• Effect of chronic oral treatment with risperidone or quetiapine on cognitive performance and neurotrophin-related signaling molecules in rats

      Poddar, Indrani; Department of Pharmacology and Toxicology (8/7/2018)
      Antipsychotic (APs) drugs are among the top selling pharmaceuticals in the world and they have a variety of important therapeutic applications for neuropsychiatric disorders. However, there are a number of controversies related to this class of agents and many of the relevant questions are difficult to prospectively address in the clinical trial environment. For example, there have been multiple clinical trials for pro-cognitive agents in schizophrenia that have failed; however, the question of how chronic prior treatment with APs might influence the response to a pro-cognitive agent was not addressed. Moreover, there is clinical evidence that chronic treatment with some APs may lead to impairments in cognition, however, this issue and the potential molecular mechanisms of the deleterious effects have been not been prospectively addressed. Accordingly, the purpose of the work described in this dissertation was to prospectively address each of these issues in animals (specifically rats) were environmental conditions can be rigorously controlled. In each of the manuscripts included in this dissertation, two of the most commonly prescribed APs, risperidone and quetiapine were evaluated. In the work conducted in Manuscript 1, we established a therapeutic relevant dosing approach for rats (oral administration in drinking water) and reinforced the argument that these two APs are not pro-cognitive agents. Moreover, we determined that alpha-7 nicotinic acetylcholine receptor (nAChR) ligand like tropisetron has potential as an adjunctive medication in schizophrenia since the pro-cognitive effect was maintained in the presence of chronic AP treatment. In Manuscript 2, we concluded that chronic treatment with risperidone or quetiapine in rats can lead to impairments in a domain of cognition (recognition memory) that is commonly altered in neuropsychiatric disorders. Moreover, the negative effects of the APs appeared to be exacerbated over time. In Manuscript 3, we concluded that risperidone and quetiapine when administered chronically to rats have the potential to adversely affect neurotrophin-related signaling molecules that support synaptic plasticity and cognitive function. These data would suggest that the extensive prescribing of these APs across multiple conditions in patients ranging in age from the very young to the very old should be carefully reexamined. Key Words: antipsychotic, cognition, brain volume, schizophrenia, neurotrophin
    • A Novel Function of ADP-Ribosylation Factor 1 in Prostate Cancer Cell Proliferation through Activating the Mitogen-Activated Protein Kinase Pathway

      Davis, Jason E.; Department of Pharmacology and Toxicology (8/23/2016)
      The enhanced activation of the mitogen-activated protein kinase (MAPK) Raf-MEK-ERK1/2 pathway directly correlates with the growth, androgen-independence, and poor prognosis of prostate cancer. However, the underlying molecular mechanisms remain poorly understood. Here, we have demonstrated that ADP-ribosylation factor 1 (ARF1), a Ras-like small GTPase, was highly expressed in human prostate cancer cells and tissues. In addition, ARF1 was markedly activated in prostate cancer cells. More interestingly, oncogenic G protein-coupled receptors (GPCRs) strongly activated ARF1 and the activation was mediated through Gβγ subunits. These data indicate that GPCRs and heterotrimeric G proteins are the upstream activators of ARF1 in prostate cancer cells. Next, we determined the role of ARF1 in the MAPK activation and proliferation in prostate cancer cells. Lentiviral-mediated overexpression of ARF1 remarkably enhanced, whereas shRNA-mediated depletion of ARF1 dramatically reduced ERK1/2 activation in prostate cancer cells. In addition, disruption of both the Golgi localization of ARF1 and the Golgi structure substantially attenuated ERK1/2 activation in prostate cancer cells. In parallel with their effects on the MAPK activation, ARF1 overexpression greatly enhanced and ARF1 knockdown inhibited the proliferation of prostate cancer cells. These data suggest that ARF1, by its ability to activate the MAPK pathway likely at the Golgi, controls prostate cancer cell proliferation. We then investigated the consequence of pharmacologically inhibiting ARF1 activation. Small molecule inhibitors including brefeldin A, golgicide A, and Exo2 that specifically target Golgi-localized ARF1 markedly reduced both ERK1/2 activation and proliferation in prostate cancer cells. These results further indicate an important role of ARF1 activation in regulating the MAPK pathway and prostate cancer cell proliferation. Altogether, our data suggest a possible GPCR-G-ARF1-MAPK signaling pathway, which may be responsible for the hyperactivation of the MAPK ERK1/2 in prostate cancer and contributes to prostate cancer progression. Our results also imply a novel approach for prostate cancer therapy by targeting ARF1 activation
    • Calpain-2 Activates Akt via the TGF~ 1-mTORC2 Pathway in Pulmonary Artery Smooth Muscle Cells

