Serotonin (5-HT) Transporter Function Research Articles

Glycogen synthase kinase-3ß supports serotonin transporter function and trafficking in a phosphorylation-dependent manner.

Serotonin (5-HT) transporter (SERT) plays a crucial role in serotonergic transmission in the central nervous system, and any aberration causes serious mental illnesses. Nevertheless, the cellular mechanisms that regulate SERT function and trafficking are not entirely understood. Growing evidence suggests that several protein kinases act as modulators. Here, we delineate the molecular mechanisms by which glycogen synthase kinase-3ß (GSK3ß) regulates SERT. When mouse striatal synaptosomes were treated with the GSK3α/ß inhibitor CHIR99021, we observed a significant increase in SERT function, Vmax , surface expression with a reduction in 5-HT Km and SERT phosphorylation. To further study how the SERT molecule is affected by GSK3α/ß, we used HEK-293 cells as a heterologous expression system. As in striatal synaptosomes, CHIR99021 treatment of cells expressing wild-type hSERT (hSERT-WT) resulted in a time and dose-dependent elevation of hSERT function with a concomitant increase in the Vmax and surface transporters because of reduced internalization and enhanced membrane insertion; silencing GSK3α/ß in these cells with siRNA also similarly affected hSERT. Converting putative GSK3α/ß phosphorylation site serine at position 48 to alanine in hSERT (hSERT-S48A) completely abrogated the effects of both the inhibitor CHIR99021 and GSK3α/ß siRNA. Substantiating these findings, over-expression of constitutively active GSK3ß with hSERT-WT, but not with hSERT-S48A, reduced SERT function, Vmax , surface density, and enhanced transporter phosphorylation. Both hSERT-WT and hSERT-S48A were inhibited similarly by PKC activation or by inhibition of Akt, CaMKII, p38 MAPK, or Src kinase. These findings provide new evidence that GSK3ß supports basal SERT function and trafficking via serine-48 phosphorylation.

Bacterial Lipopolysaccharide Increases Serotonin Metabolism in Both Medial Prefrontal Cortex and Nucleus Accumbens in Male Wild Type Rats, but Not in Serotonin Transporter Knockout Rats.

It is well known that bacterial lipopolysaccharides (LPS) both increases proinflammatory cytokines and produces sickness behavior, including fatigue and anhedonia (i.e., the inability to experience pleasure). Previously, we have shown that intraperitoneally (i.p.) administered LPS increased extracellular monoamine metabolite levels in the nucleus accumbens (NAc) and medial prefrontal cortex (mPFC), which was completely, or at least partly, prevented by pretreatment with a triple reuptake inhibitor that also blocks the serotonin (5-HT) transporter (SERT). This suggests indirectly, that LPS may enhance SERT transporter activity, and consequently, increase removal of 5-HT from the synaptic cleft, and increase metabolism of 5-HT. In the present study, we focus more specifically on the role of SERT in this increased metabolism by using rats, that differ in SERT expression. Therefore, the effects of an intraperitoneal LPS injection on extracellular concentrations of 5-HT and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) were investigated by in vivo microdialysis in the NAc and mPFC of wild type (SERT+/+), heterozygous (SERT+/−) and knockout (SERT−/−) rats. Here, we show that LPS-induced 5-HIAA formation in male rats, is significantly increased in SERT+/+ rats in both the NAc and mPFC, whereas this increase is partly or totally abolished in SERT+/− and SERT−/− rats, respectively. Thus, the present study supports the hypothesis that systemic LPS in male rats increases SERT function and consequently enhances 5-HT uptake and metabolism in both the NAc and mPFC.

Adrenal serotonin derives from accumulation by the antidepressant-sensitive serotonin transporter

Adrenal chromaffin cells comprise the neuroendocrine arm of the sympathetic nervous system and secrete catecholamines to coordinate the appropriate stress response. Deletion of the serotonin (5-HT) transporter (SERT) gene in mice (SERT−/− mice) or pharmacological block of SERT function in rodents and humans augments this sympathoadrenal stress response (epinephrine secretion). The prevailing assumption is that loss of CNS SERT alters central drive to the peripheral sympathetic nervous system. Adrenal chromaffin cells also prominently express SERT where it might coordinate accumulation of 5-HT for reuse in the autocrine control of stress-evoked catecholamine secretion. To help test this hypothesis, we have generated a novel mouse model with selective excision of SERT in the peripheral sympathetic nervous system (SERTΔTH), generated by crossing floxed SERT mice with tyrosine hydroxylase Cre driver mice. SERT expression, assessed by western blot, was abolished in the adrenal gland but not perturbed in the CNS of SERTΔTH mice. SERT-mediated [3H] 5-HT uptake was unaltered in midbrain, hindbrain, and spinal cord synaptosomes, confirming transporter function was intact in the CNS. Endogenous midbrain and whole blood 5-HT homeostasis was unperturbed in SERTΔTH mice, contrasting with the depleted 5-HT content in SERT−/− mice. Selective SERT excision reduced adrenal gland 5-HT content by ≈ 50% in SERTΔTH mice but had no effect on adrenal catecholamine content. This novel model confirms that SERT expressed in adrenal chromaffin cells is essential for maintaining wild-type levels of 5-HT and provides a powerful tool to help dissect the role of SERT in the sympathetic stress response.

