Taar1 Knockout Mice Research Articles

AlphaFold accelerated discovery of psychotropic agonists targeting the trace amine-associated receptor 1.

Artificial intelligence is revolutionizing protein structure prediction, providing unprecedented opportunities for drug design. To assess the potential impact on ligand discovery, we compared virtual screens using protein structures generated by the AlphaFold machine learning method and traditional homology modeling. More than 16 million compounds were docked to models of the trace amine-associated receptor 1 (TAAR1), a G protein-coupled receptor of unknown structure and target for treating neuropsychiatric disorders. Sets of 30 and 32 highly ranked compounds from the AlphaFold and homology model screens, respectively, were experimentally evaluated. Of these, 25 were TAAR1 agonists with potencies ranging from 12 to 0.03 μM. The AlphaFold screen yielded a more than twofold higher hit rate (60%) than the homology model and discovered the most potent agonists. A TAAR1 agonist with a promising selectivity profile and drug-like properties showed physiological and antipsychotic-like effects in wild-type but not in TAAR1 knockout mice. These results demonstrate that AlphaFold structures can accelerate drug discovery.

Mice Lacking TAAR1 Show No Early Behavioral Response to Acute Restraint Stress

The role of the TAAR1 receptor, one of the trace amine-associated receptors (TAARs) family, in the formation of the behavioral component of the stress response was studied. The behavior of female TAAR1 knockout mice and wild-type (WT) mice was investigated in tests of elevated plus maze and elevated zero maze (EPM and EZM) and forced swimming test (FST) under normal conditions and after uncontrolled restraint stress exposure for 30 min. In the EPM test, the initial level of locomotor and exploratory activity, as well as the anxiety, was identical in both groups of mice. In the EZM test, the initial indicators of anxiety in female TAAR1 KO mice compared to female WT mice were higher, and locomotor activity was lower. When testing mice in the EZM 30 minutes after the end of stress exposure, it was found that the anxiety in female WT mice sharply increased, and the indicators of locomotor activity and exploratory behavior significantly decreased. The behavioral indicators in the EZM test in TAAR1 KO mice before and after stress were identical. A pronounced behavioral component of the stress response was observed in both TAAR1 KO and WT mice during testing in EPM. There were no significant differences between TAAR1 KO and WT mice during testing in EPM four hours after stress exposure. In the FST test the latency to the first immobility was initially longer in TAAR1 KO mice compared to the WT mice, but 24 h after the stress this indicator has significantly decreased. As a result, TAAR1 KO and WT mice no longer differed in all behavioral indicators in the FST. Three weeks after acute restraint stress, both TAAR1 KO and WT groups showed a significant increase in immobility duration and a decrease in latency to the first immobility, however no difference between the both groups of animals were found. Thereby, we found the complete absence of behavioral change immediately after stressor exposure in TAAR1 KO compared to the WT mice.

Protein Metabolism Changes and Alterations in Behavior of Trace Amine-Associated Receptor 1 Knockout Mice Fed a High-Fructose Diet

Trace amines and their receptors are a family of G protein-coupled receptors widely distributed in the central nervous system and periphery. The trace amine-associated receptor 1 (TAAR1) plays a significant role as a therapeutic target for schizophrenia, depression, diabetes, and obesity. In this study, TAAR1 knockout mice and WT groups were tested in conditions of a high-fructose diet. The consumption of a high-fructose diet may be due to the influence on the metabolism processes by dopamine in the brain, neuromotor function, and level of anxiety of TAAR1 knockout mice. During a comparative analysis of behavioral, biochemical, and morphological parameters, significant differences were found between liver and biochemical parameters, the regulation of protein metabolism (AST/ALT ratio, creatine kinase activity, urea), and alterations in behavior. An elevated plus maze analysis showed the influence of fructose and genetic factors on the level of anxiety. A new marker of the grooming microstructure (depression ratio) was tested, which showed high efficiency as a marker of depression-like behavioral changes and a possible association with dopamine-dependent regulation of protein metabolism. These results confirm a possible association of the TAAR1 gene knockout with an increase in catabolic reaction levels by AST/ALT-dependent and possible dopamine-mediated protein metabolism regulation and depression-like behavior.

Enhanced Aggression, Reduced Self-Grooming Behavior and Altered 5-HT Regulation in the Frontal Cortex in Mice Lacking Trace Amine-Associated Receptor 1 (TAAR1).