      Abeyrathna, Prasanna; Deparment of Pharmacology and Toxicology (8/23/2016)
      Calpain is a family of calcium-dependent nonlysosomal neutral cysteine endopeptidases. Akt is a serine/threonine kinase that belongs to the AGC kinases and plays important roles in cell survival, growth, proliferation, angiogenesis, and cell metabolism. Both calpain and Akt are downstream signaling molecules of platelet-derived growth factor (PDGF) and mediate PDGF-induced collagen synthesis and proliferation of pulmonary artery smooth muscle cells (PASMCs) in pulmonary vascular remodeling. We found that inhibition of calpain-2 using the calpain inhibitor MDL28170 and calpain-2 siRNA attenuated Akt phosphorylation at serine-473 (S473) and threonine-308 (T308) as well as collagen synthesis and cell proliferation ofPASMCs induced by PDGF. Overexpression of calpain-2 in PASMCs induced dramatic increases in Akt phosphorylation at S4 73 and T308. Moreover, knockout of calpain attenuated Akt phosphorylation at S473 and T308 in smooth muscle of pulmonary arterioles of mice with chronic hypoxic pulmonary hypertension. The cell-permeable specific TGF~ receptor inhibitor SB431542 attenuated Akt phosphorylation at both S473 and T308 induced by PDGF and overexpressed calpain- 2 in PASMCs. Moreover, SB-431452 and knock down of ALK5 significantly reduced PDGF-induced collagen synthesis and cell proliferation of PASMCs. Nevertheless, neutralizing extracellular TGF~l using a cell-impermeable TGF~l neutralizing antibody did not affect PDGF-induced Akt phosphorylation at S473 and T308. Further, inhibition of mTORC2 by knocking down its component protein Rictor prevented Akt phosphorylation at S473 and T308 induced by PDGF and overexpressed calpain-2. These data provide the first evidence supporting that calpain-2 up-regulates PDGF-induced Akt phosphorylation via an intracrine TGF~ 1/mTORC2 mechanism.
    • Attenuating the Interaction Between Delta Protein Kinase C and the "d" Subunit of FIFo ATp Synthase Protects Against Cardiac Ischemia/Repferusion injury

      Walker, Matthew; Deparment of Pharmacology and Toxicology (6/3/2016)
      Cardiac ischemia / reperfusion (IR) injury most often results from the thrombotic blockade of the coronary arteries and is the most frequent cause of death in humans. Despite the significant role energy deprivation plays in cardiac IR injury, few studies have targeted the IR-induced impairment of the mitochondrial F1Fo ATP synthase. We have previously demonstrated delta protein kinase C (δPKC) involvement in cardiac myocyte energy deprivation via its interaction with the “d” subunit of F1Fo ATP synthase (dF1Fo) and have developed a peptide inhibitor [NH2YGRKKRQRRMLATRALSLIGKRAISTSVCAGRKLALKTIDWVSFDYKDDDDK- COOH] of this interaction. It targets to the mitochondrial matrix / inner membrane. The inhibitor peptide contains a FLAG epitope which allowed confirmation of its uptake into cardiac mitochondria. Our early studies in neonatal cardiac myocytes (NCMs) led us to the hypothesis that PKC inhibits ATP production in vivo via an interaction with dF1Fo to exacerbate cardiac IR injury. To directly test our hypothesis, we first utilized the Langendorff isolated heart model to show that PKC co-immunoprecipitates (co-IPs) with antisera to dF1Fo in myocardium exposed to simulated IR injury. Administration of the inhibitor peptide to the isolated rat hearts prior to cardiac IR attenuated the co-IP of 􀁇PKC with dF1Fo, improved recovery of contractility, diminished levels of tissue t-carbonyls and 4-hydroxy-2-nonenal (HNE), and reduced myocardial infarct size (as assessed by 2, 3, 5 triphenyltetrazolium chloride (TTC) staining) following simulated IR exposures. Additionally, this peptide enhanced ATP levels 2.1 fold, improved ADP-stimulated mitochondrial respiration, and attenuated Ca++-induced mitochondrial swelling in ischemic myocardium. We next evaluated the inhibitor peptide in an in situ rat coronary ligation model for its ability to protect live rats from cardiac IR injury. A 10 min coronary ligation increased the PKC-dF1Fo co-IP in the region at risk (RAR) by 5-fold which was attenuated by 71% with intravenous infusion of the inhibitor peptide. This response correlated with an enhancement of ATP levels, a 2-fold reduction in oxidative stress markers, improvement in systolic cardiac function, and a reduction in TTC monitored myocardial infarct size in the RAR. These results support further development of this peptide as a first-in-class-translational therapeutic for the treatment of cardiac IR injury.
    • Profiling the HCA Receptor Family through BRET Analysis of GPCR-G-Protein and GPCR-Arrestin Interactions

