Neurotransmitter Transporters: Fruitful Targets for CNS Drug Discovery L Iversen

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Molecular Psychiatry (2000) 5, 357–362  2000 Macmillan Publishers Ltd All rights reserved 1359-4184/00 $15.00 www.nature.com/mp MILLENNIUM ARTICLE Neurotransmitter transporters: fruitful targets for CNS drug discovery L Iversen

Department of Pharmacology, University of Oxford, Mansﬁeld Road, Oxford OX1 3QT, UK

More than 20 members have been identiﬁed in the neurotransmitter transporter family. These include the cell surface re-uptake mechanisms for monoamine and amino acid neurotransmitters and vesicular transporter mechanisms involved in neurotransmitter storage. The norepinephrine and serotonin re-uptake transporters are key targets for antidepressant drugs. Clini- cally effective antidepressants include those with selectivity for either NE or serotonin uptake, and compounds with mixed actions. The dopamine transporter plays a key role in mediating the actions of cocaine and the amphetamines and in conferring selectivity on dopamine neurotoxins. The only clinically used compound to come so far from research on amino acid transporters is the antiepileptic drug tiagabine, a GABA uptake inhibitor. Molecular Psychiatry (2000) 5, 357–362. Keywords: neurotransmitter transporters; vesicular transporters; antidepressants; serotonin; norepinephrine; dopamine; cocaine; amphetamines

