Pitavastatin attenuates hypercholesterolemia-induced decline in serotonin transporter availability.

INTRODUCTION: Hypercholesterolemia is associated with increased inflammation and impaired serotonin neurotransmission, potentially contributing to depressive symptoms. However, the role of statins, particularly pitavastatin, in modulating serotonin transporter (SERT) function within this context remains underexplored. This study aimed to investigate whether pitavastatin counteracts the neurobiological effects of hypercholesterolemia. METHODS: Low-density lipoprotein receptor knockout (LDLR-/-) mice on a C57BL/6 background were assigned to three groups: a control group fed a standard chow diet, a group fed a high-fat diet (HFD), and a third group fed a high-fat diet supplemented with pitavastatin (HFD + Pita). We evaluated the effects of HFD with or without pitavastatin on lipid profiles, inflammatory markers, and SERT availability using small-animal positron emission tomography (PET) scans with the radioligand 4-[18F]-ADAM over a 20-week period. RESULTS: Pitavastatin treatment in HFD-fed mice significantly reduced both total cholesterol and LDL cholesterol levels in HFD-fed mice compared to those on HFD alone. Elevated inflammatory markers such as IL-1α, MCP-1/CCL2, and TNF-α in HFD mice were notably decreased in the HFD + Pita group. PET scans showed reduced SERT availability in the brains of HFD mice; however, pitavastatin improved this in brain regions associated with mood regulation, suggesting enhanced serotonin neurotransmission. Additionally, the sucrose preference test showed a trend towards increased preference in the HFD + Pita group compared to the HFD group, indicating a potential reduction in depressive-like behavior. CONCLUSION: Our findings demonstrate that pitavastatin not only lowers cholesterol and reduces inflammation but also enhances SERT availability, suggesting a potential role in alleviating depressive symptoms associated with hypercholesterolemia. These results highlight the multifaceted benefits of pitavastatin, extending beyond its lipid-lowering effects to potentially improving mood regulation and neurotransmitter function.

Serotonin transporter knockout in rats reduces beta- and gamma-band functional connectivity between the orbitofrontal cortex and amygdala during auditory discrimination.

The orbitofrontal cortex and amygdala collaborate in outcome-guided decision-making through reciprocal projections. While serotonin transporter knockout (SERT-/-) rodents show changes in outcome-guided decision-making, and in orbitofrontal cortex and amygdala neuronal activity, it remains unclear whether SERT genotype modulates orbitofrontal cortex-amygdala synchronization. We trained SERT-/- and SERT+/+ male rats to execute a task requiring to discriminate between two auditory stimuli, one predictive of a reward (CS+) and the other not (CS-), by responding through nose pokes in opposite-side ports. Overall, task acquisition was not influenced by genotype. Next, we simultaneously recorded local field potentials in the orbitofrontal cortex and amygdala of both hemispheres while the rats performed the task. Behaviorally, SERT-/- rats showed a nonsignificant trend for more accurate responses to the CS-. Electrophysiologically, orbitofrontal cortex-amygdala synchronization in the beta and gamma frequency bands during response selection was significantly reduced and associated with decreased hubness and clustering coefficient in both regions in SERT-/- rats compared to SERT+/+ rats. Conversely, theta synchronization at the time of behavioral response in the port associated with reward was similar in both genotypes. Together, our findings reveal the modulation by SERT genotype of the orbitofrontal cortex-amygdala functional connectivity during an auditory discrimination task.

DNA Sequence Variations Affecting Serotonin Transporter Transcriptional Regulation and Activity: Do They Impact Alcohol Addiction?

