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P.4.021 MicroRNAs as novel antidepressant targets in refractory depression: converging effects of ketamine and electroconvulsive shock therapy
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Depression: towards new drug targets
in the left flank region with 0.15 ml of 0.3×106 viable S91 cells suspension on day 0 of the experiment. Fluoxetine − a selective serotonin reuptake inhibitor (10 mg/kg) − or saline were administered intraperitoneally (i.p.) daily by three weeks after tumor cells inoculation. Control animals (without tumor cells) received fluoxetine (10 mg/kg) or saline. First tumors appeared on day 12th of the experiment. Tumors were measured every second day, their diameters were assessed according to the formula (a×b×c)1/ 3 (a − tumor length, b − tumor width, c − tumor height). Animals were sacrificed on day 24th, and tumors weight was assessed. The level of IL-10 and IFN-g produced by unstimulated and stimulated (2.5 mg/ml concanavalin A − ConA) splenocytes was determined (ELISA assay). The results were statistically assessed by analysis of variance (ANOVA) with Scheffe post hoc test. Differences between means were considered significant if p < 0.05. The main observations of the present paper are: • chronic fluoxetine treatment after tumor cells inoculation inhibited solid S91 melanoma growth (ANOVA: F[2, 29] = 22.77, p < 0.001, post hoc analysis showed that fluoxetine significantly decreased tumor weight, by 84%, p < 0.001); • the production of IFN-g by Concanavalin A-stimulated splenocytes increased in tumor-bearing mice after fluoxetine treatment (ANOVA: F[2, 27] = 4.65, p < 0.02, post hoc analysis showed that fluoxetine significantly stimulated IFN-g production, by 30%, p < 0.03). Fluoxetine not only affects serotonin regulation, but also could have significant direct and indirect actions on tumor cells. As TNF-a, IL-6, IFN-g, and IL-10 synthesis depends on intracellular serotonin level [1], it is not surprising the observed increase in IFN-g level after fluoxetine treatment. It should be noted that IFN-g is a Th1 cytokine witch play a key role in generating antitumor responses, by activating natural killer cells, T-cytolytic cells and macrophages. Taking into account the above findings, as well as our previous [2] and other [3] studies it is postulated that fluoxetine via its cytotoxic abilities and modulation of the immune system is able to inhibit the progression of certain types of cancer. This study was supported by grant N40109732/2074 from Poland’s Ministry of Science and Higher Education. Reference(s) [1] Janssen, D.G., Caniato, R.N., Verster, J.C., Baune, B.T., 2010 A psychoneuroimmunological review on cytokines involved in antidepressant treatment response, Hum Psychopharmacol 25, 201–215. [2] Kirkova, M., Tzvetanova, E., Vircheva, S., Zamfirova, R., Grygier, B., Kubera, M., 2010 Antioxidant activity
of fluoxetine: studies in mice melanoma model, Cell Biochem Funct 28, 497–502. [3] Frick, L.R., Palumbo, M.L., Zappia, M.P., Brocco, M.A., Cremaschi, G.A., Genaro, A.M., 2008 Inhibitory effect of fluoxetine on lymphoma growth through the modulation of antitumor T-cell response by serotonin-dependent and independent mechanism, Biochem Pharmacol 75, 1817–1826.
