Browsing by Subject "Pharmacology"
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Item AB affects apoE transcriptionally through the activation of B-AR, cAMP and AP-2(2009-09) Rossello, Ximena SerenellaTwo key players in the development of Alzheimer’s disease (AD) are amyloid beta protein (Aβ) and apolipoprotein E (apoE). We and others have reported that Aβ elevates apoE protein levels in astrocytes, which in turn could alter lipid trafficking and cell function. The mechanism for the Aβ-induced increase in apoE levels is not clearly understood. We propose that Aβ affects apoE transcriptionally through the activation of the beta-adrenergic receptor (βAR), cAMP and the activator protein 2 (AP-2). To test this hypothesis it was first determined if the stimulation of apoE protein levels by Aβ was triggered by an upregulation of apoE mRNA, in contrast to changes in secretion or degradation. The results show a time-dependent increase in apoE mRNA expression levels with peak expression reached after 1 hour of Aβ treatment. βAR antagonists were used to evaluate the involvement of the βAR. The antagonists significantly inhibited the Aβ-induced stimulation of apoE mRNA and protein levels. In order to further understand the mechanism behind these results we assessed cAMP role in the proposed Aβ-apoE pathway. This second messenger has been associated with AD and has been shown to elevate apoE message and secretion levels. The data shows an Aβ-dependent elevation in cAMP levels as well as an increase in apoE levels after dBcAMP treatment, confirming the activation of a cAMP-dependent pathway. In addition, I provide evidence that confirms the participation of the transcription factor AP-2, specifically that of AP-2β. AP-2 is known to be unregulated by cAMP and to bind to the apoE promoter. I report an increase in AP-2β translocation to the nucleus after both cAMP and Aβ treatment and confirm its participation in the activation of the apoE promoter. In conclusion, my work reveals a novel pathway for Aβ stimulation of apoE abundance in astrocytes involving βAR and the transcription factor AP-2β. These findings not only help clarify the relationship between Aβ and apoE but also help understand AD progression and possibly show a mechanism that could aid in the fight against this fast growing disease.Item Agonist-dependent mechanism of Mu-opioid receptor desensitization.(2009-12) Chu, JiDesensitization of the μ-opioid receptor (MOR) has been implicated as an important regulatory process in the development of tolerance to opiates. Desensitization of G-protein coupled receptor (GPCR) is thought to involve receptor phosphorylation and subsequent recruitment of βArrestins (βArrs). However, the roles of receptor phosphorylation and βArr in morphine-induced MOR desensitization remain to be demonstrated; this may result from the insensitivity of the methods used to study receptor function. Using MOR-induced intracellular Ca2+ ([Ca2+]i) release to monitor receptor activation, [D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO) induced MOR desensitization in a receptor phosphorylation- and βArr-dependent manner. DAMGOinduced desensitization was blunted in HEK293 cells expressing the MORS375A mutant and was eliminated in MEF cells isolated from βArr2 knockout mice expressing the wild type MOR. However, although morphine induced a more rapid desensitization of [Ca2+]i release than DAMGO did and could induce the phosphorylation of the Ser375 residue of MOR, morphine-induced desensitization was not influenced by mutating MOR phosphorylation sites or in MEF cells lacking βArr1 and 2. In contrast, morphine induced MOR desensitization via protein kinase C (PKC). By using subtype-specific inhibitors, PKCε was shown to be the PKC subtype activated by morphine and the subtype responsible for morphine-induced desensitization. Meanwhile, DAMGO did not increase PKCε activity and DAMGO-induced MOR desensitization was not affected by a PKCε inhibitor. Among the various proteins within the receptor signaling complex, Gαi2 was phosphorylated by morphine-activated PKCε. Moreover, mutating v three putative PKC phosphorylation sites, Ser44, Ser144 and Ser302 on Gαi2 to Ala attenuated morphine-induced, but not DAMGO-induced desensitization. In addition, pretreatment with morphine desensitized cannabinoid receptor CB1 agonist WIN 55212-2-induced [Ca2+]i release, and this desensitization could be reversed by pretreating with a PKCε inhibitor or overexpressing of Gαi2 with the putative PKC phosphorylation sites mutated. Thus, depending on the agonist, activation of MOR could lead to heterologous desensitization and probable crosstalk between MOR and other Gαi-coupled receptors such as the CB1 receptor.Item Agonist-selective signaling and MOR.(2009-12) Zheng, HuiOpioids are potent analgesics, but their application is limited by the development of tolerance (the increase in doses required to achieve the same effect) after chronic or repetitive usage. Because tolerance developed much more easily for the analgesia effect than for side effects (e.g. respiration depression), it is difficult to be overcome by simply increasing the doses of opioids. On the other hand, the identification of opioid receptors and endogenous agonists suggests the involvement of opioid pathways in the central nervous system. Thus exploring the mechanisms of tolerance development has been the focus of a vast number of laboratories for decades. Several hypotheses on tolerance development have been proposed. For example, because of the correlation between receptor internalization and tolerance development, receptor internalization has been considered as an inhibitor of tolerance. In addition, the involvement of δ-opioid receptor preproenkephalin, Ca2+/calmodulin-dependent protein kinase II, Protein Kinase C and β-arrestin2 has been suggested by knockout experiments. However, there is no universal explanation for tolerance development. The long-term goal of my studies is to understand the mechanism of tolerance development. Taking