Dopamine D2 receptor signalling is strongly implicated in the aetiology of schizophrenia. We have recently characterized the function of three DRD2 SNPs: rs12364283 in the promoter affecting total D2 mRNA expression; rs2283265 and rs1076560, respectively in introns 5 and 6, shifting mRNA splicing to two functionally distinct isoforms, the short form of D2 (D2S) and the long form (D2L). These two isoforms differentially contribute to dopamine signalling in prefrontal cortex and in striatum. We performed a case–control study to determine association of these variants and of their main haplotypes with several schizophrenia-related phenotypes. We demonstrate that the minor allele in the intronic variants is associated with reduced expression of %D2S of total mRNA in post-mortem prefrontal cortex, and with impaired working memory behavioural performance, both in patients and controls. However, the fMRI results show opposite effects in patients compared with controls: enhanced engagement of prefronto-striatal pathways in controls and reduced activity in patients. Moreover, the promoter variant is also associated with working memory activity in prefrontal cortex and striatum of patients, and less robustly with negative symptoms scores. Main haplotypes formed by the three DRD2 variants showed significant associations with these phenotypes consistent with those of the individual SNPs. Our results indicate that the three functional DRD2 variants modulate schizophrenia phenotypes possibly by modifying D2S/D2L ratios in the context of different total D2 density.
dopamine; D2 receptor; working memory; prefrontal cortex; striatum
Personality traits related to emotion processing are, at least in part, heritable and genetically determined. Dopamine D2 receptor signaling is involved in modulation of emotional behavior and activity of associated brain regions such as the amygdala and the prefrontal cortex. An intronic single nucleotide polymorphism within the D2 receptor gene (DRD2, rs1076560, guanine>thymine - G>T) shifts splicing of the two protein isoforms (D2 short, D2S, mainly presynaptic, and D2 long, D2L) and has been associated with modulation of memory performance and brain activity. Here, our aim was to investigate the association of DRD2 rs1076560 genotype with personality traits of emotional stability and with brain physiology during processing of emotionally relevant stimuli. DRD2 genotype and Big Five Questionnaire scores were evaluated in 134 healthy subjects demonstrating that GG subjects have reduced ‘emotion control’ compared with GT subjects. fMRI in a sample of 24 individuals indicated greater amygdala activity during implicit processing and greater dorsolateral prefrontal cortex (DLPFC) response during explicit processing of facial emotional stimuli in GG subjects compared with GT. Other results also demonstrate an interaction between DRD2 genotype and facial emotional expression on functional connectivity of both amygdala and dorsolateral prefrontal regions with overlapping medial prefrontal areas. Moreover, rs1076560 genotype is associated with differential relationships between amygdala/DLPFC functional connectivity and emotion control scores. These results suggest that genetically determined D2 signaling may explain part of personality traits related to emotion processing and individual variability in specific brain responses to emotionally relevant inputs.
amygdala; DRD2; dopamine; emotion; fMRI; prefrontal cortex
The dopamine transporter (DAT) plays a critical role in terminating the action of dopamine by rapid reuptake into the presynaptic neuron. Previous studies have revealed that the DAT carboxyl terminus (DAT-CT) can directly interact with other cellular proteins and regulate DAT function and trafficking.
Here, we have identified that carboxypeptidase E (CPE), a prohormone processing exopeptidase and sorting receptor for the regulated secretory pathway, interacts with the DAT-CT and affects DAT function. Mammalian cell lines coexpressing CPE and DAT exhibited increased DAT-mediated dopamine uptake activity compared to cells expressing DAT alone. Moreover, coexpression of an interfering DAT-CT minigene inhibited the effects of CPE on DAT. Functional changes caused by CPE could be attributed to enhanced DAT expression and subsequent increase in DAT cell surface localization, due to decreased DAT degradation. In addition, CPE association could reduce the phosphorylation state of DAT on serine residues, potentially leading to reduced internalization, thus stabilizing plasmalemmal DAT localization.
Taken together, our results reveal a novel role for CPE in the regulation of DAT trafficking and DAT-mediated DA uptake, which may provide a novel target in the treatment of dopamine-governed diseases such as drug addiction and obesity.
Variation of the gene coding for D2 receptors (DRD2) has been associated with risk for schizophrenia and with working memory deficits. A functional intronic SNP (rs1076560) predicts relative expression of the two D2 receptors isoforms, D2S (mainly pre-synaptic) and D2L (mainly post-synaptic). However, the effect of functional genetic variation of DRD2 on striatal dopamine D2 signaling and on its correlation with prefrontal activity during working memory in humans is not known.