P.4.021 MicroRNAs as novel antidepressant targets in refractory depression: converging effects of ketamine and electroconvulsive shock therapy R.M. O’Connor1 ° , T.G. Dinan2 , J.F. Cryan3 . 1 University College Cork, School of Pharmacy, Cork City, Ireland; 2 University College Cork, Psychiatry Alimentary Pharmabiotic Centre, Cork City, Ireland; 3 University College Cork, Anatomy and Neuroscience Alimentary Pharmabiotic Centre, Cork City, Ireland Depression is a devastating mental illness placing a severe burden on individuals and society. Moreover, the World Health Organisation predicts by the year 2020, depression will be the leading cause of disease burden in developed countries. Despite being of benefit to a large number of patients, current antidepressants are hampered by a slow onset of action, a significant percentage of nonresponders and side effects. Hindering the development of novel therapeutics is the fact that large gaps remain in our knowledge surrounding the molecular pathophysiology underlying depression and in the therapeutically relevant molecular mechanisms of antidepressants. Recently, acute administration of the NMDA receptor antagonist ketamine has been shown to induce a rapid and persistent antidepressant effect with increased efficacy in treatment-resistant depression [1]. Similar to ketamine, electroconvulsive shock therapy (ECS) is an antidepressant strategy which also is effective in cases of treatmentresistant depression [2]. The molecular mechanisms underlying the therapeutic action of these treatments are not fully understood. MicroRNAs (miRNAs) are a recently discovered small regulatory RNA species which negatively regulate mRNA translation in a sequence specific manner. They are increasingly seen as attractive drug targets given their ability to regulate multiple genes [3]. Moreover, they have been shown to be involved in a host of neuronal processes affecting behaviour and are downstream targets of several psychoactive drugs. Currently the effects of ketamine and ECS on miRNAs remains completely unexplored. We
Depression: towards new drug targets hypothesised that certain miRNAs would be differentially and others similarly regulated by ketamine and ECS. To this end male Sprague Dawley rats were administred 10 days ECS (0.85 mA/0.5 ms), acute ketamine (10 mg/kg i.p.) and for comparison chronic (21 days) treatment with the prototypical selective serotonin reuptake inhibitor (SSRI), fluoxetine (10 mg/kg/day i.p). 24 hours following final treatment the hippocampus was dissected out, RNA isolated and microarray based miRNA profiling was conducted. Fluoxetine, ECS and ketamine altered two, ten and fifteen hippocampal miRNAs respectively with all three antidepressant treatments sharing one common miRNA target, suggesting this may be an important molecular change involved the antidepressant response. Interestingly, ECS and ketamine possess the highest number of common miRNA targets, altering four common miRNAs in a similar manner. This indicates these therapies may mediate their therapeutic benefit through converging downstream molecular pathways. Interestingly, bioinformatic analysis revealed that the gene targets for these miRNAs are involved in a variety of processes including cell signalling and gene regulation. This study demonstrates changes to hippocampal miRNA expression may represent part of the therapeutic molecular mechanisms employed by antidepressants. Moreover, this is the first study to our knowledge which shows treatment with ketamine and ECS possess the capacity to alter miRNAs and interestingly do so in a convergent manner. This highlights miRNAs as novel targets for the development of novel antidepressants which have the potential to possess a more rapid therapeutic onset and greater efficacy. Reference(s) [1] Greenhalgh, J., Knight, C., Hind, D., Beverley, C., Walters, S., 2005. Clinical and cost-effectiveness of electroconvulsive therapy for depressive illness, schizophrenia, catatonia and mania: systematic reviews and economic modelling studies. Health Technol Assess 9, 1–156, iii-iv. [2] O’Connor, R.M., Dinan, T.G., Cryan, J.F., 2011. Little Things on Which Happiness Depends: microRNAs as Novel Therapeutic Targets for the Treatment of Anxiety and Depression. Molecular psychiatry In press. [3] Zarate, C.A., Jr., Singh, J.B., Carlson, P.J., Brutsche, N.E., Ameli, R., Luckenbaugh, D.A., Charney, D.S., Manji, H.K., 2006. A randomized trial of an N-methyl-D-aspartate antagonist in treatmentresistant major depression. Arch Gen Psychiatry 63, 856–864.
