ARTICLE Increased Phosphorylation of CREB at Ser133 in the Dentate Gyrus Reverses Depressive Behavior in Rodents

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1 The HMB300H1S Journal, Winter 2015, 4(1):A1-A4 ARTICLE Increased Phosphorylation of CREB at Ser133 in the Dentate Gyrus Reverses Depressive Behavior in Rodents Ariba Alam 1 1 Human Biology Program, University of Toronto, Toronto, CA. Submission ID: (turnitin) Depression is a mental disorder that impair an individual s ability to concentrate resulting in individuals feeling hopeless for short or long periods of time. According to the World Health Organization, depression by 2030 is hypothesized to be the second leading cause of disability in the world. In 2012, there have been 350 million cases reported globally of people that suffer from depression. Fortunately, there is research going on in this field and this disorder has both psychosocial treatment as well as treatment by medicine. Depression is studied in labs through use of behavioral paradigms such as unpredicted chronic mild stress (UCMS), tail suspension test (TST), sucrose preference test, or forced swim test (FST). These studies induce stress on the animal by a foot shock or social isolation to induce depression like behaviors on the rodent. These depressive symptoms are reversed by the administration of antidepressants that target the CREB- BDNF pathway which essentially lead to the overexpression of CREB in the hippocampus. Many studies have reported the dentate gyrus to be the most involved in behavioral symptoms that rodents experience, and CREB upregulation in this part of the hippocampus has reversed depressive behavior the most compared to other areas of the hippocampus such as CA3 and CA1 pyramidal cells. CREB is a protein that is integrated in a pathway of other factors involved such as BDNF, Trk receptor, kinases that must be functional in order for CREB to be expressed in the hippocampus. Although the use of antidepressants that target upregulation of CREB-BDNF pathway, the causality of depression still has to be determined. These proteins are correlated with depression but the cause is still unknown. There has to be further studies that explore the core role of these proteins that will lead to a better understanding of depression. Key words: Depression; camp response elementbinding protein (CREB); Brain Derived Neurotrophic Factor (BDNF); Dentate Gyrus; Hippocampus; Behavioral Test Paradigms; Antidepressent Drugs I.BACKGROUND Studies have provided evidence that stress induces depression like behavior in mice. Depression is implicated to be due to an imbalance of CREB in the brain, particularly in the hippocampus. Depression was thought to be based on the monoamine hypothesis where deficiency in serotonin or noradrenaline caused depressive behavior (Mutlu et al., 2014; Chen, Shirayama, Shin, Neve & Duman, 2001; Gronli et al., 2006; Gass & Riva, 2007). However, this hypothesis does not explain all components of depression therefore is not the sufficient model that is followed (Mutlu et al., 2014). Alternatively, a recent hypothesis based on the CREB BDNF pathway linked to depression specifically in the hippocampus is more commonly studied (Mutli et al., 2014; Chen, Shirayama, Shin, Neve & Duman, 2001; Gronli et al., 2006, Xiao et al., 2011; Gass & Riva, 2007). CREB is involved in neuronal plasticity and regulating transcription of genes that are associated with stress responses (Guan et al., 2013; Xiao et al., 2011; Gass & Riva, 2007). Stress prevents the phosphorylation of CREB (Ser133) in the dentate gyrus of the hippocampus. CREB therefore cannot act on the Brain Derived Neurotrophic Factor (BDNF) gene downstream in the pathway therefore preventing the expression of this gene. BDNF has the capability to phosphorylate CREB Ser133,that is further responsible for activating genes that code for cyclic AMP (Gronli et al., 2006; Xiao et al., 2011). Studies have also shown that knocking out one allele for BDNF does not cause depression, but if that is linked with an inhibitor for mitogen-activated protein kinase (MAPK/MEK) that also acts on CREB by phosphorylating it would cause depression in the animal (Duman, Schlesinger, Kodama, Russell & Duman, 2007). Therefore BDNF which aids neuron survival, synaptic transmission, and synaptic plasticity complements the functions of CREB. Post-mortem brains of depressed patients have showed decrease of CREB protein in the hippocampus (Chen, Shirayama, Shin, Neve & Duman, 2001; Gass & Riva, 2007). This paper will focus on the expression of CREB protein in the hippocampus and it s correlation with depression like behavior in rodents. Primary Paper Introduction: A study done by Guan L., Jia X., Zhao X., Zhang X., et al. (2013) analyzed the expression levels of CREB/ERK/Bcl-2 in rat brains and their immobility time through a swimming test after experiencing prenatal stress (PS). These levels were measured in the hippocampus, prefrontal cortex and striatum. The prenatal stress that was given was social isolation. The motive of the article is to understand the role of the ERK-CREB pathway and its changes that occur after PS exposure and how that reflects in a swimming test.

