Fifty years ago, increased whole-blood serotonin levels, or hyperserotonemia, first linked disrupted 5-HT homeostasis to Autism Spectrum Disorders (ASDs). The 5-HT transporter (SERT) gene (SLC6A4) has been associated with whole blood 5-HT levels and ASD susceptibility. Previously, we identified multiple gain-of-function SERT coding variants in children with ASD. Here we establish that transgenic mice expressing the most common of these variants, SERT Ala56, exhibit elevated, p38 MAPK-dependent transporter phosphorylation, enhanced 5-HT clearance rates and hyperserotonemia. These effects are accompanied by altered basal firing of raphe 5-HT neurons, as well as 5HT 1A and 5HT 2A receptor hypersensitivity. Strikingly, SERT Ala56 mice display alterations in social function, communication, and repetitive behavior. Our efforts provide strong support for the hypothesis that altered 5-HT homeostasis can impact risk for ASD traits and provide a model with construct and face validity that can support further analysis of ASD mechanisms and potentially novel treatments. development | monoamine | neurotransmitter A utism spectrum disorder (ASD) is a male-predominant disorder that is characterized by deficits in social interactions and communication, as well as repetitive behavior (1). Hyperserotonemia, or increased whole-blood serotonin [i.e., 5-hydroxytryptamine (5-HT)], is a well replicated biomarker that is present in approximately 30% of subjects with ASD (2, 3). Some data suggest an association of hyperserotonemia with stereotyped or self-injurious behavior, but results have been inconsistent (4, 5). Despite the high heritability of whole-blood 5-HT levels (6), a mechanistic connection between hyperserotonemia and specific components of the pathophysiology of ASD remains enigmatic. In blood, 5-HT is contained almost exclusively in platelets (7) that lack 5-HT biosynthetic capacity but accumulate the monoamine via the antidepressant-sensitive serotonin transporter (SERT; 5-HTT). A genome-wide study of whole-blood 5-HT as a quantitative trait found association with the SERT-encoding gene SLC6A4, as well as with ITGB3, which encodes the SERT-interacting protein integrin β3. In both cases, the strongest evidence for association was found in males (8-10). Linkage studies in ASD also implicate the 17q11.2 region containing SLC6A4, again with stronger evidence in males (11, 12).As common SLC6A4 variants are only modestly associated with ASD (13), we and our colleagues previously screened SLC6A4 for rare variants in multiplex families that demonstrate strong linkage to 17q11.2. In this effort, we identified five rare SERT coding variants, each of which confers increased 5-HT transport in transfected cells as well as in lymphoblasts derived from SERT variant-expressing probands (11,14,15). We found the most common of these variants, Ala56 (allele frequency in subjects of European ancestry of 0.5-1%), to be overtransmitted to autism probands, and to be associated with both rigid-compulsive behavior and sensory aversion (11,16). N...

The indoleamine 5-hydroxytryptamine (serotonin, 5-HT) 1 plays a pivotal, modulatory role in a variety of centrally controlled physiological processes, including respiration, arousal, aggression, and mood, and in the periphery supports gastrointestinal, platelet, and placental function (1). 5-HT is inactivated following vesicular release by a presynaptic, antidepressant-sensitive 5-HT transporter (SERT, 5-HTT), a member of the Na ϩ /Cl Ϫ -dependent solute transporter family (SLC6A4) (2-4). SERT knock-out mice display altered presynaptic 5-HT homeostasis, modified 5-HT receptor sensitivities, and stressdependent behavioral modulation as well as altered responses to psychostimulants (5). In humans, altered SERT gene expression and/or transport function have been linked to multiple disorders including autism, obsessive-compulsive disorder, depression, and suicide (6 -11).Previous studies have demonstrated that both genetic and posttranscriptional processes regulate SERT activity (12, 13). SERT activity in native cells and transfected models can be rapidly (in minutes) modulated by multiple signaling pathways (14, 15). Observations with transfected HEK cells expressing human SERT (hSERT) demonstrated that protein kinase C activators or protein phosphatase 1/2A inhibitors trigger hSERT phosphorylation and a parallel decrease in hSERT cell surface density, effects that can be attenuated by . Protein kinase A and protein kinase G (PKG) activation can also trigger SERT phosphorylation (17), although the functional consequences of these stimuli are only beginning to be appreciated. SERTs appear to form homomultimers at the plasma membrane (19) and also interact with a growing list of associated proteins, including syntaxin 1A (20 -22)

The essential contribution of the antidepressant-sensitive serotonin (5-HT) transporter SERT (which is encoded by the SLC6A4 gene) to platelet 5-HT stores suggests an important role of this transporter in platelet function. Here, using SERT-deficient mice, we have established a role for constitutive SERT expression in efficient ADPand thrombin-triggered platelet aggregation. Additionally, using pharmacological blockers of SERT and the vesicular monoamine transporter (VMAT), we have identified a role for ongoing 5-HT release and SERT activity in efficient human platelet aggregation. We have also demonstrated that fibrinogen, an activator of integrin αIIbβ3, enhances SERT activity in human platelets and that integrin αIIbβ3 interacts directly with the C terminus of SERT. Consistent with these findings, knockout mice lacking integrin β3 displayed diminished platelet SERT activity. Conversely, HEK293 cells engineered to express human SERT and an activated form of integrin β3 exhibited enhanced SERT function that coincided with elevated SERT surface expression. Our results support an unsuspected role of αIIbβ3/SERT associations as well as αIIbβ3 activation in control of SERT activity in vivo that may have broad implications for hyperserotonemia, cardiovascular disorders, and autism.