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MiR-16 Targets the Serotonin Transporter: A New Facet for Adaptive Responses to Antidepressants

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Science  17 Sep 2010:
Vol. 329, Issue 5998, pp. 1537-1541
DOI: 10.1126/science.1193692

Abstract

The serotonin transporter (SERT) ensures the recapture of serotonin and is the pharmacological target of selective serotonin reuptake inhibitor (SSRI) antidepressants. We show that SERT is a target of microRNA-16 (miR-16). miR-16 is expressed at higher levels in noradrenergic than in serotonergic cells; its reduction in noradrenergic neurons causes de novo SERT expression. In mice, chronic treatment with the SSRI fluoxetine (Prozac) increases miR-16 levels in serotonergic raphe nuclei, which reduces SERT expression. Further, raphe exposed to fluoxetine release the neurotrophic factor S100β, which acts on noradrenergic cells of the locus coeruleus. By decreasing miR-16, S100β turns on the expression of serotonergic functions in noradrenergic neurons. Based on pharmacological and behavioral data, we propose that miR-16 contributes to the therapeutic action of SSRI antidepressants in monoaminergic neurons.

Transporters selective for serotonin [5-hydroxytryptamine (5-HT)] (SERTs), noradrenaline (NETs), or dopamine ensure the reuptake of monoamines at the synaptic cleft and thereby sustain the action of therapeutic agents in the treatment of a variety of psychiatric disorders (1). Dysfunction of serotonergic neurotransmission has been implicated in depression as well as obsessive-compulsive disorder, anxiety, and suicidal behavior (2). Selective serotonin reuptake inhibitors (SSRIs) are beneficial in the treatment of all these neuropsychiatric conditions. A still enigmatic observation is that SSRIs need to be administered for long time periods to yield clinical improvement (3).

The distribution of SERT in the brain mirrors that of serotonergic neuronal cell bodies and innervating fibers (4, 5). Serotonergic raphe neurons project to most parts of the central nervous system and coordinate the physiology of the whole brain (2). Chronic SSRI antidepressant treatment promotes reductions in SERT binding and protein levels but does not affect SERT mRNA levels (6), suggesting that SSRIs may interfere with SERT translation. This control of translation could be exerted by microRNAs (miRNAs), which have emerged as crucial modulators of gene expression (7, 8). Although the roles of miRNAs in cell fate decision, differentiation, maintenance of cell identity, survival, and neuronal plasticity are being uncovered (9, 10), their targets remain largely unknown.

To investigate whether miRNAs provide a mechanism for adaptive changes in SERT expression in monoaminergic neurons, we first exploited the 1C11 neuroectodermal cell line, which can differentiate into either serotonergic (1C115-HT) or noradrenergic (1C11NE) neuronal cells (fig. S1A) (11). 1C11 neuroectodermal cells express transcripts encoding SERT and neurotransmitter-related markers before their choice of cell fate (Fig. 1A and fig. S1B). Because these transcripts remain at roughly constant levels during serotonergic or noradrenergic differentiation, miRNAs may participate in the posttranscriptional mechanisms that prevent illegitimate mRNA translation according to each program.

Fig. 1

miR-16 targets SERT in the 1C11 cell line. (A) Reverse transcription polymerase chain reaction (PCR) analysis of SERT and NET transcripts during serotonergic and noradrenergic differentiation. Glyceraldehyde phosphate dehydrogenase mRNA was used as a control. (B) Overexpression of miR-16 in 1C115-HT cells reduces SERT expression. The level of SERT was determined by [125I]-RTI-55 and [3H]-paroxetine binding in 1C115-HT day 4 cells untreated (control) or treated with miR-16 or anti–miR-16 at day 2 of serotonergic differentiation. (C to E) miR-16 reduction in 1C11NE cells induces SERT expression and SSRI antidepressant binding, while not affecting NET expression. 1C11NE cells were treated with miR-16 or anti–miR-16 at day 10 of the noradrenergic program. At day 12, NET and SERT expression were measured in cell homogenates using (C) [3H]-nisoxetine, a selective inhibitor of noradrenaline uptake; (D) [3H]-paroxetine, a selective inhibitor of 5-HT uptake; and (E) [125I]-RTI-55, which recognizes NET and SERT. (F to I) miR-16 reduction in 1C11NE cells unlocks the expression of serotonergic markers. Cell extracts from 1C11NE day 12 cells untreated (control) or treated with miR-16 or anti–miR-16 at day 10 of differentiation were used to assess (F) TPH activity, (G) 5-HT intracellular content, (H) 5-hydroxyindoleacetic acid (5-HIAA) concentration, and (I) the amount of 5-HT2B receptors ([3H]-LY 266097 binding). Data are means ± SEM of seven independent experiments, (B) *P < 0.01 versus control, (E) *P < 0.01, and [(D) and (F) to (I)] *P < 0.001 versus control and miR-16.

