Gregory M. Miller

The Dopamine Transporter and Attention-Deficit/Hyperactivity Disorder.

The high incidence of attention-deficit/hyperactivity disorder (ADHD) and escalating use of ADHD medications present a compelling case for clarifying the pathophysiology of, and developing laboratory or radiologic tests for, ADHD. Currently, the majority of specific genes implicated in ADHD encode components of catecholamine signaling systems. Of these, the dopamine transporter (DAT) is a principal target of the most widely used antihyperactivity medications (amphetamine and methylphenidate); the DAT gene is associated with ADHD, and some studies have detected abnormal levels of the DAT in brain striatum of ADHD subjects. Medications for ADHD interfere with dopamine transport by brain-region- and drug-specific mechanisms, indirectly activating dopamine- and possibly norepinephrine-receptor subtypes that are implicated in enhancing attention and experiential salience. The most commonly used DAT-selective ADHD medications raise extracellular dopamine levels in DAT-rich brain regions. In brain regions expressing both the DAT and the norepinephrine transporter (NET), the relative contributions of dopamine and norepinephrine to ADHD pathophysiology and therapeutic response are obfuscated by the capacity of the NET to clear dopamine as well as norepinephrine. Thus, ADHD medications targeting DAT or NET might disperse dopamine widely and consign dopamine storage and release to regulation by noradrenergic, as well as dopaminergic neurons.

Modafinil Occupies Dopamine and Norepinephrine Transporters in Vivo and Modulates the Transporters and Trace Amine Activity in Vitro

2-[(Diphenylmethyl) sulfinyl]acetamide (modafinil), prescribed principally to treat narcolepsy, is undergoing assessment for other neuropsychiatric disorders and medical conditions. The neurochemical substrates of modafinil are unresolved. We postulated that modafinil enhances wakefulness by modulating dopamine (DAT), norepinephrine (NET), or serotonin (SERT) transporter activities. In vivo, we determined DAT and NET occupancy by modafinil by positron emission tomography imaging; in vitro, we determined modafinil activity at the DAT, NET, SERT, and rhesus monkey trace amine receptor 1 (TA1). In rhesus monkey, modafinil occupancy of striatal DAT was detected by [11C]2β-carbomethoxy-3β-4-(fluorophenyl)tropane and of thalamic NET by [11C](S,S)-2-(α-(2-methoxyphenoxy)-benzyl)morpholine. In vitro, modafinil effects in DAT-human embryonic kidney (HEK), NET-HEK, and SERT-HEK cells were investigated alone or combined with the TA1 receptor. Modafinil (i.v.) occupied striatal DAT sites (5 mg/kg: 35 ± 12%, n = 4; 8 mg/kg: 54 ± 3%, n = 3). In thalamus, modafinil occupied NET sites (5 mg/kg: 16 ± 7.8%, n = 6; 8 mg/kg: 44 ± 12%; n = 2). In vitro, modafinil inhibited [3H]dopamine (IC50 = 6.4 μM), [3H]norepinephrine (IC50 = 35.6 μM), and [3H]serotonin (IC50 > 500 μM) transport via the human DAT, NET, and SERT. Modafinil did not activate the TA1 receptor in TA1-HEK cells, but it augmented a monoamine transporter-dependent enhancement of phenethylamine activation of TA1 in TA1-DAT and TA1-NET cells, but not in TA1-SERT cells. The present data provide compelling evidence that modafinil occupies the DAT and NET in living brain of rhesus monkeys and raise the possibility that modafinil affects wakefulness by interacting with catecholamine transporters in brain.

Balancing selection and the evolution of functional polymorphism in Old World monkey TRIM5α

Retroviral restriction factor TRIM5α exhibits a high degree of sequence variation among primate species. It has been proposed that this diversity is the cumulative result of ancient, lineage-specific episodes of positive selection. Here, we describe the contribution of within-species variation to the evolution of TRIM5α. Sampling within two geographically distinct Old World monkey species revealed extensive polymorphism, including individual polymorphisms that predate speciation (shared polymorphism). In some instances, alleles were more closely related to orthologues of other species than to one another. Both silent and nonsynonymous changes clustered in two domains. Functional assays revealed consequences of polymorphism, including differential restriction of a small panel of retroviruses by very similar alleles. Together, these features indicate that the primate TRIM5α locus has evolved under balancing selection. Except for the MHC there are few, if any, examples of long-term balancing selection in primates. Our results suggest a complex evolutionary scenario, in which fixation of lineage-specific adaptations is superimposed on a subset of critical polymorphisms that predate speciation events and have been maintained by balancing selection for millions of years.

