Trace amine-associated receptor 1: a multimodal therapeutic target for

neuropsychiatric diseases.

Michael D. Schwartz1, Juan J. Canales2, Riccardo Zucchi3, Stefano Espinoza4, Ilya Sukhanov5, Raul R.

Gainetdinov6,7

1. Center for Neuroscience, SRI International, Menlo Park CA, USA

2. Division of Psychology, School of Medicine, College of Health and Medicine, University of Tasmania,

Private Bag 30, Hobart, TAS 7001, Australia

3. Fondazione Istituto Italiano di Tecnologia, Neuroscience and Brain Technologies Dept., Via Morego 30,

16163 Genoa, Italy

4. Department of Pathology, University of Pisa, Pisa, Italy

5. Institute of Pharmacology, Pavlov Medical University, St. Petersburg, Russia

6. Institute of Translational Biomedicine, St. Petersburg State University, 199034 St. Petersburg, Russia

7. Skolkovo Institute of Science and Technology, 143025 Moscow, Russia.

Disclaimer:

Support: IS is supported by the Russian Science Foundation Grant № 17-75-20177; RRG is supported

by the Russian Science Foundation Grant № 14-50-00069.

Word count:

Abstract: 198

Body text: 5964

Figures: 0

Abstract

Introduction. The trace amines, endogenous amines closely related to the biogenic amine neurotransmitters,

have been known to exert physiological and neurological effects for decades. The recent identification of a

trace amine-sensitive G-protein coupled receptor, trace amine-associated receptor 1 (TAAR1), and subsequent

development of TAAR1-selective small-molecule ligands, has renewed research into the therapeutic

possibilities of trace amine signaling.

Areas covered. Recent efforts in elucidating the neuropharmacology of TAAR1, particularly in

neuropsychiatric and neurodegenerative disease, addiction, and regulation of arousal state, will be discussed.

Focused application of TAAR1 mutants, synthetic TAAR1 ligands and endogenous biomolecules such as 3-

iodothyronamine (T1AM) has yielded a basic functional portrait for TAAR1, despite a complex biochemistry

and pharmacology. The close functional relationship between TAAR1 and dopaminergic signaling is likely to

underlie many of its CNS effects. However, TAAR1’s influences on serotonin and glutamate

neurotransmission will also be highlighted.

Expert opinion. TAAR1 holds great promise as a therapeutic target for mental illness, addiction, and sleep

disorders. A combination of preclinical and translationally-driven studies has solidified TAAR1 as a key node

in the regulation dopaminergic signaling. Continued focus on the mechanisms underlying TAAR1’s regulation

of serotonin and glutamate signaling, as well as dopamine, will yield further disease-relevant insights.

1. Introduction

1.1. Trace amines and trace amine-associated receptor 1 (TAAR1)

The trace amines, endogenous amines closely related to the biogenic amine neurotransmitters (eg.

dopamine (DA), serotonin (5-hydroxytryptamine; 5-HT) and norepinephrine (NE)) have been known to exert

physiological and neurological effects since the early 20th century [1]. However, the lack of an identifiable

endogenous receptor for these molecules, coupled with their markedly low in vivo concentrations, led in part to

the idea that trace amines were “false neurotransmitters” [2]. In 2001, this conventional wisdom was

challenged with the identification of a vertebrate G-protein coupled receptor (GPCR) that preferentially

responded to trace amines [3, 4]. This receptor, trace amine-associated receptor 1 (TAAR1), is part of a large

and evolutionarily diverse family of TAARs with 6 functional members in humans [5]. Several TAARs act as

olfactory receptors [6]. In the mammalian brain, the finding that TAAR1 powerfully modulates monoaminergic

neurotransmission [7, 8, 9], has rejuvenated research efforts into the function and therapeutic implications of

TAAR1 and its ligands.

1.2. TAAR1 expression and function

In the brain, Taar1 expression is enriched throughout the limbic and aminergic systems, encompassing

the dopaminergic ventral tegmental area (VTA)/substantia nigra and serotonergic dorsal raphe nucleus (DRN)

[1, 10, 11], and is therefore ideally positioned to regulate the activity of these neurotransmitter systems. Indeed,

transgenic mice lacking TAAR1 exhibit markedly elevated discharge rates of DA and 5-HT neurons [12],

suggesting that TAAR1 activation down-regulates monoaminergic neurotransmission. The strategic

neuroanatomical location of TAAR1 and its remarkable ability to regulate aminergic neurotransmission suggest

that this receptor could serve as a target to develop more effective, new generation pharmacotherapies for

neuropsychiatric diseases, addiction and sleep disorders.

This review will highlight recent efforts in elucidating the neurological and neurophysiological effects

and potential therapeutic utility of TAAR1 activation via recently-developed TAAR1-specific small molecules,

as well as endogenous biomolecules such as 3-iodothyronamine. While beyond the scope of this review, there

is also significant peripheral TAAR1 expression in pancreas, stomach and leukocytes, suggesting potential for

TAAR1-based drugs in diabetes, obesity and possibly immune disorders [2].