The Trace Amine-Associated Receptor 1 (TAAR1) is one of the six functional receptors belonging to the family of monoamine-related G protein-coupled receptors (TAAR1-TAAR9) found in humans. However, the exact biological mechanisms of TAAR1 central and peripheral action remain to be fully understood. TAAR1 is widely expressed in the prefrontal cortex and several limbic regions, interplaying with the dopamine system to modulate the reward circuitry. Recent clinical trials suggest the efficacy of TAAR1 agonists as potential novel antipsychotic agents. Here, we characterize behavioral and neurochemical phenotypes of TAAR1 knockout mice, focusing on aggression and self-grooming behavior that both strongly depend on the monoaminergic signaling and cortico-striatal and cortico-limbic circuits. Overall, we report increased aggression in these knockout mice in the resident-intruder test, accompanied by reduced self-grooming behavior in the novelty-induced grooming test, and by higher cortical serotonin (5-HT) tissue levels. Further studies are necessary to explore whether TAAR1-based therapies can become potential novel treatments for a wide range of neuropsychiatric disorders associated with aggression.

TAAR1 dependent and independent actions of the potential antipsychotic and dual TAAR1/5-HT1A receptor agonist SEP-363856

SEP-363856 (SEP-856) is a novel antipsychotic under clinical development. It displays a unique pattern of receptor interaction, with only weak (partial agonist) activity at dopamine D2 receptors, yet more potent agonist activity at the trace amine associated receptor (TAAR1) and 5-hydroxytryptamine 1 A receptor (5-HT1A). Nonetheless, these observations await independent confirmation and more detailed characterization of the in vitro and in vivo actions of SEP-856 at TAAR1 and 5-HT1A receptors would be instructive. Herein, we employed luminescence complementation technology in heterologous live cell systems, confocal microscopy, voltage clamp electrophysiology, behavioral readouts and TAAR1 knockout (KO) mice to study SEP-856 in further detail. We provide evidence for the ability of SEP-856 to activate TAAR1 at the surface plasma membrane, and show that this interaction results in Gαs recruitment (pEC50: 6.08 ± 0.22 EMAX: 96.41% ± 15.26) and by extension, to G-protein inwardly rectifying potassium (GIRK) channel activation. Using TAAR1-KO mice, we find TAAR1 to be indispensable for SEP-856 control of body temperature, baseline locomotion reduction and for “antipsychotic-like” efficacy as characterized by a reversal of dizocilipine (MK-801) mediated disruption of pre-pulse inhibition. Conversely, the inhibition by SEP-856 of MK-801 induced locomotion was unaffected in TAAR1 KO mice. SEP-856 behaved as a low-potency, partial agonist at the 5-HT1A receptor, while it partially inhibited recruitment of D2 receptor-coupled Gα and GIRK by DA and acted as a weak partial agonist with low potency at the same receptor when applied alone. Our findings corroborate and extend previous observations on the molecular substrates engaged by this unique, dual TAAR1/5-HT1A receptor agonist and potential antipsychotic that could prove to have major advantages in the treatment of schizophrenia and other psychotic disorders.

Sensory Gating in TAAR1 Knockout Mice

Trace amines (TA) are a family of endogenous compounds structurallysimilar to classical biogenic amines that may be involved in thepathogenesis of a number of neuropsychiatric disorders. One of themost studied and perspective member of the TA associated receptors (TAARs)family is the TAAR1. The aim of the present study was to investigatethe sensory gating (SG) in freely moving TAAR1 knockout mice ina chronic experiment. The study of SG was conducted in the paired-clickparadigm. The SG indices were calculated as an absolute value by subtractingthe second stimulus response amplitude from the first stimulus responseamplitude (S1–S2) and as a relative value calculated by dividingthe S2 amplitude by the response amplitude on S1 (S2/S1). As a result,a significant decrease in the amplitude of the N40 component wasfound in TAAR1 knockout mice compared to wild-type mice. In addition,the absolute value of sensory gating calculated by the S1–S2 methodwas also reduced, but the relative value of sensory gating denotedas S1/S2 ratio remained unchanged. Thus, the data obtained indicatethe involvement of TAAR1 in the generation of auditory evoked potentialsand the potential involvement of the trace amine system in the dosingand filtering of sensory information.

Evaluation of Approach to a Conspecific and Blood Biochemical Parameters in TAAR1 Knockout Mice.