Genetic features of alcohol dependence have been extensively investigated in recent years. A large body of studies has underlined the important role of genetic variants not only in metabolic pathways but also in the neurobiology of alcohol dependence, mediated by the neuronal circuits regulating reward and craving. Serotonin transporter (5-HTT), encoded by the SLC6A4 gene (Solute carrier family 6-neurotransmitter transporter-member 4), is targeted by antidepressant drugs such as selective serotonin reuptake inhibitors (SSRIs) and plays a pivotal role in serotoninergic transmission; it has been associated with psychiatric diseases and alcohol dependence. Transcriptional regulation and expression of 5-HTT depend not only on epigenetic modifications, among which DNA methylation (CpG and non-CpG) is primarily involved, but also on sequence variations occurring in intron/exon regions and in untranslated regions in 5' and 3', being the first sequences important for the splicing machinery and the last for the binding of transcription factors and micro RNAs. This work intends to shed light on the role of sequence variations known to affect the expression or function of 5-HTT in alcohol-dependent individuals. We found a statistically significant difference in the allelic (p = 0.0083) and genotypic (p = 0.0151) frequencies of the tri-allelic polymorphism, with higher function alleles and genotypes more represented in the control population. Furthermore, we identified three haplotypes more frequent in subjects with AUD (p < 0.0001) and one more frequent in the control population (p < 0.0001). The results obtained for the tri-allelic polymorphism in alcohol dependence confirm what is already present in part of the literature. The role of haplotypes requires further studies to be clarified.

Morphometric analysis and functional insights into the serotonergic system of Girardia tigrina (Tricladida, Platyhelminthes).

Using immunocytochemistry, serotonergic nerve elements were documented in the nervous system of the planarian Girardia tigrina. Serotonin-immunopositive components were observed in the brain, ventral, dorsal and longitudinal nerve cords, transverse nerve commissures connecting the nerve cords, and in the nerve plexus. Whole-mount preparations of G. tigrina were analyzed by fluorescent and confocal laser scanning microscopy. An essential quantitative morphometric measurement of serotonin-immunopositive structures was conducted in three body regions (anterior, middle, and posterior) of the planarian. The number of serotonin neurons was maximal in the head region. The ventral nerve cords gradually decreased in thickness from anterior to posterior body ends. Physiological action of exogenously applied serotonin was studied in G. tigrina for the first time. It was found that serotonin (0.1 and 1 µmol L-1) accelerated eye regeneration. The transcriptome sequencing performed for the first time for the planarian G. tigrina revealed the transcripts of the tryptophan hydroxylase (trph), amino acid decarboxylase (aadc) and serotonin transporter (sert) genes. The data obtained indicate the presence of the components of serotonin pathway in G. tigrina. The identified transcripts can take part in serotonin turnover and participate in the realization of biological effects of serotonin in planarians, associated with eyes regeneration and differentiation.

Evolution of threat response-related polymorphisms at the SLC6A4 locus in callitrichid primates.

Variation in an upstream repetitive region at the SLC6A4 locus, which encodes the serotonin transporter, is associated with anxiety-related behaviour in a few primate species, including humans and rhesus macaques, and has been suggested to be related to ecological adaptability among macaques. In this study, we investigate evolution of SLC6A4 polymorphisms associated with anxiety-related behaviour in common marmosets (Callithrix jacchus). Assaying variation in the SLC6A4 repeat region across 14 species in eight genera of callitrichid primates (marmosets and tamarins), we find large interspecific variation in the number of repeats present (24-43). The black tufted-ear marmoset (C. penicillata) has sequence polymorphisms similar to those found in the common marmoset, which is its sister species, and no other species has intraspecific variation at these sites. We conclude that, similar to humans and macaques, the functional polymorphism at SLC6A4 in common marmosets has a recent evolutionary origin, and that the anxiety-related allele is evolutionarily derived. Common/black tufted-ear marmosets and rhesus/bonnet macaques share high ecological adaptability and behavioural flexibility that we propose may be related to the maintenance of the polymorphism.

Oligomerization of Monoamine Transporters.

Transporters of the monoamine transporter (MAT) family regulate the uptake of important neurotransmitters like dopamine, serotonin, and norepinephrine. The MAT family functions using the electrochemical gradient of ions across the membrane and comprises three transporters, dopamine transporter (DAT), serotonin transporter (SERT), and norepinephrine transporter (NET). MAT transporters have been observed to exist in monomeric states to higher-order oligomeric states. Structural features, allosteric modulation, and lipid environment regulate the oligomerization of MAT transporters. NET and SERT oligomerization are regulated by levels of PIP2 present in the membrane. The kink present in TM12 in the MAT family is crucial for dimer interface formation. Allosteric modulation in the dimer interface hinders dimer formation. Oligomerization also influences the transporters' function, trafficking, and regulation. This chapter will focus on recent studies on monoamine transporters and discuss the factors affecting their oligomerization and its impact on their function.

Connection Failure: Differences in White Matter Microstructure Are Associated with 5-HTTLPR but Not with Risk Seeking for Losses.

