6 ion channel inhibitors upregulates microRNA expression in a mouse model of chronic mild stress

Accepted Manuscript Title: Treatment with TREK1 and TRPC3/6 Ion Channel Inhibitors Upregulates microRNA Expression in a Mouse Model of Chronic Mild Stress ¨ Authors: ˙Ilay Buran, Ebru Onalan Etem, Ahmet Tektemur, Halit Elyas PII: DOI: Reference:

S0304-3940(17)30579-7 http://dx.doi.org/doi:10.1016/j.neulet.2017.07.017 NSL 32957

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Neuroscience Letters

Received date: Revised date: Accepted date:

26-4-2017 4-7-2017 12-7-2017

¨ Please cite this article as: ˙Ilay Buran, Ebru Onalan Etem, Ahmet Tektemur, Halit Elyas, Treatment with TREK1 and TRPC3/6 Ion Channel Inhibitors Upregulates microRNA Expression in a Mouse Model of Chronic Mild Stress, Neuroscience Lettershttp://dx.doi.org/10.1016/j.neulet.2017.07.017 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Treatment with TREK1 and TRPC3/6 Ion Channel Inhibitors Upregulates microRNA Expression in a Mouse Model of Chronic Mild Stress

İlay Burana , [email protected] Ebru Önalan Etema , [email protected] Ahmet Tektemura , [email protected] Halit Elyasa , [email protected]

Fırat University, Faculty of Medicine, Departmant of Medical Biology, 23000, Elazığ, Turkey a

Ebru Önalan ETEM Firat University, Faculty of Medicine, Department of Medical Biology,23119, Elazig/TURKEY e-mail: [email protected] Tlf: 090 4242370000/4656

Highlights

 

It is the first study to show that ion channel inhibitors known as norgestimate and anadamide have antidepressive effects. Ion channel inhibitors have been shown to increase miRNA very rapidly.

ABSTRACT Depression is a common mental disorder characterized by the mood of deep sadness. Recent studies have demonstrated that microRNAs and ion channels have significant roles in the etiopathogenesis of depression. Therefore, we investigated the effects of the TREK1 ion channel inhibitor anandamide and the TRPC3/6 inhibitor norgestimate on microRNA expression and antidepressant effect in the mouse chronic mild stress (CMS) model of depression. Male BALB/c mice were divided into groups as control, CMS, CMS+sertraline, CMS+anandamide, CMS+sertraline+anandamide, CMS+norgestimate and

CMS+sertraline+norgestimate. Forced swim test (FST) and Sucrose Preference Test (SPT) were utilized to assess depression levels. Anandamide and norgestimate were administered subcutaneously (5mg/kg/day), and sertraline was applied intraperitoneally (10mg/kg/day) for two days during FST. miRNA and ion channel gene expression levels in the prefrontal cortex were assessed with qRT-PCR. qRT-PCR results demonstrated that there was a significant increase in miR-9-5p, miR-128-1-5p, and miR-382-5p, and a significant decrease in miR-165p, miR-129-5p, and miR-219a-5p in the CMS group compared with the control group. Generally, anandamide and norgestimate significantly increased all miRNA expression. It was also determined that anandamide and norgestimate had an antidepressant action in FST when used alone and especially when used in conjunction with sertraline. Based on the study results, it could be argued that an increase in miR-9-5p and miR-128-1-5p, consistent with the literature, could play significant roles in the etiopathogenesis of depression. The antidepressant action of anandamide and norgesimate in FST showed for the first time that these inhibitors could be used as in conjuction with sertraline in depression treatment.

Key Words: TREK1 (Kcnk2), TRPC3/6, Depression, Chronic Mild Stress, Forced Swimming Test

1. Introduction Major depressive disorder (MDD) is a prevalent disease worldwide and has serious negative effects on quality of life [1]. The role of biogenic amine defects in the brain is well known in the etiopathogenesis of depression. However, there are several other obscure mechanisms, including changes in expression of ion channels and activity related to the disease. Calcium (Ca2+) and potassium (K+) channels and a wide range of cellular functions such as neural proliferation, development, learning, and memory are changed in depression [2,3]. Generally, stimulation of open potassium and calcium channels inhibits cellular

excitability, decreases exciter influx, and causes hyperpolarization of the cell membrane. Inhibition of these channels could have therapeutic effects for various diseases such as epilepsy, multiple sclerosis, dementia, anxiety, stroke, and depression [4]. Some preclinical studies have demonstrated that certain potassium channel inhibitors such as tetraethyl ammonium (TEA), apamin, charybdotoxin, gliquidone, glibenclamide, and the calcium

channel inhibitor nifedipine had strong antidepressant action in a forced swimming test (FST) [5]. TREK1 (Tandem of pore domains in a weak inward rectifying K + channel (TWIK)-

