Serotonin Transporter Activity Research Articles

Dextromethorphan moderates reward deficiency associated with central serotonin transporter availability in 3,4-methylenedioxy-methamphetamine-treated animals.

The neurotoxicity of 3,4-methylenedioxy-methamphetamine (MDMA) to the serotonergic system is well-documented. Dextromethorphan (DM), an antitussive drug, decreased morphine- or methamphetamine (MA)-induced reward in rats and may prevent MDMA-induced serotonergic deficiency in primates, as indicated by increased serotonin transporter (SERT) availability. We aimed to investigate the effects of DM on reward, behavioral sensitization, and neurotoxicity associated with loss of SERT induced by chronic MDMA administration in rats. Conditioned place preference (CPP) and locomotor activity tests were used to evaluate drug-induced reward and behavioral sensitization; 4-[ 18 F]-ADAM/animal-PET and immunohistochemistry were used to explore the effects of DM on MDMA-induced loss of SERT. MDMA significantly reduced SERT binding in the rat brain; however, co-administration of DM significantly restored SERT, enhancing the recovery rate at day 14 by an average of ~23% compared to the MDMA group. In confirmation of the PET findings, immunochemistry revealed MDMA reduced SERT immunoactivity in all brain regions, whereas DM markedly increased the serotonergic fiber density after MDMA induction. Behavioral tests and in vivo longitudinal PET imaging demonstrated the CPP indexes and locomotor activities of the reward system correlate negatively with PET 4-[ 18 F]ADAM SERT activity in the reward system. Our findings suggest MDMA induces functional abnormalities in a network of brain regions important to decision-making processes and the motivation circuit. DM may exert neuroprotective effects to reverse MDMA-induced neurotoxicity.

Most patients with disorders of gut-brain interaction receive pharmacotherapy with major or moderate drug-gene interactions.

How variations predicted by pharmacogenomic testing to alter drug metabolism and therapeutic response affect outcomes for patients with disorders of gut- brain interaction is unclear. To assess the prevalence of pharmacogenomics-predicted drug-gene interactions and symptom outcomes for patients with disorders of gut-brain interaction. Patients who were treated in our clinical practice for functional dyspepsia/bowel disorder underwent pharmacogenomic testing. The change in symptoms from baseline to 6 months was compared for patients with variations in CYP2D6 and CYP2C19, which metabolize neuromodulators, and SLC6A4, which encodes the sodium- dependent serotonin transporter. At baseline, 79 of 94 participants (84%) had at least one predicted major drug- gene interaction, and all 94 (100%) had at least one predicted moderate interaction. For the 44 participants who completed a survey of their symptoms at 6 months, the mean (SD) irritable bowel syndrome-symptom severity score decreased from 284 (71) at baseline to 231 (95) at 6 months (p < 0.001). Among patients taking selective serotonin reuptake inhibitors, the decrease in symptom severity (p = 0.03) and pain (p = 0.002) scores from baseline to 6 months was greater for patients with a homozygous SLC6A4 long/long genotype (n = 30) (ie, increased serotonin transporter activity) than for patients with homozygous short/short or heterozygous long/short genotypes (n = 64). Symptom outcomes were not affected by CYP2D6 or CYP2C19 variations. The homozygous SLC6A4 long/long genotype confers better symptom resolution for patients with disorders of gut-brain interaction who take selective serotonin reuptake inhibitors than do the homozygous short/short or heterozygous long/short genotypes.

Palmitoylation Regulates Human Serotonin Transporter Activity, Trafficking, and Expression and Is Modulated by Escitalopram.

In the central nervous system, serotonergic signaling modulates sleep, mood, and cognitive control. During serotonergic transmission, the synaptic concentration of serotonin is tightly controlled in a spatial and temporal manner by the serotonin transporter (SERT). Dysregulation of this process is implicated in the pathogenesis of major-depressive, obsessive-compulsive, and autism-spectrum disorders, which makes SERT a primary target for prescription therapeutics, most notably selective serotonin reuptake inhibitors (SSRIs). S-Palmitoylation, the reversible addition of a 16-carbon fatty acid to proteins, is an increasingly recognized dynamic post-translational modification responsible for modulating protein kinetics, trafficking, and localization patterns in response to physiologic/cellular stimuli. In this study, we reveal that human SERTs are a target for palmitoylation, and using the irreversible palmitoyl acyltransferase inhibitor 2-bromopalmitate (2BP), we have identified several associated functions. Using a lower dose of 2BP in shorter time frames, inhibition of palmitoylation was associated with reductions in SERT Vmax, without changes in Km or surface expression. With higher doses of 2BP for longer time intervals, inhibition of palmitoylation was consistent with the loss of cell surface and total SERT protein, suggesting palmitoylation is an important mechanism in regulating SERT trafficking and maintenance of SERT protein through biogenic or anti-degradative processes. Additionally, we have identified that treatment with the SSRI escitalopram decreases SERT palmitoylation analogous to 2BP, reducing SERT surface expression and transport capacity. Ultimately, these results reveal that palmitoylation is a major regulatory mechanism for SERT kinetics and trafficking and may be the mechanism responsible for escitalopram-induced internalization and ultimately decreased cellular SERT protein levels.