advantage of the observations that opioids have different abilities to induced tolerance and signaling events, I have proposed is that agonists induce different levels of tolerance by inducing different signaling events (agonist-selective signaling). Because of the inconsistency between the time courses of signaling cascades (usually seconds to hours) and the tolerance development (usually hours to days), It is suggested that the agonist-selective regulation on gene expression transduces the signals from the agonist-selective signaling to agonist-selective tolerance development. Hence, in my studies, the different abilities of agonists to initiate signaling events (ERK phosphorylation, receptor desensitization on intracellular calcium release) were compared. Then several determinants (receptor phosphorylation, cholesterol-rich lipid raft microdomain, receptor palmitoylation) for the agonist-selective signaling were identified. The final portion of my studies is to explore how agonist-selective regulation on gene expression (NeuroD and miR-190) results from the agonist-selective signaling and to determine whether the agonist-selective regulation on gene expression can contribute to the different levels of tolerance induced by agonists.Item Changes in endocannabinoid signaling contribute to the anti-hyperalgesic effect of URB597 in a murine model of persistent inflammation.(2011-09) Lindberg, Amy ElizabethModulation of endocannabinoid neurotransmission has a therapeutic benefit in the treatment of inflammatory pain. Studies in this thesis investigated endocannabinoid signaling in a murine model of persistent, peripheral inflammation. Specifically, the ability of URB597, an inhibitor of fatty acid amide hydroxylase (FAAH), which degrades the endogenous cannabinoid anandamide, to reduce mechanical hyperalgesia associated with inflammation was determined. The first study tested whether local injection of URB597 dose-dependently reduced mechanical hyperalgesia associated with persistent inflammation. Inflammation was induced by injection of Complete Freund's Adjuvant (CFA) in the hind paw of mice and mechanical hyperalgesia was determined using a series of von Frey filaments. The first part of the study resolved that local injection of URB597 dose-dependently reduced mechanical hyperalgesia associated with persistent inflammation and decreased mechanical sensitivity in naïve mice. However, injection of URB597 did not result in increased endocannabinoid content in the plantar skin ipsilateral to the injection as would be expected based on the known mechanism of action of URB597. The second and third studies investigated the effect of inflammation on levels of FAAH, endocannabinoids and cannabinoid (CB)-1 receptor in naïve and CFA-injected mice to understand the neurochemistry underlying the anti-hyperalgesic effect of URB597. Levels FAAH mRNA decreased and enzyme activity trended toward a decrease in the plantar skin of the inflamed hind paw compared to tissue from naïve mice, but inflammation did not alter level of anandamide in plantar skin ipsilateral to the injections. In contrast, an increase in FAAH mRNA was accompanied by a decrease in the level of anandamide in dorsal root ganglia (DRGs) ipsilateral to the inflamed hind paw compared to naïve mice. In addition, there is an upregulation of functional CB1 receptors in DRGs ipsilateral to the inflamed hind paw in CFA-injected mice compared to DRGs from naïve mice. Together, these data support a model in which reduced synthesis of AEA in the primary afferent neurons may contribute to the mechanical hyperalgesia associated with peripheral inflammation, and upregulation of CB1 receptors on the primary afferent neurons affected by inflammation may be a compensatory response to decreased basal activation of AEA.Item Chronic morphine treatment-modulated trafficking of AMPA receptors: a potential mechanism for drug addiction(2012-08) Kam, Yuet FongMorphine is the benchmark analgesic for treating chronic pain. However, its clinical uses are hindered by its highly addictive nature, as chronic treatment with the drug will produce physical and psychological dependence upon the cessation of use. Drug craving is the main driving force for relapse after prolonged periods of abstinence, and represents an enormous challenge for the treatment of drug addiction. Since addiction is a long-term behavioral alteration, it is believed that addictive drugs produce reorganization of specific neural circuits and adjustment of synaptic strength. The underlying mechanisms of these neural adaptations may represent a promising target for prevention and/or treatment of addiction, but the detailed mechanisms of these processes remain unclear. Therefore, the main goals of this work are to delineate signaling pathways controlling morphine-induced neural adaptations and investigate their functional role in opiate addictive behaviors. Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are postsynaptic glutamate receptors, and are responsible for mediating most excitatory synaptic transmission under normal conditions. More importantly, the dynamic localization of AMPA receptors plays a critical role for modifying synaptic strength and synaptic morphology. Here, I hypothesized that regulation of AMPA receptor trafficking by morphine treatment underlies the drug-induced neural modulation implicated in the addiction process. Hence, the first part of this dissertation research examined whether and how chronic exposure to morphine modulates trafficking of surface GluR1 (a subunit of AMPA receptors) in primary hippocampal neurons. Using live-cell imaging techniques together with biochemical studies, I demonstrated that chronic exposure to morphine induced a significant loss of synaptic and extrasynaptic GluR1 by internalization. In mechanistic studies, I found that the GluR1 internalization was attributed to dephosphorylation of the receptor subunit at Ser845 following morphine treatment, but it did not result from altered neural network or NMDA