Serotonin Transporter Protein Levels Research Articles

Biomarkers of Affective Dysregulation Associated with In Utero Exposure to EtOH.

Children with fetal alcohol spectrum disorders (FASD) exhibit behavioral and affective dysregulation, including hyperactivity and depression. The mechanisms are not known, but they could conceivably be due to postnatal social or environmental factors. However, we postulate that, more likely, the affective dysregulation is associated with the effects of EtOH exposure on the development of fetal serotonergic (5-HT) and/or dopaminergic (DA) pathways, i.e., pathways that in postnatal life are believed to regulate mood. Many women who use alcohol (ethanol, EtOH) during pregnancy suffer from depression and take selective serotonin reuptake inhibitors (SSRIs), which might influence these monoaminergic pathways in the fetus. Alternatively, monoaminergic pathway abnormalities might reflect a direct effect of EtOH on the fetal brain. To distinguish between these possibilities, we measured their expressions in fetal brains and in fetal brain-derived exosomes (FB-Es) isolated from the mothers' blood. We hypothesized that maternal use of EtOH and/or SSRIs during pregnancy would be associated with impaired fetal neural development, detectable as abnormal levels of monoaminergic and apoptotic biomarkers in FB-Es. Fetal brain tissues and maternal blood were collected at 9-23 weeks of pregnancy. EtOH groups were compared with unexposed controls matched for gestational age (GA). The expression of 84 genes associated with the DA and 5-HT pathways was analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) on microarrays. FB-Es also were assayed for serotonin transporter protein (SERT) and brain-derived neurotrophic factor (BDNF) by enzyme-linked immunosorbent assay (ELISA). Six EtOH-exposed human fetal brain samples were compared to SSRI- or polydrug-exposed samples and to unexposed controls. EtOH exposure was associated with significant upregulation of DA receptor D3 and 5-HT receptor HTR2C, while HTR3A was downregulated. Monoamine oxidase A (MAOA), MAOB, the serine/threonine kinase AKT3, and caspase-3 were upregulated, while mitogen-activated protein kinase 1 (MAPK1) and AKT2 were downregulated. ETOH was associated with significant upregulation of the DA transporter gene, while SERT was downregulated. There were significant correlations between EtOH exposure and (a) caspase-3 activation, (b) reduced SERT protein levels, and (c) reduced BDNF levels. SSRI exposure independently increased caspase-3 activity and downregulated SERT and BDNF. Early exposure to EtOH and SSRI together was associated synergistically with a significant upregulation of caspase-3 and a significant downregulation of SERT and BDNF. Reduced SERT and BDNF levels were strongly correlated with a reduction in eye diameter, a somatic manifestation of FASD. Maternal use of EtOH and SSRI during pregnancy each was associated with changes in fetal brain monoamine pathways, consistent with potential mechanisms for the affective dysregulation associated with FASD.

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.

Antidepressant effects of p-coumaric acid isolated from Vaccinium bracteatum leaves extract on chronic restraint stress mouse model and antagonism of serotonin 6 receptor in vitro

