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UNIVERSITY OF CALIFORNIA, SAN DIEGO

Molecular and Pharmacological Evidence for the Role of Glucocorticoid Receptors in

Alcohol Dependence

A Thesis submitted in partial satisfaction for the requirements for the degree

Master of Science

Biology

Lauren Grace Macshane

Committee in charge:

Professor George F. Koob, Chair Professor Eduardo R. Macagno, Co-Chair Professor Kathleen A. French

2014

Lauren Grace Macshane, 2014

The Thesis of Lauren Grace Macshane is approved, and it is acceptable in quality and form for publication on microfilm and electronically:

______

______Co-Chair

______Chair

University of California, San Diego 2014

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DEDICATION

This is dedicated to the following:

To my parents for being there and supporting me through all of my schooling.

TABLE OF CONTENTS

Signature Page...... iii

Dedication...... iv

Table of Contents...... v

List of Figures...... vi

List of Tables...... vii

Acknowledgements...... viii

Abstract...... ix

Introduction...... 1

Materials and Methods...... 8

Results...... 13

Discussion...... 17

Figures and Tables...... 24

References...... 33

LIST OF FIGURES

Figure 1: Deaths Attributed to Alcohol Abuse World Wide...... 24

Figure 2: GR Signaling...... 24

Figure 3: Operant Chamber...... 25

Figure 4: Vapor Chambers...... 25

Figure 5: GR Phosphorylation in Alcohol Dependent and Nondependent Rats..... 26

Figure 6: Alcohol Self-Administration after Intra-CeA Mifepristone Injections...... 26

Figure 7: Systemic Mifepristone Effects on Alcohol Self-Administration...... 27

Figure 8: Systemic CORT113176 Effects on Alcohol Self-Administration...... 28

Figure 9: Systemic CORT113885 Effects on Alcohol Self-Administration...... 29

Figure 10: Systemic CORT108297 Effects on Alcohol Self-Administration...... 30

Figure 11: Systemic Mifepristone, CORT113176, and CORT113885 Effects on

Saccharin Self-Administration...... 30

Figure 12: Conversion Between Active Cortisol and Inactive Cortisone...... 31

Figure 13: CORT Bound GR Complex and Responses...... 31

LIST OF TABLES

Table 1: Mifepristone, CORT113176, CORT118335, and CORT108297 characterization in in vitro assays...... 32

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ACKNOWLEDGMENTS

I would like to acknowledge Dr. George F. Koob and Dr. Olivier George for giving me the opportunity to work in Koob Lab and earn a Master's degree.

I would also like to acknowledge Dr. Leandro Vendruscolo for guiding me through pharmacological experiments, answering all of my questions, helping me revise my thesis, and for his incredible patience and kindness. He ran the statistical analysis of the intra-CeA and effects of Mifepristone, CORT113176, CORT118335, and CORT108297 on alcohol self-administration.

I would also like to acknowledge Dr. Scott Edwards for his input on the GR phosphorylation study and helping to edit my thesis.

I would also like to acknowledge Dr. Carrie Wade for listening to and helping me clarify my thesis presentation.

I would also like to acknowledge Dr. Timothy Whitfield for guiding me through rat addiction experimentation.

I also would like to acknowledge Yanabel Grant for guiding me periodically with behavioral testing, and generally how to handle rats.

I also would like to acknowledge Dr. Joel E. Schlosburg for helping me with dilutions and molecular binding assays.

I would also like to acknowledge Eva Zamora-Martinez for teaching and introducing me to Western blot analysis, among other things.

The Materials and Methods in full, Results in full, and Figures 5 – 11are being prepared for submission for publication of the material. Leandro F. Vendruscolo, Scott

Edwards, Lauren G. Macshane, Joel E. Scholsburg, M. Adrienne McGinn, Eva R.

Zamora-Martinez, Vez Rapute-Canonigo, Marian L. Logrip, Joseph K. Belanoff, Hazel J.

viii

Hunt, Pietro P. Sanna, and George F. Koob. The dissertation author is an author for this paper.

Figure 5 was produced by Dr. Scott Edwards.

Figures 6, 7, 8, 9, 10, and 11 were produced by Dr. Leandro Vendruscolo.

Table 1 was produced by Dr. Hazel Hunt.

ABSTRACT OF THE THESIS

Molecular and Pharmacological Evidence for the Role of Glucocorticoid Receptors in

Alcohol Dependence

Lauren Grace Macshane

Master of Science in Biology

University of California, San Diego, 2014

Professor George F. Koob, Chair

Professor Eduardo R. Macagno, Co-Chair

alcohol exposure and withdrawal sensitize brain stress systems. Glucocorticoid receptors (GR), activated by stress hormones (cortisol in humans; corticosterone in rodents), have been shown to be dysregulated in stress/reward-related brain regions.

We report increased GR activation (indexed by GR phosphorylation) in the central amygdala (CeA), a stress-related brain region, in alcohol-dependent compared with nondependent rats. Consistently, GR antagonism with mifepristone injected directly into the CeA reduces alcohol self-administration in dependent rats compared with nondependent rats. Systemic GR antagonism with mifepristone also reduces alcohol self-administration specifically in dependent rats. Mifepristone, however, also inhibits progesterone receptors, which may participate in alcohol-related behaviors. Therefore, more selective drugs (CORT113176, CORT118335, and CORT108297) that do not block progesterone receptors but do bind GR were tested on alcohol self-administration.

CORT113176 significantly reduced alcohol self-administration in dependent rats, providing unambiguous evidence for a functional role of GR in alcohol dependence.

Finally, the GR antagonist CORT118335 reduced self-administration in both dependent and nondependent rats, whereas CORT108297 had no effect on alcohol self- administration in either dependent or nondependent groups. These findings suggest that behavioral outcomes depend on distinct conformational and signaling changes produced by each antagonist when associated with GR. The present results provide compelling evidence for GR dysregulation in alcohol dependence and suggest that GR constitutes a viable pharmacological target for alcohol dependence.

