The rare DAT coding variant Val559 perturbs DA neuron function, changes behavior, and alters in vivo responses to psychostimulants

Abstract

Despite the critical role of the presynaptic dopamine (DA) transporter (DAT, SLC6A3) in DA clearance and psychostimulant responses, evidence that DAT dysfunction supports risk for mental illness is indirect. Recently, we identified a rare, nonsynonymous Slc6a3 variant that produces the DAT substitution Ala559Val in two male siblings who share a diagnosis of attention-deficit hyperactivity disorder (ADHD), with other studies identifying the variant in subjects with bipolar disorder (BPD) and autism spectrum disorder (ASD). Previously, using transfected cell studies, we observed that although DAT Val559 displays normal total and surface DAT protein levels, and normal DA recognition and uptake, the variant transporter exhibits anomalous DA efflux (ADE) and lacks capacity for amphetamine (AMPH)-stimulated DA release. To pursue the significance of these findings in vivo, we engineered DAT Val559 knock-in mice, and here we demonstrate in this model the presence of elevated extracellular DA levels, altered somatodendritic and presynaptic D2 DA receptor (D2R) function, a blunted ability of DA terminals to support depolarization and AMPHevoked DA release, and disruptions in basal and psychostimulant-evoked locomotor behavior. Together, our studies demonstrate an in vivo functional impact of the DAT Val559 variant, providing support for the ability of DAT dysfunction to impact risk for mental illness.T he neurotransmitter dopamine (DA) plays a key role in regulating brain circuits that control reward, attention, and locomotor activity (1-3), Accordingly, dopaminergic dysfunction is believed to contribute to several neuropsychiatric disorders including Parkinson's disease (4), bipolar disorder (BPD) (5), drug abuse and addiction (6), and attention-deficit hyperactivity disorder (ADHD) (7,8). The presynaptic DA transporter (DAT) is the primary mechanism for terminating DA signaling at the synapse (9) and is the primary target for several psychostimulant drugs including cocaine (COC), methylphenidate (MPH), and amphetamine (AMPH). COC and MPH are DAT antagonists, elevating extracellular DA levels by preventing DAT-mediated DA reuptake (10). AMPH actions are more complex (11). AMPH is structurally similar to DA and, as a result, is transported by DAT, competing with DA during the reuptake process. AMPH also induces DAT-mediated nonvesicular release, also termed "DA efflux," a process that involves the actions of intracellular signaling proteins such as CamKIIα (12-16) and , alterations in interactions with DAT-associated proteins and phospholipids (12,14,20), and changes in DAT phosphorylation (12,16,(21)(22)(23)) that biases the transporter toward an efflux-competent mechanism. Despite their mechanistic differences, MPH and AMPH both rapidly elevate DA in the CNS and are components of the most frequently prescribed medications for ADHD, Ritalin and Adderall, respectively. The DA modulatory actions of MPH and AMPH reinforce hypotheses derived from brain imaging studies (24) and the analysis of common genetic variation (25-30)...

Kinase-dependent Regulation of Monoamine Neurotransmitter Transporters

“…These findings indicate that DAT Val559 exists in a state where D2-dependent PKCb action drives transporter-mediated DA export in a manner that is normally impeded with wild-type DAT, either because D2 receptors are not continually flooded by DA leak or because other inhibitory processes are in place (that must then also be lost in the variant). Recently, Mergy et al (2014) reported studies with DAT Val559 mice, providing evidence of both excessive extracellular DA and tonic presynaptic D2 receptor stimulation. The availability of the DAT Val559 mouse model provides an unprecedented opportunity to define the significance of D2 receptor mediated PKCb signaling for DAT modulation in vivo.…”

“…Indeed, AMPH lacks the ability to trigger DA efflux in the DAT Val559 mutant, serving rather to attenuate elevated basal efflux (Mazei-Robison et al, 2008). Recently, Mergy et al (2014) provided in vivo evidence via microdialysis studies of knock-in mice that the DAT Val559 variant supports a tonic elevation in basal extracellular DA, consistent with chronic DATmediated DA efflux, although whether CaMKII sustains DAT Val559 efflux activity in vivo has yet to be addressed.…”

Regulation of the Dopamine and Vesicular Monoamine Transporters: Pharmacological Targets and Implications for Disease

“…However, humans that are homozygous for loss-offunction DAT (SLC6A3) alleles present with a complex disorder that resembles PD, rather than ADHD (see section V.B; Kurian et al, 2011;Ng et al, 2014). Instead, clinical studies that genetically screen individuals with ADHD, as well as preclinical studies using cell lines and genetic mouse models, have suggested that variations in the genes encoding for DAT, rather than simple loss of function, and/or DA receptors are associated with risk for ADHD (Gill et al, 1997;Bobb et al, 2005;Mazei-Robison et al, 2005;Sakrikar et al, 2012;Mergy et al, 2014). Importantly, only a few human case studies of DAT variants have been implicated in ADHD to date.…”

Interactions of Cathinone NPS with Human Transporters and Receptors in Transfected Cells

“…Furthermore, in vitro experiments with transfected cells formed the basis for many genetic mutations that were later engineered in mice, which now serve for in vivo investigations of psychoactive drugs or as preclinical models of mental disorders (Henry et al, 2006;Mazei-Robison et al, 2008;Prasad et al, 2005). For example, in vitro experiments allowed the construction of a transgenic mouse model with a 5-hydroxytryptamine (5-HT [serotonin]) transporter (SERT) mutation for the in vivo assessment of SERT-mediated effects of antidepressants or cocaine (Prosser et al, 2014;Thompson et al, 2011) or to shed light on functional abnormalities of the DAT variant Val559, which is being investigated as a potential mouse model of attention-deficit hyperactivity disorder (Mergy et al, 2014).…”

“…We initially followed this protocol (Hysek et al, 2012c) but later adapted it according to the principles reported by Scholze et al (Scholze et al, 2000), who used a superfusion system. The superfusion system is preferentially used for rodent tissue slices that are preloaded with radioactive transporter substrates (Mergy et al, 2014), but it can also be adapted for transfected cells (Pifl et al, 1995;Scholze et al, 2000). Transfected cells are grown on coverslips and loaded with radioactive substrates.…”