Related Potassium Channel 1) potassium channel and TRPC3/6 (The Canonical Transient Receptor Potential Channel 3/6) calcium channels are expressed especially in the prefrontal cortex and hippocampus regions of the brain [6,7,8]. Although some calcium and potassium ion channel inhibitors have been shown to have therapeutic effects in rodent depression models, the therapeutic effects of the TRPC3/6 inhibitor anandamide and the TREK1 inhibitor norgestimate have not been investigated. Additionally, there are a limited number of studies that examined the changes in TREK1 and TRPC3/6 gene expressions in depression. microRNAs (miRNA) are 18–24 nucleotide small non-protein-coding RNA molecules that regulate gene expression through translational repression or mRNA degradation by binding to complementary sequences in the 3’ untranslated region (UTR) of mRNA [9]. In brain samples from human depression patients and rodent depression models characterized by synaptic and cognitive function disorders, changes miRNA expression are observed [10]. The activation of various intracellular signal pathways by growth factors, neurotransmitters, inorganic ions, pH, oxygen, temperature and osmosis can cause sharp upregulation and downregulation of miRNA [11,12]. Recent studies determined that microRNAs could target various ion channel genes. There are no studies that research how miRNA synthesis is affected by ion channel activation changes induced by specific ion channel activators or inhibitors. The objective of the present study, planned in light of the above-mentioned information, was to scrutinize the effects of the TREK1 inhibitor anandamide and the TRPC3/6 inhibitor norgestimate on depressive behavior in mice, as well as to evaluate microRNA expression in the prefrontal cortex, in the depression model induced with chronic mild stress in mice. 2. Materials and Methods 2.1. Animals Experiments were performed on 3-month-old male BALB/c mice obtained from Fırat University Experimental Research Center. The study was initiated after approval by Fırat University Animal Experiments Ethical Committee Directorate (2013/07-103). Animals were housed in polycarbonate cages (50cm×30cm×20cm) under standard laboratory

conditions (22 ± 3°C, 12 h light/dark cycles) with ad libitum access to food and water. While the control group mice lived under the above conditions during the experiment, other groups underwent the treatments mentioned in the methodology section. Seven groups of seven mice each were designated. The groups were set as control (C), chronic mild stress (CMS), CMS+Sertraline (CMS+S), CMS+Anandamide (CMS+A), CMS+Sertraline+Anandamide (CMS+S+A), CMS+Norgestimate (CMS+N), and CMS+Sertraline+Norgestimate (CMS+S+N). Depression in mice was induced with chronic mild stress (CMS), and the forced swimming test (FST) and Sucrose Preference Test (SPT) were used in the assessment of depression level. 2.2. Drugs 1.5 mg of norgestimate (USP), a TRPC3/6 inhibitor, in powder form was dissolved in 600µl pure ethanol and 100µl corn oil mixture and administered to the animals in 5 mg/kg doses subcutaneously [13]. Anandamide (Cayman Chemical Company), a TREK1 inhibitor, was injected subcutaneously at 5 mg/kg doses after it was dissolved in 10% DMSO [14]. Sertraline HCl tablets (Lustral, Pfizer), an antidepressant used to treat depression, were powdered in a mortar and homogenized in 1:1 methanol/physiological saline solution; its pH was set at approximately 11.5 with 0.5 N NaOH. The suspension was stirred overnight and then applied intraperitoneally to the animals at a 10 mg/kg dose [15,16]. Each mouse weighed about 40 g. Physiological saline solution was administered to control group mice as placebo.