It is known that the trace amine-associated receptor 1 (TAAR1) receptor is involved in limbic brain functions by regulating dopamine transmission and putative reward circuitry. Moreover, other TAARs are expressed in the olfactory system of all studied vertebrate species, sensing innate socially-relevant odors, including pheromones. Therefore, one can assume that TAARs may play a role in rodent social and sexual behavior. A comparative behavioral and biochemical analysis of TAAR1 knockout (TAAR1-KO) and wild-type mice is also important for the preliminary evaluation of the potential side effects of future TAAR1-based therapies. In our studies, we adapted a sexual incentive motivation test for mice to evaluate the sexual behavior of TAAR1-KO and wild-type mice. Previously, similar methods were primarily applied to rats. Furthermore, we measured testosterone and other biochemical parameters in the blood. As a result, we found only minimal alterations in all of the studied parameters. Thus, the lack of TAAR1 does not significantly affect sexual motivation and routine lipid and metabolic blood biochemical parameters, suggesting that future TAAR1-based therapies should have a favorable safety profile.

3,4‐methylenedioxymethamphetamine (MDMA) stimulates activation of TAAR1 and subsequent neurotransmitter transporter internalization in serotonin neurons

The use of 3,4‐methylenedioxymethamphetamine (MDMA) has recently gained interest as a potential therapeutic tool for the treatment of post‐traumatic stress disorder (PTSD). However, the mechanism of action of this drug has not yet been fully elucidated. Here we demonstrate that MDMA acts by targeting the trace amine associated receptor 1 (TAAR1), an intracellular G‐protein coupled receptor (GPCR) expressed throughout the brain and in peripheral tissues. In previous studies, we have shown that a closely‐related compound, amphetamine (AMPH), enters neurons through the dopamine and norepinephrine transporters (DAT and NET) and activates TAAR1. This activates the small GTPase RhoA that in turn increases internalization of the DAT and NET. We have also found that a neuronal glutamate transporter, EAAT3, is also internalized. These actions result in an increase in extracellular DA, NE and glutamate concentrations. In this study, we show that MDMA enters serotonin neurons through the 5‐HT transporter (SERT) and activates a similar signaling cascade.We isolated brain tissue encompassing the Raphe nucleus from E15 mice and prepared primary cultures of serotonin (5‐HT) neurons. 5‐HT neurons were identified by immunocytochemistry for 5‐HT. Functional SERT activity was confirmed by citalopram sensitive 3H‐5‐HT uptake. Pre‐treatment of these cultures for thirty minutes with 1 mM MDMA resulted in a decrease of SERT transport activity. This loss of function was blocked by an inhibitor of RhoA, indicating that MDMA regulates SERT through a Rho‐dependent signaling pathway.Using total internal reflection fluorescence (TIRF) microscopy in HEK293 cells we confirmed that the loss in SERT activity produced by MDMA was due to internalization of the SERT. The application of 1 mM MDMA led to a decrease in GFP‐tagged SERT at the cell membrane. Similarly, GFP‐tagged EAAT3 was also internalized in response to MDMA. Parallel studies in HEK293 cells in which the TAAR1 gene was deleted by CRISPR‐Cas9 showed no effects of MDMA treatment on either SERT or EAAT3 trafficking and confirmed that TAAR1 is an obligate target for the actions of MDMA,To examine the effects of MDMA on endogenous SERT and EAAT3 in situ, we prepared acute brain slices from wildtype and TAAR1 knockout mice. Slices containing the Raphe nucleus were treated with either vehicle control or MDMA (1 mM) and subsequently treated with a cell‐impermeable biotin reagent that would label all surface proteins. By western blot analysis, we observed internalization of SERT and EAAT3 in response to the MDMA treatment. However, neither transporter exhibited any change in cell‐surface expression in response to MDMA in the TAAR1 knockout tissue.Our data indicate that in serotonergic neurons MDMA potentiates the action of both serotonin and glutamate by activating TAAR1 signaling and triggering SERT and EAAT3 internalization. These observations point to new mechanisms to target for the development of drugs to treat PTSD.

Trace Amine-Associated Receptor 1 Contributes to Diverse Functional Actions of O-Phenyl-Iodotyramine in Mice but Not to the Effects of Monoamine-Based Antidepressants.

Trace Amine-Associated Receptor 1 (TAAR1) is a potential target for the treatment of depression and other CNS disorders. However, the precise functional roles of TAAR1 to the actions of clinically used antidepressants remains unclear. Herein, we addressed these issues employing the TAAR1 agonist, o-phenyl-iodotyramine (o-PIT), together with TAAR1-knockout (KO) mice. Irrespective of genotype, systemic administration of o-PIT led to a similar increase in mouse brain concentrations. Consistent with the observation of a high density of TAAR1 in the medial preoptic area, o-PIT-induced hypothermia was significantly reduced in TAAR1-KO mice. Furthermore, the inhibition of a prepulse inhibition response by o-PIT, as well as its induction of striatal tyrosine hydroxylase phosphorylation and elevation of extracellular DA in prefrontal cortex, were all reduced in TAAR1-KO compared to wildtype mice. O-PIT was active in both forced-swim and marble-burying tests, and its effects were significantly blunted in TAAR1-KO mice. Conversely, the actions on behaviour and prefrontal cortex dialysis of a broad suite of clinically used antidepressants were unaffected in TAAR1-KO mice. In conclusion, o-PIT is a useful tool for exploring the hypothermic and other functional antidepressant roles of TAAR1. By contrast, clinically used antidepressants do not require TAAR1 for expression of their antidepressant properties.

