Vesicular Monoamine Transporter-2 Inhibition Research Articles

Structural mechanisms for VMAT2 inhibition by tetrabenazine

The vesicular monoamine transporter 2 (VMAT2) is a proton-dependent antiporter responsible for loading monoamine neurotransmitters into synaptic vesicles. Dysregulation of VMAT2 can lead to several neuropsychiatric disorders including Parkinson’s disease and schizophrenia. Furthermore, drugs such as amphetamine and MDMA are known to act on VMAT2, exemplifying its role in the mechanisms of actions for drugs of abuse. Despite VMAT2’s importance, there remains a critical lack of mechanistic understanding, largely driven by a lack of structural information. Here, we report a 3.1 Å resolution cryo-electron microscopy (cryo-EM) structure of VMAT2 complexed with tetrabenazine (TBZ), a non-competitive inhibitor used in the treatment of Huntington’s chorea. We find TBZ interacts with residues in a central binding site, locking VMAT2 in an occluded conformation and providing a mechanistic basis for non-competitive inhibition. We further identify residues critical for cytosolic and lumenal gating, including a cluster of hydrophobic residues which are involved in a lumenal gating strategy. Our structure also highlights three distinct polar networks that may determine VMAT2 conformational dynamics and play a role in proton transduction. The structure elucidates mechanisms of VMAT2 inhibition and transport, providing insights into VMAT2 architecture, function, and the design of small-molecule therapeutics.

Structural mechanisms for VMAT2 inhibition by tetrabenazine.

The vesicular monoamine transporter 2 (VMAT2) is a proton-dependent antiporter responsible for loading monoamine neurotransmitters into synaptic vesicles. Dysregulation of VMAT2 can lead to several neuropsychiatric disorders including Parkinson's disease and schizophrenia. Furthermore, drugs such as amphetamine and MDMA are known to act on VMAT2, exemplifying its role in the mechanisms of actions for drugs of abuse. Despite VMAT2's importance, there remains a critical lack of mechanistic understanding, largely driven by a lack of structural information. Here, we report a 3.1 Å resolution cryo-electron microscopy (cryo-EM) structure of VMAT2 complexed with tetrabenazine (TBZ), a non-competitive inhibitor used in the treatment of Huntington's chorea. We find TBZ interacts with residues in a central binding site, locking VMAT2 in an occluded conformation and providing a mechanistic basis for non-competitive inhibition. We further identify residues critical for cytosolic and lumenal gating, including a cluster of hydrophobic residues which are involved in a lumenal gating strategy. Our structure also highlights three distinct polar networks that may determine VMAT2 conformational dynamics and play a role in proton transduction. The structure elucidates mechanisms of VMAT2 inhibition and transport, providing insights into VMAT2 architecture, function, and the design of small-molecule therapeutics.

Protection against β-N-methylamino-l-alanineꟷinduced vesicular monoamine transporter 2 inhibition by hydroxyl-containing proteinogenic amino acids

β-N-methylamino-l-alanine (BMAA) has been shown to inhibit vesicular monoamine transporter 2 (VMAT2), thereby preventing the uptake of monoaminergic neurotransmitters into platelet dense granules and synaptic vesicles. The inhibition is hypothesized to be through direct association of BMAA with hydroxyl groupꟷcontaining amino acid residues in VMAT2. This study evaluated whether BMAA-induced inhibition of VMAT2 could be prevented directly by co-incubation of BMAA with amino acids, and if this protection was specific for BMAA inhibition of VMAT2. l-tyrosine, and to a lesser extent l-serine, was able to prevent BMAA-induced VMAT2 inhibition in a concentration-dependent manner, whereas neither l-threonine nor amino acids without side chain hydroxyl groups could reduce this inhibition. Reserpine-induced VMAT2 inhibition was unaffected by any of the amino acids. These data support the hypothesized interaction between BMAA and hydroxyl groupꟷcontaining amino acids and suggests that this interaction might be leveraged to protect against the toxicity of BMAA.