2.3.Chronic Mild Stress Chronic mild stress (CMS) is a highly reliable method commonly used in animal model studies [17]. There is no standard CMS protocol, and CMS has been implemented in different forms in different laboratories. In the present study, a CMS protocol was designed with various modifications based on the studies of Wilner [18]. To induce chronic stress, mice were exposed to 2-3 random stress factors daily for 7 weeks as indicated Table 1. 2.4. Sucrose Preference Test

The reduction in preference for sucrose in mice in which chronic mild stress was applied is interpreted as anhedonia, which is one of the basic symptoms of depression. Reduction of sucrose preference levels below 65% is considered as anhedonia [19]. Sucrose preference test was conducted before and after the 7-week chronic mild stress application in the study. For the test, 1% sucrose solution was prepared. Groups were administered 2 bottles of sucrose solution for 24 hours in the training phase. Later on, one of the bottles was replaced with water, and consumption of both water and sucrose solution were measured after 15 hours. The sucrose preference was calculated according to the following formula: Sucrose preference (SP) = sucrose intake (g)/(sucrose intake (g) + water intake (g)) × 100%. Test was initiated in the active phase, and water and food limitation was not implemented before the test [20].

2.5. Forced Swim Test In depression studies conducted with rodents, and especially in antidepressant treatment surveys, the commonly used animal model with the highest reliability is the forced swim test [21]. The procedures implemented in FST were designed based on the behavior test defined by Porsolt et al. [22]. A Plexiglas cylindrical container of 27 cm diameter and 50 cm height was filled with 24-26°C water until the water level reached 30 cm. The subjects were allowed to swim for 15 minutes the first day. Anandamide (5 mg/kg) and norgestimate (5 mg/kg) were administered subcutaneously, while sertraline (10 mg/kg) was administered intraperitoneally after the swimming test. The day after (24 hours later), second dose was administered to the subjects 5 hours before the swimming test, and the third dose was administered to the subjects 1 hour before the swimming procedure. The second and third doses were same as the dose administered the first day. The control group was injected with physiological saline solution at the same dose and times. After this application, mice were left for forced swimming for 6 minutes. Video was recorded to calculate the immobility (where the head was above the water level and the body was motionless), swimming, and climbing parameters. Records were scored for 5-second intervals for swimming, climbing, and immobility by a neutral observer [23].

Immediately after the behavior tests, the brains of the mice were rapidly opened up under anesthesia. Prefrontal cortex region was separated. Tissues were transported in a liquid nitrogen tank and stored at -80°C. 2.6. qRT-PCR Analysis RNA isolation from prefrontal cortex was performed with Tri Reagent (Bioshop, Canada). cDNA mix for gene expression analysis was prepared using Applied Biosystems High Capacity RNA-to-cDNATM Kit (Applied Biosystems, Foster City, CA, USA). cDNA mix for miRNA was prepared with Applied Biosystems TaqMan MicroRNA Reverse Transcription Kit. The mixture required for gene expression was prepared with Applied Biosystems TaqMan® Gene Expression Master Mix. Gene IDs are given in Table 2. Gene expression levels were measured with Applied Biosystems 7500 Real-Time PCR system. In the study, snoRNA-202 (for miRNAs) and GAPDH (for TRPC3/6 and TREK1) were used as control genes (housekeeping). At the end of RT-PCR analyses, 2-∆∆CT method was used to calculate the differences in gene expression. 2.7.Statistics In the statistical evaluation of the present study, IBM SPSS 22.0 (Statistical Package for the Social Sciences) package program (193.255.124.131) licensed by Fırat University was used. If data satisfied the suppositions of normality and homogeneity of parametric analyses (one-way independent measure ANOVAs and t-tests), variance was performed along with Tukey’s HSD post-hoc test if it was appropriate. Statistical significance level was set at p < 0.05. 3. Results

3.1. Sucrose Preference Test Findings Decrease of sucrose preference in mice is the most significant indicator of depressive behavior. In the present study, it was determined that there was no significant difference between depression and control groups before chronic mild stress (data not shown). However, at the end of the 7-week chronic mild stress application before drug treatment, a significant decrease in sucrose preference of the CMS group was observed when compared to the control group (p=0.00) (Fig 1).