BackgroundVaccinium bracteatum Thunb. leaves (VBL) are used in traditional herbal medicines to treat various biological diseases. p-coumaric acid (CA), the main active component of VBL, has neuroprotective effects against corticosterone-induced damage in vitro. However, the effects of CA on immobility induced by chronic restraint stress (CRS) in a mouse model and 5-HT receptor activity have not been investigated. Hypothesis/PurposeWe investigated the antagonistic effects of VBL, NET-D1602, and the three components of Gαs protein-coupled 5-HT receptors. Additionally, we identified the effects and mechanism of action of CA, the active component of NET-D1602, in the CRS-exposed model. MethodsFor in vitro analyses, we used 1321N1 cells stably expressing human 5-HT6 receptors and CHO-K1 expressing human 5-HT4 or 5-HT7 receptors cell lines to study the mechanism of action. For in vivo analyses, CRS-exposed mice were orally administered CA (10, 50, or 100 mg/kg) daily for 21 consecutive days. The effects of CA were analyzed by assessing behavioral changes using a forced swim test (FST), measuring levels of hypothalamic-pituitary-adrenal (HPA) axis-related hormones in ntial therapeutic effects as 5-HT6 receptor antagonists for neurodegenerative diseases and depressioserum, and acetylcholinesterase (AChE), monoamines, including 5-HT, dopamine, and norepinephrine, using enzyme-linked immunosorbent assay kits. The underlying molecular mechanisms of the serotonin transporter (SERT), monoamine oxidase A (MAO-A), and extracellular signal-regulated kinase (ERK)/protein kinase B (Akt)/mTORC1 signaling were detected using western blotting. ResultsCA was confirmed to be an active component in the antagonistic effects of NET-D1602 on 5-HT6 receptor activity through decreases in cAMP and ERK1/2 phosphorylation. Moreover, CRS-exposed mice treated with CA showed a significantly reduced immobility time in the FST. CA also significantly decreased corticosterone, corticotropin-releasing hormone (CRH), and adrenocorticotropic hormone (ACTH) levels. CA enhanced 5-HT, dopamine, and norepinephrine levels in the hippocampus (HC) and prefrontal cortex (PFC) but decreased MAO-A and SERT protein levels. Similarly, CA significantly upregulated the ERK, Ca2+/calmodulin-dependent protein kinase II (CaMKII), Akt/mTOR/p70S6K/S6 signaling pathways in both HC and the PFC. ConclusionCA contained in NET-D1602 may play the antidepressant effects against CRS-induced depression-like mechanism and the selective antagonist effect of 5-HT6 receptor.

Duloxetine attenuates pain in association with downregulation of platelet serotonin transporter in patients with burning mouth syndrome and atypical odontalgia.

The aim of this study was evaluation of the association between severity of pain and expression of total or ubiquitinated serotonin transporter (SERT) protein in patients with burning mouth syndrome and atypical odontalgia (BMS/AO), who were treated by duloxetine. Patients with BMS/AO were assessed for severity of pain using the visual analog scale (VAS), and expression of total and ubiquitinated SERT protein in platelets before (baseline) and 12 weeks after duloxetine-treatment. The expression of total and ubiquitinated SERT protein at baseline in all patients (n=33) were higher and lower, respectively, compared to those in healthy controls. 12 weeks after duloxetine-treatment, there was no difference in the total SERT protein levels between patients (n=21) and healthy controls. In the 16 patients who could be measured, mean VAS scores and total SERT protein levels were significantly decreased after the treatment, compared to those at baseline. There was tendency for a positive correlation between total SERT protein levels and VAS scores in these patients. Our findings indicate that duloxetine relieves pain in association with downregulation of platelet SERT expression in patients with BMS/AO.

Chronic restraint stress induces serotonin transporter expression in the rat adrenal glands

Chronic restraint stress (CRS) magnifies restraint-induced corticosterone secretion through a mechanism involving increased adrenocortical 5-HT content and turnover. We analysed the impact of CRS on serotonin transporter (SERT) expression and distribution in rat adrenal glands. Male Wistar rats were submitted to CRS (20 min/day) or undisturbed control conditions for 14 days. Exposure to CRS induced a remarkable increase in SERT-like immunoreactivity in the adrenal cortex, which closely matched that of chromogranin A immunostaining, along with a significant increase in SERT protein and mRNA levels in whole adrenals as determined by immunohistochemistry, Western blot and RT-PCR assays, respectively; all these CRS-induced changes occurred almost exclusively in left adrenals. Closely similar results were obtained in animals that received a 14-day chronic corticosterone treatment. These results unravel an interesting association between chronic stress exposure and SERT expression in adrenocortical chromogranin A-positive cells, which seems to be a glucocorticoid-dependent phenomenon.

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.

