Serotonin Reuptake Inhibitors (SRIs)-type antidepressants, among which Prozac is the most known, are used from the 80's to treat depressive states. The molecular basis of the beneficial effect afforded by SRIs consumption as well as mechanisms of resistance to SRIs are still enigmatic. The aim is to identify such mechanisms to improve therapeutic approaches to combat depression.
Depression concerns more than 350 millions people over the world according to WHO data, placing this pathology as a major concern for public health. Depressive states are associated to a decrease in the brain of the level of serotonin, which regulates mood, anxiety and appetite. SRI Antidepressants (Prozac) act on serotonergic neurons to counteract serotonin deficit. Our previous works showed that Prozac, acting on serotonergic neurons of the raphé nuclei, stimulate another brain region, the locus coeruleus, which contains neurons that synthesize another neurotransmiiter, norepinephrine. Noradrenergic nerons become capable to synthesize and release serotonin, which creates a novel source of serotonin in the brain, at the root of the beneficial effect of Prozac action. Unlocking serotonergic function in noradrenergic neurons depends on reduction of the expression level of a small RNA, the microRNA mir-16. Upon Prozac exposure, serotonergic neurons release a signal molecule, the S100b factor, which acts on noradrenergic neurons, induces decrease of mir-16 and unlocks serotonin synthesis. <br />The global aim of the miR-Dep project is to delineate the mechanisms wherby SRI antidepressants act on serotonergic neurons as well as noradrenergic neurons through S100b. In view of the resistance to antidepressants of some patients, identifying the protagonists (microRNAs, signal molecules, receptors, enzymes...) involved in the Prozac response would permit to improve therapeutic strategies to combat depression.
We exploit a neuronal stem cells endowed with the capacity to differentiate into serotonergic or noradrenergic neurons to identify cellular and molecular mechanisms whereby Prozac exerts direct effects on serotonergic neurons and indirect effects on noradrenergic neurons. In view of the diversity of signals in brain, these homogeneous cell culture models allow to decipher the nature and sequence of events, that is molecular readouts, underlying the benefiical effects of antidepressants. The relevance of our in vitro results is probed in vivo in mice models with depression treated or not with Prozac.
In serotonergic neurons, Prozac interacts with the serotonin transport and blocks its transport activity of serotonin, which thereby increases external concentration of serotonin that counteracts serotonin deficit measured in depressed subjects. Our current work indicates that this increase in external serotonin induced by Prozac stimulates a serotonergic recpetor (the 5-HT2B receptor subtype) present at the membrane of serotonergic neurons, which in turn promotes the release of serotonin. This receptor also govern the release of the S100b signal molecule. The 5-HT2B receptor thus relays the Prozac response of serotonergic neurons. This result has been confirmed in vivo as mice depleted for 5-HT2B receptor do not respond to Prozac treatment.
S100b secreted by serotonergic neurons in response to Prozac promotes in noradrenergic neurons the decrease of miR-16 but also of two other microRNAs, which could contribute to the onset of serotonergic functions. Finally, our work unravels a cascade of reactions in which Prozac, via S100b, activates in noradrenergic neurons a protein factor (hnRNPK) that contributes to the expression of serotonergic functions. Activated hnRNPK seems to counteract the inhibitory effect of miR-16 on the expression of serotonergic functions in noradrenergic neurons and to unlock the synthesis of serotonin.
Our work identifies new actors involved in the Raphe-locus coeruleus dialog induced by Prozac. This provides new readouts to probe the action and efficiency of antidepressants, that is a prerequisite to optimize therapeutic protocols to combat depression. Beyond depression, our data may account for some deleterious events associated to neurodegenerative Alzheimer's disease (AD). The locus coeruleus is one of the brain structure firstly affected by AD. High levels of S100b were measured in the brain of AD individuals. Our preliminary data reveal that high doses of S100b drastically alter noradrenergic functions due to concomitant increases of miR-16 and two other microRNAs. Understanding the functional relationship between S100b, microRNAs, neuronal functions within a «Prozac'« context will thus authorize to grasp some mechanisms of neurodegeneration associated to AD and to define potential therapeutic targets.