Introduction for some time unchanged. A key observation was that the uptake of 3H-noradrenaline into the heart was vir- The concept that neurotransmitters are inactivated by tually eliminated in animals in which the sympathetic uptake of the released chemical into the nerve terminal innervation had been destroyed by surgical removal of from which it had been released or into adjacent cells the superior cervical ganglion.3 This led Hertting and is less than 40 years old. Prior to this it was generally Axelrod4 to propose that the reuptake of noradrenaline assumed that the inactivation of noradrenaline and the by the same nerves from which it had been released other monoamine neurotransmitters after their release might represent a novel mechanism for inactivating from nerves was likely to involve rapid enzymatic this neurotransmitter. breakdown, akin to that seen with acetylcholinesterase. The discovery of noradrenaline uptake was followed The degradation of monoamines by the enzyme by the finding that similar but distinct transporters monoamine oxidase was known early on, and in the were involved in the inactivation of 5-HT and dopa- 1950s a second enzyme catechol-O-methyl transferase mine, and that similar mechanisms existed for the (COMT) was discovered and was thought to play a key inactivation of the amino acid neurotransmitters role in inactivating noradrenaline and other cat- GABA, glycine and l-glutamate.5,6 Much research echolamines. interest has focused on these mechanisms, including in It was not until high specific activity tritium-labelled recent years the identification and cloning of the genes radioactive catecholamines became available in the encoding the transporter proteins involved and the late 1950s, however, that experiments could be perfor- development of knock-out strains of genetically engi- med using quantities of monoamine small enough to neered mice lacking one or other of these gene pro- mimic the very low concentrations of adrenaline or ducts. The family of neurotransmitter transporters has noradrenaline normally encountered in body fluids. turned out to be far more extensive than previously When the first experiments were performed in the imagined, with more than 20 different members (for Axelrod laboratory at the National Institutes of Health review see Masson et al).7 with 3H-adrenaline1 and later with 3H-noradrenaline2 The neurotransmitter transporters have also proved they yielded an unexpected result. Although in labora- to be very important targets for CNS drug discovery, tory animals most of the injected dose of labelled cat- particularly for antidepressant drugs. Virtually all of echolamine was rapidly metabolised (mainly by the modern generation of antidepressants act as inhibi- COMT), a substantial proportion of the injected tors of one or other of the monoamine transporters. monoamine (30–40%) was removed from the circu- Fluoxetine and the related serotonin selective reuptake lation by a rapid uptake into tissues, where it remained inhibitors (SSRIs) are among the most widely used and commercially valuable CNS drugs of all time, with Correspondence: L Iversen, Department of Pharmacology, Univer- annual sales of this group of compounds currently in sity of Oxford, Mansfield Rd, Oxford OX1 3QT, UK excess of $5 billion. This brief review will attempt to Received 10 December 1999; accepted 20 December 1999 give an overview of what has become a large and rapidly growing area of neuroscience research. Neurotransmitter transporters L Iversen 358 The family of neurotransmitter transporters cells in peripheral organs, while VMAT-2 is the pre- dominant form found in monaminergic neurons in The molecular characterisation of the neurotransmitter CNS. It is also expressed in the histamine-containing transporter family began with the cloning of the rat cells of the stomach, and in the adrenal medulla and GABA transporter by Radian and Kanner in 1986.8 in blood cells. When the norepinephrine transporter was indepen- Cholinergic neurons possess their own vesicular ace- dently cloned by Pacholczyk et al in 1991,9 it became tylcholine transporter, and a fourth vesicular trans- apparent that there was a high degree of sequence hom- porter is expressed in GABA and glycine-containing ology between the GABA and NE transporters, and this neurons. led to the recognition that they belonged to larger gene The uptake of monoamines into non-neuronal family whose other members were soon identified tissues, first described as Uptake is a mechanism that (Table 1). Separate transporters exist for GABA, 2 has a particularly high affinity for O-methylated cat- GABA/betaine, NE, serotonin, dopamine, glycine, taur- echolamine metabolites and is sensitive to inhibition ine and proline. In addition homology cloning has by steroids.5,6 Uptake was at first thought to be identified four ‘orphan’ transporter genes, although the 2 mediated by a member of the organic cation carrier transport functions of these or their substrates remain family, but it is now known to have its own steroid- unknown.7 All of these related proteins are dependent sensitive transporter molecule.10 on sodium and chloride ions for their function. They Immunocytochemical and in situ hybridization tech- use the electrochemical gradient of sodium between niques have been used to study the cellular distri- the outside and inside surfaces of the cell membrane to bution of the transporters.7 Whereas NET, DAT and provide the thermodynamic energy required to pump PROT are expressed exclusively in neurons many neurotransmitters from low concentrations outside the members of the family are expressed in non-neural cell to the much higher concentrations inside the cell. cells and several in both neurons and glia (eg SERT). Chloride ions accompany the entry of neurotransmitter Among the various GABA transporters GAT1 is local- and sodium, and there is a net movement of positively ised exclusively to nerve terminals while other mem- charged ions into the cell, although not in sufficient bers of this group are found in glial cells. Among the amounts to appreciably alter the resting membrane l-glutamate transporters EAAT-1 and 2 are expressed potential of the cell. exclusively in the brain, while other members of the A further family of five transporters was later disco- group are also found in some peripheral tissues. In the vered which mediate the uptake of l-glutamate into brain EAAT-3 and 4 are present only in neurons, while neurons and glial cells; these proteins require the pres- EAAT-1 and 2 are exclusively expressed in glial cells. ence of both sodium and potassium ions for their func- There are differences in the proportions of the various tion, but are not chloride-dependent.7 glutamate transporters found in different brain regions. The vesicular neurotransmitter transporters rep- Overall they are strategically placed to control the resent another family7 whose function is to maintain extracellular levels of this important excitatory amino the very high concentrations of monoamine and amino acid in the CNS. acid neurotransmitters in storage vesicles. They use the + + The Na /Cl -dependent transporters and the vesicu- proton gradient that exists across the vesicular mem- lar transporters are membrane proteins consisting of a brane as the motive force. The vesicular monoamine single polypeptide chain of 5–600 amino acid residues, transporters (VMAT) recognise serotonin, dopamine, with 12 ␣-helical membrane-spanning domains.7 The NE, epinephrine and histamine. VMAT-1 is present + + Na /K -dependent glutamate transporters are proteins chiefly in amine-containing endocrine and paracrine of similar length but the ␣-helical regions are less clearly defined and estimates of the number of mem- Table 1 Neurotransmitter transporters brane-spanning domains range from six to ten.11 The molecular mechanisms underlying the function of the Substrate Subtypes/ Name neurotransmitter transporters remain unclear. Unlike isoforms flux through an open ion channel, there must be a gat- ing cycle every time solute is transported, but the exact Norepinephrine NET molecular details of this are not understood. No doubt Dopamine DAT selective mutations of amino acids in the transporter Serotonin SERT or 5-HTT molecules will throw light on these questions in the GABA four GAT1–GAT4 future.12 GABA/betaine BGT-1 Glycine two GLYT1 & GLYT2 Taurine RB16a Neurotransmitter transporters as targets for CNS Proline PROT drugs l-Glutamate five EAAT1–EAAT5 Vesicular monoamines two VMAT-1 & 2 Monoamine transporters—NET and SERT Vesicular acetyl choline VAChT By far the most important group of CNS drugs that tar- Vesicular GABA/glycine VGAT get the NE and serotonin neurotransmitter transporters is the tricyclic antidepressants and their modern For more detailed information see Masson et al, 1999.7 counterparts. The discovery by the Axelrod group in