3.2.Forced Swim Test Behavior Findings Swimming, climbing, and immobility durations (sec) of mice in each group were assessed in the forced swim test. While there was no significant difference between swimming behavior of control and CMS groups (p=0.44), there was a significant decrease in climbing behavior (p=0.001) and a significant increase in immobility times of the CMS groups (p=0.001). Comparison of the CMS and sertraline groups demonstrated that there was a significant increase in the sertraline group’s swimming behavior (p=0.003) and a significant decrease in immobility time (p=0.004). Comparing with the CMS group, there was a significant increase in climbing behavior of CMS+A, CMS+S+A, and CMS+S+N groups (p=0.4, p=0.013, p=0,001, respectively). Furthermore, it was determined that there was a significant decrease in immobility of CMS+S+A and CMS+S+N groups (p=0.01, p=0.001, respectively) (Fig 2). 3.3.miRNA, TRPC3/6, and TREK1 Expression Findings miRNA expression changes are given in Table 3. Comparison of miRNAs for control and CMS groups revealed that there were significant increases in miR-9-5p, miR-128-1-5p, and miR-382-5p, and significant decreases in miR-16-5p, miR-129-5p, and miR-219a-5p (p=0.01, p=0.001, p=0.005, p=0.001, p=0.001, p=0.003, respectively). There were no statistical differences in miR-30c-5p, miR-34a-5p, miR-132-3p, miR-139-5p, and miR-4975p between CMS and control groups (p>0.05). There was a significant decrease in miR-1295p (p=0.04) and a significant increase in miR-219a-5p (p=0.02) in CMS+S group compared to CMS group, while there were no significant changes in other miRNAs (p>0.05). Comparison of CMS and CMS+A groups demonstrated that there was a statistically significant increase in miR-16-5p, miR-30c-5p, miR-34a-5p, miR-128-1-5p, miR-129-5p, miR-132-3p, miR-139-5p, miR-219a-5p, miR-382-5p and miR-497-5p (p=0.007, p=0.027, p=0.002, p=0.024, p=0.011, p=0.016, p=0.008, p=0.007, p=0.008, p=0.008, respectively). There was a statistically significant increase in miR-16-5p, miR-30c-5p, miR-34a-5p, miR128-1-5p, miR-129-5p, miR-132-3p, miR-139-5p, miR-219a-5p, and miR-497-5p in CMS+S+A group comparing with CMS group (p=0.001, p=0.009, p=0.001, p=0.003, p=0.001, p=0.002, p=0.003, p=0.001, p=0.004, respectively). There was a statistically significant increase in miR-16-5p, miR-30c-5p, miR-34a-5p, miR-128-1-5p, miR-129-5p,

miR-132-3p, miR-139-5p, miR-219a-5p, and miR-497-5p in CMS+N group compared to CMS group (p=0.003, p=0.034, p=0.011, p=0.006, p=0.011, p=0.01, p=0.027, p=0.002, p=0.021, respectively). There was a statistically significant increase in miR-9-5p, miR-165p, miR-34a-5p, miR-128-1-5p, miR-129-5p, miR-139-5p, miR-219a-5p, and miR-497-5p in CMS+S+N group compared to CMS group (p= 0,001, p=0.001, p=0.004, p=0.001, p=0.001, p=0.003, p=0.001, p=0.002, respectively). No significant change between all groups was observed in TRPC3/6 and TREK1 ion channel expression (p >0.05).

Data are presented as mean ± standard deviation. 2-fold decreases or increases were considered significant (p<0.05). CMS: Chronic mild stress, S: Sertralin, A: Anandamide, N: Norgestimate. a: versus control; b: versus CMS; c: versus CMS+S; d: versus CMS+A; e: versus CMS+S+A; f: versus CMS+N

4. Discussion Chronic mild stress is a reliable animal model, often utilized in depression studies, where basic symptoms of depression could realistically be observed [24]. Three main findings were identified in the present study. First, there was a significant increase in miRNAs synthesized in the brain, such as miR-9-5p, miR-128-1-5p, and miR-382-5p, and a significant decrease in miR-16-5p, miR-129-5p, and miR-219a-5p; however, there was no significant change in certain miRNAs such as miR-30c-5p, miR-34a-5p, miR-132-3p, miR-139-5p, and miR-497-5p. It was determined that, especially for miR-9-5p and miR-128-1-5p levels, the increases were consistent with the reports in the literature [25,26,27]. Second, it was found that ion channel inhibitors generally increased miR-16-5p, miR-30c-5p, miR-34a-5p, miR128-1-5p, miR-132-3p, miR-139-5p, miR-219a-5p and miR-497-5p expression. Third, it was determined that the ion channel inhibitors anandamide and norgestimate utilized in this study had antidepressant action in the forced swimming test. Furthermore, it was identified that the antidepressant action significantly increased in FST in groups where sertraline and inhibitors were used together when compared to the groups where sertraline was used alone. Activation changes in potassium and calcium ion channels participating in cell signaling mechanisms cause various neurologic diseases including depression [7,28,29,30]. Smolin et al. [31] determined that there was an increase in KCNN3 and KCNS3 potassium channel expression in postmortem brain tissues of major depressive disorder (MDD) cases.