TAAR1-Dependent and -Independent Actions of Tyramine in Interaction With Glutamate Underlie Central Effects of Monoamine Oxidase Inhibition

BackgroundMonoamine oxidase inhibitors (MAOIs) exert therapeutic actions by elevating extracellular levels of monoamines in the brain. Irreversible MAOIs cause serious hypertensive crises owing to peripheral accumulation of tyramine, but the role of tyramine in the central effects of MAOIs remains elusive, an issue addressed herein. To achieve robust inhibition of MAOA/B, the clinically used antidepressant tranylcypromine (TCP) was employed. MethodsBehavioral, histological, mass spectrometry imaging, and biosensor-mediated measures of glutamate were conducted with MAOIs in wild-type and TAAR1-knockout (KO) mice. ResultsBoth antidepressant and locomotion responses to TCP were enhanced in TAAR1-KO mice. A recently developed fluoromethylpyridinium-based mass spectrometry imaging method revealed robust accumulation of striatal tyramine on TCP administration. Furthermore, tyramine accumulation was higher in TAAR1-KO versus wild-type mice, suggesting a negative feedback mechanism for TAAR1 in sensing tyramine levels. Combined histoenzymological and immunohistological studies revealed hitherto unknown TAAR1 localization in brain areas projecting to the substantia nigra/ventral tegmental area. Using an enzyme-based biosensor technology, we found that both TCP and tyramine reduced glutamate release in the substantia nigra in wild-type but not in TAAR1-KO mice. Moreover, glutamate measures in freely moving animals treated with TCP demonstrated that TAAR1 prevents glutamate accumulation in the substantia nigra during hyperlocomotive states. ConclusionsThese observations suggest that tyramine, in interaction with glutamate, is involved in centrally mediated behavioral, transcriptional, and neurochemical effects of MAOIs.

3-Iodothyronamine Induces Diverse Signaling Effects at Different Aminergic and Non-Aminergic G-Protein Coupled Receptors

The thyroid hormone metabolite 3-iodothyronamine (3-T1AM) exerts diverse physiological reactions such as a decrease of body temperature, and negative inotropic and chronotropic effects. This observed pleomorphic effect in physiology can be barely explained by interaction with only one target protein such as the trace-amine receptor 1 (TAAR1), a class A G-protein coupled receptor (GPCR). Moreover, Taar1 knock-out mice still react to 3-T1AM through physiological responses with a rapid decrease in body temperature. These facts propelled our group and others to search for further targets for this molecule.The group of TAARs evolved early in evolution and, according to sequence similarities, they are closely related to adrenoceptors and other aminergic receptors. Therefore, several of these receptors were characterized by their potential to interplay with 3-T1AM. Indeed, 3-T1AM acts as a positive allosteric modulator on the beta2-adrenoceptor (ADRB2) and as a biased agonist on the serotonin receptor 1B (5HT1b) and the alpha2-adrenoceptor (ADRA2A). In addition, 3-T1AM was reported to be a weak antagonist at a non-aminergic muscarinic receptor (M3).These findings impressively reflect that such trace amines can unselectively and simultaneously function at different receptors expressed by one cell or at different tissues. In conclusion, the role of 3-T1AM is hypothesized to concert the fine-tuning of specific cell reactions by the accentuation of certain pathways dependent on distinct receptors. 3-T1AM acts as a regulator of signals by blocking, modulating, or inducing simultaneously distinct intracellular signaling cascades via different GPCRs.

Activation of Trace Amine-Associated Receptor 1 Stimulates an Antiapoptotic Signal Cascade via Extracellular Signal-Regulated Kinase 1/2.

