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Biogenic monoamines—vital transmitters orchestrating neurological, endocrinal and immunological functions 1 – 5 —are stored in secretory vesicles by vesicular monoamine transporters (VMATs) for controlled quantal release 6 , 7 . Harnessing proton antiport, VMATs enrich monoamines around 10,000-fold and sequester neurotoxicants to protect neurons 8 – 10 . VMATs are targeted by an arsenal of therapeutic drugs and imaging agents to treat and monitor neurodegenerative disorders, hypertension and drug addiction 1 , 8 , 11 – 16 . However, the structural mechanisms underlying these actions remain unclear. Here we report eight cryo-electron microscopy structures of human VMAT1 in unbound form and in complex with four monoamines (dopamine, noradrenaline, serotonin and histamine), the Parkinsonism-inducing MPP + , the psychostimulant amphetamine and the antihypertensive drug reserpine. Reserpine binding captures a cytoplasmic-open conformation, whereas the other structures show a lumenal-open conformation stabilized by extensive gating interactions. The favoured transition to this lumenal-open state contributes to monoamine accumulation, while protonation facilitates the cytoplasmic-open transition and concurrently prevents monoamine binding to avoid unintended depletion. Monoamines and neurotoxicants share a binding pocket that possesses polar sites for specificity and a wrist-and-fist shape for versatility. Variations in this pocket explain substrate preferences across the SLC18 family. Overall, these structural insights and supporting functional studies elucidate the mechanism of vesicular monoamine transport and provide the basis to develop therapeutics for neurodegenerative diseases and substance abuse. Monoamines and neurotoxicants share a binding pocket in VMAT1 featuring polar sites for specificity and a wrist-and-fist shape for versatility, and monoamine enrichment in storage vesicles arises from dominant import via favoured lumenal-open transition of VMAT1 and protonation-precluded binding during its cytoplasmic-open transition.

Vesicular monoamine transporters (VMATs) are responsible for the packaging of neurotransmitters such as dopamine, serotonin, norepinephrine, and epinephrine into synaptic vesicles. These proteins evolved from precursors in the major facilitator superfamily of transporters and are among the members of the toxin extruding antiporter family. While the primary function of VMATs is to sequester neurotransmitters within vesicles, they can also translocate toxicants away from cytosolic sites of action. In the case of dopamine, this dual role of VMAT2 is combined--dopamine is more readily oxidized in the cytosol where it can cause oxidative stress so packaging into vesicles serves two purposes: neurotransmission and neuroprotection. Furthermore, the deleterious effects of exogenous toxicants on dopamine neurons, such as MPTP, can be attenuated by VMAT2 activity. The active metabolite of MPTP can be kept within vesicles and prevented from disrupting mitochondrial function thereby sparing the dopamine neuron. The highly addictive drug methamphetamine is also neurotoxic to dopamine neurons by using dopamine itself to destroy the axon terminals. Methamphetamine interferes with vesicular sequestration and increases the production of dopamine, escalating the amount in the cytosol and leading to oxidative damage of terminal components. Vesicular transport seems to resist this process by sequestering much of the excess dopamine, which is illustrated by the enhanced methamphetamine neurotoxicity in VMAT2-deficient mice. It is increasingly evident that VMAT2 provides neuroprotection from both endogenous and exogenous toxicants and that while VMAT2 has been adapted by eukaryotes for synaptic transmission, it is derived from phylogenetically ancient proteins that originally evolved for the purpose of cellular protection.

The major monoamine neurotransmitters, serotonin (5-HT) and catecholamines (i.e., norepinephrine (NE), epinephrine (E), and dopamine (DA)), are critical to the nervous system function, and imbalances of the neurotransmitters have been connected to a variety of diseases, making their measurement useful in a clinical setting. A simple, rapid, robust, sensitive, and specific LC-MS/MS method has been developed and validated for the simultaneous quantitation of urinary serotonin and catecholamines with low cost, which is ideal for routine clinical applications. A simple extraction from complex urine was accomplished using tailored solid phase extraction incorporating phenylboronic acid complexation on a 96-well HLB microplate for the sample extraction and resulted in significantly improved throughput, selectivity, and extraction recovery. Compared to 1–10 mL of urine typically used, this method required only 10 μL. A rapid chromatographic elution with a total cycle time of 6 min per sample compared to reported run times of 19–75 min was achieved on a PFP column. The sensitivity of l and 2 ng mL −1 for the detection of low abundant E and NE combined with the high coverage of 1024 ng mL −1 for DA enabled the multi-analyte detection of these biogenic amines in a single run. Good linearity (2.0–512, 1.0–512, 4.0–1024, and 4.0–1024 ng mL −1 for NE, E, DA, and 5-HT, respectively), accuracy (87.6–104.0%), precision (≤8.0%), extraction recovery (69.6–103.7%), and matrix effect (87.1–113.1% for catecholamines and 63.6–71.4% for 5-HT) were obtained. No autosampler carryover was observed. The analytes were stable for 5 days at 20 °C, 14 days at 4 °C, and 30 days at −20 °C and five freeze–thaw cycles. The easy sample preparation, rapid LC, and multi-analyte MS detection allow two 96-well plates of samples to be extracted within 2 h and analyzed on an LC-MS/MS system within 24 h. The applicability and reliability of the assay were demonstrated by assessment of the reference interval for authentic urine specimens from 90 healthy individuals. Graphical abstract A simple, rapid, robust, sensitive and specific LC-MS/MS method combined with a dual functional solid phase extraction has been developed and validated for the simultaneous extraction and quantitation of monoamine neurotransmitters in human urine with low cost