Depression and suicidality with VMAT2 inhibitors in tardive dyskinesia A signal detection from the FDA Adverse Events Reporting System

Depression and suicidality with VMAT2 inhibitors in tardive dyskinesia A signal detection from the FDA Adverse Events Reporting System

Pharmacokinetic and Pharmacologic Characterization of the Dihydrotetrabenazine Isomers of Deutetrabenazine and Valbenazine.

Valbenazine and deutetrabenazine are vesicular monoamine transporter 2 (VMAT2) inhibitors approved for tardive dyskinesia. The clinical activity of valbenazine is primarily attributed to its only dihydrotetrabenazine (HTBZ) metabolite, [+]-α-HTBZ. Deutetrabenazine is a deuterated form of tetrabenazine and is metabolized to four deuterated HTBZ metabolites: [+]-α-deuHTBZ, [+]-β-deuHTBZ, [-]-α-deuHTBZ, and [-]-β-deuHTBZ. An open-label, crossover study characterized the pharmacokinetic profiles of the individual deuHBTZ metabolites, which have not been previously reported. VMAT2 inhibition and off-target interactions of the deuHTBZ metabolites were evaluated using radioligand binding. The only valbenazine HTBZ metabolite, [+]-α-HTBZ, was a potent VMAT2 inhibitor, with negligible affinity for off-target dopamine, serotonin, and adrenergic receptors. Following deutetrabenazine administration, [-]-α-deuHTBZ represented 66% of circulating deuHTBZ metabolites and was a relatively weak VMAT2 inhibitor with appreciable affinity for dopamine (D2S , D3 ) and serotonin (5-HT1A , 5-HT2B , 5-HT7 ) receptors. [+]-β-deuHTBZ was the most abundant deuHTBZ metabolite that potently inhibited VMAT2, but it represented only 29% of total circulating deuHTBZ metabolites. The mean half-life of [+]-α-HTBZ (22.2hours) was ∼3× longer than that of [+]-β-deuHTBZ (7.7hours). These findings are similar to studies with tetrabenazine, in that deutetrabenazine is metabolized to four deuHTBZ stereoisomers, the most abundant of which has negligible interaction with VMAT2 in vitro and appreciable affinity for several off-target receptors. In contrast, valbenazine's single HTBZ metabolite is a potent VMAT2 inhibitor in vitro with no discernible off-target activity. Determination of the effects of intrinsic/extrinsic variables on deutetrabenazine's safety/efficacy profile should incorporate assessment of the effects on all deuHTBZ metabolites.

Inhibition of VMAT2 by β2-adrenergic agonists, antagonists, and the atypical antipsychotic ziprasidone

Vesicular monoamine transporter 2 (VMAT2) is responsible for packing monoamine neurotransmitters into synaptic vesicles for storage and subsequent neurotransmission. VMAT2 inhibitors are approved for symptomatic treatment of tardive dyskinesia and Huntington’s chorea, but despite being much-studied inhibitors their exact binding site and mechanism behind binding and inhibition of monoamine transport are not known. Here we report the identification of several approved drugs, notably β2-adrenergic agonists salmeterol, vilanterol and formoterol, β2-adrenergic antagonist carvedilol and the atypical antipsychotic ziprasidone as inhibitors of rat VMAT2. Further, plausible binding modes of the established VMAT2 inhibitors reserpine and tetrabenazine and hit compounds salmeterol and ziprasidone were identified using molecular dynamics simulations and functional assays using VMAT2 wild-type and mutants. Our findings show VMAT2 as a potential off-target of treatments with several approved drugs in use today and can also provide important first steps in both drug repurposing and therapy development targeting VMAT2 function.

β-N-methylamino-l-alanine is a non-competitive inhibitor of vesicular monoamine transporter 2

The neurotoxic, non-proteinogenic amino acid β-N-methylamino-l-alanine (BMAA) has been implicated in the development of neurodegenerative diseases; however, the mechanism(s) and mode(s) of toxicity remain unclear. Similarities in the neuropathology and behavioural deficits of neonatal rats exposed to either BMAA or reserpine, a known vesicular monoamine transporter 2 (VMAT2) inhibitor, suggest a similar mode of action. The aims of this study were therefore to determine if BMAA could prevent the uptake of serotonin into dense granules via inhibition of VMAT2, and, if so, the type of inhibition caused by BMAA. Exposing platelet dense granules to BMAA resulted in a concentration-dependent reduction in serotonin uptake. The inhibition of VMAT2 was non-competitive. The findings from this study support previous reports that BMAA-associated neuropathologies in a neonatal rat model may be due to VMAT2 inhibition during critical periods of neurogenesis.

