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Proteomic Profile of a Chronic Binge-Ethanol Exposure Model Phillip Starski, Lee Peyton, Alfredo Oliveros, Carrie J. Heppelmann, Surendra Dasari, and Doo-Sup Choi J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.9b00394 • Publication Date (Web): 22 Jul 2019 Downloaded from pubs.acs.org on July 23, 2019

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Journal of Proteome Research

Proteomic Profile of a Chronic Binge-Ethanol Exposure Model Phillip Starski,1 Lee Peyton,2 Alfredo Oliveros,3 Carrie J. Heppelmann,4 Surendra Dasari,5 and Doo-Sup Choi, *,1, 2, 6 1Neuroscience

Program, 2Department of Molecular Pharmacology and Experimental

Therapeutics, 3Department of Neurologic Surgery, 4Proteomics Research Center, 5Division

of Biomedical Statistics and Informatics, 6Department of Psychiatry and

Psychology, Mayo Clinic, Rochester, Minnesota 55905, USA

*Correspondence: Dr. Doo-Sup Choi, Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic College of Medicine, 200 First Street SW, Rochester, Minnesota 55905, USA, Tel: 507-284-5602, Fax: 507-284-1762, [email protected]

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ABSTRACT Chronic binge alcohol drinking is known to increase risky decision through pathological impulsive behaviors. Recently, we established a novel rodent model of ethanol-induced waiting impulsivity using 5-choice serial reaction time task (5-CSRTT) in mice. However, molecular mechanisms underlying the chronic binge ethanol-induced waiting impulsivity is not well characterized. Among brain regions involved in impulsivity, the anterior cingulate cortex (ACC) is a major neural substrate for mediating the 5-CSRTT-based waiting impulsivity. Thus, we sought to determine the ACC proteomic profile using labelfree proteomics of mice exhibiting ethanol-induced impulsivity. Ingenuity pathway analysis revealed that impulsivity-related proteins involved in ion channel complexes such as KCNIP3 (potassium voltage-gated channel interacting protein 3) and CACNG2 (calcium voltage-gated channel auxiliary subunit gamma 2) are downregulated in the ACC. We identified significant protein expression changes in the mechanistic target of rapamycin (mTOR) canonical pathway between control and ethanol-induced impulsive mice. Impulsive mice showed over 60 percent of proteins involved in the mTOR canonical pathway have been altered. This pathway has been previously implicated in the neuroadaptation in drugs of abuse and impulsivity. We found substantial changes in the protein levels involved in neurological disorders such as schizophrenia and Alzheimer’s disease. Our findings provide a neuroproteomic profile of ethanol-induced impulsive mice.

Key Word: binge alcohol, proteomics, alcoholism, impulsivity, 5-choice serial reaction time task (5-CSRTT)


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INTRODUCTION Impulsivity is a natural phenomenon that allows for quick decisions without forethought. However, this quick decision making can lead to significant adverse consequences. Recently, impulsivity has been correlated with AUD, in which highly impulsive individuals are more likely to develop AUD1,2. Although impulsivity is regarded as an inherited or innate property, studies have begun to support the notion that ethanol may exacerbate impulsivity3,4. This is a critical hypothesis that alcohol increases natural baseline impulsivity. Thereby, highly impulsive individuals are at risk of increasing their impulsive behavior through binge ethanol episodes. However, the underlying molecular mechanisms of repeated or chronic binge ethanol drinking of exposure-induced impulsivity are understudied. One way to observe a multitude of potential molecular influencers of impulsivity is through proteomic and pathway analysis. Proteomics is one of several global approaches toward understanding the molecular mechanisms of behavioral disorders. Previously, we have revealed and validated several alcohol seeking behavior-related proteomes in multiple brain regions using iTRAQ 5 and label free proteomics6,7. We recently established a novel experimental paradigm that uses repeated binge ethanol exposure to show ethanol’s potentiating effect on impulsive behavior within the 5-choice serial reaction time task (5-CSRTT)8. Thus, we sought to investigate the proteomic profile of a critical brain region, the anterior cingulate cortex (ACC), known to be implicated in 5-CSRTT-based impulsive behavior9. The ACC is an important brain region responsible for various functions including attention, reward anticipation, and decision10. The ACC has also been attributed to impulsivity and several studies have described its relationship to ethanol and addiction11-14. Specifically, lesion studies have shown that damage to the ACC increases premature responding within the 5CSRTT9,15. Importantly, elucidating the effects of ethanol on impulsivity neuronal circuitry would be an impactful observation for further studies attempting to reduce the development of AUD. In the present study, a novel paradigm previously established in our lab was used to observe maladaptive impulsivity in mice through repeated binge-like ethanol vapor exposure. Mice were trained to use the 5-CSRTT to identify impulsivity through 3 ACS Paragon Plus Environment


