Individual differences in initial sensitivity to ethanol are strongly related to the heritable risk of alcoholism in humans. To elucidate key molecular networks that modulate ethanol sensitivity we performed a systems genetics analysis of ethanol-responsive gene expression in brain regions of the mesocorticolimbic reward circuit (prefrontal cortex, nucleus accumbens and ventral midbrain) across the BXD RI panel, a highly diverse family of isogenic mouse strains before and after treatment with ethanol. Acute ethanol altered the expression of ~2,750 genes in one or more regions and 400 transcripts were jointly modulated in all three. Ethanol-responsive gene networks were extracted with a powerful graph theoretical method that efficiently summarized ethanol's effects. These networks correlated with acute behavioral responses to ethanol and other drugs of abuse. As predicted, networks were heavily populated by genes controlling synaptic transmission and neuroplasticity. Several of the most densely interconnected network hubs, including Kcnma1 and Gsk3-beta, are known to influence behavioral or physiological responses to ethanol, validating our overall approach. Other major hub genes like Grm3 and Nrg3 represent novel targets of ethanol effects. Networks were under strong genetic control by variants that we mapped to a small number of chromosomal loci. Using a novel combination of genetic, bioinformatic and network-based approaches, we identified high priority cis-regulatory candidate genes, including Scn1b, Gria1, Sncb and Nell2. The ethanol-responsive gene networks identified here represent a previously uncharacterized intermediate phenotype between DNA variation and ethanol sensitivity in mice. Networks involved in synaptic transmission were strongly regulated by ethanol and could contribute to behavioral plasticity seen with chronic ethanol. Our novel finding that hub genes and a small number of loci exert major influence over the ethanol response of gene networks could have important implications for future studies regarding the mechanisms and treatment of alcohol use disorders.

ANTINOCICEPTIVE EFFECTS OF MONOAMINE REUPTAKE INHIBITORS IN ASSAYS OF PAIN-STIMULATED AND PAIN-DEPRESSED BEHAVIOR By Marisa B. Rosenberg A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science at Virginia Commonwealth University. Virginia Commonwealth University, 2012 Advisor: Sidney Stevens Negus, Ph.D. Professor, Department of Pharmacology/Toxicology Noxious stimuli can produce pain-related stimulation of some behaviors (e.g. withdrawal responses) and depression of other behaviors (e.g. feeding, locomotion, responding maintained by many types of positive reinforcement). Monoamine reuptake inhibitors are used clinically to treat depression and to manage some types of pain. This study examined the antinociceptive properties of a variety of monoamine reuptake inhibitors selective for SERT, NET and DAT in complementary assays of acute pain-stimulated and pain-depressed behaviors. Intraperitoneal injection of dilute lactic acid (1.8% in a volume of 1ml/kg) was used as a noxious stimulus to stimulate a stretching response and to depress intracranial self-stimulation (ICSS) of the median forebrain bundle. All eight monoamine reuptake inhibitors produced an antinociception-like blockade of acid-stimulated stretching, but only compounds with prominent DA reuptake inhibition (SDRIs RTI-113 and bupropion and the TRI RTI-112) were able to block acid-depressed ICSS, although these effects were produced only at doses that also produced an abuse-related facilitation of control ICSS. Selective or mixed-action inhibitors of 5-HT and NE failed to block acid-induced depression of ICSS. In a separate group of rats, citalopram was also tested using a repeated dosing regimen (10 mg/kg x 3 doses) shown previously to produce antidepressant effects in a forced-swim test in rats. As with acute administration, repeated citalopram decreased acid-stimulated stretching but failed to block acid-induced depression of ICSS. Taken together, these results suggest that SSRIs, SNRIs and S+NRIs produce relatively non-selective depression of all behavior rather than a selective blockade of sensory sensitivity to noxious stimuli. Conversely, dopamine reuptake may be able to block sensory detection of noxious stimuli. Additionally, these results suggest that assays of pain-depressed behavior can provide new insights on analgesia-related effects of monoamine reuptake inhibitors.

