ABSTRACT
Title
Analysis of MiRNome expression profiles in hippocampus of rats treated with different antidepressants
Authors
M. Pelizzari1, D. Tardito1, A. Mallei1, S. Corna1, G. Treccani1, L. Bocchio-Chiavetto2, G. Racagni1,2, M. Popoli1
1 Center of Neuropharmacology, Dept. of Pharmacological Sciences, University of Milano, Italy
2 Neuropsychopharmacology Unit, IRCCS San Giovanni di Dio Fatebenefratelli, Brescia, Italy.
1 Center of Neuropharmacology, Dept. of Pharmacological Sciences, University of Milano, Italy
2 Neuropsychopharmacology Unit, IRCCS San Giovanni di Dio Fatebenefratelli, Brescia, Italy.
Abstract
Major Depression (MD) is a severe and chronic mental disorder that represents one of major causes of disability worldwide. Less than 50% of all patients treated with the currently available antidepressants (AD) show full remission and the therapeutic effects are observed after chronic administration. Despite the clear need for better therapies, recent efforts to develop novel AD have been relatively unsuccessful. A main reason for this is the still the incomplete knowledge of the pathogenetic mechanisms and understanding of AD mechanisms of action. MicroRNAs (miRNAs) have recently emerged as key regulators of complex time and spatial patterns of gene/protein expression changes. Converging evidence has suggested that the expression of the miRNome (the complex of all miRNAs expressed in a tissue at a given time) is highly regulated in a time- and region-specific manner both in the central nervous system and in peripheral tissues. It has also been shown that the expression and the function of brain miRNAs is influenced by external cues, including pharmacological agents. Pilot studies reported alterations in the miRNAs regulation in psychiatric disorders, such as schizophrenia and bipolar disorder. Moreover, it has been reported that some miRNAs and their effectors are modulated by the mood stabilizers lithium and valproate and that fluoxetine modulates miR-16 expression (Zhou et al., 2009; Chen et al., 2009; Baudry et al., 2010). However, up to date no studies have explored the effects of different ADs on the miRNome in rat hippocampus.
On the basis of this rationale the aim of the study was to analyze the modulation of the miRNome expression in hippocampus of rats after treatment with two ADs endowed with different primary mechanism of action: a selective serotonin reuptake inhibitor (SSRI), fluoxetine (FLX), and a tricyclic antidepressant with predominant action on the noradrenaline reuptake, desipramine (DMI). Both AD (10 mg/Kg) or vehicle (sterile water) were administered by i.p. injection. In order to evaluate the time course of AD effects, rats were treated for different time length: 3, 7 and 14 days. The Forced Swimming Test (FST) was used to assess the behavioral response to ADs. FST was carried out on the last two days of treatment.
Total RNA including miRNAs was isolated from each hemi-hippocampus using mirVana miRNA Isolation Kit and reverse transcription (RT) reaction was performed using the TaqMan MicroRNA Reverse Transcription Kit and the Megaplex™ RT Primers (Applied Biosystem). Quantitative Real Time PCR (qRT-PCR) amplification was carried out using TaqMan Array rodent MicroRNA A+B Cards Set v3.0 using the comparative CT (δδCT) method on Applied Biosystems Fast 7900HT. The array enables quantitation of gene expression levels of up to 380 miRNAs and controls.
The analysis of immobility in the Porsolt test showed a significant effect of AD treatments. Indeed, the two-way ANOVA showed a significant effect of treatment
(F2,71 = 14.15; p < 0.0001), but not of time or interaction. DMI, but not FLX, significantly reduced the immobility after all time points of treatment
The analysis of miRNome expression profiles after AD treatments showed evidence for significant effects of AD at the different time points assessed. A bioinformatic analysis (Integromics’ RealTime StatMiner) of the data obtained allowed the identification of a list of miRNAs differentially expressed as a result of the different drugs and lengths of treatment. The results of this work demonstrated that AD treatments induce significant changes in the miRnome and, because a single miRNA may affect the expression of hundreds of genes, suggest a way whereby the changes in pathways and effectors induced by these drugs may be faithfully described.
On the basis of this rationale the aim of the study was to analyze the modulation of the miRNome expression in hippocampus of rats after treatment with two ADs endowed with different primary mechanism of action: a selective serotonin reuptake inhibitor (SSRI), fluoxetine (FLX), and a tricyclic antidepressant with predominant action on the noradrenaline reuptake, desipramine (DMI). Both AD (10 mg/Kg) or vehicle (sterile water) were administered by i.p. injection. In order to evaluate the time course of AD effects, rats were treated for different time length: 3, 7 and 14 days. The Forced Swimming Test (FST) was used to assess the behavioral response to ADs. FST was carried out on the last two days of treatment.
Total RNA including miRNAs was isolated from each hemi-hippocampus using mirVana miRNA Isolation Kit and reverse transcription (RT) reaction was performed using the TaqMan MicroRNA Reverse Transcription Kit and the Megaplex™ RT Primers (Applied Biosystem). Quantitative Real Time PCR (qRT-PCR) amplification was carried out using TaqMan Array rodent MicroRNA A+B Cards Set v3.0 using the comparative CT (δδCT) method on Applied Biosystems Fast 7900HT. The array enables quantitation of gene expression levels of up to 380 miRNAs and controls.
The analysis of immobility in the Porsolt test showed a significant effect of AD treatments. Indeed, the two-way ANOVA showed a significant effect of treatment
(F2,71 = 14.15; p < 0.0001), but not of time or interaction. DMI, but not FLX, significantly reduced the immobility after all time points of treatment
The analysis of miRNome expression profiles after AD treatments showed evidence for significant effects of AD at the different time points assessed. A bioinformatic analysis (Integromics’ RealTime StatMiner) of the data obtained allowed the identification of a list of miRNAs differentially expressed as a result of the different drugs and lengths of treatment. The results of this work demonstrated that AD treatments induce significant changes in the miRnome and, because a single miRNA may affect the expression of hundreds of genes, suggest a way whereby the changes in pathways and effectors induced by these drugs may be faithfully described.