Pharmacological Reports 67 (2015) 345–348
Contents lists available at ScienceDirect
Pharmacological Reports journal homepage: www.elsevier.com/locate/pharep
Short communication
Effect of desipramine on gene expression in the mouse frontal cortex – Microarray study Joanna Solich *, Magdalena Kolasa, Maciej Kus´mider, Agata Faron-Go´recka, Paulina Pabian, Kinga Szafran, Dariusz Z˙urawek, Marta Dziedzicka-Wasylewska Department of Pharmacology, Institute of Pharmacology Polish Academy of Sciences, Krako´w, Poland
A R T I C L E I N F O
Article history: Received 18 March 2014 Received in revised form 3 September 2014 Accepted 8 September 2014 Available online 22 September 2014 Keywords: Desipramine Gene expression Microarray Mice
A B S T R A C T
Background: These studies aimed to identify the genes differentially expressed in the frontal cortex of mice treated repeatedly with either saline or desipramine (DMI). Methods: Differences in gene expression in the mouse frontal cortex were studied using a wholegenome microarray approach. Results: The analyses revealed a group of 88 transcripts (18 genes) that were differentially expressed between the mice treated with saline and those treated with DMI. These genes include Spnb2, Mef2c, Ncam1, Hsp90ab1, Kif1b, Ddx6 and Gsk3b, which were connected in the gene relationship network. Conclusions: It appears that one week of DMI administration measurably altered the expression of a small number of genes, including genes connected with neuroplasticity and cytoskeletal changes, the regulation of calcium levels in the cell or translation processes. ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
Introduction Major depression is the most commonly observed psychiatric illness (more than 350 million people of all ages suffer from depression; WHO, 2012). Despite extensive studies, the etiology of the disease or the mechanism of action of antidepressant drugs is still not clear. The meta-analyses carried out to date have identified some genes associated with depression or antidepressant drugs (especially the selective serotonin reuptake inhibitors) as putative biomarkers of the disease or therapy [1– 4]. There were some genes connected with drug pharmacokinetics, the monoaminergic system, the stress hormone system, inflammation and neuroplasticity. Several genetic polymorphisms have been identified in genes coding for the cytochrome P450 (CYP) gene family, including CYP2D6, CYP2C19 and CYP1A2 [5]. It was also demonstrated that polymorphisms in genes connected with serotonergic and dopaminergic systems are associated with treatment response. These include the serotonin transporter gene (SLC6A4), tryptophan hydroxylase (TPH1), serotonin receptor genes 1B and 2A (HTR1B, HTR2A) and D4
* Corresponding author. E-mail address: [email protected] (J. Solich).
dopamine receptor (D4DR) [6–9]. Moreover, the norepinephrine transporter gene (SLC6A2) has been associated with the effects of antidepressant drugs [2]. Because the dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis is often observed during major depression [10], some genes related to the HPA axis and stress response, glucocorticoid receptor-a isoform (GRa), FK506 binding protein 51 (FKBP5) and corticotropin-releasing hormone (CRH) [11–13], were also studied in this context. Additionally, it is often postulated that inflammation affects the development of depressive symptoms. The genes coding cytokines were shown to be dysregulated during this illness – interleukin 1 beta (Il-1 b), macrophage migration inhibitory factor (MIF), tumor necrosis factor (TNFa), interleukin 6 (IL-6), interferon gamma (INF-g), as well as other genes like: cyclooxygenase-2 (COX-2), myeloperoxidase (MPO), nitric oxide synthase (iNOS), phospholipase A2 type IIA (sPLA2-IIA) and apolipoprotein E (ApoE) [14–16]. Major depression is also characterized by reduced neuronal plasticity, while antidepressant drugs normalize the condition of neurons. The genes that differ in depressed or antidepressant-treated patients from healthy controls were brain-derived neurotrophic factor (BDNF), VGF nerve growth factor (VGF), cAMP responsive element binding protein 1 (CREB1), glial cell derived neurotrophic factor (GDNF), artemin (ARTN) and neurotrophin 3 (NT-3) [6,17,18].