Thirty-seven healthy subjects were genotyped for rs1076560 (G>T) and underwent SPECT with [123I]IBZM (which binds primarily to post-synaptic D2 receptors) and with [123I]FP-CIT (which binds to pre-synaptic dopamine transporters, whose activity and density is also regulated by pre-synaptic D2 receptors), as well as BOLD fMRI during N-Back working memory.
Subjects carrying the T allele (previously associated with reduced D2S expression) had striatal reductions of [123I]IBZM and of [123I]FP-CIT binding. DRD2 genotype also differentially predicted the correlation between striatal dopamine D2 signaling (as identified with factor analysis of the two radiotracers) and activity of the prefrontal cortex during working memory as measured with BOLD fMRI, which was positive in GG subjects and negative in GT.
Our results demonstrate that this functional SNP within DRD2 predicts striatal binding of the two radiotracers to dopamine transporters and D2 receptors as well as the correlation between striatal D2 signaling with prefrontal cortex activity during performance of a working memory task. These data are consistent with the possibility that the balance of excitatory/inhibitory modulation of striatal neurons may also affect striatal outputs in relationship with prefrontal activity during working memory performance within the cortico-striatal-thalamic-cortical pathway.
Dopamine and dopamine transporters (DAT, which regulate extracellular dopamine in the brain) are implicated in the modulation of attention but their specific roles are not well understood. Here we hypothesized that dopamine modulates attention by facilitation of brain deactivation in the default mode network (DMN). Thus, higher striatal DAT levels, which would result in an enhanced clearance of dopamine and hence weaker dopamine signals, would be associated to lower deactivation in the DMN during an attention task.
For this purpose we assessed the relationship between DAT in striatum (measured with positron emission tomography and [11C]cocaine used as DAT radiotracer) and brain activation and deactivation during a parametric visual attention task (measured with blood oxygenation level dependent functional magnetic resonance imaging) in healthy controls. We show that DAT availability in caudate and putamen had a negative correlation with deactivation in ventral parietal regions of the DMN (precuneus, BA 7) and a positive correlation with deactivation in a small region in the ventral anterior cingulate gyrus (BA 24/32). With increasing attentional load, DAT in caudate showed a negative correlation with load-related deactivation increases in precuneus.
These findings provide evidence that dopamine transporters modulate neural activity in the DMN and anterior cingulate gyrus during visuospatial attention. Our findings suggest that dopamine modulates attention in part by regulating neuronal activity in posterior parietal cortex including precuneus (region involved in alertness) and cingulate gyrus (region deactivated in proportion to emotional interference). These findings suggest that the beneficial effects of stimulant medications (increase dopamine by blocking DAT) in inattention reflect in part their ability to facilitate the deactivation of the DMN.
The default mode network (DMN) comprises a set of brain regions with “increased” activity during rest relative to cognitive processing. Activity in the DMN is associated with functional connections with the striatum and dopamine (DA) levels in this brain region. A functional single-nucleotide polymorphism within the dopamine D2 receptor gene (DRD2, rs1076560 G > T) shifts splicing of the 2 D2 isoforms, D2 short and D2 long, and has been associated with striatal DA signaling as well as with cognitive processing. However, the effects of this polymorphism on DMN have not been explored. The aim of this study was to evaluate the effects of rs1076560 on DMN and striatal connectivity and on their relationship with striatal DA signaling. Twenty-eight subjects genotyped for rs1076560 underwent functional magnetic resonance imaging during a working memory task and 123 55 I-Fluoropropyl-2-beta-carbomethoxy-3-beta(4-iodophenyl) nortropan Single Photon Emission Computed Tomography ([123I]-FP-CIT SPECT) imaging (a measure of dopamine transporter [DAT] binding). Spatial group-independent component (IC) analysis was used to identify DMN and striatal ICs. Within the anterior DMN IC, GG subjects had relatively greater connectivity in medial prefrontal cortex (MPFC), which was directly correlated with striatal DAT binding. Within the posterior DMN IC, GG subjects had reduced connectivity in posterior cingulate relative to T carriers. Additionally, rs1076560 genotype predicted connectivity differences within a striatal network, and these changes were correlated with connectivity in MPFC and posterior cingulate within the DMN. These results suggest that genetically determined D2 receptor signaling is associated with DMN connectivity and that these changes are correlated with striatal function and presynaptic DA signaling.
