Motility phenotype in a zebrafish vmat2 mutant

In the present study, we characterize a novel zebrafish mutant of solute carrier 18A2 (slc18a2), also known as vesicular monoamine transporter 2 (vmat2), that exhibits a behavioural phenotype partially consistent with human Parkinson´s disease. At six days-post-fertilization, behaviour was analysed and demonstrated that vmat2 homozygous mutant larvae, relative to wild types, show changes in motility in a photomotor assay, altered sleep parameters, and reduced dopamine cell number. Following an abrupt lights-off stimulus mutant larvae initiate larger movements but subsequently inhibit them to a lesser extent in comparison to wild-type larvae. Conversely, during a lights-on period, the mutant larvae are hypomotile. Thigmotaxis, a preference to avoid the centre of a behavioural arena, was increased in homozygotes over heterozygotes and wild types, as was daytime sleep ratio. Furthermore, incubating mutant larvae in pramipexole or L-Dopa partially rescued the motor phenotypes, as did injecting glial cell-derived neurotrophic factor (GDNF) into their brains. This novel vmat2 model represents a tool for high throughput pharmaceutical screens for novel therapeutics, in particular those that increase monoamine transport, and for studies of the function of monoamine transporters.

Bile Acids Gate Dopamine Transporter Mediated Currents

Bile acids (BAs) are molecules derived from cholesterol that are involved in dietary fat absorption. New evidence supports an additional role for BAs as regulators of brain function. Sterols such as cholesterol interact with monoamine transporters, including the dopamine (DA) transporter (DAT) which plays a key role in DA neurotransmission and reward. This study explores the interactions of the BA, obeticholic acid (OCA), with DAT and characterizes the regulation of DAT activity via both electrophysiology and molecular modeling. We expressed murine DAT (mDAT) in Xenopus laevis oocytes and confirmed its functionality. Next, we showed that OCA promotes a DAT-mediated inward current that is Na+-dependent and not regulated by intracellular calcium. The current induced by OCA was transient in nature, returning to baseline in the continued presence of the BA. OCA also transiently blocked the DAT-mediated Li+-leak current, a feature that parallels DA action and indicates direct binding to the transporter in the absence of Na+. Interestingly, OCA did not alter DA affinity nor the ability of DA to promote a DAT-mediated inward current, suggesting that the interaction of OCA with the transporter is non-competitive, regarding DA. Docking simulations performed for investigating the molecular mechanism of OCA action on DAT activity revealed two potential binding sites. First, in the absence of DA, OCA binds DAT through interactions with D421, a residue normally involved in coordinating the binding of the Na+ ion to the Na2 binding site (Borre et al., J. Biol. Chem., 2014, 289, 25764–25773; Cheng and Bahar, Structure, 2015, 23, 2171–2181). Furthermore, we uncover a separate binding site for OCA on DAT, of equal potential functional impact, that is coordinated by the DAT residues R445 and D436. Binding to that site may stabilize the inward-facing (IF) open state by preventing the re-formation of the IF-gating salt bridges, R60-D436 and R445-E428, that are required for DA transport. This study suggests that BAs may represent novel pharmacological tools to regulate DAT function, and possibly, associated behaviors.

Interaction Profiles of Central Nervous System Active Drugs at Human Organic Cation Transporters 1–3 and Human Plasma Membrane Monoamine Transporter

Many psychoactive compounds have been shown to primarily interact with high-affinity and low-capacity solute carrier 6 (SLC6) monoamine transporters for norepinephrine (NET; norepinephrine transporter), dopamine (DAT; dopamine transporter) and serotonin (SERT; serotonin transporter). Previous studies indicate an overlap between the inhibitory capacities of substances at SLC6 and SLC22 human organic cation transporters (SLC22A1–3; hOCT1–3) and the human plasma membrane monoamine transporter (SLC29A4; hPMAT), which can be classified as high-capacity, low-affinity monoamine transporters. However, interactions between central nervous system active substances, the OCTs, and the functionally-related PMAT have largely been understudied. Herein, we report data from 17 psychoactive substances interacting with the SLC6 monoamine transporters, concerning their potential to interact with the human OCT isoforms and hPMAT by utilizing radiotracer-based in vitro uptake inhibition assays at stably expressing human embryonic kidney 293 cells (HEK293) cells. Many compounds inhibit substrate uptake by hOCT1 and hOCT2 in the low micromolar range, whereas only a few substances interact with hOCT3 and hPMAT. Interestingly, methylphenidate and ketamine selectively interact with hOCT1 or hOCT2, respectively. Additionally, 3,4-methylenedioxymethamphetamine (MDMA) is a potent inhibitor of hOCT1 and 2 and hPMAT. Enantiospecific differences of R- and S-α-pyrrolidinovalerophenone (R- and S-α-PVP) and R- and S-citalopram and the effects of aromatic substituents are explored. Our results highlight the significance of investigating drug interactions with hOCTs and hPMAT, due to their role in regulating monoamine concentrations and xenobiotic clearance.