http://dx.doi.org/10.1016/j.pharep.2014.09.001 1734-1140/ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
346
J. Solich et al. / Pharmacological Reports 67 (2015) 345–348
Many investigations of transcriptional profiling in depressive or antidepressant-treated patients were performed using whole blood samples. The present study was performed directly in the mouse prefrontal cortex, which is an important region of the brain associated with stress disorders [19] and administration of antidepressant drugs. A previous study by Orsetti and colleagues [20] demonstrated a group of genes that changed during chronic mild stress in the frontal cortex. The results of their studies confirmed the downregulation of Itga6 (integrin, alpha 6), Camk2a (calcium/calmodulin-dependent protein kinase II, alpha subunit), Plcb1 (phospholipase C, beta 1), Cart (cocaine and amphetamine regulated transcript), Gad1 (glutamate decarboxylase 1), Homer1 (homer homologue 1) and Th (tyrosine hydroxylase), but Egr2 (early growth response 2) and Ptgs2 (prostaglandin-endoperoxide synthase 2) were upregulated. Ga˛ska and colleagues [21] also detected a group of genes altered in the rat prefrontal cortex following desipramine (DMI) administration, and found alterations in the expression of genes encoding adrenergic receptors, transcription factors (i.e., early response genes), neuropeptides and genes associated with stress. To further elucidate the effect of DMI on gene expression, the present study was aimed at identifying the genes that were differentially expressed in the frontal cortex of mice treated repeatedly with DMI. Materials and methods Animals Male C57Bl/6 mice were used. The animals had free access to food and water and were kept at constant ambient temperature (24 8C) under a 12-h light/dark cycle. The animals were maintained according to the policies of the Ethics Committee (No. 372; 710/ 2010). Microarray studies The mice were injected with desipramine (ip; 20 mg/kg; Sigma, USA) or saline (ip) once a day for 7 consecutive days. The drug was dissolved in saline. The dose of desipramine was chosen based on the results of previous studies [22,23]. Following 7 days of treatment (24 h after last injection), the mice were sacrificed, their brains were dissected, frozen on dry ice and stored at 80 8C until use. The frontal cortex, cut off the brain (Bregma 2.46 mm) was homogenized in TRI Reagent (Sigma, USA) with a Tissue Lyser (Qiagen, Germany). The RNA was purified with TRI Reagent and an RNeasy Plus Micro Kit (Qiagen, Germany) according to the manufacturer’s instructions. The amount of purified RNA was determined using a Nano-Drop Spectrophotometer ND-1000 (Thermoscientific, USA), and the quality was checked with an Automated Electrophoresis Station – Experion (Bio-Rad, USA) using an Experion RNA StdSens Analysis Kit (Bio-Rad) according to the provided procedure. The reverse transcription, labeling, and hybridization to the GeneChip Mouse Genome 430 2.0 Array were performed using the standard procedures provided by Asuragen (Austin, USA). The CEL files resulting from the Affymetrix Chip scanner were analyzed using ChipInspector (version 2.1; Genomatix). This program analyzes the expression level of a single probe. The statistical algorithm in ChipInspector is a T-test with a permuted artificial background and is based on the enhanced original SAM (Significance Analysis of Microarrays) algorithm. The analysis was performed according to the manufacturer’s recommendations. Positive delta was set 0.3 while negative delta was set 0.37. Positive FDR and negative FDR was set
Contents lists available at ScienceDirect
Pharmacological Reports journal homepage: www.elsevier.com/locate/pharep
Short communication
Effect of desipramine on gene expression in the mouse frontal cortex – Microarray study Joanna Solich *, Magdalena Kolasa, Maciej Kus´mider, Agata Faron-Go´recka, Paulina Pabian, Kinga Szafran, Dariusz Z˙urawek, Marta Dziedzicka-Wasylewska Department of Pharmacology, Institute of Pharmacology Polish Academy of Sciences, Krako´w, Poland
A R T I C L E I N F O
Article history: Received 18 March 2014 Received in revised form 3 September 2014 Accepted 8 September 2014 Available online 22 September 2014 Keywords: Desipramine Gene expression Microarray Mice
A B S T R A C T
Background: These studies aimed to identify the genes differentially expressed in the frontal cortex of mice treated repeatedly with either saline or desipramine (DMI). Methods: Differences in gene expression in the mouse frontal cortex were studied using a wholegenome microarray approach. Results: The analyses revealed a group of 88 transcripts (18 genes) that were differentially expressed between the mice treated with saline and those treated with DMI. These genes include Spnb2, Mef2c, Ncam1, Hsp90ab1, Kif1b, Ddx6 and Gsk3b, which were connected in the gene relationship network. Conclusions: It appears that one week of DMI administration measurably altered the expression of a small number of genes, including genes connected with neuroplasticity and cytoskeletal changes, the regulation of calcium levels in the cell or translation processes. ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
Introduction Major depression is the most commonly observed psychiatric illness (more than 350 million people of all ages suffer from depression; WHO, 2012). Despite extensive studies, the etiology of the disease or the mechanism of action of antidepressant drugs is still not clear. The meta-analyses carried out to date have identified some genes associated with depression or antidepressant drugs (especially the selective serotonin reuptake inhibitors) as putative biomarkers of the disease or therapy [1– 4]. There were some genes connected with drug pharmacokinetics, the monoaminergic system, the stress hormone system, inflammation and neuroplasticity. Several genetic polymorphisms have been identified in genes coding for the cytochrome P450 (CYP) gene family, including CYP2D6, CYP2C19 and CYP1A2 [5]. It was also demonstrated that polymorphisms in genes connected with serotonergic and dopaminergic systems are associated with treatment response. These include the serotonin transporter gene (SLC6A4), tryptophan hydroxylase (TPH1), serotonin receptor genes 1B and 2A (HTR1B, HTR2A) and D4
* Corresponding author. E-mail address: [email protected] (J. Solich).