      Saj, Dalia; Department of Biological Sciences; Department of Pharmacology & Toxicology (Augusta University, 2020-05)
      Increasing obesity rates have put the American population at higher risk for developing obesity-related medical conditions such as hypertension, heart disease, and diabetes. The hydroxycarboxylic acid (HCA) receptor family is a family of G protein-coupled receptors (GPCRs) that are expressed in adipose tissue and function as metabolic sensors, making them potential pharmaceutical targets in the treatment of obesity and other metabolic disorders. The HCA receptor family consists of the HCA1, HCA2, and HCA3 receptors, which are activated by hydroxycarboxylic acids such as lactate and 3-hydroxybutyric acid. We utilized bioluminescence resonance energy transfer (BRET) to study agonist-induced coupling of luciferase-tagged HCA receptors to Venus fluorescent protein-tagged G protein heterotrimers or arrestins. Our results indicate that the three HCA receptors couple to the Gαi/o subfamily of G proteins. The data additionally confirms a lack of coupling to the other G protein subfamilies (Gαs, Gαq, and Gα12/13), and lacks evidence of arrestin recruitment to HCA receptors. Overall, our study highlights the use of BRET as a powerful tool for analysis of GPCR signaling and demonstrates its possible use for future studies to determine the potency of potential drugs targeting HCA receptors as a therapy for health-related problems such as obesity.
    • SELECTIVITY AND PRODUCTIVITY OF GPCR-G PROTEIN INTERACTIONS

      Okashah, Najeah; Department of Pharmacology and Toxicology (Augusta University, 2020-03)
      Hundreds of human G protein-coupled receptors (GPCRs) converge on activation of four families of heterotrimeric G proteins. Individual receptors select a subset of G proteins in order to produce appropriate cellular responses. While the precise mechanisms of coupling selectivity are uncertain, the G alpha subunit carboxy (C) terminus is believed to be the primary region recognized by GPCRs. We directly assessed coupling between 14 representative GPCRs and 16 G alpha subunits, including one wild-type G alpha subunit from each of the four families and 12 chimeras with exchanged C termini. We found that Gi-coupled receptors were relatively selective for Gi1 heterotrimers, while Gs-, Gq-, and G12- coupled receptors were more promiscuous and always coupled in some measure to Gi1 heterotrimers. Our tests with G alpha subunit chimeras show that the G alpha subunit core and C terminus both play important roles in selectivity. This suggests that the key G protein determinants of selectivity vary widely, even for different receptors that couple to the same G protein. While promiscuous GPCR-G protein coupling is often observed. These interactions behave as expected with receptor-G protein coupling and activation being almost synonymous. Agonist bound GPCRs activate the G protein heterotrimers they interact with, while ignoring G protein subtypes that they cannot activate. However, we have shown that GPCRs can form unproductive complexes with G12 heterotrimers. Vasopressin 2 receptor (V2R) forms agonist-dependent complexes with G12 heterotrimers. Unlike V2R complexes with cognate Gs heterotrimers, V2R-G12 complexes do not dissociate when GDP or GTP is present. Stimulating V2R with arginine vasopressin (AVP) does not activate signaling responses downstream of G12 activation. Evaluation of several G12-coupled receptors demonstrated that agonist induced GPCR-G12 complexes have a wide range resistance to GDP. Like V2R receptors, formyl peptide 2 receptors (FPR2) and smoothened receptors (SMOR) formed complexes with G12 heterotrimers that were relatively resistant to GDP. Our results indicate that several GPCRs can form agonist-dependent unproductive complexes with G12 heterotrimers that are relatively resistant to GDP. Suggesting that for some GPCRs agonist-dependent association with G12 heterotrimers is weakly coupled to nucleotide exchange
    • The Role of T Cells in High Fat Diet Induced Hypertension and Vascular Dysfunction in Female Rats

      Taylor, Lia; Department of Pharmacology and Toxicology (2019-01-08)
      Hypertension is a leading risk factor for the development of cardiovascular disease (CVD) worldwide, affecting ~86 million adults in the United States alone. In general, the prevalence of hypertension is lower in premenopausal women than in age-matched men. Despite having lower risk than men, however, women represent more than half of all hypertension cases, and CVD is the leading cause of death in women worldwide. Women are also more likely than men to be obese and two-thirds of hypertension cases in the U.S. positively correlate with excessive weight gain due to the consumption of diets high in saturated fat. Dahl salt-sensitive rats (DSS) have traditionally been used to study salt-sensitive hypertension. However, previously, it has been shown that male DSS also exhibit a high fat diet (HFD)-induced increase in blood pressure (BP) at normal salt intake, which is attenuated by treatment with the lymphocyte inhibitor mycophenolate mofetil (MMF), suggesting a role for B and T cells in HF-induced hypertension in male animals. However, there is limited data on the role of HFD and T cell activation on BP in females. We hypothesized that HFD will promote hypertension and vascular dysfunction in female DSS and that these cardiovascular changes will be mediated by T cells. We demonstrate that female DSS are susceptible to HFD-induced hypertension and vascular T cell infiltration and activation similar to that of males. Since local inflammation in perivascular adipose tissue (PVAT) is linked to HFD-induced increases in BP and vascular dysfunction in males, we further investigated the impact of HFD on vascular function and the buffering capacity of PVAT in female DSS. HFD alone did not impair vascular function compared to normal fat diet (NFD) in female DSS. Interestingly, the buffering capacity of PVAT was also maintained in female DSS in response to a HFD despite increases in BP and vascular inflammation, likely due to overproduction of nitric oxide (NO). To directly assess the role of T cells on HFD-induced alterations of BP and vascular function in female DSS, we utilized female CD247-/- rats on the DSS background. HFD-induced increases in BP were completely abolished in CD247-/- . Further, the buffering capacity of PVAT was preserved in CD247-/- in response to a HFD. We conclude that T cells underlie the susceptibility of female DSS to HFD-induced hypertension. Further, vascular function and PVAT buffering capacity are maintained in female DSS despite fat-induced increases in BP and vascular inflammation, suggesting an adaptive response in the early stages of diet-induced obesity in which nitric oxide (NO) may play a key role.
    • Epigenetic Regulation of Adipogenic Differentiation And Lipid Metabolism: Role of Enhancer of Zeste Homolog 2 (EZH2)

