Agomelatine and fl uoxetine treatments induce different and time-dependent modulation of rat hippocampal miRNome Mara Seguini1, Daniela Tardito1, Alessandra Mallei1, Ivan Merelli2, Dario Corrada3, Luciano Milanesi2, Giorgio Racagni1, Maurizio Popoli1 1Laboratory of Neuropsychopharmacology and Functional Neurogenomics, Department of Pharmacological and Biomolecular Sciences, University of Milano, Milano, Italy 2Institute for Biomedical Technologies, National Research Council, Segrate, Italy; 3Department of Earth and Environmental Sciences, University of Milano-Bicocca, Milano, Italy [email protected] P 2a.006 INTRODUCTION

MicroRNAs (miRNAs) play a key role in post-transcriptional regulation of expression in almost every biological process. By interacting with complementary regions mainly within the 3’-UTR of target mRNAs, miRNAs interfere with translation and/ or stability of the mRNA. A single mRNA may be regulated by multiple miRNAs and, on the other hand, a single miRNA can regulate several mRNAs, thus modulating expression of several different simultaneously [1]. MiRNAs have a fundamental role in nervous system development and function, with major involvement in neuroplasticity, neuronal differentiation and survival, as well as in neurogenesis [2]. Recent studies suggest a possible contribution of miRNAs in the pathophysiology of neuropsychiatric disorders, including major depression, as well as into the action of psychotropic drugs, such as the mood stabilizers lithium and valproate or antidepressants (ADs) [3].

Aim of this study was to verify whether hippocampal miRNome expression profi le could be affected by treatment for different time lengths (3/7 days) with two different antidepressants: the SSRI fl uoxetine and agomelatine, a MT1/MT2 agonist and 5-HT2C receptor antagonist [4]. MATERIALS AND METHODS

RNA extraction 1- ANIMALS and qRT-PCR Real-Time PCR qRT-PCR was carried out MirVana miRNA isolation kit by using the comparative 3 days: (Life Technologies) 9 agomelatine /fl uoxetine/ vehicle CT (∆∆CT) method. Raw Ct Retrotranscription values were normalized by the 7 days: (150 ng of total RNA) ∆Ct method on endogenous 9 agomelatine /fl uoxetine/vehicle Sprague Dawley male rats controls U6B, U87, Y1 and Preamplifi cation snoRNA135. Agomelatine - 40 mg/kg/die i.p. Hippocampus (MegaPlex PreAmp Primers, TaqMan Low-Density Arrays Fluoxetine - 10 mg/kg/die i.p. (randomly right or left) Life Technologies) Card A+B ≈750 miRNAs 2- EXPRESSION ANALYSES Statistical analysis was carried out with SAM Bioinformatic analyses were performed in order to identify miRNA Validation of selected targets (Signifi cance Analysis of Microarrays software, putative target genes and molecular pathways potentially involved [5]. and analysis of miRNAs version 4.0, Stanford University, http://www-stat. For each miRNAs the 100 most signifi cant targets were selected and included biogenesis (by means of qRT- stanford.edu/≈tibs/SAM/). False Discovery Rate in the annotation analysis performed with subcategories PCR and western blot). (FDR) for multiple testing was set at <5%. (Biological Process, Molecular Function and Cellular

RESULTS miRNA EXPRESSION ANALYSIS

AGOMELATINE FLUOXETINE MiRNAs signifi cantly modulated at both time lenghts after AGOMELATINE treatment 3 days 7 days 3 days 7 days 3 days 7 days MiRNA names Fold Change Fold Change

rno-mir-632 -1.57 -344.03

hsa-let-7* -1.73 -4.11

Fold change Fold change Fold mmu-mir-136* -2.09 -2.01

mmu-mir-463 -2.02 -5.21

hsa-mir-190b -2.28 12.98

After 3 days of treatment, miR-338 and miR-291a-3p were similarly modulated by both drugs (red bars), while after 7 days of treatment miR-488* and miR-382* were modulated by Regarding fl uoxetine, no miRNAs were found to be modulated by both time lenghts both agomelatine and fl uoxetine in the opposite way (green bars) MiRNA target prediction- PATHWAY ANALYSIS (results selection) 3 days of agomelatine treatment 7 days of fl uoxetine treatment Gene Ontology: molecular functions Gene Ontology: biological processes Selected pathways enriched in miRNA putative target genes (out of 29 identifi ed) Selected pathways enriched in miRNA putative target genes (out of 71 identifi ed)