MDMA (Ecstasy) and human dopamine, norepinephrine, and serotonin transporters: implications for MDMA-induced neurotoxicity and treatment

Rationale3,4-Methylenedioxymethamphetamine (MDMA, designated as “Ecstasy” if illicitly marketed in tablet form) induces significant decrements in neuronal serotonin (5-HT) markers in humans, nonhuman primates, and rats as a function of dosing and dosing regimen. In rats, MDMA-mediated effects are attributed, in part, to selective high-affinity transport of MDMA into 5-HT neurons by the 5-HT transporter (SERT), followed by extensive 5-HT release.ObjectivesTo clarify whether SERT-selective effects of MDMA at human monoamine transporters can account for the reported MDMA-induced selective toxicity of serotonin neurons in primate brain.MethodsWe investigated the interaction of [3H](±, RS)- (+, S)- and (−, R)-MDMA with the human SERT, dopamine (DA) transporter (DAT), and norepinephrine (NE) transporter (NET) in stably transfected human embryo kidney (HEK)-293 cells.ResultsThe human DAT, NET, and SERT actively transported [3H]RS(±)-MDMA saturably, stereoselectively, and in a temperature-, concentration-, and transporter-dependent manner. MDMA exhibited the highest affinity for the NET≫SERT≥DAT, the same rank order for MDMA inhibition of [3H]DA, [3H]NE, and [3H]5-HT transport and stimulated release of the [3H]monoamines, which differed from reports derived from rodent monoamine transporters. The extent of MDMA-induced release of 5-HT was higher compared with release of DA or NE.ConclusionsThe affinity of MDMA for the human SERT in transfected cells does not clarify the apparent selective toxicity of MDMA for serotonin neurons, although conceivably, its higher efficacy for stimulating 5-HT release may be a distinguishing factor. The findings highlight the need to investigate MDMA effects in DAT-, SERT-, and NET-expressing neurons in the primate brain and the therapeutic potential of NET or DAT inhibitors, in addition to SERT-selective inhibitors, for alleviating the pharmacological effects of MDMA.

The Emerging Role of Trace Amine Associated Receptor 1 in the Functional Regulation of Monoamine Transporters and Dopaminergic Activity

J. Neurochem. (2011) 116, 164–176.

Cloning of dopamine, norepinephrine and serotonin transporters from monkey brain: relevance to cocaine sensitivity

We used RT-PCR to clone monoamine transporters from Macaca mulatta, Macaca fasicularis and Saimiri sciureus (dopamine transporter; DAT) and Macaca mulatta (norepinephrine transporter; NET and serotonin transporter; SERT). Monkey DAT, NET and SERT proteins were >98% homologous to human and, when expressed in HEK-293 cells, displayed drug affinities and uptake kinetics that were highly correlated with monkey brain or human monoamine transporters. In contrast to reports of other species, we discovered double (leucine for phenylalanine 143 and arginine for glutamine 509; Variant I) and single (proline for leucine 355; Variant II) amino acid variants of DAT. Variant I displayed dopamine transport kinetics and binding affinities for various DAT blockers (including cocaine) versus [3H] CFT (WIN 35, 428) that were identical to wild-type DAT (n=7 drugs; r(2)=0.991). However, we detected a six-fold difference in the affinity of cocaine versus [3H] cocaine between Variant I (IC(50): 488+/-102 nM, SEM, n=3) and wild-type DAT (IC(50): 79+/-8.2 nM, n=3, P<0.05). Variant II was localized intracellularly in HEK-293 cells, as detected by confocal microscopy, and had very low levels of binding and dopamine transport. Also discovered was a novel exon 5 splice variant of NET that displayed very low levels of transport and did not bind cocaine. With NetPhos analysis, we detected a number of highly conserved putative phosphorylation sites on extracellular as well as intracellular loops of the DAT, NET, and SERT, which may be functional for internalized transporters. The homology and functional similarity of human and monkey monoamine transporters further support the value of primates in investigating the role of monoamine transporters in substance abuse mechanisms, neuropsychiatric disorders and development of diagnostic and therapeutic agents.

A mu-opioid receptor single nucleotide polymorphism in rhesus monkey: association with stress response and aggression

Variations in the human mu-opioid receptor gene have driven exploration of their biochemical, physiological and pathological relevance. We investigated the existence of variations in the nonhuman primate mu-opioid receptor gene to determine whether nonhuman primates can model genotype/phenotype associations of relevance to humans. Similar to the A118G single nucleotide polymorphism (SNP) in the human mu-opioid receptor gene, a SNP discovered in the rhesus monkey mu-opioid receptor gene (C77G) alters an amino acid in the N-terminal arm of the receptor (arginine for proline at position 26). Two mu-opioid receptor coding regions isolated from a single heterozygous (C77/G77) rhesus monkey brain were expressed in HEK-293 cells and characterized in radioreceptor assays. Paralleling the findings of increased affinity of β-endorphin by the A118G allele in the human, the rhesus monkey mu-opioid receptor protein derived from the G77-containing clone demonstrated a 3.5-fold greater affinity for β-endorphin than the receptor derived from the C77-containing clone. An assay developed to assess the incidence of the C77G SNP in a behaviorally and physiologically characterized cohort of rhesus monkeys (n=32) indicated that 44% were homozygous for C77-containing alleles, 50% were heterozygous and 6% were homozygous for G77-containing alleles. The presence of G77-containing alleles was associated with significantly lower basal and ACTH-stimulated plasma cortisol levels (P<0.03–0.05 and P<0.02, respectively) and a significantly higher aggressive threat score (P<0.05) in vivo. In a cohort of 20 monkeys, a trend towards an inverse correlation between aggressive threat and plasma cortisol levels was observed. The findings suggest that mu-opioid receptor haplotypes in monkeys can contribute to individual variability in stress response and related aggression. The data support the use of nonhuman primates to investigate mu-opioid receptor genotype/phenotype relations of relevance to humans.

