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Serotonin Transporter-Independent Actions of the Antidepressant Vortioxetine As Revealed Using the SERT Met172 Mouse.
Nackenoff AG, Simmler LD, Baganz NL, Pehrson AL, Sánchez C, Blakely RD
(2017) ACS Chem Neurosci 8: 1092-1100
MeSH Terms: Animals, Antidepressive Agents, Behavior, Animal, Depression, Disease Models, Animal, Hindlimb Suspension, Hippocampus, Mice, Neurogenesis, Piperazines, Serotonin, Serotonin Plasma Membrane Transport Proteins, Sulfides, Synaptosomes, Vortioxetine
Show Abstract · Added August 31, 2018
Selective serotonin (5-HT, SERT) reuptake inhibitors (SSRIs) are the most commonly prescribed treatments for depression. However, they have delayed efficacy and can induce side-effects that can encourage discontinuation. Recently, agents have been developed, including vortioxetine (Trintellix), that augment SERT blockade with interactions at other targets. At therapeutic doses, vortioxetine interacts with SERT as well as 5-HT, 5-HT, 5-HT, and 5-HT receptors. We assessed the SERT-dependency of vortioxetine action using the SERT Met172 mouse model, which disrupts high-affinity interactions of many antidepressants with the transporter. We demonstrate that the SERT Met172 substitution induces an ∼19-fold loss in vortioxetine potency for SERT inhibition in midbrain synaptosomes. Moreover, in these mice, we observed reduced SERT occupancy, a diminished ability to prolong 5-HT clearance, and a reduced capacity to elevate extracellular 5-HT. Despite reduced interactions with SERT, vortioxetine maintained its ability to enhance mobility in tail suspension and forced swim tests, reduce consumption latency in the novelty induced hypophagia test, and promoted proliferation and survival of subgranular zone hippocampal stem cells. Our findings suggest that the antidepressant actions of vortioxetine may be SERT-independent, and encourage consideration of agents that mimic one or more actions of the drug in the development of improved depression treatments.
1 Communities
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15 MeSH Terms
Identification and characterization of ML352: a novel, noncompetitive inhibitor of the presynaptic choline transporter.
Ennis EA, Wright J, Retzlaff CL, McManus OB, Lin Z, Huang X, Wu M, Li M, Daniels JS, Lindsley CW, Hopkins CR, Blakely RD
(2015) ACS Chem Neurosci 6: 417-27
MeSH Terms: Animals, Benzamides, Choline, Dose-Response Relationship, Drug, Enzyme Inhibitors, Gene Expression Regulation, HEK293 Cells, Hemicholinium 3, Humans, Isoxazoles, Male, Membrane Potentials, Membrane Transport Proteins, Mice, Mice, Inbred C57BL, Models, Biological, Mutation, Neural Inhibition, Prosencephalon, Protein Binding, Rats, Rats, Sprague-Dawley, Synaptosomes
Show Abstract · Added February 12, 2015
The high-affinity choline transporter (CHT) is the rate-limiting determinant of acetylcholine (ACh) synthesis, yet the transporter remains a largely undeveloped target for the detection and manipulation of synaptic cholinergic signaling. To expand CHT pharmacology, we pursued a high-throughput screen for novel CHT-targeted small molecules based on the electrogenic properties of transporter-mediated choline transport. In this effort, we identified five novel, structural classes of CHT-specific inhibitors. Chemical diversification and functional analysis of one of these classes identified ML352 as a high-affinity (Ki = 92 nM) and selective CHT inhibitor. At concentrations that fully antagonized CHT in transfected cells and nerve terminal preparations, ML352 exhibited no inhibition of acetylcholinesterase (AChE) or cholineacetyltransferase (ChAT) and also lacked activity at dopamine, serotonin, and norepinephrine transporters, as well as many receptors and ion channels. ML352 exhibited noncompetitive choline uptake inhibition in intact cells and synaptosomes and reduced the apparent density of hemicholinium-3 (HC-3) binding sites in membrane assays, suggesting allosteric transporter interactions. Pharmacokinetic studies revealed limited in vitro metabolism and significant CNS penetration, with features predicting rapid clearance. ML352 represents a novel, potent, and specific tool for the manipulation of CHT, providing a possible platform for the development of cholinergic imaging and therapeutic agents.
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23 MeSH Terms
Differential localization of G protein βγ subunits.