premature responses. The ACC of the ethanol-exposed (EE) and air-exposed (AE) mice was extracted immediately following their final impulsivity testing session and flash frozen. This tissue was used to perform label free neuroproteomics to elucidate the potential underpinnings of maladaptive impulsive behavior caused by ethanol. Our results revealed changes within several canonical pathways, including the mechanistic target of mTOR, as well as proteins associated with various psychiatric disorders.


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MATERIALS AND METHODS Animals Six-week old male C57BL/6J mice were purchased from the Jackson Laboratories Inc. Mice were group-housed in standard Plexiglas cages with ad libitum access to food and we started to use mice when they reached eight weeks of age. Mice were maintained on a 12h light and dark cycle. Animal care and handling procedures were approved by the Mayo Clinic Institutional Animal Care and Use Committees in accordance with NIH guidelines. 5-Choice Serial Reaction Time Task (5-CSRTT) As described in our recent article8, we used the same procedure to observe maladaptive waiting impulsivity in mice. Briefly, (A) Training. Mice were placed on a food-restriction schedule to maintain their body weight at 85% of their ad libitum weight. All operant training procedures were performed in computer-controlled mouse operant chambers (MED-NP5M-D1, Med Associates, St. Alban, VT) equipped with a rear 3W house light (HL) with an automated mechanical dipper (ENV-302M-S, Med Associates) that holds 20 l of the solution in a reward-aperture. Opposite the HL and rewardaperture are five nose-poke response apertures. (1) Acclimation: All five apertures and reward-aperture were baited with 40 l of 15% sucrose solution via pipette. Mice were then placed within the 5-CSRTT chambers for a 15-minute period for two consecutive days with the HL on with no consequences for nose-pokes into an aperture. Nosepokes and reward-aperture entries were not recorded during this time. (2) Magazine Training: After acclimation, mice underwent Pavlovian conditioning sessions for two days to associate a tone (65dB for 0.25s duration; ENV-323HAM, 4500Hz Sonalert, Med-Associates) with the delivery of the reward. The five apertures were blocked with a screen and rewards were randomly delivered with a simultaneous illumination of the reward-aperture. Rewards were available for a 5s duration, after which the reward aperture light turned off and the dipper retracted. Magazine training sessions lasted for 30 min and total responses for reward-aperture entries were recorded. (3) 5-aperture Fixed Ratio-1 Training: To establish an association between the nose-poke apertures and delivery of reward in the 5-CSRTT, a nose-poke into any of the five illuminated 5 ACS Paragon Plus Environment


apertures activated a 0.5s tone and illumination of the reward-aperture which engaged the mechanical dipper to deliver 20 l of 15% sucrose reinforcement. Following reward retrieval (reward-aperture head entry detection), the reward-aperture remained illuminated for 5s, after which the light turned off and the dipper retracted. Fixed Ratio-1 training was repeated for 3 consecutive days, with session termination contingent on the attainment of 30 rewards for day 1, and 50 rewards for days 2 and 3 or 60 min of any of the days, which ever came first. Session duration, the total number of aperture nosepokes, rewards attained, and the total number of reward-aperture entries was recorded by the computer. (B) 5-CSRTT Acquisition. Mice were exposed to 5-CSRTT training to assess the effect of ethanol vapor on impulsive reward-seeking behavior. Training sessions were divided into trials and inter-trial intervals (ITIs). The beginning of a session was indicated by the HL turning on, illumination of the reward aperture and simultaneous delivery of a free reward. Following the next immediate head entry into the reward-aperture, reward-aperture illumination persisted for 5s after which the HL turned off and the dipper retracted. A fixed ITI began and a nose-poke on any of the apertures during this time delayed aperture illumination and was recorded as a time-out (TO) response. Termination of the ITI was signaled by illumination of the HL and indicated the onset of a trial. Trials were composed of a fixed time period preceding illumination of any one of the apertures, where a nose-poke into any aperture was recorded as a premature response and resulted in the extinction of the HL, a TO duration (5s), and then restarting the trial. If no premature nose-pokes were made, one of the 5 apertures randomly illuminated for a set duration (light duration, LD). Any nose-poke response into an unlit aperture was recorded as an incorrect trial, which turned off the illumination of the correct aperture and terminated the trial. A nose-poke into an illuminated aperture was recorded as a correct trial and allowed for delivery of a reward. Nose-pokes into a previously illuminated aperture occurring within 5s following the extinction of that light (limited hold, LH) was recorded as a correct trial and resulted in reward delivery. Correct trial responses were signaled by a 0.5s sound cue, illumination of the reward-aperture and delivery of reward. Additional responses on the correct response aperture or any of the other apertures, prior to reward retrieval were recorded as perseverative responses. Following a correct response and detection of the next immediate head entry into the 6 ACS Paragon Plus Environment