Methamphetamine (MA) is highly addictive and neurotoxic, causing cell death in humans and in rodent models. MA, along with many of its analogs, is an agonist at the G protein-coupled trace amine-associated receptor 1 (TAAR1). TAAR1 activation protects against MA-induced degeneration of dopaminergic neurons, suggesting that TAAR1 plays a role in regulating MA-induced neurotoxicity. However, the mechanisms involved in TAAR1's role in neurotoxicity and cell death have not been described in detail. In this study, we investigated the apoptosis pathway in Taar1 wild-type (WT) and knockout (KO) mice and in cells expressing the recombinant receptor. Bcl-2, an antiapoptotic protein, was upregulated ∼3-fold in the midbrain area (substantial nigra and ventral tegmental area) in Taar1 KO compared with WT mice, and MA significantly increased Bcl-2 expression in WT mice but decreased Bcl-2 expression in KO mice. The proapoptotic protein Bax did not differ across genotype or in response to MA. Bcl-2 expression was significantly upregulated by the TAAR1 agonist RO5166017 ((S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine) in cells expressing the recombinant mouse TAAR1. Additionally, activation of TAAR1 by RO5166017 increased phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, and protein kinase B (AKT), but only inhibition of ERK1/2 phosphorylation prevented TAAR1-induced increases in Bcl-2 levels, indicating that TAAR1 activation increases Bcl-2 through an ERK1/2-dependent pathway. All changes to ERK1/2 pathway intermediates were blocked by the TAAR1 antagonist, N-(3-ethoxyphenyl)-4-(1-pyrrolidinyl)-3-(trifluoromethyl) benzamide. These findings suggest that TAAR1 activation protects against MA-induced cell apoptosis and TAAR1 may play a role in cell death in neurodegenerative diseases. SIGNIFICANCE STATEMENT: Methamphetamine stimulates TAAR1, a G protein-coupled receptor. The role and mechanisms for TAAR1 in methamphetamine-induced neurotoxicity are not known. Here, we report that, in genetic mouse models and cells expressing the recombinant receptor, TAAR1 activates the ERK1/2 pathway but not the AKT pathway to upregulate the antiapoptotic protein Bcl-2, which protects cells from drug-induced toxicity.

Minimal Age-Related Alterations in Behavioral and Hematological Parameters in Trace Amine-Associated Receptor 1 (TAAR1) Knockout Mice.

Since the discovery in 2001, the G protein-coupled trace amine-associated receptor 1 (TAAR1) has become an important focus of research targeted on evaluation of its role in the central nervous system (CNS). Meanwhile, impact of TAAR1 in the peripheral organs is less investigated. Expression of TAAR1 was demonstrated in different peripheral tissues: pancreatic β-cells, stomach, intestines, white blood cells (WBC), and thyroid. However, the role of TAAR1 in regulation of hematological parameters has not been investigated yet. In this study, we performed analysis of anxiety-related behaviors, a complete blood count (CBC), erythrocyte fragility, as well as FT3/FT4 thyroid hormones levels in adult and middle-aged TAAR1 knockout mice. Complete blood count analysis was performed on a Siemens Advia 2120i hematology analyzer and included more than 35 measured and calculated parameters. Erythrocyte fragility test evaluated spherocytosis pathologies of red blood cells (RBC). No significant alterations in essentially all these parameters were found in mice without TAAR1. However, comparative aging analysis has revealed a decreased neutrophils level in the middle-aged TAAR1 knockout mouse group. Minimal alterations in these parameters observed in TAAR1 knockout mice suggest that future TAAR1-based therapies should exert little hematological effect and thus will likely have a good safety profile.

The Role of Biogenic Amine Transporters in Trace Amine–Associated Receptor 1 Regulation of Methamphetamine-Induced Neurotoxicity

Methamphetamine (MA) impairs vesicular monoamine transporter 2 (VMAT2) and dopamine transporter (DAT) function and expression, increasing intracellular DA levels that lead to neurotoxicity. The trace amine-associated receptor 1 (TAAR1) is activated by MA, but when the receptor is not activated, MA-induced neurotoxicity is increased. To investigate interactions among TAAR1, VMAT2, and DAT, transporter function and expression were measured in transgenic Taar1 knockout (KO) and wild-type (WT) mice 24 hours following a binge-like regimen (four intraperitoneal injections, 2 hours apart) of MA (5 mg/kg) or the same schedule of saline treatment. Striatal synaptosomes were separated by fractionation to examine the function and expression of VMAT2 localized to cytosolic and membrane-associated vesicles. DAT was measured in whole synaptosomes. VMAT2-mediated [3H]DA uptake inhibition was increased in Taar1 KO mice in synaptosomal and vesicular fractions, but not the membrane-associated fraction, compared with Taar1 WT mice. There was no difference in [3H]dihydrotetrabenazine binding to the VMAT2 or [125I]RTI-55 binding to the DAT between genotypes, indicating activation of TAAR1 does not affect VMAT2 or DAT expression. There was also no difference between Taar1 WT and KO mice in DAT-mediated [3H]DA uptake inhibition following in vitro treatment with MA. These findings provide the first evidence of a TAAR1-VMAT2 interaction, as activation of TAAR1 mitigated MA-induced impairment of VMAT2 function, independently of change in VMAT2 expression. Additionally, the interaction is localized to intracellular VMAT2 on cytosolic vesicles and did not affect expression or function of DAT in synaptosomes or VMAT2 at the plasmalemmal surface, i.e., on membrane-associated vesicles. SIGNIFICANCE STATEMENT: Methamphetamine stimulates the G protein-coupled receptor TAAR1 to affect dopaminergic function and neurotoxicity. Here we demonstrate that a functional TAAR1 protects a specific subcellular fraction of VMAT2, but not the dopamine transporter, from methamphetamine-induced effects, suggesting new directions in pharmacotherapeutic development for neurodegenerative disorders.