PATHWAY DESCRIPTION ENRICHED TARGET GENES P-VALUE PATHWAY DESCRIPTION ENRICHED TARGET GENES P-VALUE Transforming growth factor beta Cholesterol effl ux Abcg5, Abcg8, Srb1, Cav1, Soat2, Npc2, Abca5, Apoc2, Stx12 0,0004 så Synaptic transmission TGFß3, Smad2, TGFßr3, Smurf2, Lefty1, TGFß2 0.0020 receptor binding så R h y t h m i c e x c i t a t i o n såSingle-stranded DNA binding Icmt, Edn1, Ednra1, Eno1, Pdgfrb, Sp1, Tpm1, Foxa2, Arnt2, Nog, Hmgn3, Egr2, Ebf1, Arp2, Ets1, Gfi 1, Jund, Sp1, Hnf1a, Stat1, Stat3, Sycp1, så Regulation of neuronal såDNA binding In utero embryonic development Notch1, Plcg1, TGFßR1,Cul3, Wnt9b, Ercc2, Dnmt3l, Prdm1, 0,0037 DNA binding Hnf1b, Polq, Cdt1, Neurod1, Hdac1, N-, Myf5, Sox8, Ezh1, Bcl6, E2f6, 0.0021 Map3k7ip1, Slit2, Nrf2a, Runx1, Gata4, Sfrs1, BMPR1A synaptic plasticity Dmrta1, Dnmt1, Nr4a2, Irf6, Klf7, Foxp4, Pax9, Zic2, Mcm2 sååDouble-stranding DNA Bcl2, Drd2, Snap25, Cntn2, Notch1, Ptprz1, Igf1r, Slitrk6, Shootin1, Jund, Sp1, Hnf1a, Foxa2, Cebpg, Hmg1, Nr3c2, Gata6, Smad2, Tef, Neurod1, binding Axonogenesis 0,0068 Double-stranded DNA binding 0.0057 Map1b, Ntng1, Prg12,Catna2, Slitrk1, Ndn, Ntng2, Slit2, Klf7, Dclk1 Hif1a, Xrcc5, Purb, Rad512, Maf1, Neurog3, Dnmt1, Rfc1 Uba3, Bcl2, Lca, Rb1, Gadd45, Mnt, Gadd45g, Ccng2, Skp2,Sgk, Regulation of cell cycle 0,0085 Tbp, herp1, Egr2, Csx, Mrg1, Pax3, Six1, Ebf, Pou2f3, Arp2, Dbp, Ets1, Foxg1, Bop1, Bap1, Ccne2, , Pten, Ccnd1, Nucks, Rb2, Cdk4 Sequence-specifi c DNA binding Jund, Sp1, Hnf1a, Foxa2, Stat1, Stat3, Pgr, Hmg1, Msx2, Hnf1b, Smad1, Nr3c2, 0.0060 P2rx2, Stx1a, Nrxn3, Nrxn2, Cacnb2, Vglut1, Ap2b1, Cacn1c, Glut, activity Pparb, Lmx1a, Erf, Gata6, Neurod, Hdac12, Nmyc, Sox8, Znf287, Elk4, Irf9, Synaptic transmission 0,0142 Grm2, Grm4, Stx1b, Sstr1, Chat, Glur4, Glur7, Sstr2, Csp, Htr3 så Pituitary gland development Dmrt2, Meis2, Pbx3, Lhx6, Otx3, E2f6, Elk3, Klf7, Irf6, Neurog3, Bcl32, Sox11 Regulation of neuronal synaptic plasticity Egr2, Rnf39, Cntn2, Bcan, Snca, Cpeb1,Vgf, Bhlhb2 0,0143 så O r g a n m o r p h o g e n e s i s Calcium-transporting ATPase Atp2c1, Atp2b1, Atp2a3, Atp2b1, Atp2b4 0.0064 såHistone deacetylase binding activity Cytoplasmic mRNA processing body så B r a i n m o r p h o g e n e s i s såSyntaxin binding Dnch1, Limd1, Lsm14a 0,0241 SMAD binding Acvr1c, Foxa2, TGFßR1, TGFßR3, Rnf111, Smurf2, Purb, Axin, BMPR1A, Col3a1 0.0090 assembly Specifi c transcriptional repressor Pax3, Hnf1a, Ube2i, Hdac12, Akirin2, Fbxw11, Ncor2, Zpf57, Cbfa,. Purb, Eid1, såTranscription factor binding Negative regulation of interleukin-8 0.0105 IL6R, Bpi, Adam17 0,0241 activity Tcf7l2, Axin1, Rfc1 såSMAD binding production