DRD2; dopamine; default mode network; functional magnetic resonance imaging; single-photon emission computerized tomography
The dopamine transporter (DAT) regulates the temporal and spatial actions of dopamine by reuptaking this neurotransmitter into the presynaptic neurons. We recently generated transgenic mice overexpressing DAT (DAT-tg) that have a 3-fold increase in DAT protein levels which results in a 40% reduction of the extracellular DA concentration in the striatum. The aim of this study was to examine the effect of this reduction in dopaminergic tone on postsynaptic responses mediated by dopamine receptors. We report here that DAT-tg mice have increased levels of striatal D1 (30%) and D2 (~60%) dopamine receptors with D1 receptor signaling components not significantly altered as evidenced by unaffected basal or stimulated levels of phospho-GluR1 (Ser845) and phospho-ERK2. However, the novel D2 mediated Akt signaling is markedly altered in DAT-tg animals. In particular, there is a 300% increase in the basal levels of phospho-Akt in the striatum of DAT-tg, reflecting the reduced extracellular dopamine tone in these animals. This increase in basal pAkt levels can be pharmacologically recapitulated by partial dopamine depletion in WT mice treated with the selective tyrosine hydroxylase inhibitor alpha-methyl-para-tyrosine (α-MPT). Behaviorally, DAT-tg animals demonstrate an augmented synergistic interaction between up-regulated D1 and D2 receptors which results in increased climbing behavior in transgenic mice after stimulation with either apomorphine or a co-administration of selective D1 and D2 receptor agonists. In sum, our study reveals that hypodopaminegia caused by up-regulation of DAT results in significant alterations at postsynaptic receptor function with most notable dysregulation at the level of D2 receptor signaling.
Dopamine transporter (DAT); BAC Transgenic mice; hypodopaminergia; dopamine receptors; Akt; apomorphine
Dopamine has been hypothesized to provide the basis for the interaction between motivational and cognitive control. However, there is no evidence for this hypothesis in humans. We fill this gap by using fMRI, a novel behavioral paradigm and a common polymorphism in the DAT1 gene (SLC6A3). Carriers of the 9-repeat (9R) allele of a 40 base pair repeat polymorphism in the 3′ untranslated region of DAT1, associated with high striatal dopamine, showed greater activity in the ventromedial striatum during reward anticipation than homozygotes for the 10-repeat allele, replicating previous genetic imaging studies. The crucial novel finding is that 9R carriers also exhibited a greater influence of anticipated reward on switch costs, as well as greater activity in the dorsomedial striatum during task switching in anticipation of high reward relative to low reward. These data establish a crucial role for human striatal dopamine in the modulation of cognitive flexibility by reward anticipation, thus, elucidating the neurochemical mechanism of the interaction between motivation and cognitive control.
DAT polymorphism; SLC6A3; fMRI; ventral striatum; reward; task switching; Cognition; Dopamine; Neurogenetics; Catecholamines; Task switching; DAT polymorphism; SLC6A3; fMRI; ventral striatum; reward
Complex cognitive tasks such as visual working memory (WM) involve networks of interacting brain regions. Several neurotransmitters, including an appropriate dopamine concentration, are important for WM performance. A number of gene polymorphisms are associated with individual differences in cognitive task performance. COMT, for example, encodes catechol-o-methyl transferase the enzyme primarily responsible for catabolizing dopamine in the prefrontal cortex. Striatal dopamine function, linked with cognitive tasks as well as habit learning, is influenced by the Taq-Ia polymorphism of the DRD2/ANKK1 gene complex; this gene influences the density of dopamine receptors in the striatum. Here, we investigated the effects of these polymorphisms on a WM task requiring the maintenance of 4 or 6 items over delay durations of 1 or 5 seconds. We explored main effects and interactions between the COMT and DRD2/ANKK1-Taq-Ia polymorphisms on WM performance. Participants were genotyped for COMT (Val158Met) and DRD2/ANKK1-Taq-Ia (A1+, A1−) polymorphisms. There was a significant main effect of both polymorphisms. Participants' WM reaction times slowed with increased Val loading such that the Val/Val homozygotes made the slowest responses and the Met/Met homozygotes were the fastest. Similarly, WM reaction times were slower and more variable for the DRD2/ANKK1-Taq-Ia A1+ group than the A1− group. The main effect of COMT was only apparent in the DRD2/ANKK1-Taq-Ia A1− group. These findings link WM performance with slower dopaminergic metabolism in the prefrontal cortex as well as a greater density of dopamine receptors in the striatum.
