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...

Salvinorin A (SalA), a selective κ-opioid receptor (KOR) agonist, produces dysphoria and pro-depressant like effects. These actions have been attributed to inhibition of striatal dopamine release. The dopamine transporter (DAT) regulates dopamine transmission via uptake of released neurotransmitter. KORs are apposed to DAT in dopamine nerve terminals suggesting an additional target by which SalA modulates dopamine transmission. SalA produced a concentration-dependent, nor-binaltorphimine (BNI)- and pertussis toxin-sensitive increase of ASP+ accumulation in EM4 cells coexpressing myc-KOR and YFP-DAT, using live cell imaging and the fluorescent monoamine transporter substrate, trans 4-(4-(dimethylamino)-styryl)-N-methylpyridinium) (ASP+). Other KOR agonists also increased DAT activity that was abolished by BNI pretreatment. While SalA increased DAT activity, SalA treatment decreased serotonin transporter (SERT) activity and had no effect on norepinephrine transporter (NET) activity. In striatum, SalA increased the Vmax for DAT mediated DA transport and DAT surface expression. SalA up-regulation of DAT function is mediated by KOR activation and the KOR-linked extracellular signal regulated kinase-½ (ERK1/2) pathway. Co-immunoprecipitation and BRET studies revealed that DAT and KOR exist in a complex. In live cells, DAT and KOR exhibited robust FRET signals under basal conditions. SalA exposure caused a rapid and significant increase of the FRET signal. This suggests that the formation of KOR and DAT complexes is promoted in response to KOR activation. Together, these data suggest that enhanced DA transport and decreased DA release resulting in decreased dopamine signaling may contribute to the dysphoric and pro-depressant like effects of SalA and other KOR agonists.

Brain-derived neurotrophic factor (BDNF) is critical in synaptic plasticity and in the survival and function of midbrain dopamine neurons. In this study, we assessed the effects of a partial genetic deletion of BDNF on motor function and dopamine (DA) neurotransmitter measures by comparing Bdnf +/− with wildtype mice (WT) at different ages. Bdnf +/− and WT mice had similar body weights until 12 months of age; however, at 21 months, Bdnf +/− mice were significantly heavier than WT mice. Horizontal and vertical motor activity was reduced for Bdnf +/− compared to WT mice, but was not influenced by age. Performance on an accelerating rotarod declined with age for both genotypes and was exacerbated for Bdnf +/− mice. Body weight did not correlate with any of the three behavioral measures studied. Dopamine neurotransmitter markers indicated no genotypic difference in striatal tyrosine hydroxylase, DA transporter (DAT) or vesicular monoamine transporter 2 (VMAT2) immunoreactivity at any age. However, DA transport via DAT (starting at 12 months) and VMAT2 (starting at 3 months) as well as KCl-stimulated DA release were reduced in Bdnf +/− mice and declined with age suggesting an increasingly important role for BDNF in the release and uptake of DA with the aging process. These findings suggest that a BDNF expression deficit becomes more critical to dopaminergic dynamics and related behavioral activities with increasing age.

The serotonin (5-HT) transporter (SERT) regulates serotoninergic neurotransmission by clearing 5-HT released into the synaptic space. Phosphorylation of SERT on serine and threonine mediates SERT regulation. Whether tyrosine phosphorylation regulates SERT is unknown. Here, we tested the hypothesis that tyrosine-phosphorylation of SERT regulates 5-HT transport. In support of this, alkali-resistant 32 P-labeled SERT was found in rat platelets, and Src-tyrosine kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo [3,4,d]pyrimidine (PP2) decreased platelet SERT function and expression. In human placental trophoblast cells expressing SERT, PP2 reduced transporter function, expression, and stability. Although siRNA silencing of Src expression decreased SERT function and expression, coexpression of Src resulted in PP2-sensitive increases in SERT function and expression. PP2 treatment markedly decreased SERT protein stability. Compared with WT-SERT, SERT tyrosine mutants Y47F and Y142F exhibited reduced 5-HT transport despite their higher total and cell surface expression levels. Moreover, Src-coexpression increased total and cell surface expression of Y47F and Y142F SERT mutants without affecting their 5-HT transport capacity. It is noteworthy that Y47F and Y142F mutants exhibited higher protein stability compared with WT-SERT. However, similar to WT-SERT, PP2 treatment decreased the stability of Y47F and Y142F mutants. Furthermore, compared with WT-SERT, Y47F and Y142F mutants exhibited lower basal tyrosine phosphorylation and no further enhancement of tyrosine phosphorylation in response to Src coexpression. These results provide the first evidence that SERT tyrosine phosphorylation supports transporter protein stability and 5HT transport.

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A new metal organic framework containing high density of active Cu sites demonstrates 100% oxidative conversion of CO to CO(2).

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