1.3. Synthetic TAAR1 agonists and antagonists

Hoffmann-La Roche investigators performed a large-scale effort to derivatize adrenergic ligands, which

were screened for TAAR1 activation by cAMP assays in heterologous cells expressing TAAR1, and for

specificity via radioligand binding experiments involving over a hundred different proteins. This effort yielded

several full (e.g. RO5166017 and RO5256390) and partial (e.g. RO5203648 and RO5263397) TAAR1

agonists[11, 13, 14], that to date have been successfully used in experimental models of neurological diseases

such as drug addiction, schizophrenia, and Parkinson’s disease. In general, the “RO compounds” are over 100-

fold selective for TAAR1 vs other aminergic receptors, but the Ki’s for some other receptors – namely α2

adrenergic, 5-HT2 5-HTergic, µ and κ opioid, and I1 imidazoline receptors – are in the nanomolar range, so

additional effects on different targets cannot be excluded in all cases.

Pharmacological research has also aimed at developing selective TAAR1 antagonists. Screening of

about 700,000 Roche compounds led to the identification of N-(3-Ethoxy-phenyl)-4-pyrrolidin-1-yl-3-

trifluoromethyl-benzamide (EPPTB) [10, 15]. This benzamide derivative had high selectivity and affinity for

mouse TAAR1 (Ki = 0.9 nM), although the affinity for human TAAR1 was in the micromolar range. In

particular, EPPTB was critical in revealing the constitutive background activity of the TAAR1 system, since it

caused a significant increase in the firing rate of mouse VTA DA neurons [10].

2. 3-Iodothyronamine: an endogenous TAAR1 ligand

2.1. Biochemistry and pharmacology of T1AM

3-iodothyronamine (T1AM) is an endogenous compound whose chemical structure is related to thyroid

hormones [16]. The differences consist in the absence of the carboxyl group and of all iodine atoms except one.

It is thought to be synthesized from 3,5,3’-triiodothyronine (T3) through the sequential action of deiodinases

and amino acid decarboxylases (possibly ornithine decarboxylase) [17], but the precise biosynthetic pathway

and the physiological site(s) of production are still unclear. T1AM has been identified in rodent and human

blood, and in most rodent organs including the brain, where its average endogenous level is on the order of a

few pmoles per g [16, 18, 19, 20, 21, 22, 23].

In 2004, T1AM was reported to be a powerful activator of TAAR1 [16]. Its EC50 for a biochemical

response (cAMP production) in human neoplastic cell lines expressing rat or mouse TAAR1 averaged 14 nM

and 112 nM, respectively, and so it was lower than observed for endogenous trace amines, namely tyramine and

β-phenylethylamine. With human TAAR1, the affinity for T1AM was in the micromolar range, but it was still

higher than observed for tyramine and β-phenylethylamine [24]. So, T1AM qualifies as a bone fide

physiological TAAR1 agonist.

2.2. Neurophysiological effects of T1AM

T1AM modulates several integrative functions, particularly feeding behavior, sleep, and cognition

(reviewed by [25]). In fed animals, i.c.v. T1AM administration increased food intake at dosages as low as 1.2

nmol/Kg, and a similar effect was observed after arcuate nucleus injection [26]. However, in fasting animals

the response was biphasic: dosages in the low nanomolar range were anorexic, while higher dosages (51

nmol/Kg) had the opposite effect [27]. T1AM injection (3 µg) in the preoptic region enhanced locomotor

activity and increased wakefulness, while decreasing non-rapid eye movement (NREM) sleep time [28]. i.c.v.

T1AM injection (1.32 – 4 µg/Kg) elicited prolearning and antiamnestic effects in the passive avoidance

paradigm, as well as increased curiosity in the novel object recognition task [21].

2.3. Mechanisms of action

2.3.1. Neuromodulatory actions of T1AM

The basic cellular processes targeted by T1AM remain to be determined, but are proposed to be

neuromodulatory in nature. Manni et al [21] reported that active dosages of T1AM increased average brain

T1AM concentration by about 30-fold, consistent with a physiological role of endogenous T1AM. T1AM

applied locally in rat locus coeruleus modulated the activity of adrenergic neurons with EC50 = 2.7 µM [29].

T1AM’s pro-learning and anti-amnestic effects may depend on histaminergic activity, since they were

dampened or abolished by histamine receptor antagonists and in transgenic mice lacking histidine

decarboxylase, the key enzyme regulating histamine biosynthesis [21, 22]. Modulation of adrenergic and

histaminergic activity could also underlie wake promotion since both neurotransmitters are robustly wake -

promoting [30, 31, 32].

2.3.2. T1AM and TAAR1

Preliminary evidence for a TAAR1-mediated effect has been recently reported by electrophysiological

recordings performed in rat entorhinal cortex. In this model, T1AM rescued long-term potentiation in the

presence of toxic concentrations of beta amyloid, and the effect was abolished by the TAAR1 antagonist

EPPTB [33]. This exciting observation suggests additional therapeutic utility for TAAR1 agonists because of

the putative role of beta amyloid in Alzheimer’s disease and other forms of cognitive impairment.

However, it is presently unclear whether all the actions produced by T1AM are mediated by TAAR1.

Other potential targets include other TAARs (particularly TAAR5), α2A adrenergic receptors, transient receptor

potential channels (particularly TRPM8), and monoamine transporters [25, 34]. The presence of multiple

physiological targets is not unusual for a chemical messenger, but it casts doubts on a TAAR1-specific

mechanism for T1AM. Conversely, some effects observed after T1AM administration could actually be

mediated by 3-iodothyroacetic acid, the product of T1AM oxidative deamination. For example, histamine and

3-iodothyroacetic acid are involved in the accelerated response to the hot plate test, suggesting reduced pain

threshold [20, 22]. Further investigation with TAAR1 antagonists and/or KO mice are necessary to resolve this

crucial issue.

