Human serotonin transporter variants display altered sensitivity to protein kinase G and p38 mitogen-activated protein kinase

Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, TN 37232-8548, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 09/2005; 102(32):11545-50. DOI: 10.1073/pnas.0501432102
Source: PubMed


Human serotonin [5-hydroxytryptamine (5-HT)] transporters (hSERT, 5HTT, and SLC6A4) inactivate 5-HT after release and are prominent targets for therapeutic intervention in mood, anxiety, and obsessive-compulsive disorders. Multiple hSERT coding variants have been identified, although to date no comprehensive functional analysis of these variants has been reported. We transfected hSERT or 10 hSERT coding variants and examined total and surface protein expression, antagonist recognition, and transporter modulation by posttranslational, regulatory pathways. Two variants, Pro339Leu and Ile425Val, demonstrated significant changes in surface expression supporting alterations in 5-HT transport capacity (V(max)). Regardless of basal transport activity, all SERT variants displayed a capacity for rapid, phorbol ester-triggered down-regulation. Remarkably, five variants (Thr4Ala, Gly56Ala, Glu215Lys, Lys605Asn, and Pro612Ser) demonstrated no capacity for 5-HT uptake stimulation after acute protein kinase G (PKG)/p38 mitogen-activated protein kinase (MAPK) activation. Epstein-Barr virus (EBV)-transformed lymphocytes natively expressing the most common of these variants (Gly56Ala) exhibited a similar loss of 5-HT uptake stimulation by PKG/p38 MAPK activators. HeLa cells transfected with the Gly56Ala variant demonstrated elevated basal phosphorylation and, unlike hSERT, could not be further phosphorylated after 8-bromo cGMP (8BrcGMP) treatments. These studies reveal cellular phenotypes associated with naturally occurring human SERT coding variants and suggest that altered transporter regulation by means of PKG/p38 MAPK-linked pathways may influence risk for disorders attributed to compromised 5-HT signaling.