Efficacy of Vesicular Monoamine Transporter 2 Inhibition and Synergy with Antipsychotics in Animal Models of Schizophrenia.

Antipsychotic medications function by blocking postsynaptic dopaminergic signaling in the central nervous system. Dopamine transmission can also be modulated presynaptically by inhibitors of vesicular monoamine transporter 2 (VMAT2), which inhibit loading of dopamine into presynaptic vesicles. Here we investigated the combination of these mechanisms in animal models of schizophrenia and weight gain (a primary side effect of antipsychotics). When dosed alone, the highly selective VMAT2 inhibitor RRR-dihydrotetrabenazine (RRR-DHTBZ, also known as [+]-α-HTBZ) elicited efficacy comparable to conventional antipsychotics in prepulse inhibition and conditioned avoidance models without eliciting weight gain. In combination experiments, synergy was observed: subthreshold doses of RRR-DHTBZ and risperidone or olanzapine produced robust efficacy, and in dose response experiments, RRR-DHTBZ increased the antipsychotic potency in the efficacy models but did not affect weight gain. The combinations did not affect plasma compound concentrations. The synergy is consistent with VMAT2 inhibition blocking the counterproductive presynaptic stimulation of dopamine by antipsychotics. These results suggest a therapeutic strategy of adding a VMAT2 inhibitor to lower the antipsychotic dose and reduce the side effect burden of the antipsychotic while maintaining and potentially enhancing its therapeutic effects. SIGNIFICANCE STATEMENT: Antipsychotics are often necessary and life-changing medications that reduce psychotic symptoms; however, these benefits come with a high side effect burden. This study shows that combining these postsynaptic dopaminergic modulators with a presynaptic dopamine modulator (vesicular monoamine transporter 2 [VMAT2] inhibitor) potentiates efficacy synergistically in animal models of schizophrenia without potentiating weight gain. Our data suggest that adding a VMAT2 inhibitor may be a viable therapeutic strategy for reducing antipsychotic side effects by lowering antipsychotic dose while maintaining therapeutic efficacy.

ApoE4 inhibition of VMAT2 in the locus coeruleus exacerbates Tau pathology in Alzheimer's disease.

ApoE4 enhances Tau neurotoxicity and promotes the early onset of AD. Pretangle Tau in the noradrenergic locus coeruleus (LC) is the earliest detectable AD-like pathology in the human brain. However, a direct relationship between ApoE4 and Tau in the LC has not been identified. Here we show that ApoE4 selectively binds to the vesicular monoamine transporter 2 (VMAT2) and inhibits neurotransmitter uptake. The exclusion of norepinephrine (NE) from synaptic vesicles leads to its oxidation into the toxic metabolite 3,4-dihydroxyphenyl glycolaldehyde (DOPEGAL), which subsequently activates cleavage of Tau at N368 by asparagine endopeptidase (AEP) and triggers LC neurodegeneration. Our data reveal that ApoE4 boosts Tau neurotoxicity via VMAT2 inhibition, reduces hippocampal volume, and induces cognitive dysfunction in an AEP- and Tau N368-dependent manner, while conversely ApoE3 binds Tau and protects it from cleavage. Thus, ApoE4 exacerbates Tau neurotoxicity by increasing VMAT2 vesicle leakage and facilitating AEP-mediated Tau proteolytic cleavage in the LC via DOPEGAL.

GZ-11608, a Vesicular Monoamine Transporter-2 Inhibitor, Decreases the Neurochemical and Behavioral Effects of Methamphetamine.

