G protein-biased LPAR1 agonism of prototypic antidepressants: Implication in the identification of novel therapeutic target for depression

Abstract

Prototypic antidepressants, such as tricyclic/tetracyclic antidepressants (TCAs), have multiple pharmacological properties and have been considered to be more effective than newer antidepressants, such as selective serotonin reuptake inhibitors, in treating severe depression. However, the clinical contribution of non-monoaminergic effects of TCAs remains elusive. In this study, we discovered that amitriptyline, a typical TCA, directly binds to the lysophosphatidic acid receptor 1 (LPAR1), a G protein-coupled receptor, and activates downstream G protein signaling, while exerting a little effect on β-arrestin recruitment. This suggests that amitriptyline acts as a G protein-biased agonist of LPAR1. This biased agonism was specific to TCAs and was not observed with other antidepressants. LPAR1 was found to be involved in the behavioral effects of amitriptyline. Notably, long-term infusion of mouse hippocampus with the potent G protein-biased LPAR agonist OMPT, but not the non-biased agonist LPA, induced antidepressant-like behavior, indicating that G protein-biased agonism might be necessary for the antidepressant-like effects. Furthermore, RNA-seq analysis revealed that LPA and OMPT have opposite patterns of gene expression changes in the hippocampus. Pathway analysis indicated that long-term treatment with OMPT activated LPAR1 downstream signaling (Rho and MAPK), whereas LPA suppressed LPAR1 signaling. Our findings provide insights into the mechanisms underlying the non-monoaminergic antidepressant effects of TCAs and identify the G protein-biased agonism of LPAR1 as a promising target for the development of novel antidepressants.

Fig. 1. Tricyclic antidepressants directly bind to LPAR1.

A Scheme of affinity purification experiment of LPAR1 with TCA-beads. B Immunoblots from affinity-purified lysates using TCA-beads or control beads. The amount of nortriptyline immobilized on the beads was 19.3 nmol/mg (+) or 27.3 nmol/mg (++). Two cell lysates were used for affinity purification, namely lysates from human LPAR1-overexpressed RH7777 cells (left) and those from FLAG-tagged human LPAR1-transfected HeLa cells (right). Each parent cell lysate was used as a negative control lysate. C Competitive inhibition of LPAR1 binding to TCA-beads. LPAR1-overexpressed RH7777 cell lysates were preincubated with the indicated concentrations of nortriptyline or LPA (shown as a ratio to the amount of nortriptyline immobilized on the TCA-beads) and then eluted with TCA-beads. Representative immunoblots are shown above the graph. D Amitriptyline and clomipramine (6.67-fold concentration relative to the amount of nortriptyline immobilized on the TCA-beads) inhibit LPAR1 binding to TCA-beads. LPAR1-overexpressed RH7777 cell lysates were used. A representative immunoblot is shown above the graph. N = 3–4. Data are presented as the means ± SEM. Statistical significance was calculated using one-way ANOVA with Dunnett’s multiple comparisons test (**P < 0.01, ***P < 0.001 vs vehicle).

Fig. 2. Tricyclic and tetracyclic antidepressants, but not other antidepressants, are G protein-biased LPAR1 agonists.

A Schematic representation of TGFα shedding assay and B β-arrestin recruitment assay. Dose-response curves of LPA and amitriptyline for the LPAR1-specific C G protein activation and D β-arrestin recruitment. N = 3–4. Data are presented as means ± SEM. E Possible LPAR1 downstream pathways activated by amitriptyline. F E_max values (top; E_max of LPA = 100%) for each antidepressant calculated from dose-response curves and LogRAi values (bottom) for each antidepressant that showed agonist activity (E_max > 25% and EC₅₀ < 100 μM). N = 3–4. Data are presented as the means ± SEM. *P < 0.05 (Unpaired t-test).

Fig. 3. LPAR1 mediates the behavioral effects of amitriptyline (Ami).

A Timeline and group design of the experiment is shown in panel B. B Immobility time in the forced swim test (FST). Mice were treated with Ami (160 mg/L), added to their drinking water, and were injected daily with Ki16425 (10 mg/kg/day, i.p.) or vehicle for 14 days. Repeated FST was performed on Day –1, 7, and 14. N = 36. Data are presented as means ± SEM. Statistical significance was calculated using one-way ANOVA with Sidak’s multiple comparisons test (*P < 0.05, ***P < 0.001, ns: not significant). C Timeline and group design of the experiment shown in panel D. D Immobility time in the FST. Wild type (WT) and LPAR1 heterozygous (HET) mice were treated with Ami (160 mg/L) in drinking water for 14 days. Repeated FST was performed on days –1, 7, and 14. N = 22 (vehicle in WT), 19 (Ami in WT), 17 (vehicle in HET), and 16 (Ami in HET). Statistical significance was calculated using one-way ANOVA with Sidak’s multiple comparisons test (*P < 0.05, ns: not significant). E Timeline and group design of the corticosterone (CORT)-treated mice. F Percentage of sucrose preference and G total water intake. Mice were treated with CORT (35 mg/L) or vehicle (Veh), added to their drinking water, for 7 weeks. During the last 3 weeks of CORT treatment, mice were injected daily with amitriptyline (10 mg/kg/day, i.p.) and/or Ki16425 (10 mg/kg/day, i.p.). N = 15 (vehicle and CORT+Ki16425) and 17 (CORT, CORT+Ami, and CORT+Ami+Ki16425). Statistical significance was calculated using the Kruskal–Wallis test with Dunn’s multiple comparisons test (*P < 0.05, ***P < 0.001, ns: not significant).

Fig. 4. G protein-biased LPAR1 agonist induces antidepressant-like effects.

A Dose-response curves and B logRAi values of LPAR1 agonists (LPA, VPC, pAEA, and OMPT) for the LPAR1-specific G protein activation and β-arrestin recruitment. N = 3. Data are presented as means ± SEM. *P < 0.05, ***P < 0.001 (Unpaired t-test). C Timeline of the experiments using mice treated with LPA or OMPT intrahippocampally. D Immobility time in the forced swim test (FST) and E time in the center and distance traveled in the open field test (OFT). OMPT or LPA was continuously infused into the hippocampi for 2 weeks using osmotic pumps (concentration in pump: 15 nM, delivery rate: 0.11 μL/h). Repeated FST was performed on days –1, 7, and 14, and the OFT was performed on Day 15. N = 24. *P < 0.05, ns: not significant (Unpaired t-test).

Fig. 5. Transcriptional characterization by continuous administration of LPA or OMPT.

A Comparison of RRHO expression patterns in the hippocampus of mice intrahippocampally treated for 2 weeks with LPA or OMPT. B Canonical pathways enriched with discordantly overlapping genes are presented in panel A. Pathways with positive z-scores indicate pathways that are predicted to be activated by OMPT. C Comparisons of RRHO expression patterns in the hippocampus of mice treated with LPA (Y-axis) and chronic social defeat stress models (susceptible and resilient) treated with or without imipramine (X-axis). D Comparisons of RRHO expression patterns in the hippocampus of mice treated with OMPT (Y-axis) and chronic social defeat stress models treated with or without imipramine (X-axis).