S/S carriers of 5-HTTLPR have been found to be more risk seeking for losses compared to L/L carriers. This finding may be the result of reduced top-down control from the frontal cortex due to altered signal pathways involving the amygdala and ventral striatum. The serotonergic system is known to be involved in neurodevelopment and neuroplasticity. Therefore, the aim of this study was to investigate whether structural differences in white matter can explain the differences in risk-seeking behaviour. Lower structural connectivity in S/S compared to L/L carriers and a negative relationship between risk seeking for losses and connectivity were assumed. Diffusion-weighted imaging was used to compute diffusion parameters for the frontostriatal and uncinate tract in 175 genotyped individuals. The results showed no significant relationship between diffusion parameters and risk seeking for losses. Furthermore, we did not find significant differences in diffusion parameters of the S/S vs. L/L group. There were only group differences in the frontostriatal tract showing stronger structural connectivity in the S/L group, which is also reflected in the whole brain approach. Therefore, the data do not support the hypothesis that the association between 5-HTTLPR and risk seeking for losses is related to differences in white matter pathways implicated in decision-making.

Non-Synonymous Substitutions in Cadherin 13, Solute Carrier Family 6 Member 4, and Monoamine Oxidase A Genes are Associated with Personality Traits in Thoroughbred Horses.

Retraining retired racehorses for various purposes can help correct behavioral issues. However, ensuring efficiency and preventing accidents present global challenges. Based on the hypothesis that a simple personality assessment could help address these challenges, the present study aimed to identify genetic markers associated with personality. Eight genes were selected from 18 personality-related candidate genes that are orthologs of human personality genes, and their association with personality was verified based on actual behavior. A total of 169 Thoroughbred horses were assessed for their tractability (questionnaire concerning tractability in 14 types of situations and 3 types of impressions) during the training process. Personality factors were extracted from the data using principal component analysis and analyzed for their association with single nucleotide variants as non-synonymous substitutions in the target genes. Three genes, CDH13, SLC6A4, and MAOA, demonstrated significant associations based on simple linear regression, marking the identification of these genes for the first time as contributors to temperament in Thoroughbred horses. All these genes, as well as the previously identified HTR1A, are involved in the serotonin neurotransmitter system, suggesting that the tractability of horses may be correlated with their social personality. Assessing the genotypes of these genes before retraining is expected to prevent problems in the development of a racehorse's second career and shorten the training period through individual customization of training methods, thereby improving racehorse welfare.

Microbiota influence on behavior: Integrative analysis of serotonin metabolism and behavioral profile in germ-free mice.

Previous studies on germ-free (GF) animals have described altered anxiety-like and social behaviors together with dysregulations in brain serotonin (5-HT) metabolism. Alterations in circulating 5-HT levels and gut 5-HT metabolism have also been reported in GF mice. In this study, we conducted an integrative analysis of various behaviors as well as markers of 5-HT metabolism in the brain and along the GI tract of GF male mice compared with conventional (CV) ones. We found a strong decrease in locomotor activity, accompanied by some signs of increased anxiety-like behavior in GF mice compared with CV mice. Brain gene expression analysis showed no differences in HTR1A and TPH2 genes. In the gut, we found decreased TPH1 expression in the colon of GF mice, while it was increased in the cecum. HTR1A expression was dramatically decreased in the colon, while HTR4 expression was increased both in the cecum and colon of GF mice compared with CV mice. Finally, SLC6A4 expression was increased in the ileum and colon of GF mice compared with CV mice. Our results add to the evidence that the microbiota is involved in regulation of behavior, although heterogeneity among studies suggests a strong impact of genetic and environmental factors on this microbiota-mediated regulation. While no impact of GF status on brain 5-HT was observed, substantial differences in gut 5-HT metabolism were noted, with tissue-dependent results indicating a varying role of microbiota along the GI tract.

Genetic polymorphisms in the 5-HT and endocannabinoid systems moderate the association between childhood trauma and burnout in the general occupational population.