Serotonin Transporter Activity in Mouse Oocytes Is a Positive Indicator of Follicular Growth and Oocyte Maturity.

Serotonin (5-hydroxytryptamine, 5-HT) is known to be a regulator of oocyte maturation in a large number of animal species. In maturing mammalian oocytes, the accumulation of exogenous, maternal serotonin occurs due to the activity of the membrane transporter SERT. In this work, we investigated how SERT activity in oocytes correlates with indicators of follicular selection and oocyte maturity. An immunohistochemical study showed that the difference in the 5-HT intake activity in oocytes does not correlate with the marker of apoptosis in follicular cells, but positively correlates with markers of follicular growth, such as granulosa proliferation and follicle size. Functional analysis of oocytes at different stages of maturation showed that the expression and activity of SERT increases with oocyte maturation. An in vivo experiment on administration of the selective serotonin reuptake inhibitor fluoxetine (20 mg/kg) for 7 days showed a significant decrease in the content of serotonin in both growing GV-oocytes and ovulated mature MII-oocytes. The data obtained clearly indicate that the mechanism of specific membrane transport of serotonin normally ensures the accumulation of serotonin in maturing oocytes, and can be considered as a promising positive marker of their mature status.

Serotonergic and dopaminergic control of impulsivity in gambling disorder.

Gambling disorder (GD) is major public health issue. The disorder is often characterized by elevated impulsivity with evidence from analogous substance use disorders underlining prominent roles of brain monoamines in addiction susceptibility and outcome. Critically, GD allows the study of addiction mechanisms without the confounder of the effects of chronic substances. Here, we assessed the roles of striatal dopamine transporter binding and extrastriatal serotonin transporter binding in GD as a function of impulsivity using [123 I]FP-CIT SPECT imaging in 20 older adults with GD (DSM-5 criteria; mean age 64 years) and 40 non-GD age- and sex-matched controls. We focused on GD in older individuals because there are prominent age-related changes in neurotransmitter function and because there are no reported neuroimaging studies of GD in older adults. Volume-of-interest-based and voxelwise analyses were performed. GD patients scored clearly higher on impulsivity and had higher tracer binding in the ventromedial prefrontal cortex than controls (p < 0.001), likely reflecting serotonin transporter activity. The binding in the medial prefrontal cortex positively correlated with impulsivity over the whole sample (r = 0.62, p < 0.001) as well as separately in GD patients (r = 0.46, p = 0.04) and controls (r = 0.52, p < 0.001). Striatal tracer binding, reflecting dopamine transporter activity was also positively correlated with impulsivity but showed no group differences. These findings highlight the role of prefrontal serotonergic function in GD and impulsivity. They identify cerebral coordinates of a potential target for neuromodulation for both GD and high impulsivity, a core phenotypic dimensional cognitive marker in addictions.

Decreased serotonin transporter activity in the mitral valve contributes to progression of degenerative mitral regurgitation.

Degenerative mitral valve (MV) regurgitation (MR) is a highly prevalent heart disease that requires surgery in severe cases. Here, we show that a decrease in the activity of the serotonin transporter (SERT) accelerates MV remodeling and progression to MR. Through studies of a population of patients with MR, we show that selective serotonin reuptake inhibitor (SSRI) use and SERT promoter polymorphism 5-HTTLPR LL genotype were associated with MV surgery at younger age. Functional characterization of 122 human MV samples, in conjunction with in vivo studies in SERT-/- mice and wild-type mice treated with the SSRI fluoxetine, showed that diminished SERT activity in MV interstitial cells (MVICs) contributed to the pathophysiology of MR through enhanced serotonin receptor (HTR) signaling. SERT activity was decreased in LL MVICs partially because of diminished membrane localization of SERT. In mice, fluoxetine treatment or SERT knockdown resulted in thickened MV leaflets. Similarly, silencing of SERT in normal human MVICs led to up-regulation of transforming growth factor β1 (TGFβ1) and collagen (COL1A1) in the presence of serotonin. In addition, treatment of MVICs with fluoxetine not only directly inhibited SERT activity but also decreased SERT expression and increased HTR2B expression. Fluoxetine treatment and LL genotype were also associated with increased COL1A1 expression in the presence of serotonin in MVICs, and these effects were attenuated by HTR2B inhibition. These results suggest that assessment of both 5-HTTLPR genotype and SERT-inhibiting treatments may be useful tools to risk-stratify patients with MV disease to estimate the likelihood of rapid disease progression.