Key Points About four decades ago, Julius Axelrod proposed that reuptake is an important mechanism for inactivating neurotransmitters. The genes that code for the transporters that are responsible for monoamine uptake were identified in the early 1990s. Monoamine transporters have been shown to be involved both in the regulation of the extracellular concentrations of monoamines and in the homeostatic maintenance of presynaptic function. Recent studies indicate that their expression and activity is tightly regulated. Transporters for dopamine, noradrenaline and 5-hydroxytryptamine (named DAT, NET and SERT, respectively) represent established targets for many pharmacological agents that affect brain function, including psychostimulants, antidepressants and neurotoxins. Monoamine transporters are polytopic membrane proteins, containing 12 putative transmembrane domains (TMDs). Conservation of amino acid sequences seems to be highest in the TMDs, whereas the least conserved regions are at the amino and carboxyl termini. The mechanism by which transporter proteins mediate monoamine uptake involves sequential binding and co-transport of Na + and Cl − ions. DAT transports two Na + ions and one Cl − ion with its substrate, whereas NET and SERT co-transport their substrates with one Na + and one Cl − ion. Although most models of transporter function have assumed that they function as single subunits, early studies using radiation inactivation indicated that monoamine transporters might exist as oligomers, and this has recently been confirmed. Transporter activity is regulated at the post-translational level, through modifications such as phosphorylation and N-linked glycosylation. There is also evidence that monoamine transporters undergo regulated trafficking in cells. The disruption of monoamine transporter genes in mice by knockout technologies has provided an opportunity to investigate the physiological role of these proteins in vivo . The identification of DAT as the cocaine receptor enhanced our understanding of the basic mechanisms of addictive processes, and provided strong support for the dopamine theory of addiction. Monoamine transporter genes have also received considerable attention as candidate genes for psychiatric and neurological disorders. There are several outstanding questions in the field of monoamine transporters. We only have a partial knowledge of the proteins that are associated with monoamine transporters and of the factors that contribute to transporter regulation. Also, what is the physiological significance of their channel-like activity? The classical biogenic amine neurotransmitters — dopamine, noradrenaline, and 5-hydroxytryptamine — control a variety of functions including locomotion, autonomic function, hormone secretion, and the complex behaviours that are associated with affect, emotion and reward. A key step that determines the intensity and duration of monoamine signalling at synapses is the reuptake of the released transmitter into nerve terminals through high-affinity plasma membrane transporters. In recent years, molecular, pharmacological and genetic approaches have established the importance of monoamine transporters in the control of monoamine homeostasis and have provided insights into their regulation.

Extensive phytochemical analysis of different root fractions of Jatropha pelargoniifolia Courb. (Euphorbiaceae) has resulted in the isolation and identification of 22 secondary metabolites. 6-hydroxy-8-methoxycoumarin-7-O-β-d-glycopyranoside (15) and 2-hydroxymethyl N-methyltryptamine (18) were isolated and identified as new compounds along with the known diterpenoid (1, 3, 4, and 7), triterpenoid (2 and 6), flavonoid (5, 11, 13, 14, and 16), coumarinolignan (8–10), coumarin (15), pyrimidine (12), indole (17, 18), and tyramine-derived molecules (19–22). The anti-inflammatory, analgesic, and antipyretic activities were evaluated for fifteen of the adequately available isolated compounds (1–6, 8–11, 13, 14, 16, 21, and 22). Seven (4, 6, 10, 5, 13, 16, and 22) of the tested compounds showed a significant analgesic effect ranging from 40% to 80% at 10 mg/kg in two in vivo models. Compound 1 could also prove its analgesic property (67.21%) when it was evaluated on a third in vivo model at the same dose. The in vitro anti-inflammatory activity was also recorded where all compounds showed the ability to scavenge nitric oxide (NO) radical in a dose-dependent manner. However, eight compounds (1, 4, 5, 6, 10, 13, 16, and 22) out of the fifteen tested compounds exhibited considerable in vivo anti-inflammatory activity which reached 64.91% for compound 10 at a dose of 10 mg/kg. Moreover, the tested compounds exhibited an antipyretic effect in a yeast-induced hyperthermia in mice. The activity was found to be highly pronounced with compounds 1, 5, 6, 10, 13, and 16 which decreased the rectal temperature to about 37 °C after 2 h of the induced hyperthermia (~39 °C) at a dose of 10 mg/kg. This study could provide scientific evidence for the traditional use of J. pelargoniifolia as an anti-inflammatory, analgesic, and antipyretic.

