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Concept: Monoamine transporter

190

We describe a disease encompassing infantile-onset movement disorder (including severe parkinsonism and nonambulation), mood disturbance, autonomic instability, and developmental delay, and we describe evidence supporting its causation by a mutation in SLC18A2 (which encodes vesicular monoamine transporter 2 [VMAT2]). VMAT2 translocates dopamine and serotonin into synaptic vesicles and is essential for motor control, stable mood, and autonomic function. Treatment with levodopa was associated with worsening, whereas treatment with direct dopamine agonists was followed by immediate ambulation, near-complete correction of the movement disorder, and resumption of development.

Concepts: Parkinson's disease, Neurotransmitter, Serotonin, Transport, Dopamine, Norepinephrine, Monoamine transporter, Monoamine neurotransmitter

27

The nonmedical use of synthetic cathinones is increasing on a global scale. 4-Methyl-N-methylcathinone (mephedrone) is a popular synthetic cathinone that is now illegal in the United States and other countries. Since the legislative ban on mephedrone, a number of ‘second generation’ analogs have appeared in the street drug marketplace, including 4-methyl-N-ethylcathinone (4-MEC) and 4-methyl-α-pyrrolidinopropiophenone (4-MePPP). Here we characterized the interactions of 4-MEC and 4-MePPP with transporters for 5-HT (SERT) and dopamine (DAT) using molecular, cellular and whole animal methods. In vitro transporter assays revealed that 4-MEC displays unusual ‘hybrid’ activity as a SERT substrate (i.e., 5-HT releaser) and DAT blocker, whereas 4-MePPP is a blocker at both transporters but more potent at DAT. In vivo microdialysis experiments in rat brain demonstrated that 4-MEC (1-3 mg/kg, i.v.) produced large increases in extracellular 5-HT, small increases in dopamine, and minimal motor stimulation. By contrast, 4-MePPP (1-3 mg/kg, i.v.) produced selective increases in dopamine and robust motor stimulation. Consistent with its activity as a SERT substrate, 4-MEC evoked inward current in SERT-expressing Xenopus oocytes, while 4-MePPP was inactive in this regard. To examine drug-transporter interactions at the molecular level, we modeled the fit of 4-MEC and 4-MePPP into the binding pockets for DAT and SERT. Subtle distinctions in ligand-transporter binding were found that account for the differential effects of 4-MEC and 4-MePPP at SERT. Collectively, our results provide key information about the pharmacology of newly-emerging mephedrone analogs, and give clues to structural requirements that govern drug selectivity at DAT versus SERT.Neuropsychopharmacology accepted article preview online, 15 December 2014. doi:10.1038/npp.2014.325.

Concepts: Pharmacology, Signal transduction, In vivo, In vitro, Serotonin, Agonist, Amphetamine, Monoamine transporter

23

Misuse of (±)-threo-methylphenidate (methyl-2-phenyl-2-(piperidin-2-yl)acetate; Ritalin®, MPH) has long been acknowledged, but the appearance of MPH analogs in the form of ‘research chemicals’ has only emerged in more recent years. 4-Fluoromethylphenidate (4 F-MPH) is one of these recent examples and this study presents the identification and analytical characterization of two powdered 4 F-MPH products that were obtained from an online vendor in 2015. Interestingly, the products appeared to have originated from two distinct batches given that one product consisted of (±)-threo-4 F-MPH isomers whereas the second sample consisted of a mixture of (±)-threo and (±)-erythro 4 F-MPH. Monoamine transporter studies using rat brain synaptosomes revealed that the biological activity of the 4 F-MPH mixture resided with the (±)-threo- and not the (±)-erythro isomers based on higher potencies determined for blockage of dopamine uptake (IC50 4 F-MPHmixture  = 66 nM vs. IC50 (±)-threo = 61 nM vs. IC50 (±)-erythro = 8,528 nM) and norepinephrine uptake (IC50 4 F-MPHmixture  = 45 nM vs. (±)-threo = 31 nM vs. IC50 (±)-erythro = 3,779 nM). In comparison, MPH was three times less potent than (±)-threo-4 F-MPH at the dopamine transporter (IC50  = 131 nM) and around 2.5-times less potent at the norepinephrine transporter (IC50  = 83 nM). Both substances were catecholamine selective with IC50 values of 8,805 nM and >10,000 nM for (±)-threo-4 F-MPH and MPH at the serotonin transporter. These findings suggest that the psychostimulant properties of (±)-threo-4 F-MPH might be more potent in humans than MPH.