Introduction

Alcoholism or alcohol dependence is characterized by a compulsion to seek and ingest alcohol, uncontrolled intake, and emergence of a negative emotional state (e.g., dysphoria, anhedonia, anxiety) during withdrawal (Koob et al., 2014). Initially, drug intake in a nondependent state (not addicted) provides a positive, or pleasurable/rewarding emotional response (positive reinforcement) characterized by a general “high.” Positive reinforcement is defined as adding an appetitive or pleasant stimulus (e.g., alcohol) to increase the probability of a certain behavior or response to reoccur. As addiction (dependence) develops, the drug’s pleasant effect decreases and the brain stress system becomes sensitized (Koob and Le Moal, 1997, 2008; Koob et al.,

2014). Drug intake becomes compulsive and is needed regularly to reach a normal emotional state and reduce/prevent negative emotions associated with drug withdrawal.

In this case, negative reinforcement comes to predominate, i.e., the removal/relief of an unpleasant emotional state increases the likelihood of drug use.

Among all drugs of abuse, alcohol is one of the most harmful and lethal. Fig. 1 illustrates the burden of disease caused by alcohol. Harmful use of alcohol leads to 2.5 million deaths globally each year (WHO, 2011) and about 80,000 deaths per year in the

U.S. (National Center for Health Statistics, 2004). Alcohol abuse is the leading cause of morbidity and mortality in many countries (e.g., Mexico, Brazil, Russia) and the third in the US (WHO, 2011). Alcohol contributes to other diseases, injuries, and death such as neuropsychiatric disorders, liver cirrhosis, cardiovascular diseases, high blood pressure, car accidents, violence, and suicides. Diseases caused by alcoholism also include pancreatic diseases (Lesher and Lee 1998), increased risk of breast cancer in women

(Smith-Warner et al., 1998), and in extreme cases, dementia (Korsakoff’s Syndrome)

(Corrao et al., 2004). In addition, one of the leading preventable causes of birth defects

in the U.S. is drinking during pregnancy (American Academy of Pediatrics, 2000; CDC,

1993). Therefore, it is critical to understand the biological and environmental factors that contribute to vulnerability or resilience to alcoholism in order to develop better strategies of prevention, diagnosis, and treatment of this severe mental disease.

The enormous impact of alcohol on an individual with alcoholism and society in general has driven research to understand the problem and develop new treatments.

There are available drugs to treat alcoholism, but these have displayed a limited success rate. Some of the most common drugs used to attenuate the desire for alcohol in alcoholics include acamprosate, a modulator of stimulatory and inhibitory receptors dysregulated by alcoholism, naltrexone, an opioid receptor antagonist that reduces relapse rates, and disulfiram (antiabuse drug), an inhibitor of alcohol metabolism that causes discomfort when alcohol is ingested. In a study by Laaksonen et al. (2008), disulfiram was found to be most effective in delaying heavy drinking and number of days of abstinence. Nonetheless, these drugs are known to produce side effects such as nausea, vomiting, skin itchiness, headaches, dizziness, and sexual dysfunction, which cause low compliance to the treatment. Even with drugs and behavioral/cognitive therapy, relapse rates are high (Kreek et al., 2005). Therefore, seeking new, more efficacious treatments has become a major topic of interest in neurobiological disease research.

To find treatments with fewer side effects, the specific biological dysregulation in alcohol dependence must be better understood. As mentioned above, one of the most ubiquitous symptoms of any addictive process is a sensitization of brain stress systems and the predominance of negative reinforcement in which the drug is used to reduce stress symptoms during withdrawal. Withdrawal symptoms, which mark the negative affective stage of dependence, comprise feelings of anxiety, anhedonia, depressed

mood, pain, and dysphoria, and lead to craving and compulsive drinking (Koob et al.,

2014). One of the key stress systems dysregulated by addiction, particularly alcoholism, is the hypothalamic-pituitary-adrenal (HPA) axis. In fact, human studies have shown that even nondependent individuals with a family history of alcoholism show significantly greater HPA activity and cortisol levels in response to ethanol or stressors as well as a positive correlation with increased vulnerability for developing alcoholism later in life

(Schuckit & Smith 1996; Wand et al. 1998, 1999a,b, 2001; Hernandez-Avila et al. 2002;

Zimmermann et al. 2004; Uhart et al. 2006).

Alcohol intoxication in nondependent individuals stimulates the HPA axis in laboratory animals and in humans (Lovallo et al., 2000; Adinoff et al., 1990; 2003; Uhart and Wand, 2009; Sinha et al., 2011; Richardson et al., 2008). HPA axis activation leads to the release of corticotrophin releasing factor (CRF) from the periventricular nucleus

(PVN) of the hypothalamus. CRF, via the pituitary portal system, stimulates the release of adrenocorticotropic hormone (ACTH). ACTH, via the bloodstream, induces the release of glucocorticoids (i.e., cortisol in humans or the rat homolog corticosterone

[CORT]) from the adrenal glands. Upon release, CORT binds mineralocorticoid receptors (MR or type I), which have a high affinity for CORT, and glucocorticoid receptors (GR or type II), which have a low affinity for CORT and only activate in the presence of high circulating CORT levels (McEwen et al., 1968, McEwen, 2007). High blood CORT levels bind and activate GR in the brain, which regulates the transcription and expression of CRF and other genes. Increased activation of GR reduces CRF release in the hypothalamus to shut off HPA axis activity (negative feedback). However, repeated cycles of alcohol intoxication and withdrawal produce a sensitization of extrahypothalamic stress systems (Heilig and Koob 2007; Vendruscolo et al., 2012).

