Signaling by cAMP within Postsynaptic Nanodomains

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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.