Abstract The stilbene derivative, cis-3, 4’, 5-trimethoxy-3’-aminostilbene (stilbene 5c), is a potentially potent antitumor agent that acts via binding to the colchicine-binding pocket in microtubules. Earlier studies have shown that stilbene 5c induces cell death in ovarian cancer cells and leukemic cells. The present study was designed to investigate the effectiveness of this microtubule poison against the HCT-116 human colon cancer cell line and its mechanisms of action. Time course studies demonstrated that stilbene 5c produces a biphasic decrease in cell viability. The capacity of the cells to proliferate was not restored upon removal of the drug after 6 days of exposure. Consistent with the results of the time course studies, β-galactosidase staining indicated that treatment with stilbene 5c also promotes senescence. In addition to senescence, stilbene 5c-treated HCT-116 cells displayed formation of autophagic vesicles by acridine orange staining, which was supported by fluorescence-activated cell sorting (FACS). Further evidence of autophagy was derived from treatment of HCT116 cells carrying an RFP-LC3 construct with stilbene 5c, in which LC3 puncta formation increased in a time-dependent manner. DAPI staining, TUNEL, and Annexin 5 staining indicated that apoptosis is also occurring in stilbene 5c-treated HCT-116 cells. Cell cycle analysis demonstrated growth arrest at both G1 and G2/M, and an increase in the subG1 population at days 3 and 5, which correspond to senescence and apoptosis respectively. Interestingly, DAPI and Hoechst staining revealed morphological changes in the cell nuclei (binucleated and micronucleated cells), which suggest that mitotic catastrophe may also serve as a mode of cell death after treatment with stilbene 5c. However, our studies indicated that stilbene 5c works in a p53-independent manner. Exposure of P53-null HCT116 cells to stilbene resulted in a similar sensitivity as in p53-wild type HCT116 cells. We found that autophagic vacuoles were formed in response to stilbene 5c in p53-null HCT116 cells as well. Consistent with previous studies in other experimental cancer models, this work indicates that stilbene 5c could potentially be effective against colon cancer through the promotion of multiple modes of cell death.

The high co-morbidity between alcohol (ethanol) and nicotine abuse suggests that nicotinic acetylcholine receptors (nAChRs), which are thought to underlie nicotine dependence, may also be involved in alcohol dependence. A genomic region that encodes the Alpha5* nAChR subtype has recently been shown to be associated with alcohol dependence phenotypes in humans. Therefore, the aim of this study was to determine the role of Alpha5* nAChRs in ethanol-responsive behaviors upon acute administration in mice as well as in their drinking behavior. We conducted tests in mice lacking the Alpha5 coding gene (Chrna5) in ethanol-induced hypothermia, hypnosis, anxiolysis, and conditioned place preference. We also assessed drinking behavior in these mice using models of voluntary ethanol consumption, two-bottle choice preference and intermittent access, as well as acute binge drinking behavior in the Drinking-in-the-Dark paradigm. Our results showed that deletion of the Alpha5 gene enhanced acute behaviors, including ethanol-induced hypothermia, hypnosis recovery time, and the anxiolytic-like response in mice. We also found that Alpha5 gene deletion resulted in decreased ethanol CPP, but had no effect on ethanol consumption in either model of drinking behavior tested under normal conditions. However, we discovered that under conditions of stress from multiple daily injections of saline or nicotine, Drinking-in-the-Dark intake was reduced in Alpha5 null mutant mice. We also examined the role of Beta2* nAChRs due to the tendency of the Beta2 subunit to be co-expressed with this subtype, which also plays an important role in nicotine dependence. Our results showed that pharmacological and genetic manipulation of Beta2* nAChRs modulated some acute alcohol-responsive behaviors, namely, hypnosis, recovery-time and the anxiolytic-like response produced by ethanol, but did not modulate ethanol drinking behavior in mice. These studies provide evidence that Alpha5* subtypes and Beta2* subtypes, which play a critical role in nicotine dependence, also play a role in acute ethanol-responsive behaviors in vivo, thus supporting studies in humans that nicotine and alcohol dependence share common genetic components.