receptor activation. Moreover, dephosphorylation of GluR1 at Ser845 was found to require morphine-evoked calcineurin activation. Therefore, calcineurin-dependent dephosphorylation of AMPA receptor and subsequent AMPA receptor internalization provides a novel mechanism for opioid-induced neural adaptations. The second part of this dissertation research attempted to link morphine's effects on GluR1 phosphorylation and endocytosis to addictive behavior, especially formation of memory for the environmental context of the drug experience, because recall of this memory by encountering the drug-paired cues triggers relapse to drug seeking. In this approach, a mutant mouse line was used, in which GluR1 at Ser845 was mutated to Ala (S845A) leading to an absence of morphine-induced GluR1 endocytosis. A behavioral test, conditioned place preference (CPP), was carried out to assess the ability of morphine to produce a positive association with environmental cues. I found that S845A mice were significantly slower to acquire morphine-induced CPP when compared to wild types (WT). This decreased sensitivity to morphine CPP in mutants was neither related to contextual memory deficits or abnormal locomotor activity, as there was no difference between WT and S845A mice in the contextual memory acquisition in the Morris water maze test or locomotion with or without morphine injection. To examine the persistence of morphine-associated contextual memory in the mutant mice, I also performed extinction tests on mice conditioned with 10 mg/kg morphine for four sessions, by which both WT and S845A mice exhibited similar CPP responses. Interestingly, a prolonged extinction was observed in S845A mutant mice, suggesting the S845A mutation either impaired the learning of the new conditioning or prolonged the retention of the old conditioning. Nevertheless, these results suggest that an alteration in GluR1 phosphorylation at Ser845 and subsequent receptor endocytosis/insertion are involved in acquisition and extinction of morphine CPP. Altogether, the present findings indicate that calcineurin-mediated GluR1-S845 dephosphorylation is required for morphine-induced internalization of GluR1-containing AMPA receptors, providing a molecular basis for the drug-induced neural modulation. This work also suggests that this regulation of GluR1 phosphorylation and trafficking by morphine is involved in the modulation of the drug-associated contextual memory, which reflects the involvement of AMPA receptor trafficking in the mechanisms underlying opiate-seeking behaviors.Item Comparative Studies of HTLV-1 Particle Assembly and Morphology(2017-12) Martin, Jessica L.Human T-cell leukemia virus type 1 (HTLV-1; deltaretrovirus genus; Retroviridae) was the first human retrovirus discovered. As the etiological agent of adult T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis, HTLV-1 is responsible for a considerable amount of morbidity and mortality worldwide. Between 5 and 10 million people are infected with HTLV-1, and approximately 500,000 suffer from HTLV-1-related pathologies. Previous observations have implicated HTLV-1 assembly having unique attributes compared with other retroviruses, including human immunodeficiency virus type 1 (HIV-1). The overarching hypothesis under investigation in this dissertation was that the HTLV-1 capsid (CA) domain of Gag encodes the primary determinants that impact immature particle morphology and the process of Gag-Gag oligomerization. To test this hypothesis, experiments were conducted utilizing virus-like particle (VLP) model systems, which demonstrated: 1) HTLV-1 immature particle morphology is unique among retroviral genera, particularly with flat regions of electron density that did not follow viral membrane curvature; 2) the HTLV-1 CA amino-terminal domain (NTD) can functionally replace the HIV-1 CA carboxy-terminal domain (CTD), but the HIV-1 CA NTD cannot replace the HTLV-1 CA CTD, indicating that the HTLV-1 CA subdomains provide distinct contributions to Gag-Gag oligomerization, particle morphology, and particle biogenesis; 3) the discovery of HTLV-1 CA amino acid residues identified by alanine-scanning mutagenesis (i.e., M17, Q47/F48, and Y61) that are important for Gag oligomerization and particle assembly. Taken together, these observations provide new insights into HTLV-1 particle assembly and contributes information that will aid efforts directed towards the discovery of therapeutic targets for intervention in order to prevent the HTLV-1 transmission and pathology in endemic populations.Item Conceptual Modeling of Adaptive Therapy Dosing for Chemotherapeutic Administration in Cancer Allows for the Direct Comparison of Continuous and Adaptive Dosing Regimes(2022-05) McGehee, CordeliaAdaptive therapy of cytotoxic (cell killing) chemotherapy has been proposed as a method to prolong progression-free survival in certain cancers when underlying cell-cell competition between sensitive and resistant cancer cells is present. Traditionally, cytotoxic chemotherapy dosing is administered at the maximal tolerated dose with the goal of rapidly shrinking tumor growth. In the case of a tumor where underlying intratumoral cell-cell competition between a drug sensitive and drug resistant population leads to competition for resources, it is hypothesized that maximally killing the sensitive cell population allows for competitive release of the resistant cell population and outgrowth of a chemotherapy resistant tumor. In adaptive therapy, chemotherapy is administered when a tumor reaches a certain upper threshold and then is discontinued when the tumor shrinks to a specified lower threshold. The purpose of this strategy is to use the sensitive cell population to inhibit the growth of the resistant cell population and increase the length of time to competitive release and outgrowth of the resistant cell population. In this thesis, a modified Lotka-Volterra competition model is explored across competition parameters in order to analytically address 1) the optimality of