      YIEW, KAN HUI; Department of Pharmacology and Toxicology (2017)
      Adipose tissue expansion in obesity promotes cardiometabolic disease, for which there is an urgent need to elucidate disease mechanisms and develop novel and effective medical therapies. In obesity, adipose tissues can potentially expand through adipocyte hypertrophy and/or hyperplasia, with the latter being a healthier mechanism of fat expansion. Adipocyte hyperplasia (via adipogenic differentiation) is inexplicably restrained in diet-induced obesity (DIO), however, and hypertrophy ensues, leading to inflammation, insulin resistance, and dysregulated adipose tissue lipid metabolism, which together contribute to cardiometabolic disease. The mechanisms of impaired adipogenic differentiation and lipid metabolism during DIO are unclear, with prior studies suggesting epigenetic dysregulation of histone deacetylase 9 (HDAC9, an endogenous repressor of adipogenic differentiation). The overall goal of my dissertation project is to investigate the role of Enhancer of Zeste Homolog 2 (EZH2), a histone methyltransferase, in the aforementioned processes. In Aim 1, we hypothesized that EZH2 promotes adipogenic differentiation by repressing HDAC9. EZH2 recruitment and histone 3 lysine 27 trimethylation (H3K27me3) modification were elevated at the HDAC9 gene promoter (p<0.05) concurrent with dramatic downregulation of HDAC9 mRNA levels (p<0.05) during adipogenic differentiation of primary human preadipocytes. This suggested a role for EZH2 in silencing HDAC9 gene expression. Counterintuitively, a highly selective EZH2 pharmacological inhibitor (GSK126), at a concentration that effectively blocked H3K27me3, led to increased lipid accumulation (p<0.05) in human adipocytes, without inhibiting adipocyte marker gene expression. Consistently, mice with adipose-specific EZH2 deletion (cKO) displayed significantly elevated body weight, adipose tissue mass, and adipocyte cell size. These phenotypic alterations could not be explained by differences in feeding behavior, locomotor activity, or metabolic energy expenditure, thereby suggesting that EZH2 regulates lipid metabolism. This hypothesis was explored in Aim 2. Human adipocytes treated with either an EZH2 inhibitor or vehicle exhibited comparable rates of de novo lipogenesis (DNL), fatty acid (FA) uptake, and basal/stimulated lipolysis. Consistently, cKO and littermate control mice displayed comparable in vivo and ex vivo basal/stimulated adipose lipolysis. EZH2’s function in other important metabolic pathways such as glycolysis and β-oxidation remain to be investigated. Collectively, our findings suggest a potential role of EZH2 in regulating adipocyte lipid metabolism.
    • A Data-Mining Strategy That Identifies Drugs and Genes Associated With Anti-Cancer Drug Sensitivity

      Schleifer, Robert John; Department of Pharmacology and Toxicology (2017)
      The success of cancer therapy for patients often hinges on the responsiveness of the cancer cells to therapeutics. Drug resistance to anti-cancer therapeutics, both intrinsic and acquired, has important clinical and scientific significance. Identification of drug resistance genes using traditional methodologies and translation of those findings to the clinic has proven challenging. We developed a predictive data mining-based bioinformatic framework using public patient data and high-throughput cancer cell drug screening data. This information was used for genome-wide rankings of putative drug resistance genes. Prominent drug resistance genes (e.g. ABCB1, EGFR, and AXL) were successfully identified by the pipeline, additional genes hypothesized to be novel drug resistance genes were then investigated. Experimental confirmation of the novel genes using knockdown technologies indicated a propensity for of decreased proliferation/viability of cancer cells and increased sensitivity for anticancer compounds after knockdown much like known drug resistance genes. We then assessed the potential of each gene as an anti-cancer therapeutic target by exploring how gene knockdown behaved with clinical anticancer compounds. A second arm of the data-mining pharmaco-genomic strategy involved identification of candidate compounds that decrease expression of drug resistance genes. Using the drug resistance gene AXL as a proof-of-concept, three compounds were identified that decreased AXL expression at sub-micromolar concentrations. These compounds were characterized using microarray and cell signaling studies and found to decrease cell cycle signaling as well as activity of the Akt, mTOR, and ERK pathways. This study illustrates a novel approach for rapid and efficient identification of drug sensitivity genes or gene expression altering compounds utilizing bioinformatic data-mining.
    • Circadian Clock in Angiotensin II Induced Hypertension and Vascular Disease