Research integrating neuroimaging and molecular genetics has yielded important insights into how variability in brain chemistry predicts individual differences in brain function, behavior and related risk for psychopathology. However, existing studies have been limited by their focus on the independent effects of single polymorphisms with modest impact on brain chemistry. Here, we explored the effects of five functional polymorphisms affecting dopamine (DA) signaling on reward-related ventral striatum (VS) reactivity, measured with BOLD fMRI, in a sample of 69 Caucasians. We also compiled individual multilocus genetic profile scores reflecting the additive effects of alleles conferring relatively increased DA signaling across the five polymorphic loci: DAT1 9-repeat, DRD4 7-repeat, DRD2 -141C Del, DRD2 Taq1A C (A2), and COMT 158Met. These multilocus DA profile scores accounted for 10.9% of the inter-individual variability in reward-related VS reactivity. In contrast, none of the individual polymorphisms accounted for significant variability. Our results show that biologically informed multilocus genetic profiles have unique promise as indices of variability in brain chemistry that may yield advances in mapping individual differences in behaviorally relevant brain function. In turn, such genetic profiles may fuel gene–environment interactions research establishing trajectories of risk for psychopathology.
genetic profile; dopamine; ventral striatum; reward; fMRI; dopamine; neurogenetics; neurotransmitters; biological psychiatry; genetic profile; ventral striatum; reward; fMRI
The dopamine transporter (DAT) regulates synaptic dopamine (DA) in striatum and modulation of DAT can affect locomotor activity. Thus, in Parkinson’s disease (PD), DAT loss could affect DA clearance and locomotor activity. The locomotor benefits of L-DOPA may be mediated by transport through monoamine transporters and conversion to DA. However, its impact upon DA reuptake is unknown and may modulate synaptic DA. Using the unilateral 6-OHDA rat PD model, we examined [3H]DA uptake dynamics in relation to striatal DAT and tyrosine hydroxylase (TH) protein loss compared with contralateral intact striatum. Despite >70% striatal DAT loss, DA uptake decreased only ∼25% and increased as DAT loss approached 99%. As other monoamine transporters can transport DA, we determined if norepinephrine (NE) and serotonin (5-HT) differentially modulated DA uptake in lesioned striatum. Unlabeled DA, NE, and 5-HT were used, at a concentration that differentially inhibited DA uptake in intact striatum, to compete against [3H]DA uptake. In 6-OHDA lesioned striatum, DA was less effective, whereas NE was more effective, at inhibiting [3H]DA uptake. Furthermore, norepinephrine transporter (NET) protein levels increased and desipramine was ∼two-fold more effective at inhibiting NE uptake. Serotonin inhibited [3H]DA uptake, but without significant difference between lesioned and contralateral striatum. L-DOPA inhibited [3H]DA uptake two-fold more in lesioned striatum and inhibited NE uptake ∼five-fold more than DA uptake in naïve striatum. Consequently, DA uptake may be mediated by NET when DAT loss is at PD levels. Increased inhibition of DA uptake by L-DOPA and its preferential inhibition of NE over DA uptake, indicates that NET-mediated DA uptake may be modulated by L-DOPA when DAT loss exceeds 70%. These results indicate a novel mechanism for DA uptake during PD progression and provide new insight into how L-DOPA affects DA uptake, revealing possible mechanisms of its therapeutic and side effect potential.
Twin-studies suggest that a significant portion of individual differences in the propensity to take risks resides in people’s genetic make-up and there is evidence that variability in dopaminergic systems relates to individual differences in risky choice. We examined the link between risk taking in a risk taking task (the Balloon Analogue Risk Task, BART) and a variable number tandem repeat (VNTR) polymorphism in the 3′UTR of the dopamine transporter gene (SLC6A3/DAT1). Behavior in BART is known to be associated with activity in striatal reward-processing regions, and DAT1 is assumed to modulate striatal dopamine levels. We find that carriers of DAT1 alleles, which presumably result in lower striatal dopamine availability, showed more risk taking, relative to carriers of the alleles associated with higher striatal dopamine availability. Our analyses suggest that the mechanism underlying this association is diminished sensitivity to rewards among those who take more risks. Overall, our results support the notion that a behavioral genetic approach can be helpful in uncovering the basis of individual differences in risk taking.
