Organic cation transporter 3: Keeping the brake on extracellular serotonin in serotonin-transporter-deficient mice.
ABSTRACT: Mood disorders cause much suffering and are the single greatest cause of lost productivity worldwide. Although multiple medications, along with behavioral therapies, have proven effective for some individuals, millions of people lack an effective therapeutic option. A common serotonin (5-HT) transporter (5-HTT/SERT, SLC6A4) polymorphism is believed to confer lower 5-HTT expression in vivo and elevates risk for multiple mood disorders including anxiety, alcoholism, and major depression. Importantly, this variant is also associated with reduced responsiveness to selective 5-HT reuptake inhibitor antidepressants. We hypothesized that a reduced antidepressant response in individuals with a constitutive reduction in 5-HTT expression could arise because of the compensatory expression of other genes that inactivate 5-HT in the brain. A functionally upregulated alternate transporter for 5-HT may prevent extracellular 5-HT from rising to levels sufficiently high enough to trigger the adaptive neurochemical events necessary for therapeutic benefit. Here we demonstrate that expression of the organic cation transporter type 3 (OCT3, SLC22A3), which also transports 5-HT, is upregulated in the brains of mice with constitutively reduced 5-HTT expression. Moreover, the OCT blocker decynium-22 diminishes 5-HT clearance and exerts antidepressant-like effects in these mice but not in WT animals. OCT3 may be an important transporter mediating serotonergic signaling when 5-HTT expression or function is compromised.
Project description:The neurotransmitter serotonin (5-HT) plays a vital regulatory role in both the brain and gut. 5-HT is crucial for regulating mood in the brain as well as gastrointestinal motility and secretion peripherally. Alterations in 5-HT transmission have been linked to pathological symptoms in both intestinal and psychiatric disorders and selective 5-HT transporter (5-HTT) inhibitors, affecting the 5-HT system by blocking the 5-HT transporter (5-HTT) have been successfully used to treat CNS- and intestinal disorders. Humans that carry the short allele of the 5-HTT-linked polymorphic region (5-HTTLPR) are more vulnerable to adverse environmental stressors, in particular early life stress. Although, early life stress has been shown to alter the composition of the gut microbiota, it is not known whether a lower 5-HTT expression is also associated with an altered microbiome composition. To investigate this, male and female wild type (5-HTT+/+), heterozygous (5-HTT+/-), and knockout (5-HTT-/-) 5-HT transporter rats were maternally separated for 6 h a day from postnatal day 2 till 15. On postnatal day 21, fecal samples were collected and the impact of 5-HTT genotype and maternal separation (MS) on the microbiome was analyzed using high-throughput sequencing of the bacterial 16S rRNA gene. MS showed a shift in the ratio between the two main bacterial phyla characterized by a decrease in Bacteroidetes and an increase in Firmicutes. Interestingly, the 5-HTT genotype caused a greater microbal dysbiosis (microbial imbalance) compared with MS. A significant difference in microbiota composition was found segregating 5-HTT-/- apart from 5-HTT+/- and 5-HTT+/+ rats. Moreover, exposure of rats with 5-HTT diminished expression to MS swayed the balance of their microbiota away from homeostasis to 'inflammatory' type microbiota characterized by higher abundance of members of the gut microbiome including Desulfovibrio, Mucispirillum, and Fusobacterium, all of which are previously reported to be associated with a state of intestinal inflammation, including inflammation associated with MS and brain disorders like multiple depressive disorders. Overall, our data show for the first time that altered expression of 5-HTT induces disruptions in male and female rat gut microbes and these 5-HTT genotype-related disruptions are augmented when combined with early life stress.