Unbound, inactive GR resides in the cytoplasm, complexed with chaperone proteins, which are released upon CORT binding. CORT binding increases GR phosphorylation (pGR). GR then dimerizes and the new complex is translocated into the nucleus (Oakley and Cidlowski, 2013). In the nucleus, GR binds directly to glucocorticoid response elements (GRE), upstream of the promoter in various genes, including the gene that codes for CRF (Beato et al., 1987; Freedman, 1992). A GRE is either activated, in which case the gene will be expressed (Barnes, 2006; Oakley and

Cidlowski, 2013; Fig. 2), or repressed (Uhlenhaut et al., 2013) and thus inhibiting transcription. GR-dependent repression of genes is also mediated by a negative GRE

(nGRE), a sequence slightly different from the GRE sequence in which gene transcription would be inhibited. GR-induced activation or repression is largely dependent on GR coregulators, including steroid receptor coactivator 1 (SRC-1) and nuclear receptor corepressor 1 (NCoR1). Different coregulators are recruited under various conditions and in distinct regions of the body.

To study dysregulation of the neuroendocrine stress system, the use of animals has been paramount. Difficulties of human studies (in contrast to animal studies) include subject compliance, polydrug use, as well as ethical issues. Also, animals dependent on alcohol display anxiety-like behavior, anhedonia, and physical and motivational withdrawal symptoms similar to humans (Edwards and Koob, 2010). Furthermore, similar to humans (Adinoff et al., 1990; 2003; Lovallo et al., 2000; Uhart and Wand,

2009; Sinha et al., 2011), alcohol dependence in rats has been associated with a dysregulated neuroendocrine stress system (Rasmussen et al., 2000; Zorrilla et al.,

2001; Richardson et al., 2008; Vendruscolo et al., 2012).

Alcohol dependence in rodents can be modeled via chronic, intermittent alcohol vapor exposure (Gilpin et al., 2008). In this model, rats exhibit increased intake of and

motivation for alcohol, compulsive alcohol drinking despite punishment (for a review see

Vendruscolo and Roberts, 2013), somatic withdrawal signs, and negative emotional symptoms as reflected by anxiety-like responses, elevated brain reward thresholds, and hyperalgesia (Schulteis et al., 1995; Roberts et al., 2000; Valdez et al., 2002; Rimondini et al., 2003; O'Dell et al., 2004; Funk et al., 2006; Richardson et al., 2008; Zhao et al.,

2007; Sommer et al., 2008; Edwards et al., 2012). Additionally, similarly to humans, alcohol-dependent rodents display increased stress responses as measured behaviorally and via alterations in stress-related neurotransmitters and neurocircuit activity. Therefore, in the present set of studies, experiments were performed in rats made dependent on alcohol by vapor exposure and were compared with rats not exposed to alcohol vapor (nondependent controls) for the purpose of modeling limited, recreational drinking.

Vendruscolo et al. (2012) investigated corticosterone-dependent plasticity in several brain regions of vapor-exposed and control rats. Because CORT binds both GR and MR, the study measured mRNA levels of both receptors in dependent and nondependent rats. GR mRNA levels significantly differed between nondependent and dependent rats during acute withdrawal in several stress/reward brain systems, including the prefrontal cortex, nucleus accumbens, and bed nucleus of the stria terminalis. MR mRNA levels, however, showed no significant difference between groups. Importantly, a functional role for GR in compulsive alcohol drinking was observed using pharmacological studies. Chronic GR antagonism with mifepristone (RU-486) significantly attenuated escalation of alcohol intake and compulsive-like alcohol drinking in rats exposed to alcohol vapor, whereas the same treatment did not affect alcohol drinking in nondependent rats.

Evidence has indicated an integral participation of the CeA in mediating the effects of acute alcohol withdrawal. As mentioned above, CRF expression is increased in the CeA after chronic alcohol exposure (Roberto et al., 2010), and antagonizing CRF activity in the CeA has been shown to significantly attenuate alcohol self-administration in dependent rats (Funk et al., 2007) as well as abrogate the negative effects of withdrawal such as anxiety-like behavior and hyperalgesia (Edwards et al., 2012).

Therefore, it is hypothesized that GR signaling in the CeA is dysregulated and leads to altered CRF expression. However, GR function and the effect of GR antagonism in the

CeA on alcohol dependence remain to be determined.

Despite the effectiveness of mifepristone in decreasing compulsive alcohol drinking in rats, one caveat for use of mifepristone is that, in addition to blocking GR, it also antagonizes progesterone receptors (Peeters et al., 2004). Progesterone receptor antagonism can facilitate abortion, trigger the birthing process at any point in fetal development (Couzinet et al., 1986; El-Refaey et al., 1995), and/or potentially affect a vast array of processes in both male and female endocrine function, thus limiting its usefulness. Progesterone itself has been found to have neuroregenerative and protective effects (Brinton et al., 2008). Also, while expression may vary across different regions, progesterone receptors are broadly distributed throughout the brain.

Progesterone receptor levels have been observed to change in alcohol-dependent rats

(Janis et al., 1998). Whether blocking progesterone receptors is efficacious in the attenuation of alcoholism symptomatology is unknown. However, the development of GR antagonists with low or no affinity for progesterone receptors will represent a major advancement not only in understanding the role of GR in alcohol dependence but will also assist the development of more specific pharmacological targets to treat alcohol dependence.

Our overarching hypothesis is that sensitized GR signaling, not progesterone receptor signaling, is a critical mediator of dysregulated stress systems and promotes compulsive alcohol drinking in alcohol dependence. To test this hypothesis, we used the model of alcohol vapor exposure to produce dependence in association with molecular and pharmacological. We tested our hypothesis as described in the following specific aims:

1. To determine GR activation and expression levels, phosphorylated and total GR

were measured in the CeA of dependent and nondependent rats.

2. To test the significance of CeA GR dysregulation in producing excessive drinking

behavior, GR antagonism was accomplished via injecting mifepristone directly

into the CeA.

3. To sort apart the role of GR and progesterone receptor signaling in alcohol

dependence, three novel GR specific antagonists were systemically administered

to dependent and nondependent rats before alcohol self-administration sessions.