Using in silico computational target prediction, we identified miR-16 as a miRNA with complementarity to the 3′ untranslated region of the SERT mRNA (fig. S2A) and then validated miR-16 as a SERT-targeting miRNA with a luciferase assay (fig. S2B). Consistent with a putative role of miR-16 as a negative regulator of SERT translation, we found that 1C11 neuroectodermal cells expressed a low level of miR-16, which increased along the noradrenergic pathway, whereas the level did not vary along the serotonergic program (fig. S2C).

If the SERT is indeed a target of miR-16, then miR-16 overexpression in 1C115-HT cells should decrease SERT protein levels. During serotonergic differentiation, SERT translation starts at day 2 (12). Functional 5-HT uptake and antidepressant recognition occur at day 4. Among monoamine transporters, only SERT has the capacity to bind SSRI antidepressants [such as paroxetine and fluoxetine (Prozac)] (13). The SERT further recognizes the cocaine congener [125I]-RTI-55, which also binds to NET (12). Using these pharmacological tools, we quantified the level of SERT expression in 1C115-HT cells after transfection with miR-16. miR-16 overexpression reduced the number of [3H]-paroxetine or [125I]-RTI-55 binding sites by 40% (Fig. 1B). In contrast, the number of [3H]-paroxetine and [125I]-RTI-55 binding sites remained unchanged when 1C115-HT cells were transfected with miR antisense oligonucleotides (anti–miR-16) (Fig. 1B), suggesting that SERT translation is insensitive to the endogenous basal level of miR-16 in 1C115-HT cells.

1C11NE noradrenergic cells selectively implement a functional NET at day 12 of their program (11). Although they express SERT mRNAs, SERT molecules are undetectable (Fig. 1, A and D). Assuming that the up-regulation of miR-16 during noradrenergic differentiation (fig. S2C) may have a role in silencing SERT transcripts, we exposed 1C11NE cells to anti–miR-16. Binding was measured with three drugs: [3H]-nisoxetine, which is selective for NET; [125I]-RTI-55, which binds NET and SERT; and [3H]-paroxetine, which recognizes SERT only. NET expression was insensitive to a reduction of miR-16 (Fig. 1C). In contrast, anti–miR-16 induced the appearance of paroxetine binding sites in 1C11NE cells (Fig. 1D) and increased the number of RTI binding sites to the sum of nisoxetine and paroxetine binding sites (Fig. 1E). The number of newly induced RTI sites equaled the number of paroxetine binding sites and may thus be ascribed to de novo expressed SERT molecules. Hence, the inhibition of miR-16 unlocks SERT protein expression in 1C11NE cells and renders noradrenergic cells competent to recognize SSRI antidepressants.

Next, we investigated whether decreasing miR-16 levels in 1C11NE cells would promote changes in noradrenergic phenotypic parameters distinct from NET and/or allow the implementation of serotonergic functions, in addition to that of SERT. Neutralization of endogenous miR-16 with anti–miR-16 had no impact on noradrenergic-associated functions in 1C11NE cells (fig. S3). In contrast, the cells acquired a complete serotonergic metabolism, as defined by the ability to synthesize, store, and degrade 5-HT, and they expressed 5-HT2B receptors (Fig. 1, F to I). These results show that miR-16 acts as a global repressor of the expression of serotonergic-specific functions in 1C11NE cells.

