 GluA1 的表面插入（免疫荧光显微镜；IF）和 NE 依赖性 LTP 被 OCT3 和 PMAT 的抑制剂和 KO 废除，这将首次提供缺乏但重要的内容。神经元中 NE 的细胞内信号转导的证据目标 2 是测试 cAMP 是否水解 PDE4A5。通过 β2AR - cAMP - PKA 信号传导抑制 AMPAR 的上调我们将测试是否取代 PDE4A5。 GluA1 与多肽通过 PKA 升高 GluA1 磷酸化以及 HC 中 GluA1 的表面表达 (IF) 和突触后 AMPAR 反应（mEPSC、EPSC）目标 3 是确定 AMPAR - CaV1.2 相互作用。位点并测试 AMPAR - CaV1.2 超复合物形成的功能相关性。通过突变和急性肽应用相互作用以确定其在 Ca2+ 流入树突棘中的作用这项工作将定义尚未探索的基本分子。 NE 如何调节突触后功能的机制它将阐明可能的新分子细节。阿尔茨海默氏病等神经系统疾病的影响，这至少部分是由于 CaV1.2 和 β2AR 信号转导的谷氨酸受体也与 ADHD 和 PTSD 相关。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药物的潜在有效和特定靶点破坏其中一些相互作用，同时不影响其他相互作用（例如，我们的肽 DSPL 和 Pep2 专门取代 AMPAR 中的 β2AR，但不取代 CaV1.2，反之亦然，分别为 EMBO J 35,1330）。第1页