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和神经系统障碍药物治疗的主要目标的核心。去甲肾上腺素（NE）调节注意力和机敏性。这？肾上腺素能受体（？ar）正在作为谷氨酸能突触中普遍的突触后NE效应子，与AMPAR，NMDAR和突触后L型CA2+通道CAV1.2相互作用。这些复合物还包含GS，腺苷循环酶（ACS）和PKA，即下游效应子？ AR，对于CAMP（例如，我们在科学293，98; Science 293，2205; Embo J 29，482）中似乎是高度局部的信号传导（例如，我们在科学领域的工作）。这种空间限制会解释对某些目标的特定调节？ AR -GS -AC -CAMP -PKA级联，尤其是AMPAR，NMDAR和CAV1.2。该项目利用了由树突状刺形成的谷氨酸能后突触部位的独特特征。 AMPAR，NMDAR和CAV1.2通过蛋白网络（蛋白质网状），突触后密度（PSD）定位在脊柱头部，该密度很小（〜300 nm），可以通过生化分离。 AIM 1是在分子水平上检验的假设是特定的急性或遗传破坏？ AR-AMPAR/NMDAR协会影响？这些受体的AR诱导的磷酸化，但没有在非常相同的PSD中共定位的CAV1.2（PSD将用针对AMPAR，NMDAR或CAV1.2的抗体进行免疫沉淀，以进行所有3个通道的随后磷酸分析）。这？ AR-CAV1.2结合将被破坏以测试反向。 AIM 2可以通过高分辨率CA2+成像来进行功能监测吗？ AR刺激的Ca2+通过NMDAR和CAV1.2在同一棘突中具有破坏的假设？ AR -NMDAR结合只会抑制？ AR刺激的Ca2+通过NMDAR而不是CAV1.2？ AR（反之亦然）。 AIM 3是在系统级别上测试是否？ AR与谷氨酸受体结合，与CAV1.2，或两者都对调节由5 Hz（内源性theta节奏）诱导的LTP形式很重要180 s，需要刺激？ AR和CAV1.2活性。这项工作将定义NE如何调节突触后功能的未开发的基本分子机制。因此，它将创建一个框架，以理解诸如阿尔茨海默氏病这样的神经系统疾病，至少部分是由于CAV1.2和NMDAR的失调？ AR信号传导和中风诱导的神经元损伤，至少部分是由于Ca2+渗透性AMPAR的上调，而AMPAR的上调又是针对突触后部位的？ AR信号传导。 NE信号传导也与PTSD和抑郁症有关。控制PKA介导的AMPAR，NMDAR和CAV1.2的特定信号成分的突触后组装构成了破坏其中一些相互作用但不影响其他相互作用的药物的潜在有效和特定的靶标。最后，这项工作将解决局部cAMP信号传导的问题，鉴于突触后的大小较小，该问题可能小于100 nm。因为？ ARS还与心脏，平滑肌和胰腺中的Cav1.2相关联，在空间限制的cAMP信号传导中广泛关注其在大脑中的作用。公共卫生相关性：该项目是为了调查蛋白质之间的物理相互作用的作用，这些蛋白质在突触下通过去甲肾上腺素的信号传导，神经元之间的接触点通常通过谷氨酸传输信号。去甲肾上腺素信号，谷氨酸受体和Ca2+通道CAV1.2的异常功能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氏病。定义NE信号的新分子方面将确定重要的新药物来治疗这些疾病。