Canonical TSH Regulation of Cathepsin-Mediated Thyroglobulin Processing in the Thyroid Gland of Male Mice Requires Taar1 Expression.

Trace amine-associated receptor 1 (Taar1) has been suggested as putative receptor of thyronamines. These are aminergic messengers with potential metabolic and neurological effects countering their contingent precursors, the thyroid hormones (THs). Recently, we found Taar1 to be localized at the primary cilia of rodent thyroid epithelial cells in vitro and in situ. Thus, Taar1 is present in a location of thyroid follicles where it might be involved in regulation of cathepsin-mediated proteolytic processing of thyroglobulin, and consequently TH synthesis. In this study, taar1 knock-out male mice (taar1-/-) were used to determine whether Taar1 function would entail differential alterations in thyroid states of young and adult animals. Analyses of blood serum revealed unaltered T4 and T3 concentrations and unaltered T3-over-T4 ratios upon Taar1 deficiency accompanied, however, by elevated TSH concentrations. Interestingly, TSH receptors, typically localized at the basolateral plasma membrane domain of wild type controls, were located at vesicular membranes in thyrocytes of taar1-/- mice. In addition, determination of epithelial extensions in taar1-/- thyroids showed prismatic cells, which might indicate activation states higher than in the wild type. While gross degradation of thyroglobulin was comparable to controls, deregulated thyroglobulin turnover in taar1-/- mice was indicated by luminal accumulation of covalently cross-linked thyroglobulin storage forms. These findings were in line with decreased proteolytic activities of thyroglobulin-solubilizing and -processing proteases, due to upregulated cystatins acting as their endogenous inhibitors in situ. In conclusion, Taar1-deficient mice are hyperthyrotropinemic in the absence of respective signs of primary hypothyroidism such as changes in body weight or TH concentrations in blood serum. Thyrocytes of taar1-/- mice are characterized by non-canonical TSH receptor localization in intracellular compartments, which is accompanied by altered thyroglobulin turnover due to a disbalanced proteolytic network. These finding are of significance considering the rising popularity of using TAAR1 agonists or antagonists as neuromodulating pharmacological drugs. Our study highlights the importance of further evaluating potential off-target effects regarding TSH receptor mislocalization and the thyroglobulin processing machinery, which may not only affect the TH-generating thyroid gland, but may emanate to other TH target organs like the CNS dependent on their proper supply.

The Trace Amine-Associated Receptor 1 Agonist 3-Iodothyronamine Induces Biased Signaling at the Serotonin 1b Receptor.

Trace amine-associated receptors (TAARs) belong to the class A G-protein-coupled receptors (GPCR) and are evolutionary related to aminergic receptors. TAARs have been identified to mediate effects of trace amines. TAAR1 signaling is mainly mediated via activation of the Gs/adenylyl cyclase pathway. In addition to classical trace amines, TAAR1 can also be activated by the thyroid hormone derivative 3-iodothyronamine (3-T1AM). Pharmacological doses of 3-T1AM induced metabolic and anapyrexic effects, which might be centrally mediated in the hypothalamus in rodents. However, the observed anapyrexic effect of 3-T1AM persists in Taar1 knock-out mice which raises the question whether further GPCRs are potential targets for 3-T1AM and mediate the observed physiological effect. Anapyrexia has been observed to be related to action on aminergic receptors such as the serotonin receptor 1b (5-HT1b). This receptor primarily activates the Gi/o mediated pathway and PLC signaling through the Gβγ of Gi/o. Since the expression profiles of TAAR1 and 5-HT1b overlap, we questioned whether 3-T1AM may activate 5-HT1b. Finally, we also evaluated heteromerization between these two GPCRs and tested signaling under co-expressed conditions. In this study, we showed, that 3-T1AM can induce Gi/o signaling through 5-HT1b in a concentration of 10 μM. Strikingly, at 5-HT1b the ligand 3-T1AM only activates the Gi/o mediated reduction of cAMP accumulation, but not PLC activation. Co-stimulation of 5-HT1b by both ligands did not lead to additive or synergistic signaling effects. In addition, we confirmed the capacity for heteromerization between TAAR1 and 5-HT1b. Under co-expression of TAAR1 and HTR1b, 3-T1AM action is only mediated via TAAR1 and activation of 5-HT1b is abrogated. In conclusion, we found evidence for 5-HT1b as a new receptor target for 3-T1AM, albeit with a different signaling effect than the endogenous ligand. Altogether, this indicates a complex interrelation of signaling effects between the investigated GPCRs and respective ligands.