Spatiotemporal SERT expression in cortical map development

The cerebral cortex is organized into morphologically distinct areas that provide biological frameworks underlying perception, cognition, and behavior. Profiling mouse and human cortical transcriptomes have revealed temporal-specific differential gene expression modules in distinct neocortical areas during cortical map establishment. However, the biological roles of spatiotemporal gene expression in cortical patterning and how cortical topographic gene expression is regulated are largely unknown. Here, we characterize temporal- and spatial-defined expression of serotonin (5-HT) transporter (SERT) in glutamatergic neurons during sensory map development in mice. SERT is transiently expressed in glutamatergic thalamic neurons projecting to sensory cortices and in pyramidal neurons in the prefrontal cortex (PFC) and hippocampus (HPC) during the period that lays down the basic functional neural circuits. We previously identified that knockout of SERT in the thalamic neurons blocks 5-HT uptake by their thalamocortical axons, resulting in excessive 5-HT signaling that impairs sensory map architecture. In contrast, here we show that selective SERT knockout in the PFC and HPC neurons does not perturb sensory map patterning. These data suggest that transient SERT expression in specific glutamatergic neurons provides area-specific instructions for cortical map patterning. Hence, genetic and pharmacological manipulations of this SERT function could illuminate the fundamental genetic programming of cortex-specific maps and biological roles of temporal-specific cortical topographic gene expression in normal development and mental disorders.

Genetic background modulates phenotypes of serotonin transporter Ala56 knock-in mice

BackgroundPreviously, we identified multiple, rare serotonin (5-HT) transporter (SERT) variants in children with autism spectrum disorder (ASD). Although in our study the SERT Ala56 variant was over-transmitted to ASD probands, it was also seen in some unaffected individuals, suggesting that associated ASD risk is influenced by the epistatic effects of other genetic variation. Subsequently, we established that mice expressing the SERT Ala56 variant on a 129S6/S4 genetic background display multiple biochemical, physiological and behavioral changes, including hyperserotonemia, altered 5-HT receptor sensitivity, and altered social, communication, and repetitive behavior. Here we explore the effects of genetic background on SERT Ala56 knock-in phenotypes.MethodsTo explore the effects of genetic background, we backcrossed SERT Ala56 mice on the 129 background into a C57BL/6 (B6) background to achieve congenic B6 SERT Ala56 mice, and assessed autism-relevant behavior, including sociability, ultrasonic vocalizations, and repetitive behavior in the home cage, as well as serotonergic phenotypes, including whole blood serotonin levels and serotonin receptor sensitivity.ResultsOne consistent phenotype between the two strains was performance in the tube test for dominance, where mutant mice displayed a greater tendency to withdraw from a social encounter in a narrow tube as compared to wildtype littermate controls. On the B6 background, mutant pup ultrasonic vocalizations were significantly increased, in contrast to decreased vocalizations seen previously on the 129 background. Several phenotypes seen on the 129 background were reduced or absent when the mutation was placed on the B6 background, including hyperserotonemia, 5-HT receptor hypersensivity, and repetitive behavior.ConclusionsOur findings provide a cogent example of how epistatic interactions can modulate the impact of functional genetic variation and suggest that some aspects of social behavior may be especially sensitive to changes in SERT function. Finally, these results provide a platform for the identification of genes that may modulate the risk of ASD in humans.