Molecular Psychiatry Neurotransmitter transporters L Iversen 359 196113 that imipramine potently inhibited the uptake Table 3 Additional indications for SSRI antidepressants of norepinephrine led to the first understanding of the mechanism of action of the first generation tricyclic Compound Additional indications antidepressants. Following the discovery of the serotonin uptake system in brain it soon became apparent Fluoxetine Bulimia nervosa, Obsessive compulsive that the classical tricyclic drugs imipramine and ami- disorder (OCD), Pre-menstrual syndromea triptyline were almost equally potent as inhibitors of Fluvoxamine OCD NE and 5-HT uptake (Table 2). This reinforced the Paroxetine OCD, Panic attacks, Social anxiety disordera monoamine hypothesis of depression as a monoamine Sertraline OCD, Panic attacks, Post-traumatic stress a deficiency state, and stimulated much further research syndrome in the pharmaceutical industry to discover new inhibi- a tors of monoamine uptake. The debate as to whether Clinical trials successfully completed, approval awaited. inhibition of NE or 5-HT was the most important in conferring antidepressant efficacy has swung one way the question of whether genetic defects in SERT or in and the other over the past 40 years and there is no the regulation of its expression might explain the etiol- definitive answer to this question. A fascinating ogy of mood disorders. The gene coding for human account of the historical development of research in SERT is localized on chromosome 17q11.2. It spans this field is given by David Healy in his monograph over 35 kilobases and is organised in 14 introns. No ‘The Antidepressant Era’.14 An early effort to improve genetic variations have been found in the coding region the selectivity of antidepressants was made in the of the SERT gene in depressed patients, but a number 1970s by scientists at the CIBA-GEIGY Company in of studies have found that certain variants in a poly- Switzerland, who developed the selective NE uptake morphic region flanking the 5Ј region or in the second inhibitor maprotiline. This proved to be clinically intron are associated with depressive illness, anxiety- effective as an antidepressant but it was not a great suc- related personality traits or suicidal alcoholism.7 cess commercially and had few clear advantages over Ironically, some of the most recently introduced anti- the classical tricyclics. This idea was also swept away depressants hark back to the non-selective compounds by the wave of enthusiasm for serotonin selective reup- of the earlier era. Thus the new entrants venlafaxine take inhibitors (SSRIs) that started with the success of and milnacipran are described as drugs that combine fluoxetine.15 Fluoxetine and the related group of SSRIs both NE and serotonin re-uptake inhibition17 (although have proved to be a remarkably important advance in in vitro binding data show that venlafaxine binds with psychopharmacology. Although these drugs have no some 300 times higher affinity to human SER than to greater antidepressant efficacy than the classical tri- NET).16 The compound sibutramine is also an inhibitor cyclics, they have a greatly reduced cardiac toxicity of both NE and serotonin uptake, but it has been and lack the cholinergic properties of the older drugs. approved for use as an anti-obesity agent rather than The SSRIs exhibit some adverse side effects, notably in an antidepressant.18 At the same time reboxetine is the impaired sexual function in both men and women, but first antidepressant drug in a new class of NET-selec- because they are relatively safe physicians have been tive inhibitors. It is claimed to be particularly suitable less inhibited about using them. This has extended the for depressed patients who ‘feel tired all the time’.17 clinical recognition and treatment of depression to a Reboxetine is reported to be as effective as the SSRIs much greater number of people than hitherto. At the or older tricylics, but is not associated with sexual dys- same time new indications have been demonstrated function.19 The older antidepressant bupropion, which and officially approved for the SSRIs, including syn- acts as a weak inhibitor of NE and dopamine uptake, dromes associated with anxiety and panic, extending with little effect on serotonin uptake has had only still further the wide use of these agents in psychophar- modest success as an antidepressant, but has been macology (Table 3). approved in the US as an aid to smoking cessation.17 The remarkable success of the SSRIs has prompted What are we to make of these twists and turns? How can drugs that are selective NE uptake inhibitors be equally effective as those that selectively target sero- Table 2 Antidepressant amine uptake inhibitors tonin? In practice it is difficult to know how selective the monoamine uptake inhibitors are in vivo. None of Mixed NET/SERT NET-selective SERT-selective the antidepressants is completely selective for NE or inhibitors inhibitors inhibitors (SSRIs) SERT. The SSRIs have some affinity for NET, and some (eg paroxetine) are quite potent inhibitors of NET.16 In Amitriptyline Nortripyline Citalopram some cases the formation of active metabolites alters Imipramine Desipramine Fluoxetine Dothiepin Maprotiline Fluvoxamine the drug selectivity profile. Thus the non-selective Nefazodone Reboxetine Paroxetine compounds imipramine and lofepramine are exten- Milnacipran Sertraline sively metabolised to desipramine, a highly potent and Duloxetine Indalpine selective NE uptake inhibitor. Similarly whereas ami- Venlafaxine triptyline has little selectivity for NET or SERT, the metabolite nortriptyline is a selective NET inhibitor. It For more detailed information see Owens et al.16 seems likely that both NE-selective and SSRIs exert

