PROTEOMIC ANALYSIS OF THE EFFECTS OF CEFTRIAXONE ON COCAINE INDUCED MITOCHONDRIAL DYSFUNCTION IN THE NUCLEUS ACCUMBENS By LESLIE HOWARD A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FUL FILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2018
2018 Leslie R. Howard
To my grandmother, Yvonne George
4 ACKNOWLEDGMENTS This research was funded by DA0372 70 from the National Institute of Health ( NIDA) awarded to Lori Knackstedt. I thank Lizhen Wu and Peter Hamor for their assistance on this project. Also, a thank you to Dr. Lori Knackstedt, for her guidance during the project, as well as Dr. Kevin Wang and Dr. Marek Schwendt for advising via committee membership.
5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 6 LIST OF FIGURES ................................ ................................ ................................ .......... 7 LIST OF ABBREVIATIONS ................................ ................................ ............................. 8 ABSTRACT ................................ ................................ ................................ ..................... 9 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 11 The Operant Self administration, Extinction reinstatement Model .......................... 11 Glutamate and Addiction ................................ ................................ ......................... 13 Glutamate and Ceftr iaxone ................................ ................................ ..................... 14 Proteomics ................................ ................................ ................................ .............. 18 2 MATERIALS AND METHODS ................................ ................................ ................ 21 Subjects ................................ ................................ ................................ .................. 21 Drugs ................................ ................................ ................................ ...................... 21 Surgical Procedures ................................ ................................ ............................... 21 Cocaine Self Administration Extinction Training ................................ ..................... 22 Proteomic Assays ................................ ................................ ................................ ... 23 Western Blotting ................................ ................................ ................................ ..... 24 Statistical Ana lysis ................................ ................................ ................................ .. 24 3 RESULTS ................................ ................................ ................................ ............... 27 Self Administration and Extinction ................................ ................................ .......... 27 Proteom ics ................................ ................................ ................................ .............. 27 Western Blots ................................ ................................ ................................ ......... 30 4 DISCUSSION ................................ ................................ ................................ ......... 45 Mitochondria and Cocaine ................................ ................................ ...................... 48 Conclusions ................................ ................................ ................................ ............ 51 LIST OF REFERENCES ................................ ................................ ............................... 55 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 61
6 LIST OF TABLES Table page 2 1 Primary and Secondary Antibodies. ................................ ................................ ... 26 3 1 Molecular weight, Mascot Score, Protein Abundance and Abundance ratio fo r ATP5A, Cox6c, and Cox4i1 proteins in Saline, Cocaine, Ceftriaxone, and Cocaine ceftriaxone groups ................................ ................................ ................ 33 3 2 A selection of t he expression values of dysregulated proteins across the Cocaine saline, Saline ceftriaxone and Cocaine ceftriaxone groups by fold change, relative to Saline vehicle controls ................................ ........................ 34 4 1 Protein Abundance of Dysregulated Proteins Mitochondrial Complex V ............ 54 4 2 Dysregulated Proteins Mitochondrial Complex IV ................................ ............... 54
7 LIST OF FIGURES Figure page 3 1 Behavioral Data. ................................ ................................ ................................ 35 3 2 Protein Abundance ................................ ................................ .......................... 36 3 3 Top Canonical Pathways for each group ordered from most activated to least. ................................ ................................ ................................ ................... 37 3 4 Mitochondrial Dysfunction Pathway Cocaine. ................................ ..................... 38 3 5 Mitochondrial Dysfunction Pathway Ceftriaxone.. ................................ ............... 39 3 6 Mitochondrial Dysfunction Pathway Cocaine Ceftriaxone.. ................................ 40 3 7 Long Term Potentiation Pathway extracted to visualize directional change in altered proteins of this pathway.. ................................ ................................ ........ 41 3 8 Western blot analysis of ATP5a expression for Cocaine saline, Cocaine ceftriaxone, and Saline control groups. ................................ .............................. 42
8 LIST OF ABBREVIATIONS Cox Cytochrome C Oxidase EXT IM Kg/mg MGluR2/3 Extinction IntraMuscular Kilograms/milligram Group II metabotropic glutamate receptors NAc Nucleus Accumbens Core PFC Prefrontal Cortex PSD Postsynaptic Density SA Self Administration SPSS Statistical Package for the Social Sciences TTX Tetrodotoxin xCt Cystine glutamate Exchanger
9 Abstract of Thesis Presented to the G raduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science PROTEOMIC ANALYSIS OF THE EFFECTS OF CEFTRIAXONE ON COCAINE INDUCED MITOCHONDRIAL DYSFUNCTION IN THE NUCLEUS ACCUMBE NS By Leslie R. Howard May 2018 Chair: Lori Knackstedt Major: Psychology C ocaine self administration followed by extinction leads to a decrease in basal extracellular glutam ate in the NAc of the rat brain. Relapse to cocaine seeking is accompanied b y gl utamate efflux in the NAc. Ceftriaxone prevents the decrease of extracellular glutamate during extinction and attenuates re instatement to cocaine seeking while preventi ng glutamate efflux in the NAc. C eftriaxone holds great promise as a pharmacotherapy fo r cocaine relapse but little is known about protein interactions it targets. via label free proteomic analysis 607 proteins displayed altered expression as a function of cocaine self administration, 721 as a function of ceftriaxone and 728 as a function of cocaine ceftriaxone R esults indicated proteins in the Mitochondrial Dysfunction canonical pathway were most significantly altered. Three p r oteins from this pathway were validat ed with western blot s : Cox6c, Cox4I1, and ATP5a. Results revealed increased ATP5a expression in both cocaine groups relative to controls, in contrast with the prot eomic analysis pattern where ATP 5a had the highest abundance for control s and the lowest for the ceftriaxone group. Western blot r esults suggest that protein expression for Cox6c is altered in both cocaine groups compared to controls
10 and that Cox6c expression is signific antly decreased after cocaine. Although inconsistencies are shown between pro teomic and western blot data, overall, results indicate ceftriaxone treatment following cocaine self administration and extinction does not restore Cox4i1 protein levels to normal Exploration of these protein changes helps elucidate the role of mitochondr ia in cocaine addiction and understand underl ying connections of ceftriaxone.