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P.4.022 Analysis of miRNome expression profiles in hippocampus of rats treated with antidepressants M. Pelizzari1 ° , D. Tardito1 , A. Mallei1 , S. Corna1 , G. Treccani1 , L. Bocchio-Chiavetto2 , G. Racagni1 , M. Popoli1 . 1 University of Milan, Dept. of Pharmacological Sciences, Milan, Italy; 2 IRCCS San Giovanni di Dio Fatebenefratelli, Neuropsychopharmacology Unit, Brescia, Italy Purpose: MicroRNAs (miRNAs) have recently emerged as key regulators of complex patterns of gene/protein expression changes in both the central nervous system and peripheral tissues. Pilot studies reported alterations in miRNAs regulation in psychiatric disorders, such as schizophrenia, bipolar disorder, and more recently also in depression [1]. It has also been shown that some miRNAs and their effectors are modulated by the mood stabilizers lithium and valproate and that fluoxetine (FLX, a selective serotonin reuptake inhibitor) modulates miR-16 expression [1]. On these basis the aim of the present study was to analyze the modulation of the miRNome expression in hippocampus of rats after treatment, for different time length (3, 7 and 14 days), with two antidepressants (ADs) endowed with different primary mechanism of action: the SSRI fluoxetine (FLX) and desipramine (DMI, a tricyclic antidepressant with predominant action on the noradrenaline reuptake). Methods: Total RNA including miRNAs was isolated from each hemi-hippocampus of AD and vehicle treated rats and reverse transcribed. Quantitative Real Time PCR (qRT-PCR) amplification was carried out using TaqMan Array rodent MicroRNA A+B Cards Set v3.0 using the ddCT method on Applied Biosystems Fast 7900HT. Bioinformatic analysis were performed in order to identify miRNA target genes and molecular pathways potentially altered by the expression of single or multiple miRNAs by means of the most known prediction tools for miRNA targets recognition: miRanda, TargetScan, DIANA-microT. Results: A mean of about 450 miRNAs were detected in all samples (mean Ct value <35). The expression analysis showed a significant time-associated effect of treatments. Although major changes were found after seven days of FLX or DMI treatment, the miRNome expression profiles were significantly modulated also after 3 and 14 days of treatment with both ADs. In particular, we found that 7 days of FLX treatment significantly increased the expression of 13 miRNAs (Fold Change 2) and downregulated 2 miRNAs (Fold Change 0.5), while DMI up-regulates 28 miRNAs and down-regulates 3 miRNAs.
Depression: towards new drug targets
in the left flank region with 0.15 ml of 0.3×106 viable S91 cells suspension on day 0 of the experiment. Fluoxetine − a selective serotonin reuptake inhibitor (10 mg/kg) − or saline were administered intraperitoneally (i.p.) daily by three weeks after tumor cells inoculation. Control animals (without tumor cells) received fluoxetine (10 mg/kg) or saline. First tumors appeared on day 12th of the experiment. Tumors were measured every second day, their diameters were assessed according to the formula (a×b×c)1/ 3 (a − tumor length, b − tumor width, c − tumor height). Animals were sacrificed on day 24th, and tumors weight was assessed. The level of IL-10 and IFN-g produced by unstimulated and stimulated (2.5 mg/ml concanavalin A − ConA) splenocytes was determined (ELISA assay). The results were statistically assessed by analysis of variance (ANOVA) with Scheffe post hoc test. Differences between means were considered significant if p < 0.05. The main observations of the present paper are: • chronic fluoxetine treatment after tumor cells inoculation inhibited solid S91 melanoma growth (ANOVA: F[2, 29] = 22.77, p < 0.001, post hoc analysis showed that fluoxetine significantly decreased tumor weight, by 84%, p < 0.001); • the production of IFN-g by Concanavalin A-stimulated splenocytes increased in tumor-bearing mice after fluoxetine treatment (ANOVA: F[2, 27] = 4.65, p < 0.02, post hoc analysis showed that fluoxetine significantly stimulated IFN-g production, by 30%, p < 0.03). Fluoxetine not only affects serotonin regulation, but also could have significant direct and indirect actions on tumor cells. As TNF-a, IL-6, IFN-g, and