Striatal Tyrosine Hydroxylase Is Stimulated via TAAR1 by 3-Iodothyronamine, But Not by Tyramine or β-Phenylethylamine.

The trace amine-associated receptor 1 (TAAR1) is expressed by dopaminergic neurons, but the precise influence of trace amines upon their functional activity remains to be fully characterized. Here, we examined the regulation of tyrosine hydroxylase (TH) by tyramine and beta-phenylethylamine (β-PEA) compared to 3-iodothyronamine (T1AM). Immunoblotting and amperometry were performed in dorsal striatal slices from wild-type (WT) and TAAR1 knockout (KO) mice. T1AM increased TH phosphorylation at both Ser19 and Ser40, actions that should promote functional activity of TH. Indeed, HPLC data revealed higher rates of L-dihydroxyphenylalanine (DOPA) accumulation in WT animals treated with T1AM after the administration of a DOPA decarboxylase inhibitor. These effects were abolished both in TAAR1 KO mice and by the TAAR1 antagonist, EPPTB. Further, they were specific inasmuch as Ser845 phosphorylation of the post-synaptic GluA1 AMPAR subunit was unaffected. The effects of T1AM on TH phosphorylation at both Ser19 (CamKII-targeted), and Ser40 (PKA-phosphorylated) were inhibited by KN-92 and H-89, inhibitors of CamKII and PKA respectively. Conversely, there was no effect of an EPAC analog, 8-CPT-2Me-cAMP, on TH phosphorylation. In line with these data, T1AM increased evoked striatal dopamine release in TAAR1 WT mice, an action blunted in TAAR1 KO mice and by EPPTB. Mass spectrometry imaging revealed no endogenous T1AM in the brain, but detected T1AM in several brain areas upon systemic administration in both WT and TAAR1 KO mice. In contrast to T1AM, tyramine decreased the phosphorylation of Ser40-TH, while increasing Ser845-GluA1 phosphorylation, actions that were not blocked in TAAR1 KO mice. Likewise, β-PEA reduced Ser40-TH and tended to promote Ser845-GluA1 phosphorylation. The D1 receptor antagonist SCH23390 blocked tyramine-induced Ser845-GluA1 phosphorylation, but had no effect on tyramine- or β-PEA-induced Ser40-TH phosphorylation. In conclusion, by intracellular cascades involving CaMKII and PKA, T1AM, but not tyramine and β-PEA, acts via TAAR1 to promote the phosphorylation and functional activity of TH in the dorsal striatum, supporting a modulatory influence on dopamine transmission.

Deletion of Trace Amine-Associated Receptor 1 Attenuates Behavioral Responses to Caffeine.

