Optical Imaging Demonstrates Tissue-Specific Metabolic Perturbations in Mblac1 Knockout Mice.

OBJECTIVE: Metabolic changes have been extensively documented in neurodegenerative brain disorders, including Parkinson's disease and Alzheimer's disease (AD). Mutations in the C. elegans swip-10 gene result in dopamine (DA) dependent motor dysfunction accompanied by DA neuron degeneration. Recently, the putative human ortholog of swip-10 (MBLAC1) was implicated as a risk factor in AD, a disorder that, like PD, has been associated with mitochondrial dysfunction. Interestingly, the AD risk associated with MBLAC1 arises in subjects with cardiovascular morbidity, suggesting a broader functional insult arising from reduced MBLAC1 protein expression and one possibly linked to metabolic alterations. METHODS: Our current studies, utilizing Mblac1 knockout (KO) mice, seek to determine whether mitochondrial respiration is affected in the peripheral tissues of these mice. We quantified the levels of mitochondrial coenzymes, NADH, FAD, and their redox ratio (NADH/FAD, RR) in livers and kidneys of wild-type (WT) mice and their homozygous KO littermates of males and females, using 3D optical cryo-imaging. RESULTS: Compared to WT, the RR of livers from KO mice was significantly reduced, without an apparent sex effect, driven predominantly by significantly lower NADH levels. In contrast, no genotype and sex differences were observed in kidney samples. Serum analyses of WT and KO mice revealed significantly elevated glucose levels in young and aged KO adults and diminished cholesterol levels in the aged KOs, consistent with liver dysfunction. DISCUSSION/CONCLUSION: As seen with C. elegans swip-10 mutants, loss of MBLAC1 protein results in metabolic changes that are not restricted to neural cells and are consistent with the presence of peripheral comorbidities accompanying neurodegenerative disease in cases where MBLAC1 expression changes impact risk.

Optical Imaging Reveals Liver Metabolic Perturbations in Mblac1 Knockout Mice.

Metabolic changes have been extensively documented in brain tissue undergoing neurodegeneration, including Parkinson's disease and Alzheimer's disease (AD). Mutations in the C. elegans swip-10 gene result in dopamine (DA) dependent motor dysfunction accompanied by DA neuron degeneration. Recently, the putative human ortholog of swip-10 (MBLAC1) was implicated as a risk factor in AD, that like PD, has been associated with mitochondrial dysfunction. Interestingly, the AD risk associated with MBLAC1 arises in subjects with cardiovascular morbidity, suggesting the possibility of a broader functional insult arising from reduced MBLAC1 protein expression, and one possibly linked to metabolic alterations. Our current studies, utilizing Mblac1 knockout (KO) mice, seeks to determine whether mitochondrial respiration is affected in peripheral tissues of these animals in this model. To initiate these studies, we quantified the levels of mitochondrial coenzymes, NADH, FAD, and their redox ratio (NADH/FAD, RR) in the livers of wild type (WT) mice and their homozygous KO littermates, using 3D optical cryo-imaging. We found that Mblac1 KO mice exhibited a greater oxidized redox state compared to WT mice. When compared to the WT group, the redox ratio of KO mice was decreased by 46.32%, driven predominantly by significantly lower NADH levels (more oxidized state). We speculate that, as seen with C. elegans swip-10 mutants, that loss of MBLAC1 protein results in deficits in tricarboxylic acid cycle (TCA) production of NADH and FAD TCA that leads to diminished cellular ATP production and oxidative stress. Such observations are consistent with changes that in the central nervous system (CNS) could support neurodegeneration and in the periphery account for comorbidities.

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.

There's no place like home? Return to the home cage triggers dopamine release in the mouse nucleus accumbens.

Various stimuli have been employed as reinforcers in preclinical rodent models to elucidate the underpinnings of reward at a molecular and circuit level, with the release of dopamine (DA) in the nucleus accumbens (NAc) as a well-replicated, physiological correlate. Many factors, however, including strain differences, sex, prior stress, and reinforcer administration protocols can influence reward responding and DA release. Although previous evidence indicates that access to the home cage can be an effective reinforcer in behavioral tasks, whether this simple environmental manipulation can trigger DA release in the NAc has not been demonstrated. Here, using fiber photometric recordings of in vivo NAc dopamine release from a genetically-encoded DA sensor, we show that the movement of animals from the home cage to a clear, polycarbonate recording chamber evokes little to no DA release following initial exposure whereas returning animals from the recording chamber to a clean, home-like cage or to the home cage robustly triggers the release of DA, comparable in size to that observed with a 10 mg/kg i.p. Cocaine injection in the recording chamber. Although DA release can be evoked in moving mice to a clean cage, this release was significantly augmented when moving animals from the clean cage to the home cage. Our data provide direct evidence that home cage return from a foreign environment results in a biochemical change consistent with that of a rewarding stimulus. This simple environmental manipulation provides a minimally invasive approach to study the reward circuitry underlying an ethologically relevant reinforcer, return to the safe confines of "home". The home cage - DA release paradigm may also represent a biomarker-driven paradigm for the evaluation of genetic and experiential events that underlie anhedonic states, characteristic of major mood disorders, and to present new opportunities to identify their treatments.