      Pati, Paramita; Department of Pharmacology and Toxicology (2015)
      Hypertension remains a major risk factor for cardiovascular disease and death. While clinical studies and guideline recommendations underscore the benefits of reducing sodium intake in the treatment of high blood pressure, recent human data suggest that underlying conditions of disease may confound these positive effects of low salt diets. Herein, we examined the influence of circadian dysfunction during experimental hypertension caused by angiotensin II (Ang II), a key peptide in blood pressure regulation. While a low salt diet caused the expected decrease in blood pressure in wild type (WT) mice, mice with disruption of the circadian clock exhibited a paradoxical response to low salt. Mice with disruption in the circadian clock component Period (Period-knockout/KO mice), were abolished in blood pressure rhythm due to an increase in daytime blood pressure. This impairment in blood pressure rhythm in Per-KO mice on the low salt diet was restored to rhythmic oscillation by the angiotensin receptor blocker losartan. Similarly, exogenous administration of Ang Il caused a non-dipping blood pressure phenotype in the Per-KO mice on a normal salt diet, which resulted in pathological thickening of the vasculature indicative of vascular disease. These effects were related to circadian rhythm as impairment in blood pressure caused by low salt was recapitulated in WT mice induced to circadian derangement by a shortened light cycle. Further thickening of the vasculature and increased renin levels were observed in Per-KO mice on a chronic low salt diet but not in WT mice. Moreover, disruption of the Period gene altered ATI receptor expression and other components of the renin-angiotensin system. These data suggest that circadian dysfunction may compromise the benefits of a low salt diet and support recent clinical data that raise caution to sodium restriction as a therapy for hypertension.
    • Involvement of arginase upregulation in diabetes- and angiotensin II-induced vascular dysfunction

      Bhatta, Anil; Department of Pharmacology and Toxicology (2015)
      Cardiovascular disease (CVD) is the number 1 killer of men and women in the United States and the world. Diabetes, hypertension, obesity, and aging are some of the risk factors for CVD. A major cause of morbidity and mortality in CVD is vascular dysfunction, which progresses rapidly as the risk factors progress. Vascular dysfunction is characterized by a constellation of blood flow reducing pathologies, including impaired vasorelaxation and elevated arterial stiffening. The mechanisms leading to these vascular abnormalities are not well understood. We tested the hypothesis that arginase, an enzyme in the urea cycle, mediates vascular dysfunction in hypertension and obesity related diabetes. Arginase (ARG) can compete with nitric oxide (NO) synthase for their common substrate, L-arginine. Increased arginase can also provide more ornithine for synthesis of polyamines via ornithine decarboxylase (ODC) and proline/collagen via ornithine aminotransferase (OAT), leading to vascular cell proliferation and collagen formation, respectively. We hypothesized that elevated arginase activity is involved in Ang II-induced vascular dysfunction and that limiting its activity can prevent these changes. We tested this by studies in C57BL/6J mice lacking one copy of the ARG1 gene that were treated with Ang II (1 mg/kg/day, 4 weeks). We demonstrated that Ang II induces smooth muscle cell proliferation, collagen synthesis, and arterial fibrosis and stiffness via a mechanism involving increased arginase activity. Furthermore, we examined the role of arginase in vascular dysfunctions and pathologies associated with obesity-related type 2 diabetes in mice fed with high-fat/high-sucrose (HFHS) diet for 6 months. This model produced a clinical presentation and pathophysiological relevance to the human condition in obesity related type 2 diabetes. We demonstrated that HFHS diet impaired endothelial dependent vasorelaxation and increased arterial stiffness in WT mice, but not in mice treated with arginase inhibitor ABH. Endothelial cell specific knockout of ARG1 (EC-A1-/-) in mice also prevented HFHS induced vascular dysfunctions. Aortic perivascular collagen deposition was significantly higher in HFHS mice compared to normal diet. Furthermore, marked increase in vascular cell adhesion molecule expression and macrophage infiltration into the aortic walls was observed with HFHS diet. Additionally, plasma lipid peroxidase activity, a measure of systemic oxidative stress, was also markedly increased in HFHS mice. These changes were prevented in ABH treated mice and EC-A1-/- mice. These studies suggest that enhanced ARG1 activity promotes vascular dysfunctions associated with elevated Ang II levels or obesity related diabetes.
    • Transient Self-Association of Beta2-Adrenergic Receptors