11 CHAPTER 1 INTRODUCTION Cocaine is one of the most widely abused illicit drugs in the United States. According to the National Survey on Drug Use and Health, in 2016 an estimate d 14.4 percent of people ages 12 or older used cocaine in their lifetime. Adults aged 18 25 years old have the highest rate of current cocaine use compared to all other age groups, with 1.6 percent reporting cocaine use in the past month and 5.6 percent reporting cocaine use in the past year (NIDA 2017) Cocaine U s e D isorder (CUD) is characterized as a chronic relapsing disorder, as the recidivism rate for cocaine abuse is very high. For example, one study looking at 5 year follow up treatment outcomes for cocaine addiction in the United States reported that 42% of subjects had used cocaine in the past year (Simpson et al. 2002) Other studies consistently report cocaine to account for the majority of admissions for drug use treatment and notably, 40 50% of these patients return for treatment (Anglin et al. 1997; Hser et al. 1998) The Operant Self administration, Extinction reinstatement Model Animal mo dels of relapse to drug seeking have been developed to investigate neurobiological mechanisms underlying human relapse as well as to elucidate potential pharmacological treatments for relapse to drugs of abuse. Animal models of human drug taking should me et the following criteria: behaviors are contingent upon drug delivery, behaviors are provoked and sustained by drug delivery, and drug delivery increases the frequency of those behaviors. The self administration paradigm meets these criteria and appropr iately models the reinforcing effect of drugs seen in humans (Lovinger 2011) In this model, animals first go through operant drug self administration in which drug delivery is con tingent upon an operant response (e.g. lever press) and
12 paired with a cue (light and tone). A behavior of interest commonly studied is drug relapse after a period of abstinence; a widely used animal model of this behavior is the extinction reinstatement m odel. After acquisition of self administration, animals go through extinction training in which no drugs or cues are offered upon performance of the operant response in order to extinguish the drug seeking behavior. After animals meet extinction criteria the animals undergo a reinstatement test which can include exposure to stress, previously associated drug related cues, or the drug itself. Drug seeking is quantified by the number of responses (lever presses) during the reinstatement test which is thou ght to mimic drug seeking behavior seen in human relapse (de Wit and Stewart 1981; Stewart and de Wit 1987) Pharmacologically based studies often use this model and administer experimental treatment during extinction training in order to test its effect on drug seeking behaviors (lever pressing) seen during the reinstatement testing. Additionally, reinstateme nt models allow for methodological variations which primed reinstatement, lever pressing (operant response) during testing lead to contingent presentations of the tone light (discrete) cue but no drug delivery (Davis and Smith n.d.; Shaham et al. 2003) Non contingent discrete cue presentation variations have proven to be less effective (Meil and See 1996) Drug primed reinstatement tests involve the non contingent chamber where lever presses result in no drug or cue delivery. There is good correspondence between the events that provoke relapse in humans and the events that induce reinstatement in laboratory rodents (Shaham et al. 2003)
13 Glutamate and A ddiction Glutamate is the most common excitatory neurotrans mitter in the brain but previous literature regarding addiction research mainly focuses on reward circuitry in dopaminergic pathways. However, of late, a large body of evidence has shed light on the involvement of glutamatergic pathways in the brain as me diators of drug seeking behavior. Accordingly, the glutamate hypothesis of addiction states that chronic cocaine use alters glutamatergic transmission in cortical regions associated with drug seeking behaviors present during relapse, specifically prefront al cortex projections to the nucleus accumbens core (NAc) (Kalivas 2004; Nadler 2012) Chronic cocain e exposure initiates changes in fu nction and activity of system xC and glial glutamate transpor ter GLT 1 in the NAc. System xC is a sodium independent, cystine glutamate exchanger located primarily in astrocytes. System xC takes up one molecule of extr acellular cysteine for every one molecule of intracellular glutamate exported into the extrasynaptic space (McBean and Flynn 2001) Baker et al. (2002) hypothesized the cysteine glutamate exchange r to be a primary, non vesicular regulator of extrasynaptic glutamate in the rat NAc; results p rovided evidence of such as a blockade of the exchanger in this region lead to a 60% decrease in extrasynaptic glutamate This study also investigated the relat ionship between the cystine glutamate exchanger and group II metabotropic glutamate receptors (mGluR2/3) w hich are predominantly involved in presynaptic inhibition. Extracellular glutamate levels increased with the infusion of a mGluR2/3 antagonist, while blockade of the exchanger disallowed this antagonist induced accumulation of gluta mate in the extracellular space. T herefore, this suggests that not only is the cystine glutamate exchanger the primary source of extracellular glutamate, but that the gluta mate it releases modulates
14 glutamatergic transmission by providing intrinsic tone on mGluR2/ 3 (Baker et al., 2002). Following extinction from cocaine self administration, extracellular basal levels of glutamate in the NAc of the rat brain are decreased (Baker et al. 2002) The decline in extracellular glutamate seen in cocaine withdrawn animals is associated with a downregulation of the cystine glutamate exchanger, as cocaine self administration has bee n found to reduce the expression of xCT, the catalytic subunit of the cystine glutamate exchanger (Knackstedt et al., 2010). Glutamate has five sodium dependent excitatory amino acid transporters encompassing the solute carrier 1 protein family (SLC1) which participate in its reuptake: EAAT1/GLAST (SLC1A3), EAAT2/GLT 1 (SLC1A2), EAAT3/EAAC1 (SLC1A1), EAAT4 (SLC1A6), and EAAT5 (SLC1A7). The human homologue of GLT 1, EAAT2, is predominantly detected in astrocytes and detected less frequently in n euronal profiles (Spencer and Kalivas 2017; Shigeri et al. 2004) Additionally, GLT 1 is responsible for approximately 90% of extracellular glutamate clearance (Tanaka et al. 1997; Shigeri et al. 2004) Cocai ne extinguished rats show decreased GLT 1 expression in the NAc (Knackstedt et al. 2010). Together this evidence bolsters the hypothesis that cocaine induced changes to key modulators of the glutamate homeostatic control mechanism lead to an imbalance betw een extrasynaptic and synaptic glutamate, in turn affecting plasticity and circuitry (Reissner and Kalivas 2010) Glutamate and Ceftriaxone Ceftriaxone is a beta lactam antibiotic that has been used clinically for bacterial infecti ons such as meningitis for years and is now being used in multiple preclinical animal studies to explore its restorative effect on glutamate homeostasis following drug addiction. Recent evidence suggests that antibiotics belonging to the beta lactam family
15 demonstrate beneficial effects far beyond their known antimicrobial actions; in particular, it was found that some, but not all, beta lactam antibiotics protect against glutamate dysfunction by activating the expression of glutamate transporters (Rothstein et al. 2005) Rothstein et al., 2005 was the first to demonstrate an effect of ceftriaxone on GLT 1. Rodents were given five to seven days of ceftriaxone therapy (200 mg per kg, i.p. daily,) and GLT 1 protein expression was fou nd to have increased by threefold as a result of ceftriaxone treatment. Not only did this study reveal increased GLT 1 protein expression, it also showed that 7 day ceftriaxone treatment led to increased GLT 1 mediated glutamate transport in a dose depend ent fashion in cultured spinal cord slices. In contrast, glutamate transporters GLAST, EAAC1 and EAAT4 were unaffected by ceftriaxone therapy (Rothstein et al. 2005) Knackstedt et al. (2010) tested the effects of ceftriaxone on glutamate uptake, protein expression of GLT 1 and xCT after cocaine self administration and extinction training, as well as reinstatement to cocaine seeking in rats. Rats self administered cocaine for two hour sessions once per day for two weeks follow ed by 3 weeks of extinction training. During extinction rats were treated with ceftriaxone (200 mg/kg) or vehicle. The results of this study showed that glutamate uptake and expression of xCT and GLT 1 were reduced by cocaine self administration and that ceftriaxon e restored xCT and GLT 1 levels. A dditionally results showed that ceftriaxone treatment attenuated cue and drug primed reinstatement of cocaine seeking. Therefore, the mechanism of ceftriaxone is thought to involve an upregulation of xCT on g lial cells, normalizing extrasynaptic glutamate levels. Multiple lines of evidence converge on the findings that ceftriaxone treatment increases the activity of xCT and GLT 1 in the rat
16 NAc and prevents the reduction in basal extracellular glutamate level s seen during extinction from repeated cocaine self administration. In addition, ceftriaxone was shown to prevent glutamate efflux in the NAc during reinstatement compared to rats who did not receive ceftriaxone (Trantham Davidson et al. 2012; Knackstedt et al. 2010) A study by LaCrosse et. al (2016) revealed that ceftriaxone did not significantly effect GLT 1 protein expression in the rat PFC but did increase GLT 1 in the rat NAc. Although the ceftriaxone induced increase in NAc GLT 1 expression was not sufficient to prevent glutamate efflux during relapse, ceftriaxone successfully attenuated relapse in rats after cocaine self administration and abstinence. This study also found that ceftriaxone reduced GluA1 receptor expression in the rat NAc following cocaine, but GluA2 receptors were unaffected. Ultimately, these result suggest that underlying uld be a modification of post synaptic AMPA receptor subunit composition (LaCrosse et al. 2016) LaCrosse et al (2017) used the extinction reinstatement mo del and infused antisense to decrease GLT 1 or xCT protein expression in the NAc in order to determine the role of these proteins in cue induced reinstatement of cocaine seeking and its attenuation by Ceftriaxone. This portion of the study included four g roups all of which self administered cocaine: Veh Ctrl, Veh xCT AS, Cef Ctrl, and Cef xCTAS. The only group that did not reinstate lever pressing during testing was the Cef Ctrl group, which indicates that xCT knockdown prevents the ability of ceftriaxon e to attenuate reinstatement to cocaine seeking. The same experimental procedures were followed to knockdown GLT 1 with results again showing the Cef Ctrl group to be the only one susceptible to the attenuating effects of ceftriaxone on drug seeking and i ndicating the vital role of GLT 1 in its effectiveness. Ceftriaxone
17 did not attenuate reinstatement and did not upregulate GLT 1 for intra NAc xCT knockdown rats but did yield an increase in AMPA receptor subunits GluA1 and GluA2 expression. Ceftriaxone also failed to attenuate reinstatement and upregulate xCT expression in intra NAc GLT 1 knockdown rats while there was no effect on GluA1 and GluA2 expression in this group. xCT knockdown in the NAc, without cocaine or ceftriaxone treatment, resulted in a n increased expression of GluA1 and GluA2 receptor subunits, while GLT 1 expression was unaffected. This study also found that ceftriaxone does not increase GLT 1 and xCT expression via transcriptional mechanisms (LaCrosse et al. 2017) Further evidence supporting the necessity of GLT 1 for the efficacy of ceftriaxone com es from a study conducted by Fischer et al. (2013) in which it was shown that blocking GLT 1 in the rat NAc reverses treatment induced attenuation of cocaine seeking (Fischer et al. 2013) Ceftriaxone has proven to be an effective treatment for a number of substance use disorders other th an cocaine use disorder. Alcohol craving has been consistently associated with increased basal extracellular glutamate in the NAc. One study determined Ceftriaxone treatment administered to ethanol preferring rats induced upregulation of xCT and GLT 1, w hich ensued an approximate five fold reduction of ethanol intake (Rao et al. 2015) Ceftriaxone has been shown to reduce reinstatement to morphine, methamphetam ine and nicotine as well (Alajaji et al. 2013; Fan et al. 2012; Abulseoud et al. 2012; Weiland et al. 2015) Opiate dependence, similarly to cocaine, is mediated by increased glutamatergic activity. One group of researchers tested the eff icacy of ceftriaxone to inhibit development of morphine physical dependence in rats. In this study, morphine (20mg/kg) or saline was injected two times per day for 10 days.
