Enhanced activity of human serotonin transporter variants associated with autism

Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, TN 37232-8548, USA.
Philosophical Transactions of The Royal Society B Biological Sciences (Impact Factor: 7.06). 11/2008; 364(1514):163-73. DOI: 10.1098/rstb.2008.0143
Source: PubMed


Rare, functional, non-synonymous variants in the human serotonin (5-hydroxytryptamine, 5-HT) transporter (hSERT) gene (SLC6A4) have been identified in both autism and obsessive-compulsive disorder (OCD). Within autism, rare hSERT coding variants associate with rigid-compulsive traits, suggesting both phenotypic overlap with OCD and a shared relationship with disrupted 5-HT signalling. Here, we document functional perturbations of three of these variants: Ile425Leu; Phe465Leu; and Leu550Val. In transiently transfected HeLa cells, the three variants confer a gain of 5-HT transport phenotype. Specifically, enhanced SERT activity was also observed in lymphoblastoid lines derived from mutation carriers. In contrast to previously characterized Gly56Ala, where increased transport activity derives from catalytic activation, the three novel variants exhibit elevated surface density as revealed through both surface antagonist-binding and biotinylation studies. Unlike Gly56Ala, mutants Ile425Leu, Phe465Leu and Leu550Val retain a capacity for acute PKG and p38 MAPK regulation. However, both Gly56Ala and Ile425Leu demonstrate markedly reduced sensitivity to PP2A antagonists, suggesting that deficits in trafficking and catalytic modulation may derive from a common basis in perturbed phosphatase regulation. When expressed stably from the same genomic locus in CHO cells, both Gly56Ala and Ile425Leu display catalytic activation, accompanied by a striking loss of SERT protein.

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Available from: James S Sutcliffe, Jun 20, 2014
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    • "We also found that the most common of these variants, SERT Ala56, was significantly associated with rigid-compulsive behavior and sensory aversion. Lending credence to a functional contribution of the SERT variants identified, each of the variants was found to confer increased 5-HT transport activity in cell models relative to wildtype SERT [14,20,21]. In our study, the Ala56 variant showed an approximately 2:1 transmission bias to affected versus unaffected children [14]. "
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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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    • "Recent studies have revealed a central role for biogenic amine transporter function in neuropsychiatric disorders, including ADHD (Mazei-Robison et al., 2008) and autism (Prasad et al., 2009). "
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    ABSTRACT: Dopaminergic signaling and plasticity are essential to numerous CNS functions and pathologies, including movement, cognition, and addiction. The amphetamine- and cocaine-sensitive dopamine (DA) transporter (DAT) tightly controls extracellular DA concentrations and half-life. DAT function and surface expression are not static but are dynamically modulated by membrane trafficking. We recently demonstrated that the DAT C terminus encodes a PKC-sensitive internalization signal that also suppresses basal DAT endocytosis. However, the cellular machinery governing regulated DAT trafficking is not well defined. In work presented here, we identified the Ras-like GTPase, Rin (for Ras-like in neurons) (Rit2), as a protein that interacts with the DAT C-terminal endocytic signal. Yeast two-hybrid, GST pull down and FRET studies establish that DAT and Rin directly interact, and colocalization studies reveal that DAT/Rin associations occur primarily in lipid raft microdomains. Coimmunoprecipitations demonstrate that PKC activation regulates Rin association with DAT. Perturbation of Rin function with GTPase mutants and shRNA-mediated Rin knockdown reveals that Rin is critical for PKC-mediated DAT internalization and functional downregulation. These results establish that Rin is a DAT-interacting protein that is required for PKC-regulated DAT trafficking. Moreover, this work suggests that Rin participates in regulated endocytosis.
    Full-text · Article · Sep 2011 · The Journal of Neuroscience : The Official Journal of the Society for Neuroscience
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