Chen et al. [32] reported that KCNB1 (another potassium channel) expression in a CMS rat model increased in frontal cortex region, and expression levels recover to normal after fluoxetine application. However, in our study, it was determined that TREK1, TRPC3, and TRPC6 ion channel expressions did not change significantly in CMS mice compared to control mice. It is possible that, beyond TRPC3/6 and TREK1 channel expression changes, the changes in their activities could have caused ion imbalances in neuron cells and thus could have resulted in differences in expression of certain microRNAs. miRNAs are expressed in neurons at high levels. Neuronal miRNA pathways could create an extremely powerful mechanism to regulate the protein content of dynamic neuronal activities without a need for new gene transcription [33,34]. Acute and chronic stress used to create depression in rodents have been found to cause miRNA expression changes. For instance, acute stress causes an increase in certain miRNAs (miR-9, miR-9*, miR-26b, miR29b, miR-30b, miR-30c, miR-30e, miR-125a, miR-126-3p, miR-129-3p, miR-207, miR-212, miR-351, miR-423, miR-487b, miR-494, miR-690, miR-691, miR-709, miR-711, and Let-7 a-e) in the prefrontal cortex region of mice [25]. Of acute and chronic stress in a rat study conducted by Meerson et al. [35] was demonstrated to cause the downregulation or upregulation of miR-134, miR-183, miR-132, Let-7a-1, miR-9-1 and miR-124a-1 in hippocampus region. Cao et al. [36] identified a decrease in miR-298, miR-130b, miR-135a, miR-323, miR-503, miR-15b, miR-532, and miR-125a, and an increase in miR-7a, miR-212, miR-124, miR-139 and miR-182 in the hippocampus of rats to which were applied chronic mild stress combined with separation. Among these miRNAs, miR-125a and miR-182 returned to their normal levels with traditional herbal antidepressant administration. In the present study, we demonstrated that upregulated miRNAs include miR-9-5p, mir-128-1-5p and miR-382-5p in CMS. miR-9 targets genes that are related to depression pathogenesis such as DRD2 (Dopamine Receptor D2), CLOCK (Clock Circadian Regulator), and MAP2K (Mitogen-activated protein kinase kinases) [37]. miR-128-1-5p targets ERK2 (Extracellular Signal-Regulated Kinase2), which is known to be a significant signaling pathway and plays a role in depression pathogenesis. Dwivedi et al. [38] observed that ERK activity decreased in certain regions of the brains of depression patients. miR-382-5p targets BDNF (Brain Derived Neurotrophic Factor), which is known to play a crucial role in depression pathogenesis. It was considered possible that upregulation of miR-382-5p could contribute to mechanisms responsible for BDNF levels that decrease with depression [39]. In our study, it was observed that there was a significant decrease in miR-16-5p, miR-129-5p and mir-219a-

5p expression in CMS. miR-16 targets SERT (Serotonin transporter), 5-HTR2A (5Hydroxytryptamıne Receptor 2A), BDNF, GRM7 (Glutamate Metabotropic Receptor 7), and MAP2K1, which play roles in depression [37,40]. In particular, since high miR-16 levels decrease BDNF expression, depression-like results could be expected. However, in the present study and similarly in a study performed by Bai et al. [41], an increase in miR-16 levels was not observed in CMS. miR-129-5p, which displayed a significant decrease in the present study, targets CAMK2A, which increases in depression [42]. Similarly, miR-219a-5p, which displayed a significant decrease in our study, targets CAMK2G, which is known to play a role in antidepressant activity and depression pathogenesis [43,44]. (Prediction of mentioned miRNA targets have been determined using TargetScanS and miRDB online database). The effects of certain miRNAs on ion channel synthesis have been demonstrated. In a study conducted by Luo et al. [45], it was determined that miR-1 downregulation increased CACNB2 protein expression levels in atrial fibrillation patients. In the same way, in a study conducted with rats that induced neuropathic chronic pain model, it was observed that miR103 regulated CACNA1C expression [46]. In another study conducted by Parthasarathy et al. [47], it was identified that extreme miR-16 expression resulted in a decrease in ENaC