18 Ceftriaxone (150, 200 mg/kg) was given to both groups daily during morphine expos ure. Naloxone (10mg/kg) was given to each rat 1, 48, and 96 hours after the last morphine injection. Naloxone induced withdrawal symptoms that were inhibited by ceftriaxone, even after the Naloxone injection at hour 96. These results suggest that ceftri axone inhibits development of morphine physical dependence possibly due to its actions on glutamatergic transmission in the CNS (Rawls et al. 2010) Ceftriaxone has also shown disease, stroke, and epilepsy due to its elevating actions on synaptic glutamate (Leung et al. 2012; Thne Reineke et al. 2008; Jelenkovic et al. 2008) Despite promising evidence of the behavioral efficacy of ceftriaxone treatment, the ne urobiological mechanism of its action underlying attenuation to drug seeking behaviors is yet to be established and therefore, identifying specific, high affinity targets for ceftriaxone could greatly influence therapeutic development for addiction and oth er neurological behavioral disorders. Lacrosse et al., (2017) showed that upregulation of xCT and GLT 1 by ceftriaxone is not a result of increased transcription. Therefore, this study seeks to detect neurobiological targets of ceftriaxone aside from xCT and GLT 1 using proteomic assays to reveal differences in protein expression. Proteomics Proteomics is the study of proteomes and their function. While much extant literature has assessed gene and consequent protein expression as a result of cocaine a dministration in humans and in animals, there are significantly less studies evaluating drug induced comprehensive changes in protein expression patterns for specific brain regions using high throughput proteomic techniques. New proteomic technology has a llowed for researchers to evaluate drug induced changes across thousands of proteins
19 in defined brain regions and to generate expression profiles of susceptible neuroanatomical substrate s. For example, Hemby et al. (2006), performed an integrative proteom ic analysis of the NAc in rhesus monkeys following cocaine self administration. This study used two dimensional difference in gel electrophoresis (2D DIGE) to compare protein abundance in the NAc between cocaine self administering monkeys and controls and found increased protein levels of protein kinase C isoform, adenylate kinase isoenzyme 5, mitochondrial related proteins and others in the cocaine exposed monkeys. Whereas cocaine soluble N ethylmaleimide sensiti ve factor attachment protein and both neural and non neural enolase. A complimentary proteomic approach wherein MALDI TOF TOF was utilized for protein identity confirmation, identified 15 cocaine altered phosphoproteins. Significantly upregulated protein s included glutathione S transferase, brain type aldolase and others. In contrast to the aforementioned assay, the proteomic approach revealed a significant reduction of several mitochondrial proteins. Altogether, these studies as well as others investig ating the effects of cocaine with proteomics indicate corresponding dysregulation of many proteins related to cell metabolism, mitochondrial function, and signaling (Tannu et al. 2010) Reissner & Uys et al. (2011) also explored NAc proteomic changes, but in rats, after cocaine self administration followed by extinction training. An 8 plex iTRAQ (isobaric tag for relative and absolute quantitation) proteomic screen was used to compare the postsynaptic density enriched subfraction of the NAc between cocaine trained animals and yoked saline control subjects; 42 proteins in this region were significantly different. A number of proteins related to cell signaling and metabolism were dysregu lated as a function of cocaine self
20 administration, including the following: Cox5b, ATP5a1, Cox6b, Cox6, NDUfa10, Na + /K + ATPase a3 subunit, Cox5a, VDAC1, and AKAP79/150. AKAP79/150 (human ortholog AKAP5), one of 27 upregulated proteins in the experiment al group, was selected for a functional analysis as AKAPs (A kinase anchor proteins) contribute to PKA signaling which is vital to the acquisition of many addiction related behaviors such as self administration (Hyman et al. 2006) Inhibition of AKAP signaling in the NAc via downregulation of AKAP79/150 reduced reinstatement to drug seeking and showed a significant reduction in the p ostsynaptic density matter of protein kinase A as well as surface expression of GluR1 in cocaine self administering animals (Reissner et al. 2011) Work is currently underway to assimilate proteomic and genomic databases in a syste mic way, as the application of these techniques can fill in our understanding of the biological mechanism leading to cocaine substance use disorder and help elucidate novel therapeutic targets for treatment of addiction (Hemby 2006) The mechanism of action of ceftriaxone and how it regulates glutamate homeostasis is presently unknown. Here we are the first to investiga te the effects of chronic ceftriaxone administration in rodent NAc using high throughput proteomic technology.
