The presynaptic serotonin (5-hydroxytryptamine, 5-HT) transporter (SERT) dictates 5-HT inactivation following release in the brain and periphery and is a major target for antidepressant medications as well as psychostimulants, including cocaine and 3,4-methylenedioxymethamphetamine (MDMA, "Ecstasy"). Studies over the past two decades by our group have progressively illuminated the structural basis for 5-HT clearance and the mechanisms resident within serotonergic neurons and terminals that provide for control of membrane SERT activity, including evidence of trafficking-dependent and independent pathways. Furthermore, we have identified multiple, naturally occurring SERT coding variants (e.g. Gly56Ala) that disrupt SERT regulation in vitro. In the prior iteration of this project, we identified protein kinase G subtype 1 (PKG1) and p38 MAPKa as critical determinants of receptor-driven SERT trafficking and catalytic activation, respectively. In this renewal application, we propose to advance these studies through an integrated series of in vitro, ex vivo and in vivo analyses capitalizing on our increased understanding of receptor systems activating these pathways and the development of novel transgenic mouse models. Through this effort we seek to not only explore the fundamental biology underlying SERT regulation, but also to provide proof of concept for novel therapeutic approaches to treat 5-HT associated brain disorders.
In Specific Aim I, we seek to establish the structural basis of SERT regulation by A3A adenosine receptors (A3ARs) and IL-1 receptors (IL-1Rs) that provide for rapid SERT modulation. Specifically, we investigate the hypothesis that these receptors participate in, and signal through physical complexes with SERT and rely on cGMP-dependent SERT phosphorylation as well as altred membrane compartmentation. Finally, we pursue domains and residues that establish the differential control of SERT imparted by these signaling pathways.
In Specific Aim II, we elucidate the impact in vitro of naturaly- occuring, human SERT protein variation on receptor- and kinase-mediated SERT regulation and extend our efforts to ask whether coding variation in A3ARs and IL-1Rs mimics the effects of SERT coding variation.
In Specific Aim III, we seek to translate our understanding of A3AR, IL-1R and p38 MAPKa pathways to examine the impact on 5-HT homeostasis, 5-HT clearance and synaptic 5-HT signaling in brain preparations.
This Aim will make use of both pharmacological approaches as well as a novel line displaying adult, 5-HT-neuron- specific elimination of p38 MAPKa.
In Specific Aim I V, we.examine a novel line of mice engineered to expess a disease-associated, hyperphosphorylated coding variant of SERT (SERT Gly56Ala) for effects on phosphorylation, protein complexes, 5-HT clearance and synaptic 5-HT signaling in native preparations. Finally, these mice are explored for impact on behavioral effects of the psychostimulants cocaine and MDMA.

Public Health Relevance

Mental illnesses ranging from autism to addiction are believed to arise from a complex interplay of environment and compromised biological mechanisms that dictate brain development, function and plasticity. The neurotransmitter serotonin has long been understood to play an important, modulatory role in brain signaling and is itself under constant regulatory control. Building from the project leader's experience and expertise in the study of the serotonin transporter (SERT), the brain's chief mechanism for serotonin inactivation and a critical target for antidepressant and addictive drugs, this proposal seeks answers to fundamental questions of SERT regulation and their relationship to synaptic signaling and disease mechanisms.

Agency
National Institute of Health (NIH)
Institute
National Institute of Mental Health (NIMH)
Type
Research Project (R01)
Project #
5R01MH094527-23
Application #
8586356
Study Section
Special Emphasis Panel (ZRG1-MDCN-F (02))
Program Officer
Nadler, Laurie S
Project Start
1994-08-01
Project End
2015-08-31
Budget Start
2013-12-01
Budget End
2014-11-30
Support Year
23
Fiscal Year
2014
Total Cost
$439,659
Indirect Cost
$157,762
Name
Vanderbilt University Medical Center
Department
Neurosciences
Type
Schools of Medicine
DUNS #
004413456
City
Nashville
State
TN
Country
United States
Zip Code
37212
Prosser, R A; Stowie, A; Amicarelli, M et al. (2014) Cocaine modulates mammalian circadian clock timing by decreasing serotonin transport in the SCN. Neuroscience 275:184-93
Ye, R; Carneiro, A M D; Airey, D et al. (2014) Evaluation of heritable determinants of blood and brain serotonin homeostasis using recombinant inbred mice. Genes Brain Behav 13:247-60
Rudnick, Gary; Kramer, Reinhard; Blakely, Randy D et al. (2014) The SLC6 transporters: perspectives on structure, functions, regulation, and models for transporter dysfunction. Pflugers Arch 466:25-42
Kerr, Travis M; Muller, Christopher L; Miah, Mahfuzur et al. (2013) Genetic background modulates phenotypes of serotonin transporter Ala56 knock-in mice. Mol Autism 4:35
Campbell, Nicholas G; Zhu, Chong-Bin; Lindler, Kathryn M et al. (2013) Rare coding variants of the adenosine A3 receptor are increased in autism: on the trail of the serotonin transporter regulome. Mol Autism 4:28
Baganz, Nicole L; Blakely, Randy D (2013) A dialogue between the immune system and brain, spoken in the language of serotonin. ACS Chem Neurosci 4:48-63
Veenstra-VanderWeele, Jeremy; Blakely, Randy D (2012) Networking in autism: leveraging genetic, biomarker and model system findings in the search for new treatments. Neuropsychopharmacology 37:196-212
Zhu, Chong-Bin; Lindler, Kathryn M; Campbell, Nicholas G et al. (2011) Colocalization and regulated physical association of presynaptic serotonin transporters with A? adenosine receptors. Mol Pharmacol 80:458-65
Ye, R; Blakely, R D (2011) Natural and engineered coding variation in antidepressant-sensitive serotonin transporters. Neuroscience 197:28-36