Concepts: Serotonin, Solute carrier family, Dopamine, Norepinephrine, Norepinephrine transporter, Cocaine, Dopamine transporter, Monoamine transporter

4

The firing activity of serotonergic neurons in raphe nuclei is regulated by negative feedback exerted by extracellular serotonin (5-HT)o acting through somatodendritic 5-HT1A autoreceptors. The steady-state [5-HT]o, sensed by 5-HT1A autoreceptors, is determined by the balance between the rates of 5-HT release and reuptake. Although it is well established that reuptake of 5-HTo is mediated by 5-HT transporters (SERT), the release mechanism has remained unclear. It is also unclear how selective 5-HT reuptake inhibitor (SSRI) antidepressants increase the [5-HT]o in raphe nuclei and suppress serotonergic neuron activity, thereby potentially diminishing their own therapeutic effect. Using an electrophysiological approach in a slice preparation, we show that, in the dorsal raphe nucleus (DRN), continuous nonexocytotic 5-HT release is responsible for suppression of phenylephrine-facilitated serotonergic neuron firing under basal conditions as well as for autoinhibition induced by SSRI application. By using 5-HT1A autoreceptor-activated G protein-gated inwardly rectifying potassium channels of patched serotonergic neurons as 5-HTo sensors, we show substantial nonexocytotic 5-HT release under conditions of abolished firing activity, Ca(2+) influx, vesicular monoamine transporter 2-mediated vesicular accumulation of 5-HT, and SERT-mediated 5-HT transport. Our results reveal a cytosolic origin of 5-HTo in the DRN and suggest that 5-HTo may be supplied by simple diffusion across the plasma membrane, primarily from the dense network of neurites of serotonergic neurons surrounding the cell bodies. These findings indicate that the serotonergic system does not function as a sum of independently acting neurons but as a highly interdependent neuronal network, characterized by a shared neurotransmitter pool and the regulation of firing activity by an interneuronal, yet activity-independent, nonexocytotic mechanism.

Concepts: Nervous system, Neuron, Action potential, Serotonin, Selective serotonin reuptake inhibitor, Dopamine, Monoamine transporter, Raphe nuclei

4

Parkinson disease (PD) is the most common movement disorder and, although the exact causes are unknown, recent epidemiological and experimental studies indicate that several environmental agents may be significant risk factors. To date, these suspected environmental risk factors have been man-made chemicals. In this report, we demonstrate via genetic, biochemical, and immunological studies that the common volatile fungal semiochemical 1-octen-3-ol reduces dopamine levels and causes dopamine neuron degeneration in Drosophila melanogaster. Overexpression of the vesicular monoamine transporter (VMAT) rescued the dopamine toxicity and neurodegeneration, whereas mutations decreasing VMAT and tyrosine hydroxylase exacerbated toxicity. Furthermore, 1-octen-3-ol also inhibited uptake of dopamine in human cell lines expressing the human plasma membrane dopamine transporter (DAT) and human VMAT ortholog, VMAT2. These data demonstrate that 1-octen-3-ol exerts toxicity via disruption of dopamine homeostasis and may represent a naturally occurring environmental agent involved in parkinsonism.

Concepts: Cell, Signal transduction, Cell membrane, Parkinson's disease, Neurotransmitter, Drosophila melanogaster, Dopamine, Monoamine transporter