Diacylglycerol kinase δ destabilizes serotonin transporter protein through the ubiquitin-proteasome system

Brain-specific diacylglycerol kinase (DGK) δ-knockout mice exhibited serotonin transporter (SERT) inhibitor-sensitive obsessive-compulsive disorder-like behaviors. Moreover, SERT protein levels were markedly increased in the DGKδ-deficient brain. However, its molecular mechanisms remain unclear. We found that the catalytic subdomain-a and the coiled-coil structure-containing region of DGKδ interacted with the C-terminal cytoplasmic region (CTC) of SERT. Moreover, the protein levels of full-length SERT and SERT-CTC alone were significantly decreased by DGKδ in a catalytic activity-dependent manner. A proteasome inhibitor, MG-132, inhibited DGKδ-dependent SERT degradation. Notably, DGKδ interacted with MAGE-D1 adaptor protein and Praja-1 E3 ubiquitin-protein ligase, and enhanced the ubiquitination of SERT through Praja-1. Taken together, these results indicate that DGKδ interacts with SERT and induces SERT degradation in an activity-dependent manner through the Praja-1 ubiquitin ligase-proteasome system. These new findings provide novel insights into serotonergic system regulation and the pathophysiology/therapeutics of serotonin-/SERT-related diseases such as obsessive-compulsive disorder, depression, autism and schizophrenia.

Increased Serotonin Transporter Expression in Huntington's Disease Patients Is Not Consistently Replicated in Murine Models.

Selective serotonin reuptake inhibitors (SSRIs) target the serotonin transporter (SERT) and are commonly prescribed for depression in Huntington's disease (HD) patients. However, SERT expression in HD has not been carefully evaluated in patients or mouse models. In this study, we investigated SERT levels in HD patients and HD mouse models. We obtained HD patient brain striatal samples and matched controls, as well as brain tissue from CAG140 and R6/2 mice. SERT mRNA and protein levels were analyzed using quantitative RT-PCR and immunoblotting. We found that SERT protein, but not mRNA is markedly increased in grade 4 HD patient striatal tissue. These findings suggest posttranscriptional or translational SERT dysregulation as a possible etiologic factor modulating psychopathology in HD. Interestingly, SERT expression is variable in mouse models of the disease. Increased SERT levels are demonstrated in the brain of CAG140 mice, a full-length knock-in mouse model of the disease, but not in the striatum of the R6/2 fragment murine model of the disease. Based on this parameter, the CAG140 huntingtin knock-in mouse model is more suitable than the R6/2 model for the study of serotonergic pathway pathology in Huntington's disease.

Potential Mechanism for HIV-Associated Depression: Upregulation of Serotonin Transporters in SIV-Infected Macaques Detected by 11C-DASB PET.

Purpose: Increased incidence of depression in HIV+ patients is associated with lower adherence to treatment and increased morbidity/mortality. One possible underlying pathophysiology is serotonergic dysfunction. In this study, we used an animal model of HIV, the SIV-infected macaque, to longitudinally image serotonin transporter (SERT) expression before and after inoculation, using 11C-DASB (SERT ligand) PET imaging. Methods: We infected seven rhesus macaques with a neurovirulent SIV strain and imaged them at baseline and multiple time points after inoculation (group A). Pyrosequencing methylation analysis of the SERT promoter region was performed. We also measured SERT mRNA/protein in brain single-cell suspensions from another group (group B) of SIV-infected animals (n = 13). Results: Despite some animals showing early fluctuations, 86% of our group A animals eventually showed a net increase in midbrain/thalamus binding potential (BPND) over the course of their disease (mean increased binding between last time point and baseline = 30.2% and 32.2%, respectively). Repeated-measures mixed-model analysis showed infection duration to be predictive of midbrain BPND (p = 0.039). Thalamic BPND was statistically significantly associated with multiple CSF cytokines (P < 0.05). There was higher SERT protein levels in the second group (group B) of SIV-infected animals with SIV encephalitis (SIVE) compared to those without SIVE (p = 0.014). There were no longitudinal changes in SERT gene promoter region percentage methylation between baselines and last time points in group A animals. Conclusion: Upregulated SERT leading to lower synaptic levels of serotonin is a possible mechanism of depression in HIV+ patients, and extrapolating our conclusions from SIV to HIV should be sought using translational human studies.