2 These proteins were studied due to their important functions. CREB is involved in neuronal plasticity and regulating transcription of genes that are associated with stress response. ERK responds to extracellular stimuli by regulating gene expression, cellular growth, synthesis of new proteins in order to protect cells. Bcl-2 is regulated by CREB and is an anti-apoptotic factor. It is involved in regulating cell death, cell plasticity and growth of new neurons. ERK is activated by NMDA receptor excitation. MK-801 is a drug used in the study as it is been demonstrated to be affiliated with antidepressant properties. It is a non-competitive antagonist of NMDA receptor therefore blocks glutamate from binding to this receptor. Saline was used alongside to MK-801. Introduction to Additional Studies: Many sorts of behavioral paradigms have been used in studies that test for depression. The more frequently used include a tail suspension test, forced swimming test, sucrose preference, and unpredicted chronic mild stress (UCMS) or chronic mild stress (CMS). The unpredictable chronic mild stress model stresses out the animal that mimics natural stress which would parallel with human stress. These include food starvation, light-dark cycle altered, empty water bottle in their cage, etc. After exposing these mice to such stresses, they are given an antidepressant: agomelatine or melatonin for treatment (Mutlu et al., 2014). Another study used foot shocks and then was given imipramine, fluoxetine, isofluran gas, amitriptyline (AMI) or saline to see the effects on the rodent (Chen, Shirayama, Shin, Neve & Duman, 2001; Gronli et al., 2006; Gourley et al., 2008). One study compared the results between antidepressants and a transgenic experiment where they injected Herpes Simplex Virus carrying CREB in the dentate gyrus. They wanted to see the effect of overexpressing CREB has on the animal (Chen, Shirayama, Shin, Neve & Duman, 2001). The animal can also be treated with MK-801, fluoxetine, desipramine, tranylcypromine, reboxetine that fixes CREB levels in the hippocampus after the animal experiences stress (Guan et al., 2013; Tardito et al., 2009). Acupuncture studies have also reversed depression behavior in rodents there is also used in many studies that study CREB-BDNF pathway related to stress (Yang et al., 2013; Sun et al., 2014). After the animals have been exposed to atleast one of these treatments, their CREB and BDNF and ERK levels are measured in the hippocampus and compared with the control group that was given no stress or the saline group. The saline group is given stress, but administering saline does not help them with their depressive behavior, therefore it s represented as another reassurance of the experiment. Major results have indicated that these antidepressants indeed increase the levels of CREB, and BDNF, and ERK in the brain after these treatments and thus reversing depressive behavior. II. RESEARCH OVERVIEW SUMMARY OF MAJOR RESULTS Method/Experiment of Primary Paper: The method that was utilized by Guan L., Jia X., Zhao X., Zhang X., et al. (2013) consisted of mating sexually active male rats to females over night within a room temperature of degrees, humidity 60% and with a 12 hour day/night cycle (control group). Each pregnant rat was than separated into a control group, PS-Saline group, and PS- MK-801 group. The control group was not given any stress, but the other two groups were socially isolated 3 times a day for 45 minutes. The rats were given either saline or MK-801 on days of pregnancy. The researchers than did a forced swimming test (FST) on the offspring to see the effects of PS on them. Also, RNA was extracted from the three brain areas (hippocampus, striatum and prefrontal cortex) and went through an RT-PCR to examine the intensity of ERK, CREB, and Bcl-2 mrna expression in each. Results from Primary Paper: A2 The results from Guan L., Jia X., Zhao X., Zhang X., et al. (2013) showed PS rats showed an increased immobility time. There was lower expression of ERK2 mrna, CREB mrna, Bcl-2 mrna in the hippocampus, prefrontal cortex of PS rats with saline compared to control and MK-801 (modified the expression). The CREB levels were fixed by MK-801 as the levels of CREB were similar to