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对突触后信号的影响 摘要 去甲肾上腺素(NE)调节注意力和警觉性。β-2AR肾上腺素能受体(β-2AR)是目前流行的 谷氨酸能突触上的突触后NE效应器，在那里它与AMPAR和突触后L相互作用 型钙通道(LTCC)CaV1.2。这些复合体还含有β2AR(Gs，腺苷)的所有下游效应物 细胞周期酶(Acs)和cAMP依赖的蛋白激酶PKA)，通过cAMP进行高度定位的信号传递(参见我们的 科学开创性工作293，98；科学293,2205；EMBO J 29,482；EMBO J 35,1330；Science 信号10、东距9659和东距9647)。我们令人兴奋的初步数据表明：1)去甲肾上腺素进入神经元并 通过NE转运体Oct3和pmAt刺激细胞内β2AR信号转导并驱动AMPAR 2)cAMP降解磷酸二酯酶PDE4A5，在睡眠中增加 剥夺并调节由此导致的记忆损害，通过PSD与β2AR-AMPAR复合体联系在一起。 95；3)CaV1.2与AMPAR形成超复合体，从而在突触过程中有效地激活CaV1.2 在有NE存在的突触后部位的传递。目的1是测试细胞内β2ARs的激活是否在 Oct3和pmAt摄取NE触发AMPAR磷酸化和表面递送，从而促进LTP AS 已知PKA可刺激AMPAR表面插入。我们将测试去甲肾上腺素诱导的磷酸化 (免疫印迹；IB)和GluA1(免疫荧光显微镜；IF)和NE依赖的LTP的表面插入 被抑制剂和Oct3和pmAt的KO所废除。这将是第一次提供缺乏但重要的 去甲肾上腺素在神经元细胞内传递信号的证据。目的2是测试cAMP水解物PDE4A5 通过β2AR-cAMP-PKA信号抑制AMPAR的上调。我们将测试是否将PDE4A5从 含多肽的GluA1增加PKA对GluA1的磷酸化及Hcs(IF)表面GluA1的表达 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超复合体形成的功能相关性。我们将扰乱 突变和急性多肽应用的相互作用确定其在钙离子流入树突棘中的作用 和GluA1表面的活体成像插入。这项工作将定义尚未探索的基本分子 NE调节突触后功能的机制。它将阐明新的分子细节，可能是 受神经疾病的影响，如阿尔茨海默病，这至少部分是由于 CaV1.2和谷氨酸受体通过β-2AR信号转导。NE信号也与ADHD和PTSD有关。这个 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潜在有效和特异的药物靶点 干扰其中一些相互作用，而不影响其他相互作用(例如，我们的多肽DSP1和Pep2 将β2AR从AMPAR中移开，但不从CAV1.2中移开，反之亦然，分别为EMBO J 35,1330)。 第1页