Tbp, Bcl2, Csx, Mycd, Rb1, Sp1, Hnf1a, Vhl, Stat3, Cebpg, Hmg1, Ube2i, Parp1, Regulation of microtubule polymerization Clasp2, Fam33a, Ska1 0,0241 Transcription factor binding Lek1, Creg1, gata6, Smad2, Neurod1, Hif1a, Hdac12, Myst3, Sox17, Trib2, 0.0113 or depolymerization så Schwan cell differentiation Myod1, Runx2, med1, Dnmt1 Response to cortisol stimulus Igfbp7, Slit2,Slit3 0,0241 Type II transforming growth factor så Neuron differentiation TGFß3, TGFßr3, TGFß2 0.0191 Rhythmic excitation Edn1, Chat, Scn9a 0,0241 beta receptor binding Schwann cell differentiation Egr2, Nab2, Nab1 0,0241 så Cell morphogenesis involved Nerve growth factor binding Ngfr, Pace4, Furin, Nt3 0.0201 så Type II transforming growth Thiamine-containing compound metabolic in differentiation Slc19a2, Thtpa, Tpk1 0,0241 P2y6, Gir, Oldr1, Sstr3, Adra2b, Vegfr3, Par2, D1a, Ednra1, Grin2b, Grm1, Grm2, factor beta receptor binding process Receptor activity IL6Ra, Il9r, Acvr1c, Nrp1, Pdgfra, Adora3, Il7r, Gabra1, Gabra5, Sema4g, Atrn, 0.0221 så A x o n o g e n e s i s så Transforming growth factor Brain morphogenesis Gad1, Catna2, Bbs4, Bbs1, Pafah1b1 0,0243 Bcam, Sort1, Ptprr, Grid2, Cb2, BMPR1A, Gpr107 så Dendrite morphogenesis Herp1, Prkcl2, Mycd, Sp1, Gmnn, Nudt21, Hif1a, Hdac12, Cbx5, Kat2a, Nrip1, beta receptor binding Inhibition of adenylate cyclase activity Histone deacetylase binding 0.0271 Ncor2, Dnmt1 så MHC class I protein binding by metabotropic glutamate receptor Grm2, Grm3, Grm4, Glur7 0,0267 signaling pathway Trprp, Vegfc, Il2, Igf1, TGFa, INHßB, Hdgfrp3, Ambn, Lep, Il7, TGFß3, Nodal, Artn, så Nuclear Growth factor activity 0.0325 Regulation of phosphoprotein Lefty1, Ctgf, Bmp8a, Gdf10, TGFß2, Bmp7, Vegfb Ppp1r2, Drd2, Rcan1, Hsp90b1 0,0267 binding phosphatase activity Syntaxin binding Stxbp3, Vamp2, Cav1, Stxbp1, Nsf, Snap23 0.0346 så Growth factor activity Nsp, Map2k1, Gpc2, Atp2b2,Rb1, Casp3, Notch1, Ppia, Chat, Vgf, Pigt, så C h o l e s t e r o l e f fl u x Kcnh2, Kcnc3, Kcnb2, Kcn15, Kcnq2, Cacna1c, Cacnb2, Tpcn1, Scn2a1, Neuron differentiation 0,0405 så Ligand- dependent nuclear Narc1, Itm2c, Otx2, Bhlhb5, En2, Runx1, Rxrg, Gas7, Ptprr, Efna1 Voltage-gated ion channel activity Kcnc1, Kir2.4, Kcnv2, Scn11a, Kcng4, Kcna7, Shal, Kv4.3, Scn9a, Scn1a, 0.0442 så S t e r o l t r a n s p o r t Kir6.2 receptor binding Triglyceride biosynthetic process Acs3, Lpl, Acas, Dgat2, Cic1, Agpat9, Elovl3, Elovl4, Agpat1 0,0473 så Cholesterol transport Single-stranded DNA binding Csbp, Hmg1, Peo1, Sssb4, Purb, Rpa2, Rad512 0.0448 Dendrite morphogenesis Sdc2, Slc11a2,Catna2,Klf7,Dclk1 0,0496 MiRNA putative target genes validation