Despite escalating methamphetamine use and high relapse rates, pharmacotherapeutics for methamphetamine use disorders are not available. Our iterative drug discovery program had found that R-N-(1,2-dihydroxypropyl)-2,6-cis-di-(4-methoxyphenethyl)piperidine hydrochloride (GZ-793A), a selective vesicular monoamine transporter-2 (VMAT2) inhibitor, specifically decreased methamphetamine's behavioral effects. However, GZ-793A inhibited human-ether-a-go-go-related gene (hERG) channels, suggesting cardiotoxicity and prohibiting clinical development. The current study determined if replacement of GZ-793A's piperidine ring with a phenylalkyl group to yield S-3-(4-methoxyphenyl)-N-(1-phenylpropan-2-yl)propan-1-amine (GZ-11608) diminished hERG interaction while retaining pharmacological efficacy. VMAT2 inhibition, target selectivity, and mechanism of GZ-11608-induced inhibition of methamphetamine-evoked vesicular dopamine release were determined. We used GZ-11608 doses that decreased methamphetamine-sensitized activity to evaluate the potential exacerbation of methamphetamine-induced dopaminergic neurotoxicity. GZ-11608-induced decreases in methamphetamine reinforcement and abuse liability were determined using self-administration, reinstatement, and substitution assays. Results show that GZ-11608 exhibited high affinity (Ki = 25 nM) and selectivity (92-1180-fold) for VMAT2 over nicotinic receptors, dopamine transporter, and hERG, suggesting low side-effects. GZ-11608 (EC50 = 620 nM) released vesicular dopamine 25-fold less potently than it inhibited VMAT2 dopamine uptake. GZ-11608 competitively inhibited methamphetamine-evoked vesicular dopamine release (Schild regression slope = 0.9 ± 0.13). GZ-11608 decreased methamphetamine sensitization without altering striatal dopamine content or exacerbating methamphetamine-induced dopamine depletion, revealing efficacy without neurotoxicity. GZ-11608 exhibited linear pharmacokinetics and rapid brain penetration. GZ-11608 decreased methamphetamine self-administration, and this effect was not surmounted by increasing methamphetamine unit doses. GZ-11608 reduced cue- and methamphetamine-induced reinstatement, suggesting potential to prevent relapse. GZ-11608 neither served as a reinforcer nor substituted for methamphetamine, suggesting low abuse liability. Thus, GZ-11608, a potent and selective VMAT2 inhibitor, shows promise as a therapeutic for methamphetamine use disorder. SIGNIFICANCE STATEMENT: GZ-11608 is a potent and selective vesicular monoamine transporter-2 inhibitor that decreases methamphetamine-induced dopamine release from isolated synaptic vesicles from brain dopaminergic neurons. Results from behavioral studies show that GZ-11608 specifically decreases methamphetamine-sensitized locomotor activity, methamphetamine self-administration, and reinstatement of methamphetamine-seeking behavior, without exhibiting abuse liability. Tolerance does not develop to the efficacy of GZ-11608 to decrease the behavioral effects of methamphetamine. In conclusion, GZ-11608 is an outstanding lead in our search for a therapeutic to treat methamphetamine use disorder.

Concurrent Inhibition of Vesicular Monoamine Transporter 2 Does Not Protect Against 3,4-Methylenedioxymethamphetamine (Ecstasy) Induced Neurotoxicity.