Functional characterization of the human TPH2 5' regulatory region: untranslated region and polymorphisms modulate gene expression in vitro.

Tryptophan hydroxylase-2 (TPH2) is a recently identified TPH isoform responsible for neuronal serotonin (5-HT) synthesis, and TPH2 polymorphisms are associated with a range of behavioral traits and psychiatric disorders. This study characterized cis-acting elements and three common polymorphisms (−703G/T, −473T/A, and 90A/G) in the 5′ regulatory region of human TPH2 by using luciferase reporter assay, quantitative real-time PCR, and electrophoretic mobility shift assay (EMSA). The core promoter of human TPH2 was localized to the region between −107 and +7, and the segment of +8 to +53 within the 5′-UTR was found to exert a potent inhibitory effect on gene expression at both transcriptional and post-transcriptional levels. In both RN46A and HEK-293 cell lines, the TTA (−703T/−473T/90A) haplotype of the three polymorphisms showed the lowest gene expression compared with other haplotypes, and the −703G/T and −473T/A polymorphisms tended to exert a synergic effect on gene expression dependent upon the sequence of the 5′-UTR. In RN46A, the 90A/G polymorphism significantly increased luciferase activity and mRNA level irrespective of the other two polymorphisms, while in HEK-293 cells the effect of 90A/G was dependent on the alleles at loci −703 and −473. EMSA showed that all the three polymorphisms potentially alter DNA–protein interactions, while the 90A/G polymorphism predictably alters the 5′-UTR secondary structure of mRNA and influences RNA–protein interactions. In conclusion, our present study demonstrates that both the 5′-UTR and common polymorphisms (especially the 90A/G) in the 5′ regulatory region of human TPH2 have a significant impact on gene expression.

Analysis of copy number variation in the rhesus macaque genome identifies candidate loci for evolutionary and human disease studies

Copy number variants (CNVs) are heritable gains and losses of genomic DNA in normal individuals. While copy number variation is widely studied in humans, our knowledge of CNVs in other mammalian species is more limited. We have designed a custom array-based comparative genomic hybridization (aCGH) platform with 385 000 oligonucleotide probes based on the reference genome sequence of the rhesus macaque (Macaca mulatta), the most widely studied non-human primate in biomedical research. We used this platform to identify 123 CNVs among 10 unrelated macaque individuals, with 24% of the CNVs observed in multiple individuals. We found that segmental duplications were significantly enriched at macaque CNV loci. We also observed significant overlap between rhesus macaque and human CNVs, suggesting that certain genomic regions are prone to recurrent CNV formation and instability, even across a total of approximately 50 million years of primate evolution ( approximately 25 million years in each lineage). Furthermore, for eight of the CNVs that were observed in both humans and macaques, previous human studies have reported a relationship between copy number and gene expression or disease susceptibility. Therefore, the rhesus macaque offers an intriguing, non-human primate outbred model organism with which hypotheses concerning the specific functions of phenotypically relevant human CNVs can be tested.

Trace Amine-Associated Receptor 1 Is a Modulator of the Dopamine Transporter

Trace amine-associated receptor 1 (TAAR1) is a G protein-coupled receptor activated by a broad range of monoamines and amphetamine-related psychostimulants. Recent studies demonstrated wide distribution of TAAR1 in brain, coexpression of TAAR1 with dopamine transporter (DAT) in a subset of dopamine neurons in both mouse and rhesus monkey substantia nigra, and monoamine transporter-modulated activation. This study explored whether TAAR1 could influence DAT-mediated dopamine uptake and efflux. Rhesus monkey TAAR1 expressed with DAT in human embryonic kidney 293 cells was dose-dependently activated by dopamine or (+)-methamphetamine. This activation resulted in large cAMP increases and a transient reduction in [3H]dopamine accumulation within the cells, which was similar to the effect of dopamine D1 receptor (D1) or forskolin treatment. In addition, TAAR1 effects on dopamine uptake could be blocked by a protein kinase A or protein kinase C (PKC) inhibitor. [3H]Dopamine efflux assays performed in Dulbeccos modified Eagles medium displayed a TAAR1-dependent spontaneous [3H]dopamine efflux that was dose-dependently augmented by dopamine or (+)-methamphetamine and that was blocked by either methylphenidate or a PKC inhibitor. DAT cells in Krebs-HEPES buffer had an apparent spontaneous [3H]dopamine loss, but it could not be blocked by either methylphenidate or a PKC inhibitor. Taken together, this study provides evidence that TAAR1 is involved in functional regulation of DAT and suggests that TAAR1 is a potentially important target for therapeutics for methamphetamine addiction.