BACKGROUND: Interactions between the serotonin (5-HT) and endocannabinoid (eCB) systems have been reported in the psychopathology of stress-related symptoms, while their interplay in regulating the relationship between childhood trauma and burnout remains unclear. In this study, we investigated the interaction of childhood trauma with genetic polymorphisms in these two systems in predicting burnout. METHODS: Burnout, childhood trauma, and job stress were assessed using rating scales in 992 general occupational individuals. Genetic polymorphisms including HTR2A rs6313, 5-HTT rs6354 and FAAH rs324420, were genotyped. Linear hierarchical regression analysis and PROCESS macro in SPSS were used to examine two- and three-way interactions. RESULTS: There were significant interactions of job stress × HTR2A rs6313 and childhood abuse × FAAH rs324420 on reduced personal accomplishment. Moreover, we found significant three-way interactions of childhood abuse × FAAH rs324420 × HTR2A rs6313 on cynicism and reduced personal accomplishment, childhood abuse × FAAH rs324420 × 5-HTT rs6354 on emotional exhaustion, and childhood neglect × FAAH rs324420 × 5-HTT rs6354 on reduced personal accomplishment. These results suggest that the FAAH rs324420 A allele carriers, when with some specific genetic polymorphisms of 5-HT system, would show more positive associations between childhood trauma and burnout. CONCLUSIONS: Genetic polymorphisms in the 5-HT and eCB systems may jointly moderate the impact of childhood trauma on burnout.

Long-term Environmental Enrichment Normalizes Schizophrenia-like Abnormalities and Promotes Hippocampal Slc6a4 Promoter Demethylation in Mice Submitted to a Two-hit Model.

Here, we explored the impact of prolonged environmental enrichment (EE) on behavioral, neurochemical, and epigenetic changes in the serotonin transporter gene in mice subjected to a two-hit schizophrenia model. The methodology involved administering the viral mimetic PolyI:C to neonatal Swiss mice as a first hit during postnatal days (PND) 5-7, or a sterile saline solution as a control. At PND21, mice were randomly assigned either to standard environment (SE) or EE housing conditions. Between PND35-44, the PolyI:C-treated group was submitted to various unpredictable stressors, constituting the second hit. Behavioral assessments were conducted on PND70, immediately after the final EE exposure. Following the completion of behavioral assessments, we evaluated the expression of proteins in the hippocampus that are indicative of microglial activation, such as Iba-1, as well as related to neurogenesis, including doublecortin (Dcx). We also performed methylation analysis on the serotonin transporter gene (Slc6a4) to investigate alterations in serotonin signaling. The findings revealed that EE for 50 days mitigated sensorimotor gating deficits and working memory impairments in two-hit mice and enhanced their locomotor and exploratory behaviors. EE also normalized the overexpression of hippocampal Iba-1 and increased the expression of hippocampal Dcx. Additionally, we observed hippocampal demethylation of the Slc6a4 gene in the EE-exposed two-hit group, indicating epigenetic reprogramming. These results contribute to the growing body of evidence supporting the protective effects of long-term EE in counteracting behavioral disruptions caused by the two-hit schizophrenia model, pointing to enhanced neurogenesis, diminished microglial activation, and epigenetic modifications of serotonergic pathways as underlying mechanisms.

No association between peripheral serotonin-gene-related DNA methylation and brain serotonin neurotransmission in the healthy and depressed state.

BACKGROUND: Methylation of serotonin-related genes has been proposed as a plausible gene-by-environment link which may mediate environmental stress, depressive and anxiety symptoms. DNA methylation is often measured in blood cells, but little is known about the association between this peripheral epigenetic modification and brain serotonergic architecture. Here, we evaluated the association between whole-blood-derived methylation of four CpG sites in the serotonin transporter (SLC6A4) and six CpG sites of the tryptophan hydroxylase 2 (TPH2) gene and in-vivo brain levels of serotonin transporter (5-HTT) and serotonin 4 receptor (5-HT4) in a cohort of healthy individuals (N = 254) and, for 5-HT4, in a cohort of unmedicated patients with depression (N = 90). To do so, we quantified SLC6A4/TPH2 methylation using bisulfite pyrosequencing and estimated brain 5-HT4 and 5-HTT levels using positron emission tomography. In addition, we explored the association between SLC6A4 and TPH2 methylation and measures of early life and recent stress, depressive and anxiety symptoms on 297 healthy individuals. RESULTS: We found no statistically significant association between peripheral DNA methylation and brain markers of serotonergic neurotransmission in patients with depression or in healthy individuals. In addition, although SLC6A4 CpG2 (chr17:30,236,083) methylation was marginally associated with the parental bonding inventory overprotection score in the healthy cohort, statistical significance did not remain after accounting for blood cell heterogeneity. CONCLUSIONS: We suggest that findings on peripheral DNA methylation in the context of brain serotonin-related features should be interpreted with caution. More studies are needed to rule out a role of SLC6A4 and TPH2 methylation as biomarkers for environmental stress, depressive or anxiety symptoms.