2.4. Synthetic T1AM analogs

To address these questions, synthetic T1AM analogs have been developed by modifying the

thyronamine scaffold. The first two series of analogs consisted of phenyltyramine derivatives [35, 36], while

more recently halogen-free biaryl-methane thyronamine analogs (the so called “SG compounds”) have been

synthesized [37, 38]. Some of these compounds were equipotent or even more potent than T1AM, as measured

by cAMP induction. They also reproduced the in vivo effects of T1AM on glucose homeostasis and cognitive

function in mouse. However, their selectivity has not yet been specifically evaluated, so the same questions

raised for T1AM regarding interaction with different targets apply here. Systematic evaluation of T1AM

analogs is expected to clarify several features of T1AM/TAAR1 interactions and structure-activity relationships

[39], and provides a valuable background for further research in the TAAR1 pharmacology.

3. TAAR1 in neuropsychiatric disorders

3.1. Trace amines, DA and neuropsychiatric disease

Trace amine dysregulation has long been associated with several psychiatric and neurological diseases.

For example, elevated PEA content has been documented in schizophrenia [40, 41, 42], whereas decreased PEA

levels were associated with depression [43, 44, 45]. However, the lack of an identifiable endogenous receptor

and dearth of suitable investigative tools limited advancement on this front for some time [2]. The

identification of TAAR1 [3, 4] and subsequent demonstration that TAAR1 modulates DA and 5-HT

neurotransmission [7, 8, 9] has renewed interest in this association. Brain DA is critically involved in the

etiology and pathogenesis of neuropsychiatric disorders including schizophrenia, Attention Deficit and

Hyperactivity Disorder (ADHD) and Parkinson’s disease; DA dysregulation is proposed to contribute to

Obsessive-Compulsive and Related Disorders (OCD), bipolar disorder, major depression and dyskinesias [46].

Most of these conditions have been previously linked to dysregulated endogenous trace amines [1, 47]. With

the development of TAAR1-specific mutant mice [48, 49, 50] and selective pharmacological compounds[10,

11, 13, 14, 15], these advances have vastly enhanced understanding of TAAR1’s role in neuropsychiatric

disorders and its potential therapeutic applications.

3.2. TAAR1 and schizophrenia

3.2.1. Dopaminergic dysregulation and schizophrenia

The DA theory of schizophrenia asserts that increased dopaminergic tone or D2 receptor sensitivity

resulting in dysregulated DA signaling, underlies the development of schizophrenia, particularly its positive

symptoms (e.g. hallucinations, delusions and disordered thoughts and speech). This theory has a strict

predictive validity, since all known clinically effective antipsychotics are D2 receptor antagonists [51].

Hyperactivity induced by dopaminergic psychostimulants is considered a behavioral manifestation of increased

dopaminergic activity in the mesolimbic pathway [52], and potentiated psychostimulant-induced activity is used

as an animal correlate of positive symptoms [53]. Accordingly, the ability of a drug to antagonize this

hyperactivity has been used for decades as a preclinical screening tool to identify novel antipsychotics [54, 55].

3.2.2. TAAR1 and Dopaminergic tone

Taar1 KO mice do not differ in size, weight and temperature from wild type (WT) littermates and

perform normally in behavioral tests including motor coordination, visual acuity, grip test, nociception and

locomotor activity in the open field [49, 50, 56]. However, these mice exhibit increased locomotor responses to

dopaminergic psychostimulants such as amphetamine, methamphetamine or MDMA compared to WTs [48, 50,

57, 58], as well as high doses of the selective D2/ D3 agonist quinpirole [59]. Taar1 KO mice were also more

sensitive to locomotor sensitization induced by repeated amphetamine and methamphetamine administration

[57, 60]. TAAR1 agonists prevented cocaine- and amphetamine-induced hyperlocomotion in WT mice [11, 14,

61] and Wistar rats [13] and enhanced olanzapine (atypical antipsychotic)-induced inhibition of locomotor

activity following cocaine [14]. Conversely, amphetamine-induced hyperactivity was strongly attenuated in

TAAR1-overexpressing (OE) mice, and ‘rescued’ by the selective partial agonist RO5073012 [61]. Together,

these findings indicate TAAR1 as a viable locus for treatment of positive psychotic symptoms, likely mediated

by modulation of dopaminergic signaling. Consistent with this hypothesis, TAAR1 agonism blocks

hyperlocomotion in hyperdopaminergic DA transporter knockout (DAT) KO mice and rats [11, 13, 62].

3.2.3. TAAR1 and NMDA hypofunction

N-methyl-D-aspartate (NMDA) receptor antagonists also increase motor activation [63, 64], and this

hyperactivity is similarly reversed by antipsychotics [65]. As with dopaminergic psychostimulants, TAAR1

agonists reversed hyperlocomotion induced by the NMDA antagonists L-687,414 and phencyclidine [11, 14].

The partial TAAR1 agonist RO5203648 also blocked hyperlocomotion in NMDA receptor-deficient mice [13].

However, L-687,414-induced hyperactivity was not further potentiated in Taar1 KO mice [11], in contrast to

the exaggerated response to dopaminergic stimulants in these animals. Future studies should examine the

respective contributions of dopaminergic versus glutamatergic pathways to the expression of hyperactivity vis a

vis TAAR1.