An article on the Prozac action on serotonergic and noradrenergic neurons is in preparation.
Achieving a better understanding of the physiopathology of depression and of the therapeutic action of antidepressants is mandatory to design more potent and selective therapies to treat Major Depression Disorders (MDD). Because nearly all antidepressants available to date target the serotonergic and/or noradrenergic systems, a thorough knowledge of the action of these drugs on serotonergic and noradrenergic neurons would represent an important advance. Elucidating the cellular and molecular responses of neurons to antidepressants in vivo represents a very arduous task, due to the multiplicity of cell types and signals at play. Recently, our teams jointly reported on the use of the 1C11 neuronal cell line, able to differentiate into either serotonergic or noradrenergic neurons with a frequency of nearly 100% and in a mutually exclusive manner, to uncover an unsuspected mode of action of the antidepressant fluoxetine (Prozac). Fluoxetine belongs to the class of serotonin reuptake inhibitor (SRI) antidepressants, known to block the function of the serotonin transporter (SERT). We demonstrated that, in serotonergic neurons, fluoxetine promotes an increase in the microRNA miR-16, which, in turn, negatively controls the translation of its target, the SERT-encoding mRNA. Fluoxetine further triggers the release of several signaling molecules: S100beta, BDNF, Wnt2 and 15dPGJ2, which can relay the action of fluoxetine at distance from serotonergic neurons. We found that S100beta operates on noradrenergic neurons in vitro by decreasing the level of miR-16 and unlocking the expression of SERT and that of other serotonergic functions, while not altering noradrenergic functions. Noradrenergic neurons thus become a new source of serotonin and are rendered sensitive to SRIs. Importantly, we were able to validate our in vitro findings in vivo in raphe and the locus coeruleus and to demonstrate a beneficial effect of the fluoxetine-dependent regulation of miR-16 in these 2 brain regions in a mouse model of depression. We further gained evidence that BDNF, Wnt2 and 15dPGJ2 act in synergy to promote a decrease in miR-16 levels in the hippocampus, which triggers hippocampal neurogenesis. Finally, we established the translational relevance of the fluoxetine-induced release of BDNF, Wnt2 and 15dPGJ2 by showing that these 3 molecules are increased in the CSF of mice or depressed patients in response to fluoxetine.
We now aim to build upon the identification of these effectors and pathways to decipher the integrated response of serotonergic neurons to antidepressants and to assess their long-term adaptation and the reversibility of the fluoxetine-induced changes. Our project also focuses on the mechanisms behind the plasticity of noradrenergic neurons, which adapt to the connection with fluoxetine-exposed serotonergic neurons by implementing serotonergic functions themselves. A key question we propose to address is whether noradrenergic neurons can maintain a mixed serotonergic-noradrenergic phenotype over a long time-period and whether permanent input (S100beta) signals are required.
Our experimental strategy will rely primarily on the serotonergic and noradrenergic derivatives of the 1C11 cell line to probe effectors (receptors, signal transduction intermediates, extracellular signaling molecules) and signalling pathways to establish molecular scenarios to be further tested in vivo in mice or, when appropriate, in the CSF of depressed patients.
A main asset of the project is the complementarity of the 2 teams, which has already proved successfull in yielding the overall results at the origin of the present proposal. By shedding further light on the molecular pathways that are recruited by SRI antidepressants in serotonergic and, indirectly, noradrenergic neurons, our project should have implications as to (1) the identification of molecular readouts to probe the efficacy of drug response and (2) the optimization of therapeutic protocols.
Madame Odile KELLERMANN (INSERM UMR-S 747 Lab. Cellules Souches, Signalisation et Prions (Eq. 5))
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
INSERM UMR-S 747 Lab. Cellules Souches, Signalisation et Prions (Eq. 5)
INSERM Laboratoire Biomarqueurs de l'Insuffisance Cardiaque
Help of the ANR 297,800 euros
Beginning and duration of the scientific project: October 2013 - 42 Months