Anxiolytic- and Antidepressant-Like Effects of Fish Oil-Enriched Diet in Brain-Derived Neurotrophic Factor Deficient Mice.

Despite significant advances in the understanding of the therapeutic activity of antidepressant drugs, treatment-resistant depression is a public health issue prompting research to identify new therapeutic strategies. Evidence strongly suggests that nutrition might exert a significant impact on the onset, the duration and the severity of major depression. Accordingly, preclinical and clinical investigations demonstrated the beneficial effects of omega-3 fatty acids in anxiety and mood disorders. Although the neurobiological substrates of its action remain poorly documented, basic research has shown that omega-3 increases brain-derived neurotrophic factor (BDNF) levels in brain regions associated with depression, as antidepressant drugs do. In contrast, low BDNF levels and hippocampal atrophy were observed in animal models of depression. In this context, the present study compared the effects of long-lasting fish oil-enriched diet, an important source of omega-3 fatty acids, between heterozygous BDNF+/- mice and their wild-type littermates. Our results demonstrated lower activation of Erk in BDNF+/- mice whereas this deficit was rescued by fish oil-enriched diet. In parallel, BDNF+/- mice displayed elevated hippocampal extracellular 5-HT levels in relation with a local decreased serotonin transporter protein level. Fish oil-enriched diet restored normal serotonergic tone by increasing the protein levels of serotonin transporter. At the cellular level, fish oil-enriched diet increased the pool of immature neurons in the dentate gyrus of BDNF+/- mice and the latter observations coincide with its ability to promote anxiolytic- and antidepressant-like response in these mutants. Collectively, our results demonstrate that the beneficial effects of long-term exposure to fish oil-enriched diet in behavioral paradigms known to recapitulate diverse abnormalities related to the depressive state specifically in mice with a partial loss of BDNF. These findings contrast with the mechanism of action of currently available antidepressant drugs for which the full manifestation of their therapeutic activity depends on the enhancement of serotoninergic and BDNF signaling. Further studies are warranted to determine whether fish oil supplementation could be used as an add-on strategy to conventional pharmacological interventions in treatment-resistant patients and relevant animal models.

Serum Serotonin/Tryptophan Ratio Correlates with Inflammation in Crohn's Disease Patients

Inflammatory bowel diseases (IBD), which primarily consist of ulcerative colitis and Crohn's disease (CD) are characterized by persistent inflammation of the gastrointestinal (GI) tract. The current standard for diagnosis of IBD utilizes invasive methods including endoscopy and tissue biopsy, with blood tests being less specific for IBD. Substantial evidence has implicated involvement of the neurohormone serotonin (5‐hydroxytryptamine, 5‐HT) in the pathophysiology of IBD. Both mRNA and protein levels of serotonin transporter (SERT) have been consistently shown to be decreased in the intestinal mucosa of IBD patients. However, there is disagreement between previous studies on whether mucosal 5‐HT is increased or decreased in IBD. Furthermore, serum levels, rather than tissue levels, of 5‐HT in patients with IBD have not yet been examined. The current study examined 5‐HT, tryptophan (TRP), and kynurenine (KYN) levels in CD patients to test the hypothesis that 5‐HT serum levels correlate with inflammation.MethodsSerum samples were obtained from a German cohort of 45 CD patients and were categorized into one of three groups based upon their CD activity index (CDAI) and disease history. 15 patients were in remission (CDAI &lt; 150), 15 patients had active disease (CDAI &gt; 450), and 15 patients had chronic disease characterized by persistence of clinical symptoms and no lasting remission after adequate drug therapy. Age, gender, and race were not significantly different between the three groups. LC‐MS was used to measure 5‐HT, TRP, and KYN levels in the serum samples and Luminex Multiplex ELISA was used to measure cytokine levels. Results are shown as 95% confidence intervals of the mean.ResultsTRP and its derivatives 5‐HT and KYN were measured in CD patient serum: 5‐HT (0.65 – 0.93 μM), TRP (50.93 – 59.67 μM), and KYN (1.94 – 2.55 μM). To normalize for differences in body TRP levels, data were calculated as a ratio between 5‐HT and TRP (5‐HT/TRP) and between KYN and TRP (KYN/TRP). There were no significant differences in 5‐HT, TRP, and KYN levels between remission, active, and chronic groups. However, the 5‐HT/TRP ratio was significantly elevated in CD patients with active disease (0.014 – 0.034, p &lt; 0.01) as compared either to patients in remission (0.009 – 0.017) or with chronic disease (0.009 – 0.018) with no difference in KYN/TRP ratio. Further, the 5‐HT/TRP ratio positively correlated with serum C‐reactive protein levels (r = 0.47, p &lt; 0.01), serum IL‐1β levels (r = 0.52, p &lt; 0.01), and serum TNF‐α (r = 0.36, p &lt; 0.05). In conclusion, we have shown that the serum 5‐HT/TRP ratio can discriminate between active disease and chronic disease or remission among CD patients. In addition, the 5‐HT/TRP ratio correlates with established serum markers of inflammation in CD patients, emphasizing the important role that 5‐HT plays in the pathophysiology of IBD.Support or Funding InformationNIDDK NIH R01 DK098170, NIDDK NIH F30 DK113703This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effect of Lactobacillus rhamnosus GG supernatant on serotonin transporter expression in rats with post-infectious irritable bowel syndrome.