3.2.4. Caveats and questions

Some variability is reported in the direct locomotor effects of TAAR1 agonists themselves. Some

studies reported that TAAR1 agonists decreased locomotor activity in intact animals [13, 14] while others did

not find any effects of these drugs when administered alone [61, 66, 67, 68]. Intriguingly, TAAR1 deletion

greatly attenuated climbing and other stereotypy behaviors induced by high doses of the mixed D1/D2 agonist

apomorphine [69], likely due to a direct agonistic action of apomorphine at TAAR1 [4]. Since apomorphine-

induced stereotypies are also used to screen for antipsychotics [70, 71, 72], these findings imply a need for care

when interpreting apomorphine-induced behaviors in rodents.

Whereas all clinically used antipsychotics augment haloperidol-induced catalepsy, partial TAAR1

agonists actually reduce this phenomenon [13, 14] and do not themselves induce catalepsy [14]. Intriguingly,

the catalepsy induced by haloperidol was significantly reduced in Taar1 KO mice [8].

3.2.5. Cognitive symptoms of schizophrenia

Memory, attention, and other cognitive deficits form another important aspect of the symptomatology of

schizophrenia [73]. Taar1 KO mice showed impairment in prepulse inhibition of the acoustic startle response

[50], indicating a sensorimotor gating deficit. This abnormality is commonly used to demonstrate

schizophrenia-like phenotypes in animals because human schizophrenic patients exhibit an analogous deficit

[74]. By contrast, spatial working memory in the forced alternation test was intact in Taar1 KO mice [50]. The

TAAR1 agonists RO5256390, RO5203648, and RO5263397 increased accuracy in the object retrieval task in

cynomolgus macaques [13, 14], and RO5256390 (1.0 and 3.0 mg/kg) fully reversed executive function deficits

induced by repeated PCP treatment (5.0 mg/kg) in the attention set shift task in rats [14]. Further assessment of

procognitive effects of TAAR1 agonists is therefore warranted, particularly in key cognitive domains such as

attention and learning.

In general, the loss of TAAR1 induces elevated DA neurotransmission in the mesolimbic pathway in

mice. Specific TAAR1 agonists ameliorate pharmacologically- and genetically-induced hyperlocomotion

without the undesirable motor side effects characteristic of D2 receptor-blocking antipsychotics. Moreover,

TAAR1 agonists may be effective in treating cognitive deficits associated with schizophrenia.

3.3. TAAR1 and Parkinson’s disease

Loss of nigrostriatal dopaminergic neurotransmission is the key point in pathogenesis of Parkinson’s

disease. L-DOPA, the chemical precursor of DA, is the first and the most widely used treatment of Parkinson’s

disease. TAAR1-KO mice in which dopaminergic neurons were unilaterally lesioned with the neurotoxin 6-

OHDA had increased sensitivity to L-DOPA-induced rotational behavior and dyskinesia compared to WT

littermates [75]. Thus, it would be important to determine whether partial TAAR1 agonists ameliorate L-

DOPA-associated side-effects such as dyskinesia. Intriguingly, Sotnikova and colleagues observed

“antiparkinsonian” effects of amphetamines and MDMA in DA-depleted DAT KO mice [76], but the same

effect persisted in double knockout mice lacking both DAT and TAAR1, indicating that this potential

“antiparkinsonian” action of amphetamines is TAAR1-independent [58].

3.4. TAAR1 and ADHD

Evidence increasingly suggests that DAT dysfunction is involved in the pathogenesis of ADHD [77, 78,

79]; indeed, DAT KO mice are a known genetic animal model of ADHD [80]. In a novel environment, these

mice exhibit profound hyperactivity [81, 82]. TAAR1 agonists blocked hyperlocomotion in these mice [11],

mimicking the calming effect of amphetamine and methylphenidate [83], the drugs clinically used to treat

ADHD patients. Similar effects of the partial TAAR1 agonist RO5203648 were observed in DAT KO rats (Leo

et al., 2018). Conversely, double DAT/Taar1 KO mice demonstrated further increased hyperactivity over DAT

KO mice [13], providing additional support for TAAR1’s role in dopaminergic control.

ADHD patients are also characterized by increased impulsivity. A recent study has shown that lack of

TAAR1 led to perseverative and impulsive behaviors that correlated with deficient prefrontal cortical

glutamatergic transmission [84]. Furthermore, RO5166017 and RO5203648 decreased premature responding in

a fixed interval conditioning schedule in WT mice [84]. RO5256390 and RO5263397 also increased the

number of reinforcers earned in a differential reinforcement of low response rates test in cynomolgus macaques

[13]. In a recent paper, RO5263397 reduced hyperimpulsivity (5CSRTT) in methamphetamine-treated rats, but

did not affect the number of premature responses in 5CSRTT and choice of large reward in delay of reward test

in vehicle-treated rats [81].

3.5. TAAR1 and OCD

Although selective 5-HT reuptake inhibitors (SSRI) form the first line of drugs to treat OCD [85] a role

for DA function in OCD pathogenesis has been actively studied in recent years (for review see [86]).

Antipsychotics form a second line of OCD treatment since about 50% OCD patients are resistant to SSRI

therapy. RO5263397 reduced obsessive drinking in schedule-induced polydipsia, a popular preclinical model

of compulsive behavior [87]. Additionally, the full TAAR1 agonist RO5256390 blocked compulsive eating in

rats [88]. Thus, studies on TAAR1’s involvement in animal models of compulsivity and OCD, while

promising, require further exploration.

3.6. TAAR1 and affective disorders

RO5263397 and RO5203648, but not RO5256390 demonstrated antidepressant action in the forced

swim test [13, 14, 88], a test which has high predictive validity for identification of new antidepressants [89].