Human personality traits have a considerable genetic component. Cloninger et al. were the first to postulate that certain personality traits, such as novelty seeking, are related to the dopamine neurotransmitter system. In this study, we investigated the associations between dopamine receptor D4 (DRD4) exon III and dopamine transporter (DAT1) polymorphisms and personality traits. The DRD4 and DAT1 gene polymorphisms were genotyped in 214 healthy Korean subjects, whose personality traits were assessed with the Temperament and Character Inventory (TCI). There were no significant differences between scores of TCI temperament dimensions (novelty seeking, harm avoidance, reward dependence, and persistence) and DRD4 gene polymorphism. The DAT1 gene polymorphisms also showed no significant association with any of the temperament subscales of the TCI. These data suggest that DRD4 and DAT1 gene polymorphism may not associated with personality traits in a Korean population.
Dopamine receptor D4; dopamine transporter gene; polymorphism; personality traits; temperament and character inventory; Korean
Alterations in working memory, default-mode network (DMN), and dopamine transporter have all been proposed as endophenotypes for Attention-Deficit Hyperactivity/Disorder (ADHD). Despite evidence that these systems are interrelated, their relationship to each other has never been studied in the context of ADHD. In order to understand the potential mediating effects of task-positive and task-negative networks between DAT1 and diagnosis, we tested effects of genotype and diagnosis on regions of positive and negative BOLD signal change (as measured with fMRI) in 53 adults with ADHD and 38 control subjects during a working memory task. We also examined the relationship of these responses to ADHD symptoms. Our results yielded four principal findings: 1) association of 9R with adult ADHD, 2) marginal DAT1 association with task-related suppression in left medial PFC, 3) marginal genotype × diagnosis interaction in the dorsal anterior cingulate cortex, and 4) correlation of DMN suppression to ADHD symptoms. These findings replicate the association of the 9R allele with adult ADHD. Further, we show that DMN suppression is likely linked to DAT1 and to severity of inattention in ADHD. DMN may therefore be a target of DAT1 effects, and lie on the path between the gene and inattention in ADHD.
fMRI; default-mode network; attention
Preclinical and clinical evidence suggest an association between alcoholism and the primary regulator of extracellular dopamine concentrations, the dopamine transporter (DAT). However, the nature of this association is unclear. We determined if ten days of voluntary alcohol self-administration followed by withdrawal could directly alter DAT function, or if genetically-mediated changes in DAT function and/or availability could influence vulnerability to alcohol abuse. Heterozygous (DAT+/-) and homozygous mutant (DAT-/-) and wildtype (DAT+/+) mice were allowed to consume 5% alcohol in a schedule-induced polydipsia (SIP) task. In vivo fixed potential amperometry in anesthetized mice was used to (1) identify functional characteristics of mesoaccumbens dopamine neurons related to genotype, including dopamine autoreceptor (DAR) sensitivity, DAT efficiency, and DAT capacity, (2) determine if any of these characteristics correlated with alcohol drinking observed in DAT+/+ and DAT+/- animals, and (3) determine if SIP-alcohol self-administration altered DAR sensitivity, DAT efficiency, and DAT capacity by comparing these characteristics in wildtype (DAT+/+) mice that were SIP-alcohol naïve, with those that had undergone SIP-alcohol testing. DAT-/- mice consumed significantly less alcohol during testing and this behavioral difference was related to significant differences in DAR sensitivity, DAT efficiency, and DAT capacity. These functional characteristics were correlated to varying degrees with g/kg alcohol consumption in DAT+/+ and DAT+/- mice. DAR sensitivity was consistently reduced and DAT efficiency was enhanced in SIP-alcohol experienced DAT+/+ mice in comparison to naïve animals. These results indicate that DAR sensitivity is reduced by SIP-alcohol consumption and that DAT efficiency is modified by genotype as well as SIP-alcohol exposure. DAT capacity appeared to be strictly associated with SIP-alcohol consumption.
dopamine transporter; dopamine autoreceptor; schedule-induced polydipsia; fixed potential amperometry; alcohol self-administration
We investigated the functional relationship between the SNARE protein syntaxin 1A (syn 1A) and the dopamine transporter (DAT) by treating rat striatal tissue with Botulinum Neurotoxin C (BoNT/C) and co-transfecting syn 1A with DAT in non-neuronal cells, followed by analysis of DAT activity, phosphorylation, and regulation. Treatment of striatal slices with BoNT/C resulted in elevated dopamine (DA) transport Vmax and reduced DAT phosphorylation, while heterologous co-expression of syn 1A led to reduction in DAT surface expression and transport Vmax. Syn 1A was present in DAT immunoprecipitation complexes, supporting a direct or indirect interaction between the proteins. Phorbol ester regulation of DA transport activity was retained in BoNT/C-treated synaptosomes and syn 1A transfected cells, demonstrating that PKC and syn 1A effects occur through independent processes. These findings reveal a novel mechanism for regulation of DAT activity and phosphorylation, and suggest the potential for syn 1A to impact DA neurotransmission through effects on reuptake.