AIMTo evaluate the effect of Lactobacillus rhamnosus GG supernatant (LGG-s) on the expression of serotonin transporter (SERT) in rats with post-infectious irritable bowel syndrome (PI-IBS).METHODSCampylobacter jejuni 81-176 (1010 CFU/mL) was used to induce intestinal infection to develop a PI-IBS model. After evaluation of the post-infectious phase by biochemical tests, DNA agarose gel electrophoresis, abdominal withdrawal reflex (AWR) test, and the intestinal motility test, four PI-IBS groups received different concentrations of LGG-s for 4 wk. The treatments were maintained for 1.0, 2.0, 3.0 or 4.0 wk during the experiment, and the colons and brains were removed for later use each week. SERT mRNA and protein levels were detected by real-time PCR and Western blot, respectively.RESULTSThe levels of SERT mRNA and protein in intestinal tissue were higher in rats treated with LGG-s than in control rats and PI-IBS rats gavaged with PBS during the whole study. Undiluted LGG-s up-regulated SERT mRNA level by 2.67 times compared with the control group by week 2, and SERT mRNA expression kept increasing later. Double-diluted LGG-s was similar to undiluted-LGG-s, resulting in high levels of SERT mRNA. Triple-diluted LGG-s up-regulated SERT mRNA expression level by 6.9-times compared with the control group, but SERT mRNA expression decreased rapidly at the end of the second week. At the first week, SERT protein levels were basically comparable in rats treated with undiluted LGG-s, double-diluted LGG-s, and triple-diluted LGG-s, which were higher than those in the control group and PBS-treated PI-IBS group. SERT protein levels in the intestine were also comparable in rats treated with undiluted LGG-s, double-diluted LGG-s, and triple-diluted LGG-s by the second and third weeks. SERT mRNA and protein levels in the brain had no statistical difference in the groups during the experiment.CONCLUSIONLGG-s can up-regulate SERT mRNA and protein levels in intestinal tissue but has no influence in brain tissue in rats with PI-IBS.

Lactobacillus acidophilus and Bifidobacterium longum supernatants upregulate the serotonin transporter expression in intestinal epithelial cells.

Background/Aims:Probiotics play a role in relieving irritable bowel syndrome (IBS); however, the underlying mechanism is yet unclear. The aim of the study was to investigate the effects of the supernatants of Lactobacillus acidophilus and Bifidobacterium longum on the expression of serotonin transporter (SERT) messenger ribonucleic acid (mRNA) and protein.Materials and Methods:HT-29 and Caco-2 cells were treated with different concentrations of L. acidophilus and B. longum supernatants for 12 h and 24 h, respectively. SERT mRNA and proteins levels were detected by real-time polymerase chain reaction (real-time PCR) and Western-blotting.Results:The mRNA levels of SERT in HT-29 and Caco-2 cells treated with different concentrations of L. acidophilus or B. longum supernatants for 12 h and 24 h, each, were higher than that in the control groups. In addition, the expression of the protein in both cells was also upregulated, which was approximately similar to that of the corresponding mRNA.Conclusions:L. acidophilus and B. longum supernatants can upregulate SERT mRNA and protein levels in intestinal epithelial cells.