3, 4-Methylenedioxymethamphetamine (MDMA) is a hallucinogenic amphetamine derivative. The acute effects of MDMA are hyperthermia, hyperactivity, and behavioral changes, followed by long-term serotonergic neurotoxicity in rats and primates. However, the underlying mechanisms of MDMA neurotoxicity remain elusive. We reported that pretreatment of rats with Ro 4-1284, a reversible inhibitor of the vesicular monoamine transporter 2 (VMAT2), reduced MDMA-induced hyperactivity in rats, abolished the hyperthermic response, and the long-term neurotoxicity. Current studies focused on the effects of co- and/or postinhibition of VMAT2 on the acute and chronic effects of MDMA and on the dose-response relationship between MDMA-induced elevations in body temperature and subsequent reductions in indolamine concentrations. Sprague Dawley rats were treated with MDMA (20, 25, or 27.5 mg/kg sc), and either co- and/or posttreatment with the VMAT2 inhibitor (10 mg/kg ip). Rats simultaneously treated with Ro 4-1284 and MDMA exhibited a more rapid increase in body temperature compared to just MDMA. However, the duration of the elevated body temperature was significantly shortened (approximately 3 h vs approximately 8 h, respectively). A similar body temperature response was observed in rats posttreated (7 h after MDMA) with Ro 4-1284. Despite decreases in the area under the curve (Δtemp X time) of body temperature caused by Ro 4-1284, there were no significant differences in the degree of indolamine depletion between any of the MDMA-treated groups. The results suggest that the neuroprotective effects of VMAT2 inhibition is likely due to the indirect monoamine depleting effects of the Ro 4-1284 pretreatment, rather than by the direct inhibition of VMAT2 function.

New Scaffold for Lead Compounds to Treat Methamphetamine Use Disorders.

Despite increased methamphetamine use worldwide, pharmacotherapies are not available to treat methamphetamine use disorder. The vesicular monoamine transporter-2 (VMAT2) is an important pharmacological target for discovery of treatments for methamphetamine use disorder. VMAT2 inhibition by the natural product, lobeline, reduced methamphetamine-evoked dopamine release, methamphetamine-induced hyperlocomotion, and methamphetamine self-administration in rats. Compared to lobeline, lobelane exhibited improved affinity and selectivity for VMAT2 over nicotinic acetylcholine receptors. Lobelane inhibited neurochemical and behavioral effects of methamphetamine, but tolerance developed to its behavioral efficacy in reducing methamphetamine self-administration, preventing further development. The lobelane analog, R-N-(1,2-dihydroxypropyl)-2,6-cis-di-(4-methoxyphenethyl)piperidine hydrochloride (GZ-793A), potently and selectively inhibited VMAT2 function and reduced neurochemical and behavioral effects of methamphetamine. However, GZ-793A exhibited potential to induce ventricular arrhythmias interacting with human-ether-a-go-go (hERG) channels. Herein, a new lead, R-3-(4-methoxyphenyl)-N-(1-phenylpropan-2-yl)propan-1-amine (GZ-11610), from the novel scaffold (N-alkyl(1-methyl-2-phenylethyl)amine) was evaluated as a VMAT2 inhibitor and potential therapeutic for methamphetamine use disorder. GZ-11610 was 290-fold selective for VMAT2 over dopamine transporters, suggesting that it may lack abuse liability. GZ-11610 was 640- to 3500-fold selective for VMAT2 over serotonin transporters and nicotinic acetylcholine receptors. GZ-11610 exhibited > 1000-fold selectivity for VMAT2 over hERG, representing a robust improvement relative to our previous VMAT2 inhibitors. GZ-11610 (3-30mg/kg, s.c. or 56-300mg/kg, oral) reduced methamphetamine-induced hyperactivity in methamphetamine-sensitized rats. Thus, GZ-11610 is a potent and selective inhibitor of VMAT2, may have low abuse liability and low cardiotoxicity, and after oral administration is effective and specific in inhibiting the locomotor stimulant effects of methamphetamine, suggesting further investigation as a potential therapeutic for methamphetamine use disorder.

Varenicline and GZ-793A differentially decrease methamphetamine self-administration under a multiple schedule of reinforcement in rats.

Methamphetamine is a potent psychostimulant with high abuse rates. Currently, there is no Food and Drug Administration-approved pharmacotherapy for methamphetamine addiction. Ideally, a pharmacotherapy should selectively decrease methamphetamine self-administration without affecting responding for other reinforcers. One way to test this is with the use of a multiple schedule of reinforcement, in which drug and food are available in alternating components within a session. The present study evaluated GZ-793A, a vesicular monoamine transporter-2 inhibitor, and varenicline, a partial agonist at α4β2 and full agonist at α7 nicotinic acetylcholine receptors, for their ability to decrease methamphetamine and food self-administration using a multiple schedule of reinforcement. Male Sprague-Dawley rats self-administered methamphetamine (0.03 mg/kg/intravenous infusion) and food pellets under a multiple schedule of reinforcement. GZ-793A or varenicline was administered before multiple schedule sessions. GZ-793A (5 and 20 mg/kg) significantly decreased methamphetamine intake compared with saline and did not alter food-maintained responding. In contrast, varenicline decreased methamphetamine intake less specifically across time. The results suggest that vesicular monoamine transporter-2 inhibition may be a viable pharmacological target for the treatment of methamphetamine-use disorders.