Overlap and Specificity in the Substrate Spectra of Human Monoamine Transporters and Organic Cation Transporters 1, 2, and 3

Human monoamine transporters (MATs) are cation transporters critically involved in neuronal signal transmission. While inhibitors of MATs have been intensively studied, their substrate spectra have received far less attention. Polyspecific organic cation transporters (OCTs), predominantly known for their role in hepatic and renal drug elimination, are also expressed in the central nervous system and might modulate monoaminergic signaling. Using HEK293 cells overexpressing MATs or OCTs, we compared uptake of 48 compounds, mainly phenethylamine and tryptamine derivatives including matched molecular pairs, across noradrenaline, dopamine and serotonin transporters and OCTs (1, 2, and 3). Generally, MATs showed surprisingly high transport activities for numerous analogs of neurotransmitters, but their substrate spectra were limited by molar mass. Human OCT2 showed the broadest substrate spectrum, and also the highest overlap with MATs substrates. Comparative kinetic analyses revealed that the radiotracer meta-iodobenzylguanidine had the most balanced uptake across all six transporters. Matched molecular pair analyses comparing MAT and OCT uptake using the same methodology could provide a better understanding of structural determinants for high cell uptake by MATs or OCTs. The data may result in a better understanding of pharmacokinetics and toxicokinetics of small molecular organic cations and, possibly, in the development of more specific radiotracers for MATs.

The Cellular Effects of Nicotine and Tobacco Particulate Matter on Monoamine Transporters

<p>Cigarette smoking causes nearly 6 million deaths worldwide every year (WHO, 2011). Current smoking cessation therapies available to the public are only marginally effective (Jorenby, 2006; Balfour et al., 2000), partly due to our incomplete understanding of the molecular biology of smoking addiction. The majority of studies examining the molecular biology of smoking addiction have focused on nicotine alone. However, there is a growing body of evidence that non-nicotinic components of cigarette smoke contribute to smoking addiction. Nicotine has previously been shown to modulate the function of the monoamine transporters, but studies in the literature are often contradictory and this effect is not completely understood (see Danielson et al., 2011 for review). Furthermore, very few studies have examined the effects of non-nicotinic components of tobacco smoke on the monoamine transporters. This thesis has examined the effects of nicotine and a tobacco extract (TPM) on the dopamine, serotonin, and norepinephrine transporters (DAT, NET, and SERT). Changes in monoamine transporter function, protein expression, and mRNA expression were measured ex vivo in discrete regions of the rat brain following chronic and acute in vivo nicotine and TPM treatment, and in vitro nicotine and TPM treatment. We found that nicotine and TPM affect monoamine transporter function, in a time- and dose-dependent manner, and that intact whole brain circuitry is required for these effects to be seen. In particular, nicotine (0.35 mg/kg) and TPM (containing 0.35 mg/kg nicotine) significantly decreased DAT function in the NAc at 30 min. This effect did not result in a corresponding decrease in DAT protein expression and was mediated by nicotinic receptors containing β2 subunits. Furthermore, TPM caused some changes in monoamine transporter function and mRNA expression that were not observed with nicotine alone. In functional studies this effect was particularly seen in the striatum of rats treated with nicotine (0.35 mg/kg) or TPM (containing 0.35 mg/kg nicotine). Overall these data demonstrate that nicotine affects monoamine transporter function in a nicotinic receptor-dependent manner, and that nicotine and TPM have different effects on monoamine transporter function and expression. This is the first study to examine the effects of TPM on monoamine transporter function, and supports previous evidence of a contribution of non-nicotinic components of cigarette smoke to neuroadaptations related to smoking. Findings from this study contribute to knowledge on the molecular biology of smoking addiction, which could in future lead to the development of more effective smoking cessation therapies.</p>

The Cellular Effects of Nicotine and Tobacco Particulate Matter on Monoamine Transporters