Betke KM, Rose KL, Friedman DB, Baucum AJ, Hyde K, Schey KL, Hamm HE
(2014) Biochemistry 53: 2329-43
MeSH Terms: Amino Acid Sequence, Animals, Chromatography, Liquid, GTP-Binding Protein beta Subunits, GTP-Binding Protein gamma Subunits, Male, Mice, Mice, Inbred C57BL, Molecular Sequence Data, Protein Isoforms, Signal Transduction, Subcellular Fractions, Synaptosomes, Tandem Mass Spectrometry
Show Abstract · Added May 27, 2014
G protein βγ subunits play essential roles in regulating cellular signaling cascades, yet little is known about their distribution in tissues or their subcellular localization. While previous studies have suggested specific isoforms may exhibit a wide range of distributions throughout the central nervous system, a thorough investigation of the expression patterns of both Gβ and Gγ isoforms within subcellular fractions has not been conducted. To address this, we applied a targeted proteomics approach known as multiple-reaction monitoring to analyze localization patterns of Gβ and Gγ isoforms in pre- and postsynaptic fractions isolated from cortex, cerebellum, hippocampus, and striatum. Particular Gβ and Gγ subunits were found to exhibit distinct regional and subcellular localization patterns throughout the brain. Significant differences in subcellular localization between pre- and postsynaptic fractions were observed within the striatum for most Gβ and Gγ isoforms, while others exhibited completely unique expression patterns in all four brain regions examined. Such differences are a prerequisite for understanding roles of individual subunits in regulating specific signaling pathways throughout the central nervous system.
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14 MeSH Terms
Transgenic overexpression of the presynaptic choline transporter elevates acetylcholine levels and augments motor endurance.
Holmstrand EC, Lund D, Cherian AK, Wright J, Martin RF, Ennis EA, Stanwood GD, Sarter M, Blakely RD
(2014) Neurochem Int 73: 217-28
MeSH Terms: Acetylcholine, Animals, Behavior, Animal, Choline, Cholinergic Agents, Chromosomes, Artificial, Bacterial, Gene Dosage, Hemicholinium 3, Membrane Transport Proteins, Mice, Mice, Inbred C57BL, Mice, Transgenic, Physical Endurance, Receptors, Presynaptic, Synaptosomes
Show Abstract · Added December 2, 2013
The hemicholinium-3 (HC-3) sensitive, high-affinity choline transporter (CHT) sustains cholinergic signaling via the presynaptic uptake of choline derived from dietary sources or from acetylcholinesterase (AChE)-mediated hydrolysis of acetylcholine (ACh). Loss of cholinergic signaling capacity is associated with cognitive and motor deficits in humans and in animal models. Whereas genetic elimination of CHT has revealed the critical nature of CHT in maintaining ACh stores and sustaining cholinergic signaling, the consequences of elevating CHT expression have yet to be studied. Using bacterial artificial chromosome (BAC)-mediated transgenic methods, we generated mice with integrated additional copies of the mouse Slc5a7 gene. BAC-CHT mice are viable, appear to develop normally, and breed at wild-type (WT) rates. Biochemical studies revealed a 2 to 3-fold elevation in CHT protein levels in the CNS and periphery, paralleled by significant increases in [(3)H]HC-3 binding and synaptosomal choline transport activity. Elevations of ACh in the BAC-CHT mice occurred without compensatory changes in the activity of either choline acetyltransferase (ChAT) or AChE. Immunohistochemistry for CHT in BAC-CHT brain sections revealed markedly elevated CHT expression in the cell bodies of cholinergic neurons and in axons projecting to regions known to receive cholinergic innervation. Behaviorally, BAC-CHT mice exhibited diminished fatigue and increased speeds on the treadmill test without evidence of increased strength. Finally, BAC-CHT mice displayed elevated horizontal activity in the open field test, diminished spontaneous alteration in the Y-maze, and reduced time in the open arms of the elevated plus maze. Together, these studies provide biochemical, pharmacological and behavioral evidence that CHT protein expression and activity can be elevated beyond that seen in wild-type animals. BAC-CHT mice thus represent a novel tool to examine both the positive and negative impact of constitutively elevated cholinergic signaling capacity.
Copyright © 2013 Elsevier Ltd. All rights reserved.
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15 MeSH Terms
Serotonin transporter and integrin beta 3 genes interact to modulate serotonin uptake in mouse brain.