21 CHAPTER 2 MATERIALS AND METHODS Subjects Thirty six individually housed male Sprague Dawley rats (Charles River Laboratories) weighing 250 300g in a temperature controlled vivarium on a 12h light/dark cycle were used in this study. Twelve rats divided into four groups were used for the proteomic analysis with n = 3 per group : Coca ine Ceftriaxone, Cocaine Vehichle Saline Ceftriaxone, and Saline Control The remaining 24 rats were divided equally between Cocaine Vehichle, Cocaine Ceftriaxone, and Saline C ontrol groups and used for western blot analysis. Our lab previously showed that ceftriaxone treatment has no effect on xCt and GLT 1 when giv en to rats who do not go through cocaine self administration; therefore, western blots were not performed on tissue from Saline ceftriaxone grouped rats. Animals were provided 20g of food/day with ad libitum water. All methods used comply with the Nation al Institute of Health Guide for the Care and Use of Laboratory Animals Drugs Cocaine hydrochloride was dissolved in 0.9% physiological saline at a concentration of 4 mg/mL. During self administration, subjects received 0.35 mg cocaine/infusion. Ceftri axone (Nova Plus, Irving, TX) was dissolved in saline; rats were treated with 200 mg/kg IP. Surgical Procedures Immediately prior to jugular vein catheter implantation surgeries, rats were anesthetized with ketamine HCl (87.5 mg/kg, IM) and xylazine (5mg/k g, IM) and administered Ketorolac (3 mg/kg, IP) for analgesia. Catheters, SILASTIC silicon tubing
22 (Dow Corning, Midland, MI), with an inner diameter of 0.51mm and an outer diameter of 0.94 mm were implanted into the right jugular vein. The catheter was s ecured with sutures, exited the vein, and passed subcutaneously between the scapulae where it exited through the skin on the back. Stainless steel cannulas held stable within harnesses (Instech, Plymouth Meeting, PA) were then attached to the catheter. P roceeding surgery, catheters were immediately flushed with 0.1mL Heparin (100 units/mL). Ketorolac (2 mg/kg, IP) was post operatively administered for 3 days for pain management. The antibiotic, Cefazolin (100 mg/mL), was post operatively administered vi a catheter for 5 days. Rats were allowed to recover for 5 days before the initiation of the experimental protocol. Cocaine Self Administration Extinction Training Rats were trained to self administer cocaine in standard, two lever operant chambers (30 x 24 x 30 cm; Med Associates, St. Albans, VT) on a FR 1 schedule of reinforcement in which each active lever press (right lever) yielded an intravenous infusion of cocaine (.35 mg/infusion) (or yoked saline for control counterparts) paired with the delive ry of an auditory (tone; 2900 Hz) and a visual (stimulus light; above active lever) cue. The stimulus light signified the time out period of 20 seconds during which active lever presses were recorded but did not result in drug delivery. Inactive lever pr esses (left lever) were recorded but never reinforced. Self administration sessions 2h session for 12 days to be included in further data analysis. Rats then began ext inction training in which active lever presses did not yield drug reward and did not produce drug paired cues. Heparin was also used to flush the catheters after post operative medications and before and after each self administration session for catheter
23 patency. Rats next unde rwent extinction training for 13 days. Ceftriaxone (200 mg/kg IP) was given to half of the cocaine rats and half of the saline rats immediately following the session during the last 6 days of extinction, while the remaining rats w ere injected with vehicle (Veh; 0.9% physiological saline, 1 mL/kg, IP). Animals were euthanized via rapid decapitation 24h after the last ceftriaxone/vehicle injection which is in accordance with our previous behavioral and molecular assessments of ceftr iaxone treatment (Bechard et al. 2017; LaCrosse et al. 2017; LaCrosse et al. 2016) As discussed in detail below, the NAc was dissected and frozen for global proteomic analysis or western blotting. Our rats did not undergo reinstatement testing because the aim of this study was not to explore the behavioral efficacy of treatment but rather to explore the proteomic effect of chronic cocaine us e and whether or not treatment, previously proven to be effective in behavioral tests (reinstatement), could reverse respective protein alterations. Proteomic A ssays The NAc was dissected and frozen for global proteomic analysis. Samples were processed using in StageTip method for digestion and peptide purification. A label free proteomics analysis was performed using nanoelectrospray ionization (ESI) tandem MS with a LTQ Orbitrap Elite mass spectrometer, controlled by Xcalibur software. Mass spectra pro cessing was performed using Proteome Discoverer 126.96.36.1998. The generated de isotoped peak list was submitted to an in house Mascot Server 2.2.07 for searching against the rattus SwissProt database (TaxID=10090, released 2017 05 10. 52,539 sequences). Masco t search parameters were set as follows: species, rattus; enzyme: trypsin; maximal two missed cleavage; mass tolerance: 20 ppm for precursor peptide ions and 0.4 dalton tolerance.
24 Western Blotting Proteins were separated using 4 20% SDS PAGE and transfer red to PVDF membrane. The membrane was blocked in 5% milk/Tris buffered saline with 0.1% Tween20 and probed overnight at 4C with primary antibody against ATP5a (1:5000, abcam ab110273), Cox6c (1:200, abcam ab110267), or Cox41i (1:500, Protein Tech 11242 1 AP). Membranes were next washed with TBS Tween and incubated with anti goat mouse (Cox6c and ATP5a) or rabbit (Cox4i1) secondary antibody (1:7000) at room temperature for 2h. Calnexin was used a loading control for all proteins. ImageJ software was use d to analyze band density. Table 2 1 contains antibody information. Statistical Analysis SPSS (IBM, Armonk, NY) was used to analyze behavioral and molecular data, with alpha set to p<0.05. Inactive and active lever presses during self administratio n and extinction as well as number of infusions during self administration were compared between Groups using repeated measures (RM) analysis of variance (ANOVA), with Time as the within subjects factor. Western blot data was analyzed using one way ANOVA. For all proteomic analyses, only proteins with a mascot score >30 were included. The mascot score includes different measures of protein identification to score how well the experimental data match the database sequences. Greater mascot scores indicate a greater confidence that identified proteins are true proteins. Peptide identification can be used to signify approximate protein quantity in the sample protein abundance gauges with the number of identified peptides. The protein abundance index is the number of peptides observed by Mass Spectrometry divided by the number of peptides that are theoretically observable Ishihama et al. (2005) showed that the
25 quantity of protein in a sample being studied is correlated with the exponential of the protein a bundance index (Ishihama et al. 2005)
26 Table 2 1. Primary and Secondary Antibodies Primary Origin Cat. # Conc. Secondary Conc. ATP5A ABCam 110273 1:5000 Mouse 1:7000 COX6C ABCam 110267 1:2000 Mouse 1:7000 COX4i1 Protein Tech 11242 1 1:5000 Rabbit 1:7000 Calnexin 1:5 0 000 Rabbit 1:40 000
27 CHAPTER 3 RESULTS Self Administration and E xtinction RM ANOVA of self administration inactive lever presses revealed no significant effect of time (F(3,13)=.2093, p=.115) or group X time interact ion (F(3,13)=.822, p=.491). There was a significant effect of group, (F(1,13)=8.072, p=.014). RM ANOVA of self administration active lever presses ( Fig. 3 1A ) revealed a significant effect of time (F(3,13) =3.827, p=.014 and no significant effects for gr oup X time interaction (F(3,13) =1.82, p=.330) or group (F(1,13)=.001, p=.972). RM ANOVA of self administration number of infusions ( Fig. 3 1B ) revealed a significant effect of time (F(3,13)=10.004, p=.000 and no significant effects of group X time intera ction (F(3,13)=.916, p=.448) or group (F(1,13)=.041, p=.843). There were no significant differ ences in lever presses between C ocaine Vehichle and Cocaine C eftriaxone groups during extinction ( Fig. 3 1 C ). RM ANOVA of extinction inactive lever presses reve aled a significant effect of time (F(2,13)=13.768, p=.000 and no significant effects of group X time interaction (F(2,13)=.376, p=.729) or group (F(1,13)=.001, p=.971). RM ANOVA of extinction active lever presses revealed a significant effect of time (F(2 ,13)= 6.431 p=.005 and no significant effects of group X time interaction (F(2,13)=.335, p=.728) or group (F(1,13)=.671, p=.427). Proteomics Table 3 1 is a selected compilation of significantly dysre gulated proteins for the Cocaine Vehicle, Saline Ce ftriaxone, and Cocaine C eftriaxone groups. Overall, 607 proteins displayed altered expression and had a mascot score >30 as a function of cocaine self administration, 721 proteins as a function of ceftriaxone and 728 proteins