4. To test whether GR antagonism is specific in alcohol dependence and not reward

in general, rats were given systemic injections of GR antagonists and tested for

self-administration of saccharin sweetened-water.

Materials and Methods

Animals

Adult male Wistar rats (Charles River, Raleigh, NC), weighing 225-275 g at the beginning of the experiments, were housed in groups of 2-3 per cage in a temperature- controlled (22ºC) vivarium on a 12 h/12 h light/dark cycle (lights on at 8:00 PM) with ad libitum access to food and water except during behavioral testing. All behavioral tests were conducted during the dark phase of the light/dark cycle. All procedures adhered to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of The Scripps

Research Institute.

Operant self-administration

Self-administration sessions were conducted in standard operant conditioning chambers (Med Associates, St. Albans, VT; Fig. 3). The rats were first trained to self- administer alcohol using a fixed-ratio 1 (FR1) schedule of reinforcement (i.e., each operant response was reinforced with 0.1 ml of solution) as previously reported

(Vendruscolo et al., 2012). First, the rats were given free-choice access to alcohol (10% w/v) and water for 1 day in their home cages to habituate them to the taste of alcohol.

Second, the rats were subjected to an overnight session in the operant chambers with access to one lever (right lever) that delivered water (FR1). Food was available ad libitum during this training. Third, after 1 day off, the rats were subjected to a 2 h session

(FR1) for 1 day and a 1 h session (FR1) the next day, with one lever delivering alcohol

(right lever). All of the subsequent sessions lasted 30 min, and two levers were available

(left lever: water; right lever: alcohol). Upon stable levels of intake were reached animals

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were split in two groups: vapor exposed (dependent; dep) and air exposed

(nondependent; nondep).

Alcohol vapor chambers

The rats were made dependent by chronic, intermittent exposure to alcohol vapors as previously described (O’Dell et al., 2004; Gilpin et al., 2008; Fig. 4). They underwent cycles of 14 h on (blood alcohol levels during vapor exposure ranged between 150 and 250 mg%) and 10 h off, during which behavioral testing occurred (i.e.,

6-8 h after vapor was turned off when brain and blood alcohol levels are negligible;

Gilpin et al., 2009). Nondependent rats were not exposed to alcohol vapor but were concomitantly tested with dependent rats.

Western blot analysis

Quantitative analysis of total GR and phosphoprotein densities was conducted as previously described (Edwards et al., 2013). Brains were collected from a group of dependent and nondependent rats during acute withdrawal to match the time point for behavioral testing. Brains were snap frozen and stored at -80ºC until processing. Central and basolateral (BLA) amygdala tissue samples were homogenized by sonication in lysis buffer (320 mM sucrose, 5 mM HEPES, 1 mM EGTA, 1 mM EDTA, 1% SDS, with

Protease Inhibitor Cocktail and Phosphatase Inhibitor Cocktails II and III diluted 1:100;

Sigma), heated at 100°C for five minutes, and stored at -80°C until determination of protein concentration by a detergent-compatible Lowry method (Bio-Rad). Samples of protein (15 g) were subjected to SDS-polyacrylamide gel electrophoresis on 10% acrylamide gels using a Tris/Glycine/SDS buffer system (Bio-Rad), followed by electrophoretic transfer to PVDF membranes (polyvinylidene; GE Healthcare).

Membranes were blocked overnight in 5% nonfat milk at 4°C, and then incubated in primary antibody recognizing the Ser211 (human)/Ser232 (rat) phosphorylated form of

GR (1:1000, 5% nonfat milk, Cell Signaling). Membranes were washed and labeled with species-specific peroxidase-conjugated secondary antibody (1:10K, Bio-Rad) for one hour at room temperature. Following chemiluminescence detection (SuperSignal West

Pico, Thermo Scientific), blots were stripped for 20 min at room temperature (Restore,

Thermo Scientific) and re-probed for total protein levels of GR (1:2000, Thermo

Scientific). Immunoreactivity was quantified by densitometry (ImageJ 1.45S, NIH) under linear exposure conditions. Separate gel sets composed of control and experimental animals are processed in parallel for each region. Density values were expressed as a percentage of the mean of control values for each gel to normalize data across blots.

Individual phosphoprotein levels were normalized to individual total protein levels to generate pGR/GR ratio values for statistical comparison.

Mifepristone CeA injection

A separate group of dependent and nondependent rats were implanted with bilateral guide cannulae aimed at the CeA (-2.6 AP, 4.2 ML, and -6.6 DV from skull) and bilaterally infused with mifepristone (0, 10, 30 µg/side) dissolved in dimethyl sulfoxide

(DMSO) (0.5 ml) 90 min prior to operant tests during acute withdrawal in a within subjects Latin-square design. Infusions occurred over a period of 2 min plus 1 min for diffusion.

Systemic drug treatment

Mifepristone was purchased from Sigma-Aldrich (St. Louis, MO, USA).

CORT108297, CORT118335 and CORT113176 were provided by Corcept Inc. (Menlo

Park, CA, U.S.). Different cohorts of dependent and nondependent rats were intraperitoneally (IP) injected with mifepristone (0, 30, and 60 mg/kg), CORT108297 (0,

5, 15, 30, and 60 mg/kg), CORT118335 (0, 1, 3, and 10 mg/kg), or CORT113176 (0, 10,

30, and 100 mg/kg) 90 min prior to self-administration sessions. All drugs were dissolved in 10% dimethylformamide (DMF) (Sigma-Aldrich, St. Louis, MO, USA)/10% Emolphor

(Rhodia, New Brunswick, NJ, USA) and diluted in saline. The volume of injections was 3 ml/kg. The doses of each compound were administered following a within-subjects Latin

Square design.

