Raphe-mediated signals control the hippocampal response to SRI antidepressants via miR-16

Abstract

Serotonin reuptake inhibitor (SRI) antidepressants such as fluoxetine (Prozac), promote hippocampal neurogenesis. They also increase the levels of the bcl-2 protein, whose overexpression in transgenic mice enhances adult hippocampal neurogenesis. However, the mechanisms underlying SRI-mediated neurogenesis are unclear. Recently, we identified the microRNA miR-16 as an important effector of SRI antidepressant action in serotonergic raphe and noradrenergic locus coeruleus (LC). We show here that miR-16 mediates adult neurogenesis in the mouse hippocampus. Fluoxetine, acting on serotonergic raphe neurons, decreases the amount of miR-16 in the hippocampus, which in turn increases the levels of the serotonin transporter (SERT), the target of SRI, and that of bcl-2 and the number of cells positive for Doublecortin, a marker of neuronal maturation. Neutralization of miR-16 in the hippocampus further exerts an antidepressant-like effect in behavioral tests. The fluoxetine-induced hippocampal response is relayed, in part, by the neurotrophic factor S100β, secreted by raphe and acting via the LC. Fluoxetine-exposed serotonergic neurons also secrete brain-derived neurotrophic factor, Wnt2 and 15-Deoxy-delta12,14-prostaglandin J2. These molecules are unable to mimic on their own the action of fluoxetine and we show that they act synergistically to regulate miR-16 at the hippocampus. Of note, these signaling molecules are increased in the cerebrospinal fluid of depressed patients upon fluoxetine treatment. Thus, our results demonstrate that miR-16 mediates the action of fluoxetine by acting as a micromanager of hippocampal neurogenesis. They further clarify the signals and the pathways involved in the hippocampal response to fluoxetine, which may help refine therapeutic strategies to alleviate depressive disorders.

Figure 1

Infusion of fluoxetine into raphe decreases miR-16 in the hippocampus, which in turn, increases serotonin transporter (SERT) and bcl-2 levels, promotes neurogenesis and exerts an antidepressant effect. (a–e) Mice received a chronic perfusion of fluoxetine into raphe (1 μ, 2 μl min⁻¹, 3 days) in combination (n=11) or not (n=6) with direct injection of miR-16 (1 μl, 2 μ, every 36 h) into the hippocampus. Alternatively, a direct injection of anti-miR-16 (1 μl, 2 μ, every 36 h, n=6 mice) alone into the hippocampus was performed. Scrambled miRNAs (n=7) or anti-miRNAs (n=10) were used as controls. Control values were obtained in n=13 mice. All measurements were made on hippocampus samples: miR-16 level (real-time PCR) (a), SERT expression ([³H]-paroxetine binding) (b), Bcl-2 protein expression (western blot) (c) and neurogenesis (Doublecortin (Dcx) immunolabeling) (d, e). (f) Immunolabeling of SERT (red) in hippocampal cells positive for V-GLUT 1 (bottom) or V-GLUT-2 (top) (green) after infusion of fluoxetine into raphe for 3 days. (g) Injection of fluoxetine into raphe or anti-miR16 into the hippocampus similarly reduced the time of immobility in the forced swimming test. (h–j) Six-week unpredictable chronic mild stress (UCMS)-induced deterioration of the coat state score (h) and reductions in body weight gain (i) and sucrose preference (j) were alleviated by injection of fluoxetine into raphe or anti-miR16 into the hippocampus (n=9 mice in each group). Values are means±s.e.m. ^*P<0.01 and ^**P<0.05 vs control, ^#P<0.01 and ^##P<0.05 vs vehicle UCMS.

Figure 2

S100β released by raphe upon fluoxetine treatment does not act directly on the hippocampus but partially relays the fluoxetine response via the locus coeruleus. (a–c) Mice were chronically exposed to fluoxetine for 20 days (daily intraperitoneal injection, 5 mg kg⁻¹, ‘basal' n=7). During the treatment, two groups of mice also received stereotaxic injection into the raphe of S100β-siRNA (2 μg, 1 μg μl⁻¹, n=8) or scrambled oligonucleotides every 36 h (n=9). Control values were obtained in n=13 mice. The downregulation of miR-16 (real-time PCR) (a) and the upregulation of SERT ([³H]-paroxetine binding) (b) and bcl-2 protein levels (western blot analysis) (c) in the hippocampus after a 20-day intraperitoneal injection of fluoxetine in mice were partially abolished by siRNA-mediated knockdown of S100β in raphe. (d–f) Mice received a stereotaxic injection of S100β (1 n, 2 μl min⁻¹, 1 day, n=7) in the locus coeruleus (LC). Alternatively, fluoxetine was perfused into the raphe either alone (‘basal' n=8) or combined with injection of S100β antibodies (1 μg ml⁻¹) into the hippocampus (n=8) or the LC (n=7), or with degeneration of noradrenergic fibers with the DSP4 neurotoxin (n=7). Control values were obtained in n=12 mice. Hippocampal extracts from these different groups of mice were collected to quantify the miR-16 level (d), SERT (e) and bcl-2 (f) protein expression. The values are the means±s.e.m, ^*P<0.01 and ^**P<0.05 vs control, ^§P<0.05 vs scramble, ^#P<0.01 and ^##P<0.05 vs basal fluoxetine in raphe.

Figure 3

Brain-derived neurotrophic factor (BDNF), Wnt2 and 15-Deoxy-delta12,14-prostaglandin J2 (15d-PGJ2) are secreted by serotonergic neurons in response to fluoxetine and are increased in cerebrospinal fluid (CSF) of mice or depressed patients following fluoxetine treatment. (a–c) Treatment of 1C11^5−HT cells with fluoxetine (50 n, 2 days) induced the release of BDNF (a), Wnt2 (b) and 15d-PGJ2 (c) (n=6). (d–i), Treatment of mice (d–f) or depressed patients (g–i) with fluoxetine induced an increase of BDNF (d, g), Wnt2 (e, h) and 15d-PGJ2 (f, i) in CSF. The values are the means±s.e.m (n=5 for mice, n=7 for patients), ^*P<0.01 vs control.

Figure 4

Brain-derived neurotrophic factor (BDNF), Wnt2 and 15-Deoxy-delta12,14-prostaglandin J2 (15d-PGJ2) act synergistically on the hippocampus by decreasing miR-16 and increasing serotonin transporter (SERT) and bcl-2 levels. (a–c) Mice received a chronic perfusion of fluoxetine (fluox) into raphe (n=6) or a direct stereotaxic perfusion of BDNF, Wnt2 or 15d-PGJ2 into the hippocampus (n=7). Combined injections were carried out using concentrations selected according to the combination index method of Chou-Talalay. An optimal response was reached with a combination of 50 ng ml⁻¹ BDNF, 1.5 ng ml⁻¹ Wnt2 and 0.25 μ 15d-PGJ2. (a) The miR-16 level (real-time PCR), (b) SERT expression ([³H]-paroxetine binding) and (c) bcl-2 protein expression (western blot analysis) were measured in hippocampus extracts. Control values were obtained in n=9 mice. The values are the means±s.e.m. ^*P<0.01 and ^**P<0.05 vs control. (d) Prozac treatment induces the secretion of S100β, BDNF, Wnt2 and 15d-PGJ2 from raphe serotonergic neurons. These signals relay the action of fluoxetine by downregulating miR-16, which acts as a micromanager in the hippocampal response to SRI antidepressants.