3

Neurotransmitter transporters play an important role in termination of synaptic transmission by mediating reuptake of neurotransmitter, but the molecular processes behind translocation are still unclear. The crystal structures of the bacterial homologue, LeuT, provided valuable insight into the structural and dynamic requirements for substrate transport. These structures support the existence of gating domains controlling access to a central binding site. On the extracellular side, access is controlled by the ‘thin gate’ formed by an interaction between Arg-30 and Asp-404. In the human dopamine transporter (DAT) the corresponding residues are Arg-85 and Asp-476. Here, we present results supporting the existence of a similar interaction in DAT. The DAT R85D mutant has a complete loss of function, but the additional insertion of an arginine in opposite position (R85D/D476R), causing a charge reversal, results in a rescue of binding sites for the cocaine analogue [(3)H]CFT. Also, the coordination of Zn(2+) between introduced histidines (R85H/D476H) caused a ~2.5-fold increase in [(3)H]CFT binding (BMAX). Importantly, Zn(2+) also inhibited [(3)H]dopamine transport in R85H/D476H suggesting that a dynamic interaction is required for the transport process. Furthermore, cysteine-reactive chemistry shows that mutation of the gating residues causes a higher proportion of transporters to reside in the outward facing conformation. Finally we show that charge reversal of the corresponding residues (R104E/E493R) in the serotonin transporter also rescues [(3)H]S-citalopram binding, suggesting a conserved feature. Taken together, these data suggest that the extracellular thin gate is present in monoamine transporters and that a dynamic interaction is required for substrate transport.

Concepts: DNA, Mutation, Neurotransmitter, Serotonin, Transport, Dopamine, Cocaine, Monoamine transporter

3

Nerve functions require phosphatidylinositol-4,5-bisphosphate (PIP2) that binds to ion channels, thereby controlling their gating. Channel properties are also attributed to serotonin transporters (SERTs); however, SERT regulation by PIP2 has not been reported. SERTs control neurotransmission by removing serotonin from the extracellular space. An increase in extracellular serotonin results from transporter-mediated efflux triggered by amphetamine-like psychostimulants. Herein, we altered the abundance of PIP2 by activating phospholipase-C (PLC), using a scavenging peptide, and inhibiting PIP2-synthesis. We tested the effects of the verified scarcity of PIP2 on amphetamine-triggered SERT functions in human cells. We observed an interaction between SERT and PIP2 in pull-down assays. On decreased PIP2 availability, amphetamine-evoked currents were markedly reduced compared with controls, as was amphetamine-induced efflux. Signaling downstream of PLC was excluded as a cause for these effects. A reduction of substrate efflux due to PLC activation was also found with recombinant noradrenaline transporters and in rat hippocampal slices. Transmitter uptake was not affected by PIP2 reduction. Moreover, SERT was revealed to have a positively charged binding site for PIP2. Mutation of the latter resulted in a loss of amphetamine-induced SERT-mediated efflux and currents, as well as a lack of PIP2-dependent effects. Substrate uptake and surface expression were comparable between mutant and WT SERTs. These findings demonstrate that PIP2 binding to monoamine transporters is a prerequisite for amphetamine actions without being a requirement for neurotransmitter uptake. These results open the way to target amphetamine-induced SERT-dependent actions independently of normal SERT function and thus to treat psychostimulant addiction.

Concepts: Cell membrane, Neurotransmitter, Serotonin, Dopamine, Norepinephrine, Amphetamine, Monoamine transporter, Serotonin transporter

3

Mood disorders cause much suffering and lost productivity worldwide, compounded by the fact that many patients are not effectively treated by currently available medications. The most commonly prescribed antidepressant drugs are the selective serotonin (5-HT) reuptake inhibitors (SSRIs), which act by blocking the high-affinity 5-HT transporter (SERT). The increase in extracellular 5-HT produced by SSRIs is thought to be critical to initiate downstream events needed for therapeutic effects. A potential explanation for their limited therapeutic efficacy is the recently characterized presence of low-affinity, high-capacity transporters for 5-HT in brain [i.e., organic cation transporters (OCTs) and plasma membrane monoamine transporter], which may limit the ability of SSRIs to increase extracellular 5-HT. Decynium-22 (D-22) is a blocker of these transporters, and using this compound we uncovered a significant role for OCTs in 5-HT uptake in mice genetically modified to have reduced or no SERT expression (Baganz et al., 2008). This raised the possibility that pharmacological inactivation of D-22-sensitive transporters might enhance the neurochemical and behavioral effects of SSRIs. Here we show that in wild-type mice D-22 enhances the effects of the SSRI fluvoxamine to inhibit 5-HT clearance and to produce antidepressant-like activity. This antidepressant-like activity of D-22 was attenuated in OCT3 KO mice, whereas the effect of D-22 to inhibit 5-HT clearance in the CA3 region of hippocampus persisted. Our findings point to OCT3, as well as other D-22-sensitive transporters, as novel targets for new antidepressant drugs with improved therapeutic potential.