that of the control even after experiencing stress prenatally. There was no significant difference seen in expression of mrna expression among the three proteins in the striatum. These changes were all reflected in the increased immobility timing correlated to depressive behaviour. Results from Additional Studies: The model size ranged from 9 (Chen, Shirayama, Shin, Neve & Duman, 2001; Gronli et al., 2006; Duman, Schlesinger, Kodama, Russell & Duman, 2007; Tardito et al., 2009) to 15 (Mutlu et al., 2014, Guan et al., 2013; Xiao et al., 2011). The largest experimental size used was 159 (Gourley et al., 2008). The studies showed a decrease in antidepressant behavior after administering an antidepressant such as Agomelatine (Mutlu et al., 2014). This antidepressant allowed the upregulation of BDNF- CREB in the hippocampus to decrease immobility time and also to increase memory of the animal (Mutlu et al., 2014). Another study injected a Herpes Simplex Virus (HSV)- CREB in the dentate gyrus of the hippocampus, which also resulted in less immobility time, and decrease in escape failures (Chen, Shirayama, Shin, Neve & Duman, 2001). This study compared the effect of antidepressants compared to a transgenic experiment where CREB was

3 The HMB300H1F Journal, Fall 2013, 3(1):A1-A4 A3 overexpressed. The results demonstrated that antidepressents decreased escape failures by 65% and CREB overexpression by 45% (Chen, Shirayama, Shin, Neve & Duman, 2001). However, in a swim test they demonstrated 35% reduced immobility time after administering imipramine in the dentate gyrus in comparison with 45% decrease in immobility time through CREB overexpression (Chen, Shirayama, Shin, Neve & Duman, 2001). Therefore, depending on what stress the animal was given, different techniques to treat the depression would result in better outcomes. Phosphorylation of Ser-133 on CREB in one study also demonstrated a higher preference for sucrose (Gronli et al., 2006). One study used the chronic unpredictable stress model which produced depressive behavior in mice. The results exhibited a 53% reduction of granule cell neurogenesis just after receiving seven days of stress, but this result was evident after a month of observation (Gronli et al., 2006). Although these studies used different methods, the end result was an increase in CREB expression that allowed the depressive behaviors to be reversed. binds to CRE on the DNA to activate BDNF is involved in depressive symptoms if down-regulated. CREB is phosphorylated by upstream factors that include Protein Kinase A, CaMKIV, and RSK proteins. The more these proteins are activated will allow this pathway to be expressed, and BDNF to get transcribed. Alterations in this pathway is correlated with depressive or antidepressive behavior. CONCLUSIONS AND DISCUSSION After inducing different types of stresses in rodents, and then administering antidepressants or transgenically overexpression of CREB in the hippocampus resulted in expected results of increased sucrose preference, decreased immobility time during swimming tests and decreased escape failures (Mutlu et al., 2014; Chen, Shirayama, Shin, Neve & Duman, 2001; Gronli et al., 2006; Guan et al., 2013; Xiao et al., 2011; Tardito et al., 2009; Yang et al., 2013). One common factor in all of these studies is the CREB-BDNF pathway that is upregulated specifically in the dentate gyrus which results in antidepressant behavior in rodents (Gass & Riva, 2007). Despite, the drugs improving the performance of mice in these various experiments does not provide information on how long these drugs can be dependent upon. Would the mice ever become unresponsive to them or would a higher dose have to be given to sustain the antidepressant behavior? Therefore, are these drugs only for short term use? The protein CREB that is upregulated is influenced by many factors such as camp, BDNF, and kinases that allow CREB to be phosphorylated (Gass & Riva, 2007). Although, the involvement of CREB is evident in depression, but the role of this protein still has to be determined especially in all areas of the hippocampus (Gass & Riva, 2007). Figure 1. This figure from the paper by Gass & Riva (2007) show the pathway involved in depressant behavior. Protein CREB that Figure 2 Figure 3