28 as a function of cocaine and ceftriaxone treatment combined; in all cases a 2 fold change cutoff is applied to select differentially expressed proteins between groups. The abundance of proteins of interest for each group are presented in ( Fig. 3 2 ). Our data showed the top tw elve canonical pathways of the proteins significantly dysregulated by cocaine, ceftriaxone, and cocaine ceftriaxone groups ( Fig. 3 3 ). The Mitochondrial Dysfunction pathway was most significantly changed in all three groups, prompting our further investiga p ) > 16 ; Fig. 3 3 A p ) > 17 ; Fig. 3 3 B ], and cocaine ceftriaxone group p ) > 20 ; Fig. 3 3 C ]. This pathway was the most significantly affected in the cocaine ceftriaxone group. Altho ugh Mitochondrial Dysfunction showed the most significant changes in proteome compared to other canonical pathways, the results indicated no direction to the change, meaning a proportionate number of pro teins in the pathway were up versus downregulated F igures 3 4 3 5, and 3 6 display changes in protein expression by color for the Mitochondrial Dysfunction pathway. As it can be seen in the aforementioned figures, an overall trend of protein upregulation (red) nor protein downregulation (green) is presen t; neither color is more represented than the other for any of the three groups. Interestingly, however, many top ranked canonical pathways show directional change induced by ceftriaxone. In figure 3 3, orange represents a positive z score meaning protei ns in these pathways showed significantly increased activation; whereas blue represents a negative z score meaning proteins in these pathways showed significantly decreased activation compared to Saline Controls. As it can be seen in figure 3 3, many path ways showing increased activity (orange) for the Cocaine Vehicle group compared to the Saline Control group (3 3 A ) such as
29 Synaptic Long Term Potentiation, Protein Kinase A Signaling, CREB Signaling in Neurons, and others showed decreased activity (blue) in the Saline Ceftriaxone group (3 3 B ) as well as the Cocaine Ceftriaxone group (3 3 C ) compared to the Saline control group. For instance, the same pattern of change for the Synaptic Long Term Potentiation pathway can also be observed in figure 3 7 which shows the specific proteins that increased (red) or decreased (green) in expression; overall, with each group shown in in the figure being compared to the Sal Cntl group, specific proteins that were upregulated in the Coc Veh group such as PKC and PLC were down regulated in the Sal Cef group, and most importantly, also downregulated in the Coc Cef group. These results indicate that not only does ceftriaxone work inversely on the same pathways as coca of cocaine, which speaks to the effectiveness of ceftriaxone as a therapeutic agent for cocaine addiction. Specific protein alterations in the Mitochondrial Dysfunction pathway were extracted in order to explore change directionality for individual protei ns in this pathway. As aforementioned, many proteins in the Mitochondrial Dysfunction pathway were changed across all groups; therefore, to aid in protein selection for validation via western blotting, Table 3 2 was created to visualize all the proteins s ignificantly changed in each of the three groups or either differentially changed between the treatment (Coc Cef) and vehicle groups (C oc Veh & Sal C ntl ). ATP Synthase subunit 5 (ATP5) or Complex V of the electron transport chain (ETC), responsible for co nverting ADP to ATP in the presence of a proton gradient across the membrane, has many individual subunits. The independent effect of cocaine and ceftriaxone on ATP5 specific
30 subunit expression varied between the two groups, but portrayed approximate bala nce within groups between increased and decreased expression of subunits (Table 3 2) However, for the cocaine ceftriaxone group, there is an overall trend of increased expression for individual subunits of the ATP5 complex. These generalizations are dis played in Table 3 2 in which negative numbers indicate protein down regulation. It can be seen t hat in the first two columns (C oc V eh ) and ( S al C ef)) that there are almost equal amounts of ATP5 subunits upregulated as there are downregulated within each c olumn. Conversely, in the 3 rd column (Coc C ef), it is shown that all ATP5 subunits are upregulated with the exception of subunit ATP5H. For Cytochrome C Oxidase subunits, specifically Cox6c and Cox4i1, there is a cocaine induced downregulation and ceftri axone induced upregulation. Notably, these proteins are still upregulated in the cocaine plus ceftriaxone group meaning that the effect of ceftriaxone is more powerful than the effect of cocaine on these particular proteins (Table 3 2) Western Blot s A one way ANOVA with a Greenhouse Geisser correction was conducted to compare protein density of ATP5a between all groups and revealed a significant main effect of group (F(2,23)=25. 882, p=.000). Bonferroni Post H oc test showed a significant difference between the Cocaine Vehicle group (n=7) and the Saline Control group (n=9), with a mean difference of 2.01, (p = .0 0 ) There was also a significant difference between the Cocaine Ceftriaxone group (n=8) and the Saline Control group, with a mean difference o f 2.23, (p = .0 0 ) However, Bonferroni Post Hoc test revealed no significant difference between the Cocaine Vehicle group and the Cocaine C eftriaxone group with a mean difference of .216, (p =1 ). These results suggest that cocaine and cocaine plus ceftriax one alter protein expression compared to controls ( Fig. 3 6 ).
31 ATP5a in increased after cocaine. This data indicates that ceftriaxone does not normalize altered ATP5a protein expression after cocaine self administration and extinction. A one way ANOVA wit h a Greenhouse Geisser correction was conducted to compare protein density of Cox6c between all groups and revealed a significant m ain effect of group (F(2,19 )=16.622, p=.000). Bonferroni Post H oc test showed a significant difference between the Cocaine V ehicle group (n= 7 ) and the Saline Control group (n= 8 ) with a mean difference of 0. 51 ( p= .00 ) There was also a significant difference between the Cocaine Ceftriaxone group (n=7 ) and the Saline Control group, with a mean difference of 0.27 ( p = .0 2 ) Bonf erroni Post Hoc test also revealed a significant difference between the Cocaine Vehicle group and the Cocaine C eftriaxone group with a mean difference of 0.24 ( p = .04 ). These results suggest that protein expression for Cox6c is altered in both cocaine tr eated groups (Coc Veh and Coc Cef) compared to controls and that Cox6c expression is significantly decreased after cocaine. Although ceftriaxone treatment did not restore Cox6c protein levels to normal, Cox6c expression significantly increased in rats wh o were treated with ceftriaxone after cocaine self administration compared to rats who did not receive treatment (fig 3 7). A one way ANOVA with a Greenhouse Geisser correction was conducted to compare protein density of Cox4i1 between all groups and reve aled a signific ant main effect of group (F(2,20 )= 4.234, p<.05 ). Bonferroni Post H oc test showed a significant difference between the Cocaine Ceftriaxone group (n=7 ) and the Saline Control group (n=9 ), with a mean difference of 0.28 ( p = .0 3 ) Bonferroni Po st Hoc test revealed no significant difference between the Cocaine Vehicle group (n=7 ) and the Saline Control
32 group, with a mean difference of 0.21 ( p = 15 ) Bonferroni Post Hoc test also revealed no significant difference between the Cocaine Vehicle group and the Cocaine C eftriaxone group with a mean difference of 0.06, ( p =1 ). These results suggest that p rotein expression for Cox4i1 is decreased after cocaine, and further decreased by ceftriaxone ( Fig. 3 8 ). This indicates ceftriaxone treatment followin g cocaine self administration and extinction does not restore Cox4i1 proteins levels to normal
33 Table 3 1. Molecular weight, Mascot Score, Protein Abundance and Abundance ratio for ATP5A, Cox6c, and Cox4i1 proteins in Saline, Cocaine, Ceftriaxone, and Coc aine ceftriaxone groups Description MW [kDa] Mascot Score F1: Saline F2: Cocaine F3: Ceftriaxone F4: Coc Cef Abundance Ratio: (F4) / (F1) Atp5a1 59.7 14041 1.08E+09 4.67E+08 1.37E+08 7.11E+08 0.66 Cox6c2 8.4 265 2.71E+07 6.52E+06 5.44E+06 9.96E+08 36. 75 Cox4i1 19.5 511 1.07E+07 4.62E+06 1.10E+07 5.15E+08 48.28