continuous fixed dose versus adaptive dosing schedules and 2) the role of drug dose and mechanism of action in the choice of dosing regime. Using this model, several novel results are shown. First, for certain parameters, complete tumor eradication can be achieved in the presence of a resistant subpopulation under adaptive cytotoxic or continuous antiproliferative (decreasing growth rate) dosing schedules. Second, in this parameter space, fixed dose antiproliferative dosing schedules are more robust than cytotoxic adaptive regimes to uncertainty in initial conditions. Third, in parameter spaces where eradication of the resistant cell population is not feasible, both fixed dose antiproliferative schedules and cytotoxic adaptive therapy schedules may result in delayed resistant cell outgrowth over maximum tolerated dose and are comparable in their benefits. Overall, these results indicate that both antiproliferative continuous fixed dose therapy and cytotoxic adap-tive therapy can be used for tumor management in the case of underlying intratumoral competition between chemotherapy sensitive and chemotherapy resistant cells.Item Downregulation of natriuretic peptide receptor-A: evidence for clathrin- and dynamin-independent internalization.(2009-12) Flora, Darcy RaeAtrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) are endogenous cardiac hormones that are essential for cardiovascular homeostasis. To decrease blood pressure and cardiac hypertrophy, ANP and BNP bind to the transmembrane guanylyl cyclase natriuretic peptide receptor-A (NPR-A). Activation of NPR-A leads to the synthesis of the intracellular second messenger cGMP, which mediates the physiological effects of the natriuretic peptides. Under cardiovascular stress serum ANP and BNP concentrations are elevated. Initially, these cardiac peptides stimulate compensatory hemodynamic functions, but over time their cardiac unloading effects wane despite continued elevation of serum ANP and BNP levels. Unfortunately, the underlying molecular mechanisms responsible for the diminished effect are poorly understood. Transaortic banding was performed to induce congestive heart failure in mice; failed hearts had both reduced ANP-dependent guanylyl cyclase activity and NPR-A protein levels compared to control hearts, indicating that NPR-A is downregulated in the failed heart. Consistent with the in vivo studies, cell culture experiments demonstrated that prolonged ANP exposure resulted in degradation of NPR-A in multiple cell lines. To investigate potential mechanisms involved in NPR-A downregulation, a novel antibody-based intracellular accumulation assay was developed. The assay revealed that NPR-A is basally internalized by a relatively slow clathrin- and dynamin-independent process that is stimulated by ANP. Dynamin inactivation increased intracellular accumulation of NPR-A at long, but not short, time periods after initiation of internalization, which is consistent with the notion of NPR-A recycling in a dynamin-dependent process. Surprisingly, the rate of NPR-A internalization was accelerated in clathrin-depleted cells. Understanding the molecular mechanisms underlying NPR-A downregulation will aid in the development of potential therapeutic strategies that disrupt this process and prolong the beneficial compensatory effects of natriuretic peptides in patients with cardiovascular disease.Item Dynamic regulation of R7BP (R7 Binding Protein) containing R7 RGS (R7 Regulators of G protein Signaling) protein complexes: role in controlling neuronal dopamine and opioid signaling in the striatum.(2010-02) Anderson, Garret R.G protein-coupled receptor (GPCR) signaling pathways mediate the transmission of signals from the extracellular environment to the generation of cellular responses, a process that is critically important for neurons and neurotransmitter action. The ability to promptly respond to rapidly changing stimulation requires timely inactivation of G proteins, a process controlled by a family of specialized proteins known as regulators of G protein signaling (RGS). The R7 group of RGS proteins (R7 RGS) has received special attention due to their pivotal roles in the regulation of a range of crucial neuronal processes such as vision, motor control, reward behavior and nociception in mammals. One member of the R7 RGS family, RGS9-2 has been previously implicated as an essential modulator of signaling through neuronal dopamine and opioid G protein coupled receptors. RGS9-2 is specifically expressed in striatal neurons where it forms complexes with R7BP (R7 RGS Binding Protein), which we have found to ultimately affect several critical properties of RGS9-2. First, it is this interaction with R7BP which is necessary for determining the subcellular targeting of RGS9-2 to the plasma membrane and to the specialized neuronal compartment of excitatory synapses, the postsynaptic density. Secondly, R7BP plays a selective role amongst the R7 RGS family in determining the proteolytic stability of RGS9-2. Further characterization of R7 RGS complexes in the striatum revealed that two equally abundant R7 RGS proteins, RGS9-2 and RGS7, are unequally coupled to the R7BP subunit which is present in complex predominantly with RGS9-2 rather than with RGS7. However, upon changes in neuronal activity the subunit composition of these complexes in the striatum undergoes rapid and extensive remodeling. Changes in the neuronal excitability or oxygenation status result in extracellular calcium entry, uncoupling RGS9-2 from R7BP, triggering its selective degradation. Concurrently, released R7BP binds to cytoplasmic RGS7 and recruits it to the plasma membrane and the postsynaptic density. These observations introduce activity dependent remodeling of R7 RGS complexes as a new molecular plasticity mechanism in striatal neurons and suggest a general model for achieving rapid posttranslational subunit rearrangement in multi-subunit complexes. The physiological consequence of this remodeling process appears to play a role in determining