Mutation of the Drosophila melanogaster serotonin transporter dSERT impacts sleep, courtship, and feeding behaviors.

The Serotonin Transporter (SERT) regulates extracellular serotonin levels and is the target of most current drugs used to treat depression. The mechanisms by which inhibition of SERT activity influences behavior are poorly understood. To address this question in the model organism Drosophila melanogaster, we developed new loss of function mutations in Drosophila SERT (dSERT). Previous studies in both flies and mammals have implicated serotonin as an important neuromodulator of sleep, and our newly generated dSERT mutants show an increase in total sleep and altered sleep architecture that is mimicked by feeding the SSRI citalopram. Differences in daytime versus nighttime sleep architecture as well as genetic rescue experiments unexpectedly suggest that distinct serotonergic circuits may modulate daytime versus nighttime sleep. dSERT mutants also show defects in copulation and food intake, akin to the clinical side effects of SSRIs and consistent with the pleomorphic influence of serotonin on the behavior of D. melanogaster. Starvation did not overcome the sleep drive in the mutants and in male dSERT mutants, the drive to mate also failed to overcome sleep drive. dSERT may be used to further explore the mechanisms by which serotonin regulates sleep and its interplay with other complex behaviors.

Genetics and epigenetics: paternal adolescent ethanol consumption in serotonin transporter knock-out rats and offspring sensitivity to ethanol

RationaleAlcohol use disorder (AUD) is shown to have an overall heritability of around 50%. One of the genes associated with AUD is SLC6A4 (solute carrier family 6 member A4) which codes for the serotonin transporter (SERT). The study looked at serotonin dysfunction on ethanol consumption in adolescents and the subsequent intergenerational effects of drinking by using a rat model: SERT+/+ (regular functioning), SERT+/− (50% transporter reduction) and SERT−/− (complete reduction).ObjectivesWe investigated sex and genotype differences in ethanol consumption in SERT knock-out Wistar rats (F0) followed by studying behaviour in the offspring (F1) of the male drinkers to assess effects of paternal alcohol consumption.MethodsAn intermittent access two-bottle choice paradigm (IA2BC) was used to yield ethanol drinking behaviour in F0 adolescent Wistar rats. The highest drinking males were mated to alcohol-naive females and their offspring were compared with controls. Drinking behaviour (IA2BC) and ethanol-induced motor coordination effects (via rotarod) were measured in the F1s.ResultsF0 drinking saw no SERT genotype differences in males. However, females consumed higher volumes of ethanol compared to males, with SERT−/− females showing the highest intake. A clearer genotype effect was seen in the F1 animals, with reduction in SERT activity leading to enhanced ethanol intake in both sexes. Importantly, paternal exposure to ethanol significantly reduced the ethanol induced motor side effects in offspring, independent of sex and genotype.ConclusionsThese indicate a difference in the way genetic factors may act across sexes and suggest the involvement of epigenetic mechanisms in the intergenerational effects of alcohol.

Intestinal serotonergic system is modulated by Toll-like receptor 9

Intestinal serotonergic system is a key modulator of intestinal homeostasis; however, its regulation is still unclear. Toll-like receptor 9 (TLR9), an innate immune receptor, detects different external agents in the intestine, preserving intestinal integrity. Since little is known about TLR9 role in the intestine, our aim was to address the potential regulation between TLR9 and intestinal serotonergic system. Caco-2/TC7 cell line and intestinal tract of Tlr9−/− mice were used in this study. Serotonin uptake studies were performed, and molecular expression of different serotonergic components was analyzed by western blot and real-time PCR. Our results show that TLR9 activation inhibits serotonin transporter activity and expression, involving p38/MAPK and ERK/MAPK intracellular pathways, and reciprocally, serotonin increases TLR9 expression. Supporting this interaction, serotonin transporter, serotonin receptors and serotonin producer enzymes were found altered in intestinal tract of Tlr9−/− mice. We conclude that TLR9 could contribute to intestinal homeostasis by modulation of intestinal serotonergic system.

