Autism gene variant causes hyperserotonemia, serotonin receptor hypersensitivity, social impairment and repetitive behavior.

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.

5-HTTLPR and gender moderate changes in negative affect responses to tryptophan infusion.

Expression of the serotonin transporter is affected by the genotype of the 5-HTTLPR (short and long forms) as well as the genotype of the SNP rs25531 within this region. Based on the combined genotypes for these polymorphisms, we designated each allele as a high or low expressing allele according to established expression levels-resulting in HiHi, HiLo, & LoLo genotype groups for analysis. We evaluated effects of gender and the promoter genotype on induction of negative affect by intravenous infusion of L: -tryptophan (TRP). The protocol consisted of a day-1 sham saline infusion and a day-2 active TRP infusion. Models assessed 5-HTTLPR composite genotype and gender as predictors of change in ratings of negative emotion during TRP infusion. During sham infusion there were no significant changes from baseline in mood ratings. During TRP infusion all negative affect ratings increased significantly from baseline (P's < .02). The genotype x gender interaction was a significant predictor of depression-dejection (P = .013), and trended towards predicting anger-hostility (P = .084). Males in the HiHi group had greater increases in negative affect during infusion, compared to all groups except LoLo females, who also showed increased negative affect.

Analysis of neuroanatomical differences in mice with genetically modified serotonin transporters assessed by structural magnetic resonance imaging.

Background: The serotonin (5-HT) system has long been implicated in autism spectrum disorder (ASD) as indicated by elevated whole blood and platelet 5-HT, altered platelet and brain receptor and transporter binding, and genetic linkage and association findings. Based upon work in genetically modified mice, 5-HT is known to influence several aspects of brain development, but systematic neuroimaging studies have not previously been reported. In particular, the 5-HT transporter (serotonin transporter, SERT; 5-HTT) gene, Slc6a4, has been extensively studied. Methods: Using a 7-T MRI and deformation-based morphometry, we assessed neuroanatomical differences in an Slc6a4 knockout mouse on a C57BL/6 genetic background, along with an Slc6a4 Ala56 knockin mouse on two different genetic backgrounds (129S and C57BL/6). Results: Individually (same sex, same background, same genotype), the only differences found were in the female Slc6a4 knockout mouse; all the others had no significant differences. However, an analysis of variance across the whole study sample revealed a significant effect of Slc6a4 on the amygdala, thalamus, dorsal raphe nucleus, and lateral and frontal cortices. Conclusions: This work shows that an increase or decrease in SERT function has a significant effect on the neuroanatomy in 5-HT relevant regions, particularly the raphe nuclei. Notably, the Slc6a4 Ala56 knockin alone appears to have an insignificant, but suggestive, effect compared to the KO, which is consistent with Slc6a4 function. Despite the small number of 5-HT neurons and their localization to the brainstem, it is clear that 5-HT plays an important role in neuroanatomical organization.

Impact of Maternal Serotonin Transporter Genotype on Placental Serotonin, Fetal Forebrain Serotonin, and Neurodevelopment.

Biomarker, neuroimaging, and genetic findings implicate the serotonin transporter (SERT) in autism spectrum disorder (ASD). Previously, we found that adult male mice expressing the autism-associated SERT Ala56 variant have altered central serotonin (5-HT) system function, as well as elevated peripheral blood 5-HT levels. Early in gestation, before midbrain 5-HT projections have reached the cortex, peripheral sources supply 5-HT to the forebrain, suggesting that altered maternal or placenta 5-HT system function could impact the developing embryo. We therefore used different combinations of maternal and embryo SERT Ala56 genotypes to examine effects on blood, placenta and embryo serotonin levels and neurodevelopment at embryonic day E14.5, when peripheral sources of 5-HT predominate, and E18.5, when midbrain 5-HT projections have reached the forebrain. Maternal SERT Ala56 genotype was associated with decreased placenta and embryonic forebrain 5-HT levels at E14.5. Low 5-HT in the placenta persisted, but forebrain levels normalized by E18.5. Maternal SERT Ala56 genotype effects on forebrain 5-HT levels were accompanied by a broadening of 5-HT-sensitive thalamocortical axon projections. In contrast, no effect of embryo genotype was seen in concepti from heterozygous dams. Blood 5-HT levels were dynamic across pregnancy and were increased in SERT Ala56 dams at E14.5. Placenta RNA sequencing data at E14.5 indicated substantial impact of maternal SERT Ala56 genotype, with alterations in immune and metabolic-related pathways. Collectively, these findings indicate that maternal SERT function impacts offspring placental 5-HT levels, forebrain 5-HT levels, and neurodevelopment.