Allosteric Inhibition and Pharmacochaperoning of the Serotonin Transporter by the Antidepressant Drugs Trazodone and Nefazodone.

The antidepressants trazodone and nefazodone were approved some 4 and 3 decades ago, respectively. Their action is thought to be mediated, at least in part, by inhibition of the serotonin transporter [SERT/solute carrier (SLC)-6A4]. Surprisingly, their mode of action on SERT has not been characterized. Here, we show that, similar to the chemically related drug vilazodone, trazodone and nefazodone are allosteric ligands: trazodone and nefazodone inhibit uptake by and transport-associated currents through SERT in a mixed-competitive and noncompetitive manner, respectively. Contrary to noribogaine and its congeners, all three compounds preferentially interact with the Na+-bound outward-facing state of SERT. Nevertheless, they act as pharmacochaperones and rescue the folding-deficient variant SERT-P601A/G602A. The vast majority of disease-associated point mutations of SLC6 family members impair folding of the encoded transporter proteins. Our findings indicate that their folding defect can be remedied by targeting allosteric sites on SLC6 transporters. SIGNIFICANCE STATEMENT: The serotonin transporter is a member of the solute carrier-6 family and is the target of numerous antidepressants. Trazodone and nefazodone have long been used as antidepressants. Here, this study shows that their inhibition of the serotonin transporter digressed from the competitive mode seen with other antidepressants. Trazodone and nefazodone rescued a folding-deficient variant of the serotonin transporter. This finding demonstrates that folding defects of mutated solute carrier-6 family members can also be corrected by allosteric ligands.

Modulation of Serotonin-Related Genes by Extracellular Vesicles of the Probiotic Escherichia coli Nissle 1917 in the Interleukin-1ß-Induced Inflammation Model of Intestinal Epithelial Cells.

Inflammatory bowel disease (IBD) is a chronic inflammatory condition involving dysregulated immune responses and imbalances in the gut microbiota in genetically susceptible individuals. Current therapies for IBD often have significant side-effects and limited success, prompting the search for novel therapeutic strategies. Microbiome-based approaches aim to restore the gut microbiota balance towards anti-inflammatory and mucosa-healing profiles. Extracellular vesicles (EVs) from beneficial gut microbes are emerging as potential postbiotics. Serotonin plays a crucial role in intestinal homeostasis, and its dysregulation is associated with IBD severity. Our study investigated the impact of EVs from the probiotic Nissle 1917 (EcN) and commensal E. coli on intestinal serotonin metabolism under inflammatory conditions using an IL-1ß-induced inflammation model in Caco-2 cells. We found strain-specific effects. Specifically, EcN EVs reduced free serotonin levels by upregulating SERT expression through the downregulation of miR-24, miR-200a, TLR4, and NOD1. Additionally, EcN EVs mitigated IL-1ß-induced changes in tight junction proteins and oxidative stress markers. These findings underscore the potential of postbiotic interventions as a therapeutic approach for IBD and related pathologies, with EcN EVs exhibiting promise in modulating serotonin metabolism and preserving intestinal barrier integrity. This study is the first to demonstrate the regulation of miR-24 and miR-200a by probiotic-derived EVs.

Serotonin 2C receptor in a rat model of temporal lobe epilepsy: From brainstem expression to pharmacological blockade in relation to ventilatory function.