This effect could be mediated via a TAAR1-dependent enhancement of serotonergic signaling since the

antidepressant effect was only seen with partial agonists, which increase 5-HT firing [11, 13, 14]. However,

several lines of evidence also support dopaminergic involvement in affective disorders (for review see [90]).

Further studies aimed at the neurochemical basis for the antidepressive effects of TAAR1 agonism are therefore

warranted.

4. TAAR1 and addiction therapeutics

4.1. Therapeutic challenges in psychostimulant addiction

Drug addiction is a multifaceted neuropsychiatric disorder with widespread medical and societal

implications. Improving the treatment, support and rehabilitation of those affected by drug addiction continues

to be an important research agenda. Although behavioral and cognitive therapy, combined with psychosocial

support and community interventions, constitute irreplaceable initiatives to aid in recovery, there exists

considerable agreement among psychiatrists and health professionals specializing in addiction that novel

pharmacological approaches are needed to treat the disorder more effectively [91, 92]. Chiefly, new

medications are required to better manage withdrawal symptoms and craving in the early days and weeks after

drug discontinuation. Such medications could facilitate compliance and engagement in behavioral therapy,

multiplying the beneficial effects of non-pharmacological approaches. In this context, addiction to stimulant

drugs, such as cocaine and methamphetamine, is particularly problematic due to the lack of effective treatment

options.

4.2. The biogenic amines and addiction

Research into the neurobiological mechanisms that contribute to drug addiction suggests that the

classical biogenic amines, including DA, norepinephrine and 5-HT, and their corresponding receptor targets,

play a critical role [93]. The DA substitution approach in stimulant addiction involves the use of a competing

Dopaminergic agonist to potentially suppress withdrawal and drug craving in abstinent individuals [94].

Although this is still an avenue under investigation, compounds that act directly at the DA transporter (e.g.

slow-acting transporter blockers), or at DA receptors, are themselves more likely to have abuse potential and

long-term side effects. This liability justifies the search for new receptor targets to indirectly modulate DA

transmission through the ups and downs of the addiction cycle. Due to its unique association with ascending

Dopaminergic projections and key associated limbic circuits, TAAR1 has emerged as one of the most promising

targets for the treatment of neuropsychiatric disorders, especially addiction.

4.3. TAAR1 & stabilization of dysregulated DA signaling

The development of synthetic TAAR1 ligands have proven critical in elucidating its physiological and

behavioral functions. While both full and partial TAAR1 agonists decrease stimulant-induced DA overflow in

the nucleus accumbens (NAcb) [7, 95, 96], their effects on DA neuron firing rate can be different. In patch

clamp preparations, the full agonist RO5256390 attenuated neuronal firing in the VTA [11], whereas the partial

agonist RO5263397 augmented the firing frequency as did the antagonist EPPTB [10]. This suggests that

TAAR1 is constitutively active and/or tonically activated by endogenous ligands at the level of midbrain such

that partial agonism results in antagonistic-like effects. Consequently, the use of a partial agonist may be more

advantageous in situations where neurochemical imbalance (e.g., induced by drug exposure) leads to

insufficient or excessive TAAR1 stimulation, providing a means to “stabilize” TAAR1 activity. In addition,

through induction of pacemaker activation, partial agonism may contribute to “normalizing” DA neuron cell

discharge, which is known to be dysregulated following chronic cocaine exposure [97, 98]. In agreement with

these physiological observations, and supporting the notion that TAAR1 activation may indeed dampen DA

transmission under certain conditions, Taar1 KO mice exhibited increased sensitivity to amphetamine and

increased striatal DA release [49], whereas brain-specific TAAR1 overexpression reduced the psychomotor

stimulant effects of amphetamine [61].

The development of TAAR1-selective agonists has since allowed the accumulation of compelling

evidence in support of TAAR1 as a candidate for the design of addiction medications. Motor sensitization, a

process that evolves following repeated psychomotor stimulant treatment and that involves plasticity changes in

the mesolimbic DA system, was attenuated by the partial TAAR1 agonists RO5203648 [99] and RO5263397

[100, 101]. Self-administration models are the gold standard in addiction research, allowing the study of a

variety of behavioral processes. TAAR1 activation with the partial agonist, RO5203648, dose-dependently

decreased cocaine self-administration [13], with similar reductions being observed in methamphetamine self-

administration [99, 100]. Importantly, RO5203648 was able to block stimulant self-administration without

concomitant decreases in response rates for food self-administration, thus ruling out motor or motivational

deficits. Subsequent work demonstrated that both RO5203648 and the full TAAR1 agonist, RO5256390,

flattened the dose-response curve for cocaine self-administration, indicating that TAAR1 activation effectively

decreased the reinforcing effects of cocaine [102].

4.4. TAAR1 regulates reward mechanisms

In addition to perturbing motor behavior and promoting reinforcement learning, stimulants are known to

increase brain reward and recruit motivational mechanisms to instigate their procurement. TAAR1 activation

regulates reward and motivational processes induced by stimulant drugs. Using an intracranial self-stimulation

paradigm, Pei et al. (2015) showed that both RO5263397 and RO5256390 lowered cocaine self-stimulation

thresholds, thus suggesting reduced cocaine reward. Moreover, in a progressive ratio schedule of

reinforcement, RO5203648 dose-dependently shifted both cocaine’s and methamphetamine’s response rate

curve rightward and delayed the time to reach break point, while elevating the break point for food self-

administration [95, 96]. These data clearly indicate that TAAR1 activation reduces stimulant reward and the

motivation to seek and self-administer stimulant drugs.