Trace amines (TAs), endogenous amino acid metabolites that are structurally similar to the biogenic amines, are endogenous ligands for trace amine-associated receptor 1 (TAAR1), a GPCR that modulates dopaminergic, serotonergic, and glutamatergic activity. Selective TAAR1 full and partial agonists exhibit similar pro-cognitive, antidepressant- and antipsychotic-like properties in rodents and non-human primates, suggesting TAAR1 as a novel target for the treatment of neurological and psychiatric disorders. We previously reported that TAAR1 partial agonists are wake-promoting in rats and mice, and that TAAR1 knockout (KO) and overexpressing mice exhibit altered sleep-wake and EEG spectral composition. Here, we report that locomotor and EEG spectral responses to the psychostimulants modafinil and caffeine are attenuated in TAAR1 KO mice. TAAR1 KO mice and WT littermates were instrumented for EEG and EMG recording and implanted with telemetry transmitters for monitoring locomotor activity (LMA) and core body temperature (Tb). Following recovery, mice were administered modafinil (25, 50, 100 mg/kg), caffeine (2.5, 10, 20 mg/kg) or vehicle p.o. at ZT6 in balanced order. In WT mice, both modafinil and caffeine dose-dependently increased LMA for up to 6 h following dosing, whereas only the highest dose of each drug increased LMA in KO mice, and did so for less time after dosing. This effect was particularly pronounced following caffeine, such that total LMA response was significantly attenuated in KO mice compared to WT at all doses of caffeine and did not differ from Vehicle treatment. Tb increased comparably in both genotypes in a dose-dependent manner. TAAR1 deletion was associated with reduced wake consolidation following both drugs, but total time in wakefulness did not differ between KO and WT mice. Furthermore, gamma band EEG activity following both modafinil and caffeine treatment was attenuated in TAAR1 KO compared to WT mice. Our results show that TAAR1 is a critical component of the behavioral and cortical arousal associated with two widely used psychostimulants with very different mechanisms of action. Together with our previous findings, these data suggest that TAAR1 is a previously unrecognized component of an endogenous wake-modulating system.

ПРЕИМПУЛЬСНОЕ ТОРМОЖЕНИЕ У МЫШЕЙ-НОКАУТОВ ПО TAAR1 РЕЦЕПТОРУ, "Российский физиологический журнал им. И.М. Сеченова"

Преимпульсное торможение (PPI) рассматривается как эндофенотип шизофрении, что позволяет использовать животные модели как для выявления фармакологических эффектов, так и для выяснения механизмов функционирования нейронных сетей. В последнее время большое внимание уделяется изучению так называемых TAARs (TAAR1-TAAR9) рецепторов следовых аминов, которые могут иметь отношение к патогенезу различных видов нейропсихиатрических расстройств, включая шизофрению. Исследование проводили на самцах мышей линий C57BL/6 (n = 16), дикий тип WT (n = 29) и нокаутные мыши (knockout, KO) по TAAR1 (n = 19). Исходными для WT и KO являлись мыши линий 129S1/Sv и C57BL/6. Результаты данного исследования показали, что преимпульсное торможение хорошо выражено у всех исследованных линий. Кроме того, амплитуда ответа на стимул (SS) в паре стимул с престимулом (PP-SS) у животных линии KO достоверно выше по сравнению с ответом у животных линии WT.

0014 TRACE AMINE-ASSOCIATED RECEPTOR 1 REGULATES WAKEFULNESS, BEHAVIORAL ACTIVATION AND EEG SPECTRAL COMPOSITION

Trace amines (TAs) are endogenous amino acid metabolites that are structurally similar to the biogenic amines. TAs are endogenous ligands for trace amine-associated receptor 1 (TAAR1), a GPCR that modulates dopaminergic, serotonergic, and glutamatergic activity. Selective TAAR1 agonists have been shown to have pro-cognitive, antipsychotic-like, anti-addiction, stress-reducing, weight-reducing, glucose-lowering and wake-promoting activities. We used Taar1 knockout (KO) and over-expressing (OE) mice and TAAR1 agonists to elucidate the role of TAAR1 in sleep/wake. EEG, EMG, body temperature (Tb) and locomotor activity (LMA) were recorded in Taar1 KO, OE and WT mice. Following a 24h recording to characterize baseline sleep/wake, mice were sleep-deprived (SD) for 6h. In separate experiments, mice were given three doses of the TAAR1 partial agonist RO5263397, caffeine, modafinil or vehicle p.o. Baseline wakefulness was modestly increased in OE compared to WT mice. Baseline theta (4.5-9Hz) and low gamma (30-60Hz) activity was elevated in KO compared to OE mice in NREM and REM sleep. Following SD, both KO and OE mice exhibited a homeostatic sleep rebound. In WT mice, RO5263397 increased waking and reduced NREM and REM sleep, decreased gamma power during wake and NREM, and decreased Tb without affecting LMA; these effects were absent in KO mice and potentiated in OE mice. By contrast, caffeine increased wake time, NREM gamma power, and LMA in all strains compared to vehicle; this effect was attenuated in KO and potentiated in OE mice. Subsequent studies confirmed that motor activation and gamma band activity increases induced by caffeine or modafinil are attenuated in KO mice compared to WT. TAAR1 overexpression increases wakefulness whereas TAAR1 partial agonism strongly increases wakefulness and reduces NREM and REM sleep. These results indicate a modulatory role for TAAR1 in sleep/wake and cortical activity and suggest TAAR1 as a novel target for wake-promoting therapeutics. NIH R01 NS082876.