Signaling by cAMP within Postsynaptic Nanodomains

突触后纳米结构域内的 cAMP 信号传导

基本信息

批准号：
8439852
负责人：
JOHANNES W HELL
金额：
$ 38.62万
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：
2012
资助国家：
美国
起止时间：
2012-08-15 至 2017-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8439852
关键词：
Action Potentials Acute Address Adenylate Cyclase Adrenergic Receptor Affect Albuterol Alzheimer&apos s Disease Antibodies Attention Autistic Disorder Back Binding Binding Sites Brain Caliber Cardiac Cells Complex Cyclic AMP Cyclic AMP-Dependent Protein Kinases Data Dendritic Spines Diffusion Disease Drug Delivery Systems Event G-Protein-Coupled Receptors Genetic Glutamate Receptor Glutamates Grant Head Heart Image Immunoblotting Individual Ion Channel Lead Link Maintenance Mediating Membrane Mental Depression Mental disorders Modeling Molecular Monitor Mus Mutate Neurons Norepinephrine PKA inhibitor Pancreas Peptides Phospho-Specific Antibodies Phosphorylation Point Mutation Post-Traumatic Stress Disorders Production Prosencephalon Proteins Psyche structure Receptor Signaling Regulation Resolution Role Sampling Science Signal Transduction Site Slice Smooth Muscle Stimulus Stroke Synapses Testing Tetanus Theta Rhythm Training Up-Regulation Vertebral column Work alertness density experience interest nervous system disorder novel postsynaptic presynaptic density protein 95 prevent receptor receptor binding response

项目摘要

DESCRIPTION (provided by applicant): Synapses are central to neuronal signaling and prime targets for drug treatments of neurological and mental disorders. Norepinephrine (NE) regulates attention and alertness. The ? adrenergic receptor ( ? AR) is emerging as the prevalent postsynaptic NE effector at glutamatergic synapses, where it interacts with AMPAR, NMDAR and the postsynaptic L-type Ca2+ channel Cav1.2. These complexes also contain Gs, adenylyl cyclases (ACs) and PKA, the downstream effectors of ? AR, for what appears to be highly localized signaling (within 100 nm) by cAMP (e.g., our work in Science 293, 98; Science 293, 2205; EMBO J 29, 482). Such spatial restriction would explain specific regulation of certain targets of the ? AR - Gs - AC - cAMP - PKA cascade and especially of AMPAR, NMDAR and Cav1.2. This project takes advantage of unique features of glutamatergic postsynaptic sites, which are formed by dendritic spines. AMPAR, NMDAR and Cav1.2 are localized at spine heads by a protein meshwork, the postsynaptic density (PSD), which is small (~300 nm) and can be isolated biochemically. Aim 1 is to test on a molecular level the hypothesis that specific acute or genetic disruption of the ? AR-AMPAR/NMDAR association affects ? AR-induced phosphorylation of these receptors but not of Cav1.2 that is co-localized within the very same PSDs (PSDs will be immunoprecipitated with antibodies against AMPAR, NMDAR or Cav1.2 for subsequent phospho-analysis of all 3 channels). The ? AR- Cav1.2 binding will be disrupted to test the reverse. Aim 2 will functionally monitor by high resolution Ca2+ imaging ? AR-stimulated Ca2+ influx through NMDAR and Cav1.2 within same spines with the hypothesis that disrupting ? AR - NMDAR binding will only inhibit ? AR-stimulated Ca2+ influx through NMDAR but not Cav1.2 ? AR (and vice versa). Aim 3 is to test on a systemic level whether ? AR binding to glutamate receptors, to Cav1.2, or both are important for regulation of a form of LTP induced by a tetanus of 5 Hz (endogenous theta rhythm) for 180 s that requires stimulation of the ? AR and Cav1.2 activity. This work will define unexplored fundamental molecular mechanisms of how NE regulates postsynaptic functions. It will thereby create a framework for understanding neurological diseases such as Alzheimer's disease, which is at least in part due to dysregulation of Cav1.2 and NMDAR by ? AR signaling, and stroke induced neuronal damage, which is at least in part due to upregulation of Ca2+ permeable AMPAR, which in turn are targeted to postsynaptic sites by ? AR signaling. NE signaling is also relevant for PTSD and depression. The postsynaptic assembly of specific signaling components that control PKA-mediated phosphorylation of AMPAR, NMDAR and Cav1.2 constitutes a potentially effective and specific target for drugs that disrupt some of these interactions while not affecting others. Finally, this work will address the question of how localized cAMP signaling can be, which might be <100 nm given the small size of postsynaptic sites. Because ? ARs also associate with Cav1.2 in heart, smooth muscle and pancreas, spatially restricted cAMP signaling is of wide interest beyond its role in the brain. PUBLIC HEALTH RELEVANCE: This project is to investigate the role of physical interactions between proteins that mediate signaling by norepinephrine at synapses, the contact points between neurons where they transmit their signals, typically via glutamate. Aberrant functioning of norepinephrine signaling, of glutamate receptors, and of the Ca2+ channel Cav1.2 are implicated in mental and neurological diseases such as posttraumatic stress disorder, autism, depression, and Alzheimer's disease. Defining new molecular aspects of NE signaling will identify important new drug targets for treatment of these diseases.