Hyperhomocysteinemia (hHcys), an important pathogenic factor contributing to the progression of end-stage renal disease (ESRD), has been shown to activate NOD-like receptor protein 3 (NLRP3) inflammasomes and cause podocyte dysfunction and glomerular sclerosis. hHcys induces aggregation of the three inflammasome components – NLRP3, apoptosis-associated speck-like protein (ASC), and caspase-1 – and its activation is indicated by increased caspase-1 activity and secretion of interleukin-1β (IL-1β). The aims of the present study sought to elucidate the role of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-mediated redox signaling in hHcys-induced NLRP3 inflammasome activation, to dissect the contribution of common endogenous reactive oxygen species (ROS) including superoxide (O2•−), hydrogen peroxide (H2O2), peroxynitrite (ONOO−), and hydroxyl radical (•OH), and to explore the molecular mechanisms by which the NLRP3 inflammasome senses changes in oxidative stress through thioredoxin-interacting protein (TXNIP). Specific inhibition of the gp91phox subunit of NADPH oxidase markedly reduced Hcys-induced caspase-1 activity and IL-1β production in cultured podocytes. Concurrently, gp91phox−/− or administration of a gp91ds-tat peptide also exhibited diminished glomerular inflammasome formation and activation in mice fed a folate-free (FF) diet to induce hyperhomocysteinemia and displayed glomerular protection as shown by prevention of hHcys-induced proteinuria, albuminuria and glomerular sclerosis. Interestingly, dismutation of O2•− by 4-Hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl and administration of H2O2 decomposer catalase either in cultured podocytes or hyperhomocysteinemic mice inhibited hHcys-induced NLRP3 inflammasome aggregation and activation. Hyperhomocysteinemic mice also demonstrated a significant increase in glomerular TXNIP binding to NLRP3, confirmed by confocal microscopy, size-exclusion chromatography, and co-immunoprecipitation studies. Blockade of TXNIP by genetic interference or by the calcium channel blocker verapamil prevented this hHcys-induced TXNIP-NLRP3 binding, NLRP3 inflammasome formation and activation, as well as protected hyperhomocysteinemic mice from glomerular dysfunction and damaged morphology. In conclusion, hHcys-induced NADPH oxidase activation is importantly involved in the switching on of NLRP3 inflammasomes in podocytes, where NADPH oxidase-derived O2•− and H2O2 primarily contribute to NLRP3 inflammasome activation. TXNIP binding to NLRP3 is a key signaling mechanism allowing NLRP3 inflammasome to sense these changes in oxidative stress. These findings greatly enhance our understanding of the early pathogenesis of hHcys-induced glomerular sclerosis, which may identify new therapeutic targets for prevention or treatment of ESRD.

A DIRECT AND INDIRECT MECHANISM FOR CCR5 IN OPIOID AND HIV-1 MEDIATED NEURODEGENERATION By Elizabeth M. Podhaizer, Ph.D. A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Virginia Commonwealth University. Virginia Commonwealth University, 2014 Major Director: Kurt F. Hauser, Ph.D., Professor of Pharmacology & Toxicology Human immunodeficiency virus (HIV)-1 infection currently affects over 34 million people worldwide, and despite the use of cART, the prevalence of HIV-1 associated neurocognitive impairments (HAND) has not declined. Additionally, other co-morbid factors such as the abuse of injection drugs (i.e. heroin, morphine) increase both the frequency and the speed by which patients progress to AIDS. To begin to understand the mechanisms, we chose to examine a pathway, through CCR5, which may act as a convergence point for opioids and HIV-1 proteins. C-C chemokine receptor 5 (CCR5) is an immune receptor involved in physiological processes in the brain in addition to mediating neuroinflammatory signaling events, and it is a co-receptor for HIV-1. CCR5 interacts directly with gp120 to facilitate HIV-1 infection, and may interact indirectly with HIV-1 Tat through convergent signaling mechanisms. Additionally, CCR5 is modified by opioid responses, and so may be central to opioid-HIV-1 interactions that are seen in our model. We hypothesized that CCR5 would mediate the opioid-HIV-1 interaction. We examined both HIV-1 gp120 and HIV-1 Tat, both for interactions with opioids and modification by the CCR5 antagonist, maraviroc. HIV-1 gp120ADA was neurotoxic on its own, but showed no interactions with morphine. However, further probing revealed that morphine can in fact modify the neurotoxic effects of gp120, but that the response is dependent on gp120 strain. We did, however, find that morphine did enhance the neurotoxicity of Tat, which we’ve shown previously, as well as that inhibition of CCR5 can prevent this interactive effect. Additionally, use of CCR5 knockout glia or neurons modified the response and suggests that neurons and glia play different roles in the integration of opioid and HIV-1 signals. Sublethal effects of morphine and Tat were also dampened by maraviroc pretreatment or use of knockout cells, as was the secretion of chemokine ligands. Manipulation of CCR5 showed utility in preventing neurodegenerative effects both to HIV-1 proteins alone as well as to the interactive opioid-HIV-1 signaling responses and suggests that maraviroc, a cART therapeutic used to prevent viral entry, may also aid in reducing the chronic inflammatory state of the CNS that leads to the persistent neurocognitive complications.