Because of its involvement in breathing control and neuronal excitability, dysregulation of the serotonin (5-HT) 2C receptor (5-HT2C) might play a key role in sudden unexpected death in epilepsy. Seizure-induced respiratory arrest is thus prevented by a 5-HT2B/C agonist in different seizure model. However, the specific contribution of 5-HT2C in chronic epilepsy-related respiratory dysfunction remains unknown. In a rat model of temporal lobe epilepsy (EPI rats), in which we previously reported interictal respiratory dysfunctions and a reduction of brainstem 5-HT tone, quantitative reverse transcriptase polymerase chain reaction showed overexpression of TPH2 (5-HT synthesis enzyme), SERT (5-HT reuptake transporter), and 5-HT2C transcript levels in the brainstem of EPI rats, and of RNA-specific adenosine deaminase (ADAR1, ADAR2) involved in the production of 5-HT2C isoforms. Interictal ventilation was assessed with whole-body plethysmography before and 2 h after administration of SB242084 (2 mg/kg), a specific antagonist of 5-HT2C. As expected, SB242084 administration induced a progressive decrease in ventilatory parameters and an alteration of breathing stability in both control and EPI rats. However, the size of the SB242084 effect was lower in EPI rats than in controls. Increased 5-HT2C gene expression in the brainstem of EPI rats could be part of a compensatory mechanism against epilepsy-related low 5-HT tone and expression of 5-HT2C isoforms for which 5-HT affinity might be lower.

Alterations in the brain serotonin system and serotonin-regulated behavior during aging in zebrafish males and females.

The brain serotonin (5-HT) system performs a neurotrophic function and supports the plasticity of the nervous system, while its age-related changes can increase the risk of senile neurodegeneration. Zebrafish brain is highly resistant to damage and neurodegeneration due to its high regeneration potential and it is a promising model object in searching for molecular factors preventing age-related neurodegeneration. In the present study alterations in 5-HT-related behavior in the home tank and the novel tank diving test, as well as 5-HT, 5-HIAA levels, tryptophan hydroxylase (TPH), monoamine oxidase (MAO) activity and the expression of genes encoding TPH, MAO, 5-HT transporter and 5-HT receptors in the brain of 6, 12, 24 and 36 month old zebrafish males and females are investigated. Marked sexual dimorphism in the locomotor activity in the novel tank test is revealed: females of all ages move slower than males. No sexual dimorphism in 5-HT-related traits is observed. No changes in 5-HT and 5-HIAA levels in zebrafish brain during aging is observed. At the same time, the aging is accompanied by a decrease in the locomotor activity, TPH activity, tph2 and htr1aa genes expression as well as an increase in the MAO activity and slc6a4a gene expression in their brain. These results indicate that the brain 5-HT system in zebrafish is resistant to age-related alterations.

Gene-Environment Interactions in Irrational Beliefs: The Roles of Childhood Adversity and Multiple Candidate Genes.

Cognitive behavioral therapy is based on the view that maladaptive thinking is the causal mechanism of mental disorders. While this view is supported by extensive evidence, very limited work has addressed the factors that contribute to the development of maladaptive thinking. The present study aimed to uncover interactions between childhood maltreatment and multiple genetic differences in irrational beliefs. Childhood maltreatment and irrational beliefs were assessed using multiple self-report instruments in a sample of healthy volunteers (N = 452). Eighteen single-nucleotide polymorphisms were genotyped in six candidate genes related to neurotransmitter function (COMT; SLC6A4; OXTR), neurotrophic factors (BDNF), and the hypothalamic-pituitary-adrenal axis (NR3C1; CRHR1). Gene-environment interactions (G×E) were first explored in models that employed one measure of childhood maltreatment and one measure of irrational beliefs. These effects were then followed up in models in which either the childhood maltreatment measure, the irrational belief measure, or both were substituted by parallel measures. Consistent results across models indicated that childhood maltreatment was positively associated with irrational beliefs, and these relations were significantly influenced by COMT rs165774 and OXTR rs53576. These results remain preliminary until independent replication, but they represent the best available evidence to date on G×E in a fundamental mechanism of psychopathology.

A Conserved Intramolecular Ion-Pair Plays a Critical but Divergent Role in Regulation of Dimerization and Transport Function among the Monoamine Transporters.