Regulation of autism-relevant behaviors by cerebellar-prefrontal cortical circuits.

Cerebellar dysfunction has been demonstrated in autism spectrum disorders (ASDs); however, the circuits underlying cerebellar contributions to ASD-relevant behaviors remain unknown. In this study, we demonstrated functional connectivity between the cerebellum and the medial prefrontal cortex (mPFC) in mice; showed that the mPFC mediates cerebellum-regulated social and repetitive/inflexible behaviors; and showed disruptions in connectivity between these regions in multiple mouse models of ASD-linked genes and in individuals with ASD. We delineated a circuit from cerebellar cortical areas Right crus 1 (Rcrus1) and posterior vermis through the cerebellar nuclei and ventromedial thalamus and culminating in the mPFC. Modulation of this circuit induced social deficits and repetitive behaviors, whereas activation of Purkinje cells (PCs) in Rcrus1 and posterior vermis improved social preference impairments and repetitive/inflexible behaviors, respectively, in male PC-Tsc1 mutant mice. These data raise the possibility that these circuits might provide neuromodulatory targets for the treatment of ASD.

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.

Functional coding variation in the presynaptic dopamine transporter associated with neuropsychiatric disorders drives enhanced motivation and context-dependent impulsivity in mice.

Recent genetic analyses have provided evidence that clinical commonalities associated with different psychiatric diagnoses often have shared mechanistic underpinnings. The development of animal models expressing functional genetic variation attributed to multiple disorders offers a salient opportunity to capture molecular, circuit and behavioral alterations underlying this hypothesis. In keeping with studies suggesting dopaminergic contributions to attention-deficit hyperactivity disorder (ADHD), bipolar disorder (BPD) and autism spectrum disorder (ASD), subjects with these diagnoses have been found to express a rare, functional coding substitution in the dopamine (DA) transporter (DAT), Ala559Val. We developed DAT Val559 knock-in mice as a construct valid model of dopaminergic alterations that drive multiple clinical phenotypes, and here evaluate the impact of lifelong expression of the variant on impulsivity and motivation utilizing the 5- choice serial reaction time task (5-CSRTT) and Go/NoGo as well as tests of time estimation (peak interval analysis), reward salience (sucrose preference), and motivation (progressive ratio test). Our findings indicate that the DAT Val559 variant induces impulsivity behaviors that are dependent upon the reward context, with increased impulsive action observed when mice are required to delay responding for a reward, whereas mice are able to withhold responding if there is a probability of reward for a correct rejection. Utilizing peak interval and progressive ratio tests, we provide evidence that impulsivity is likely driven by an enhanced motivational phenotype that also may drive faster task acquisition in operant tasks. These data provide critical validation that DAT, and more generally, DA signaling perturbations can drive impulsivity that can manifest in specific contexts and not others, and may rely on motivational alterations, which may also drive increased maladaptive reward seeking.

Sympathetic neuron-associated macrophages contribute to obesity by importing and metabolizing norepinephrine.

The cellular mechanism(s) linking macrophages to norepinephrine (NE)-mediated regulation of thermogenesis have been a topic of debate. Here we identify sympathetic neuron-associated macrophages (SAMs) as a population of cells that mediate clearance of NE via expression of solute carrier family 6 member 2 (SLC6A2), an NE transporter, and monoamine oxidase A (MAOA), a degradation enzyme. Optogenetic activation of the sympathetic nervous system (SNS) upregulates NE uptake by SAMs and shifts the SAM profile to a more proinflammatory state. NE uptake by SAMs is prevented by genetic deletion of Slc6a2 or inhibition of the encoded transporter. We also observed an increased proportion of SAMs in the SNS of two mouse models of obesity. Genetic ablation of Slc6a2 in SAMs increases brown adipose tissue (BAT) content, causes browning of white fat, increases thermogenesis, and leads to substantial and sustained weight loss in obese mice. We further show that this pathway is conserved, as human sympathetic ganglia also contain SAMs expressing the analogous molecular machinery for NE clearance, which thus constitutes a potential target for obesity treatment.

