Signaling by cAMP within postsynaptic nano domains R01NS078792

描述(申请人提供)：突触是神经元信号传递的中心，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。2肾上腺素能受体是谷氨酸能突触中广泛存在的突触后去甲肾上腺素效应分子，它与AMPAR、NMDAR和突触后L型钙通道Cav1.2相互作用。这些复合体还包含Gs、腺苷酸环化酶(ACS)和PKA，它们是ç2 AR的下游效应器，似乎是cAMP高度定位的信号(在100 nm内)(例如，我们在科学293，98；科学293,2205；EMBO J 29,482上的工作)。这种空间限制可以解释B2AR-Gs-AC-cAMP-PKA级联反应中某些靶点的特定调控，特别是AMPAR、NMDAR和Cav1.2的调控。这个项目利用了由树突棘形成的谷氨酸能突触后部位的独特特征。Ampar、NMDAR和Cav1.2通过突触后密度(PSD)的蛋白质网络定位于脊柱头部，突触后密度很小(~300 nm)，可以用生化方法分离。目标1是在分子水平上检验以下假设：AR-AMPAR/NMDAR关联的特定急性或遗传中断会影响 ç2 AR诱导这些受体的磷酸化，但不能诱导共定位于相同PSD中的Cav1.2的磷酸化(PSD将与抗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)。这个 ？2 AR-Cav1.2结合将被中断以测试相反的结合。AIM 2将通过高分辨率的钙成像对相同脊椎内通过NMDAR和Cav1.2刺激的钙内流进行功能监测，并假设破坏？2 AR-NMDAR结合只会抑制？2 AR刺激的钙内流，而不会抑制Cav1.2？2 AR的钙内流(反之亦然)。目的3是在系统水平上测试β2 AR与谷氨酸受体或Cav1.2的结合，或两者对调节由5赫兹(内源性Theta节律)的180 S诱导的一种形式的LTP是否重要，这需要刺激？2 AR和Cav1.2的活动。这项工作将定义尚未探索的NE如何调节突触后功能的基本分子机制。因此，它将创建一个了解阿尔茨海默病等神经系统疾病的框架，这至少部分是由于B 2 AR信号对Cav1.2和NMDAR的调节失调，以及中风引起的神经元损伤，这至少部分是由于钙离子通透性AMPAR的上调，而AMPAR又通过B 2 AR信号靶向突触后部位。NE信号也与创伤后应激障碍和抑郁有关。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一个潜在的有效和特异的药物靶点，这些药物可以破坏其中一些相互作用，而不影响其他相互作用。最后，这项工作将解决cAMP信号的局部化程度问题，考虑到突触后站点的小尺寸，cAMP信号可能是&lt；100 nm。由于在心脏、平滑肌和胰腺中也与Cav1.2相关，空间受限的cAMP信号除了在大脑中的作用外，还具有广泛的意义。

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.