Enteropathogenic infections modulate intestinal serotonin transporter (SERT) function by activating Toll-like receptor 2 (TLR-2) in Crohn\u2019s disease

Serotonin (5-hydroxytryptamine [5-HT]) is an intestinal neuromodulator that regulates several essential enteric physiological functions such as absorption or secretion of fluids, and peristaltic reflexes. Availability of the intestinal 5-HT is dependent on serotonin transporter (SERT), which uptakes 5-HT and facilitates its degradation. Interestingly, Toll-like receptor 2 (TLR-2) is co-localized with 5-HT, which suggests a possible impact of neuroendocrine cells in the inflammatory response through TLR-2 activation. Serum 5-HT levels were measured in 80 Crohn’s disease (CD) patients and 40 healthy control subjects. Additionally, fully differentiated Caco-2 monolayers were infected with Mycobacteria paratuberculosis (MAP), L. monocytogenes, or M. smegmatis in the presence of exogenous 5-HT at different concentrations. Cells were subsequently harvested and used for measuring SERT activity, RNA isolation followed by RT-PCR, protein quantification, and tissue damage markers (DHE, LDH, GSH and MDA). TLR-2 intracellular signaling pathways were assessed by pre-incubating Caco-2 monolayers with selective blockers of ERK, cAMP/PKA, p38 MAPK, and 5-HT3 receptors. MAP-infected CD patients (N = 40) had higher serum 5-HT levels (462.95 ± 8.55 ng/mL, N = 40) than those without MAP infection (385.33 ± 10.3 ng/mL, N = 40). TLR-2 activation by enteropathogenic bacteria inhibited SERT activity in the presence of exogenous 5-HT by up to 50%. These effects were increasing gradually over 72 h, and MAP infection had the greatest effect on SERT inhibition when cells were exposed to 5-HT in a concentration dependent manner. Additionally, inhibition of SERT activity was accompanied with higher levels of pro-inflammatory cytokines (TNF-α, IL-6, IL-8) and oxidative stress markers (DHE, LDH and MDA), whereas SERT expression and protein level were downregulated. Consequently, inhibition of TLR-2 and p38 MAPK signaling or blocking 5-HT3 receptors restored SERT activity and reduced the production of pro-inflammatory cytokines, as reflected by the downregulation of oxidative stress and tissue damage markers. The involvement of TLR-2 in the intestinal pathology might be concluded not only from its innate immune role, but also from its effect on modulating the intestinal serotonergic response. Ultimately, regulating the intestinal serotonergic system can be therapeutically exploited to mitigate other enteropathogenic infections, which will help in understanding the gut-microbiome-brain connection.

S928 Enteropathogenic Infections Modulate Intestinal Serotonin Transporter (SERT) Function by Activating Toll-Like Receptor 2 (TLR-2) in Crohn’s Disease

Introduction: Availability of intestinal serotonin (5-HT) is dependent on serotonin transporter (SERT), which uptakes 5-HT and facilitates its degradation. Interestingly, Toll-like receptor 2 (TLR-2) is co-localized with 5-HT, which suggests a possible impact of neuroendocrine cells in the inflammatory response through TLR-2 activation. Whether modulating SERT activity and intestinal 5-HT accumulation contributes to tissue injury caused by enteropathogenic infections is unknown. Methods: Serum 5-HT levels were measured in 80 CD patients and 40 healthy control subjects. Additionally, fully differentiated Caco-2 monolayers were infected with Mycobacteria paratuberculosis (MAP), L. monocytogenes or M. smegmatis in the presence of exogenous 5-HT at different concentrations. Cells were harvested and used for measuring SERT activity, RNA isolation followed by RT-PCR, protein quantification, and tissue damage markers (DHE, LDH, GSH and MDA). TLR-2 intracellular signaling pathways were assessed by pre-incubating Caco-2 monolayers with selective blockers of ERK, cAMP/PKA, p38 MAPK, and 5-HT3 receptors. Results: Serum 5-HT levels were significantly higher among CD patients in comparison to healthy subjects [424.14 ± 9.42 ng/mL vs 211.69 ± 8.11 ng/mL, respectively]. TLR-2 activation by enteropathogenic bacteria inhibited SERT activity in the presence of exogenous 5-HT by up to 50%. These effects were increasing gradually over 72 hours, and MAP infection had the greatest effect on SERT inhibition when cells were exposed to 5-HT in a concentration dependent manner. Additionally, inhibition of SERT activity was accompanied with higher level of pro-inflammatory cytokines (TNF-α, IL-6, IL-8) and oxidative stress markers (DHE, LDH and MDA), whereas SERT expression and protein level were downregulated. Consequently, inhibition of TLR-2 and p38 MAPK pathways or blocking 5-HT3 restored SERT activity and reduced the production of pro-inflammatory cytokines by about 2 folds, which was reflected on downregulating oxidative stress and tissue damage markers. Conclusion: The involvement of TLR-2 in intestinal pathology might be concluded not only from its innate immune role, but also from its effect on modulating the intestinal serotonergic response. Ultimately, regulating the intestinal serotonergic system could provide therapeutic approaches in enteropathogenic infections, which will help in understanding the gut-microbiome-brain connection.Figure 1.: Proposed mechanism for increasing intestinal and serum 5-HT among CD patients. Enterochromaffin cells release 5-HT into the intestinal lumen. Under normal conditions, SERT activity is able to reuptake 5-HT back into the intracellular compartment, which regulates intestinal 5-HT availability. However, enteropathogenic infections cause inflammation by activating TLR-2, which reduces SERT expression and function, leading to accumulation of 5-HT and disruption of the intestinal tight junctions. This results in paracellular movement of 5-HT across the intestinal epithelium to the blood vessels, where it can cause further systemic complications.