Saccharin self-administration

Another group of nondependent rats was trained to lever press for saccharin self- administration under an FR1 schedule using identical conditions as described for alcohol self-administration except that a saccharin (0.004%, w/v) solution was used. A submaximal rewarding saccharin concentration was chosen based on previous studies

(Vendruscolo et al., 2010) to prevent reaching a “ceiling effect” in any group and maintain similar response rates as alcohol. Mifepristone, CORT118335 and

CORT113176, which were effective in reducing alcohol self-administration, were IP injected 90 min prior to saccharin self-administration in a within-subjects Latin Square design.

Statistical analysis

The data are expressed as mean and SEM. The data were analyzed using

ANOVA with repeated measures, with dose (or drug) as the within-subjects factor and group (dependent vs. nondependent) as the between-subjects factor. When appropriate, post hoc comparisons were performed using Fisher’s Least Significant Difference (LSD) test. Western blot data was analyzed using Student’s t test. The accepted level of significance for all tests was p < 0.05.

The Materials and Methods in full, Results in full, and Figures 5 – 11are being prepared for submission for publication of the material. Leandro F. Vendruscolo, Scott

Edwards, Lauren G. Macshane, Joel E. Scholsburg, M. Adrienne McGinn, Eva R.

Zamora-Martinez, Vez Rapute-Canonigo, Marian L. Logrip, Joseph K. Belanoff, Hazel J.

Hunt, Pietro P. Sanna, and George F. Koob. The dissertation author is an author for this paper.

Results

Glucocorticoid receptor phosphorylation was increased in the CeA (t10=2.6; p<0.05) but not BLA (t(10)=0.25; p=0.8) in alcohol-dependent compared with nondependent rats (Fig. 5).

Fig. 6 shows the results of intra-CeA mifepristone injections on alcohol self- administration. The two-way repeated measures ANOVA revealed a group by dose interaction (F(2,18)=3.9; p < 0.05). As expected, vehicle-treated dependent rats self- administered more alcohol compared with vehicle-treated nondependent rats (p <

0.005). Mifepristone treatment (10 g/side) significantly decreased alcohol self- administration specifically in dependent rats (p < 0.05).

Table 1 shows the profile of the different antagonists on GR-related in vitro assays. A binding assay provided the affinity of the drug for the receptor (first three rows of Table 1). The order of GR binding affinity for the different antagonists was mifepristone > CORT108297 > CORT113176 > CORT118335. A reporter gene assay measured the activity of the receptor induced by the drug indicating gene transcription

(fourth and fifth rows of Table 1). The order of gene activity was: mifepristone <

CORT113176 < CORT108297 < CORT118335.

The last six rows of Table 1 represent the results of the drugs on GR-related assays. These in vitro biological systems are sensitive to GR agonism and antagonism.

In the assays 1, 2, 4, and 6, dexamethasone, a synthetic GR agonist, was used to activate the biological systems and the drugs were tested to antagonize the dexamethasone effect. Assays 3 and 5 were conducted in agonist mode, i.e., dexamethasone was not added and the effect produced by the drugs represent their agonistic effect. 1. Tyrosine aminotransferase (TAT), an enzyme present in the liver that catalyzes the conversion of tyrosine to 4-hydroxyphenylpyruvate, expression is

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increased when stimulated by increased CORT levels. The antagonistic effect of the drugs on this system was mifepristone > CORT113176 > CORT108297 > CORT118335.

The liver Hep G2 cells are human hepatocytes from a perpetual cell line (hepatocellular carcinoma) and are commonly used in research. 2. A similar test was done in rat H4 cells; antagonism was seen in the order of mifepristone = CORT113176 > CORT118335

> CORT108297. 3. Rat H4 TAT cells were tested in agonist mode and results indicate the following agonistic effect: CORT118335 > mifepristone > CORT108297 >

CORT113176 had no agonistic effect. 4. A549 cells are adenocarcinomic human alveolar basal epithelial cells and were used to study the anti-inflammatory effects of GR agonists. GR agonists inhibit the inflammatory cytokines secreted by these cells when stimulated. Interleukin (IL)-1b was used to stimulate secretion of cytokines, one of which

(IL-6) was measured. Glucocorticoid receptor antagonism was tested by IL-1b stimulation and adding the different antagonists to discover if the drugs reduce the agonistic effects of glucocorticoids. The antagonist potency in this assay was mifepristone > CORT113176 > CORT118335 > CORT108297. 5. The same test was performed in agonistic mode and the effect was CORT108297 > CORT118335 > mifepristone > CORT113176. 6. The assay in PBMCs (peripheral blood mononuclear cells), standard immune system blood cells used here to measure the production of a tumor necrosis factor (TNF, a cytokine) that causes apoptosis and increases in response to LPS (lipopolisaccharide), an inflammatory agent. Lower values in this test indicate GR antagonism and the results were mifepristone > CORT113176 > CORT108297 >

CORT118335.

For systemic injections of mifepristone, the two-way repeated measure ANOVA revealed a group by dose interaction (F(2,40)=4.7; p < 0.05). The post hoc comparisons indicated that dependent rats self-administered more alcohol compared with non-

dependent rats (p < 0.0001). Mifepristone dose-dependently decreased alcohol self- administration in dependent rats (for 30 mg/kg: p < 0.05; for 60 mg/kg: p < 0.0001), without altering alcohol responding of nondependent rats. No effects were found for water self-administration (Fig. 7).

The results for CORT113176 are shown in Fig. 7. The ANOVA revealed a significant interaction between group and dose (F(3,42)=5.2; p < 0.005). The post hoc comparisons indicated that dependent rats displayed increased alcohol self- administration compared with nondependent rats (p < 0.0001) and the doses of 10 (p <

0.05) and 30 (p < 0.0001) decreased alcohol self-administration in dependent rats only, whereas 100 mg/kg decreased alcohol intake for both dependent (p < 0.0001) and nondependent (p < 0.01) animals. No significant differences were found for water intake.