botulinum neurotoxin C; synaptosomes; phorbol ester; transport regulation; synprint domain
Dopaminergic therapy in Parkinson's disease (PD) can improve some cognitive functions while worsening others. These opposite effects might reflect different levels of residual dopamine in distinct parts of the striatum, although the underlying mechanisms remain poorly understood. We used functional magnetic resonance imaging (fMRI) to address how apomorphine, a potent dopamine agonist, influences brain activity associated with working memory in PD patients with variable levels of nigrostriatal degeneration, as assessed via dopamine-transporter (DAT) scan. Twelve PD patients underwent two fMRI sessions (Off-, On-apomorphine) and one DAT-scan session. Twelve sex-, age-, and education-matched healthy controls underwent one fMRI session. The core fMRI analyses explored: (1) the main effect of group; (2) the main effect of treatment; and (3) linear and nonlinear interactions between treatment and DAT levels. Relative to controls, PD-Off patients showed greater activations within posterior attentional regions (e.g., precuneus). PD-On versus PD-Off patients displayed reduced left superior frontal gyrus activation and enhanced striatal activation during working-memory task. The relation between DAT levels and striatal responses to apomorphine followed an inverted-U-shaped model (i.e., the apomorphine effect on striatal activity in PD patients with intermediate DAT levels was opposite to that observed in PD patients with higher and lower DAT levels). Previous research in PD demonstrated that the nigrostriatal degeneration (tracked via DAT scan) is associated with inverted-U-shaped rearrangements of postsynaptic D2-receptors sensitivity. Hence, it can be hypothesized that individual differences in DAT levels drove striatal responses to apomorphine via D2-receptor-mediated mechanisms.
Cognition; DAT; dopamine-agonist; fMRI; Parkinson's disease; working memory
Epistatic gene–gene interactions could contribute to the heritability of complex multigenic disorders, but few examples have been reported. Here, we focus on the role of aberrant dopaminergic signaling, involving the dopamine transporter DAT, a cocaine target, and the dopamine D2 receptor, which physically interacts with DAT. Splicing polymorphism rs2283265 of DRD2, encoding D2 receptors, were shown to confer risk of cocaine overdose/death (odds ratio ∼3) in subjects and controls from the Miami Dade County Brain Bank.1 Risk of cocaine-related death attributable to the minor allele of rs2283265 was significantly enhanced to OR=7.5 (P=0.0008) in homozygous carriers of the main 6-repeat allele of DAT rs3836790, a regulatory VNTR in intron8 lacking significant effect itself. In contrast, carriers of the minor 5-repeat DAT allele showed no significant risk (OR=1.1, P=0.84). DAT rs3836790 and DRD2 rs2283265 also interacted by modulating DAT protein activity in the ventral putamen of cocaine abusers. In high-linkage disequilibrium with the VNTR, DAT rs6347 in exon9 yielded similar results. Assessing the impact of DAT alone, a rare DAT haplotype formed by the minor alleles of rs3836790 and rs27072, a regulatory DAT variant in the 3′-UTR, occurred in nearly one-third of the cocaine abusers but was absent in African American controls, apparently conferring strong risk. These results demonstrate gene–gene–drug interaction affecting risk of fatal cocaine intoxication.
cocaine-related death; DAT; DRD2; epistasis; gene–gene interaction; gene regulation; haplotype
The biological regulating factors of physical activity in animals are not well understood. This study investigated differences in central mRNA expression of seven dopamine genes (Drd1, Drd2, Drd3, Drd4, Drd5, TH, and DAT) between high active C57/LJ (n=17) male mice and low active C3H/HeJ (n=20) male mice, and between mice with access to a running wheel and without running wheel access within strain. Mice were housed with running wheels interfaced with a computer for 21 days with distance and duration recorded every 24 hours. On day 21, the striatum and nucleus accumbens were removed during the active period (∼9pm) for dopaminergic analysis. On average, the C57L/J mice with wheels ran significantly farther (10.25±1.37 km/day vs. 0.01±0.09 km/day, p<0.001), longer (329.73±30.52 mins/day vs. 7.81±6.32 mins/day, p<0.001), and faster (31.27±3.13 m/min vs. 11.81±1.08 m/min, p<0.001) than the C3H/HeJ mice with wheels over the 21 day period. No differences in gene expression were found between mice in either strain with wheels and those without wheels suggesting that access to running wheels did not alter dopaminergic expression. In contrast, relative expression for two dopamine genes was significantly lower in the C57L/J mice compared to the C3H/HeJ mice. These results indicate that decreased dopaminergic functioning is correlated with increased activity levels in C57L/J mice and suggests that D1-like receptors as well as Tyrosine Hydroxylase (an indicator of dopamine production), but not D2-like receptors may be associated with the regulation of physical activity in inbred mice.