Effects of intestinal microbiota of patients with chronic constipation on the expression of serotonin transporter and the bowel movement in mice

Objective To investigate the effects of intestinal microbiota of patients with chronic constipation on the expression of serotonin transporter (SERT) and the bowel movement in mice. Methods Fecal samples of patients with slow transit constipation met Rome Ⅲ criteria and healthy normal controls were collected and made into fecal microbiota solution. Twenty specfic pathogen free (SPF) mice were divided into experiment group and control group. The mice of two groups were both pre-treated with streptomycin to establish the germ-free mice model. The mice of control group were gavaged with mixed fecal microbiota solution of healthy normal controls and the mice of experiment group were gavaged with mixed fecal microbiota solution of patients with chronic constipation. Mice were sacrificed after fed for 15 days. Defecation parameters and ink discharge time of mice were detected. The expressions of SERT mRNA and SERT protein in mice intestinal tissues were detected with real time-polymerase chain reaction and Western blotting. The 5-hydroxytryptamine (5-HT) levels of mice intestinal tissues were evaluated by enzyme-linked immunosorbent assay (ELISA) and double immunofluorescent staining. The methods of t test and Chi-square test were performed for statistical analysis. Results On the 15thday, the total number of the feces within 2 h of the experiment group and control group was 8.55±1.83 and 12.14±2.90, respectively, and the difference was statistically significant (t=-3.33, P<0.05). The weight of feces were (151.90±32.42) mg and (246.72±64.01) mg, respectively, and the difference was statistically significant (t=4.18, P<0.01). The dry weight of feces were (65.52±11.76) mg and (92.93±23.07) mg, respectively, and the difference was statistically significant (t=3.37, P<0.05). The water content of feces were (56.63±3.01)% and (61.95±3.70)%, respectively, and the difference was statistically significant (t=3.57, P<0.05). The defecating time of first black feces of the experiment group and control group were (83.24±11.31) min and (69.06±2.72) min, respectively, and the difference was statistically significant (t=-2.74, P<0.05). The expressions of SERT mRNA and SERT protein levels in mice intestine tissues of the experiment group were significantly higher than those of the control group (t=2.61, -6.89; both P<0.05). 5-HT level of mice intestinal tissues of the experimental group and control group were (151.69±10.18) ng/mL and (198.77±25.99) ng/mL, respectively, and the difference was statistically significant (t=-4.13, P<0.01). Conclusion Intestinal microbiota of patients with chronic constipation may influence the expression of SERT in the mice intestinal tissues, and then decrease the level of 5-HT, slowing the bowel movement in mice. Key words: Chronic constipation; Intestinal microbiota; Serotonin transporter; 5-hydroxytryptamine; Bowel movement; mice

The ubiquitination of serotonin transporter in lymphoblasts derived from fluvoxamine-resistant depression patients