dopamine receptor (D4DR) [6–9]. Moreover, the norepinephrine transporter gene (SLC6A2) has been associated with the effects of antidepressant drugs [2]. Because the dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis is often observed during major depression [10], some genes related to the HPA axis and stress response, glucocorticoid receptor-a isoform (GRa), FK506 binding protein 51 (FKBP5) and corticotropin-releasing hormone (CRH) [11–13], were also studied in this context. Additionally, it is often postulated that inflammation affects the development of depressive symptoms. The genes coding cytokines were shown to be dysregulated during this illness – interleukin 1 beta (Il-1 b), macrophage migration inhibitory factor (MIF), tumor necrosis factor (TNFa), interleukin 6 (IL-6), interferon gamma (INF-g), as well as other genes like: cyclooxygenase-2 (COX-2), myeloperoxidase (MPO), nitric oxide synthase (iNOS), phospholipase A2 type IIA (sPLA2-IIA) and apolipoprotein E (ApoE) [14–16]. Major depression is also characterized by reduced neuronal plasticity, while antidepressant drugs normalize the condition of neurons. The genes that differ in depressed or antidepressant-treated patients from healthy controls were brain-derived neurotrophic factor (BDNF), VGF nerve growth factor (VGF), cAMP responsive element binding protein 1 (CREB1), glial cell derived neurotrophic factor (GDNF), artemin (ARTN) and neurotrophin 3 (NT-3) [6,17,18].
http://dx.doi.org/10.1016/j.pharep.2014.09.001 1734-1140/ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
346
J. Solich et al. / Pharmacological Reports 67 (2015) 345–348
Many investigations of transcriptional profiling in depressive or antidepressant-treated patients were performed using whole blood samples. The present study was performed directly in the mouse prefrontal cortex, which is an important region of the brain associated with stress disorders [19] and administration of antidepressant drugs. A previous study by Orsetti and colleagues [20] demonstrated a group of genes that changed during chronic mild stress in the frontal cortex. The results of their studies confirmed the downregulation of Itga6 (integrin, alpha 6), Camk2a (calcium/calmodulin-dependent protein kinase II, alpha subunit), Plcb1 (phospholipase C, beta 1), Cart (cocaine and amphetamine regulated transcript), Gad1 (glutamate decarboxylase 1), Homer1 (homer homologue 1) and Th (tyrosine hydroxylase), but Egr2 (early growth response 2) and Ptgs2 (prostaglandin-endoperoxide synthase 2) were upregulated. Ga˛ska and colleagues [21] also detected a group of genes altered in the rat prefrontal cortex following desipramine (DMI) administration, and found alterations in the expression of genes encoding adrenergic receptors, transcription factors (i.e., early response genes), neuropeptides and genes associated with stress. To further elucidate the effect of DMI on gene expression, the present study was aimed at identifying the genes that were differentially expressed in the frontal cortex of mice treated repeatedly with DMI. Materials and methods Animals Male C57Bl/6 mice were used. The animals had free access to food and water and were kept at constant ambient temperature (24 8C) under a 12-h light/dark cycle. The animals were maintained according to the policies of the Ethics Committee (No. 372; 710/ 2010). Microarray studies The mice were injected with desipramine (ip; 20 mg/kg; Sigma, USA) or saline (ip) once a day for 7 consecutive days. The drug was dissolved in saline. The dose of desipramine was chosen based on the results of previous studies [22,23]. Following 7 days of treatment (24 h after last injection), the mice were sacrificed, their brains were dissected, frozen on dry ice and stored at 80 8C until use. The frontal cortex, cut off the brain (Bregma 2.46 mm) was homogenized in TRI Reagent (Sigma, USA) with a Tissue Lyser (Qiagen, Germany). The RNA was purified with TRI Reagent and an RNeasy Plus Micro Kit (Qiagen, Germany) according to the manufacturer’s instructions. The amount of purified RNA was determined using a Nano-Drop Spectrophotometer ND-1000 (Thermoscientific, USA), and the quality was checked with an Automated Electrophoresis Station – Experion (Bio-Rad, USA) using an Experion RNA StdSens Analysis Kit (Bio-Rad) according to the provided procedure. The reverse transcription, labeling, and hybridization to the GeneChip Mouse Genome 430 2.0 Array were performed using the standard procedures provided by Asuragen (Austin, USA). The CEL files resulting from the Affymetrix Chip scanner were analyzed using ChipInspector (version 2.1; Genomatix). This program analyzes the expression level of a single probe. The statistical algorithm in ChipInspector is a T-test with a permuted artificial background and is based on the enhanced original SAM (Significance Analysis of Microarrays) algorithm. The analysis was performed according to the manufacturer’s recommendations. Positive delta was set 0.3 while negative delta was set 0.37. Positive FDR and negative FDR was set