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reward- aperture, mice had 5s to retrieve the reward before the reward- aperture light turned off, the dipper retracted, and the next trial began. Failure to nose-poke on any illuminated or non-illuminated aperture during a trial was recorded as an omission trial. Sessions were terminated after 50 trials or when the session timer reached 30 min. (C) 5-CSRTT Test Schedule: Mice were tested every day in the 5-CSRTT. After initial, no treatment, baseline training (10s LD, 5s ITI), mice were pre-screened for baseline impulsiveness by increasing the ITI to 7s for three sessions and averaging percentage of premature responses. The 7s ITI sessions were separated by two 5s ITI sessions to bring the mice back toward normal behavior16. Mice were separated into treatment groups to give an even distribution of baseline impulsiveness, with outliers being excluded from the study. LD decreased to 5s for 1-2 weeks of training, then decreased to 2s LD for the final training weeks. After the final training week, the animals were tested using a variable ITI. Ethanol Vapor: Mice were given one week of baseline testing (10s LD, 5s ITI) to show stable performance. Immediately following the last session, mice were injected (i.p.) with 1.5 g/kg ethanol (EE mice) or saline solution (AE mice) before being placed into an ethanol vapor chamber set to a level of 0.19-0.25 g/dl as tested on a standard breathalyzer (Intoximeters Inc, St. Louis MO), with the saline mice being placed into a chamber with clean air. Mice spent 4 hours in the chamber before being placed back into animal housing. Blood alcohol levels reached up to 200 mg/dl during ethanol treatment and after removal from the vapor chamber. An ethanol vapor treatment occurred each day after behavioral testing (from 1:00 p.m. to 5:00 p.m.) to avoid ethanol-intoxicating effects on 5-CSRTT, which were continued for thirty-five days while mice were in the acquisition phase as well as for the seven days of impulsivity testing. Importantly, approximately sixteen hours had elapsed from the time of exiting the ethanol vapor before the next of 5-CSRTT session (9:00 a.m. to 12:00 p.m.). Label Free Proteomics (A)Gel Electrophoresis and Trypsin digest: Animals were sacrificed approximately 30 minutes after their final 5-CSRTT impulsivity testing session. The ACC tissue was immediately extracted after sacrifice and snap frozen using dry ice. The frozen tissue 7 ACS Paragon Plus Environment