Genetic Variation in Serotonin Transporter Modulates Tactile Hyperresponsiveness in ASD.

Several lines of evidence implicate dysfunction of the serotonin (5-HT) system in autism spectrum disorder (ASD). Specifically, the serotonin transporter (5-HTT, SERT) has been scrutinized as an ASD candidate risk gene. SERT plays key roles in the development of circuits that underlie sensory function, particularly in the somatosensory system. One previous study in ASD found association of a rare, hyperfunctional SERT variant with sensory aversion, but studies of common SERT variants have never examined sensory symptoms in ASD. Using standardized caregiver assessments of sensory function in children, we evaluated patterns of sensory responsiveness in 47 children with ASD and 38 typically developing (TD) children. Study participants were genotyped for the functional SERT promoter polymorphisms, 5-HTTLPR and rs25531, to test the hypothesis that the higher expressing genotypes would be associated with hyperresponsiveness to touch, a common sensory aversion in ASD. All measures of sensory hypo- and hyperresponsiveness were increased in children with ASD, with hyporesponsive sensory patterns negatively correlated to age and hyperresponsive sensory patterns positively correlated to repetitive behavior. Strikingly, high-expressing SERT genotypes were associated with increased tactile hyperresponsiveness in the ASD group. Our findings indicate genetic variation that increases SERT function may specifically impact somatosensory processing in ASD.

A novel behavioral paradigm to assess multisensory processing in mice.

Human psychophysical and animal behavioral studies have illustrated the benefits that can be conferred from having information available from multiple senses. Given the central role of multisensory integration for perceptual and cognitive function, it is important to design behavioral paradigms for animal models to provide mechanistic insights into the neural bases of these multisensory processes. Prior studies have focused on large mammals, yet the mouse offers a host of advantages, most importantly the wealth of available genetic manipulations relevant to human disease. To begin to employ this model species for multisensory research it is necessary to first establish and validate a robust behavioral assay for the mouse. Two common mouse strains (C57BL/6J and 129S6/SvEv) were first trained to respond to unisensory (visual and auditory) stimuli separately. Once trained, performance with paired audiovisual stimuli was then examined with a focus on response accuracy and behavioral gain. Stimulus durations varied from 50 ms to 1 s in order to modulate the effectiveness of the stimuli and to determine if the well-established "principle of inverse effectiveness" held in this model. Response accuracy in the multisensory condition was greater than for either unisensory condition for all stimulus durations, with significant gains observed at the 300 ms and 100 ms durations. Main effects of stimulus duration, stimulus modality and a significant interaction between these factors were observed. The greatest behavioral gain was seen for the 100 ms duration condition, with a trend observed that as the stimuli became less effective, larger behavioral gains were observed upon their pairing (i.e., inverse effectiveness). These results are the first to validate the mouse as a species that shows demonstrable behavioral facilitations under multisensory conditions and provides a platform for future mechanistically directed studies to examine the neural bases of multisensory integration.

Genetic background modulates phenotypes of serotonin transporter Ala56 knock-in mice.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Absence of preference for social novelty and increased grooming in integrin ß3 knockout mice: initial studies and future directions.

Elevated whole blood serotonin 5-HT, or hyperserotonemia, is a common biomarker in autism spectrum disorder (ASD). The integrin ß3 receptor subunit gene (ITGB3) is a quantitative trait locus for whole blood 5-HT levels. Recent work shows that integrin ß3 interacts with the serotonin transporter (SERT) in both platelets and in the midbrain. Furthermore, multiple studies have now reported gene-gene interaction between the integrin ß3 and SERT genes in association with ASD. Given the lack of previous data on the impact of integrin ß3 on brain or behavioral phenotypes, we sought to compare mice with decreased or absent expression of the integrin ß3 receptor subunit (Itgb3 +/- and -/-) with wildtype littermate controls in behavioral tasks relevant to ASD. These mice did not show deficits in activity level in the open field or anxiety-like behavior on the elevated plus maze, two potential confounds in the evaluation of mouse social behavior. In the three-chamber social test, mice lacking integrin ß3 were shown to have normal sociability but did not show a preference for social novelty. Importantly, the absence of integrin ß3 did not impair olfaction or the ability to recall familiar social odors. Additionally, mice lacking integrin ß3 showed increased grooming behavior in novel environments. These preliminary studies reveal altered social and repetitive behavior in these mice, which suggests that the integrin ß3 subunit may be involved in brain systems relevant to ASD. Further work is needed to fully characterize these behavioral changes and the underlying brain mechanisms.