4.5. Preventing drug relapse

There is a myriad of catalysts that can trigger drug relapse, one of the most insidious problems

associated with drug addiction. Models of relapse have been employed in the laboratory to investigate the

potential of TAAR1 agonists to regulate relapse to drug seeking behavior. Data have been similarly convincing

in that both RO5203648 and RO5263397 dose-dependently prevented context-induced cocaine relapse in a

model of forced abstinence [95] and cue- and cocaine prime-induced reinstatement of cocaine and

methamphetamine seeking after extinction training [95, 96, 101]. These observations support the notion that

TAAR1 agonists may be useful in relapse prevention and management of rehabilitation processes in addiction.

4.6. TAAR1 and the molecular mechanisms of addiction

The molecular mechanisms and signaling pathways through which TAAR1 exerts such remarkable

effects on stimulant-induced behaviors are still poorly understood. TAAR1 distribution is predominantly

intracellular [4, 103], stimulating both accumulation of cAMP, via Gαs-adenylyl cyclase activation which

promotes PKA and PKC phosphorylation [3, 4, 104], and a G protein-independent, β-arrestin2-dependent

pathway involving a DA D2 receptor-regulated protein kinase B (AKT)/glycogen synthase kinase (GSK-3)

[105]. To uncover the mechanisms through which TAAR1 prevents cocaine effects on DA transmission,

Asif-Malik et al. (2017) recently conducted in vitro fast-scan cyclic voltammetry experiments, elegantly

demonstrating a new pathway to control cocaine’s neurochemical actions that involves TAAR1. Upon

TAAR1 stimulation, such pathway recruits D2 autoreceptors functionally linked to TAAR1 and downstream

molecular targets converging on GSK-3, but not on PKA or PKC [7]. It is worth noting that GSK-3 has

been previously implicated in cocaine sensitization [106] and cocaine reward memory [107]. These results

open new avenues to further explore such complex molecular interactions, with a view to optimize TAAR1-

based drug development in the area of addiction treatment.

4.7. Summary

Although changes in DA transmission are undoubtedly important, it is now recognized that the spiral of

cycles of abstinence and relapse that characterizes stimulant addiction is associated with widespread metabolic

changes in the brain and alterations in the way that different brain regions connect, communicate, and function.

These connectivity problems, especially loss of prefrontal-to-striatal functional connectivity, have been linked

to impaired “top-down” control and impulsivity trait, which predict drug escalation and increased relapse to

drug abuse. Recent data showed that Taar1 KO mice exhibited impulsive behavior and dysregulated function in

the prefrontal cortex, whereas pharmacological activation of TAAR1 with selective agonists reduced premature

impulsive responses [84]. In agreement with these findings, RO5263397 attenuated methamphetamine-induced

impulsive behavior [108]. This evidence suggests that TAAR1 also exerts control over addiction-related

circuits and behaviors that extend beyond the DA system and its associated functions.

In conclusion, the evidence reviewed in this section suggests that TAAR1 is uniquely placed to exert a

decisive influence over key neurochemical processes and behaviors associated with drug effects and addiction.

Indeed, both neurochemical and behavioral observations demonstrate the ability of TAAR1 to regulate not only

the effects of cocaine and methamphetamine on DA transmission, but also a wide range of behavioral,

motivational and cognitive processes that are affected by chronic drug exposure. As noted, the effects of

TAAR1 activation on drug self-administration, drug reward and relapse are particularly striking. Taken

together, these findings support the candidacy of TAAR1 as one of the most promising therapeutic targets in

addiction.

5. TAAR1 and wakefulness

5.1. TAAR1, the monoamines, and sleep-wake regulation

Sleep disturbances exact significant costs in terms of personal health consequences and economic

productivity [109, 110]. Sleep and circadian dysregulation are common comorbidities in neuropsychiatric and

neurodegenerative disorders [111] [112, 113] as well as addiction [114, 115]. This link is not surprising, since

the monoaminergic and glutamatergic neurotransmitter systems whose dysregulation underlies these diseases

also play fundamental roles in the regulation of sleep and wakefulness (for detailed reviews see [30, 31, 32,

116]). Investigations of TAAR1’s involvement in arousal state control suggest an important role in regulating

basal sleep and wakefulness, as well as potential therapeutic value for the sleep disorder narcolepsy.

5.2. TAAR1 mutants and sleep

5.2.1. Basal sleep-wake regulation in TAAR1 mutants

To determine the role of endogenous TAAR1 in regulating sleep and wakefulness, Taar1 KO and OE

mice were instrumented for EEG/EMG recording and compared to a common pool of WT littermates under

standard 12h light:dark (LD) cycles [117]. Circadian organization of locomotor activity, core body

temperature, sleep and waking was normal in both mutant strains, with wakefulness concentrated in the dark

phase. Total wake time was increased in OE mice relative to WTs over 24h and was associated with an

increased number of wake bouts; by contrast, KO mice exhibited decreased wakefulness and increased NREM

sleep at the lights-on transition compared to OEs and WTs. Compensatory recovery sleep following a 6h sleep

deprivation was normal in both mutants, indicating that homeostatic sleep regulation was unaffected by TAAR1

mutation. Thus, constitutive TAAR1 overexpression and deletion elicit a mild but significant increase and

decrease in basal wakefulness, respectively. These opposing effects are somewhat surprising, since both

knockout and overexpression is associated with elevated VTA DA and DRN 5-HT firing rates in vitro [11, 61],

and both DA [118, 119, 120] and 5-HT [121, 122] activity are positively correlated with wakefulness.