was then homogenized in a Storm 24 magnetic Bullet Blender for 3 min at a speed setting of 4 (next Advance Inc., Averill Park NY, USA) with 1 scoop (50 µl) of 0.5 mm ZrO2 oxide beads and 50-70 µl of Cell-lytic MT mammalian tissue extraction reagent (Sigma-Aldrich) containing 50 mM Tris buffer (pH 7.4), 2 mM EDTA, 5 mM EGTA, and 0.1% SDS. In addition, the buffer also contained Complete (Roche) protease inhibitor cocktail and phosphatase inhibitor cocktails type II and III (Sigma-Aldrich). Homogenates were then centrifuged at 16,000g for 15 min at 4⁰C. The supernatant was extracted and protein concentration was determined using Bradford assay and 20 ug of each sample was loaded onto a 4-12% Bis-Tris poly-acrylamide gel (Invitrogen, Carlsbad, CA) for electrophoresis. Gel was stained with BioSafe Coomassie according to directions supplied by company (BioRad, Hercules, CA), each gel lane was divided into 6 segments down the length of the lane. Each gel segment (6 per sample) was excised, cut into 1-2mm pieces and transferred to 0.5 ml tubes for in-gel digest. Proteins were destained with 40% acetonitrile with 50mM Tris (pH 8.1) until clear, reduced with 50 mM TCEP in 50 mM Tris (pH 8.1) for 40 minutes at 60°C, followed with alkylation using 25 mM iodoacetamide in 50 mM Tris (pH 8.1) for 40 minutes in the dark at room temperature. Proteins were digested in-situ with 0.16 ug trypsin (Promega Corporation, Madison WI) in 25 mM Tris pH 8.1 and 0.0002% Zwittergent 3-16, overnight at 37C, followed by peptide extraction with 2% trifluoroacetic acid and acetonitrile. Extractions were dried and stored at -20°C. (B) Mass Spectrometry: Dried trypsin digested samples were suspended in 0.2% formic acid/0.1% TFA/0.002% zwittergent 3-16. A portion of the sample was analyzed by nano-flow liquid chromatography electrospray tandem mass spectrometry (nanoLC-ESI-MS/MS) using a Thermo Scientific Q-Exactive Mass Spectrometer (Thermo Fisher Scientific, Bremen, Germany) coupled to a Thermo Ultimate 3000 RSLCnano HPLC system. The digest peptide mixture was loaded onto a 330 nL Halo 2.7 ES-C18 trap (Optimize Technologies, Oregon City, OR). Chromatography was performed using A solvent (98% water/2% acetonitrile/0.2 % formic acid) and B solvent (80% acetonitrile/10% isopropanol/10% water/0.2% formic acid), over a 2% to 45% B gradient for 90 minutes at 400 nL/min through a PicoFrit (New Objective, Woburn, MA) 100 m x 33 cm column hand packed with Agilent Poroshell 120 EC C18 packing (Agilent Technologies, Santa Clara, CA). Q-Exactive 8 ACS Paragon Plus Environment

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mass spectrometer was set to acquire an ms1 survey scans from 350-1600 m/z at resolution 70,000 (at 200 m/z) with an AGC target of 3e6 ions and a maximum ion inject time of 60 msec. Survey scans were followed by HCD MS/MS scans on the top 15 ions at resolution 17,500 with an AGC target of 2e5 ions and a maximum ion inject time of 60 msec. Dynamic exclusion placed selected ions on an exclusion list for 40 seconds. (C) Data Analysis: MaxQuant software (Max Planck Institute of Biochemistry, Martinsried, Germany), version 1.5.1.2, was used to database search, time align, and peak extract information from generated mass spectrometry files 17. An in-house script written in R programming language performed differential expression analysis using protein group intensities. First, protein group intensities of each sample were log2 transformed and normalized using a 5% trimmed mean method (Fig. S3). For each protein group, the normalized intensities observed in two groups of samples were modeled using a Gaussian-linked generalized linear model. A t-test was used to detect the differentially expressed proteins between pairs of experimental groups. Differential expression pvalues were FDR corrected using Benjamini-Hochberg-Yekutieli procedure. Protein groups with a p-value of < 0.05 and an absolute log2 ratio of at least 1.4 were considered as significantly differentially expressed.



Statistical Analysis 5-CSRTT testing sessions were analyzed by two-way repeated measures analysis of variance (ANOVA) followed by Bonferroni's multiple comparisons test, where appropriate. A two-way ANOVA was used to compare AE and EE fixed and variable impulsivity testing. All statistical analysis was calculated using Prism 7.0 software (GraphPad Software Inc., San Diego, CA, USA). Statistical significance was set at p < 0.05. Gene set enrichment analysis statistical significance was set at p < 0.05 and a false discovery rate (FDR) of < 0.25. Statistical analyses resulting from associations between reference and identified protein data with canonical pathways or upstream were performed by the IPA function analysis algorithm using a right-tailed Fisher’s Exact Test, where p≤ 0.05 was considered statistically significant.


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Figure 1. Label free proteomic workflow. A) Behavioral schedule and premature responding of air-exposed (AE) and ethanol-exposed (EE) mice. *p

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