The dopamine metabolite 3-methoxytyramine is a neuromodulator

Abstract

Dopamine (3-hydroxytyramine) is a well-known catecholamine neurotransmitter involved in multiple physiological functions including movement control. Here we report that the major extracellular metabolite of dopamine, 3-methoxytyramine (3-MT), can induce behavioral effects in a dopamine-independent manner and these effects are partially mediated by the trace amine associated receptor 1 (TAAR1). Unbiased in vivo screening of putative trace amine receptor ligands for potential effects on the movement control revealed that 3-MT infused in the brain is able to induce a complex set of abnormal involuntary movements in mice acutely depleted of dopamine. In normal mice, the central administration of 3-MT caused a temporary mild hyperactivity with a concomitant set of abnormal movements. Furthermore, 3-MT induced significant ERK and CREB phosphorylation in the mouse striatum, signaling events generally related to PKA-mediated cAMP accumulation. In mice lacking TAAR1, both behavioral and signaling effects of 3-MT were partially attenuated, consistent with the ability of 3-MT to activate TAAR1 receptors and cause cAMP accumulation as well as ERK and CREB phosphorylation in cellular assays. Thus, 3-MT is not just an inactive metabolite of DA, but a novel neuromodulator that in certain situations may be involved in movement control. Further characterization of the physiological functions mediated by 3-MT may advance understanding of the pathophysiology and pharmacology of brain disorders involving abnormal dopaminergic transmission, such as Parkinson's disease, dyskinesia and schizophrenia.

Figure 1. 3-MT induces behavioral activation and intracellular signaling in the striatum of DA deficient mice.

(A) Identification of motor actions of 3-MT in DDD mice. DAT-KO mice were treated with αMT (250 mg/kg, i.p.) and 1 h after αMT were challenged repeatedly with increasing concentrations of a drug (interval between treatments is 1 h). 3-MT induced abnormal activation in DDD mice after i.c.v. infusion of both 36 and 180 µg of 3-MT (visual observations), as revealed by the significant effect of 3-MT in measures of horizontal activity at dose 180 µg (cumulative horizontal activity counts for 1 h following infusion of 180 µg 3-MT is 1711.4±580.1 vs. 26.8±11.8 in vehicle-treated group; p<0.05, two-tailed Mann-Whitney U test, n = 6 per group). (B) 3-MT administered at dose of 36 µg, i.c.v. (30 min after infusion) caused significant increase in Erk2 phosphorylation in the striatal tissue of DDD mice (n = 10 per group; ** - p<0.01; Student's t-test).

Figure 2. 3-MT induces activation of human TAAR1 in cAMP assay and causes CREB and Erk2 phosphorylation in HEK-293 cells.

(A) cAMP response to tyramine and 3-MT in cells expressing hTAAR1 receptor. Dowex and Alumina column chromatography was used to measure [3H]-cAMP accumulation in HEK-293 cells transfected with the hTAAR1 receptor and treated with the concentrations of compounds shown in the Figure for 15 minutes at room temperature. Results are the mean ± SEM of two (tyramine) or three (3-MT) independent experiments performed in duplicate. EC50 for tyramine was estimated as 320±100 nM and for 3-MT as 700±180 nM. No effects of tyramine and 3-MT were observed in corresponding Mock cells expressing endogenous receptors only (data not shown). The inserted images obtained with a Zeiss LSM510 confocal microscope show the fluorescence from the immunostaining of HA epitope tagged hTAAR1 receptors expressed at the plasma membrane compartment of non permeabilized HEK-293 cells (left image), and an overlay of the fluorescence on a phase image of the same cells (right image) . (B) and (C) Time-course of effect of 3-MT (10 µM) on Erk2 (B) and CREB (C) phosphorylation in HEK-293 cells expressing hTAAR1. hTAAR1 was expressed in cells as described previously and treated with vehicle or 3-MT (10 µM). The cells were lysed and then analyzed by Western blot for Erk2 and CREB phosphorylation. 3-MT produced time dependent phosphorylation of Erk2 and CREB in cells expressing hTAAR1 while no effects were observed in vehicle-treated controls. A significant effect in comparison to untreated cells (time point 0) was observed at 2, 5, 10 and 20 min time points for ERK2 phosphorylation and at 10 and 20 min periods for CREB phosphorylation (n = 4 independent experiments per group, p<0.05, one-way ANOVA followed by Dunnet's multiple comparison test). No effect of 3-MT was observed in corresponding Mock cells without hTAAR1 expression (data not shown).

Figure 3. Behavioral effects of 3-MT are reduced in TAAR1-KO mice.

Administration of 3-MT (9 µg, i.c.v.) to WT mice (A) but not TAAR1-KO mice (B) induced abnormal behavioral activation as reflected by total distance traveled. Analysis of total distance traveled for 60 min after 3-MT administration revealed significant effect of 3-MT versus vehicle treatment (p<0.05; Student's t-test) in WT but not TAAR1-KO mice (Figure 3D). (C, D and E) Dose-dependence of 3-MT-induced complex set of abnormal movements (please see description in the text and Supplemental Figure 1S) as detected in computerized locomotor activity monitor as changes in movement time (C), total distance traveled (D) and vertical activity (E). Data are presented as cumulative counts for 60 min after 3-MT administration. Two-way ANOVA analysis revealed significant main effects of dose (p = 0.0001) and genotype (p<0.0001), but no significant dose by genotype interaction (p = 0.4) in measures of movement time (C), significant main effects of dose (p = 0.0002), genotype (p<0.0001) and dose by genotype interaction (p = 0.0198) in measures of total distance (D) and significant main effects of dose (p = 0.026) and genotype (p<0.0001) but no significant dose by genotype interaction (p = 0.321) in measures of vertical activity (E). Pair-wise comparisons conducted with post-hoc Tukey's HSD test revealed significant differences between genotypes (* - p<0.05 effect of 3-MT in WTs vs. KOs) and dose (# - p<0.05 effect of 3-MT vs. respective vehicle-treated controls). Please note, that after 36 µg a similar pattern of abnormal movements was observed in both genotypes (visual observations).

Figure 4. Striatal signaling effects of 3-MT are reduced in TAAR1-KO mice.

Effect of 3-MT infusion (18 µg, 30 min after treatment) on Erk2 (A) and CREB (B) phosphorylation in WT and TAAR1-KO mice. 3-MT induced significant phosphorylation of both Erk2 and CREB in WT mice, but not in TAAR1-KO mice (* p<0.05; Student's t-test n = 10 per group).