      Lan, Tien-Hung; Department of Pharmacology and Toxicology (2014-03)
      G-protein coupled receptors (GPCRs) constitute the largest family of cell surface receptors. Through binding to ligands such as hormones, peptides, neurotransmitters, or photons, GPCRs are activated and regulate a variety of physiological responses. It has been widely accepted that GPCRs can self-associate as dimers or higher-order oligomers even though protomeric GPCR is capable of activating heterotrimeric G proteins and recruiting arrestins. Although GPCR complexes have been suggested to possess distinct functional properties such as receptor trafficking, receptor phosphorylation, biased downstream signaling, and allosteric communication, the stability and the structural mechanisms for the dimerization of class A GPCRs have not been extensively studied. The β2 adrenergic receptor (β2AR) is a prototype of class A GPCRs and is probably the most extensively studied among the currently available high resolution crystal structures of GPCRs. However, lack of clear dimer interface and the controversy of the stability of β2AR dimeric complexes brings to the question that whether β2AR self-associate as a stable dimer.
    • A Variable Prenatal Stress Paradigm as a Valid Drug Discovery Platform for Cognitive Deficits Associated with Neuropsychiatric Disorders

      Wilson, Christina Ann; Department of Pharmacology and Toxicology (2012-10)
      Cognitive dysfunction is now recognized to be central to the functional disability of several neuropsychiatric disorders. However, treatment options for the management of cognitive symptoms are limited and the development of novel therapeutics has been made difficult by the lack of appropriate animal models. It has been suggested that variable prenatal stress (PNS) in rodents might be an etiologically appropriate model for some components of schizophrenia. Thus, the overall goal of this dissertation project was to conduct a comprehensive behavioral study of the model to assess face validity, and to make a preliminary assessment of its construct and predictive validity. Our results indicate that exposure to PNS results in elevated corticosterone levels following exposure to acute stress, increased aggressive behaviors, as well as increased locomotor activity and stereotypic behaviors. Further, PNS rats had altered innate fear responses to predator odor as well as impaired fear extinction. Additionally, PNS in rats was associated with impairments of sustained attention, inhibitory response control, and recognition memory all of which could be attenuated by the norepinephrine reuptake inhibitor, atomoxetine. Collectivity, these data support the premise that PNS in rodents is a valid model system for studying some behavioral components of neuropsychiatric disorders as well as their treatment.
    • Post Traumatic Stress Disorder: Insights from Cat Hair and Catfish

      Nalloor, Rebecca Ipe; Department of Pharmacology and Toxicology (2012-06)
      Post traumatic stress disorder (PTSD) is an anxiety disorder that develops in some, but not all, individuals following a traumatic experience. Established PTSD is difficult to treat, therefore prevention and early intervention is important to reduce prevalence. Identifying individuals susceptible to developing PTSD before trauma exposure and investigating neurophysiological processes that contribute to the disease will help develop better treatment and preventive methods. Limitations to such investigations in humans make animal models a necessary tool. Like humans, only some rats develop PTSD-like behavior after trauma but pre-trauma identification of these rats was not possible until now. We were able to reliably predict before trauma exposure which rats are susceptible (Susceptible) or resistant (Resistant) to developing two PTSD-like symptoms: impaired fear extinction and lasting elevation in acoustic startle responses. We hypothesized that Susceptible rats will have pre-existing alterations in plasticity-related responses in the hippocampus, a brain region whose altered size and function is associated with PTSD diagnosis. We also hypothesized that Susceptible rats will differ from Resistant rats in the acquisition of a traumatic event and tested this using Arc/H1a catFISH, a cellular imaging technique that detects neurons expressing plasticity-related immediate early genes (IEGs) during behavior. We found that, in Resistant rats a large proportion of the same dorsal CA1 (dCA1) neurons expressed IEGs during two identical explorations of the experimental box. This suggests that dCA1 responds to identical events with high fidelity. In Susceptible rats, however, different neuronal ensembles expressed IEGs during identical explorations suggesting a lack of fidelity in hippocampal response to identical events. In addition fewer ventral CA3 neurons expressed IEGs during the second exploration in Susceptible as compared to Resistant rats. We also examined the basolateral nucleus of the amygdala, but found no difference in IEG expression. Contrary to hypothesis, differences between Susceptible and Resistant rats during a foot shock paired exploration (traumatic event) were not pronounced. These findings show that rats susceptible to developing PTSD-like symptoms can be behaviorally identified and have altered hippocampal plasticity-related responses prior to the trauma. This study provides a frame-work for the investigation and remediation of susceptibilities.
    • hElp3 Directly Modulates the Expression of HSP70 Gene in HeLa Cells via HAT Activity

