Signaling by cAMP within Postsynaptic Nanodomains

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。的？肾上腺素能受体（？AR）作为突触后NE效应物在突触能突触中出现，在那里它与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，下游效应？AR，对于似乎是cAMP的高度局部化信号传导（在100 nm内）（例如，《科学》293，98;《科学》293，2205;《欧洲分子生物学杂志》29，482）。这种空间限制将解释特定的调节某些目标的？AR-Gs-AC-cAMP- PKA级联反应，尤其是AMPAR、NMDAR和Cav1.2.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是测试在分子水平上的假设，即特定的急性或遗传破坏的？AR-AMPAR/NMDAR关联影响？AR诱导的这些受体的磷酸化，但不诱导共定位于相同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）。什么？AR-Cav 1. 2结合将被破坏以测试反向。Aim 2将通过高分辨率Ca 2+成像进行功能监测？AR刺激的Ca ~（2+）内流通过NMDAR和Cav1.2在相同的脊椎与假设，破坏？AR - NMDAR结合只会抑制？AR刺激的Ca 2+内流通过NMDAR，但不是Cav1.2？AR（反之亦然）。目标3是在系统层面上测试是否？AR结合谷氨酸受体，Cav1.2，或两者都是重要的调节形式的LTP诱导的强直5赫兹（内源性θ节律）为180秒，需要刺激的？AR和Cav1.2活性。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，了解神经系统疾病，如阿尔茨海默氏病，这是至少部分由于失调的Cav1.2和NMDAR？AR信号，和中风引起的神经元损伤，这至少部分是由于上调钙渗透AMPAR，这反过来又有针对性的突触后位点？AR信号。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。为什么？AR还与心脏、平滑肌和胰腺中的Cav1.2相关，空间限制的cAMP信号传导除了在大脑中的作用外还受到广泛关注。

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.