Limosilactobacillus reuteri ATCC 6475 metabolites upregulate the serotonin transporter in the intestinal epithelium.

The serotonin transporter (SERT) readily takes up serotonin (5-HT), thereby regulating the availability of 5-HT within the intestine. In the absence of SERT, 5-HT remains in the interstitial space and has the potential to aberrantly activate the many 5-HT receptors distributed on the epithelium, immune cells and enteric neurons. Perturbation of SERT is common in many gastrointestinal disorders as well as mouse models of colitis. Select commensal microbes regulate intestinal SERT levels, but the mechanism of this regulation is poorly understood. Additionally, ethanol upregulates SERT in the brain and dendritic cells, but its effects in the intestine have never been examined. We report that the intestinal commensal microbe Limosilactobacillus (previously classified as Lactobacillus) reuteri ATCC PTA 6475 secretes 83.4 mM ethanol. Consistent with the activity of L. reuteri alcohol dehydrogenases, we found that L. reuteri tolerated various levels of ethanol. Application of L. reuteri conditioned media or exogenous ethanol to human colonic T84 cells was found to upregulate SERT at the level of mRNA. A 4-(4-(dimethylamino) phenyl)-1-methylpyridinium (APP+) uptake assay confirmed the functional activity of SERT. These findings were mirrored in mouse colonic organoids, where L. reuteri metabolites and ethanol were found to upregulate SERT at the apical membrane. Finally, in a trinitrobenzene sulphonic acid model of acute colitis, we observed that mice treated with L. reuteri maintained SERT at the colon membrane compared with mice receiving phosphate buffered saline vehicle control. These data suggest that L. reuteri metabolites, including ethanol, can upregulate SERT and may be beneficial for maintaining intestinal homeostasis with respect to serotonin signalling.

Sex and brain region-specific regulation of serotonin transporter activity in synaptosomes in guanine nucleotide-binding protein G(q) alpha knockout mice.

The regulation of the serotonin transporter (SERT) by guanine nucleotide-binding protein alpha (Gα) q was investigated using Gαq knockout mice. In the absence of Gαq, SERT-mediated uptake of 5-hydroxytryptamine (5HT) was enhanced in midbrain and frontal cortex synaptosomes, but only in female mice. The mechanisms underlying this sexual dimorphism were investigated using quantitative western blot analysis revealing brain region-specific differences. In the frontal cortex, SERT protein expression was decreased in male knockout mice, seemingly explaining the sex-dependent variation in SERT activity. The differential expression of Gαi1 in female mice contributes to the sex differences in the midbrain. In fact, Gαi1 levels inversely correlate with 5HT uptake rates across both sexes and genotypes. Likely due to differential SERT regulation as well as sex differences in the expression of tryptophan hydroxylase 2, Gαq knockout mice also displayed sex- and genotype-dependent alterations in total 5HT tissue levels as determined by high-performance liquid chromatography. Gαq inhibitors, YM-254890 and BIM-46187, differentially affected SERT activity in both, synaptosomes and cultured cells. YM-254890 treatment mimicked the effect of Gαq knockout in the frontal cortex. BIM-46187, which promotes the nucleotide-free form of Gα proteins, substantially inhibited 5HT uptake, prompting us to hypothesise that Gαq interacts with SERT similarly as with G-protein-coupled receptors and inhibits SERT activity by modulating transport-associated conformational changes. Taken together, our findings reveal a novel mechanism of SERT regulation and impact our understanding of sex differences in diseases associated with dysregulation of serotonin transmission, such as depression and anxiety.