Molecular Psychiatry Neurotransmitter transporters L Iversen 360 their effects through some common final pathway in tive inhibitors of dopamine uptake might be useful and the brain. Perhaps the SSRIs act indirectly to modulate free of dependence liability. One such compound, bra- noradrenergic function.20 The original monoamine sofensine, is in clinical development for the treatment hypothesis of depression as formulated by Schildkraut of Parkinson’s disease.27 Other selective DAT inhibi- in 196521 stated: tors have been proposed for the treatment of the with- drawal phase of CNS drug abuse. ‘Some, if not all, depressions are associated with an absol- Another important group of psychopharmaceuticals, ute or relative deficiency of catecholamines, particularly norepinephrine, at functionally important adrenergic the amphetamines, also serve as both substrates and receptor sites in the brain. Elation conversely may be asso- inhibitors of DAT. Amphetamines have the ability to ciated with an excess of such amines’. be taken up by dopaminergic neurons and to promote the release of dopamine. d-Amphetamine and methyl Opinion currently seems to be swinging back in sup- phenidate have become widely used in the treatment port of the view that an up-regulation of noradrenergic of children with attention-deficit hyperactivity dis- function may be the key element underlying the effi- order (ADHD) in recent years.28 The concept of admin- 20 cacy of antidepressant drugs. istering psychostimulants to hyperactive children The molecular mechanisms in the brain that are trig- might at first sight appear paradoxical. Recent findings gered by the antidepressants, however, remain obscure. in the DAT knock-out mouse model may offer new The fact that all drugs require a period of several weeks insight into the mechanisms involved. The adminis- before they become fully effective suggests that they tration of d-amphetamine or methyl phenidate at doses modify gene expression in the brain and that the that cause behavioural stimulation in normal mice had resulting altered biochemical state takes a long time to a paradoxical calming effect on the hyperactive DAT become stabilised. Many theories have been proposed, knockout animals.29 This calming effect appeared to be including alterations in the expression of alpha and due to the ability of the amphetamines to cause an beta-adrenergic receptors, changes in transcription factors and/or neurotrophic factors, and even morphologi- increased release of serotonin, as they also have cal alterations in the connectivity of monoaminergic appreciable affinity for both SERT and NET in addition nerves.20 to their affinity for DAT. It is possible that antidepressant drugs have other Studies of the human gene encoding DAT have sug- targets in addition to their actions at cell surface gested an association between polymorphisms in a 30 monoamine transporters. Al-Damluji and Kopin22 have non-coding region and ADHD. described a novel amine uptake process in peptide- The DAT is also important in mediating the actions containing hypothalamic neurons, which they named of the selective dopaminergic neurotoxins 6-hydroxy- ‘transport-P’. Like the vesicular transporters this pro- dopamine, and 1-methyl-4-phenylpiperidinium ion + cess is driven by a proton gradient, but it is distinct (MPP ). The selective accumulation of these com- from the vesicular transporters in being insensitive to pounds by DAT in dopaminergic neurons explains reserpine, but sensitive to a variety of tricyclic anti- their targeted neurotoxicity. One possible explanation depressants at micromolar concentrations.23 It is not for the selective loss of dopaminergic neurons in Park- clear, however, what role if any transport-P plays in inson’s disease is that patients may have been exposed the inactivation of the monoamine neurotransmitters. to some environmental dopaminergic neurotoxin, but the search for such a compound has so far proved Monoamine transporters—DAT fruitless. Some antidepressants, notably mazindol and bupropion, inhibit the dopamine transporter (DAT) as well as NET or SERT. The DAT is best known, however, as Vesicular monoamine transporters one of the principal sites of action of the psychostimul- The vesicular monoamine transporters represent the ant drug cocaine. Mice that are genetically engineered site of action of reserpine and tetrabenazine, which to knock out the expression of the DAT gene are pro- cause a long-lasting impairment of monoamine storage foundly hyperactive and fail to show any further stimu- resulting in the depletion of monoamines from CNS lation of activity in response to cocaine or d-ampheta- and peripheral aminergic neurons. Although reserpine 24 mine. Such animals, nevertheless, will continue to was used briefly during the 1950s as an anti-schizo- 25 self-administer cocaine, suggesting that the phrenic drug it had several adverse side effects, includ- rewarding properties of the drug cannot be explained ing the ability to cause profound depression of mood. entirely by its ability to inhibit DAT. Cocaine is also a The vesicular monoamine transporters are potent inhibitor of both serotonin and NE uptake. It important, however, as they represent a second retains some rewarding properties even in SERT 26 important target for the amphetamines. The action of knock-out mice suggesting that inhibition of NE these psychostimulants depends on their ability to be uptake may also contribute importantly to its pharma- taken up into dopaminergic and other aminergic neu- cology. rons, and by interaction with the vesicular amine trans- A corollary of the understanding that cocaine owes porters to cause a release of dopamine and other important parts of its overall CNS profile to mech- endogenous monoamines. anisms other than inhibition of DAT is that more selec-