3

Fluoxetine * p< 0.05 vs vehicle * The TGFß signaling pathway is one of the most signifi cative Agomelatine pathways putatively modulated after both 3 and 7 days of * Putative miRNA target genes validated agomelatine treatment, and after 7 day of fl uoxetine treatment. 2 by means of qRT-PCR reactions. In the last few years, studies have suggested that members of * * Smad1, Smad2, TGFßR1, TGFß3, the TGFß pathway have a key role in the central nervous system * BMPR1A, Acvr1, INHßA, INHßB, IL6 as neurotrophic and neuroprotective factors. Recent fi ndings and IL6R belongs to the TGFß/BMP have also indicated that some of these are involved Fold change signaling pathway. in the modulation of both excitatory and inhibitory synaptic 1 transmission in the adult mammalian brain as well as in the GALR1 is a putative target gene of regulation of neurite outgrowth and synapse formation [6]. mir-291a-3p (similarly modulated by both drugs after 3 days of treatment). Highlighted some of the miRNA putative target genes modulated after agomelatine or fl uoxetine treatment: 0 A A R IL6 3/7 days agomelatine – orange; 7 days fl uoxetine – red; IL6 TGFß3 ACVR1 INHß INHßB GALR1 common to both treatments - yellow. SMAD1 SMAD2 TGFßR1 BMPR1

CONCLUSIONS såBoth ADs induce early and time-dependent modifi cations in rat hippocampal miRNome expression profi le suggesting that miRNAs could represent early mediators of antidepressants action såAgomelatine and fl uoxetine showed a different profi le of miRNome modulation: agomelatine induced more marked effects after 3 days, while fl uoxetine after 7 days så2 miRNAs (mir-291a-3p and mir-338) were similarly modulated after 3 days of treatment by both drugs, thus suggesting the presence of common targets sååBioinformatic analysis suggests a signifi cant modulation of pathways involved in epigenetic mechanisms (in particular for agomelatine treatment) after 3 days, whereas major involvement of mechanisms related to neuroplasticity and neuronal functions was found after 7 days of treatment with both drugs sååPreliminary results of qRT-PCR analysis after 3 days of treatment with both drugs has shown a signifi cant modulation, in line with miRNA regulation, of putative target genes like TGFß3, Inhibin ß-B, IL6 and its receptor, known for their role as neurotrophic and neuroprotective factors, modulating synaptic plasticity, cognition and affective behavior. Although further work is needed to get further insight, these results could be of help to better clarify agomelatine and fl uoxetine mode of action and further support the involvementTgfßr1, of miRNAs in the effects of antidepressant drugs

REFERENCES: [1] O’Carrol D, Schaefer A, Neuropsychopharmacology. 2013;38(1):39-54. Acknowledgements: This research has been supported by the Institut de Recherches Internationales Servier (IRIS), Suresnes, France, and by a [2] McNeill E, Van Vactor D, Neuron 75. 2012;363-379; [3] Tardito D, et al., Expert Opin Investig grant from Fondazione Cariplo (2009- 2701) to D. Tardito. Drugs. 2013;22:217-233; [4] Racagni G, et al.,World J Biol Psychiatry. 2011;12:574-587; Disclosure: M. Popoli received support and/or has consulted for Abiogen, GlaxoSmithKline, Merck Sharp and Dohme, Servier, Abbott and Fidia. [5] Corrada D, et al., Brief Bioinform. 2011;12:588-600; [6] Krieglestein, et al., Trends Neurosci. G. Racagni has scientifi c collaboration with, and is member of the scientifi c boards of, Eli Lilly, InnovaPharma, and Servier. The other Authors 2011:34:421-429 declare no confl ict of interest. P.2.a.006 Agomelatine and fluoxetine induce different and time-dependent modulation of rat hippocampal miRNome M. Seguini1, D. Tardito1, A. Mallei1, I. Merelli2, D. Corrada3, L. Milanesi2, G. Racagni1, M. Popoli1 1University of Milana Department of Pharmacological and Biomolecular Sciencesa Milana Italy 2National Research Councila Institute for Biomedical Technologiesa Segratea Italy 3University of Milano-Bicoccaa Department of Earth and Environmental Sciencesa Milanoa Italy