Chronic restraint stress affects serotonin transporter uptake kinetics but not binding sites in the rat hippocampus

Disturbances in serotonergic neurotransmission and stressful life events have been implicated in depressive disorders (Anisman et al., 2008). The serotonin (5-HT) transporter (SERT) plays a key role in serotonergic neurotransmission by controlling synaptic 5-HT availability through the reuptake of 5-HT into presynaptic neurons (Murphy et al., 2004). While a number of animal experiments have shown increased synthesis and release of 5-HT after various stressful stimuli (Anisman et al., 2008), only a few studies have addressed the effects of acute stress on SERT function, and the impact of chronic stress on SERT activity has not previously been reported. Chronic stress paradigms in animals recapitulate many of the core behavioral characteristics of clinical depression and are fundamental for delineating stress-induced neurochemical and molecular processes implicated in depressive disorders (Willner, 2005). To increase the understanding of how chronic stress affects serotonergic signaling we examined the effects of chronic restraint stress (CRS; 1/2h/21 days and 6h/21 days) on [H]5-HT uptake kinetics and binding of [H]-S-citalopram in the rat hippocampus and prefrontal cortex; two brain regions receiving dense serotonergic innervations and of critical importance for the brain’s response to stress.

Tyrosine Phosphorylation of the Human Serotonin Transporter: A Role in the Transporter Stability and Function

The serotonin (5-HT) transporter (SERT) regulates serotoninergic neurotransmission by clearing 5-HT released into the synaptic space. Phosphorylation of SERT on serine and threonine mediates SERT regulation. Whether tyrosine phosphorylation regulates SERT is unknown. Here, we tested the hypothesis that tyrosine-phosphorylation of SERT regulates 5-HT transport. In support of this, alkali-resistant (32)P-labeled SERT was found in rat platelets, and Src-tyrosine kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo [3,4,d]pyrimidine (PP2) decreased platelet SERT function and expression. In human placental trophoblast cells expressing SERT, PP2 reduced transporter function, expression, and stability. Although siRNA silencing of Src expression decreased SERT function and expression, coexpression of Src resulted in PP2-sensitive increases in SERT function and expression. PP2 treatment markedly decreased SERT protein stability. Compared with WT-SERT, SERT tyrosine mutants Y47F and Y142F exhibited reduced 5-HT transport despite their higher total and cell surface expression levels. Moreover, Src-coexpression increased total and cell surface expression of Y47F and Y142F SERT mutants without affecting their 5-HT transport capacity. It is noteworthy that Y47F and Y142F mutants exhibited higher protein stability compared with WT-SERT. However, similar to WT-SERT, PP2 treatment decreased the stability of Y47F and Y142F mutants. Furthermore, compared with WT-SERT, Y47F and Y142F mutants exhibited lower basal tyrosine phosphorylation and no further enhancement of tyrosine phosphorylation in response to Src coexpression. These results provide the first evidence that SERT tyrosine phosphorylation supports transporter protein stability and 5HT transport.

IFN-γ and TNF-α decrease serotonin transporter function and expression in Caco2 cells

Recent studies have shown that mucosal serotonin (5-HT) transporter (SERT) expression is decreased in animal models of colitis, as well as in the colonic mucosa of humans with ulcerative colitis and irritable bowel syndrome. Altered SERT function or expression may underlie the altered motility, secretion, and sensation seen in these inflammatory gut disorders. In an effort to elucidate possible mediators of SERT downregulation, we treated cultured colonic epithelial cells (Caco2) with conditioned medium from activated human lymphocytes. Application of the conditioned medium caused a decrease in fluoxetine-sensitive [3H]5-HT uptake. Individual proinflammatory agents were then tested for their ability to affect uptake. Cells were treated for 48 or 72 h with PGE2 (10 μM), IFN-γ (500 ng/ml), TNF-α (50 ng/ml), IL-12 (50 ng/ml), or the nitric oxide-releasing agent S-nitrosoglutathione (GSNO; 100 μM). [3H]5-HT uptake was then measured. Neither PGE nor IL-12 had any effect on [3H]5-HT uptake, and GSNO increased uptake. However, after 3-day incubation, both TNF-α and IFN-γ elicited significant decreases in SERT function. Neither TNF-α nor IFN-γ were cytotoxic when used for this period of time and at these concentrations. These two cytokines also induced decreases in SERT mRNA and protein levels. By altering SERT expression, TNF-α and IFN-γ could contribute to the altered motility and expression seen in vivo in ulcerative colitis or irritable bowel syndrome.