1,4-Diphenalkylpiperidines: A new scaffold for the design of potent inhibitors of the vesicular monoamine transporter-2

A series of 1,4-diphenalkylpiperidine analogs were synthesized and evaluated for their affinity and inhibitory potency at the [3H]dihydrotetrabenazine (DTBZ) binding site and [3H]dopamine (DA) uptake site on the vesicular monoamine transporter-2 (VMAT2). Results revealed that translocation of the phenethyl side chains of lobelane from C2 and C6 to C1 and C4 around the central piperidine ring slightly reduces affinity and inhibitory potency at VMAT2 with respect to lobelane. However, methoxy and fluoro-substitution of either phenyl ring of these 1,4-diphenethyl analogs afforded VMAT2 inhibition comparable or higher (5-fold) affinity at the DTBZ binding and DA uptake sites relative to lobelane, whereas replacement of the 4-phenethyl moiety in these analogs with a 4-phenmethyl moiety markedly reduced affinity for the DTBZ binding and DA uptake sites by 3- and 5-fold, respectively. Among the twenty five 1,4-diphenethylpiperidine analogs evaluated, compounds containing a 4-(2-methoxyphenethyl) moiety exhibited the most potent inhibition of DTBZ binding and vesicular DA uptake. From this subgroup, analogs 8h, 8j and 8m exhibited Ki values of 9.3nM, 13nM and 13nM, respectively, for inhibition of [3H]DA uptake by VMAT2, and represent some of the most potent inhibitors of VMAT2 function reported thus far.

Lobelane analogues containing 4-hydroxy and 4-(2-fluoroethoxy) aromatic substituents: Potent and selective inhibitors of [3H]dopamine uptake at the vesicular monoamine transporter-2

A series of lobelane and GZ-793A analogues that incorporate aromatic 4-hydroxy and 4-(2-fluoroethoxy) substituents were synthesized and evaluated for inhibition of [3H]dopamine (DA) uptake at the vesicular monoamine transporter-2 (VMAT2) and the dopamine transporter (DAT), and [3H]serotonin uptake at the serotonin transporter (SERT). Most of these compounds exhibited potent inhibition of DA uptake at VMAT2 in the nanomolar range (Ki=30–70nM). The two most potent analogues, 7 and 14, both exhibited a Ki value of 31nM for inhibition of VMAT2. The lobelane analogue 14, incorporating 4-(2-fluoroethoxy) and 4-hydroxy aromatic substituents, exhibited 96- and 335-fold greater selectivity for VMAT2 versus DAT and SERT, respectively, in comparison to lobelane. Thus, lobelane analogues bearing hydroxyl and fluoroethoxy moieties retain the high affinity for VMAT2 of the parent compound, while enhancing selectivity for VMAT2 versus the plasmalemma transporters.

Subjective and objective evidence of low abuse potential of the peripherally-acting kappa opioid, CR845, compared with pentazocine