Whyte A, Jessen T, Varney S, Carneiro AM
(2014) Neurochem Int 73: 122-6
MeSH Terms: Animals, Brain Chemistry, Citalopram, Integrin beta3, Mice, Mice, Knockout, Serotonin, Serotonin Plasma Membrane Transport Proteins, Serotonin Uptake Inhibitors, Synapses, Synaptosomes
Show Abstract · Added February 12, 2015
Dysfunctions in serotonin (5-hydroxytryptamine, 5-HT) systems have been associated with several psychiatric illnesses, including anxiety, depression, obsessive-compulsive disorders and autism spectrum disorders. Convergent evidence from genetic analyses of human subjects has implicated the integrin β3 subunit gene (ITGB3) as a modulator of serotonergic systems via genetic interactions with the 5-HT transporter gene (SLC6A4, SERT). While genetic interactions may result from contributions of each gene at several levels, we hypothesize that ITGB3 modulates the 5-HT system at the level of the synapse, through the actions of integrin αvβ3. Here we utilized a genetic approach in mouse models to examine Itgb3 contributions to SERT function both in the context of normal and reduced SERT expression. As integrin αvβ3 is expressed in postsynaptic membranes, we isolated synaptoneurosomes, which maintain intact pre- and post-synaptic associations. Citalopram binding revealed significant Slc6a4-driven reductions in SERT expression in midbrain synapses, whereas no significant changes were observed in hippocampal or cortical projections. Expecting corresponding changes to SERT function, we also measured 5-HT uptake activity in synaptoneurosomal preparations. Itgb3 single heterozygous mice displayed significant reductions in 5-HT Vmax, with no changes in Km, in midbrain preparations. However, in the presence of both Itgb3 and Slc6a4 heterozygozity, 5-HT uptake was similar to wild-type levels, revealing a significant Slc6a4 by Itgb3 genetic interaction in the midbrain. Similar findings were observed in cortical preparations, whereas in the hippocampus, most Vmax changes were driven solely by Slc6a4. Our findings provide evidence that integrin αvβ3 is involved in the regulation of serotonergic systems in some, but not all 5-HT synapses, revealing novel contributions to synaptic specificity within the central nervous system.
Copyright © 2014 Elsevier Ltd. All rights reserved.
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11 MeSH Terms
Ex vivo identification of protein-protein interactions involving the dopamine transporter.
Hadlock GC, Nelson CC, Baucum AJ, Hanson GR, Fleckenstein AE
(2011) J Neurosci Methods 196: 303-7
MeSH Terms: Animals, Brain Chemistry, Dopamine Plasma Membrane Transport Proteins, Male, Nerve Tissue Proteins, Neurochemistry, Presynaptic Terminals, Protein Interaction Mapping, Proteomics, Rats, Rats, Sprague-Dawley, Synaptosomes
Show Abstract · Added December 7, 2012
The dopamine (DA) transporter (DAT) is a key regulator of dopaminergic signaling as it mediates the reuptake of extrasynaptic DA and thereby terminates dopaminergic signaling. Emerging evidence indicates that DAT function is influenced through interactions with other proteins. The current report describes a method to identify such interactions following DAT immunoprecipitation from a rat striatal synaptosomal preparation. This subcellular fraction was selected since DAT function is often determined ex vivo by measuring DA uptake in this preparation and few reports investigating DAT-protein interactions have utilized this preparation. Following SDS-PAGE and colloidal Coomassie staining, selected protein bands from a DAT-immunoprecipitate were excised, digested with trypsin, extracted, and analyzed by liquid chromatography tandem mass spectrometry (LC/MS/MS). From the analysis of the tryptic peptides, several proteins were identified including DAT, Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) β, CaMKII δ, protein kinase C (PKC) β, and PKC γ. Co-immunoprecipitation of PKC, CaMKII, and protein interacting with C kinase-1 with DAT was confirmed by Western blotting. Thus, the present study highlights a method to immunoprecipitate DAT and to identify co-immunoprecipitating proteins using LC/MS/MS and Western blotting. This method can be utilized to evaluate DAT protein-protein interactions but also to assess interactions involving other synaptic proteins. Ex vivo identification of protein-protein interactions will provide new insight into the function and regulation of a variety of synaptic, membrane-associated proteins, including DAT.
Published by Elsevier B.V.
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12 MeSH Terms
Transgenic elimination of high-affinity antidepressant and cocaine sensitivity in the presynaptic serotonin transporter.