Project description:Neurotransmitter regulation of bone metabolism has been a subject of increasing interest and investigation. We reported previously that osteoblastic cells express a functional serotonin (5-HT) signal transduction system, with mechanisms for responding to and regulating uptake of 5-HT. The clonal murine osteocytic cell line, MLO-Y4, demonstrates expression of the serotonin transporter (5-HTT), and the 5-HT1A, and 5-HT2A receptors by real-time RT-PCR and immunoblot analysis. Immunohistochemistry using antibodies for the 5-HTT, and the 5-HT1A and 5-HT2A receptors reveals expression of all three proteins in both osteoblasts and osteocytes in rat tibia. 5-HTT binding sites were demonstrated in the MLO-Y4 cells with nanomolar affinity for the stable cocaine analog [125I]RTI-55. Imipramine and fluoxetine, antagonists with specificity for 5-HTT, show the highest potency to antagonize [125I]RTI-55 binding in the MLO-Y4 cells. GBR-12935, a relatively selective dopamine transporter antagonist, had a much lower potency, as did desipramine, a selective norepinephrine transporter antagonist. The maximal [3H]5-HT uptake rate in MLO-Y4 cells was 2.85 pmol/15 min/well, with a Km value of 290 nM. Imipramine and fluoxetine inhibited specific [3H]5-HT uptake with IC50 values in the nanomolar range. 5-HT rapidly stimulated PGE2 release from MLO-Y4 cells; the EC50 for 5-HT was 0.1 microM, with a 3-fold increase seen at 60 min. The rate-limiting enzyme for serotonin synthesis, tryptophan hydroxylase, is expressed in MLO-Y4 cells as well as osteoblastic MC3T3-E1 cells. Thus, osteocytes, as well as osteoblasts, are capable of 5-HT synthesis, and express functional receptor and transporter components of the 5-HT signal transduction system.
Project description:Serotonin (5-HT) is a monoamine neurotransmitter and plays important roles in several of the human body's systems. Known as a primary target for psychoactive drug development, the 5-HT transporter (5-HTT, SERT) plays a critical role in the regulation of serotonergic function by reuptaking 5-HT. The allelic variation of 5-HTT expression is caused by functional gene promoter polymorphism with two principal variant alleles, 5-HTT gene-linked polymorphic region (5-HTTLPR). It has been demonstrated that 5-HTTLPR is associated with numerous neuropsychiatric disorders. The functional roles of 5-HTTLPR have been reported in human choriocarcinoma (JAR), lymphoblast and raphe cells. To date, the significance of 5-HTTLPR in gastrointestinal tract-derived cells has never been elucidated. Thus, the impact of 5-HTTLPR on 5-HTT transcription was studied in SW480 human colon carcinoma cells, which were shown to express 5-HTT. We found 42-bp fragment in long (L) allele as compared to short (S) allele, and this allelic difference resulted in 2-fold higher transcriptional efficiency of L allele (P < 0.05) as demonstrated using a functional reporter gene assay. Nevertheless, the transcriptional effect of estrogen and glucocorticoid on 5-HTT expression via 5-HTTLPR was not found in this cell line. Our study was the first to demonstrate the molecular role of this allelic variation in gastrointestinal tract cells.
Project description:Genetic association studies suggest that variations in the 5-hydroxytryptamine (5-HT; serotonin) transporter (5-HTT) gene are associated with susceptibility to psychiatric disorders such as anxiety or posttraumatic stress disorder. Individuals carrying high 5-HTT-expressing gene variants display low amygdala reactivity to fearful stimuli. Mice overexpressing the 5-HTT (5-HTTOE), an animal model of this human variation, show impaired fear, together with reduced fear-evoked theta oscillations in the basolateral amygdala (BLA). However, it is unclear how variation in 5-HTT gene expression impacts on the microcircuitry of the BLA to change behavior. We addressed this issue by investigating the activity of parvalbumin (PV)-expressing interneurons (PVINs), the biggest IN population in the basal amygdala (BA). We found that increased 5-HTT expression impairs the recruitment of PVINs (measured by their c-Fos immunoreactivity) during fear. Ex vivo patch-clamp recordings demonstrated that the depolarizing effect of 5-HT on PVINs was mediated by 5-HT2A receptor. In 5-HTTOE mice, 5-HT-evoked depolarization of PVINs and synaptic inhibition of principal cells, which provide the major output of the BA, were impaired. This deficit was because of reduced 5-HT2A function and not because of increased 5-HT uptake. Collectively, these findings provide novel cellular mechanisms that are likely to contribute to differences in emotional behaviors linked with genetic variations of the 5-HTT.