Drug and Alcohol Dependence 156 (2015) e183–e245 e211 preclinical model used to develop and test potential pharmacotherapies is the rodent intravenous (i.v.) self-administration paradigm. The aim of the current study was establish rodent i.v. self-administration under a multiple schedule with alternating components ofMETHand food reinforcement. Once behavior stabilized on this schedule we tested two potential pharmacotherapies for METH addiction: GZ-793A, a vesicular monoamine transporter 2 (VMAT2) inhibitor and varenicline, a partial agonist at a4b2 nicotinic acetylcholine receptors (nAChR). Methods: Following catheterization surgery, male SpragueDawley rats acquired both i.v. METH (0.3mg/kg/infusion) and food self-administration under a multiple schedule of reinforcement. Following stable responding pretreatments of GZ-793A (0, 10, 15, or 30mg/kg, s.c.) and varenicline (0, 0.3, 1, or 2mg/kg, s.c.) were administered in a Latin Square design with half of the animals receiving GZ-793A first while the other half received varenicline first. Results: Both GZ-793A and varenicline decreased METH self-administration. However, GZ-793A was more selective at decreasing METH intake without altering food-maintained responding. More specifically, the 20mg/kg of GZ-793A significantly decreased METH intake compared to saline without effecting food-maintained responding. Also, the lowest dose of GZ-793A significantly decreased METH intake compared to the lowest dose of varenicline, againwithout disrupting food-maintained responding. Nodoseofvarenicline significantly loweredMETH intakecompared to a saline control. Conclusions: Cumulatively, this suggests that VMAT2 inhibition, rather than nAChR partial agonism, is a more promising avenue to pursue for a potential pharmacotherapy forMETH addiction. Financial Support: This project was funded by the Ferlic Summer Research Fund to MMK. http://dx.doi.org/10.1016/j.drugalcdep.2015.07.568 Women’s intervention to stop HIV/HCV (WISH) Michele Staton-Tindall 5,∗, Matthew Webster3, Carl Leukefeld2, Jennifer R. Havens4, Carrie B. Oser1 1 Sociology, University of Kentucky, Lexington, KY, United States 2 University of Kentucky, Lexington, LA, United States 3 Behavioral Science, University of Kentucky, Lexington, KY, United States 4 Center on Drug and Alcohol Research, University of Kentucky, Lexington, KY, United States 5 Social Work, University of Kentucky, Lexington, KY,

Withdrawal from repeated administration of a low dose of reserpine induced opposing adaptive changes in the noradrenaline and serotonin system function: a behavioral and neurochemical ex vivo and in vivo studies in the rat.

Reserpine is an inhibitor of the vesicular monoamine transporter 2 (VMAT2) and monoamine releaser, so it can be used as a pharmacological model of depression. In the present paper, we investigated the behavioral and neurochemical effects of withdrawal from acute and repeated administration of a low dose of reserpine (0.2 mg/kg) in Wistar Han rats. We demonstrated the behavioral and receptor oversensitivity (postsynaptic dopamine D1) during withdrawal from chronic reserpine. It was accompanied by a significant increase in motility in the locomotor activity test and climbing behavior in the forced swim test (FST). Neurochemical studies revealed that repeated but not acute administration the a low dose of reserpine triggered opposing adaptive changes in the noradrenergic and serotonin system function analyzed during reserpine withdrawal, i.e. 48 h after the last injection. The tissue concentration of noradrenaline was significantly decreased in the hypothalamus and nucleus accumbens only after repeated drug administration (by about 20% and 35% vs. control; p<0.05, respectively). On the other hand, the concentration of its extraneuronal metabolite, normetanephrine (NM) increased significantly in the VTA during withdrawal both from acute and chronic reserpine. The serotonin concentration was significantly reduced in the VTA after chronic reserpine (by about 40% vs. the control group, p<0.05) as well as its main metabolite, 5-HIAA (by about 30% vs. control; p<0.05) in the VTA and hypothalamus. Dopamine and its metabolites were not changed after acute or chronic reserpine administration. In vivo microdialysis studies clearly evidenced the lack of the effect of a single dose of reserpine, and its distinct effects after chronic treatment on the release of noradrenaline and serotonin in the rat striatum. In fact, the withdrawal from repeated administration of reserpine significantly increased an extraneuronal concentration of noradrenaline in the rat striatum but at the same time produced a distinct fall in the extraneuronal serotonin in this brain structure. On the basis of the presented behavioral and neurochemical experiments, we suggest that chronic administration of reserpine even in such low dose which not yet acted on the release of monoamines but produced an inhibition of VMAT2 caused a long-lasting disadvantageous effect of plasticity in the brain resembling depressive disorders.

Vesicular monoamine transporter 2 and the acute and long-term response to 3,4-(±)-methylenedioxymethamphetamine.