The results for CORT118335 are illustrated in Fig. 9. The ANOVA revealed an overall group effect (F(1,22)=19.9; p < 0.0005), with dependent animals showing higher alcohol intake compared with nondependent rats. An overall dose effect was also detected (F(3,66)=9.3; p < 0.0001). Animals given 3 mg/kg (p < 0.005) and 10 mg/kg (p <

0.0001) showed decreased alcohol intake compared with vehicle controls. No effects were found for water self-administration.

Fig. 10 shows the results for CORT108297 on alcohol and water self- administration. The ANOVA showed a significant group effect only (F(1,12)=22.8; p <

0.001), with dependent rats responding more for alcohol compared with nondependent rats. No significant effects were detected for water self-administration.

Fig. 11 shows the results of mifepristone, CORT113176, and CORT118335 on saccharin (0.004%, w/v) intake. The doses that affected alcohol self-administration did not change alter self-administration of saccharin-sweetened water.

The Materials and Methods in full, Results in full, and Figures 5 – 11are being prepared for submission for publication of the material. Leandro F. Vendruscolo, Scott

Edwards, Lauren G. Macshane, Joel E. Scholsburg, M. Adrienne McGinn, Eva R.

Zamora-Martinez, Vez Rapute-Canonigo, Marian L. Logrip, Joseph K. Belanoff, Hazel J.

Hunt, Pietro P. Sanna, and George F. Koob. The dissertation author is an author for this paper.

Discussion

In the present study we used an animal model that produces several symptoms of alcohol dependence to investigate the molecular and pharmacological role of GR signaling in alcoholism. Our hypothesis was that GR function is sensitized during the transition to dependence. As expected, dependent rats (vapor exposed) exhibited significantly higher alcohol self-administration compared with nondependent rats (air exposed). Consistent with our hypothesis, we found that GR phosphorylation, a molecular marker of receptor activation, was increased in the CeA, but not BLA, of dependent rats compared with controls. In line with this neuroadaptation, specific blockade of GR within the CeA by microinjection of mifepristone significantly reduced alcohol self-administration selectively in dependent rats. Additionally, both mifepristone and the selective GR antagonist CORT113176, given systemically, had a selective effect in reducing alcohol self-administration in dependent rats, whereas CORT118335 decreased self-administration in rats regardless of alcohol history. The same treatments did not affect self-administration of saccharin sweetened-water, indicating that GR blockade was specific for alcohol reinforcement rather than reinforcement in general.

Finally, CORT108297 had no effect, indicating the complexity of GR signaling on alcohol-related behaviors. Together, these findings provide compelling evidence for a central role of GR in alcohol dependence.

Increased GR phosphorylation in the CeA of dependent rats suggests that this system is sensitized in alcohol dependence. Our preliminary results (not shown) and the results by Little et al. (2008) indicate that systemic (blood) CORT levels are not different between alcohol-dependent and nondependent rats. Therefore, an important question remains: how can GR phosphorylation be increased during alcohol dependence while

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blood CORT levels are unchanged? A number of possibilities may be advanced to account for this outcome.

Despite a lack of change in CORT blood levels, increased GR phosphorylation in the CeA of dependent rats can possibly be due to localized changes in CORT levels.

Little et al. (2008) have reported that rodents chronically exposed to alcohol displayed increased CORT levels in specific brain regions such as the prefrontal cortex, hippocampus, striatum, and midbrain, while plasma concentrations remained unchanged. CORT levels were not measured in the CeA in the study mentioned above, but it is very likely that the same increase would occur in the CeA, thus explaining increased GR activation therein.

Increased regional CORT could be due to the reductase activity of 11- hydroxysteroid dehydrogenase (HSD-1). This enzyme converts inactive cortisone to active CORT (Rajan et al., 1996; Jellinck et al., 1999). Importantly, it has been reported that HSD-1 is localized in several brain regions (Moisan et al., 1990; Sakai et al., 1992;

Seckl and Walker, 2001). Conversely, HSD-2 converts active CORT to inactive cortisone

(Wyrwoll et al., 2011), although brain levels of this enzyme are more restricted. Thus, an imbalance in the levels of HSD-1 and HSD-2 in the CeA between alcohol dependent and nondependent rats could explain the differential GR activation between these groups of rats.

Alterations in GR chaperone binding may be another reason for increased GR activity. After activation, GR dissociates from chaperones in the cytoplasm to translocate to the nucleus where it produces transcriptional changes (Fig. 2). GRs are then released back into the cytoplasm (exported from the nucleus) and associate again with chaperones. Increased levels of chaperones would increase the likelihood for the free

GR to be reattached to chaperones and to be functionally reset again (Pratt et al., 2006).

Inhibition of the GR–hsp90 heterocomplex assembly leads to a fast reduction in the amount of GR protein in a cell, a decline that is inhibited by proteasome inhibitors

(Whitesell and Cook 1996; Segnitz and Gehring 1997). Reduced amounts of hsp-90 also are known to alter GR activity (Picard et al., 1990). Future studies will investigate the specific roles of HSD-1, HSD-2, and chaperones on the differential GR activation in alcohol dependence.

To further test the role of GR in the CeA of alcohol-dependent rats, intra-CeA injections with mifepristone were carried out. These injections significantly reduced alcohol intake in dependent rats, indicating that the CeA plays a key role in escalated alcohol self-administration during alcohol dependence. It is noteworthy that CeA mifepristone only attenuated alcohol intake (albeit significantly), and did not completely eliminate excessive drinking. This finding indicates that other brain regions, yet to be explored, contribute to escalated alcohol drinking in alcohol dependence.

As mentioned in the Introduction, a caveat of using mifepristone is its action on progesterone receptors. It has been shown that progesterone receptors play a role in behavior, including alcohol-related behaviors (Janis et al., 1998). However, we found that both mifepristone and CORT113176, given systemically, significantly decreased alcohol self-administration in dependent rats, whereas alcohol intake in nondependent was not substantially affected by the treatment. Unlike mifepristone (Peeters et al.,

2004), CORT113176 has high binding affinity to GR without detectable binding to progesterone receptors (Table 1). These findings provide unambiguous evidence for a role of GR, and not progesterone receptors, in alcohol dependence.

