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.

Public Health Relevance

This project is to investigate the role of physical interactions between proteins that mediate signaling by norepinephrine at synapses, the contact points between neurons where they transmit their signals, typically via glutamate. Aberrant functioning of norepinephrine signaling, of glutamate receptors, and of the Ca2+ channel Cav1.2 are implicated in mental and neurological diseases such as posttraumatic stress disorder, autism, depression, and Alzheimer's disease. Defining new molecular aspects of NE signaling will identify important new drug targets for treatment of these diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS078792-02
Application #
8533055
Study Section
Synapses, Cytoskeleton and Trafficking Study Section (SYN)
Program Officer
Stewart, Randall R
Project Start
2012-08-15
Project End
2017-06-30
Budget Start
2013-07-01
Budget End
2014-06-30
Support Year
2
Fiscal Year
2013
Total Cost
$377,330
Indirect Cost
$127,071
Name
University of California Davis
Department
Pharmacology
Type
Schools of Medicine
DUNS #
047120084
City
Davis
State
CA
Country
United States
Zip Code
95618
Hell, Johannes W (2014) CaMKII: claiming center stage in postsynaptic function and organization. Neuron 81:249-65
Zhang, Yonghong; Matt, Lucas; Patriarchi, Tommaso et al. (2014) Capping of the N-terminus of PSD-95 by calmodulin triggers its postsynaptic release. EMBO J 33:1341-53
Murphy, Jessica A; Stein, Ivar S; Lau, C Geoffrey et al. (2014) Phosphorylation of Ser1166 on GluN2B by PKA is critical to synaptic NMDA receptor function and Ca2+ signaling in spines. J Neurosci 34:869-79
Zhang, Mingxu; Patriarchi, Tommaso; Stein, Ivar S et al. (2013) Adenylyl cyclase anchoring by a kinase anchor protein AKAP5 (AKAP79/150) is important for postsynaptic *-adrenergic signaling. J Biol Chem 288:17918-31
Harvey, Robert D; Hell, Johannes W (2013) CaV1.2 signaling complexes in the heart. J Mol Cell Cardiol 58:143-52