There is insufficient serotonergic neuronal function in the pathophysiology of major depressive disorder (MDD). Serotonin transporter (SERT) plays a critical role in terminating the function of serotoninergic neurons. SERT is linked to vulnerability to MDD and is an important target for antidepressants. The expression of SERT in lymphocytes and platelets is associated with their expression in central nervous system. Most of the clinical studies that have analyzed the role of SERT in depression have focused on absolute expression of SERT in the brain or peripheral tissue. Our study has shown that the SERT protein is ubiquitinated, which has been implicated through the SERT stability and depressive behaviors in mice. In our study, we have used lymphoblasts derived from the peripheral blood lymphocytes to quantitatively examine SERT protein expression and ubiquitination in fluvoxamine-responsive and fluvoxamine–resistant MDD patients. We found that the protein levels of SERT were higher in the fluvoxamine-resistant MDD patients. Ubiquitinated protein levels of SERT were lower in the fluvoxamine-resistant MDD patients. The proteasome inhibitor failed to increase the protein levels of SERT in both fluvoxamine-responsive and fluvoxamine-resistant MDD patients. In sum, these findings suggest that the downregulation of the ubiquitination of SERT protein induces insufficient degradation of SERT by proteasome, which resulted in the upregulation of SERT protein in fluvoxamine-resistant MDD patients. Although further studies with various populations will be required to generalize results, SERT protein expression, ubiquitination, and the responsiveness of SERT expression to proteasome inhibitor are potential biomarkers for the diagnosis of MDD and antidepressant efficacy.

NCAM-deficient mice show prominent abnormalities in serotonergic and BDNF systems in brain – Restoration by chronic amitriptyline

Mood disorders are associated with alterations in serotonergic system, deficient BDNF (brain-derived neurotrophic factor) signaling and abnormal synaptic plasticity. Increased degradation and reduced functions of NCAM (neural cell adhesion molecule) have recently been associated with depression and NCAM deficient mice show depression-related behavior and impaired learning. The aim of the present study was to investigate potential changes in serotonergic and BDNF systems in NCAM knock-out mice. Serotonergic nerve fiber density and SERT (serotonin transporter) protein levels were robustly reduced in the hippocampus, prefrontal cortex and basolateral amygdala of adult NCAM−/− mice. This SERT reduction was already evident during early postnatal development. [3H]MADAM binding experiments further demonstrated reduced availability of SERT in cell membranes of NCAM−/− mice. Moreover, the levels of serotonin and its major metabolite 5-HIAA were down regulated in the brains of NCAM−/− mice. NCAM−/− mice also showed a dramatic reduction in the BDNF protein levels in the hippocampus and prefrontal cortex. This BDNF deficiency was associated with reduced phosphorylation of its receptor TrkB. Importantly, chronic administration of antidepressant amitriptyline partially or completely restored these changes in serotonergic and BDNF systems, respectively. In conclusion, NCAM deficiency lead to prominent and persistent abnormalities in brain serotonergic and BDNF systems, which likely contributes to the behavioral and neurobiological phenotype of NCAM−/− mice.

Lactobacillus rhamnosus GG supernatant upregulates serotonin transporter expression in intestinal epithelial cells and mice intestinal tissues.

The role that probiotics play in relieving irritable bowel syndrome (IBS) has been demonstrated; however, the mechanism by which IBS is affected remains unclear. In this study, serotonin transporter (SERT) mRNA and serotonin transporter protein (SERT-P) levels in HT-29, Caco-2 cells, and mice intestinal tissues were examined after treatment with Lactobacillus rhamnosus GG supernatant (LGG-s). HT-29 and Caco-2 cells were treated with different concentrations of LGG-s for 12 and 24h and C57BL/6 mice received supplements of different concentrations for 4weeks. SERT mRNA and SERT-P levels were detected by real-time PCR and Western blotting. SERT mRNA and SERT-P levels in HT-29 and Caco-2 cells were higher than those in the control 24h after treatment. Undiluted LGG-s upregulated SERT mRNA levels by 9.4-fold in the first week, which dropped in the second week. The double-diluted LGG-s upregulated SERT mRNA by 2.07-fold in the first week; levels dropped to 1.75-fold within the second week and under base expression levels by the third week, while they again climbed to 1.56-fold in the fourth week. The triple-diluted LGG-s could not upregulate SERT mRNA expression until the end of the fourth week. The SERT-P levels in the double-diluted LGG-s group were higher than that in the control but fluctuated with time. SERT-P levels in the triple-diluted LGG-s were higher than that in the control in the last 2weeks and increased with time. LGG-s can upregulate SERT mRNA and SERT-P levels in intestinal epithelial cells and mice intestinal tissues.