Interestingly, CORT118335 reduced alcohol self-administration in both dependent and nondependent rats, whereas CORT108297 did not change alcohol intake in any group. At first, this finding appears to be contradictory. However, it is

important to note that the GR system involves interactions with multiple membrane, cytoplasmic, and nuclear partners and, depending on the conformational changes produced by different GR ligands, different behavioral outcomes can be seen. Identifying which “players” interact with GR to reduce alcohol intake in dependent subjects will help in the discovery of novel pharmacological targets to treat alcoholism.

It is noteworthy that, consistent with the behavioral effects, mifepristone and

CORT113176 present a very similar pharmacodynamic/molecular profile and strong GR antagonism, whereas CORT118335 and CORT108297 have mixed antagonistic and partial agonistic actions depending on the system in which they are being tested (Table

1). The effects of short term, active GR, are often measured in in vitro assays via increases or decreases in immune proteins levels. If a drug has an effect on active GR then the levels of such proteins would change. Because mifepristone is a prototypical

GR antagonist, its in vitro effects, were compared with CORT113176, CORT118335, and CORT108297. Interleukins or cytokines are secreted proteins and signaling molecules that are primarily known for coordinating different immune responses.

Normally, glucocorticoids downregulate the inflammatory response (Barnes, 2006;

Ayroldi and Riccardi, 2009), for example, IL6, a pro-inflammatory cytokine, is usually inhibited by glucocorticoids. However, overactivation of the HPA axis has been found to change IL levels. Alcoholism has been associated with increased blood levels of IL6 only

(Nicolaou et al., 2004; Sheron et al., 1993; González-Quintela et al., 2000).

Because CORT113176, CORT118335, and CORT108297 only bound to GR, and each drug had a different effect on rats, their interaction with GR leading up to expression must differ. Two known cofactors, steroid coactivator (SRC)-1 and nuclear receptor corepressor (NCoR), have been shown to selectively bind GR in the presence of mifepristone. The CORT-bound GR complex binds an nGRE site in the promoter of

the CRF gene to upregulate CRF expression, but in combination with SRC-1, the complex binds a negative GRE to inhibit expression (Malkoski and Dorin, 1999). SRC-1 has two splice variants, SRC-1A and SRC-1E, which exhibit opposite effects on the CRF promoter (van der Laan S. et al., 2008). SRC-1A is more highly expressed in the PVN than the CeA (Meijer et al., 2000, 2005) and its interaction with active GR has been found to increase GR-dependent repression of the CRF promoter (van der Laan S. et al.,

2008). SRC-1E, more highly expressed in the CeA (Meijer et al., 2000, 2005), however, is known to reduce GR-dependent repression of CRF expression and increase non-GR

CREB driven transcription of CRF (van der Laan et al., 2008). Mifepristone greatly antagonizes SRC-1E but not SRC-1A, allowing continued repression of CRF in the CeA

(Meijer et al., 2005). CORT108297, however, binds agonistically to SRC-1A but not

SRC-1E, suggesting that SRC-1A repression of CRF expression would be increased upon binding especially in the PVN, whereas SRC-1E continues to work in the opposite direction by upregulating CRF transcription in the CeA. Therefore, an intriguing hypothesis to explain the present results is that whereas mifepristone may decrease

CRF levels in the CeA of alcoholic rats, CRF levels in the PVN remained unchanged.

Importantly, blockade of CRF in the CeA has been shown to block escalated alcohol intake in dependent rats (Funk et al., 2007). CORT108297, however, would increase the expression of CRF in the PVN, but would not decrease CRF in the CeA (Zalachoras et al., 2013). Therefore, it is possible that the altered expression of CRF in the CeA is the determining factor for compulsive alcohol drinking during dependence.

NCoR is expressed at lower levels in the PVN than in the CeA (van der Laan et al., 2005). It does not exhibit regulatory effects on the negative GRE promoter of CRF unless it is interacting with mifepristone. Mifepristone facilitates the association of GR and NCoR in the CRF promoter and leads to repression of CRF expression.

CORT108297, however, does not associate significantly with NCoR (Zalachoras et al.,

2013). Thus, the behavioral effect of mifepristone and the lack of such an effect of

CORT108297 could be explained by different actions of these two drugs on NCoR and consequent CRF changes. CORT118335 and CORT113176 have not been examined with regard to SRC-1 or NCoR, which would be important for future studies to determine the roles of these cofactors.

Overall, it is not clear what the effect of IL level changes due to alcohol dependence has on the dysregulation of corticosteroids. Thus, more research to assess the downstream targets of these drugs is warranted. Future tests are needed to measure the expression levels of GR coregulators, as well as CRF in the PVN and CeA, in alcohol dependence. These measures would include mRNA levels of NCoR, SRC-1A, and SRC-1E, in addition to CRF in the PVN and CeA. It would also be valuable to measure these in the context of mifepristone, CORT113176, CORT118335, and

CORT108297 treatment.

From a translational perspective, different gene GR polymorphisms have been associated with alcohol abuse in humans. For instance, several GR gene polymorphisms have been found to be associated with onset of alcohol abuse in adolescents (Desrivières et al., 2011). Additionally, epigenetic GR modifications due to alcohol dependence in humans have been observed (Ponomarev et al., 2012) as well as wide spread transcriptome expression changes that have been found in the frontal lobes of cirrhotic alcoholics (Liu et al., 2007).