However, TAAR1 overexpression also elevates firing in LC noradrenergic neurons as well as VTA GABAergic

neurons [61], both of which are associated with wake promotion [123, 124], which may explain the enhanced

wakefulness seen in OE, but not KO mice. Conditional deletion/expression of TAAR1 in vivo would be of

considerable help in identifying how TAAR1 influences basal arousal states.

By contrast, TAAR1 deletion was associated with impulsivity and increased nocturnal nose-poke

activity in a goal-directed task [84], suggesting a greater perturbation of rest-activity cycles than seen in the

sleep EEG studies. Such a phenotype could arise from an interaction between the normal diurnal cycling of

extracellular DA, which peaks at lights-off [125], and dysregulated DA signaling in Taar1 KO mice [9]. This

hypothesis could explain both the temporal bias of the phenotype and its emergence in a reward-associated

context (compared to a more neutral homecage environment) but has yet to be tested.

5.2.2. EEG spectral abnormalities

Taar1 mutation elicited marked alterations in EEG spectral composition; specifically, theta (4-8 Hz) and

gamma (> 30Hz) band activity was elevated in KO compared to OE mice in both sleep and wakefulness, with

WT mice intermediate between them. Such a phenotype could arise from serotonergic dysregulation in KO

mice [11], although 5-HT suppresses gamma and theta activity [126, 127], rather than enhancing it as seen in

Taar1 KOs. Alternatively, dysregulated arousal states and EEG spectra could result from abnormal

glutamatergic regulation [84]. Enhancing glutamatergic transmission via group II metabotropic glutamate

receptors [128], particularly mGluR2 [129], as well as group I mGluR5 receptors [130, 131, 132] promotes

waking and high-frequency EEG activity (i.e. gamma power), while inhibition tends to potentiate NREM sleep

and EEG slow wave activity (i.e. delta power, 0.5-4Hz).

5.3. TAAR1 agonism and wake promotion

5.3.1. TAAR1 partial agonism

The partial agonists RO5203648 and RO5263397 increased total time awake while suppressing non-

REM and REM sleep for up to 3h in WT rats [13, 14] and mice [117]. Importantly, both RO5203648 and

RO5263397 promoted wakefulness without increasing locomotor activity, in contrast to the hyperactivity

frequently produced by psychostimulants. In mice, RO5263397 decreased mid- to high-range frequencies in the

waking and NREM EEG spectra, representing the alpha, beta and gamma bands [117]; in rats RO5263397

decreased NREM delta power. This pharmaco-EEG profile was entirely dependent on Taar1 expression, as

RO5263397 was entirely ineffectual when given to Taar1 KO mice, while wake promotion and REM

suppression was strongly potentiated in OE mice[117].

5.3.2. TAAR1 full agonism

The full agonist RO5256390 failed to increase wakefulness in WT rats and mice when administered in

the mid-dark and mid-light phase, respectively [14, 133]. In rats, RO5256390 was totally ineffective at all

doses tested, whereas in WT mice RO5256390 suppressed REM sleep [133]. This unexpected result was

hypothesized to reflect species differences in the intrinsic activity of RO5256390, increasing the likelihood of

some partial-agonist-like effects in mice compared to rats. Timing of drug administration may also have played

a part; RO5256390 was tested in rats during the dark phase, when REM sleep is normally reduced compared to

the light phase [134, 135]. On the other hand, RO5256390 elicited a similar EEG spectral profile as

RO5263397 in mice (i.e. decreased power in the theta, alpha and beta bands; M. Schwartz, unpublished

observations). As with RO5263397, all observed effects on sleep and waking following RO5256390 were

abolished in Taar1 KO mice, indicating a TAAR1-mediated effect [133].

5.3.3. Prospective mechanisms underlying TAAR1-mediated wake promotion

The wake-promoting effects of RO5203648 and RO5263397 could result from enhanced

monoaminergic signaling following partial TAAR1 agonism [14], especially since the full agonist RO5256390-

which suppresses monoaminergic signaling- failed to promote wakefulness [14, 133]. Similarly, the profound

REM-suppressing effect of TAAR1 partial agonism could be mediated via enhanced DA signaling [119, 136,

137] or via interactions with 5-HT1a and 5-HT1b receptors [11, 138, 139], both of which regulate REM sleep

[140, 141, 142]. Thus, the enhancement of wakefulness would appear to depend heavily on the “antagonist-

like” actions of the partial agonists. On the other hand, the similarity in EEG power spectral profiles induced by

the full and partial agonists suggests a common mechanism relying on the agonist-induced activation of

TAAR1. While still speculative, this striking combination of TAAR1 activation and inhibition is rarely seen

within the same assay. To help isolate the contributions of these possible mechanisms, a specific TAAR1

antagonist suitable for in vivo studies would be a welcome addition to the existing pharmacological tooklit.

5.4. TAAR1 agonism as a narcolepsy therapeutic

Narcolepsy is a sleep disorder characterized by hypersomnolence, sleep disruption, sleep paralysis and

cataplexy, a sudden loss of skeletal muscle tone during wakefulness. Narcolepsy arises from dysregulation of

the wake-promoting and –stabilizing hypocretin/orexin (Hcrt) neurons located in the lateral hypothalamus [143,

144, 145]. Current pharmacological treatments for narcolepsy, including stimulants such as amphetamines and

modafinil and the GABA agonist gamma-hydroxy butyrate (GHB), either offer limited therapeutic value (eg.