Modulation of the trafficking of the human serotonin transporter by human alpha‐synuclein

alpha-Synuclein (alpha-Syn), a protein primarily localized in the presynaptic compartment of neurons, is known to regulate dopaminergic neurotransmission by negatively modulating dopamine transporter activity and regulating its trafficking to or away from the cell surface. Given the considerable homology between dopamine transporters and the serotonin (5-HT) transporter (SERT), we examined whether alpha-Syn could similarly regulate SERT function. Increasing expression levels of human alpha-Syn gradually decreased [(3)H]5-HT uptake by human SERT in cotransfected Ltk(-) cells, by diminishing its V(max) without changing its K(m), as compared to cells expressing only SERT. Biotinylation studies to label cell-surface proteins showed that alpha-Syn decreased the levels of SERT present at the plasma membrane. alpha-Syn and SERT were able to coimmunoprecipitate (co-IP), suggesting heteromeric complexes between these two proteins through direct protein-protein interactions. The negative modulation of SERT activity by alpha-Syn occurred through the non-Abeta-amyloid component (NAC) domain of alpha-Syn (aa58-107); DNA constructs encoding this region mimicked the full-length alpha-Syn protein by decreasing [(3)H]5-HT uptake by the transporter. Furthermore, only the constructs encoding the NAC domain of alpha-Syn prevented the co-IPs between full-length alpha-Syn and SERT, in both transfected cells and in rat solubilized lysates isolated from the prefrontal cortex. These studies suggest a novel physiological role for alpha-Syn in regulating SERT activity and may be of relevance in certain mental illnesses and in depression, in which SERT function is believed to be dysregulated.

Calcium-dependent inhibition of synaptosomal serotonin transport by the alpha 2-adrenoceptor agonist 5-bromo-N-[4,5-dihydro-1H-imidazol-2-yl]-6-quinoxalinamine (UK14304).

Termination of serotonergic transmission is the function of the plasma membrane 5-hydroxytryptamine (serotonin, 5-HT) transporter (SERT), which is also a high-affinity target in vivo for antidepressants, amphetamines, and cocaine. Studies show that SERT is regulated by protein kinase- and phosphataselinked pathways. In contrast, receptor-linked modulation of SERT is only minimally defined. Because noradrenergic stimulation is reported to influence 5-HT release, we explored possible presynaptic adrenoceptor-mediated regulation of SERT. In mouse forebrain synaptosomes, alpha2-adrenoceptor agonists, particularly 5-bromo-N-[4,5-dihydro-1H-imidazol-2-yl]-6-quinoxalinamine (UK14304), triggered a concentration- and time-dependent decrease in 5-HT transport. In contrast, 5-HT uptake was unaffected by pharmacological alpha1-adrenoceptor activation. Kinetically, UK14304 significantly decreased the apparent substrate affinity, Km without altering transport capacity, Vmax. At concentrations of UK14304 supporting maximal inhibition of SERT in synaptosomes, no effect on SERT in transfected cells was observed, suggesting that UK14304 acts indirectly to reduce SERT activity. The effect of UK14304 on 5-HT uptake was not shared by other Na+ and Cl--dependent transporters. UK14304-mediated inhibition of SERT function was yohimbine-sensitive, as was inhibition triggered by norepinephrine, and was abolished in the absence of added Ca2+. Moreover, UK14304 effects were attenuated by voltage-sensitive Ca2+ channel antagonists, consistent with a role for Ca2+ in UK14304 effects. In agreement with altered 5-HT transport activity in vitro, in vivo chronoamperometry studies revealed that UK14304 significantly prolonged 5-HT clearance. Our findings suggest that UK14304 modulates SERT function in vitro and in vivo via signaling pathways, possibly supported by an influx of Ca2+ through voltage-sensitive Ca2+ channels.

The Effects of Methamphetamine on Serotonin Transporter Activity

Multiple administrations of methamphetamine (METH) rapidly decreased serotonin (5HT) transporter (SERT) function in rat striatum and hippocampus. The purpose of this study was to identify the mechanisms/ factors contributing to this METH-induced decrease in SERT function. Multiple high-dose METH injections rapidly decreased 5HT uptake without altering binding of the 5HT transporter ligand paroxetine. Hyperthermia contributed to this deficit in transporter function in striatum and hippocampus, as prevention of METH-induced hyperthermia attenuated this decrease. A role for dopamine (DA) was suggested by findings that pretreatment with the tyrosine hydroxylase inhibitor alpha-methyl-p-tyrosine, the D1 antagonist SCH-23390, or the D2 antagonist eticlopride attenuated the METH-induced decrease in striatal, but not hippocampal, SERT activity. These effects were independent of the ability of these DA-antagonizing drugs to prevent METH-induced hyperthermia. These results suggest that DA contributes to the decrease in SERT function caused by multiple METH injections in the striatum, but not hippocampus, and that hyperthermia facilitates these deficits in SERT function in both brain regions. In contrast, the response of SERT to a single administration of METH was DA and hyperthermia independent. These findings suggest that the mechanisms/ factors involved in decreasing SERT activity after a single administration of METH are distinct from that caused by multiple administrations.