The Association of 5-HTTLPR XLL Genotype with Higher Cortisol Levels in African Americans

Genetic variants of the human serotonin transporter (SERT) may contribute to HPA axis dysregulation. SERT has two promoter region polymorphisms (5-HTTLPR: VNTR and SNP: rs25531), which may alter levels of SERT protein and its function. Combining these polymorphisms creates a functional polymorphism (FN) which may modulate mRNA expression. This study examines the relationship between these genetic variants and morning and evening salivary samples of both cortisol and dehydroepiandrosterone sulfate (DHEAS) concentrations in 269 African American (AA) adults. Resultant allele frequencies for the VNTR, SNP, and FN genotypes were 70% L (2% XLL), 84% A, and 54% LA (2% XLLA), respectively. The XLL genotype was associated with significantly higher concentrations of cortisol (~3X) and DHEAS (~2X) for both VNTR and FN polymorphisms. No significant differences were found for SNP genotypes. Conclusions were that persons with VNTR and FN XLL polymorphisms had significantly higher cortisol and DHEAS concentrations than other genotypes. AAs also appear to have a higher frequency of the rare XL allele than Caucasians. Whether the XLL genotype predisposes AAs to greater health challenges will require further research to determine the implications of these findings.

Long-Term Exposure of RN46A Cells Expressing Serotonin Transporter (SERT) to a cAMP Analog Up-regulates SERT Activity and Is Accompanied by Neural Differentiation of the Cells

To examine the functional regulation of serotonin transporter (SERT) by cAMP, we examined whether SERT uptake activity was affected by dibutyryl cAMP (dbcAMP), a cAMP analog, in SERT-transfected RN46A cells derived from embryonic rat raphe neurons. Long-term exposure (> 4 h) of dbcAMP (1 mM) to SERT-expressing RN46A cells significantly up-regulated SERT activity. In addition, a selective PKA activator, but not a selective EPAC activator, increased the serotonin uptake activity of SERT, suggesting that this regulation was mainly mediated via PKA. Time-dependent up-regulation of SERT activity by dbcAMP was accompanied by neural differentiation of RN46A cells. Further investigation of dbcAMP-induced up-regulation of SERT revealed that dbcAMP elevated SERT protein levels without affecting SERT mRNA transcription. The chase assay for residual SERT protein revealed that dbcAMP slowed its degradation rate. Immunohistochemical analysis revealed that plasma membrane–localized SERT was more abundant in dbcAMP-treated cells than in non-treated cells, suggesting that dbcAMP up-regulated SERT by decreasing its degradation and increasing its plasma membrane expression. These results raise the possibility that the elevation of intracellular cAMP up-regulated SERT function through a mechanism linked to the differentiation of RN46A cells and show the importance of SERT function during the developmental process of the serotonergic nervous system.

The expression of serotonin transporter protein correlates with the severity of psoriasis and chronic stress

Psoriasis may be worsened by stress and mood disorders. There is an increased expression of the serotonin transporter protein (SERT) in involved psoriatic skin as compared to non-involved psoriatic skin and normal skin. The aim of this study was to investigate if the increased expression of SERT in psoriasis correlates with the severity of disease, chronic stress, and depression. Biopsies from involved and non-involved skin from the back of 20 patients with chronic plaque psoriasis were immunohistochemically analysed, using a monoclonal antibody to SERT. The severity of psoriasis was assessed for each patient using the Psoriasis area and severity index (PASI). Levels of depression and chronic stress were measured using Beck's Depression Inventory (BDI) and the salivary cortisol test, respectively. A positive correlation (r=0.53; p<0.05) between PASI and the numbers of SERT-positive dendritic cells in the epidermis of involved psoriatic skin was determined. We also observed a negative correlation (r=-0.46; p<0.05) between salivary cortisol ratio levels and the numbers of SERT-positive cells in the epidermis of involved psoriatic skin, indicating a correlation between SERT expression and chronic stress. The serotonergic system may be involved in the chronic inflammation evident in psoriatic skin. Through modulating the levels of SERT, there might be a therapeutic possibility for reducing chronic inflammation in psoriasis.