We hypothesized that the miR-16–dependent regulation of the SERT shown in the 1C11 cell line may have physiopathological relevance in vivo. We first quantified miR-16 in mouse serotonergic raphe nuclei (2) versus the noradrenergic locus coeruleus (14). As in the 1C11 cell line, lower levels of miR-16 were found in raphe than in the locus coeruleus (fig. S2D). Then we assessed whether SSRI antidepressant treatment could alter the levels of miR-16 in these regions of the mouse brain. When fluoxetine was infused into raphe, we observed a 2.5-fold increase in the level of miR-16 and a twofold reduction in [3H]-paroxetine binding in raphe (Fig. 2, A and B). Direct injection of miR-16 into raphe yielded a similar decrease in [3H]-paroxetine binding (Fig. 2C). Finally, [3H]-paroxetine binding was not affected after the infusion of fluoxetine together with anti–miR-16 (Fig. 2B). These data demonstrate that fluoxetine regulates SERT expression through miR-16 in raphe.

Fig. 2

Fluoxetine increases miR-16 levels in raphe by antagonizing canonical Wnt signaling. (A) Mice received chronic stereotaxic injection (2 μl/min) of fluoxetine (1 μM) into raphe for 3 days in combination or not with activators of the canonical Wnt pathway [Wnt3a (50 ng/ml), LiCl (1 mM), or SB-216763, a selective GSK-3β inhibitor (100 nM)]. The levels of miR-16 and pre/pri–miR-16 in raphe were determined by real-time PCR. Data are means ± SEM (n = 7 animals), **P < 0.01 versus control, *P < 0.05, and §P < 0.01 versus fluoxetine alone. (B and C) SERT expression ([3H]-paroxetine binding) was determined in raphe extracts of mice perfused (2 μl/min) for 3 days into the raphe with fluoxetine (1 μM) in the presence or absence of anti–miR-16 (1 μl, 2 μM) (B) or after direct injection of miR-16 (1 μl, 2 μM) (C). Data are means ± SEM of seven animals, *P < 0.01 versus control.

The fluoxetine-induced up-regulation of miR-16 in raphe nuclei may involve a pre/pri–miR-16 enhanced transcription and/or maturation. In raphe versus the locus coeruleus, the level of pre/pri–miR-16 was inversely correlated with the level of miR-16 (compare figs. S2D and S4). In addition, the fluoxetine-mediated increase in miR-16 in raphe was accompanied by a decrease in pre/pri–miR-16 (Fig. 2A), thus supporting the maturation hypothesis. Because canonical Wnt signaling may repress miR-16 maturation (15), we quantified the levels of miR-16 and pre/pri–miR-16 under combined fluoxetine treatment and activation of the Wnt pathway. The up-regulation of miR-16 and the down-regulation of pre/pri–miR-16 triggered by fluoxetine in raphe were both eliminated by either Wnt3a, LiCl, or SB-216763 (Fig. 2A). Chronic fluoxetine treatment actually interfered with canonical Wnt signaling, as inferred from the increase in glycogen synthase kinase–3β (GSK-3β) activity (fig. S5A). Hence, the SSRI fluoxetine augments the level of miR-16 in raphe by antagonizing Wnt signaling and thereby negatively regulates SERT expression.

When infused into the locus coeruleus, fluoxetine failed to induce any change in miR-16 expression (fig. S6), which is in agreement with the lack of SERT expression in noradrenergic neurons under basal conditions. In contrast, upon infusion of fluoxetine into raphe, we monitored a 30% reduction in miR-16 in the locus coeruleus (Fig. 3A), associated with a decrease in GSK3β activity (fig. S5B). This down-regulation of miR-16 was accompanied by the induction of SERT expression, as well as tryptophan hydroxylase (TPH) activity and 5-HT2B receptors (Fig. 3, B to D). Confocal microscopy confirmed that SERT induction occurred in tyrosine hydroxylase–positive neurons (fig. S7). Thus, the locus coeruleus responds to fluoxetine injection in raphe by switching on serotonergic functions. Likewise, in a more clinically relevant paradigm, 20 days after daily intraperitoneal injection of fluoxetine into mice, we measured a 27% decrease of miR-16 associated with an expression of SERT molecules in the locus coeruleus (fig. S8, A and B).