the signaling sensitivity to dopamine stimulation. Considering that upon the genetic elimination of RGS9, all available R7BP is funneled towards complex formation with RGS7, not only are RGS9 controlled GPCR signaling pathways affected, but those controlled by RGS7 as well. RGS9 knockout mice have an increased sensitivity to dopamine and opioid receptor stimulation and consequently display altered motor and reward behavior. The question arises as to the role of modulation of RGS7 function in controlling these behaviors. Since the function of RGS9-2 is controlled by its association with R7BP, we would predict that the elimination of R7BP would lead to similar alterations in striatal physiology for RGS9 controlled pathways. While at the same time, RGS7 would be largely unaffected by the elimination of R7BP, thus RGS7 controlled pathways would predictably remain unaltered. Using this rationale, we report that elimination of R7BP in mice results in motor coordination deficits and greater locomotor response to morphine administration consistent with the essential role of RGS9 in controlling these behaviors and the critical role played by R7BP in maintaining RGS9-2 expression in the striatum. However, in contrast to previously reported observations with RGS9-2 knockouts, mice lacking R7BP do not exhibit higher sensitivity to locomotor-stimulating effects of cocaine, suggesting a role for RGS7 in controlling dopamine sensitivity. Using a striatum-specific knockdown approach, we demonstrate that the sensitivity of motor stimulation to cocaine is indeed dependent on RGS7 function. These results indicate that dopamine signaling in the striatum is controlled by concerted interplay between two RGS proteins, RGS7 and RGS9-2, which are balanced by a common subunit, R7BP.Item Effects of chronic morphine treatment on tumor angiogenesis and growth.(2009-06) Koodie, LisaMorphine is one of the most effective analgesics commonly prescribed for the treatment of severe to moderate cancer pain. To date very little is known regarding the effect of long-term morphine treatment on tumor angiogenesis. At this time, the effect of morphine on tumor growth is contradictory and still inconclusive. As solid tumors grow, the formation of a blood supply or angiogenesis is essential. In previous studies, morphine inhibited vascular endothelial growth factor (VEGF) secretion from mice cardiomyocytes and human umbilical vein endothelial cells. VEGF is a highly potent pro-angiogeneic molecule and we therefore hypothesized morphine would also inhibit angiogenesis associated with tumor growth. In the first part of these studies we show that morphine inhibited the hypoxia-induced tumor cell expression of VEGF to significantly reduce tumor cell angiogenesis, and suppress tumor growth in vivo. Additional investigations supported the view that the effect of morphine was not due to a direct effect on tumor cell apoptosis, but instead indirectly through angiogenesis. Tumor, stromal and inflammatory cells within the tumor microenvironment all contribute to a large pool of chemoattractants that increase the recruitment of myeloid cells from peripheral blood circulation into the tumor tissues. These cells mature and differentiate into neutrophils, and macrophages that eventually result in a pro-inflammatory-like environment to support and maintain tumor growth. Considering that morphine is highly immuno-suppressive, we also hypothesized that morphine will inhibit immune cell recruitment and thus angiogenesis. In an in vivo model of cell migration and recruitment we found that morphine inhibited not only CD11b+ progenitors of inflammatory cells but also the recruitment of Tie2+/CD14+ endothelial cell precursors known to actively participate in vessel formation to tumor sites. These studies have allowed us to further understand the effects of a potent analgesic such as morphine in cancer growth. Our data support the use of morphine for pain associated with cancer. Our results support the view that morphine may not cause any further detriment in the cancer patients' quality of life but further suppress angiogenesis associated with tumor growth and progression.Item Endogenous modulation of addiction: chronic pain and the NMDA/NOS cascade.(2010-07) Wade, Carrie LynnOpioid treatment for chronic pain is controversial due to abuse potential and perceived addiction potential. Because of perceptions of addiction from chronic opioid treatment for pain it is important to clearly understand the biological bases for a number of factors related to opioid therapy in the context of chronic pain, including the effectiveness of opioid treatment under distinct conditions chronic pain and alterations in the effectiveness of opioid treatment under distinct conditions of chronic opioid pharmacotherapy. One way to approach this question is to study the changes that occur with chronic pain and see how those changes parallel those that occur with opioid addiction. Our approach to address the questions raised above is to apply a combination of rodent models of pain and opioid self-administration. In the first phase of this study we examine changes in oral fentanyl self-administration under distinct conditions of chronic pain including inflammatory pain, neuropathic pain and an idiopathic pain model of sickle cell anemia. The second set of studies examines the potential for an endogenous modulator of the NMDA/NOS cascade to interact with adverse opioid events such as tolerance and addiction. We observed that mice with inflammatory pain, neuropathic pain and sickle cell anemia had differential fentanyl self-administration profiles following induction of mechanical hyperalgesia. In the second set of studies we observed that agmatine reduced opioid-induced tolerance and abolished self-administration behaviors. We also found that endogenous agmatine may have a neuroprotective effect on these opioid effects.Item Examination of molecular changes in acquired tamoxifen resistance and subsequent response to anti-IGF1R therapy.