Cancer is the second most common cause of death in the US. The most frequently observed cancer type in women is breast cancer. A special type of breast cancer is triple negative (TNBC) cancer that is characterized by lacking three receptors: estrogen, progesterone and human epithelial growth factor (HER 2). The HS578t breast cell line is a model of TNBC that also has a mutation of the p53 protein. Ionizing radiation is used widely in the clinic to debulk tumors before surgery as well as post-surgery to eliminate residual tumor cells outside the surgical field. Previous studies from our laboratory showed that inhibition of autophagy does sensitize p53 wild type MCF-7 and ZR-75 breast tumor cells to radiation. However, this is not necessarily the response in all breast cell lines. The Hs578t cells did not appear to be sensitized to radiation after inhibition of autophagy using chloroquine as a pharmacological inhibitor. The present study was designed to build upon these previous findings and further confirm that the Hs578t breast cell line could not be sensitized to radiation through autophagy inhibition. Time course studies showed a reduction of viable cell number upon irradiation of Hs578t breast tumor cells and that both autophagy and senescence were induced. Acridine orange staining was used to examine the acidic vacuole formation while β-galactosidase staining indicated the promotion of senescence. Flow cytometry was used to quantify both autophagy and senescence. Inhibition of autophagy using pharmacological inhibitors such as ammonium chloride, or genetic silencing of autophagy by beclin1, which is a protein initiator of autophagy, did not sensitize Hs578t breast tumor cells to irradiation. It shows from these studies that autophagy is not necessarily cytoprotective in all breast cancer cell lines, which should be considered in current clinical trials designed to sensitize tumor cells to chemotherapy and radiation through inhibition of autophagy.

Microtubule poisons have proven to be effective in the treatment of a variety of malignancies. Although taxol-based derivatives promote microtubule stabilization, there is continuing interest in compounds that, like colchicines, act as microtubule destabilizing agents. Previous work from this laboratory showed that the novel microtubule poison, JG- 03-14, was active against breast tumor cells, promoting autophagic cell death. In the current work, we studied the influence of JG-03-14 on p53 wild type HCT116 colon carcinoma cells. A crystal violet sensitivity assay indicated that JG-03-14 induced growth inhibition, with 75% suppression of growth evident at a concentration of 500 nM. Time course studies of drug effects on cell viability indicated that JG-03-14 also produced cell killing. FACS analysis demonstrated that the HCT-116 cells arrested in the G2/M stage; furthermore, there was evidence of a hyperdiploid population that would be consistent with failure of the cells to divide despite completion of DNA synthesis. Finally, there was evidence of a small sub G0/G1cell population, indicating that the cells were not dying primarily by apoptosis, and suggesting that JG-03-14 induces an alternative mode of cell death. In contrast to cell shrinkage and nuclear fragmentation that was evident after treatment with taxol ( a positive control for apoptosis), DAPI staining of HCT-116 cells treated with JG-03-14 showed intact and enlarged nuclei, again consistent with the absence of apoptosis. Furthermore, there was no evidence of mitotic catastrophe (micronuclei in binucleated cells). Based on previous studies in MCF-7 and MDA-MB231 cell lines that demonstrated a substantial population of autophagic cells, HCT-116 cells were subjected to staining with acridine orange and monodansylcadaverine after treatment with JG-03-14. While control cells tended to show a single large autophagic vesicle closely associated with the cell nucleus, treatment with JG-03-14 resulted in extensive distribution of small acidic vesicles within the cytoplasm, indicative of autophagy. GFP-LC3 transfected cells incubated with JG-03-14 showed punctuated patterns that were also consistent with the promotion of autophagy. Finally, activation of the DNA damage response pathway was ruled out by the lack of induction of p53 and p21 in cells treated with JG-03-14. In summary, our studies indicate that the JG-03-14 induces both growth arrest and autophagic cell death in HCT116 colon carcinoma cells. The possibility of an alternative mode of cell death induced by JG-03-14 makes it a potentially usefull candidate as a chemotherapeutic drug that could be used to treat cancers resistant to apoptosis. Our result also suggested that JG-03-14 failed to induce bone marrow toxicity adding to its potential for clinical use.