3,4-(±)-Methylenedioxymethamphetamine (MDMA, Ecstasy) is a ring-substituted amphetamine derivative with potent psychostimulant properties. The neuropharmacological effects of MDMA are biphasic in nature, initially causing synaptic monoamine release, primarily of serotonin (5-HT). Conversely, the long-term effects of MDMA manifest as prolonged depletions in 5-HT, and reductions in 5-HT reuptake transporter (SERT), indicative of serotonergic neurotoxicity. MDMA-induced 5-HT efflux relies upon disruption of vesicular monoamine storage, which increases cytosolic 5-HT concentrations available for release via a carrier-mediated mechanism. The vesicular monoamine transporter 2 (VMAT2) is responsible for packaging monoamine neurotransmitters into cytosolic vesicles. Thus, VMAT2 is a molecular target for a number of psychostimulant drugs, including methamphetamine and MDMA. We investigated the effects of depressed VMAT2 activity on the adverse responses to MDMA, via reversible inhibition of the VMAT2 protein with Ro4-1284. A single dose of MDMA (20 mg/kg, subcutaneous) induced significant hyperthermia in rats. Ro4-1284 (10 mg/kg, intraperitoneal) pretreatment prevented the thermogenic effects of MDMA, instead causing a transient decrease in body temperature. MDMA-treated rats exhibited marked increases in horizontal velocity and rearing behavior. In the presence of Ro4-1284, MDMA-mediated horizontal hyperlocomotion was delayed and attenuated, whereas rearing activity was abolished. Finally, Ro4-1284 prevented deficits in 5-HT content in rat cortex and striatum, and reduced depletions in striatal SERT staining, 7 days after MDMA administration. In summary, acute inhibition of VMAT2 by Ro4-1284 protected against MDMA-mediated hyperthermia, hyperactivity, and serotonergic neurotoxicity. The data suggest the involvement of VMAT2 in the thermoregulatory, behavioral, and neurotoxic effects of MDMA.

Effects of VMAT2 inhibitors lobeline and GZ-793A on methamphetamine-induced changes in dopamine release, metabolism and synthesis in vivo.

Vesicular monoamine transporter-2 (VMAT2) inhibitors reduce methamphetamine (METH) reward in rats. The current study determined the effects of VMAT2 inhibitors lobeline (LOB; 1 or 3 mg/kg) and N-(1,2R-dihydroxylpropyl)-2,6-cis-di(4-methoxyphenethyl)piperidine hydrochloride (GZ-793A; 15 or 30 mg/kg) on METH-induced (0.5 mg/kg, SC) changes in extracellular dopamine (DA) and its metabolite dihydroxyphenylacetic acid (DOPAC) in the reward-relevant nucleus accumbens (NAc) shell using in vivo microdialysis. The effect of GZ-793A (15 mg/kg) on DA synthesis in tissue also was investigated in NAc, striatum, medial prefrontal cortex and orbitofrontal cortex. In NAc shell, METH produced a time-dependent increase in extracellular DA and decrease in DOPAC. Neither LOB nor GZ-793A alone altered extracellular DA; however, both drugs increased extracellular DOPAC. In combination with METH, LOB did not alter the effects of METH on DA; however, GZ-793A, which has greater selectivity than LOB for inhibiting VMAT2, reduced the duration of the METH-induced increase in extracellular DA. Both LOB and GZ-793A enhanced the duration of the METH-induced decrease in extracellular DOPAC. METH also increased tissue DA synthesis in NAc and striatum, whereas GZ-793A decreased synthesis; no effect of METH or GZ-793A on DA synthesis was found in medial prefrontal cortex or orbitofrontal cortex. These results suggest that selective inhibition of VMAT2 produces a time-dependent decrease in DA release in NAc shell as a result of alterations in tyrosine hydroxylase activity, which may play a role in the ability of GZ-793A to decrease METH reward.

HIV‐1 protein Tat inhibits vesicular monoamine transporter‐2 activity in rat striatum

HIV‐1 protein Tat inhibits vesicular monoamine transporter‐2 activity in rat striatum