dopamine; locomotion; running wheel; mice; dopamine receptor; striatum; nucleus accumbens
The neurotransmitter dopamine (DA) modulates brain circuits involved in attention, reward and motor activity. Synaptic DA homeostasis is primarily controlled via two presynaptic regulatory mechanisms, DA D2 receptor (D2R)-mediated inhibition of DA synthesis and release, and DA transporter (DAT)-mediated DA clearance. D2Rs can physically associate with DAT and regulate DAT function, linking DA release and reuptake to a common mechanism. We have established that the attention-deficit hyperactivity disorder (ADHD)-associated human DAT coding variant Ala559Val (hDAT A559V) results in anomalous DA efflux (ADE) similar to that caused by AMPH-like psychostimulants. Here, we show that tonic activation of D2R provides support for hDAT A559V-mediated ADE. We determine in hDAT A559V a pertussis toxin-sensitive, CaMKII-dependent phosphorylation mechanism that supports D2R-driven DA efflux. These studies identify a signaling network downstream of D2R activation, normally constraining DA action at synapses, that may be altered by DAT mutation to impact risk for DA related disorders.
dopamine; transporter; efflux; hDAT A559V; D2R; CaMKII; ADHD
Knowledge about the functional status of the frontal cortex in infancy is limited. This study investigated the effects of polymorphisms in four dopamine system genes on performance in a task developed to assess such functioning, the Freeze-Frame task, at 9 months of age. Polymorphisms in the catechol-O-methyltransferase (COMT) and the dopamine D4 receptor (DRD4) genes are likely to impact directly on the functioning of the frontal cortex, whereas polymorphisms in the dopamine D2 receptor (DRD2) and dopamine transporter (DAT1) genes might influence frontal cortex functioning indirectly via strong frontostriatal connections. A significant effect of the COMT valine158methionine (Val158Met) polymorphism was found. Infants with the Met/Met genotype were significantly less distractible than infants with the Val/Val genotype in Freeze-Frame trials presenting an engaging central stimulus. In addition, there was an interaction with the DAT1 3′ variable number of tandem repeats polymorphism; the COMT effect was present only in infants who did not have two copies of the DAT1 10-repeat allele. These findings indicate that dopaminergic polymorphisms affect selective aspects of attention as early as infancy and further validate the Freeze-Frame task as a frontal cortex task.
frontal cortex; infancy; dopamine genes; attention; frontal-subcortical circuits
Background: DAT activity is regulated by protein kinases.
Results: We identify Thr53 as a DAT phosphorylation site in rat striatum by mass spectrometry and a phospho-specific antibody; Thr53 mutation reduced dopamine influx and ablated transporter-mediated efflux.
Conclusion: Phosphorylation of DAT Thr53 is involved in transport activity.
Significance: These results identify Thr53 phosphorylation of DAT in vivo and elucidate associated functional properties.
In the central nervous system, levels of extraneuronal dopamine are controlled primarily by the action of the dopamine transporter (DAT). Multiple signaling pathways regulate transport activity, substrate efflux, and other DAT functions through currently unknown mechanisms. DAT is phosphorylated by protein kinase C within a serine cluster at the distal end of the cytoplasmic N terminus, whereas recent work in model cells revealed proline-directed phosphorylation of rat DAT at membrane-proximal residue Thr53. In this report, we use mass spectrometry and a newly developed phospho-specific antibody to positively identify DAT phosphorylation at Thr53 in rodent striatal tissue and heterologous expression systems. Basal phosphorylation of Thr53 occurred with a stoichiometry of ∼50% and was strongly increased by phorbol esters and protein phosphatase inhibitors, demonstrating modulation of the site by signaling pathways that impact DAT activity. Mutations of Thr53 to prevent phosphorylation led to reduced dopamine transport Vmax and total apparent loss of amphetamine-stimulated substrate efflux, supporting a major role for this residue in the transport kinetic mechanism.