Thompson BJ, Jessen T, Henry LK, Field JR, Gamble KL, Gresch PJ, Carneiro AM, Horton RE, Chisnell PJ, Belova Y, McMahon DG, Daws LC, Blakely RD
(2011) Proc Natl Acad Sci U S A 108: 3785-90
MeSH Terms: Animals, Antidepressive Agents, Behavior, Animal, Cocaine, Gene Knock-In Techniques, In Vitro Techniques, Kinetics, Mice, Mice, Transgenic, Presynaptic Terminals, Raphe Nuclei, Serotonin, Serotonin Plasma Membrane Transport Proteins, Serotonin Uptake Inhibitors, Synaptosomes
Show Abstract · Added July 10, 2013
Serotonin [i.e., 5-hydroxytryptamine (5-HT)]-targeted antidepressants are in wide use for the treatment of mood disorders, although many patients do not show a response or experience unpleasant side effects. Psychostimulants, such as cocaine and 3,4-methylenedioxymethamphetamine (i.e., "ecstasy"), also impact 5-HT signaling. To help dissect the contribution of 5-HT signaling to the actions of these and other agents, we developed transgenic mice in which high-affinity recognition of multiple antidepressants and cocaine is eliminated. Our animals possess a modified copy of the 5-HT transporter (i.e., SERT, slc6a4) that bears a single amino acid substitution, I172M, proximal to the 5-HT binding site. Although the M172 substitution does not impact the recognition of 5-HT, this mutation disrupts high-affinity binding of many competitive antagonists in transfected cells. Here, we demonstrate that, in M172 knock-in mice, basal SERT protein levels, 5-HT transport rates, and 5-HT levels are normal. However, SERT M172 mice display a substantial loss of sensitivity to the selective 5-HT reuptake inhibitors fluoxetine and citalopram, as well as to cocaine. Through a series of biochemical, electrophysiological, and behavioral assays, we demonstrate the unique properties of this model and establish directly that SERT is the sole protein responsible for selective 5-HT reuptake inhibitor-mediated alterations in 5-HT clearance, in 5-HT1A autoreceptor modulation of raphe neuron firing, and in behaviors used to predict the utility of antidepressants.
2 Communities
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15 MeSH Terms
Interleukin-1 receptor activation by systemic lipopolysaccharide induces behavioral despair linked to MAPK regulation of CNS serotonin transporters.
Zhu CB, Lindler KM, Owens AW, Daws LC, Blakely RD, Hewlett WA
(2010) Neuropsychopharmacology 35: 2510-20
MeSH Terms: Animals, Behavior, Animal, Brain, Depression, Disease Models, Animal, Dose-Response Relationship, Drug, Enzyme Inhibitors, Imidazoles, Lipopolysaccharides, Male, Mice, Mice, Inbred C57BL, Mice, Knockout, Pyridines, Receptors, Interleukin-1, Serotonin Plasma Membrane Transport Proteins, Synaptosomes, p38 Mitogen-Activated Protein Kinases
Show Abstract · Added July 10, 2013
Serotonin (5-hydroxytryptamine, 5-HT) has long been implicated in regulation of mood. Medications that block the neuronal 5-HT transporter (SERT) are used as major pharmacological treatment for mood disorders. Conversely, stimuli that enhance SERT activity might be predicted to diminish synaptic 5-HT availability and increase the risk for 5-HT-related CNS disorders. We have shown that the inflammatory cytokines enhance brain SERT activity in cultured serotonergic cells and nerve terminal preparations in vitro. In this study, we establish that intraperitoneal injection of the cytokine-inducer lipopolysaccharide (LPS) stimulates brain SERT activity, acting at doses below those required to induce overt motor suppression. SERT stimulation by LPS is paralleled by increased immobility in both the tail suspension test (TST) and the forced swim test (FST); antidepressant-sensitive alterations are thought to model aspects of behavioral despair. Both the stimulation of SERT activity and induced immobility are absent when LPS is administered to interleukin-1 receptor (IL-1R)-deficient mice and in the presence of SB203580, an inhibitor of IL-1R-stimulated p38 MAPK. Moreover, the ability of LPS to enhance immobility in TST is lost in SERT knockout mice. These findings reveal an ability of peripheral inflammatory stimuli to enhance brain SERT activity through IL-1R and p38 MAPK pathways in vivo and identify a requirement for SERT expression in immune-system-modulated despair behaviors. Our studies identify IL-1R- and p38 MAPK-dependent regulation of SERT as one of the mechanisms by which environmentally driven immune system activation can trigger despair-like behavior in an animal model, encouraging future analysis of the pathway for risk factors in neuropsychiatric disorders.
1 Communities
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18 MeSH Terms
Mechanisms underlying methamphetamine-induced dopamine transporter complex formation.