Genetic variation in alpha2-adrenoreceptors and heart rate recovery after exercise.

Heart rate recovery (HRR) after exercise is an independent predictor of adverse cardiovascular outcomes. HRR is mediated by both parasympathetic reactivation and sympathetic withdrawal and is highly heritable. We examined whether common genetic variants in adrenergic and cholinergic receptors and transporters affect HRR. In our study 126 healthy subjects (66 Caucasians, 56 African Americans) performed an 8 min step-wise bicycle exercise test with continuous computerized ECG recordings. We fitted an exponential curve to the postexercise R-R intervals for each subject to calculate the recovery constant (kr) as primary outcome. Secondary outcome was the root mean square residuals averaged over 1 min (RMS1min), a marker of parasympathetic tone. We used multiple linear regressions to determine the effect of functional candidate genetic variants in autonomic pathways (6 ADRA2A, 1 ADRA2B, 4 ADRA2C, 2 ADRB1, 3 ADRB2, 2 NET, 2 CHT, and 1 GRK5) on the outcomes before and after adjustment for potential confounders. Recovery constant was lower (indicating slower HRR) in ADRA2B 301-303 deletion carriers (n = 54, P = 0.01), explaining 3.6% of the interindividual variability in HRR. ADRA2A Asn251Lys, ADRA2C rs13118771, and ADRB1 Ser49Gly genotypes were associated with RMS1min. Genetic variability in adrenergic receptors may be associated with HRR after exercise. However, most of the interindividual variability in HRR remained unexplained by the variants examined. Noncandidate gene-driven approaches to study genetic contributions to HRR in larger cohorts will be of interest.

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

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 AMPH-evoked 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.

Good riddance to dopamine: roles for the dopamine transporter in synaptic function and dopamine-associated brain disorders.

The neurotransmitter dopamine (DA) plays a critical role in CNS circuits that provide for attention, executive function, reward responses, motivation and movement. DA is inactivated by the cocaine- and amphetamine-sensitive DA transporter (DAT), a protein that also provides a pathway for non-vesicular DA release. After a brief review of DAT function and psychostimulant actions, we consider the importance DAT in relation to the distinct firing patterns of DA neurons that permit awareness of novelty and reward. Finally, we review recent efforts to gather direct support for DAT-linked disorders, with a specific focus on DAT mutations recently identified in subjects with ADHD.

Genetic targeting of the amphetamine and methylphenidate-sensitive dopamine transporter: on the path to an animal model of attention-deficit hyperactivity disorder.

Alterations in dopamine (DA) signaling underlie the most widely held theories of molecular and circuit level perturbations that lead to risk for attention-deficit hyperactivity disorder (ADHD). The DA transporter (DAT), a presynaptic reuptake protein whose activity provides critical support for DA signaling by limiting DA action at pre- and postsynaptic receptors, has been consistently associated with ADHD through pharmacological, behavioral, brain imaging and genetic studies. Currently, the animal models of ADHD exhibit significant limitations, stemming in large part from their lack of construct validity. To remedy this situation, we have pursued the creation of a mouse model derived from a functional nonsynonymous variant in the DAT gene (SLC6A3) of ADHD probands. We trace our path from the identification of these variants to in vitro biochemical and physiological studies to the production of the DAT Val559 mouse model. We discuss our initial findings with these animals and their promise in the context of existing rodent models of ADHD.

Extracellular norepinephrine clearance by the norepinephrine transporter is required for skeletal homeostasis.

Changes in bone remodeling induced by pharmacological and genetic manipulation of ß-adrenergic receptor (ßAR) signaling in osteoblasts support a role of sympathetic nerves in the regulation of bone remodeling. However, the contribution of endogenous sympathetic outflow and nerve-derived norepinephrine (NE) to bone remodeling under pathophysiological conditions remains unclear. We show here that differentiated osteoblasts, like neurons, express the norepinephrine transporter (NET), exhibit specific NE uptake activity via NET and can catabolize, but not generate, NE. Pharmacological blockade of NE transport by reboxetine induced bone loss in WT mice. Similarly, lack of NE reuptake in norepinephrine transporter (Net)-deficient mice led to reduced bone formation and increased bone resorption, resulting in suboptimal peak bone mass and mechanical properties associated with low sympathetic outflow and high plasma NE levels. Last, daily sympathetic activation induced by mild chronic stress was unable to induce bone loss, unless NET activity was blocked. These findings indicate that the control of endogenous NE release and reuptake by presynaptic neurons and osteoblasts is an important component of the complex homeostatic machinery by which the sympathetic nervous system controls bone remodeling. These findings also suggest that drugs antagonizing NET activity, used for the treatment of hyperactivity disorders, may have deleterious effects on bone accrual.