Concepts: Serotonin, Antidepressant, Selective serotonin reuptake inhibitor, Major depressive disorder, Sertraline, Tricyclic antidepressant, Fluoxetine, Monoamine transporter

2

Altered concentrations of monoamine neurotransmitters and metabolites have been repeatedly found in people with Down syndrome (DS, trisomy 21). Because of the limited availability of human post-mortem tissue, DS mouse models are of great interest to study these changes and the underlying neurobiological mechanisms. Although previous studies have shown the potential of Ts65Dn mice - the most widely used mouse model of DS - to model noradrenergic changes, a comprehensive monoaminergic characterization in multiple brain regions has not been performed so far. Here, we used RP-HPLC with electrochemical detection to quantify (nor)adrenergic (NA, adrenaline and MHPG), dopaminergic (DA, HVA and DOPAC), and serotonergic compounds (tryptophan, 5-HT and 5-HIAA) in ten regionally dissected brain regions of Ts65Dn mice, as well as in Dp1Tyb mice - a novel DS mouse model. Comparing young adult aneuploid mice (2.5-5.5months) with their euploid WT littermates did not reveal generalized monoaminergic dysregulation, indicating that the genetic overload in these mice barely affected the absolute concentrations at this age. Moreover, we studied the effect of aging in Ts65Dn mice: comparing aged animals (12-13months) with their younger counterparts revealed a large number of significant changes. In general, the (nor)adrenergic system appeared to be reduced, while serotonergic compounds were increased with aging. Dopaminergic alterations were less consistent. These overall patterns appeared to be relatively similar for Ts65Dn and WT mice, though more observed changes were regarded significant for WT mice. Similar human post-mortem studies are necessary to validate the monoaminergic construct validity of the Ts65Dn and Dp1Typ mouse models.

Concepts: Neurotransmitter, Serotonin, Down syndrome, Aneuploidy, Dopamine, Monoamine transporter, Neurotransmitters, Monoamine neurotransmitter

2

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common genetic cause of Parkinson’s disease. Here, we investigated whether the G2019S LRRK2 mutation causes morphological and/or functional changes at nigro-striatal dopamine neurons. Density of striatal dopaminergic terminals, nigral cell counts, tyrosine hydroxylase protein levels as well as exocytotic dopamine release measured in striatal synaptosomes, or striatal extracellular dopamine levels monitored by in vivo microdialysis were similar between ≥12-month-old G2019S knock-in mice and wild-type controls. In vivo striatal dopamine release was insensitive to the LRRK2 inhibitor Nov-LRRK2-11, and was elevated by the membrane dopamine transporter blocker GBR-12783. However, G2019S knock-in mice showed a blunted neurochemical and motor activation response to GBR-12783 compared to wild-type controls. Western blot and dopamine uptake analysis revealed an increase in dopamine transporter levels and activity in the striatum of 12-month-old G2019S KI mice. This phenotype correlated with a reduction in vesicular monoamine transporter 2 levels and an enhancement of vesicular dopamine uptake, which was consistent with greater resistance to reserpine-induced hypolocomotion. These changes were not observed in 3-month-old mice. Finally, Western blot analysis revealed no genotype difference in striatal levels of endogenous α-synuclein or α-synuclein bound to DOPAL (a toxic metabolite of dopamine). However, Serine129-phosphorylated α-synuclein levels were higher in 12-month-old G2019S knock-in mice. Immunohistochemistry confirmed this finding, also showing no genotype difference in 3-month-old mice. We conclude that the G2019S mutation causes progressive dysfunctions of dopamine transporters, along with Serine129-phosphorylated α-synuclein overload, at striatal dopaminergic terminals, which are not associated with dopamine homeostasis dysregulation or neuron loss but might contribute to intrinsic dopaminergic terminal vulnerability. We propose G2019S knock-in mice as a presymptomatic Parkinson’s disease model, useful to investigate the pathogenic interaction among genetics, aging, and internal or environmental factors leading to the disease.

Concepts: Gene, Parkinson's disease, Substantia nigra, Neurotransmitter, Striatum, Dopamine, Dopamine transporter, Monoamine transporter