(2012-06) Fagan, Dedra HannahThe type-I insulin like growth factor (IGF1R) contributes to the proliferation, survival, and metastasis of breast cancer cells. Disruption of IGF1R signaling alone or in combination with established therapies has emerged as an important strategy in cancer therapy. Several anti-IGF1R antibodies and tyrosine kinase inhibitors (TKI's) are being evaluated in phase I, II, or III clinical trials, often in endocrine resistant populations. Thus far, clinical trials have provided less than promising results. Although preclinical studies showed promising results, these studies were performed using endocrine sensitive cell models. Here, we sought to determine the efficacy of IGF1R inhibitors using an endocrine resistant human breast cancer cell model in vitro and in vivo. The first section of this work highlights the creation and characterization of a tamoxifen-resistant (TamR) cell line. We demonstrate in two estrogen receptor positive breast cancer cell lines that TamR cells maintain estrogen receptor expression. Levels of IGF1R, a known estrogen regulated gene, were greatly reduced in TamR cells. Further, signaling, proliferation, and anchorage-independent growth through the receptor were abolished in TamR cells. Interestingly, signaling and growth through the closely related insulin receptor (a non-estrogen regulated gene) remained intact in TamR cells. The second part of this work utilizes TamR cells to examine the efficacy of IGF1R inhibitors in endocrine resistant and sensitive breast cancer cells. We show that the signaling, proliferation, and anchorage-independent growth of endocrine sensitive MCF-7 cells can be inhibited using a variety of IGF1R antibodies. TamR cells, which lack IGF1R expression, are not affected by IGF1R antibody treatment in vitro or in vivo. In contrast, tyrosine kinase inhibitors which can inhibit both IGF1R and IR were able to inhibit the signaling, proliferation, and anchorage-independent growth of both TamR and parental cells. Taken together, our data demonstrate that tamoxifen resistant cells and tamoxifen treated xenografts have reduced levels of IGF1R, making IGF1R antibody treatment ineffective. Our work highlights the importance of evaluating new therapies using a preclinical model that matches the patient population the therapy will be used in. Finally, our data suggest that inhibition of IR may be necessary to manage tamoxifen resistant breast cancer.Item Functional role of receptor-interacting protein 140 (RIP140) in adipocyte dysfunctions and inflammatory response in macrophages.(2012-03) Ho, Ping-ChihThe prevalence of metabolic diseases in modern society, including Type II diabetes mellitus (T2DM), hypertension and cardiovascular diseases, is a major burden on health care systems. Among these diseases, T2DM and its associated complications contribute to the progression of other metabolic diseases such as fatty liver diseases and atherosclerosis. Understanding the initiation and progression of T2DM is critical for developing treatments for T2DM and its associated metabolic disorders. Adipocyte dysfunctions and chronic inflammation have been shown recently to play essential roles in the progression of T2DM. Normally, adipocytes can store energy as triglycerides, fine-tune other metabolic tissues¡¦ lipid and glucose metabolism, and secreted cytokines (adipokines) to modulate immune response. In T2DM or obesity, adipocytes become dysfunctional, with increased lipolysis, an altered adipokine profile, and decreased insulin sensitivity and glucose uptake ability. These changes affect not only the adipocytes themself but also systemic glucose and lipid metabolism. In obese patients and in the high-fat diet (HFD)-fed mouse model, increased inflammatory response in macrophages also contributes to adipocyte dysfunction. The escalated inflammatory response plays pathophysiological roles in various metabolic disorders, including atherosclerosis and arthritis, and increases the incidence of septic shock. However, the underlying mechanisms for initiation of adipocyte dysfunctions and escalation of inflammatory response remain unclear. Receptor-interacting protein 140 (RIP140) is a co-regulator for various transcription factors and nuclear receptors and is expressed mainly in macrophages and metabolic tissues, including adipocytes, hepatocytes and muscle cells. RIP140 affects the progression of T2DM through its nuclear activity as shown by the resistance of knockout mice to diet-induced diabetes and its associated metabolic disorders. In my studies, I found that when I used HFD feeding to induce T2DM, RIP140 could accumulate within the cytoplasm of adipocytes. I further demonstrated that cytoplasmic RIP140 not only interacted with AS160 to impede GLUT4 vesicle trafficking and adiponectin vesicle secretion, but also formed a complex with perilipin A to enhance lipolysis. These findings suggest that HFD feeding can alter RIP140¡¦s cellular distribution, which leads to adipocyte dysfunctions including higher lipolysis, lower glucose uptake, and reduction in adiponectin secretion. I also showed that HFD feeding promoted cytoplasmic accumulation of RIP140 in adipocytes through a PKCϵ-dependent signaling pathway by enhancing intracellular lipid content (as an intrinsic stimulus) and circulating endothelin-1 (as an extrinsic stimulus). Most importantly, administration of a selective ET-1 receptor anatagonist, ambrisentan, reduced HFD-induced cytoplasmic accumulation of RIP140 in adipocytes and further ameliorate hepatic steatosis and insulin sensitivity in vivo. These findings reveal the novel roles of cytoplasmic RIP140 in adipocyte dysfunctions and provide evidence for cytoplasmic RIP140 as a promising target for treatment of T2DM. Recently, RIP140 has also been shown to affect proinflammatory cytokine production by functioning as co-activator for NF-fÛB in macrophages. I showed that HFD feeding up-regulated RIP140 expression by promoting intracellular cholesterol level which led to increased proinflammatory potential in