Regulation of serotonin transport by geranylgeranyl transferase and Ca 2+ /calmodulin‐dependent protein kinase II

The serotonin transporter (SERT) transports extracellular serotonin (5HT) into neuron terminals and serves as the primary mechanism by which 5HT is regulated in the brain. Recently, it has been shown that SERT activity is increased after treatment with cholesterol-lowering statin drugs in a manner independent of cholesterol but dependent on the cholesterol biosynthetic intermediate geranylgeranyl pyrophosphate (GGPP). GGPP and its corresponding enzyme geranylgeranyl transferase (GGT) lipidate proteins for targeting to the plasma membrane. Although statins are known to increase constitutive activity of the canonical targets of GGPP including Rac1, CDC42, and RhoA, the mechanism by which GGPP regulates SERT activity is unknown. To investigate the role of GGPP on neuronal SERT function, serotonin neurons were modeled in serotonergic RN46A-B14 cells with stably overexpressed SERT. Simvastatin or a GGT inhibitor that specifically blocks geranylgeranylation by GGPP were applied to cells for 24hr and 5HT transport was assayed by measuring 5HT uptake in cells after incubation with exogenous 5HT. Simvastatin and GGT inhibition increased 5HT uptake by 58% and 82% (Mean (SEM): Veh=2.09 (0.30), Sim=4.99 (0.53), GGT=11.84 (2.63), p<0.05), respectively, in a manner blocked by fluoxetine. Pharmacological inhibition of CDC42 or knockdown of the mRNA for RhoA did not block statin-increased 5HT uptake. Protein kinases p38 mitogen-activated protein kinase (P38MAPK) and Ca2+/calmodulin-dependent protein kinase II (CaMKII) were also investigated based on their regulation downstream of GGPP. While phosphorylation of P38MAPK was significantly increased by 37% after 24hr simvastatin treatment (Mean (SEM): Veh=100 (6.7), Sim=159 (19.0), p<0.02), inhibition of P38MAPK did not block statin-increased uptake. However, inhibition of CaMKII with CaMKII inhibitor Tat-CN21 dose-dependently blocked statin-increased 5HT uptake (Mean (SEM): Veh=100 (0), Sim=263 (25.8), Tat-CN21=119.48 (12.2), p<0.001). Ongoing studies of the inhibition of GGT on 5HT uptake are being conducted using a panel of CaMKII inhibitors including Tat-CN21, myristoylated autocamtide-2-related inhibitory peptide, and small molecule inhibitors with corresponding control agents. Our results indicate that SERT is regulated through the isoprenylation pathway in a GGPP, GGT and CaMKII-dependent manner.

Syntaxin 3 interacts with serotonin transporter and regulates its function

Herein, we investigated the functional association of the serotonin transporter (SERT) with syntaxin-3 (STX3). We first overexpressed SERT and STX3 in various cells and examined their interaction, localization, and functional association. Immunoprecipitation studies revealed that STX3 interacted with SERT when expressed in COS-7 cells. Immunocytochemical studies revealed that SERT and STX3 were colocalized in the endoplasmic reticulum (ER) and Golgi apparatus. STX3 overexpression significantly reduced the uptake activity of SERT by attenuating its plasma membrane expression, suggesting that overexpressed STX3 anchors SERT in the ER and Golgi apparatus. STX3 knockdown did not affect the uptake activity of SERT but altered its glycosylation state. To elucidate the association of STX3 with SERT under physiological conditions, rather than overexpressing cells, we investigated this interaction in polarized Caco-2 cells, which endogenously express both proteins. Immunocytochemical studies revealed that SERT and STX3 were localized in microvilli-like structures at the apical plasma membrane. STX3 knockdown marginally but significantly decreased the serotonin uptake activity of Caco-2 cells, suggesting that STX3 positively regulates SERT function in Caco-2 cells, as opposed to SERT regulation by STX3 in overexpressing cells. Collectively, STX3 may colocalize with SERT during SERT membrane trafficking and regulate SERT function in an STX3-expressing lesion-dependent manner.

Involvement of PKCβI-SERT activity in stress vulnerability of mice exposed to twice-swim stress

Stress vulnerability and pathogenic mechanisms in stress-related disorders are strongly associated with the functions of serotonin transporter (SERT). SERT phosphorylation induces a reduction of the serotonin (5-HT, 5-hydroxytryptamine) transport properties, its phosphorylation regulated by protein kinase C (PKC). However, the functional relationship between regulated SERT activity by PKC and stress vulnerability remains unclear. Here, we investigated whether the functional regulation of SERT by PKC was involved in stress vulnerability using mice exposed to twice-swim stress that exhibited the impairment of social behaviors. The mild-swim stress (6 min) given just before the social interaction test did not affect the social behaviors of mice. However, mice exposed to strong-swim stress (15 min) became vulnerable to the mild-swim stress, and subsequent social behaviors were impaired. Chelerythrine, a PKC inhibitor, exacerbated decreased sociality in mice exposed to acute mild-swim stress. Phorbol 12-myristate 13-acetate (PMA), a PKC activator, ameliorated the impairment of social behaviors in mice exposed to twice-swim stress. Phosphorylated PKCβI or SERT and 5-HT levels were decreased in the prefrontal cortex of twice-stressed mice. These decreases were attenuated by PMA. Our findings demonstrate that mice exposed to twice-swim stress developed stress vulnerability, which may be involved in the regulation of SERT phosphorylation and 5-HT levels accompanying PKCβI activity.