34 Table 3 2. A selection of the expression values of dysregulated proteins across the Cocaine saline, Saline ceftriaxone and Cocaine ceftriaxone groups by fold change, relative to Saline vehic le controls. Negative signs indicate a decrease in expression value for each protein. Cox4i1had a decrease in expression value in the Cocaine group and an increase in expression value in the Ceftriaxone and Cocaine ceftriaxone groups. Cox6c showed a decr eased expression value in the Cocaine group and an increased expression in the Cocaine Ceftriaxone group. ATP5 subunits did not show any specific patterns of increased or decreased protein expression across groups. Symbol Coc Veh Expression Value Sal Ce f Expression Value Coc Cef Expression Value Complex/ Associated Proteins COX4I1 2.041 3.361 6.611 Complex IV (cox3) COC6C 2.268 49.969 Complex IV (cox3) COX6A1 100 100 Complex IV (cox3) NDUFA5 3.04 2.747 2.71 Complex I NDUAF9 2.363 2.188 Comp lex I NDUFA10 3.1 Complex I NDUFV2 3.396 6.205 4.755 Complex I SNCA 2.256 41.135 100 ATP5C1 12.199 3.288 26.203 Complex V ATP5L 18.182 100 Complex V ATP5D 18.182 44.545 Complex V ATP5G3 5.482 7.871 2.261 Complex V ATP5J2 2.122 6.998 19.900 Complex V ATP5O 3.636 Complex V ATP5B 16.393 Complex V ATP5F1 2.006 4.385 Complex V ATP5A1 5.963 Complex V ATP5H 4.098 Complex V
35 Figure 3 1. Behavioral Data A) Self Administration Active Lever Presses. There was a significant effect o f time, (F(3,13) =3.827, p=.014) ; and no significant effect of group, (F(1,13)=.001, p=.972). This means rats did not differ in number of lever presses between groups and both groups increased operant responding over time. There was no significant interaction of group by time, (F(3,13) =1.82, p=.330). B) Self Administration Infusions. Average number of cocaine infusions during self administration. There was a significant effect of time, (F(3,13)=10.004, p=.000) ; and no significant effects for group, (F(1,13)=.041, p=.843) or group by time interaction, (F(3,13)=.916, p=.448). C) Average number of lever presses during extinction on the previously active lever. There was a significant effect of time, (F(2, 13)=13.768, p=.000)*; and no significant effects of group, (F(1,13)=.001, p=.971) or group by time interaction, (F(2,13)=.376, p=.729). Ceftriaxone or vehicle was administered on day 8 (arrow). 0 20 40 60 80 1 2 3 4 5 6 7 8 9 10 11 12 Right Lever Presses Day SA Active Lever COC-SAL COC-CEF207 0 10 20 30 40 50 1 2 3 4 5 6 7 8 9 10 11 12 Infusions Day SA Infusions COC-SAL COC-CEF207 0 50 100 1 2 3 4 5 6 7 8 9 10 11 12 13 Right Lever Presses Day Extinction Active Lever COC-SAL COC-CEF207 A B C
36 Fig ure 3 2 Protein Abundance A) Change in Protein Abundance of ATP5A, Cox6c, and Cox4i1 across Saline, Cocai ne, Ceftriaxone, and Cocaine ceftriaxone groups. B) Abundance Ratio of ATP5A, Cox6c, and Cox4i1. Protein abundance of the Cocaine ceftriaxone group dived by the protein abundance of the Saline control group. COX4I1 COX6C ATP5A1 0.00E+00 5.00E+04 1.00E+05 1.50E+05 Sal-Cntl Coc-Veh Sal-Cef Coc-Cef x 10000 Protein Abundance COX4I1 COX6C ATP5A1 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 COX4I1 COX6C ATP5A Abundance Ratio: Coc Cef/Sal Cntl A B
37 Figure 3 3 Top Can onical Pathways for each group ordered from most activated to least. log(p value) was used to order pathways. The ratio is the number of proteins that were identified in the experiment in respect to the total number of proteins in the pathway as a percen t. Orange bands represent a positive z score, meaning proteins in these pathways increased in activation; whereas blue bands represent a negative z score in which proteins in these pathways decreased in activation. Gray bands indicate that there is no ac tivity pattern available proteins changed in both directions overall. Mitochondrial Dysfunction was the most activated pathway for the Cocaine, Ceftriaxone, and Cocaine Ceftriaxone groups. No activity pattern is available for the Mitochondrial Dysfunctio n Pathway in either of the three groups. Synaptic Long Term Potentiation pathway increased in activation for the Cocaine group, but decreased in activation for the Ceftriaxone group, and for the Cocaine Ceftriaxone group as well. A)Cocaine Vehich le B)Saline Ceftriaxone C)Cocaine Ceftriaxone
38 Figure 3 4. Mitochondrial Dysfunction Pathway Cocaine Proteins/complexes in red were upregulated; proteins/complexes in green were downregulated. Overall, Cocaine slightly downr egulated mitochondrial complexes I V involved in the electron transport chain. However, some proteins, such as alpha synuclein, GRX2 and others were upregu lated as a function of cocaine.
39 Figure 3 5. Mitochondrial Dysfunction Pathway Ceftriaxone Prot eins/complexes in red were upregulated; proteins/complexes in green were downregulated. Ceftriaxone alone appears to have a similar effect as cocaine on mitochondrial complexes II and III. Ceftriaxone appears to downregulated proteins in complexes I and V more than cocaine does; however, ceftriaxone appears to have a rescuing effect on proteins in complex IV, showing upregulation where cocaine showed downregulation of specific proteins.
40 Figure 3 6. Mitochondrial Dysfunction Pathway Cocaine Ceftriaxone Proteins/complexes in red were upregulated; proteins/complexes in green were downregulated. The combination of cocaine and ceftriaxone appears to have a partially rescuing effect on proteins in complex I, IV, and V that were downregulated by cocaine, a n eutralizing effect on complex II, a similar effect to the other two groups on complex III. These results indicate that ceftriaxone has an effect on many of the same proteins effected by cocaine.
41 Figure 3 7 Long Term Potentiation Pathway extracted to v isualize directional change in altered proteins of this pathway. Proteins altered in the opposite direction for the Ceftriaxone group compared to the Cocaine group persist in the Cocaine ceftriaxone group revealing a rescuing effect of ceftriaxone on this pathway after cocaine us e. Cocaine Ceftriaxone/Cocaine Ceftriaxone
42 Figure 3 8 Western blot analysis of ATP5a expression for Cocaine Vehicle, Cocaine Ceftriaxone, and Saline C ontrol groups. A one way ANOVA revealed a signific ant main effect of group (F(2,21 )=25.882, p=.000). Bonferroni Po st Hoc test revealed a significant difference between Cocaine Vehicle (n=7) and Saline C ontrol (n=9) groups ( p<.05); as well as a significant difference between Cocaine Ceftriaxone (n=8) and Saline C ontrol groups ( p<.05). Ho wever, Bonferroni Post Hoc test revealed no significant difference between Cocaine Vehicle and Cocaine C eftriaxone groups ( p>.05). 100% 301% 323% 0% 50% 100% 150% 200% 250% 300% 350% 400% Sal-Cntl Coc-Veh Coc-Cef Normalized ATP5a Expression
43 Figure 3 7 Western blot analysis of Cox6c expression for Cocaine Vehicle, Cocaine Ceftriaxone, and Sali ne C ontrol groups. A one way ANOVA revealed a significant main effect of group (F(2,19)=16.622, p=.000). Bonferroni Post Hoc test showed a significant difference between the Cocaine Vehicle group (n=7) and the Saline Control group (n=8), ( p <.05) as well as a significant difference between the Cocaine Ceftriaxone group (n=7) and the Saline Control group, ( p<.05). Bonferroni Post Hoc test also revealed a significant difference between the Cocaine Vehicle group and the Cocaine C eftriaxone group ( p < .05). 100.00% 48.85% 73.05% 0% 20% 40% 60% 80% 100% 120% Sal-Cntl Coc-Veh Coc-Cef Normalized Cox6c Expression
44 Figure 3 8 Western blot analysis of Cox4i1 expression for Cocaine Vehicle, Cocaine Ceftriaxone, and Saline C ontrol groups. A one way ANOVA revealed a significant main effect of group (F(2,20)=4.234, p<.0 5). Bonferroni Post Hoc test showed a significant difference between the Cocaine Ceftriaxone group (n=7 ) and the Saline Control group (n=9 ), ( p<.05). Bonferroni Post Hoc test revealed no significant difference between the Cocaine Vehicle g roup (n=7 ) and the Saline Control group, ( p>.05). Bonferroni Post Hoc test also revealed no significant difference between the Cocaine Vehicle group and the Cocaine Ceftriaxone group ( p>.05). 100.00% 78.66% 72.33% 0% 20% 40% 60% 80% 100% 120% Sal-Cntl Coc-Veh Coc-Cef Normalized Cox4i1 Expression
