Behaviorally penetrant, anomalous dopamine efflux exposes sex and circuit dependent regulation of dopamine transporters.

Virtually all neuropsychiatric disorders display sex differences in prevalence, age of onset, and/or clinical symptomology. Although altered dopamine (DA) signaling is a feature of many of these disorders, sex-dependent mechanisms uniquely responsive to DA that drive sex-dependent behaviors remain unelucidated. Previously, we established that anomalous DA efflux (ADE) is a prominent feature of the DA transporter (DAT) variant Val559, a coding substitution identified in two male-biased disorders: attention-deficit/hyperactivity disorder and autism spectrum disorder. In vivo, Val559 ADE induces activation of nigrostriatal D2-type DA autoreceptors (D2ARs) that magnifies inappropriate, nonvesicular DA release by elevating phosphorylation and surface trafficking of ADE-prone DAT proteins. Here we demonstrate that DAT Val559 mice exhibit sex-dependent alterations in psychostimulant responses, social behavior, and cognitive performance. In a search for underlying mechanisms, we discovered that the ability of ADE to elicit D2AR regulation of DAT is both sex and circuit-dependent, with dorsal striatum D2AR/DAT coupling evident only in males, whereas D2AR/DAT coupling in the ventral striatum is exclusive to females. Moreover, systemic administration of the D2R antagonist sulpiride, which precludes ADE-driven DAT trafficking, can normalize DAT Val559 behavioral changes unique to each sex and without effects on the opposite sex or wildtype mice. Our studies support the sex- and circuit dependent capacity of D2ARs to regulate DAT as a critical determinant of the sex-biased effects of perturbed DA signaling in neurobehavioral disorders. Moreover, our work provides a cogent example of how a shared biological insult drives alternative physiological and behavioral trajectories as opposed to resilience.

Serotonin Transporter Ala276 Mouse: Novel Model to Assess the Neurochemical and Behavioral Impact of Thr276 Phosphorylation In Vivo.

The serotonin (5-HT) transporter (SERT) is a key regulator of 5-HT signaling and is a major target for antidepressants and psychostimulants. Human SERT coding variants have been identified in subjects with obsessive-compulsive disorder (OCD) and autism spectrum disorder (ASD) that impact transporter phosphorylation, cell surface trafficking and/or conformational dynamics. Prior to an initial description of a novel mouse line expressing the non-phosphorylatable SERT substitution Thr276Ala, we review efforts made to elucidate the structure and conformational dynamics of SERT with a focus on research implicating phosphorylation at Thr276 as a determinant of SERT conformational dynamics. Using the high-resolution structure of human SERT in inward- and outward-open conformations, we explore the conformation dependence of SERT Thr276 exposure, with results suggesting that phosphorylation is likely restricted to an inward-open conformation, consistent with prior biochemical studies. Assessment of genotypes from SERT/Ala276 heterozygous matings revealed a deviation from Mendelian expectations, with reduced numbers of Ala276 offspring, though no genotype differences were seen in growth or physical appearance. Similarly, no genotype differences were evident in midbrain or hippocampal 5-HT levels, midbrain and hippocampal SERT mRNA or midbrain protein levels, nor in midbrain synaptosomal 5-HT uptake kinetics. Behaviorally, SERT Ala276 homozygotes appeared normal in measures of anxiety and antidepressant-sensitive stress coping behavior. However, these mice displayed sex-dependent alterations in repetitive and social interactions, consistent with circuit-dependent requirements for Thr276 phosphorylation underlying these behaviors. Our findings indicate the utility of SERT Ala276 mice in evaluation of developmental, functional and behavioral consequences of regulatory SERT phosphorylation in vivo.

Human Serotonin Transporter Coding Variation Establishes Conformational Bias with Functional Consequences.

The antidepressant-sensitive serotonin (5-HT) transporter (SERT) dictates rapid, high-affinity clearance of the neurotransmitter in both the brain and periphery. In a study of families with multiple individuals diagnosed with autism spectrum disorder (ASD), we previously identified several, rare, missense coding variants that impart elevated 5-HT transport activity, relative to wild-type SERT, upon heterologous expression as well as in ASD subject lymphoblasts. The most common of these variants, SERT Ala56, located in the transporter's cytosolic N-terminus, has been found to confer in transgenic mice hyperserotonemia, an ASD-associated biochemical trait, an elevated brain 5-HT clearance rate, and ASD-aligned behavioral changes. Hyperfunction of SERT Ala56 has been ascribed to a change in 5-HT KM, though the physical basis of this change has yet to be elucidated. Through assessments of fluorescence resonance energy transfer (FRET) between cytosolic N- and C-termini, sensitivity to methanethiosulfonates, and capacity for N-terminal tryptic digestion, we obtain evidence for mutation-induced conformational changes that support an open-outward 5-HT binding conformation in vitro and in vivo. Aspects of these findings were also evident with another naturally occurring C-terminal SERT coding variant identified in our ASD study, Asn605. We conclude that biased conformations of surface resident transporters that can impact transporter function and regulation are an unappreciated consequence of heritable and disease-associated SERT coding variation.