Norepinephrine transporter variant A457P knock-in mice display key features of human postural orthostatic tachycardia syndrome.

Postural orthostatic tachycardia syndrome (POTS) is a common autonomic disorder of largely unknown etiology that presents with sustained tachycardia on standing, syncope and elevated norepinephrine spillover. Some individuals with POTS experience anxiety, depression and cognitive dysfunction. Previously, we identified a mutation, A457P, in the norepinephrine (NE; also known as noradrenaline) transporter (NET; encoded by SLC6A2) in POTS patients. NET is expressed at presynaptic sites in NE neurons and plays a crucial role in regulating NE signaling and homeostasis through NE reuptake into noradrenergic nerve terminals. Our in vitro studies demonstrate that A457P reduces both NET surface trafficking and NE transport and exerts a dominant-negative impact on wild-type NET proteins. Here we report the generation and characterization of NET A457P mice, demonstrating the ability of A457P to drive the POTS phenotype and behaviors that are consistent with reported comorbidities. Mice carrying one A457P allele (NET(+/P)) exhibited reduced brain and sympathetic NE transport levels compared with wild-type (NET(+/+)) mice, whereas transport activity in mice carrying two A457P alleles (NET(P/P)) was nearly abolished. NET(+/P) and NET(P/P) mice exhibited elevations in plasma and urine NE levels, reduced 3,4-dihydroxyphenylglycol (DHPG), and reduced DHPG:NE ratios, consistent with a decrease in sympathetic nerve terminal NE reuptake. Radiotelemetry in unanesthetized mice revealed tachycardia in NET(+/P) mice without a change in blood pressure or baroreceptor sensitivity, consistent with studies of human NET A457P carriers. NET(+/P) mice also demonstrated behavioral changes consistent with CNS NET dysfunction. Our findings support that NET dysfunction is sufficient to produce a POTS phenotype and introduces the first genetic model suitable for more detailed mechanistic studies of the disorder and its comorbidities.

Genetic variation in the presynaptic norepinephrine transporter is associated with blood pressure responses to exercise in healthy humans.

BACKGROUND: The presynaptic norepinephrine transporter (NET) mediates synaptic clearance and recycling of norepinephrine. NET-deficient transgenic mice have elevated blood pressure (BP), heart rate, and catecholamine concentrations. However, the in-vivo effects of common NET variants on cardiovascular regulation at rest and during exercise are unknown. METHODS: We studied cardiovascular responses and plasma catecholamine concentrations at rest and during bicycle exercise at increasing workloads (25, 50, and 75 W) in 145 healthy participants. We used multiple linear regressions to analyze the effect of common, purportedly functional polymorphisms in NET (rs2242446 and rs28386840) on cardiovascular measures. RESULTS: 44 and 58.9% of participants carried at least one variant allele for NET T-182C and A-3081T, respectively. Systolic BP during exercise and systolic BP-area under the curve were higher in carriers of variant NET alleles (P=0.003 and 0.009 for T-182C and A-3081T, respectively) and NET haplotype -182C/-3081T compared with -182T/-3081A (all P<0.01). Diastolic BP during exercise was also higher at lower, but not at higher exercise stages in carriers of NET -182C (P<0.01) and -3081T variants (P<0.05). NET genotypes were not associated with catecholamine concentrations or heart rate. CONCLUSION: Common genetic NET variants (-182C and -3081T) are associated with greater BP response to exercise in humans.

Novel and functional norepinephrine transporter protein variants identified in attention-deficit hyperactivity disorder.