Fig. 3

Fluoxetine injection into raphe decreases miR-16 in the locus coeruleus and turns on SERT expression, 5-HT synthesis, and 5-HT2B receptor expression in the locus coeruleus. Chronic stereotaxic injection of fluoxetine (1 μM, 2 μl/min) into the mouse raphe was carried out for 3 days. All measurements were made on locus coeruleus extracts: (A) miR-16 level using real-time PCR, (B) SERT expression ([3H]-paroxetine binding), (C) TPH activity, and (D) amount of 5-HT2B receptors ([3H]-LY 266097 binding). Values are means ± SEM of seven animals, *P < 0.01 and **P < 0.001 versus control.

The question then arises of how the response of serotonergic neurons to fluoxetine treatment is relayed to noradrenergic neurons in vivo. Reciprocal connections exist between these two brainstem monoaminergic nuclei, thus supporting communication between the two systems (16). Recently, the expression of miR-16 in monocytes was shown to be down-regulated by S100β (17), a neurotrophic protein that is up-regulated by fluoxetine treatment (18). We therefore hypothesized that the secretion of S100β increases upon exposure of raphe to fluoxetine and that this protein acts as a paracrine factor to promote the reduction in miR-16 in the locus coeruleus, in turn unlocking the expression of serotonergic functions. We first exposed 1C115-HT cells to fluoxetine and observed an accumulation of S100β in the culture medium (Fig. 4A). Although the addition of S100β slightly decreased miR-16 levels in these serotonergic cells (Fig. 4B), it did not affect SERT expression (Fig. 4C), which is in agreement with the lack of impact of miR-16 silencing on SERT in 1C115-HT cells (Fig. 1B). A larger decrease (43% of control level) of miR-16 was seen in 1C11NE cells exposed to S100β (Fig. 4B), which correlated with the appearance of SERT (Fig. 4C). In addition, after S100β treatment, 1C11NE cells acquired the ability to synthesize and store 5-HT (Fig. 4, D and E) and to express 5-HT2B receptors (Fig. 4F). These data thus validate our working hypothesis on an in vitro level. We then measured the level of S100β in raphe upon infusion of fluoxetine. Fluoxetine up-regulated S100β levels in serotonergic nuclei (133% versus control) (Fig. 5A). Further, injection of S100β into the locus coeruleus decreased (by 22.4%) miR-16 levels and turned on the expression of SERT (Fig. 5, B and C). Finally, antibody-mediated neutralization of S100β in the locus coeruleus prevented the decrease in miR-16 levels observed upon infusion of fluoxetine in raphe (Fig. 5D). In addition, the decrease in miR-16 and the onset of SERT expression observed in the locus coeruleus, upon systemic fluoxetine treatment, were both eliminated by small interfering RNA–mediated knockdown of S100β in raphe (fig. S8, A and B). The data from 1C115-HT cells (Fig. 4A) and the innervation of the locus coeruleus by raphe fibers (16) strengthen the hypothesis that secretion of S100β by serotonergic neurons, at the locus coeruleus, mediates the action of fluoxetine. Secretion of S100β by glial cells in the raphe is less likely to promote a long-range action on the locus coeruleus.