描述（由申请人提供）：突触是神经元信号传导的核心，也是神经和精神疾病药物治疗的主要靶点。去甲肾上腺素 (NE) 调节注意力和警觉性。这？肾上腺素能受体 (? AR) 正在成为谷氨酸能突触中常见的突触后 NE 效应器，与 AMPAR、NMDAR 和突触后 L 型 Ca2+ 通道 Cav1.2 相互作用。这些复合物还含有 G、腺苷酸环化酶 (AC) 和 PKA，即 ? 的下游效应子。 AR，用于 cAMP 的高度局部化信号传导（100 nm 内）（例如，我们在 Science 293, 98；Science 293, 2205；EMBO J 29, 482 中的工作）。这种空间限制可以解释对某些目标的具体监管？ AR - Gs - AC - cAMP - PKA 级联，尤其是 AMPAR、NMDAR 和 Cav1.2。该项目利用了由树突棘形成的谷氨酸能突触后位点的独特特征。 AMPAR、NMDAR 和 Cav1.2 通过蛋白质网络（即突触后密度 (PSD)）定位在脊柱头部，该蛋白质网络很小（约 300 nm），可以通过生化方法分离。目标 1 是在分子水平上检验以下假设：特定的急性或遗传破坏？ AR-AMPAR/NMDAR 关联影响 ? AR 诱导这些受体的磷酸化，但不诱导位于相同 PSD 内的 Cav1.2 的磷酸化（PSD 将使用针对 AMPAR、NMDAR 或 Cav1.2 的抗体进行免疫沉淀，以便随后对所有 3 个通道进行磷酸化分析）。这？ AR-Cav1.2 结合将被破坏以测试反向。 Aim 2 将通过高分辨率 Ca2+ 成像进行功能监测？ AR 刺激 Ca2+ 通过 NMDAR 和 Cav1.2 在同一棘内流入，假设破坏 ? AR-NMDAR结合只会抑制？ AR 刺激 Ca2+ 通过 NMDAR 流入，但不通过 Cav1.2 ？ AR（反之亦然）。目标3是在系统层面上检验是否？ AR 与谷氨酸受体、Cav1.2 或两者的结合对于调节由 5 Hz 破伤风（内源性 θ 节律）诱导 180 秒的 LTP 形式非常重要，这种形式需要刺激 ? AR 和 Cav1.2 活动。这项工作将定义 NE 如何调节突触后功能的尚未探索的基本分子机制。因此，它将创建一个理解神经系统疾病（例如阿尔茨海默病）的框架，这种疾病至少部分是由于 ? 对 Cav1.2 和 NMDAR 的失调造成的。 AR 信号传导和中风引起的神经元损伤，这至少部分是由于 Ca2+ 渗透性 AMPAR 的上调，而 AMPAR 又通过 ? 靶向突触后位点。 AR 信号。 NE 信号传导也与 PTSD 和抑郁症相关。控制 PKA 介导的 AMPAR、NMDAR 和 Cav1.2 磷酸化的特定信号成分的突触后组装构成了药物的潜在有效且特定的靶标，可破坏其中一些相互作用，同时不影响其他相互作用。最后，这项工作将解决 cAMP 信号传导如何局部化的问题，考虑到突触后位点的尺寸可能小于 100 nm。因为？ AR 还与心脏、平滑肌和胰腺中的 Cav1.2 相关，空间受限的 cAMP 信号传导除了在大脑中的作用之外还受到广泛关注。公共健康相关性：该项目旨在研究突触处介导去甲肾上腺素信号传导的蛋白质之间的物理相互作用的作用，突触是神经元之间的接触点，通常通过谷氨酸传递信号。去甲肾上腺素信号传导、谷氨酸受体和 Ca2+ 通道 Cav1.2 的功能异常与精神和神经系统疾病有关，例如创伤后应激障碍、自闭症、抑郁症和阿尔茨海默病。定义 NE 信号传导的新分子方面将确定治疗这些疾病的重要新药物靶点。