Modafinil treats the somnolence but does not improve cataplexy) or carry significant side effects (eg.

tolerance/abuse risk, sedation); thus novel therapeutics are needed [146]. To test efficacy of TAAR1 as a

therapeutic target for narcolepsy, RO5263397 and RO5256390 were given to two different mouse narcolepsy

models, the orexin-ataxin3 mouse [147] in which Hcrt neurons degenerate shortly after birth, and the

orexin/tTA- diphtheria toxin A fragment (DTA) mouse, in which Hcrt degeneration is conditionally regulated via

doxycycline access [148]. Both RO5263397 and RO5256390 reduced the number of cataplexy episodes and the

time spent in cataplexy [133], comparing favorably with the norepinephrine reuptake inhibitor desipramine, a

known anticataplectic [149]. Anticataplectic effects could be mediated via serotonergic modulation [150, 151]

and/or D2 signaling [59, 152]. At the highest dose, RO5256390 increased wakefulness in DTA but not ataxin

mice, with no further effects on NREM or REM sleep. RO5263397 suppressed REM sleep in ataxin but not

DTA mice, without altering wake or NREM sleep. As in WT mice and rats, neither drug elicited

hyperlocomotion.

5.5. Summary

In contrast to the similarity of full and partial agonism on neurobehavioral assays and studies of

addiction, the sleep studies to date highlight the complexity of TAAR1 signaling. For example, the full and

partial agonists exhibit divergent actions on wakefulness, but similar impact on EEG spectral profiles and

anticataplectic efficacy. These divergent effects likely reflect the multimodal nature of TAAR1’s actions in

vivo and highlight the complexity of manipulating endogenous TAAR1 signaling, particularly with partial

agonists that exhibit both agonist- and antagonist-like properties. Nevertheless, the studies to date suggest a

potentially key role for TAAR1 in regulating arousal state and cortical activation. TAAR1 agonism may also be

useful in treating sleep disorders, as demonstrated by the narcolepsy studies.

6. Conclusions

TAAR1 is a promising locus for treatment of neurological, neuropsychiatric and behavioral conditions

that have historically proven difficult to address, including schizophrenia, addiction, and sleep disorders. In

fact, two pharmacological companies have already initiated late-stage clinical trials of TAAR1-based drugs in

schizophrenia patients (Berry et al., 2018). While the influence of TAAR1 on Dopaminergic systems is likely

critical to its efficacy, serotonergic and glutamatergic signaling likely also play prominent roles. Indeed, such

multimodal actions could underlie the utility of TAAR1 agonists in ameliorating side effects (motor

dysregulation, weight gain), abuse potential (especially for dopaminergic drugs), and limited therapeutic profile

(eg. positive vs. negative symptoms of schizophrenia; sleep disturbance vs. cataplexy in narcolepsy).

Similarly, the T1AM studies illustrate the importance of studying endogenous trace amines, which may reveal

new therapeutic applications [33] as well as roles for other TAARs [153].

7. Expert opinion

TAAR1 is a confirmed regulator of at least three major neurotransmitter systems that are intimately involved

with psychosis, motivation, affect, impulse control and cognition, as well as integrative physiological

functions like metabolism and sleep. Pharmacological targeting of TAAR1 shows great promise in a variety

of disease models, including but not limited to schizophrenia, addiction, depression, ADHD, Parkinson

disease and OCD. To date, much of the research has centered on Dopaminergic circuits and dysregulation,

yet TAAR1 manipulation impacts the serotonergic and glutamatergic systems in addition to DA. Thus,

further novel therapeutic applications are not only conceivable, but likely.

This area has benefited immensely from the recent development of transgenic animals and new,

highly selective small-molecule ligands. However, a significant gap remains between what is known of the

cellular and molecular actions of TAAR1, and the behavioral outputs resulting from those actions . Future

efforts should work towards isolating the individual contributions of dopaminergic, serotonergic and

glutamatergic circuits to the behavioral/organismal effects seen to date. These will require novel tools and

applications, such as genetic approaches to target TAAR1-expressing neurons and selective human

antibodies. Such work is expected to clarify how TAAR1 regulates neuronal ‘tone’ and thereby modulates

how the brain experiences, and responds to, environmental stimuli in intact and pathological conditions.

Finally, clinical studies evaluating the efficacy of TAAR1 agonists in psychiatric patients are in progress;

the results of these studies will powerfully shape the direction of future research in this field.

8. Article Highlights

TAAR1 regulates DA, 5-HT, and glutamate neurotransmission by decreasing basal firing rates and

negatively modulating receptor sensitivity.

Selective full and partial TAAR1 agonists exhibit potent antipsychotic, antidepressant, anti-impulsive

and procognitive effects.

TAAR1 agonism reduces psychostimulant self-administration, reward mechanisms and relapse

potential.

T1AM, an endogenous TAAR1 agonist derived from thyroid hormone, modulates food intake, increases

wakefulness and improves cognitive performance.

TAAR1 partial, but not full agonists, promote wakefulness, while both full and partial agonists suppress

cataplexy.

Based on preclinical studies, TAAR1 agonism represents a novel strategy for treating neuropsychiatric

diseases involving dysregulated monoaminergic signaling such as schizophrenia, addiction, depression,

ADHD, Parkinson disease and OCD.

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