45 CHAPTER 4 DISCUSSION This pro ject took a novel approach to the attempt at unveiling the neurobiological expression after cocaine use. In this experiment, a comparison via proteomic analysis was conducted b etween rats that underwent cocaine sel f administration and rats that received saline during SA. During extinction, half of the rats from each group received ceftriax one treatment and the other half were administered vehicle injections This allowed us to see changes in protein expression in the NAc as a result of cocaine use and the possibility of ceftriaxone being able to reverse the effect of cocaine on some proteins. Our results are consistent with prior literature indicating an independent effect of cocaine and ceftriaxone on mitochondrial dysfunction. There are four mitochondrial complexes involved in the mitochondrial electron transport chain (ETC) which ultimately function to create ATP for cellular energy. Protein complexes I, III, and IV direc tly pump protons from the matrix into the intermembrane space. NADH deposits electrons in complex I; energy is released as electrons move through redox centers within the complex. This energy is then used for proton pumping. Complex II promotes proton p umping in complexes III and IV. FADH2 helps high energy electrons enter complex II ; however the liberated energy is not used to pump protons. Electrons from complexes I and II are transferred to complex III by coenzyme Q. One of the transferred electro ns is recycled by complex III and the other goes through two redox centers within the complex before it is carried to complex IV by Cytochrome C. Electron transport ends in complex IV when four electrons convert
46 oxygen into two water molecules and another four protons are pumped into the intermembrane space. Therefore, oxygen is the final electron acceptor in the ETC. Herein, Mitochondrial Dysfunction was the most dysregulated canonical pathway in all three groups. Therefore, proteins that were altered i n expression and specific to this pathway were extracted for further investigation; it was decided western blot analysis for validation of changes in protein expression would be conducted on proteins from this pathway. Figure s 3 4 3 5, and 3 6 use colors to represent increased versus decreased activation of specific proteins in the Mitochondrial Dysfunction pathway : red indicates an increase in expression value and green indicates a decrease in expression value. According to these figures, there is a dra stic change in protein expression for Complexes IV and V of the Mitochondrial ETC that is unique to each group. Table 4 1 shows the protein abundance of all dysregulated proteins that are in or associated with Complex V of the mitochondrial electron tran spor t chain (ETC) Complex V is mostly made up of ATP associated protein subunits. ATP synthase is made up of two complexes each containing multiple subunits: F1, the catalytic core and Fo, the membrane spanning component The expression values in Table 3 2 show the fold change in express ion for each protein after the Mitochondrial D ysfunction pathway was extracted from the initial results. By looking at these figures and tables combined, it is readily visible that there is increased activation of Compl ex V proteins in the Coc Cef group compared to all other groups This is especially interesting considering that proteins in this comple x show an overall trend of downregulation in the Coc Veh group and in the Sal Cef group; however, in the Coc Cef group, proteins in this complex show patterns of upregulation instead. These results emphasize the importance of
47 interactions taking place between cocaine and ceftriaxone that lead to different proteomic effects in the NAc. Table 4 2 shows the protein abundance of all dysregulated proteins that are in or associated with Complex I V of the mitochondrial electron transpor t chain (ETC) In humans, this complex is known to contain 16 cytochrome c oxidase subunits as well as three mitochondrially encoded cytochrome c oxidases. Table 3 2 shows that there is a decrease in expression value (by fold change) for Cox6c and Cox4i1 in the Coc Veh group compared to the Sal Cntl group. This table also shows that there is an increase in expression value for both of the protein s in the Coc Cef group compared to the Sal Cntl group. According to this table, ceftriaxone treatment and cocaine seem to have opposing effects on Cox6c and Cox4i1 expression. The unique dysregulation of proteins in Complex IV between groups can also be visualized in figures 3 4, 3 5, and 3 6. In accordance with these results, it was decided perform western blotting for a protein from Complex V and Complex IV of the Mitochondrial ETC. ATP5a1 was specifically chosen for western blot analysis due to the unique pattern of protein alteration displayed in the data. Table 4 1 shows that the Sal Cntl group has the highest pr otein abundance for ATP5a, the Coc Cef group has the next high est protein abundance with the Coc Veh group and Sal Cef groups following r espectively. It can be seen in figure 3 2 A that protein levels of ATP5a (gray) in the Coc Cef group are approaching the normalized level seen in the Sal Cntl group. The western blot analysis conducted to validate whether cocaine self administration combi ned with ceftriaxone treatment upregulate d ATP5a, presented inconsistent results compared to the results from the proteomic analysis. Western blot analysis showed that
48 ATP5a density was lowest in the Sal Cntl group with a significant increase in density for each experimental group compared to the controls. These results i ndicate that ceftriaxone does not have a restorative effect on ATP5a after chronic cocaine use. The proteins selected for validation via western blot analysis from Complex IV were Cox6c and Cox4i1. According to table 4 2, the protein abundance of Cox6c in order from highest to lowest for each group is as follows: 1) Coc Cef, 2) Sal Cntl, 3) Coc Veh, and 4) Sal Cef. The protein abundance of Cox4i1 in order from highest to lowest for eac h group is as follows: 1) Coc Cef, 2) Sal Cef, 3) Sal Cntl, and 4) Coc Veh. It should be noted that the protein abundance values for both of these Complex IV proteins are higher in the Coc Cef and Sal Cntl groups compared to the Coc Veh group which indic ates a possible restorative effect of treatment on these proteins; however according to figure 3 2 A the potential restoration of these proteins is not nearly as powerful as the restoration effect seen for ATP5a. Results from the western blot analysis for Cox6c and Cox4i1 are also inconsistent with the results obtained from the proteomic analysis. Western blot data for Cox6c shows the highest protein density in the Sal Cntl group, followed by the Coc Cef group, and lastly the Coc Veh group showed the leas t protein density. Western blot data for Cox4i1 also shows the Sal Cntl group to have the highest protein density. These results also show that the Coc Veh group has the second highest protein density of Cox4i1 and the Coc Cef group has the smallest dens ity. Inconsistencies between proteomic and western blot analyses could be due to experimental procedures and should be further explored in the future. Mitochondria and Cocaine The mitochondrial ETC creates a pH and charge gradient via pr oton pumping. Cocaine has a positive charge at physiologic pH enabling its accumulation in negatively
49 charged organelles such as mitochondria with the capacity to dissipate mitochondrial transmembrane electric potential (Preedy 2017) For example, a study by del Castillo (2009) investigated the vulnerability to cocaine addiction with a proteomic an alysis of the NAc of rats. The study followed a 2 (drug: cocaine or saline) X 2 (procedure: extinguish (E) or no extinguish (NE)) between subjects design in which all four groups underwent cocaine induced condition place preference training before extin guishing or not extinguishing the learned behavior. Directly following the last extinction test, animals were give an injection of cocaine (15mg/kg i.p.) or saline and euthanized by decapitation 10 hours later. The NAc was dissected for proteomic analysi s. The results suggest that proteins involved in the mitochondrial dysfunction pathway were most significantly altered between the groups. For example, in the E saline group compared to the E cocaine group, NDUFA10 protein was upregulated, which is a subu nit of mitochondrial complex I. NE subjects compared to E subjects, showed a down regulation of ATP5a regardless of treatment condition (saline or cocaine) which implies ATP5a could be an ideal biomarker for susceptibility to cocaine induced drug seeking behavior Bierczynska Krzysik et al., (2006) also found a reduction in ATP5a expression in the rat striatum following chronic morphine treatment (Bierczynska Krzysik et al. 2006) Additionally, Alpha synuclein was decreased in the NE cocaine group compared to the NE saline group (del Castillo et al. 2009) Alpha synuclein negatively regulates dopamine transmission; and therefore it has been hypothesized by this group of researchers that cocaine induced reduction of alpha synuclein could be correlated to the attenuation of with prior research generally confirming that brain energy metabolism is perturbed as a
