Signaling by cAMP within postsynaptic nano domains R01NS078792

突触后纳米域内 cAMP 的信号传导 R01NS078792

• 批准号: 9066462
• 负责人: JOHANNES W HELL
• 金额: $ 6.7万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-15 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9066462
• 关键词: Action Potentials; Acute; Address; Adenylate Cyclase; Adrenergic Receptor; Affect; Albuterol; Alzheimer' s Disease; Antibodies; Attention; Autistic Disorder; Back; Binding; Binding Sites; Brain; Cardiac; Cells; Complex; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Data; Dendritic Spines; Diffusion; Disease; Drug Targeting; Event; G-Protein-Coupled Receptors; Genetic; Glutamate Receptor; Glutamates; Grant; Head; Heart; Image; Immunoblotting; Individual; Ion Channel; 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; interest; nano; nervous system disorder; neurotransmission; novel; postsynaptic; presynaptic density protein 95; prevent; receptor; receptor binding; response

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 ß2 adrenergic receptor (ß2 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 ß2 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 ß2 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 ß2 AR-AMPAR/NMDAR association affects ß2 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 ß2 AR- Cav1.2 binding will be disrupted to test the reverse. Aim 2 will functionally monitor by high resolution Ca2+ imaging ß2 AR-stimulated Ca2+ influx through NMDAR and Cav1.2 within same spines with the hypothesis that disrupting ß2 AR - NMDAR binding will only inhibit ß2 AR-stimulated Ca2+ influx through NMDAR but not Cav1.2 ß2 AR (and vice versa). Aim 3 is to test on a systemic level whether ß2 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 ß2 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 ß2 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 ß2 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 ß2 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.

描述（由申请人提供）：突触是神经元信号传导的中心，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。β 2肾上腺素能受体（β 2 AR）作为突触后NE效应子出现在突触能突触上，与AMPAR、NMDAR和突触后L型Ca ~（2+）通道Cav1.2相互作用。这些复合物还含有Gs、腺苷酸环化酶（AC）和PKA，β 2 AR的下游效应物，用于通过cAMP（例如，《科学》293，98;《科学》293，2205;《欧洲分子生物学杂志》29，482）。这种空间限制可以解释β 2AR-Gs-AC-cAMP- PKA级联的某些靶点，特别是AMPAR、NMDAR和Cav1.2的特异性调节.该项目利用了由树突棘形成的突触后位点的独特功能。AMPAR、NMDAR和Cav1.2通过蛋白质网络（突触后密度（PSD））定位于棘头，所述PSD小（~300 nm）并且可以被生物化学分离。目的1是在分子水平上检验β 2 AR-AMPAR/NMDAR关联的特定急性或遗传破坏影响β 2 AR-AMPAR/NMDAR关联的假说。 β 2 AR诱导的这些受体的磷酸化，但不诱导共定位于非常相同的PSD内的Cav1.2的磷酸化（PSD将与抗AMPAR、NMDAR或Cav1.2的抗体免疫沉淀，用于所有3个通道的后续磷酸化分析）。的 β 2 AR- Cav1.2结合将被破坏以测试反向。目的2将通过高分辨率Ca 2+成像功能性地监测相同棘内通过NMDAR和Cav1.2的β 2 AR刺激的Ca 2+内流，假设破坏β 2 AR - NMDAR结合将仅抑制通过NMDAR而不是Cav1.2 β 2 AR的β 2 AR刺激的Ca 2+内流（反之亦然）。目的3是在系统水平上测试β 2 AR与谷氨酸受体、Cav1.2或两者的结合是否对调节由5 Hz强直（内源性θ节律）诱导的LTP形式重要，所述LTP形式需要β 2 AR和Cav1.2活性的刺激持续180 s。这项工作将确定NE如何调节突触后功能的未探索的基本分子机制。因此，它将创建一个框架，用于理解神经疾病，例如阿尔茨海默病，其至少部分是由于β 2 AR信号传导引起的Cav1.2和NMDAR的失调，以及中风诱导的神经元损伤，其至少部分是由于Ca 2+可渗透的AMPAR的上调，其又通过β 2 AR信号传导靶向突触后位点。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磷酸化的特定信号传导组分的突触后组装构成了药物的潜在有效和特异性靶点，这些药物破坏了其中一些相互作用，而不影响其他相互作用。最后，这项工作将解决如何定位cAMP信号传导的问题，考虑到突触后位点的小尺寸，其可能小于100 nm。由于β 2 AR也与心脏、平滑肌和胰腺中的Cav1.2相关，因此空间受限的cAMP信号传导除了在脑中的作用外还受到广泛关注。