      Li, Fen; Ma, Jixian; Ma, Yu; Hu, Yanyan; Tian, Shujuan; White, Richard E.; Han, Guichun; Department of Pharmacology and Toxicology (2011-12-21)
      Human Elongator complex, which plays a key role in transcript elongation in vitro assay, is incredibly similar in either components or function to its yeast counterpart. However, there are only a few studies focusing on its target gene characterization in vivo. We studied the effect of down-regulation of the human elongation protein 3 (hELP3) on the expression of HSP70 through antisense strategy. Transfecting antisense plasmid p1107 into HeLa cells highly suppressed hELP3 expression, and substantially reduced expression of HSP70 mRNA and protein. Furthermore, chromatin immunoprecipitation assay (ChIP Assay) revealed that hElp3 participates in the transcription elongation of HSPA1A in HeLa cells. Finally, complementation and ChIP Assay in yeast showed that hElp3 can not only complement the growth and slow activation of HSP70 (SSA3) gene transcription, but also directly regulates the transcription of SSA3. On the contrary, these functions are lost when the HAT domain is deleted from hElp3. These data suggest that hElp3 can regulate the transcription of HSP70 gene, and the HAT domain of hElp3 is essential for this function. These findings now provide novel insights and evidence of the functions of hELP3 in human cells.
    • Use-dependent Antagonism of Nicotinic Acetylcholine Receptors as a Novel Treatment for Drug Addiction

      Hall, Brandon J; Department of Pharmacology and Toxicology (2011-11)
      The contributions of nicotinic acetylcholine receptors (nAChRs) to the onset and maintenance of drug addiction are well known, but these receptors are too often overlooked as potential targets for addiction treatment. The goal of this study was to demonstrate that use-dependent antagonism of nAChRs by the compound bis (2, 2, 6, 6-tetramethyl-4-piperidinyl) sebacate (BTMPS) offers a novel approach to treatment for drug addiction, and that positive outcomes of this treatment can be demonstrated across different classes of abusive drugs, nicotine or morphine in all three phases of an animal model of what is known as the drug abuse cycle: 1) binge-intoxication, 2) withdrawal-negative affect, and 3) preoccupation-anticipation. Different groups of rats were allowed to self-administer drugs of abuse (nicotine or morphine) on a 24 hr basis for a period of 14 days to establish binge-intoxication. Upon completion of self-administration, each rat was evaluated for withdrawal-negative affect. Subsequent to acute withdrawal the rats were placed in standard housing cages for a period of six weeks. At the end of the six week period, each rat was examined for unrewarded drug seeking responses, or preoccupation-anticipation, for another 14 day period preoccupation-anticipation. Injections of vehicle or BTMPS were administered to the animals during each behavioral phase of the study. Treatment with BTMPS significantly reduced the self-administration of both nicotine and morphine compared to vehicle treated animals. BTMPS treated animals also displayed reduced acute withdrawal symptoms when compared to their vehicle treated counterparts. When intervention occurred during self-administration or acute withdrawal, BTMPS treatment resulted in a significant reduction in drug-seeking responses after a protracted period of abstinence from drug. However, delaying treatment with the compound until the drug seeking phase of the study was ineffective against reducing drug seeking behavior. Administration of BTMPS alone did not appear to elicit adverse side effects in the animals, neither affecting their motivation to obtain food nor compromising the animals' performance during the behavioral procedures in the study. Thus, the resultsof this study support the hypothesis that use-dependent antagonism of nAChRs offers the potential for an alternative approach to treatment of substance abuse and drug addiction.
    • Angiotensin II Signaling Mechanisms Involved in the Elevation of Arginase Activity/Expression and Vascular Dysfunction

      Shatanawi, Alia; Department of Pharmacology and Toxicology (2011-11)
      Vascular endothelial dysfunction is a major cause of morbidity and mortality in patients with cardiovascular diseases such as hypertension, atherosclerosis and diabetes. Nitric oxide (NO) produced by endothelial nitric oxide synthase (NOS) is needed for normal vascular function. During hypertension, diabetes or atherosclerosis, elevated levels of arginase can compete with NOS for available L-arginine thus reducing vascular NO production. Elevated angiotensin II (Ang II) is a key participant of endothelial dysfunction in many cardiovascular diseases and has been linked to elevated arginase activity. In this study we explored the signaling pathway leading to increased arginase expression/activity in responses to Ang II in bovine aortic endothelial cells (BAEC). Treatment of BAEC with Ang II (10-7 M, 24 hrs) caused a 40±6% increase in arginase activity. This was accompanied by 30±8% decrease in NO production. Our studies indicate involvement of the RhoA/ROCK-p38 mitogen activated protein kinase (MAPK) in Ang II-induced arginase upregulation and reduced NO production, as inhibitors of ROCK or p38 MAPK prevented the Ang II-induced increase in arginase activity. Our studies in mice also show involvement of p38 MAPK in Ang II-induced vascular dysfunction associated with elevated arginase activity and expression. Ang II (42 μg/kg/h) caused impaired EC-dependent vasorelaxation in mouse aorta (55±7% vs. 75±8% for control). This impairment was prevented by treatment with p38 inhibitor SB203580 (5 μg/kg/day). Ang II also caused a 6.2 fold increase in vascular arginase activity/expression that was completely prevented by p38 MAPK inhibition. Additionally, treatment of BAEC with Ang II causes phosphorylation of activating transcription factor-2 (ATF-2) and enhancement of the binding of ATF-2 to arginase promotor through an AP-1 site as evident from electrophoretic mobility shift assay experiments. Transfection of BAEC with ATF-2 siRNA prevents Ang II-induced increases in arginase activity/expression and maintains NO production. These results indicate that ATF-2 is necessary for enhanced expression of arginase by Ang II. Collectively, our results indicate that Ang II increases endothelial arginase activity/expression through a RhoA/ROCK-p38 MAPK-ATF-2 pathway leading to reduced NO production and endothelial dysfunction. Targeting these signaling steps might be therapeutic points for preventing vascular endothelial dysfunction associated with elevated arginase activity/expression.
    • Effects of Parturition on Estrogen Signaling in Resistance Arteries