Project description:The serotonin (5-HT) transporter (SERT) is a key determinant of the concentration of 5-HT available for synaptic signaling and is the target of the antidepressant-selective 5-HT reuptake inhibitors (SSRIs). In addition to the role of SERT in mediating the actions of antidepressant medications, multiple, rare gain of function coding variants in SERT have been identified in subjects with autism spectrum disorder (ASD). The Blakely lab has extensively characterized the structural and functional impact of one of these variants, SERT Ala56, in vitro and in vivo, revealing a constitutively-imposed, biased conformation that enhances transporter function and that perturbs transporter regulation. We hypothesize that SERT Ala56 disrupts the interactions of molecular networks that normally ensure proper transporter structure, localization and regulation, and whose elucidation may reveal new determinants of serotonergic dysfunction in ASD. Here, we pursue an unbiased proteomic analysis of changes in SERT interacting protein (SIPs) imposed by the SERT Ala56 variant, expressed in transgenic mice. Further investigation of the SIPs and associated networks nominated through our studies may provide new opportunities to detect and modulate brain networks that contribute to the underlying complexity of ASD.
Project description:Certain polymorphisms reduce serotonin (5-HT) reuptake transporter (5-HTT) function and increase susceptibility to psychiatric disorders. Heterozygous (5-HTT(+/-))-deficient mice, models for humans with these polymorphisms, have elevated brain 5-HT concentrations and behavioral abnormalities. As postsynaptic 5-HT(2A/2C) receptors are coupled to cytosolic phospholipase A(2) (cPLA(2)), which releases arachidonic acid (AA) from membrane phospholipid, 5-HTT-deficient mice may have altered brain AA signaling and metabolism. To test this hypothesis, signaling was imaged as an AA incorporation coefficient k(*) in unanesthetized homozygous knockout (5-HTT(-/-)), 5-HTT(+/-) and wild-type (5-HTT(+/+)), mice following saline (baseline) or 1.5 mg/kg s.c. DOI, a partial 5-HT(2A/2C) receptor agonist. Enzyme activities, metabolite concentrations, and head-twitch responses to DOI were also measured. Baseline k(*) was widely elevated by 20-70% in brains of 5-HTT(+/-) and 5-HTT(-/-) compared to 5-HTT(+/+) mice. DOI increased k(*) in 5-HTT(+/+) mice, but decreased k(*) in 5-HTT-deficient mice. Brain cPLA(2) activity was elevated in 5-HTT-deficient mice; cyclooxygenase activity and prostaglandin E(2) and F(2alpha) and thromboxane B(2) concentrations were reduced. Head-twitch responses to DOI, although robust in 5-HTT(+/+) and 5-HTT(+/-) mice, were markedly fewer in 5-HTT(-/-) mice. Pretreatment with para-chlorophenylalanine, a 5-HT synthesis inhibitor, restored head twitches in 5-HTT(-/-) mice to levels in 5-HTT(+/+) mice. We propose that increased baseline values of k(*) in 5-HTT-deficient mice reflect tonic cPLA(2) stimulation through 5-HT(2A/2C) receptors occupied by excess 5-HT, and that reduced k(*) and head-twitch responses to DOI reflected displacement of receptor-bound 5-HT by DOI with a lower affinity. Increased baseline AA signaling in humans having polymorphisms with reduced 5-HTT function might be identified using positron emission tomography.
Project description:Serotonin or 5-hydroxytryptamine (5-HT) has been shown to be essential in lots of physiological and pathological processes. It is well known that 5-HT and 5-HT transporter (5-HTT) play important roles in the pulmonary artery in pulmonary hypertension. However, little is known about the function of 5-HTT in other arteries. In this study we found that the expression of 5-HTT was elevated in injured carotid arteries and over-expression of 5-HTT induced proliferation of smooth muscle cells (SMCs); however, this phenotype could be reversed by knocking-down of 5-HTT or endothelial cells conditional medium (EC-CM). A 5-HTT inhibitor, fluoxetine, treated animals also exhibited reduced restenosis after injury. We identified that miR-195 was packaged in the extracellular vesicles from EC-CM. We further confirmed that extracellular vesicles could transfer miR-195 from ECs to SMCs to inhibit the expression of 5-HTT in SMCs and the proliferation of SMCs. These results provide the first evidence that ECs communicate with SMCs via micro-RNA195 in the regulation of the proliferation of SMCs through 5-HTT, which will contribute to a better understanding of communications between ECs and SMCs via micro-RNA. Our findings suggest a potential target for the control of vessel restenosis.