ERK; MAP Kinases (MAPKs); Mass Spectrometry (MS); Protein Kinase C (PKC); SH3 Domains; 1-Methyl-4-phenylpyridinium (MPP+); PP1/2A; cis-trans Isomerization; Phospho-specific Antibody; Proline-directed Phosphorylation
3,4-Methylendioxymethamphetamine (MDMA) has both stimulatory and hallucinogenic properties which make its psychoactive effects unique and different from those of typical psychostimulant and hallucinogenic agents. The present study investigated the effects of MDMA on extracellular dopamine (DAex) and serotonin (5-HTex) levels in the striatum and prefrontal cortex (PFC) using in vivo microdialysis techniques in mice lacking DA transporters (DAT) and/or 5-HT transporters (SERT). subcutaneous injection of MDMA (3, 10 mg/kg) significantly increased striatal DAex in wild-type mice, SERT knockout mice, and DAT knockout mice, but not in DAT/SERT double-knockout mice. The MDMA-induced increase in striatal DAex in SERT knockout mice was significantly less than in wildtype mice. In the PFC, MDMA dose-dependently increased DAex levels in wildtype, DAT knockout, SERT knockout and DAT/SERT double-knockout mice to a similar extent. In contrast, MDMA markedly increased 5-HTex in wildtype and DAT knockout mice and slightly increased 5-HTex in SERT-KO and DAT/SERT double-knockout mice. The results confirm that MDMA acts at both DAT and SERT and increases DAex and 5-HTex.
MDMA; serotonin transporter; dopamine transporter; knockout; microdialysis.
Attention-Deficit Hyperactivity Disorder (ADHD) is the most commonly diagnosed disorder of school-age children. Although genetic and brain imaging studies suggest a contribution of altered dopamine (DA) signaling in ADHD, evidence of signaling perturbations contributing to risk is largely circumstantial. The presynaptic, cocaine and amphetamine (AMPH)-sensitive DA transporter (DAT) constrains DA availability at pre- and post-synaptic receptors following vesicular release and is targeted by the most commonly prescribed ADHD therapeutics. Using polymorphism discovery approaches with an ADHD cohort, we identified a human DAT (hDAT) coding variant, R615C, located in the transporter’s distal C-terminus, a region previously implicated in constitutive and regulated transporter trafficking. Here we demonstrate that whereas wildtype DAT proteins traffic in a highly regulated manner, DAT 615C proteins recycle constitutively, and demonstrate insensitivity to the endocytic effects of AMPH and protein kinase C (PKC) activation. The disrupted regulation of DAT 615C parallels a redistribution of the transporter variant away from GM1 ganglioside- and flotillin1-enriched membranes, and is accompanied by altered calcium/calmodulin-dependent protein kinase II (CaMKII) and flotillin-1 interactions. Using C-terminal peptides derived from wildtype DAT and the R615C variant, we establish that the DAT 615C C-terminus can act dominantly to preclude AMPH regulation of wildtype DAT. Mutagenesis of DAT C-terminal sequences suggest that phosphorylation of T613 may be important in sorting DAT between constitutive and regulated pathways. Together, our studies support a coupling of DAT microdomain localization with transporter regulation and provide evidence of perturbed DAT activity and DA signaling as a risk determinant for ADHD.
ADHD; dopamine; transporter; amphetamine; PKC; trafficking
The dopamine transporter (DAT) retrieves the neurotransmitter dopamine from the synaptic cleft at dopaminergic synapses. Variations in solute carrier family 6A, member 3 (SLC6A3/DAT1), the human gene encoding DAT, have been implicated in attention deficit hyperactivity and bipolar disorders, and DAT is a prominent site of action for drugs such as amphetamines and cocaine. In this issue of the JCI, Kurian et al. report that an autosomal recessive infantile parkinsonism-dystonia is caused by loss-of-function mutations in DAT that impair dopamine reuptake (see the related article beginning on page 1595). Though this might be predicted to result in dopamine excess in the synaptic cleft, it likely also causes depletion of presynaptic dopamine stores and possibly downregulation of postsynaptic dopamine receptor function, resulting in impairments in dopaminergic neurotransmission consistent with the clinical presentation. This is the first report of a genetic alteration in DAT function underlying a parkinsonian disorder.