(Epithelial sodium channel) levels in patients with acute lung damage. Similar studies to those mentioned above focused on the effects of miRNAs on ion channels. The most significant data obtained in the present study was that norgestimate and anandamide upregulated miRNA expression levels. It is quite difficult to explain the mechanism that causes increases in microRNAs, especially in the groups given ion channel inhibitors, since there are a very limited number of studies in the field. In a study by Rüegger et al. [48], it was determined that miRNA expression decreased when epithelial growth factors were applied to the MCF10A cell lines. Krol et al. [49] reported that miR-204 and miR-211 expression decreased when transformed from light to darkness in rat retina. When serotonin was administered to Aplysia californica, a water snail, a reduction in miR-124 and miR-184 levels was observed [49]. Several pathologic heart disease studies reported that ion dynamics disorders resulted in an increase in miR-195 and a decrease in miR-133 and miR-1 [50,51]. It is clear that different molecules such as growth factors and neurotransmitters in the natural microenvironment of cells may change miRNAs expression levels [52]. It is possible that ion channel inhibitors administered as a treatment in the CMS mouse model could cause

irregularities in ion flow, contributing to microenvironment changes and affecting miRNA transcription. Recent studies demonstrated that the TREK-1 ion channel could be a target for a new antidepressant drug and bolster antidepressant applications. Although there are no studies that have investigated the effects of the TREK-1 channel inhibitor anandamide in the CMS mouse model, there are numerous studies on K+ channel antidepressant activities, and the antidepressant activity of different K+ channel inhibitors was demonstrated. Heurteaux et al. [28] stated that TREK-1 knock-out mice resisted depression. Consistent with this finding, it

was determined that fluoxetine inhibited TREK-1 flow and created antidepressant response [6,53]. In a study conducted by Tsai [54], it was determined that TREK-1 inhibition decreased

corticosterone levels under stress in mice, and the behavior of the mice was similar to mice under antidepressant administration. In a study conducted by Takahashi et al. [55], K+ channel blockers such as glibenclamide, charybdotoxin, and apamin were shown to have antidepressant-like effects in FST. It was also identified that the therapeutic effects of antidepressants such as fluoxetine, desipramine, amitriptyline, nortriptyline, clomipramine, maprotiline, citalopram, and paroxetine are displayed through K+ flow inhibition. Combined folic acid (antidepressant agent) and K+ channel blocker application in active doses to mice created an antidepressant-like effect in FST. In mice that were administered cromakalim, a channel opener, to cement the hypothesis, it was observed that it increased the reduction of immobility time in FST [55]. In the present study, in the K+ channel inhibitor anandamide administered groups, an increase in climbing behavior and a decrease in immobility time were identified. This finding demonstrates that, similar to other K+ channel inhibitors, anandamide caused an improvement in FST and had antidepressant action. Similar to K+ channels inhibitors, calcium channel inhibitors have a significant role in treatment of mood disorders. Interruption of intracellular Ca+2-calmodulin-NOS-guanylate cyclase signaling pathway could create antidepressant-like effects [56]. In the study by Aburawi et al. [57], it was determined that nifedipine, which is a calcium channel blocker, delayed immobility initiation in forced swimming test. It was realized that this antidepressant action occurred via 5-HT1A activation. In the same study, it was shown that verapamil, which is another calcium channel blocker that delays the onset of immobilization in forced swimming test, has antidepressant-like action. In our study, it was determined that the

inhibitor of TRPC3/6 calcium channels norgestimate showed antidepressant activity by increasing climbing behavior. 5. Conclusion In our study, we investigated effects of TREK1 inhibitor anandamide and the TRPC3/6 inhibitor norgestimate on microRNA expression and their therapeutic role in a depression model generated by chronic mild stress in mice. In particular, norgestimate has provided a remarkable reduction in immobility behavior and is a potential therapeutic adjunct. In the present study, the other most important finding is that groups treated with ion channel inhibitors anandamide and norgestimate generally showed a significant increase in miRNA levels compared to other groups with CMS. As a result, TRPC3/6 and TREK-1 ion channels can represent powerful new targets for antidepressant medications. The planning of new studies to be performed in this area, especially for the demonstration of similar activities of different ion channel inhibitors, will complement the information deficiencies in this area and confirm the findings of our study.

Conflict of Interest There is no conflict of interest.

6. Acknowledgment This study was supported by Fırat University Scientific Research Projects Coordination Unit. (Project No: TF.13.58)

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Fig 1: Comparison of percentage sucrose preference of control (n=7) and CMS groups (n=42). The preference index for sucrose was significantly higher in control than in CMS groups. Results are expressed as mean ± standard deviation (p < 0.05).