Serotonin transporter inhibition and 5-HT2C receptor activation drive loss of cocaine-induced locomotor activation in DAT Val559 mice.

Dopamine (DA) signaling dysfunction is believed to contribute to multiple neuropsychiatric disorders including attention-deficit/hyperactivity disorder (ADHD). The rare DA transporter (DAT) coding substitution Ala559Val found in subjects with ADHD, bipolar disorder and autism, promotes anomalous DA efflux in vitro and, in DAT Val559 mice, leads to increased reactivity to imminent handling, waiting impulsivity, and enhanced motivation for reward. Here, we report that, in contrast to amphetamine and methylphenidate, which induce significant locomotor activation, cocaine administration to these mice elicits no locomotor effects, despite retention of conditioned place preference (CPP). Additionally, cocaine fails to elevate extracellular DA. Given that amphetamine and methylphenidate, unlike cocaine, lack high-affinity interactions with the serotonin (5-HT) transporter (SERT), we hypothesized that the lack of cocaine-induced hyperlocomotion in DAT Val559 mice arises from SERT blockade and augmented 5-HT signaling relative to cocaine actions on wildtype animals. Consistent with this idea, the SERT blocker fluoxetine abolished methylphenidate-induced locomotor activity in DAT Val559 mice, mimicking the effects seen with cocaine. Additionally, a cocaine analog (RTI-113) with greater selectivity for DAT over SERT retains locomotor activation in DAT Val559 mice. Furthermore, genetic elimination of high-affinity cocaine interactions at SERT in DAT Val559 mice, or specific inhibition of 5-HT2C receptors in these animals, restored cocaine-induced locomotion, but did not restore cocaine-induced elevations of extracellular DA. Our findings reveal a significant serotonergic plasticity arising in the DAT Val559 model that involves enhanced 5-HT2C signaling, acting independently of striatal DA release, capable of suppressing the activity of cocaine-sensitive motor circuits.

Region-Specific Regulation of Presynaptic Dopamine Homeostasis by D2 Autoreceptors Shapes the In Vivo Impact of the Neuropsychiatric Disease-Associated DAT Variant Val559.

Disruptions of dopamine (DA) signaling contribute to a broad spectrum of neuropsychiatric disorders, including attention-deficit hyperactivity disorder (ADHD), addiction, bipolar disorder, and schizophrenia. Despite evidence that risk for these disorders derives from heritable variation in DA-linked genes, a better understanding is needed of the molecular and circuit context through which gene variation drives distinct disease traits. Previously, we identified the DA transporter (DAT) variant Val559 in subjects with ADHD and established that the mutation supports anomalous DAT-mediated DA efflux (ADE). Here, we demonstrate that region-specific contributions of D2 autoreceptors (D2AR) to presynaptic DA homeostasis dictate the consequences of Val559 expression in adolescent male mice. We show that activation of D2ARs in the WT dorsal striatum (DS), but not ventral striatum (VS), increases DAT phosphorylation and surface trafficking. In contrast, the activity of tyrosine hydroxylase (TH) is D2AR-dependent in both regions. In the DS but not VS of Val559 mice, tonic activation of D2ARs drives a positive feedback loop that promotes surface expression of efflux-prone DATs, raising extracellular DA levels and overwhelming DAT-mediated DA clearance capacity. Whereas D2ARs that regulate DAT are tonically activated in the Val559 DS, D2ARs that regulate TH become desensitized, allowing maintenance of cytosolic DA needed to sustain ADE. Together with prior findings, our results argue for distinct D2AR pools that regulate DA synthesis versus DA release and inactivation and offer a clear example of how the penetrance of gene variation can be limited to a subset of expression sites based on differences in intersecting regulatory networks.SIGNIFICANCE STATEMENT Altered dopamine (DA) signaling has been linked to multiple neuropsychiatric disorders. In an effort to understand and model disease-associated DAergic disturbances, we previously screened the DA transporter (DAT) in subjects with attention-deficit hyperactivity disorder (ADHD) and identified multiple, functionally impactful, coding variants. One of these variants, Val559, supports anomalous DA efflux (ADE) and in transgenic mice leads to changes in locomotor patterns, psychostimulant sensitivity, and impulsivity. Here, we show that the penetrance of Val559 ADE is dictated by region-specific differences in how presynaptic D2-type autoreceptors (D2ARs) constrain DA signaling, biasing phenotypic effects to dorsal striatal projections. The Val559 model illustrates how the impact of genetic variation underlying neuropsychiatric disorders can be shaped by the differential engagement of synaptic regulatory mechanisms.