macrophages. In this study, intracellular cholesterol level regulates RIP140 expression by decreasing microRNA-33a, which targeted RIP140 via a conserved region in 3¡¦-UTR of RIP140 mRNA. I further discovered that TLR ligands could trigger RIP140 degradation to resolve inflammation. This RIP140 degradation was modulated by RelA-recruited SCF E3 ligase and Syk-mediated phosphorylation on RIP140. My studies in macrophages demonstrate that RIP140 in macrophages can be modulated by a HFD to affect the systemic inflammatory response and further suggest that defects in RIP140 degradation may cause non-resolving inflammation which is involved in septic shock and various metabolic disorders. Taken together, my studies provide evidence for the novel functions of RIP140 in adipocyte dysfunction and inflammatory response in macrophages and determine the mechanisms by which HFD affect RIP140¡¦s distribution and expression in adipcoytes and macrophages. These findings contribute to our understanding of how HFD causes adipocyte dysfunctions and increase inflammatory response.Item Genome-wide pharmacological modulation of cap-dependent translational control(2013-12) Braziunas, Jeffrey JosephThe first step of cap-dependent translation is mediated by the mRNA cap-binding protein eukaryotic initiation factor 4E (eIF4E). Although involved in translating nearly all cellular transcripts, mRNAs vary widely in their translational response to eIF4E activity changes. Prior studies of mRNA structure revealed several features governing eIF4E responsiveness; however, most of this knowledge is based on comparison of two levels of eIF4E activity with unclear physiological relevance. To identify mRNA structural features that govern genome-wide ribosome recruitment across a full range of physiological eIF4E activities, we precisely modulated eIF4E activity using an eIF4E-inducible system together with 4Ei-1, an inhibitor of the eIF4E-5'mRNA cap association. We identified genes that were more (4E hypersensitive) or less (4E hyposensitive) responsive to eIF4E activity changes than average. Distinct characteristics associated with each class: 4E hypersensitive genes had longer 5'UTRs with higher GC content, longer 3'UTRs with lower GC content; more AU-rich elements and a higher density of unique microRNA targets sites than typical genes. Importantly, these structural characteristics predicted the translational response across the dose range of 4Ei-1. Gene ontology analysis showed an association between 4E hypersensitive genes and proliferation; and cell cycle experiments with 4Ei-1 validated this result. A search for the outcome and mechanism of this proliferative gene activation in a physiological setting revealed that abrupt gain of eIF4E function in quiescent cells first triggers G0 exit and then cell cycle transit at least partially by increasing ribosome recruitment to cyclins C and D1. Whereas cyclin C is not necessary for this effect; cyclin D1 is indispensable, although not sufficient. Our findings provide important insights into mRNA properties of eIF4E-modulated translational control.Item A heroin/morphine vaccine: mechanism of action and extending its use to other abused opioids(2013-07) Raleigh, Michael DennisHeroin is more widely used than any other illicit opioid and mortality rates among heroin users are an average of 13 times higher than the general population. Intravenous heroin use is associated with crime, social disruption, and transmission of blood-born pathogens such as human immunodeficiency virus and hepatitis C. Effective pharmacotherapies are available to treat heroin abuse but have been largely unsuccessful because they require frequent dosing, have a high abuse potential, or have low compliance. Vaccines against heroin and its metabolites (e.g. morphine) are being considered as a complementary treatment for heroin abuse because they are long-acting, selective, have no abuse potential, and may benefit those unwilling to take the current pharmacotherapies. Vaccination with morphine-conjugate vaccines can elicit a strong immune response that reduces the behavioral effects of heroin in animals, presumably by morphine-specific antibodies binding opioids in blood and reducing their distribution to brain. This thesis explores the use of M-KLH, a morphine hapten conjugated to keyhole limpet hemocyanin (KLH) using a tetraglycine linker and mixed with either Freund's or alum adjuvant for increasing the immune response. Morphine vaccines present many challenges that make translation to clinical use difficult. Heroin is sequentially metabolized to its active intermediates 6-monoacetylmorphine (6-MAM), morphine, and then to morphine-6-glucuronide (in vivo and ex vivo). Heroin enters brain and is rapidly converted to 6-MAM, which is presumed to mediate most of heroin's early effects. With regard to the mechanism of action of morphine vaccines, it is unclear whether the antibodies they generate must bind heroin, its downstream metabolites, or both to prevent opioid distribution from plasma to brain and reduce heroin's behavioral effects. However, because analytical assays to measure heroin and metabolite concentrations in tissues have used a wide range of conditions and varying degrees of stability have been reported, studying the effect of vaccination on heroin distribution is not straightforward. In addition, heroin and metabolite distribution after i.v. heroin administration, the most common route of abuse by humans, has not been well characterized in non-vaccinated rodents. Finally, blockade of heroin by vaccination may not prevent the abuse of structurally distinct opioids. The overall goal of this thesis was to better understand the mechanism of action of morphine vaccines and to extend their use to other abused opioids. The specific aims were to stabilize heroin in blood and brain tissues for subsequent pharmacokinetic studies, study distribution of heroin and its metabolites in non-vaccinated and vaccinated rats, explore the effects of vaccination on heroin-induced behaviors, and