50 result of chronic cocaine use, including modifications in glucose metabolism and dysregulated glutamate homeostas is (Cunha Oliveira et al. 2013; Kalivas 2009) However, little research has been conducted showing the effect of cocaine in brain regions associated wit h drug reward and craving on the fundamental component of brain bioenergetics, mitochondria. One such study by Cunha Oliveira (2013) explored the effect of cocaine on isolated brain mitochondria and found drug induced mitochondrial complex I dysfunction. This study compared the effect of cocaine on brain and liver mitochondria and found that brain mitochondria show more evident cocaine induced inhibition of mitochondrial complex I than liver mitochondria do. Rats used in this experiment were killed after procedures by decapitation and whole brain mitochondria were isolated using a modified version of Rosenthal et al. (1987) method (Cunha Oliveira et al. 2013) Previous studies also demonstrate that long ter m reductions in glucose metabolism induced by cocaine also affect the transcription of genes associated with mitochondrial function in the dlPFC (Lehrmann et al. 2003) Additionally, the use of proteomics has hel ped identify protein interactions related to glutamate and mitochondrial dysfunction in mood disorders such as major depressive disorder (Wang et al. 2016) and bipolar disorder (Robinson et al. 2016) 1 upregulation and ARE 1 transcriptional programs seem to cont ribute to ceftriaxone induced xC upregulation (Zhang et al. 2015; Lee et al. 2008; Lewerenz et al. 2009) but altogether, little is known about central nervous system targets responsible for interactors such as alpha synuclein (Ruzza et al. 2014) One preliminary study used
51 two dimensional difference gel electrophoresis to separate and quantify cytosolic fractions of NAc proteins in human post mortem tissue from cocaine overdose (COD) victims and control tissue; the proteins were identified by matrix assisted laser desorption/ionization time of flight mass spectroscopy (Hemby 2006) Of the identified spots, 11 proteins were upregulated in COD victims compared to healthy controls including DJ1 and ubiquitin car boxyl terminal esterase L1 which are both associated (Abou Sleiman et al. 2006; Hemby 2006) Another protein synuclein, is elevated in human COD victims (Qin et al. 2005) In all, this study makes a significant contribution to cocaine relapse prevention literature by providing knowledge of dysregulated proteins and canonical pathways following chronic cocaine use. Future directions for this work include behavioral and functional analyses of proteins differentially expressed as a result of cocaine use and ceftriaxone treatment. Conclusions In conclusion, our results are similar to previous literature, showing mitochondrial dysfunction after chronic cocaine use and showing many similar proteins within the mitochondrial dysfun ction pathway that are dysregulated. For instance, Uys et al. (2011) found Cox5b, ATP5a1, Cox6b, Cox6, NDUfa10, Na + /K + ATPase a3 subunit, Cox5a, VDAC1, and AKAP79/150 to be dysregulated by cocaine. Our results also show these proteins to be dysregulated by cocaine. Additionally, AKAP79/150 was upregulated as a function of cocaine in the Uys et al. (2011) study; whereas, our data shows AKAP5 to be altered between groups, but the Saline control group shows the highest protein abundance for AKAP5. Uys et al. (2011) also showed that
52 downregulation of AKAP79/150 reduces reinstatement to cocaine seeking. Although no behavioral tests were performed in this experiment, our data does indicate that ceftriaxone could decrease AKAP5 expression, as the protein abun dance for the ceftriaxone only group for this protein is the lowest. In the study performed by del Castillo et al. (2009), a downregulation of ATP5a was seen in animals who did not extinguish drug taking behavior regardless of treatment condition. These results help to explain our western blot findings of no difference in ATP5a expression between cocaine saline and cocaine ceftriaxone groups. We saw significantly reduced expression of ATP5a in the saline control group compared to the two experimental gro extinguished a drug seeking behavior. Results from Cunha Oliveira et al. (2013) showing inhibition of complex I activity following cocaine use bolster our findings shown in Figu re 3 4 that complex I mitochondrial proteins are downregulated after cocaine self administration. Del Castillo et al. (2009) also reported a cocaine induced reduction of alpha synuclein. However, studies on human post mortem tissue from cocaine overdose show elevated levels of alpha synuclein compared to healthy controls (Qin et al. 2005) In this experiment as seen in Figure s 3 4 3 5, and 3 6, alpha synuclein was upregulated in the cocaine group as well as the ceftriaxone and cocaine ceftriaxone groups indicating that although ceftriaxone might interact with alpha synuclein, it does not impose a restorative effect after downregulation vi a cocaine self administration Although studies looking at the effect of cocaine on alpha synuclein presently show in consistencies, the collection of data still converges on the idea that cocaine addiction
53 Together, our results and others show the mitochondria dysfunction pathways to be highly effected by chronic cocaine use. Furthermore, the role each complex of the ETC should be behaviorally explored. Although, the behavioral significance of many of the proteins we found to be dysregulated have yet to be explore d ; it is validating that the same proteins are fo und to be dysregulated by cocaine across many different studies and methods. Ceftriaxone was able to reverse the effect cocaine had on some proteins therefore making it a viable target for coc aine addiction therapy
54 Table 4 1. Protein Ab undance of Dysregulated Proteins Mitochondrial Complex V Accession Description # Peptides Score Mascot: Mascot F1: Sal Cntl F2: Coc Veh F3: Sal Cef F4: Coc Cef Ratio: (F4) / (F1) P15999 Atp5a1 14 14041 1.08E+09 4.67E+08 1.37E+08 7.11E+08 0.66 P07340 At p1b1 4 11586 4.45E+08 2.95E+08 1.81E+08 1.90E+08 0.43 P10719 Atp5b 17 22776 1.42E+09 1.16E+09 3.98E+08 6.47E+08 0.46 Q6PDU7 Atp5l 2 156 7.25E+06 2.87E+05 2.46E+07 3.39 P06685 Atp1a1 16 5614 3.22E+08 8.86E+07 2.82E+07 2.12E+07 0.07 P06686 Atp1a2 1 3 4328 3.49E+06 1.99E+07 4.19E+04 1.20E+04 0.00 D3ZAF6 Atp5j2 2 3554 7.91E+07 1.21E+08 1.69E+08 1.08E+09 13.60 P19511 Atp5f1 P 7 2067 1.11E+08 1.17E+08 1.06E+08 8.31E+07 0.75 P29419 Atp5i 3 2017 1.03E+06 5.98E+08 0.00 P31399 Atp5h 1 1102 3.87E+07 1.67E+07 1.00E+07 6.46E+06 0.17 P11608 Mt atp8 1 97 1.19E+06 7.74E+04 1.79E+06 3.27E+07 27.52 Q5FVI6 Atp6v1c1 5 935 5.75E+07 5.63E+07 4.02E+06 1.90E+07 0.33 Q06647 Atp5o 3 918 2.57E+07 5.22E+06 5.92E+06 4.21E+07 1.64 P11506 Atp2b2 10 839 1.48E+08 5 .97E+07 9.96E+06 1.10E+07 0.07 Q71S46 Atp5g3 1 269 1.42E+06 5.63E+06 3.42E+06 2.19E+06 1.54 Q63377 Atp1b3 2 172 9.73E+06 6.64E+06 1.11E+06 6.95E+06 0.71 P35435 Atp5c1 5 426 1.26E+07 8.02E+07 8.47E+06 1.56E+08 12.31 P35434 Atp5d 2 412 8.80E+06 1.47E +06 4.41E+06 2.87E+08 32.59 Table 4 2 Dysregulated Proteins Mitochondrial Complex I V Accession Description # Peptides Score Mascot: Mascot Abundance: F1: Saline Abundance: F2: Cocaine Abundance: F3: Ceftriaxone Abundance: F4: Coc Cef Abundance Ratio: (F4) / (F1) P11951 Cox6c2 3 265 2.71E+07 6.52E+06 5.44E+06 9.96E+08 3.68E+01 P10888 Cox4i1 4 511 1.07E+07 4.62E+06 1.10E+07 5.15E+08 4.83E+01 P35171 Cox7a2 1 605 1.43E+08 0.00E+00 P11240 Cox5a 5 365 2.87E+07 1.80E+07 9.47E+06 6.55E+08 2.29E+01 P62898 Cycs 4 190 1.33E+04 3.35E+08 0.00E+00 P12075 Cox5b 1 40 5.23E+04 4.09E+06 0.00E+00 P10818 Cox6a1 1 134 2.20E+04 1.80E+04 4.12E+06 2.53E+07 1.15E+03
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61 BIOGRAPHICAL SKETCH Leslie Howard completed a Bachelor of Scie nce degree from Mississippi State University with a major in psychology and a minor in chemistry. During her undergraduate career, Leslie worked in a cognitive psych ology laboratory focused on meta memory and in an analytical chemistry laboratory focused on medical technology. After completion of a Master of Science degree in psychology, Leslie plans to continue her education by pursuing a doctorate degree focused on addiction research.