Dopamine Receptor D2 and Associated microRNAs Are Involved in Stress Susceptibility and Resistance to Escitalopram Treatment

Abstract

Background: Early life stress has been demonstrated to increase the risk of developing depression in adulthood. However, the roles and associated molecular mechanisms of stresses in the onset and relapse of depression have yet to be fully elucidated.

Methods: Depression-like behaviors were induced in rats by maternal deprivation and chronic unpredictable stress. Depression- and anxiety-like behaviors of rats, dopamine receptor D2 level, and microRNAs expression in rats' brain tissues were measured.

Results: Chronic unpredictable stress alone induced depression-like behaviors in rats, but maternal deprivation enhanced the effect of chronic unpredictable stress. Escitalopram significantly decreased depression-like behaviors in chronic unpredictable stress rats but was less effective in maternal deprivation with chronic unpredictable stress rats. Maternal deprivation increased dopamine receptor D2 messenger RNA expression and decreased microRNA-9 expression in the striatum. Chronic unpredictable stress increased dopamine receptor D2 mRNA and protein levels and decreased microRNA-9 expression in the nucleus accumbens. Furthermore, maternal deprivation enhanced the effect of chronic unpredictable stress on dopamine receptor D2 gene and microRNA-9 expression. Chronic unpredictable stress increased the expression of microRNA-326 in the nucleus accumbens but decreased it in the striatum, whereas maternal deprivation elevated microRNA-326 expression in the striatum. Escitalopram normalized microRNA-326 expression but had no effect on the expression of microRNA-9, dopamine receptor D2 mRNA, and dopamine receptor D2 protein in both the nucleus accumbens and striatum. The in vitro study showed that only microRNA-9 directly targeted the 3' untranslated region of dopamine receptor D2 mRNA and inhibited dopamine receptor D2 protein expression.

Conclusion: Early life stress enhanced the susceptibility to late life stress and resistance to escitalopram treatment through decreasing microRNA-9 expression and subsequently upregulating dopamine receptor D2 expression in the nucleus accumbens. microRNA-326 may be a novel target of escitalopram.

Keywords: depression; dopamine receptor D2; microRNAs; stress.

Figure 1.

Comparison of behavioral indices. (A-E) Comparison of ambulatory distance (A), the number of vertical movements (B), fecal pellets (C), sucrose preference of rate (D), and immobility time (E) among groups. ^* P< .05 vs NOR for saline treatment; ^Δ P < .05 vs CUS for saline treatment; ^ǂ P < .05 vs MD for saline treatment; ^& P < .05 between 2 treatments.

Figure 2.

Dopamine receptor D2 (DRD2) protein and mRNA expression in the nucleus accumbens (NAc) and striatum. Comparison of DRD2 mRNA (A) and protein (B) expression in the NAc among groups. Comparison of DRD2 mRNA (C) and protein (D) expression in the striatum among groups. ^* P<.05 vs normal control (NOR) for saline treatment; ^Δ P<.05 vs chronic unpredictable stress (CUS) for saline treatment; ^ǂ P<.05 vs maternal deprivation (MD) for saline treatment. E, escitalopamine; S, saline.

Figure 3.

Expression of miRNAs in the nucleus accumbens (NAc) and striatum. Comparison of miR-9 (A) and miR-326 (B) expression in the NAc among groups. Comparison of miR-9 (C) and miR-326 (D) expression in the striatum among groups. ^* P<.05 vs normal control (NOR) for saline treatment; ^Δ P<.05 vs chronic unpredictable stress (CUS) for saline treatment; ^ǂ P<.05 vs maternal deprivation (MD) for saline treatment. ^& P<.05 between 2 treatments.

Figure 4.

microRNAs (miRNAs) regulated dopamine receptor D2 (DRD2) expression in vitro. (A) Nucleotide base of DRD2 mRNA 3’ untranslated region (3’-UTR) pairs with miR-9 and miR-326. (B) The expression of FAM (green) after transfection of miR-9 and miR-326 into SH-SY5Y cell. (C) The expression of miR-9 and miR-326 after transfection of mimics or inhibitor into SH-SY5Y cell. (D) The expression of DRD2 mRNA after transfection of mimics or inhibitor into SH-SY5Y cell. (E) Western blot of DRD2 and ß-actin after transfection of mimics and inhibitor into SH-SY5Y cell. (F) Relative luciferase activity of DRD2 (wild-type, WT) 3’UTR and its mutation (MUT) reporter after cotransfection of plasmid and miR-9 mimics (or negative control mimics) into 293T cell. *P < .05.