IL-10 synthesis depends on intracellular serotonin level [1], it is not surprising the observed increase in IFN-g level after fluoxetine treatment. It should be noted that IFN-g is a Th1 cytokine witch play a key role in generating antitumor responses, by activating natural killer cells, T-cytolytic cells and macrophages. Taking into account the above findings, as well as our previous [2] and other [3] studies it is postulated that fluoxetine via its cytotoxic abilities and modulation of the immune system is able to inhibit the progression of certain types of cancer. This study was supported by grant N40109732/2074 from Poland’s Ministry of Science and Higher Education. Reference(s) [1] Janssen, D.G., Caniato, R.N., Verster, J.C., Baune, B.T., 2010 A psychoneuroimmunological review on cytokines involved in antidepressant treatment response, Hum Psychopharmacol 25, 201–215. [2] Kirkova, M., Tzvetanova, E., Vircheva, S., Zamfirova, R., Grygier, B., Kubera, M., 2010 Antioxidant activity
of fluoxetine: studies in mice melanoma model, Cell Biochem Funct 28, 497–502. [3] Frick, L.R., Palumbo, M.L., Zappia, M.P., Brocco, M.A., Cremaschi, G.A., Genaro, A.M., 2008 Inhibitory effect of fluoxetine on lymphoma growth through the modulation of antitumor T-cell response by serotonin-dependent and independent mechanism, Biochem Pharmacol 75, 1817–1826.
P.4.021 MicroRNAs as novel antidepressant targets in refractory depression: converging effects of ketamine and electroconvulsive shock therapy R.M. O’Connor1 ° , T.G. Dinan2 , J.F. Cryan3 . 1 University College Cork, School of Pharmacy, Cork City, Ireland; 2 University College Cork, Psychiatry Alimentary Pharmabiotic Centre, Cork City, Ireland; 3 University College Cork, Anatomy and Neuroscience Alimentary Pharmabiotic Centre, Cork City, Ireland Depression is a devastating mental illness placing a severe burden on individuals and society. Moreover, the World Health Organisation predicts by the year 2020, depression will be the leading cause of disease burden in developed countries. Despite being of benefit to a large number of patients, current antidepressants are hampered by a slow onset of action, a significant percentage of nonresponders and side effects. Hindering the development of novel therapeutics is the fact that large gaps remain in our knowledge surrounding the molecular pathophysiology underlying depression and in the therapeutically relevant molecular mechanisms of antidepressants. Recently, acute administration of the NMDA receptor antagonist ketamine has been shown to induce a rapid and persistent antidepressant effect with increased efficacy in treatment-resistant depression [1]. Similar to ketamine, electroconvulsive shock therapy (ECS) is an antidepressant strategy which also is effective in cases of treatmentresistant depression [2]. The molecular mechanisms underlying the therapeutic action of these treatments are not fully understood. MicroRNAs (miRNAs) are a recently discovered small regulatory RNA species which negatively regulate mRNA translation in a sequence specific manner. They are increasingly seen as attractive drug targets given their ability to regulate multiple genes [3]. Moreover, they have been shown to be involved in a host of neuronal processes affecting behaviour and are downstream targets of several psychoactive drugs. Currently the effects of ketamine and ECS on miRNAs remains completely unexplored. We
Depression: towards new drug targets hypothesised that certain miRNAs would be differentially and others similarly regulated by ketamine and ECS. To this end male Sprague Dawley rats were administred 10 days ECS (0.85 mA/0.5 ms), acute ketamine (10 mg/kg i.p.) and for comparison chronic (21 days) treatment with the prototypical selective serotonin reuptake inhibitor (SSRI), fluoxetine (10 mg/kg/day i.p). 