The monoamine transporters, including the serotonin transporter (SERT), dopamine transporter (DAT), and norepinephrine transporter (NET), are the therapeutic targets for the treatment of many neuropsychiatric disorders. Despite significant progress in characterizing the structures and transport mechanisms of these transporters, the regulation of their transport functions through dimerization or oligomerization remains to be understood. In the present study, we identified a conserved intramolecular ion-pair at the third extracellular loop (EL3) connecting TM5 and TM6 that plays a critical but divergent role in the modulation of dimerization and transport functions among the monoamine transporters. The disruption of the ion-pair interactions by mutations induced a significant spontaneous cross-linking of a cysteine mutant of SERT and an increase in cell surface expression but with an impaired specific transport activity. On the other hand, similar mutations of the corresponding ion-pair residues in both DAT and NET resulted in an opposite effect on their oxidation-induced dimerization, cell surface expression, and transport function. Reversible biotinylation experiments indicated that the ion-pair mutations slowed down the internalization of SERT but stimulated the internalization of DAT. In addition, cysteine accessibility measurements for monitoring SERT conformational changes indicated that substitution of the ion-pair residues resulted in profound effects on the rate constants for cysteine modification in both the extracellular and cytoplasmatic substrate permeation pathways. Furthermore, molecular dynamics simulations showed that the ion-pair mutations increased the interfacial interactions in a SERT dimer but decreased it in a DAT dimer. Taken together, we propose that the transport function is modulated by the equilibrium between monomers and dimers on the cell surface, which is regulated by a potential compensatory mechanism but with different molecular solutions among the monoamine transporters. The present study provided new insights into the structural elements regulating the transport function of the monoamine transporters through their dimerization.

Serotonin transporter knockdown relieves depression-like behavior and ethanol-induced CPP in mice after chronic social defeat stress.

Patients with stress-triggered major depression disorders (MDD) can often seek comfort or temporary relief through alcohol consumption, as they may turn to it as a means of self-medication or coping with overwhelming emotions. The use of alcohol as a coping mechanism for stressful events can escalate, fostering a cycle where the temporary relief it provides from depression can deepen into alcohol dependence, exacerbating both conditions. Although, the specific mechanisms involved in stress-triggered alcohol dependence and MDD comorbidities are not well understood, a large body of literature suggests that the serotonin transporter (SERT) plays a critical role in these abnormalities. To further investigate this hypothesis, we used a lentiviral-mediated knockdown approach to examine the role of hippocampal SERT knockdown in social defeat stress-elicited depression like behavior and ethanol-induced place preference (CPP). The results showed that social defeat stress-pro depressant effects were reversed following SERT knockdown demonstrated by increased sucrose preference, shorter latency to feed in the novelty suppressed feeding test, and decreased immobility time in the tail suspension and forced swim tests. Moreover, and most importantly, social stress-induced ethanol-CPP acquisition and reinstatement were significantly reduced following hippocampal SERT knockdown using short hairpin RNA shRNA-expressing lentiviral vectors. Finally, we confirmed that SERT hippocampal mRNA expression correlated with measures of depression- and ethanol-related behaviors by Pearson's correlation analysis. Taken together, our data suggest that hippocampal serotoninergic system is involved in social stress-triggered mood disorders as well as in the acquisition and retrieval of ethanol contextual memory and that blockade of this transporter can decrease ethanol rewarding properties.

Identification of the potassium-binding site in serotonin transporter.

Clearance of serotonin (5-hydroxytryptamine, 5-HT) from the synaptic cleft after neuronal signaling is mediated by serotonin transporter (SERT), which couples this process to the movement of a Na+ ion down its chemical gradient. After release of 5-HT and Na+ into the cytoplasm, the transporter faces a rate-limiting challenge of resetting its conformation to be primed again for 5-HT and Na+ binding. Early studies of vesicles containing native SERT revealed that K+ gradients can provide an additional driving force, via K+ antiport. Moreover, under appropriate conditions, a H+ ion can replace K+. Intracellular K+ accelerates the resetting step. Structural studies of SERT have identified two binding sites for Na+ ions, but the K+ site remains enigmatic. Here, we show that K+ antiport can drive substrate accumulation into vesicles containing SERT extracted from a heterologous expression system, allowing us to study the residues responsible for K+ binding. To identify candidate binding residues, we examine many cation binding configurations using molecular dynamics simulations, predicting that K+ binds to the so-called Na2 site. Site-directed mutagenesis of residues in this site can eliminate the ability of both K+ and H+ to drive 5-HT accumulation into vesicles and, in patch clamp recordings, prevent the acceleration of turnover rates and the formation of a channel-like state by K+ or H+. In conclusion, the Na2 site plays a pivotal role in orchestrating the sequential binding of Na+ and then K+ (or H+) ions to facilitate 5-HT uptake in SERT.