Hadlock GC, Baucum AJ, King JL, Horner KA, Cook GA, Gibb JW, Wilkins DG, Hanson GR, Fleckenstein AE
(2009) J Pharmacol Exp Ther 329: 169-74
MeSH Terms: Animals, Benzazepines, Blotting, Western, Data Interpretation, Statistical, Dopamine, Dopamine Antagonists, Dopamine D2 Receptor Antagonists, Dopamine Plasma Membrane Transport Proteins, Dopamine Uptake Inhibitors, In Vitro Techniques, Male, Methamphetamine, Neostriatum, Nucleus Accumbens, Oxidopamine, Rats, Rats, Sprague-Dawley, Receptors, Dopamine D1, Receptors, Dopamine D2, Salicylamides, Sympathectomy, Chemical, Synaptosomes
Show Abstract · Added December 7, 2012
Repeated, high-dose methamphetamine (METH) administrations cause persistent dopaminergic deficits in rodents, nonhuman primates, and humans. In rats, this treatment also causes the formation of high-molecular mass (greater than approximately 120 kDa) dopamine transporter (DAT)-associated complexes, the loss of DAT monomer immunoreactivity, and a decrease in DAT function, as assessed in striatal synaptosomes prepared 24 h after METH treatment. The present study extends these findings by demonstrating the regional selectivity of DAT complex formation and monomer loss because these changes in DAT immunoreactivity were not observed in the nucleus accumbens. Furthermore, DAT complex formation was not a consequence limited to METH treatment because it was also caused by intrastriatal administration of 6-hydroxydopamine. Pretreatment with the D2 receptor antagonist, eticlopride [S-(-)-3-chloro-5-ethyl-N-[(1-ethyl-2-pyrrolidinyl)methyl]-6-hydroxy-2-methoxybenzamide hydrochloride], but not the D1 receptor antagonist, SCH23390 [R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride], attenuated METH-induced DAT complex formation. Eticlopride pretreatment also attenuated METH-induced DAT monomer loss and decreases in DAT function; however, the attenuation was much less pronounced than the effect on DAT complex formation. Finally, results also revealed a negative correlation between METH-induced DAT complex formation and DAT activity. Taken together, these data further elucidate the underlying mechanisms and the functional consequences of repeated administrations of METH on the DAT protein. Furthermore, these data suggest a multifaceted role for D2 receptors in mediating METH-induced alterations of the DAT and its function.
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22 MeSH Terms
Syntaxin 1A interaction with the dopamine transporter promotes amphetamine-induced dopamine efflux.
Binda F, Dipace C, Bowton E, Robertson SD, Lute BJ, Fog JU, Zhang M, Sen N, Colbran RJ, Gnegy ME, Gether U, Javitch JA, Erreger K, Galli A
(2008) Mol Pharmacol 74: 1101-8
MeSH Terms: Amino Acid Sequence, Amphetamine, Animals, Cell Line, Cell Membrane, Cells, Cultured, Corpus Striatum, Dopamine, Dopamine Plasma Membrane Transport Proteins, Glutathione Transferase, Humans, Kidney, Mesencephalon, Mice, Mice, Transgenic, Molecular Sequence Data, Neurons, Recombinant Fusion Proteins, Synaptosomes, Syntaxin 1, Transfection
Show Abstract · Added December 10, 2013
The soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein syntaxin 1A (SYN1A) interacts with and regulates the function of transmembrane proteins, including ion channels and neurotransmitter transporters. Here, we define the first 33 amino acids of the N terminus of the dopamine (DA) transporter (DAT) as the site of direct interaction with SYN1A. Amphetamine (AMPH) increases the association of SYN1A with human DAT (hDAT) in a heterologous expression system (hDAT cells) and with native DAT in murine striatal synaptosomes. Immunoprecipitation of DAT from the biotinylated fraction shows that the AMPH-induced increase in DAT/SYN1A association occurs at the plasma membrane. In a superfusion assay of DA efflux, cells overexpressing SYN1A exhibited significantly greater AMPH-induced DA release with respect to control cells. By combining the patch-clamp technique with amperometry, we measured DA release under voltage clamp. At -60 mV, a physiological resting potential, AMPH did not induce DA efflux in hDAT cells and DA neurons. In contrast, perfusion of exogenous SYN1A (3 microM) into the cell with the whole-cell pipette enabled AMPH-induced DA efflux at -60 mV in both hDAT cells and DA neurons. It has been shown recently that Ca2+/calmodulin-dependent protein kinase II (CaMKII) is activated by AMPH and regulates AMPH-induced DA efflux. Here, we show that AMPH-induced association between DAT and SYN1A requires CaMKII activity and that inhibition of CaMKII blocks the ability of exogenous SYN1A to promote DA efflux. These data suggest that AMPH activation of CaMKII supports DAT/SYN1A association, resulting in a mode of DAT capable of DA efflux.
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21 MeSH Terms