Fig 2: Swimming, climbing, and immobility times (sec) in the forced swim test of control and all CMS groups. Data are presented as mean ± standard deviation. CMS: Chronic mild stress, S: Sertraline, A: Anandamide, N: Norgestimate. a: versus control; b: versus CMS; c: versus CMS+S; d: versus CMS+A; e: versus CMS+S+A; f: versus CMS+N

Table 1: Schedule of CMS Stress factor

Monday

Thuesday

07.00pm Overnight illumination 07.00am Exposure to a foreign object Food deprivation

Wednesday Thursday

Friday

Saturday

Sunday

→ 07.00am → 07.00am

08.00am08.00pm 06.00am06.00pm + 06.00pm07.00pm

Water deprivation + empty bottle Damp sawdust

06.00am08.00pm 08.00pm 08.00pm

Isolation Exposure to light (2000 lx)

09.00pm12.00pm

→

01.00pm03.00pm

Exposure to 10.00amdark 01.00pm

09.00am05.00pm

Cage tilt 45◦ Tail suspansion White noise (80 dB)

06.00pm06.05pm 02.00pm03.00pm

01.00pm02.00pm

10.00am11.00am

Table 2: Assay ID of the miRNAs and ion channel genes analyzed by quantitative RT-PCR (Applied Biosystems, Foster City, CA, USA). miRNA and Ion Channel Name

Assay ID

mmu-miR-9-5p mmu-miR-16-5p mmu-miR-30c-5p mmu-miR-34a-5p mmu-miR-128-1-5p mmu-miR-129-5p mmu-miR-132-3p mmu-miR-139-5p

000583 000391 000419 000426 464067_mat 000590 000457 002289

mmu-miR-219a-5p mmu-miR-382-5p

000522 000572

mmu-miR-497-5p snoRNA202 TRPC3 TRPC6 TREK1 GAPDH

001346 001232 Mm00444690_m1 Mm01176083_m1 Mm01323942_m1 Mm99999915_g1

Table 3: miRNA, TRPC3, TRPC6, and TREK1 expression fold changes

Mice miRNAs

miR-9-5p

miRNA Relative Fold Changes

CMS

CMS+S

CMS+A

CMS+S+A

CMS+N

CMS+S+N

2.8 a

1.5 b

3.72 ac

4.19 ac

2.03 e

3.8 abcdef

p value 0.00 (F=144.8) 0.00

miR-16-5p

0.13 a

0.24 ab

0.43 ab

1.53 bcd

0.67 abce

3.1 abcdef (F=367.5) 0.00

mir-30c-5p

0.68

0.59 b

1.5 bc

2.11 abc

1.4 bc

2.6 adef (F=10.6) 0.00

miR-34a-5p

0.63

0.92 b

3.3 abc

3.83 abc

1.77 bce

4.2 abcdf (F=28.7) 0.00

miR-128-1-5p

10.9 a

12.0 ab

24.14 abc

24.9 abc

35.3 abc

45.2 abcdef (F=273.6) 0.00

miR-129-5p

0.13 a

0.2 a

0.37 ab

4.7 abcd

1.38 bcde

8.8 abcdef (F=402.5) 0.00

miR-132-3p

1.78

2.8 ab

4.46 ab

9.38 abcd

5.1ab

3.59 aef (F=21.5) 0.00

miR-139-5p

1.16

1.38 b

3.46 abc

5.12 abc

2.5 abe

4.2 abcdef (F=519.7) 0.00

miR-219a-5p

0.21 a

0.55

0.75 b

3.45 abcd

1.71 bcde

6.29 abcdf (F=37.5)

miR-382-5p

3.67 a

4.78 ab

11.3 abc

3.86 ad

2.9 ad

11.3 ad

0.00

(F=20.5) 0.00 miR-497-5p

0.75

1.19 b

2.27 abc

3.17 abc

1.75 b

4.09 abf (F=13.2)

Ion Channels

TRPC3, TRPC6, TREK1 Relative Fold Changes

TRPC3

1.49

0.240 1.44

1.73

1.06

1.47

1.27 (F=1.5) 0.152

TRPC6

0.87

0.83

0.8

0.6

0.59

0.59 (F=1.8) 0.082

TREK1

0.68

0.59

1.0

1.28

1.42

1.13 (F=2.4)