Acetylcholine (ACh) is a major neurotransmitter in the central and peripheral nervous system. The rate-limiting step in ACh synthesis is believed to be the presynaptic acquisition of the precursor choline, achieved by the high- affinity, Na+-dependent, hemicholinium-3 (HC-3)-sensitive choline transporter (CHT). Recent breakthroughs in the molecular elucidation of invertebrate and mammalian CHT genes, the development of CHT-specific antibodies, and the creation of tractable in vitro and transgenic model systems have established new opportunities to define neuronal CHT subcellular distribution, mechanisms of activity- and receptor-dependent CHT regulation, and the functional consequences of genetic manipulation of CHT. Recently, we have established that CHT is )redominantly vesicular in localization, both in vivo as well as in in vitro model systems. CHT appears to reside )n a subpopulation of cholinergic synaptic vesicles that express the vesicular ACh transporter (VAChT) and which store ACh. Preliminary studies document both a change in the localization of CHT in synaptic membranes in response to depolarization in wildtype mice and a posttranslational mechanism to achieve normal levels of choline transport and HC-3 binding despite a 50% reduction in CHT protein in CHT +/- mice. In this new application, we apply biochemical, imaging and functional methodologies using in vitro and in vivo model systems to investigate the nature of the vesicular pool harboring CHT and clarify the physical requirements for vesicular targeting and synaptic CHT trafficking. Secondly, we explore plasma membrane shuttling as a major route for activity, cell signaling and behaviorally induced changes in choline uptake and implement a yeast 2-hybrid screen for novel CHT interactors. Finally, we analyze the consequences of full and partial genetic CHT ablation in the mouse for cholinergic biochemistry, pharmacology, physiology and behavior. These studies will elucidate novel aspects of CHT regulation, clarify how CHT supports cholinergic synaptic/behavioral plasticity and provide new CHT links to brain disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of Mental Health (NIMH)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37MH073159-09
Application #
8277373
Study Section
Special Emphasis Panel (NSS)
Program Officer
Nadler, Laurie S
Project Start
2004-06-01
Project End
2014-03-31
Budget Start
2012-04-01
Budget End
2013-03-31
Support Year
9
Fiscal Year
2012
Total Cost
$386,100
Indirect Cost
$138,600
Name
Vanderbilt University Medical Center
Department
Neurosciences
Type
Schools of Medicine
DUNS #
004413456
City
Nashville
State
TN
Country
United States
Zip Code
37212
Koshy Cherian, Ajeesh; Parikh, Vinay; Wu, Qi et al. (2017) Hemicholinium-3 sensitive choline transport in human T lymphocytes: Evidence for use as a proxy for brain choline transporter (CHT) capacity. Neurochem Int 108:410-416
Sarter, Martin; Lustig, Cindy; Blakely, Randy D et al. (2016) Cholinergic genetics of visual attention: Human and mouse choline transporter capacity variants influence distractibility. J Physiol Paris 110:10-18
Iwamoto, Hideki; Calcutt, M Wade; Blakely, Randy D (2016) Differential impact of genetically modulated choline transporter expression on the release of endogenous versus newly synthesized acetylcholine. Neurochem Int 98:138-45
Bollinger, Sean R; Engers, Darren W; Ennis, Elizabeth A et al. (2015) Synthesis and structure-activity relationships of a series of 4-methoxy-3-(piperidin-4-yl)oxy benzamides as novel inhibitors of the presynaptic choline transporter. Bioorg Med Chem Lett 25:1757-1760
Ennis, Elizabeth A; Wright, Jane; Retzlaff, Cassandra L et al. (2015) Identification and characterization of ML352: a novel, noncompetitive inhibitor of the presynaptic choline transporter. ACS Chem Neurosci 6:417-27
Holmstrand, Ericka C; Lund, David; Cherian, Ajeesh Koshy et al. (2014) Transgenic overexpression of the presynaptic choline transporter elevates acetylcholine levels and augments motor endurance. Neurochem Int 73:217-28
Mergy, Marc A; Gowrishankar, Raajaram; Davis, Gwynne L et al. (2014) Genetic targeting of the amphetamine and methylphenidate-sensitive dopamine transporter: on the path to an animal model of attention-deficit hyperactivity disorder. Neurochem Int 73:56-70
Rudnick, Gary; Krämer, 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
Dong, Yu; Dani, John A; Blakely, Randy D (2013) Choline transporter hemizygosity results in diminished basal extracellular dopamine levels in nucleus accumbens and blunts dopamine elevations following cocaine or nicotine. Biochem Pharmacol 86:1084-8
Zurkovsky, Lilia; Bychkov, Evgeny; Tsakem, Elviche L et al. (2013) Cognitive effects of dopamine depletion in the context of diminished acetylcholine signaling capacity in mice. Dis Model Mech 6:171-83

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