      Royal, Crista Ruth; Department of Pharmacology and Toxicology (2011-04)
      Few studies have examined the potential effects of childbirth on the responses of the female vasculature – especially the resistance microvasculature of non-reproductive tissues. We investigated the response of mesenteric microvascular resistance vessels to 17β-estradiol (E2), an important vasoactive hormone. Vessels were obtained from either nulliparous or postpartum female Sprague-Dawley rats, and isometric tension studies were performed. We found that E2 induced a concentration-dependent, endotheliumindependent relaxation of microvessels precontracted with 10-5M phenylephrine; however, E2-induced relaxation was reduced by nearly half in vessels from postpartum animals compared to nulliparous controls. Inhibiting nitric oxide synthase activity with 10-4M L-NMMA attenuated the relaxation effect of E2 on arteries from nulliparous animals. In contrast, L-NPA (which exhibits selectivity for type 1 or nNOS) had little effect on arteries from postpartum animals, suggesting a reduced influence of nNOS after parturition. Moreover, expression of nNOS protein in microvessels was decreased 39% in the postpartum state compared to arteries from nulliparous animals. We propose that the impaired E2-induced relaxation response of microvessels from postpartum animals reflects a downregulation of NO production due to lower nNOS expressed in vascular smooth muscle cells. We measured a 73% decrease in serum E2 levels in the postpartum state compared to nulliparous animals. Because E2 has been shown to increase nNOS protein expression, we propose that lower E2 levels after parturition decrease expression of nNOS, leading to a reduced vasodilatory capacity of resistance microvessels. Impaired E2-induced relaxation in resistance arteries from postpartum rats could also be restored by inhibiting COX with indomethacin (E2 300 nM 23.5±8.9% n=5) and even more profoundly with COX-2 inhibitor celecoxib (E2 300 nM 33.9±5.5% n=8). However, many women artificially increase estrogen levels soon after giving birth by taking oral contraceptives. Little is known regarding how parturition affects estrogen signaling, especially in resistance arteries, which can contribute to blood pressure regulation. We found that inhibiting COX-2 restored E2-induced vasodilation in arteries from postpartum rats.
    • Internalization Dissociates b2-Adrenergic Receptors

      Lan, Tien-Hung; Kuravi, Sudhakiranmayi; Lambert, Nevin A.; Department of Pharmacology and Toxicology (2011-02-22)
      G protein-coupled receptors (GPCRs) self-associate as dimers or higher-order oligomers in living cells. The stability of associated GPCRs has not been extensively studied, but it is generally thought that these receptors move between the plasma membrane and intracellular compartments as intact dimers or oligomers. Here we show that b2-adrenergic receptors (b2ARs) that self-associate at the plasma membrane can dissociate during agonist-induced internalization. We use bioluminescence-resonance energy transfer (BRET) to monitor movement of β2ARs between subcellular compartments. BRET between b2ARs and plasma membrane markers decreases in response to agonist activation, while at the same time BRET between b2ARs and endosome markers increases. Energy transfer between b2ARs is decreased in a similar manner if either the donor- or acceptor-labeled receptor is mutated to impair agonist binding and internalization. These changes take place over the course of 30 minutes, persist after agonist is removed, and are sensitive to several inhibitors of arrestin- and clathrin-mediated endocytosis. The magnitude of the decrease in BRET between donor- and acceptor-labeled b2ARs suggests that at least half of the receptors that contribute to the BRET signal are physically segregated by internalization. These results are consistent with the possibility that b2ARs associate transiently with each other in the plasma membrane, or that b2AR dimers or oligomers are actively disrupted during internalization.
    • Making Structural Sense of Dimerization Interfaces of Delta Opioid Receptor Homodimers

      Johnston, Jennifer M.; Aburi, Mahalaxmi; Provasi, Davide; Bortolato, Andrea; Urizar, Eneko; Lambert, Nevin A.; Javitch, Jonathan A.; Filizola, Marta; Department of Pharmacology and Toxicology (2011-01-24)
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