4 Figure 2. This figure from the paper by Chen, Shirayama, Shin, Neve & Duman (2001) show escape failures in rodents. In the control there are low chances of the rats to encounter escape failures. The saline treated rats that were given inescapable foot shock (IES) show higher levels of escape failures. Whereas, fluoxetine and imipramine treated rats after giving them inescapable foot shocks demonstrated a significant decrease in number of escape failures compared to ones treated with only saline (which is not an antidepressant). Figure 3. Rats exposed to inescapable foot shock (IES), and then microinjected with herpes simplex virus control (IES- HSV-LacZ) or HSV-CREB that allowed CREB to be overexpressed in the hippocampus and prefrontal cortex. Overexpression levels of CREB helped determine its involvement in decreasing escape failures or reversing depression symptoms. Different parts of the hippocampus such as dentate gyrus, CA1 pyramidal layer and prefrontal cortex were examined to determine any differences between the areas. The area that was the most significant in decreasing escape failures after overexpressing CREB using Herpes Simplex virus as a transgenic experiment was the dentate gyrus region of the hippocampus. However, the escape failures did not reduce at such a significant level in either the CA1 pyramidal layer or the prefrontal cortex. A2 The upregulation of CREB only in the dentate gyrus is seen to have antidepressant like effects, and not in the CA3 or CA1 pyramidal cells of the hippocampus or even the prefrontal cortex (Chen, Shirayama, Shin, Neve & Duman, 2001; Gronli et al., 2006; Xiao et al., 2011). Therefore, is it essential to know why CREB only acts in the dentate gyrus and how that influences the behavior of the rodent. Also, the role of this protein in the other regions of the hippocampus must also be determined to have a better understanding of depression. Knowing more about the projections that the dentate gyrus has would also help to determine where CREB gets transported to and if those areas have any involvement in altering behavior in mice. A future approach can be taken on determining the role of CREB in CA1 and CA3 pyramidal cells of the hippocampus, and also the mechanism involved in depression. Depressive behavior is examined when the CREB-BDNF pathway is down regulated in the dentate gyrus. When the protein CREB is upregulated in the dentate gyrus reverses depressive behavior observed in many behavioral model paradigms. These behavioral paradigms include a sucrose preference test, tail suspension test (TST), or chronic mild stress model where the rodents are exposed to stressful scenarios that induces depressive behavior. After observing depression in rodents, an antidepressant such as MK-801 or imipramine is injected into the rodent in the hippocampus which reverses depression like behavior. These drugs are observed to act on upregulating CREB in the dentate gyrus. Conclusions Depression in people is becoming more frequent in all parts of the world according to the World Health Organization (Blendy, 2006). There is a 10-30% risk of women being affected by this, and 7-15% for men both of which are probable to occur (Blendy, 2006). The primary approach taken to treat depression was through monoamines, or Selective Serotonin Reuptake Inhibitors (SSRI), however this approach is now not the ideal way of treating depression. The monoamine hypothesis is now replaced with a CREB-BDNF upregulation pathway being involved in depression (Blendy, 2006; Gass & Riva, 2007; Chen, Shirayama, Shin, Neve & Duman, 2001; Gronli et al., 2006; Schmidt, 2011). Through various behavioral models used such as sucrose preference test, inescapable foot shock, tail suspension test, immobility test in swimming that demonstrate depressed behavior are used in studies to study depression. The mechanism in all the studies presented are similar where the rodents are presented with stress initially and then treated with saline, or antidepressents. The pathway in the brain that is targeted is the CREB-BDNF pathway that is dependent on many transcriptional factors and upstream proteins such as TrkB receptors, CRE binding region, camp, and PKA (Gass & Riva, 2007). The protein that