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。的吗?肾上腺素能受体？AR)是谷氨酸能突触中普遍存在的突触后NE效应物，它与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（ACs）和PKA，它们是？AR，似乎是cAMP高度定位的信号（在100纳米内）（例如，我们在Science 293, 98; Science 293, 2205; EMBO J 29, 482）。这种空间限制可以解释对某些目标的特定调控。AR - Gs - AC - cAMP - PKA级联，特别是AMPAR， NMDAR和Cav1.2。该项目利用了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通过突触后密度（PSD）蛋白网定位于脊柱头部，该蛋白网很小（约300 nm），可通过生物化学方法分离。目的1是在分子水平上测试特定急性或基因破坏的假设？AR-AMPAR/NMDAR关联影响？ar诱导了这些受体的磷酸化，而不是Cav1.2的磷酸化，后者在相同的psd内共定位（psd将与针对AMPAR、NMDAR或Cav1.2的抗体免疫沉淀，以便随后对所有3个通道进行磷酸化分析）。的吗?将AR- Cav1.2结合破坏，进行反向测试。Aim 2将通过高分辨率Ca2+成像功能监测？ar刺激的Ca2+内流通过同一脊柱内的NMDAR和Cav1.2，假设破坏？AR - NMDAR结合只会抑制？ar刺激Ca2+内流通过NMDAR而不是Cav1.2 ？AR（反之亦然）。目的三是在系统层面上测试是否？AR与谷氨酸受体结合，与Cav1.2结合，或两者同时结合，对于由5hz（内源性θ节奏）持续180 s的破伤风诱导的LTP形式的调节是重要的，这种LTP需要刺激？AR和Cav1.2活性。这项工作将定义未探索的NE如何调节突触后功能的基本分子机制。因此，它将为理解阿尔茨海默病等神经系统疾病创造一个框架，阿尔茨海默病至少部分是由于Cav1.2和NMDAR的失调。AR信号和中风引起的神经元损伤，这至少部分是由于Ca2+可渗透AMPAR的上调，而AMPAR反过来又被靶向到突触后位点。基于“增大化现实”技术的信号。NE信号也与创伤后应激障碍和抑郁症有关。控制pka介导的AMPAR、NMDAR和Cav1.2磷酸化的特定信号组件的突触后组装构成了药物的潜在有效和特异性靶标，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，这项工作将解决如何定位cAMP信号的问题，考虑到突触后位点的小尺寸，它可能小于100 nm。因为?ARs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.