The main psychoactive and therapeutic effects of Δ9-tetrahydrocannabinol (THC) are mediated through cannabinoid type 1 receptors (CB1Rs). The therapeutic uses of THC are mitigated by the development of tolerance to these therapeutic effects, whereas tolerance does not readily develop to some of the side-effects of THC, like motor impairment and reward. The development of tolerance occurs through adaptations at CB1Rs, which include desensitization (G-protein uncoupling) and downregulation (receptor degradation). Brain region-dependent differences in THC-mediated adaptations are proposed to explain the differences in tolerance to various THC-mediated effects. These studies focused on whether ΔFosB, a stable transcription factor, could regulate CB1R adaptations since regions resistant to CB1R adaptations, like the basal ganglia, exhibit THC-mediated ΔFosB induction. The studies in this dissertation tested the hypothesis that THC-mediated induction of ΔFosB is regulated through interactions between cannabinoid and dopamine systems and that brain region-dependent differences in ΔFosB transcriptional regulation could explain some aspects of long-term CB1R signaling and CB1R adaptations. Results determined that THC induced ΔFosB primarily in forebrain areas, like striatum, that are innervated by midbrain dopamine neurons. An inverse, brain region-dependent correlation was found between CB1R desensitization and ΔFosB induction. Studies utilizing bitransgenic mice with overexpression of ΔFosB, or its dominant negative ∆cJun, determined that ΔFosB regulates CB1R signaling and reduces CB1R desensitization. Based on this regional profile, studies determined the role of dopamine signaling in THC-mediated ∆FosB induction. Results showed that THC-mediated induction of ΔFosB required dopamine type 1 receptors, but not the dopamine-and cAMP-dependent phosphoprotein of Mr 32kDA. Finally, the functional consequences of THC-mediated ΔFosB induction were assessed by measuring expression of known targets of ΔFosB following both acute and repeated THC administration. Results found that, in prefrontal cortex, known targets of ΔFosB exhibited functionally different signaling expression patterns when comparing acute THC with THC-challenge in THC-experienced mice, which enhanced ΔFosB induction. These studies establish a role for ΔFosB in regulating long-term CB1R signaling/adaptation following repeated THC administration and could have implications for changes in the effects of THC during repeated administration, including the development of differential tolerance to motor-impairing and rewarding effects of THC versus other pharmacological effects.

Breast cancer (BC) is one of the most common diagnosed malignancies in females. Although 90% of early diagnosed women are expected to survive for at least 5 years, their quality of life is adversely affected by a cluster of symptoms which we collectively named “psychoneurological symptoms’’ (PN). Given that acquired telomere attrition has been speculated to be a causal factor in chronic diseases and the lack in the literature of mechanisms giving rise to PN symptoms, this study was performed to assess telomere length using a chromosome-specific telomere assay before receiving chemotherapy and at the first chemotherapy. We showed significant telomere attrition on the short arm of chromosome 9. In addition, we showed a negative correlation between telomere length and depression. Furthermore, we evaluated several variables as predictors of the change in telomere length and showed that chemotherapy was predictive of shortened telomere length. Taken together, one can speculate that shortened telomeres could result in epigenetic alterations in the genes juxtaposed to the telomeric region, giving rise to the development and persistence of PN symptoms. Knowledge gained from this study will offer hope for the development of therapeutic interventions that could prevent undesirable side effects and ensure a better quality of life for patients with BC.