Background: MicroRNAs (miRNAs) are small, non-coding RNAs that regulate target-gene expression by translation, inhibition or mRNA degradation. Each miRNA can regulate hundreds of target mRNAs, thus playing a crucial role into signaling and network modulation in almost every cellular process, including neuronal development and homeostasis [1]. Recent investigations suggested that dysregulation in miRNA expression may be critical for mental disorders pathophysiology, including major depression, and psychotropic drugs action [2]. Aim of this study was to verify whether hippocampal miRNome expression profile could be affected by treatment for 3 or 7 days with two different antidepressants: the SSRI fluoxetine and agomelatine, a MT1/MT2 receptor agonist and 5-HT2C receptor antagonist [3].

Methods: Rats (9 for group) were treated with vehicle, agomelatine (40mg/kg/day i.p.) or fluoxetine (10mg/kg/day i.p.) for 3 or 7 days. MiRNA expression analysis was conducted in total hippocampus by q-RT-PCR with TaqMan Array rodent MicroRNA A+B Cards Set v3.0 (Life Technologies). Statistical analysis was carried out with the SAM software, v. 4.0, with FDR for multiple testing at <5a. In order to identify miRNA putative target genes and molecular pathways potentially involved, bioinformatic analyses were performed by integrating and filtering the results of different miRNA target prediction algorithms, followed by annotation analyses with Gene Ontology subcategories and KEGG pathways (exact Fisher test with p<0.05; and multiple testing correction).

Results: The expression analysis showed that hippocampal miRNome was significantly modulated by both drugs. After 3 days, while fluoxetine down-regulated the expression of 8 miRNAs, agomelatine induced a marked effect by modulating 34 miRNAs (6 up- and 28 down-regulated); interestingly, 2 miRNAs were similarly modulated by both antidepressants. A stronger effect of fluoxetine was found after 7 days of treatment, with 35 miRNAs modulated (28 up- and 7 down-regulated), while agomelatine modified the expression of 22 miRNAs (19 up- and 3 down-regulated), five of which were modulated also after 3 days of treatment. The bioinformatic analysis suggested that agomelatine, mainly after 3 days, could modulate pathways involved in epigenetic mechanisms, inflammation, and neuroplasticity among the others. The same analysis on miRNAs modulated by fluoxetine highlighted the possible involvement of mechanisms mainly related to neuronal and synaptic plasticity and neurotransmission. Interestingly, several target genes have been previously associated to both depression pathophysiology and antidepressant action. Experiments are in progress in order to validate some of the putative target genes, by means of mRNA/protein expression studies.

Discussion: Our results show that fluoxetine and agomelatine can induce early and time-dependent modifications in rat hippocampal miRNome, although with different effects. Indeed, main effects of agomelatine were found after 3 days of treatment, whereas after 7 days of treatment with fluoxetine. The bioinformatic analyses revealed that pathways involved in epigenetic mechanisms, inflammation, neuroplasticity and neurotransmission could be affected by the modulated miRNAs. Although further work is needed to get further insight, these results suggest that miRNA might be involved in the effects of antidepressants and may explain the early improvement of some symptoms with agomelatine.

1. McNeill, E., Van Vactor, D., 2012. MicroRNAs shape the neuronal landscape. Neuron 75, 363–379.

2. Tardito, D., Mallei, A., Popoli, M., 2013. Lost in translation. New unexplored avenues for neuropsychopharmacology: epigenetics and microRNAs. Expert Opin Investig Drugs 22, 217–233.

3. Racagni, G., Riva, M.A., Molteni, R., Musazzi, L., Calabrese, F., Popoli, M., Tardito, D., 2011. Mode of action of agomelatine: synergy between melatonergic and 5-HT2C receptors. World J Biol Psychiatry 12, 574–587.

Disclosure statement: This study was supported by a research grant from Servier.

Citation: Eur Neuropsychopharmacol. 2014;24aSuppl 2):S364

Keywords Antidepressants: basic Epigenetics Gene expression