Antidepressant activities and regulative effects on serotonin transporter of Nardostachys jatamansi DC

Ethnopharmacological relevanceNardostachys jatamansi (D.Don) DC. (family Caprifoliaceae, NJ) is well-documented and commonly used in the systems of traditional medicine in China, Tibet, Nepal, Bhutan, India and Japan for curing digestive and neuropsychiatric disorders with a long history of medication. However, the possible action mechanisms of antidepressant effects of NJ remain unraveled. Aim of the studyThe aim of this study was to systematically investigate chemical substances of NJ and their effects on serotonin transporter (SERT) in antidepressant activity. Materials and methodsAntidepressant effects of total methanol extract of NJ were evaluated by tail suspension test (TST) and open field test (OFT). Then the total extract was analyzed by ultra-high-performance liquid chromatography (UHPLC) method, and its effect on SERT activity was evaluated by high content assay (HCA) to determine half maximal effective concentration (EC50). This total extract was subfractioned into twenty subfractions by preparative high-performance liquid chromatography (p-HPLC) method, and ‘subfraction-SERT activity’ relationship curve was fitted with medians of the retention time of those subfractions and their SERT activity values. Then, the fraction NJFr.01 enriched with SERT enhancers was optimized, prepared and analyzed by UHPLC method. Antidepressant effects of the fraction NJFr.01 were evaluated by TST and OFT. Further, major constituents of the total extract and fraction NJFr.01 were isolated by p-HPLC and identified by extensive nuclear magnetic resonance (NMR) analyses and comparisons with those reported data, and their SERT activities were also evaluated. Finally, antagonistic effects of chlorogenic acid and desoxo-narchinol A against fluoxetine on SERT were evaluated. ResultsResults of TST and OFT demonstrated antidepressant effects of toatal extract of NJ. The EC50 of total extract on SERT enhancement was 31.63 μg/mL. The fitted ‘subfraction-SERT activity’ relationship curve revealed that fraction NJFr.01 was enriched with SERT enhancing constituents. Both total extract and fraction NJFr.01 significantly enhanced SERT activity, while the rest fraction NJFr.02 didn't show any SERT activity. Then, antidepressant effects of fraction NJFr.01 were demonstrated by TST and OFT. Further, phytochemistry investigation and UHPLC analyses confirmed the identification of fourteen constituents in the total extract of NJ, including 7-oxonardinoperoxide (1), desoxo-narchinol A (2), kanshone B (3), narchinol B (4), nardosinonediol (5), kanshone A (6), 1-hydroxylaristolone (7), debilon (8), nardosinone (9), kanshone H (10), 1,8,9,10-tetradehydroaristolan-2-one (11), (−)-aristolone (12), 1(10)-aristolene-2-one (13) and jatamol A (14), and seven constituents in the fraction NJFr.01, including chlorogenic acid (15), 8α-dihydrogeniposide (16), 7-deoxy-8-epi-loganic acid (17), adoxosidic acid (18), 8-epi-loganic acid (19), 8α-6,7-dihydroapodantheroside acetate (20) and 6″-acetylpatrinalloside (21). Their structures were established by NMR analyses and comparisons with those reported data. HCA results of these constituents demonstrated the major components of fraction NJFr.01 enhanced SERT activity. Antagonistic results showed that chlorogenic acid and desoxo-narchinol A reversed inhibition effect of fluoxetine on SERT activity. ConclusionThis study first systematically expatiated the roles of SERT activity in antidepressant effects of NJ, including total methanol extract and the water-soluble fraction NJFr.01 enriched with SERT enhancing constituents. This is the first report of natural SERT enhancing extract and fractions with antidepressant potential in NJ.