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Available from: James S Sutcliffe
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    • "Hence SERT trafficking may be skewed in favor of SERT externalization in individuals showing elevated 5-HT blood levels, although the existence of additional factors cannot be excluded. The molecular mechanisms favouring SERT externalization involve different genes in males and females (Mei et al., 2007; Weiss et al., 2005), including for example common variants at ITGB3 (Napolioni et al., 2011; Weiss et al., 2004, 2005, 2006a, 2006b) and rare variants at SLC6A4 (Prasad et al., 2005; Veenstra-VanderWeele et al., 2012). It is also apparently modulated by sex hormones, since 5-HT blood levels are especially elevated in some autistic children, whereas after puberty this excess becomes less pronounced (McBride et al., 1998). "
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    ABSTRACT: Elevated blood serotonin (5-HT) levels were the first biomarker identified in autism research. Many studies have contrasted blood 5-HT levels in autistic patients and controls, but different measurement protocols, technologies, and biomaterials have been used through the years. We performed a systematic review and meta-analysis to provide an overall estimate of effect size and between-study heterogeneity, while verifying whether and to what extent different methodological approaches influence the strength of this association. Our literature search strategy identified 551 papers, from which 22 studies providing patient and control blood 5-HT values were selected for meta-analysis. Significantly higher 5-HT levels in autistic patients compared to controls were recorded both in whole blood (WB) [O.R.=4.6; (3.1–5.2); P=1.0×10−12], and in platelet-rich plasma (PRP) [O.R.=2.6 (1.8–3.9); P=2.7×10−7]. Predictably, studies measuring 5-HT levels in platelet-poor plasma (PPP) yielded no significant group difference [O.R.=0.54 (0.2–2-0); P=0.36]. Altogether, elevated 5-HT blood levels were recorded in 28.3% in WB and 22.5% in PRP samples of autistic individuals, as reported in 15 and 4 studies, respectively. Studies employing HPLC vs fluorometric assays yield similar cumulative effect sizes, but the former display much lower variability. In summary, despite some limitations mainly due to small study sample sizes, our results significantly reinforce the reliability of elevated 5-HT blood levels as a biomarker in ASD, providing practical indications potentially useful for its inclusion in multi-marker diagnostic panels for clinical use.
    Full-text · Article · Jun 2014 · European neuropsychopharmacology: the journal of the European College of Neuropsychopharmacology
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    • "The attenuation of serotonergic phenotypes in the B6 SERT Ala56 mice may account for the difference in findings from reports in the hSERT OE mouse. Alternatively, these discrepant findings could be due to the difference in mechanism between increased (hSERT OE) and dysregulated activity (SERT Ala56 knock-in) [20,21,23]. "
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    ABSTRACT: Previously, we identified multiple, rare serotonin (5-HT) transporter (SERT) variants in children with autism spectrum disorder (ASD). Although in our study the SERT Ala56 variant was over-transmitted to ASD probands, it was also seen in some unaffected individuals, suggesting that associated ASD risk is influenced by the epistatic effects of other genetic variation. Subsequently, we established that mice expressing the SERT Ala56 variant on a 129S6/S4 genetic background display multiple biochemical, physiological and behavioral changes, including hyperserotonemia, altered 5-HT receptor sensitivity, and altered social, communication, and repetitive behavior. Here we explore the effects of genetic background on SERT Ala56 knock-in phenotypes. To explore the effects of genetic background, we backcrossed SERT Ala56 mice on the 129 background into a C57BL/6 (B6) background to achieve congenic B6 SERT Ala56 mice, and assessed autism-relevant behavior, including sociability, ultrasonic vocalizations, and repetitive behavior in the home cage, as well as serotonergic phenotypes, including whole blood serotonin levels and serotonin receptor sensitivity. One consistent phenotype between the two strains was performance in the tube test for dominance, where mutant mice displayed a greater tendency to withdraw from a social encounter in a narrow tube as compared to wildtype littermate controls. On the B6 background, mutant pup ultrasonic vocalizations were significantly increased, in contrast to decreased vocalizations seen previously on the 129 background. Several phenotypes seen on the 129 background were reduced or absent when the mutation was placed on the B6 background, including hyperserotonemia, 5-HT receptor hypersensivity, and repetitive behavior. Our findings provide a cogent example of how epistatic interactions can modulate the impact of functional genetic variation and suggest that some aspects of social behavior may be especially sensitive to changes in SERT function. Finally, these results provide a platform for the identification of genes that may modulate the risk of ASD in humans.
    Full-text · Article · Oct 2013 · Molecular Autism
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    • "Further support for a role of these variants in ASD comes from studies reporting an Ile425Val variant that segregated in pedigrees harboring multiple psychiatric phenotypes, with Asperger syndrome (an ASD), OCD, and other anxiety disorders being the most prominent [20-22]. Functional characterization of these SERT variants revealed that each elevated 5-HT transport function, as well as altered protein kinase G (PKG) and p38 mitogen activated protein kinase (MAPK) regulation [23,24]. Our characterization of one of these variants (Gly56Ala) in knock-in transgenic mice revealed elevated 5-HT clearance and p38 MAPK-dependent transporter hyperphosphorylation in vivo accompanied by deficits in the three classical behavioral domains associated with ASD [25]. "
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    ABSTRACT: Recent discoveries highlight rare genetic variation as an important class of autism spectrum disorder (ASD) risk factors, and that such variants can implicate biological networks for further investigation. Altered serotonin (5-HT) signaling has been implicated in ASD for over 50 years, and we and others have identified multiple, rare, ASD-associated variants in the 5-HT transporter (SERT, SLC6A4) gene that lead to elevated 5-HT re-uptake and perturbed regulation. We hypothesized that loci encoding SERT regulatory proteins harbor genetic variants that impact SERT function and/or regulation and therefore could contribute to ASD risk. The adenosine A3 receptor (A3AR) regulates SERT via protein kinase G (PKG) and p38 mitogen-activated protein kinase (p38 MAPK) signaling pathways leading to enhanced SERT surface expression and catalytic activity, respectively. To test our SERT network hypothesis, we asked whether a relative increase of rare functional variants in the A3AR gene (ADORA3) was present in cases vs. controls. Discovery Sanger sequencing of ADORA3 exons and flanking DNA in a case-control sample, and subsequent analysis of a comparison sample using whole exome sequence data were conducted to test for increased functional variants in cases. We evaluated the functional impact of two variants from the discovery sample on A3AR signaling and SERT activity. Sequencing discovery showed an overall increase in rare coding variants in cases vs. controls (P=0.013). While a comparison sample from exome sequence did not show a significant enrichment (P=0.071), combined analysis strengthened evidence for association of rare, functional variants in ASD (P=0.0025). Two variants discovered in ASD cases (Leu90Val and Val171Ile) lie in or near the ligand-binding pocket, and Leu90Val was enriched individually in cases (P=0.040). In vitro analysis of cells expressing Val90 A3AR revealed elevated basal cGMP levels compared with cells expressing the wildtype receptor. Additionally, the specific A3AR agonist N6-(3-iodobenzyl)-N-methyl-5'-carbamoyladenosine (IB-MECA) induced increased cGMP levels across the full time course studied in Val90 A3AR cells, as compared with the wildtype receptor expressing cells. In Val90 A3AR/SERT co-transfected cells, IB-MECA stimulation elevated SERT activity over that seen with the wildtype receptor, with a delayed recovery of 5-HT uptake activity to baseline levels. By comparison, the Ile171 A3AR variant was unable to support IB-MECA stimulation of SERT. Although both Val90 and Ile171 were present in greater numbers in these ASD cases, segregation analysis in carrier families showed incomplete penetrance, consistent with other documented rare ASD risk alleles. Our results validate the hypothesis that the SERT regulatory network harbors rare, functional variants that impact SERT activity and regulation in ASD, and encourages further investigation of this network as a site for additional functional variation that may impact ASD risk.
    Full-text · Article · Aug 2013 · Molecular Autism
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