Fig. 4

Fluoxetine induces 1C115-HT cells to release S100β, which decreases miR-16 expression and triggers the implementation of serotonergic markers in 1C11NE cells. (A) Treatment of 1C115-HT cells with fluoxetine (50 nM) for 2 days increased the extracellular content of S100β. (B to F) 1C115-HT or 1C11NE cells were exposed to S100β (1 nM) for 2 days. (B) The level of miR-16 as analyzed by real-time PCR was decreased in 1C115-HT and 1C11NE cells. (C) S100β did not affect SERT expression in 1C115-HT cells, whereas it induced the expression of SERT in 1C11NE cells ([3H]-paroxetine binding). In 1C11NE cells, S100β triggered de novo 5-HT synthesis (detection of TPH activity) (D), 5-HT intracellular content (E), and 5-HT2B receptor expression ([3H]-LY 266097 binding) (F). Data are means ± SEM of seven independent experiments, *P < 0.01 versus control.

Fig. 5

Fluoxetine injection into raphe acts on the locus coeruleus via S100β and induces behavioral responses that are mimicked by increases in miR-16 in raphe or decreases in miR-16 in the locus coeruleus. (A) Chronic stereotaxic injection of fluoxetine (1 μM, 2 μl/min, 3 days) into mouse raphe induced S100β efflux. (B and C) Stereotaxic injection of S100β (1 nM, 2 μl/min, 1 day) into the mouse locus coeruleus decreased miR-16 (B) and induced SERT expression (C) in locus coeruleus extracts as determined by real-time PCR and [3H]-paroxetine binding, respectively. (D) Injection of antibodies against S100β (1 μg/ml, 24 hours) into the locus coeruleus prevented the down-regulation of miR-16 in this brain structure induced by chronic infusion of fluoxetine into raphe. Data are means ± SEM (n = 7 animals), *P < 0.01 versus control. (E to H) Six-week UCMS-induced deterioration of the coat state score (E), reduction of body weight gain (F), and decreases in sucrose preference (G) and locomotor activity (H) were alleviated by stereotaxic injection of fluoxetine (1 μM, 2 μl/min, in the last 5 weeks) or miR-16 (1 μl, 2 μM, every 36 hours) into mouse raphe or anti–miR-16 (1 μl, 2 μM, every 36 hours) into the locus coeruleus. The injection of scrambled miRNAs did not yield any improvement in these tests. Data are means ± SEM (n = 6 to 9 mice per group). §P < 0.01 versus control, *P < 0.05, and **P < 0.01 versus vehicle UCMS.

Finally, we demonstrated the potential benefit of the fluoxetine-induced regulation of miR-16 in two mouse models of depression: the forced swimming test (FST) (fig. S9) and the unpredictable chronic mild stress (UCMS) paradigm (19, 20). Mice exposed to a 6-week UCMS regimen exhibited a deterioration of coat state and reductions in body weight gain, sucrose preference, and locomotor activity that were alleviated to the same extent either by chronic infusion of fluoxetine or miR-16 into raphe or by anti–miR-16 into the locus coeruleus (Fig. 5, E to H) (21).

Our study identifies the SERT-targeting miRNA miR-16 as a player in relaying SSRI antidepressant action (fig. S10). Fluoxetine operates directly on serotonergic raphe nuclei by increasing the maturation of miR-16 from its precursor pre/pri–miR-16. Raphe additionally responds to chronic fluoxetine treatment by releasing S100β, which in turn acts on the noradrenergic neurons of the locus coeruleus. By lowering miR-16 levels, S100β unlocks the expression of serotonergic functions in this noradrenergic brain area. Our pharmacological and behavioral data thus posit miR-16 as a central effector that regulates SERT expression and mediates the adaptive response of serotonergic and noradrenergic neurons to fluoxetine treatment.

Supporting Online Material

www.sciencemag.org/cgi/content/full/329/5998/1537/DC1

Materials and Methods

Figs. S1 to S10

References

References and Notes

  1. Materials and methods and supporting data are available on Science Online.
  2. We thank P. Weil-Malherbe, V. Mutel, F. d’Agostini, G. Zürcher, E. Borroni, J. L. Moreau, F. Jenck, M. Bühler, and N. Pieron for skillful methodological assistance, and S. Blanquet, M. Briley, and L. Aggerbeck for critical reading of the manuscript. O.K. is a professor at Paris XI University. This work was funded by CNRS, ANR, and INSERM.
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