24 hours following final treatment the hippocampus was dissected out, RNA isolated and microarray based miRNA profiling was conducted. Fluoxetine, ECS and ketamine altered two, ten and fifteen hippocampal miRNAs respectively with all three antidepressant treatments sharing one common miRNA target, suggesting this may be an important molecular change involved the antidepressant response. Interestingly, ECS and ketamine possess the highest number of common miRNA targets, altering four common miRNAs in a similar manner. This indicates these therapies may mediate their therapeutic benefit through converging downstream molecular pathways. Interestingly, bioinformatic analysis revealed that the gene targets for these miRNAs are involved in a variety of processes including cell signalling and gene regulation. This study demonstrates changes to hippocampal miRNA expression may represent part of the therapeutic molecular mechanisms employed by antidepressants. Moreover, this is the first study to our knowledge which shows treatment with ketamine and ECS possess the capacity to alter miRNAs and interestingly do so in a convergent manner. This highlights miRNAs as novel targets for the development of novel antidepressants which have the potential to possess a more rapid therapeutic onset and greater efficacy. Reference(s) [1] Greenhalgh, J., Knight, C., Hind, D., Beverley, C., Walters, S., 2005. Clinical and cost-effectiveness of electroconvulsive therapy for depressive illness, schizophrenia, catatonia and mania: systematic reviews and economic modelling studies. Health Technol Assess 9, 1–156, iii-iv. [2] O’Connor, R.M., Dinan, T.G., Cryan, J.F., 2011. Little Things on Which Happiness Depends: microRNAs as Novel Therapeutic Targets for the Treatment of Anxiety and Depression. Molecular psychiatry In press. [3] Zarate, C.A., Jr., Singh, J.B., Carlson, P.J., Brutsche, N.E., Ameli, R., Luckenbaugh, D.A., Charney, D.S., Manji, H.K., 2006. A randomized trial of an N-methyl-D-aspartate antagonist in treatmentresistant major depression. Arch Gen Psychiatry 63, 856–864.
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P.4.022 Analysis of miRNome expression profiles in hippocampus of rats treated with antidepressants M. Pelizzari1 ° , D. Tardito1 , A. Mallei1 , S. Corna1 , G. Treccani1 , L. Bocchio-Chiavetto2 , G. Racagni1 , M. Popoli1 . 1 University of Milan, Dept. of Pharmacological Sciences, Milan, Italy; 2 IRCCS San Giovanni di Dio Fatebenefratelli, Neuropsychopharmacology Unit, Brescia, Italy Purpose: MicroRNAs (miRNAs) have recently emerged as key regulators of complex patterns of gene/protein expression changes in both the central nervous system and peripheral tissues. Pilot studies reported alterations in miRNAs regulation in psychiatric disorders, such as schizophrenia, bipolar disorder, and more recently also in depression [1]. It has also been shown that some miRNAs and their effectors are modulated by the mood stabilizers lithium and valproate and that fluoxetine (FLX, a selective serotonin reuptake inhibitor) modulates miR-16 expression [1]. On these basis the aim of the present study was to analyze the modulation of the miRNome expression in hippocampus of rats after treatment, for different time length (3, 7 and 14 days), with two antidepressants (ADs) endowed with different primary mechanism of action: the SSRI fluoxetine (FLX) and desipramine (DMI, a tricyclic antidepressant with predominant action on the noradrenaline reuptake). Methods: Total RNA including miRNAs was isolated from each hemi-hippocampus of AD and vehicle treated rats and reverse transcribed. Quantitative Real Time PCR (qRT-PCR) amplification was carried out using TaqMan Array rodent MicroRNA A+B Cards Set v3.0 using the ddCT method on Applied Biosystems Fast 7900HT. Bioinformatic analysis were performed in order to identify miRNA target genes and molecular pathways potentially altered by the expression of single or multiple miRNAs by means of the most known prediction tools for miRNA targets recognition: miRanda, TargetScan, DIANA-microT. Results: A mean of about 450 miRNAs were detected in all samples (mean Ct value <35). The expression analysis showed a significant time-associated effect of treatments. Although major changes were found after seven days of FLX or DMI treatment, the miRNome expression profiles were significantly modulated also after 3 and 14 days of treatment with both ADs. In particular, we found that 7 days of FLX treatment significantly increased the expression of 13 miRNAs (Fold Change 2) and downregulated 2 miRNAs (Fold Change 0.5), while DMI up-regulates 28 miRNAs and down-regulates 3 miRNAs.