Project description:Cocaine users consistently develop working memory (WM) impairments but the mediating molecular mechanisms are unknown so far. Recent evidence suggests that the serotonin (5-HT) system is altered by chronic cocaine use, while also being involved in WM processing. Thus, we investigated the effects of genetic variations impacting 5-HT activity and of peripheral 5-HT transporter (5-HTT) mRNA expression on WM performance in cocaine users and stimulant naive controls. Two hundred twenty participants (126 cocaine users, 94 controls) were assessed with visuospatial, spatial, and verbal WM tasks, genotyped for the length polymorphism in the promoter region of the 5-HTT (5-HTTLPR), the variable number of tandem repeats in the second intron of the 5-HTT (VNTR In2), two single-nucleotide polymorphisms (rs4570625 and rs1386497) in the tryptophan hydroxylase-2 (TPH2) gene and quantified for peripheral 5-HTT mRNA expression in whole-blood samples. Several significant gene × environment interactions between 5-HT genotypes and cocaine use on WM emerged: in cocaine users, the long/long (5-HTTLPR), 9+10/9+10 (VNTR In2) and C/C (TPH2 rs1386497) genotypes were risk alleles for WM impairments, whereas in healthy controls these polymorphisms were associated with improved WM performance. Analogously, high 5-HTT mRNA levels were associated with worse executive WM performance in cocaine users but with increased performance in controls. These gene × environment interactions suggest that the 5-HT system has an important role in the development of cognitive deficits in chronic cocaine users. Hence, pharmacological compounds targeting 5-HT neurotransmission might be promising for the treatment of cognitive deficits in cocaine dependence.
Project description:Epigenetic mechanisms may moderate genetic and environmental risk (GxE) for mood disorders. We used an experimental rhesus macaque model of early life stress to test whether epigenetic regulation of serotonin transporter (5-HTT) may contribute to GxE interactions that influence behavior and emotion. We hypothesized that peripheral blood mononuclear cell (PBMC) DNA methylation within an 800 bp cytosine-phosphate-guanosine (CpG) island that overlaps with the 5-HTT transcription initiation start site, a hypothesized model of the same genomic region in brain tissue, would mediate or moderate the effects of early life stress and a functional 5-HTT promoter polymorphism (rh5-HTTLPR) on two outcomes: PBMC 5-HTT expression and behavioral stress reactivity. Eighty-seven infant rhesus macaques (3-4 months of age) were either mother reared in large social groups (n = 70) or nursery reared (n = 17). During a maternal/social separation, infants' blood was sampled and behavioral stress reactivity recorded. PBMC DNA and RNA samples were used to determine rh5-HTTLPR genotype, 5-HTT mRNA expression using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and 5-HTT CpG methylation status using sodium bisulfite pyrosequencing. Consistent with human data, carriers of the low-expressing rh5-HTTLPR alleles exhibited higher mean 5-HTT CpG methylation, which was associated with lower PBMC 5-HTT expression. Higher 5-HTT CpG methylation, but not rh5-HTTLPR genotype, exacerbated the effects of early life stress on behavioral stress reactivity in infants. 5-HTT CpG methylation may be an important regulator of 5-HTT expression early in development and may contribute to the risk for mood disorders observed in 'high-risk'5-HTTLPR carriers.
Project description:Human gene association studies have produced conflicting findings regarding the relationship between the 5-HT transporter (5-HTT) and anxiety. In the present study genetically modified mice were utilised to examine the effects of changes in 5-HTT expression on anxiety. In addition, the influence of 5-HTT expression on two innate "species-typical" behaviours (burrowing and marble burying) and body weight was explored. Across a range of models, 5-HTT overexpressing mice displayed reduced anxiety-like behaviour whilst 5-HTT knockout mice showed increased anxiety-like behaviour, compared to wildtype controls. In tests of species-typical behaviour 5-HTT overexpressing mice showed some facilitation whilst 5-HTT knockout mice were impaired. Reciprocal effects were also seen on body weight, as 5-HTT overexpressors were lighter and 5-HTT knockouts were heavier than wildtype controls. These findings show that variation in 5-HTT gene expression produces robust changes in anxiety and species-typical behaviour. Furthermore, the data add further support to findings that variation of 5-HTT expression in the human population is linked to changes in anxiety-related personality traits.