The noncovalent complexation of monoamine neurotransmitters and related ammonium and quaternary ammonium ions by a conformationally flexible tetramethoxy glucosylcalix[4]arene was studied by electrospray ionization Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry. The glucosylcalixarene exhibited highest binding affinity towards serotonin, norepinephrine, epinephrine, and dopamine. Structural properties of the guests, such as the number, location, and type of hydrogen bonding groups, length of the alkyl spacer between the ammonium head-group and the aromatic ring structure, and the degree of nitrogen substitution affected the complexation. Competition experiments and guest-exchange reactions indicated that the hydroxyl groups of guests participate in intermolecular hydrogen bonding with the glucocalixarene.

There is some uncertainty whether the acute hyperthermia caused by MDMA (ecstasy) plays a significant role in determining the long-term neurotoxic effects on brain 5-HT systems and associated changes in mood and behaviour. The present study assessed whether long-term behavioural and cognitive changes seen in MDMA-treated rats are affected by hyperthermia at the time of drug administration. Male Wistar rats were treated with MDMA (4x5 mg/kg i.p. over 4 h on 2 consecutive days) or vehicle at either a high ambient temperature (28 degrees C) or a low ambient temperature (16 degrees C). Eight to 18 weeks later, rats were tested in behavioural measures of anxiety (social interaction and emergence tests), a test of cognition (object recognition test) and the forced swim test of depression. At the conclusion of behavioural testing the rats were killed and their brains analysed using HPLC. MDMA treatment caused a clear and consistent hyperthermia at 28 degrees C and hypothermia at 16 degrees C. Months later, rats pre-treated with MDMA at either 16 or 28 degrees C displayed increased anxiety in the social interaction and emergence tests and reduced escape attempts and increased immobility in the forced swim test. MDMA pre-treatment was also associated with poorer memory on the object recognition test, but only in rats given the drug at 28 degrees C. Rats pre-treated with MDMA showed loss of 5-HT in the hippocampus, striatum, amygdala and cortex, regardless of body temperature at the time of dosing. However, 5-HIAA loss in the amygdala and hippocampus was greater in rats pre-treated at 28 degrees C. Dopamine in the striatum was also depleted in rats given MDMA. These results indicate that hyperthermia at the time of dosing with MDMA is not necessary to produce subsequent 5-HT depletion and anxiety in rats. They also extend previous findings of long-term effects of brief exposure to MDMA in rats to include apparent \"depressive\" symptoms in the forced swim model.

Issue Title: Special Issue: Amine oxidases: structures, mechanisms and therapeutic targets

Since alterations in monoamines and monoamine oxidase (MAO) have been postulated to play a role in toxic effects of lead (Pb) on the central nervous system, we have examined the protective effects of calcium (Ca2+) and zinc (Zn2+) supplementation on Pb-induced perturbations in the levels of monoamines and the activity of MAO. Swiss albino mice were lactationally exposed to low (0.2%) and high (1%) levels of Pb-acetate via drinking water of the mother. Pb-exposure commenced on postnatal day (PND) 1, continued up to PND 21 and stopped at weaning. Ca2+ or Zn2+ (0.02% in 0.2% Pb-water or 0.1% in 1% Pb-water) was supplemented separately to the mother up to PND 21. The levels of monoamines (epinephrine, norepinephrine, dopamine and serotonin) and the activity of MAO in the brain regions such as hippocampus, cortex, cerebellum and medulla of young (1 month old) and adult (3 month old) mice were determined in the synaptosomal fractions. The synaptosomal monoamines though increased with low level (0.2%) Pb-exposure, significantly decreased with high level (1%) Pb-exposure in all the brain regions in both the age groups. In general, the young mice seem to be more vulnerable to Pb-neurotoxicity. Ca2+ or Zn2+ supplementation significantly reversed the Pb-induced perturbations both in the levels of monoamines and in the activity of MAO. However, the recovery in monoamine levels and MAO activity was more pronounced with Ca2+ supplementation as compared to Zn2+. These results provide evidence that dietary Ca2+ and/or Zn2+ provide protection against Pb-induced neurotoxic effects.