Attention-deficit hyperactivity disorder (ADHD) is a highly heritable disorder of impaired behavioral inhibition, increased motor activity, and inattention. The norepinephrine transporter (NET, SLC6A2) represents an important candidate gene for contribution to ADHD because it regulates catecholamine extracellular and tissue concentrations and contributes to executive functions disrupted in ADHD, and NET is a target for most effective ADHD therapeutics. We identified four NET coding single nucleotide polymorphisms (SNPs) in two ADHD sample sets; two SNPs produce protein variants (T283M, V245I), one of which, T283M, is a novel variant. Examination of the maternal family members through whom the T283M mutation was transmitted, provided no additional ADHD diagnoses. Given the previous identification of a NET mutation that contributes to a familial tachycardia syndrome, we examined autonomic function to reveal in the proband the highest standing-induced increase in heart rate among the ADHD subjects examined. We measured [3H]NE and [3H]dopamine transport for T283M, V245I, and a previously identified NET variant, T283R. T283M and V245I demonstrated decreased substrate transport, as did T283R, suggesting that the T283 residue is sensitive to mutation. Identification of polymorphic sites within NET, specifically those that produce functional consequences, is one critical step in elucidating the genetic variation contributing to the heritable component of diseases such as ADHD.

Choline transporter gene variation is associated with attention-deficit hyperactivity disorder.

The neurotransmitter acetylcholine (ACh) plays a critical role in brain circuits mediating motor control, attention, learning and memory. Cholinergic dysfunction is associated with multiple brain disorders including Alzheimer's Disease, addiction, schizophrenia and Attention-Deficit Hyperactivity Disorder (ADHD). The presynaptic choline transporter (CHT, SLC5A7) is the major, rate-limiting determinant of ACh production in the brain and periphery and is consequently upregulated during tasks that require sustained attention. Given the contribution of central cholinergic circuits to the control of movement and attention, we hypothesized that functional CHT gene variants might impact risk for ADHD. We performed a case-control study, followed by family-based association tests on a separate cohort, of two purportedly functional CHT polymorphisms (coding variant Ile89Val (rs1013940) and a genomic SNP 3' of the CHT gene (rs333229), affording both a replication sample and opportunities to reduce potential population stratification biases. Initial genotyping of pediatric ADHD subjects for two purportedly functional CHT alleles revealed a 2-3 fold elevation of the Val89 allele (n = 100; P = 0.02) relative to healthy controls, as well as a significant decrease of the 3'SNP minor allele in Caucasian male subjects (n = 60; P = 0.004). In family based association tests, we found significant overtransmission of the Val89 variant to children with a Combined subtype diagnosis (OR = 3.16; P = 0.01), with an increased Odds Ratio for a haplotype comprising both minor alleles. These studies show evidence of cholinergic deficits in ADHD, particularly for subjects with the Combined subtype, and, if replicated, may encourage further consideration of cholinergic agonist therapy in the disorder.

The functional impact of SLC6 transporter genetic variation.

Solute carrier 6 (SLC6) is a gene family of ion-coupled plasma membrane cotransporters, including transporters of neurotransmitters, amino acids, and osmolytes that mediate the movement of their substrates into cells to facilitate or regulate synaptic transmission, neurotransmitter recycling, metabolic function, and fluid homeostasis. Polymorphisms in transporter genes may influence expression and activity of transporters and contribute to behavior, traits, and disease. Determining the relationship between the monoamine transporters and complex psychiatric disorders has been a particular challenge that is being met by evolving approaches. Elucidating the functional consequences of and interactions among polymorphic sites is advancing our understanding of this relationship. Examining the influence of environmental influences, especially early-life events, has helped bridge the gap between genotype and phenotype. Refining phenotypes, through assessment of endophenotypes, specific behavioral tasks, medication response, and brain network properties has also improved detection of the impact of genetic variation on complex behavior and disease.

A polymorphism in the norepinephrine transporter gene alters promoter activity and is associated with attention-deficit hyperactivity disorder.

The norepinephrine transporter critically regulates both neurotransmission and homeostasis of norepinephrine in the nervous system. In this study, we report a previously uncharacterized and common A/T polymorphism at -3081 upstream of the transcription initiation site of the human norepinephrine transporter gene [solute carrier family 6, member 2 (SLC6A2)]. Using both homologous and heterologous promoter-reporter constructs, we found that the -3081(T) allele significantly decreases promoter function compared with the A allele. Interestingly, this T allele creates a new palindromic E2-box motif that interacts with Slug and Scratch, neural-expressed transcriptional repressors binding to the E2-box motif. We also found that both Slug and Scratch repress the SLC6A2 promoter activity only when it contains the T allele. Finally, we observed a significant association between the -3081(A/T) polymorphism and attention-deficit hyperactivity disorder (ADHD), suggesting that anomalous transcription factor-based repression of SLC6A2 may increase risk for the development of attention-deficit hyperactivity disorder and other neuropsychiatric diseases.