<p>Cigarette smoking causes nearly 6 million deaths worldwide every year (WHO, 2011). Current smoking cessation therapies available to the public are only marginally effective (Jorenby, 2006; Balfour et al., 2000), partly due to our incomplete understanding of the molecular biology of smoking addiction. The majority of studies examining the molecular biology of smoking addiction have focused on nicotine alone. However, there is a growing body of evidence that non-nicotinic components of cigarette smoke contribute to smoking addiction. Nicotine has previously been shown to modulate the function of the monoamine transporters, but studies in the literature are often contradictory and this effect is not completely understood (see Danielson et al., 2011 for review). Furthermore, very few studies have examined the effects of non-nicotinic components of tobacco smoke on the monoamine transporters. This thesis has examined the effects of nicotine and a tobacco extract (TPM) on the dopamine, serotonin, and norepinephrine transporters (DAT, NET, and SERT). Changes in monoamine transporter function, protein expression, and mRNA expression were measured ex vivo in discrete regions of the rat brain following chronic and acute in vivo nicotine and TPM treatment, and in vitro nicotine and TPM treatment. We found that nicotine and TPM affect monoamine transporter function, in a time- and dose-dependent manner, and that intact whole brain circuitry is required for these effects to be seen. In particular, nicotine (0.35 mg/kg) and TPM (containing 0.35 mg/kg nicotine) significantly decreased DAT function in the NAc at 30 min. This effect did not result in a corresponding decrease in DAT protein expression and was mediated by nicotinic receptors containing β2 subunits. Furthermore, TPM caused some changes in monoamine transporter function and mRNA expression that were not observed with nicotine alone. In functional studies this effect was particularly seen in the striatum of rats treated with nicotine (0.35 mg/kg) or TPM (containing 0.35 mg/kg nicotine). Overall these data demonstrate that nicotine affects monoamine transporter function in a nicotinic receptor-dependent manner, and that nicotine and TPM have different effects on monoamine transporter function and expression. This is the first study to examine the effects of TPM on monoamine transporter function, and supports previous evidence of a contribution of non-nicotinic components of cigarette smoke to neuroadaptations related to smoking. Findings from this study contribute to knowledge on the molecular biology of smoking addiction, which could in future lead to the development of more effective smoking cessation therapies.</p>

A ROLE FOR CORTICAL DOPAMINE IN THE PARADOXICAL CALMING EFFECTS OF PSYCHOSTIMULANTS

Attention deficit hyperactivity disorder (ADHD) affects young children and manifests symptoms such as hyperactivity, impulsivity and cognitive disabilities. Psychostimulants, which are the primary treatment for ADHD, target monoamine transporters and have a paradoxical calming effect, but their mechanism of action, is unclear. Studies using the dopamine (DA) transporter (DAT) knockout mice, which have elevated striatal DA levels and are considered an animal model of ADHD, have suggested that the paradoxical calming effect of psychostimulants might be through the actions on serotonin neurotransmission. On the other hand, newer non-stimulant class of drugs such as atomoxetine and Intuniv suggest that targeting the norepinephrine (NE) system in the PFC might explain this paradoxical calming effect. We sought to decipher the mechanism of this paradoxical effect of psychostimulants through an integrated approach using ex vivo monoamine efflux experiments, monoamine transporter knockout mice, drug infusions and behavior. Our ex vivo efflux experiments reveal that NE transporter (NET) blocker desipramine elevates both norepinephrine and dopamine but not serotonin levels, in PFC tissue slices from wild-type and DAT-KO but not NET KO mice. However, serotonin (5-HT) transporter (SERT) inhibitor fluoxetine elevates only serotonin in all three genotypes. Systemic administration of both desipramine and fluoxetine but local PFC infusion of only desipramine and not fluoxetine inhibits hyperactivity in the DAT-KO mice. In contrast, pharmacological norepinephrine depletion but dopamine elevation using Nepicastat also inhibits hyperactivity in DATKO mice. Together, these data suggest that elevation of PFC dopamine and not norepinephrine or serotonin as a convergent mechanism for the paradoxical psychostimulant effects observe in ADHD therapy.

Bile acids gate dopamine transporter mediated currents

Bile acids (BAs) are molecules derived from cholesterol that are involved in dietary fat absorption. New evidence supports an additional role for BAs as regulators of brain function. Interestingly, sterols such as cholesterol interact with monoamine transporters (MAT), including the dopamine (DA) transporter (DAT) which plays a key role in DA neurotransmission and reward circuitries in the brain. The present study explores interactions of the BA, obeticholic acid (OCA), with DAT and mechanistically defines the regulation of DAT activity via both electrophysiology and molecular modeling. We express murine DAT (mDAT) in Xenopus laevis oocytes and confirm that DA induces an inward current that reaches a steady-state at a negative membrane voltage. Next, we show that OCA triggers an inward current through DAT that is Na+ dependent and not regulated by intracellular calcium. OCA also inhibits the DAT-mediated Li+ leak current, a feature that parallels DA action and indicates direct binding to the transporter. Interestingly, OCA does not alter DA affinity nor the ability of DA to promote a DAT-mediated inward current, suggesting that the interaction of OCA with the transporter is non-competitive, in regard to DA. The current induced by OCA is transient in nature, returning to baseline in the continued presence of the BA. To understand the molecular mechanism of how OCA affects DAT electrical activity, we performed docking simulations. These simulations revealed two potential binding sites that provide important insights into the potential functional relevance of the OCA-DAT interaction. First, in the absence of DA, OCA binds DAT through interactions with D421, a residue normally involved in coordinating the binding of the Na+ ion to the Na2 binding site (Borre et al., 2014;Cheng and Bahar, 2015). Furthermore, we uncover a separate binding site for OCA on DAT, of equal potential functional impact, that is facilitated through the residues DAT R445 and D436. This binding may stabilize the inward-facing open (IFo) state by preventing the re-formation of the IF gating salt bridges, R60-D436 and R445-E428, that are required for DA transport. This study suggests that BAs may represent novel pharmacological tools to regulate DAT function, and possibly, associated behaviors.