Molecular Psychiatry Neurotransmitter transporters L Iversen 361

Figure 1 Cartoon depicting the role of neurotransmitter transporters at the synapse. PNT = plasma membrane neurotransmitter transporter; VNT = vesicular neurotransmitter transporter. Redrawn from Mason et al.7

Amino acid neurotransmitter transporters been fully exploited, but the re-emergence of NET-spe- A number of experimental compounds have been cific antidepressants, and the possible applications of developed that act as selective inhibitors of GABA selective inhibitors of DAT suggest that there may still uptake. As might be expected, such compounds exhibit be room for innovation even in such a crowded field. anticonvulsant properties in animal seizure models, The amino acid neurotransmitter transporters rep- but so far tiagabine is the only compound that has been resent another potentially fruitful field for future CNS approved for human use as an anti-epileptic drug.31 drug discovery, with only one useful drug approved for Tiagabine selectively inhibits the GAT-1 transporter, human use so far, the GABA uptake inhibitor tiagabine. present in both neurons and astrocytes in the brain. Inhibiting the inactivation of l-glutamate after its Inhibitors of the other GABA transporters, or the vari- release in CNS may not be without hazards, as inhibi- ous l-glutamate transporters are not yet available, nor tors of the EAAT family may be liable to provoke seiz- have the other amino acid transporters (Table 1) ure activity or to act as pro-convulsants. On the other attracted much pharmacological interest. hand this is a complex family with five distinct transporters. It is possible that the selective inhibition of one or other member of the family could yield ben- Future prospects eficial effects (eg cognitive enhancement) with little The neurotransmitter transporter family has provided hazard. many valuable targets for psychopharmacology. There A completely novel target has been discovered in seems every prospect that this will continue. It might research on the mechanisms involved in the inacti- seem that the monoamine transporters had already vation of the naturally occurring brain cannabinoid

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