Mifepristone is an FDA-approved drug and has also been shown to reduce symptoms of psychotic depression in humans, which is hypothesized to be associated with a dysregulated HPA axis (Belanoff et al., 2001, 2002; DeBattista and Belanoff,

2006). Neurocognitive function and mood improvements have also been seen in

individuals with bipolar disorder after mifepristione treatment (Young et al., 2004). In addition, attenuation of alcohol-cued craving has been demonstrated with the use of mifepristone in human subjects (Mason et al., 2012). Little and colleagues are currently conducting a double-blind, 36 month, placebo-controlled trial of mifepristone on cognitive function in alcoholics. Ultimately, GR appears to be an important target in treating alcohol dependence from a variety of conceptual angles.

In summary, the upregulated phosphorylation of GR in the CeA of alcohol dependent rats and attenuation of alcohol intake with intra-CeA mifepristone injections indicates a sensitization of this stress-related brain region in alcohol dependence. The effect of specific GR antagonists on alcohol intake indicates that this effect is mediated by GR and not progesterone receptors. Different GR antagonists can produce different behavioral effects, highlighting the complexity of the GR system. Together, these findings indicate the enormous potential of GR signaling for the development of specific drugs to treat alcohol dependence.

Figures and Tables

Figure 1. Disabilities attributed to alcohol abuse as a measure of overall disease burden (WHO, 2011).

Figure 2. General mechanism of action of GR. The Glucocorticoid receptor (GR) resides in the cytoplasm with chaperones. Upon activation, GR is translocated into the nucleus to modulate gene transcription (adapted from Oakley and Cidlowski, 2013).

24 25

Figure 3. Rat self-administration in operant chambers. Pressing one lever produces delivery of either 10% w/v EtOH or 0.04% saccharin and pressing the other lever produces delivery of water, all of which dispenses into a small dish that the rat drinks from.

Figure 4. Alcohol vapor chambers were used to induce and maintain alcohol dependence in rats. The vapor was turned on intermittently, for 14 hours daily.

Figure 5. GR phosphorylation in alcohol-dependent and nondependent rats. Phosphorylation of GR in the central amygdala (CeA) was increased in dependent compared with nondependent rats. GR phosphorylation in the basolateral amygdala (BLA) was not different between dependent and nondependent rats. Representative Western blot bands are shown on the right. *Significantly different from nondependent (p < 0.05).

Mifepristone/RU486 - Intra-CeA Ethanol Self-Administration

60 + n

i 0 µg m

50 10 µg 0

3 *

/ 40 30 µg

s r

e 30

c r

o 20

n i

e 10 R 0 nondep dep

Figure 6. Mifepristone injected directly into the CeA reduced alcohol self-administration in dependent rats only. +Different from vehicle-treated nondependent rats (p < 0.05). * Different from vehicle-treated dependent rats (p < 0.05).

Figure 7. Effects of systemic injections of mifepristone on alcohol delf-administration. Mifepristone dose-dependently decreased alcohol self-administration in dependent but not nondependent rats. Water intake was not affected by mifepristone treatment. #Different from vehicle-treated nondependent rats (p < 0.05). * Different from vehicle- treated dependent rats (p < 0.05).

Figure 8. CORT113176 dose-dependently decreased alcohol self-administration in dependent rats. The highest dose also decreased alcohol self-administration in nondependent rats. Water intake was not affected by drug treatment. #Different from vehicle-treated nondependent rats (p<0.05). * Different from vehicle-treated dependent rats (p<0.05).

Figure 9. CORT118335 dose-dependently decreased alcohol self-administration in both dependent and nondependent rats. Water intake was not affected by drug treatment. #Different from vehicle-treated nondependent rats (p<0.05). * Different from vehicle- treated dependent rats (p<0.05).

Figure 10. CORT108297 did not change alcohol or water self-administration in either dependent or nondependent rats. #Different from vehicle-treated nondependent rats (p<0.05).

Figure 11. Mifepristone, CORT113176 and CORT118335 at doses that affected alcohol intake did not alter self-administration of saccharine-sweetened water.

Figure 12. The conversion between active cortisol and inactive cortisone in the cytoplasm of cells (Oakley and Cidlowski, 2013).

Figure 13. The CORT-bound GR complex stimulates an array of physiological responses (Barnes et al., 2006).

Table 1. In vitro GR-related assays showing binding affinity, gene expression and enzyme activity for various GR antagonists. * Agonist mode. PR: progesterone receptor. MR: mineralocorticoid receptor.

Mifepristone 108297 118335 113176 GR binding Ki (nM) 0.09 0.27 0.5 0.28 PR binding Ki (nM) 1 inactive Inactive inactive MR binding Ki (nM) 5495 inactive Not tested inactive GR reporter gene Ki (nM) 0.9 8 25 4.6 MR reporter gene Ki (nM) not available inactive 148 inactive HepG2 TAT Ki (nM) 3 26 63 12 Rat H4 TAT Ki (nM) (max) 2.2 (100%) 14 (80%) 12 (65%) 4.2 (100%) > 1,000 No *Rat H4 TAT EC50 (nM) (max ) (54%) 6.3 (49%) 660 (59%) agonism A549 IL-1 induced IL-6 Ki (nM) (max) 13 (69%) 18 (21%) 95 (42%) 31 (53%) 1,800 *A549 IL-1B induced IL-6 EC50 (nM (max) 2.2 (51%) 55 (75%) (63%) 390 (32%) PBMC LPS induced TNF Ki (nM) 5.4 51 96 38

The Materials and Methods in full, Results in full, and Figures 5 – 11are being prepared for submission for publication of the material. Leandro F. Vendruscolo, Scott

Edwards, Lauren G. Macshane, Joel E. Scholsburg, M. Adrienne McGinn, Eva R.

Zamora-Martinez, Vez Rapute-Canonigo, Marian L. Logrip, Joseph K. Belanoff, Hazel J.

Hunt, Pietro P. Sanna, and George F. Koob. The dissertation author is an author for this paper.

Figure 5 was produced by Dr. Scott Edwards.

Figures 6, 7, 8, 9, 10, and 11 were produced by Dr. Leandro Vendruscolo.

Table 1 was produced by Dr. Hazel Hunt.

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