is essential for reversing depressive behavior is CREB protein, and the area that is involved in anti-depressive behavior significantly is the dentate gyrus. Criticisms and Future Directions The validity of these behavioral paradigms are questionable. Although they demonstrate behavioral symptoms in rodents, it does not necessarily parallel with human depression. Depression in humans can last for long periods or short periods depending on what the individual goes through in their life. Some humans are more susceptible to depression and some are not (Dzirasa & Covington, 2012). For instance, how does a tail suspension test (TST) or a sucrose preference test demonstrate anhedonia. Humans experience stress either early in life or later, but they are not in the form of foot shocks. Therefore, inducing foot shocks in rodents may alter different pathways in the animal compared to pathways that are altered by other forms of stress such as food starvation. Maybe the mechanism by which these mice are expressing depression symptoms is through a completely different pathway than what occurs in the brain. Therefore, mimicking the right type of stresses is essential. Moreover, upregulation of CREB has been helpful in reversing depressive symptoms, but this is not the cause of depression. CREB is a protein that is affected by another entity that yet has to be determined. The true function of this protein was not explored in all parts of the hippocampus, which is essential to understand the underlying cause of depression. Next, the temporal resolution or the amount of stress that is needed to start the production of these proteins was also not indicated in

5 The HMB300H1F Journal, Fall 2013, 3(1):A1-A4 A4 the study (Duman, Schlesinger, Kodama, Russell & Duman, 2007). It was not clearly identified to how long the rodent express antidepressant behavior after being administered the drug or overexpression of CREB in the dentate gyrus. For instance, after how much exposure to stress does the animal require to start upregulating these proteins or does the increased expression patterns of these proteins start at the same time the rat is exposed to even a little bit of stress? By looking at different approaches to resolve these questions will help gain a better understanding of the roles that these proteins play in depression and also contribute to a holistic understanding of this disease. The models used must also be easily transferred in clinical trials to improve the validity of the tests (Schmidt, 2011). Validity is conquered through integrating most essential features of depression in the animal model. Therefore, the model has to reflect practical scenarios and introduction to new models with predictive ability will aid in unveiling neurobiological mechanisms associated with depression. Furthermore, measuring guilt, mood or suicidal feelings in rodents is not easily determined, but for humans is easily equitable through questionnaires. Therefore, equating animal models to humans is very difficult. Another limitation in these studies is the effect of drugs on depressed individuals. In mice, acute administration of antidepressents has an effect on reversing depressive behavior. However, in humans, acute administration of drugs does not reverse depression in all individuals (Dzirasa & Covington, 2012). If the drug has an effect on an individual it would not be apparent until a couple of weeks of administration. Every individual is different with different responses to treatment therefore one form of model in rodents may not be enough to overcome this complex brain disorder. 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6 The HMB300H1F Journal, Fall 2013, 3(1):A1-A4 A5 April, 6, 2015 This work was supported by The Association for the Development of Undergraduate Neuroscience Education (SRA & RLN), The Endowment for Science Education (EA), and The Synaptic State Faculty Research Foundation (EA). The authors thank Mr. Spine L. Cord, Dr. Amy G. Dala, and the students in Neuroscience 101 for technical assistance, execution, and feedback on this lab exercise. Address correspondence to: Ariba Alam, Human Biology Department, University of Toronto, Toronto, CA Copyright 2015 Dr. Bill JU, Neurosciences, Human Biology Program