Expression and impact of Lsamp neural adhesion molecule in the serotonergic neurotransmission system

Limbic system associated membrane protein (Lsamp) is a neural adhesion protein which has been recently found to be differentially expressed between serotonergic neuron subtypes. We have previously shown elevated serotonin (5-HT) turnover rate in Lsamp-deficient mice. The purpose of the current study was to elucidate the role of Lsamp in serotonergic neurotransmission.Chronic (18 days) administration of serotonin reuptake inhibitor (SSRI) escitalopram (10 mg/kg) significantly increased general activity in wild-type mice in the open field and protected exploration in Lsamp−/− mice in the elevated-plus maze. An important psychopathology-related endophenotype, elevated 5-HT turnover in the brain of Lsamp-deficient mice, was reproduced in the saline group. Escitalopram restored the elevated 5-HT turnover of Lsamp-deficient mice to a level comparable with their wild-type littermates, suggesting that high 5-HT turnover in mutants is mediated by the increased activity of serotonin transporter (SERT protein encoded by Slc6a4 gene). The baseline level of Slc6a4 transcript was not changed in Lsamp-deficient mice, however, our immunohistochemical analysis showed partial co-expression of Lsamp with both SERT and Tph2 proteins in raphe. Overactivity of SERT in Lsamp−/− mice is further supported by significant elevation of Maoa transcript and increase of DOPAC, another Mao A product, specifically in the raphe. Again, elevation of DOPAC was reduced to the level of wild-type by chronic SSRI treatment. The activity of Lsamp gene promoters varied in 5-HT producing nuclei: both Lsamp 1a and 1b promoters were active in the dorsal raphe; most of the expression in the median raphe was from 1b promoter, whereas Lsamp 1a promoter was almost exclusively active in the caudal subgroup of raphe nuclei. We suggest that Lsamp may have an impact on the integrity of serotonergic synapses, which is possibly the neurochemical basis of the anxiety- and sociability-related phenotype in Lsamp-deficient mice.

Involvement of protein kinase C beta1-serotonin transporter system dysfunction in emotional behaviors in stressed mice

Selective serotonin reuptake inhibitors are the first-line antidepressants for treating major depressive and post-traumatic stress disorders. These inhibitors directly bind to the serotonin transporter (SERT). Protein kinase C (PKC) is a key regulator of SERT functions as it can attenuate SERT activity through phosphorylation and its subsequent internalization. However, whether PKC-regulated SERT functions are involved in emotional impairment in a mouse model of stress remains unclear. Using a mouse model of swim-induced stress, we investigated whether the PKC-SERT system is involved in emotional impairment and tried to identify the PKC isoforms involved in this mechanism. Mice exposed to swim stress showed enhanced immobility and decreased social interaction times compared to those in swim stress-naive mice. Moreover, significant decreases in phosphorylated PKCβI and SERT levels were observed in the prefrontal cortex of stressed mice compared to those of swim stress-naive mice. No changes in levels of other phosphorylated PKC isoforms were observed between the two groups. Phorbol 12-myristate 13-acetate (a PKC activator) administration significantly attenuated enhanced immobility and decreased social interaction time in stressed mice and increased the serotonin turnover. Further, the PKC activator increased levels of phosphorylated PKCβI or SERT and decreased cell surface localization of SERT in stressed mice. Contrary to this, chelerythrine (a PKC inhibitor) administration exacerbated the immobility and sociality of mice exposed to mild stress. Our results suggest that PKCβI activation attenuates emotional impairment by suppressing SERT function in stressed mice. Thus, PKCβI may be a key target for the development of new treatment strategies for emotional impairment in stress-related disorders.

Serotonin transporter protein in autopsied brain of chronic users of cocaine.

The long-held speculation that the brain serotonin system mediates some behavioral effects of the psychostimulant cocaine is supported in part by the high affinity of cocaine for the serotonin transporter (SERT) and by reports that the serotonin transporter (SERT), estimated by SERT binding, is increased in brain of human chronic cocaine users. Excessive SERT activity and consequent synaptic serotonin deficiency might cause a behavioral (e.g., mood) abnormality in chronic users of the drug. Previous studies focused on changes in SERT binding, which might not necessarily reflect changes in SERT protein. Therefore, we compared levels of SERT protein, using a quantitative Western blot procedure, in autopsied brain (striatum, cerebral cortices) of chronic human cocaine users (n = 9), who all tested positive for the drug/metabolite in brain, to those in control subjects (n = 15) and, as a separate drug of abuse group, in chronic heroin users (n = 11). We found no significant difference in protein levels of SERT or the serotonin synthesizing enzyme tryptophan hydroxylase-2 among the control and drug abuse groups. In the cocaine users, no significant correlations were observed between SERT and brain levels of cocaine plus metabolites, or with levels of serotonin or its metabolite 5-hydroxyindoleacetic acid. Our postmortem data suggest that a robust increase in striatal/cerebral cortical SERT protein is not a common characteristic of chronic, human cocaine users.