determine if vaccine efficacy is retained when combined with a vaccine targeting oxycodone, another commonly abused opioid. These aims were explored using clinically relevant drug doses. Heroin and metabolite degradation was significantly reduced by 1) the addition of ice-cold sodium fluoride (a general esterase inhibitor) and formate buffer (pH 3.0) in heroin-spiked tissues, 2) rapid removal of red blood cells via centrifugation, and 3) drying opioids after extraction from tissues prior to measuring their levels. Using these conditions heroin and its metabolites were stabilized in tissues for subsequent distribution studies. In non-vaccinated rats 6-MAM was the predominant metabolite in brain as early as one minute after administration of 0.26 mg/kg i.v. heroin, which is consistent with previous studies that suggest that 6-MAM mediates heroin's early behavioral effects. Vaccination with a morphine-conjugate vaccine (M-KLH) led to a reduction of 6-MAM and morphine, but not heroin, distribution to brain after heroin administration, suggesting that morphine vaccines reduce accumulation of 6-MAM in brain. The mechanism by which this occurs is likely through antibody binding of 6-MAM in plasma to prevent its distribution to brain and is consistent with very high plasma 6-MAM concentrations in vaccinated rats after i.v. heroin or 6-MAM administration. Vaccination with M-KLH led to a reduction of heroin-induced anti-nociception and locomotor activity and remained effective for up to 16 days after repeated dosing suggesting that heroin vaccines may have long-lasting efficacy. These results are consistent with findings from the distribution studies and support the hypothesis that morphine vaccines function by retaining 6-MAM in plasma and prevent its accumulation in brain. To determine whether opioid vaccines could be combined without reducing individual vaccine efficacy and prevent heroin addicts from abusing structurally distinct opioids, rats were vaccinated with M-KLH, an oxycodone-conjugate vaccine (Oxy-KLH), or the bivalent vaccine (both M-KLH and Oxy-KLH). Total morphine- and oxycodone-specific antibody titers were significantly increased in rats that received the bivalent vaccine compared to rats that received individual vaccines. Concurrent i.v. administration of 6-MAM and oxycodone in M-KLH vaccinated rats led to increased 6-MAM retention in plasma and reduced 6-MAM distribution in brain. A similar effect on oxycodone distribution was seen in Oxy-KLH vaccinated rats. There was a trend towards greater efficacy in altering both 6-MAM and oxycodone distribution in the bivalent group compared to individual vaccine groups. These data suggest that combining opioid vaccines will retain, and possibly enhance, individual vaccine efficacy and might be a viable option to prevent addicts from abusing structurally distinct opioids. These findings contribute to the understanding of how morphine vaccines elicit their effects on heroin-induced behaviors and suggest that morphine vaccines, alone or in combination with other pharmacotherapies, may benefit those seeking treatment for heroin addiction.Item Identification and characterization of novel ADP-ribosyl cyclase family members(2011-08) Hirte, Renee MarieCD38 and CD157 have been identified as mammalian forms of ADP-ribosyl cyclase (ADPRC). Recently novel membrane bound and cytosolic ADPRCs that are distinct from CD38 and CD157 have been identified in various tissues by our laboratory as well as others. Recently, evidence has indicated the presence of ADPRC in tissues lacking CD38. From this it is clear that there are multiple forms of mammalian ADPRC, many of which have not yet been identified or characterized. The overall goal of the research presented in this thesis was to identify and characterize the novel cytosolic cyclase(s) present in the heart cytosol and determine the mechanism(s) by which cytosolic cyclase(s) are regulated. Previously it has been shown that Aplysia ADPRC, CD38 and CD157 share approximately 30% sequence identity. There are two main features shared by each of these members of the ADPRC family including a conserved region near the center and ten conserved cysteine residues that can be perfectly aligned. We used a molecular approach to identify potential candidates for the cytosolic protein (or other yet unidentified ADPRCs) based on a conserved protein motif search of the mouse genome to identify proteins that shared the feature of the conserved cysteine residues. The existence of novel cyclases has implications for a broad range of cellular processes that are influenced by calcium signaling. Characterization and identification of novel ADPRCs may provide key insight into the function of the novel cyclase(s) as well as interaction and relationships to other members of the ADPRC family, which will further elucidate the complexities of calcium signaling.Item Identification of compounds inhibiting a Leishmania RNA editing reaction.(2010-05) Liang, ShuangSeveral species of Leishmania are human pathogens that afflict more than 12 million people worldwide, and the current treatment options are limited. An RNA editing reaction that is both essential and specific to the parasites is an attractive target for new drug development. The editing reaction involves the post-transcriptional modification of specific mitochondrial mRNAs through the precise deletion or insertion of uridylates. Many aspects of the editing mechanism are still unclear, and the lack of specific inhibitors to probe the reaction has hindered the field. Although high-throughput screening of chemical libraries is a powerful strategy often used to identify inhibitors, the available in vitro editing assays do not have the necessary sensitivity and format for this approach to be feasible. A novel editing assay was developed in this thesis that overcame previous limitations as it can both detect edited product in the low femtomole range and is ideal for high-throughput format. The reporter for the assay consists of an RNA editing substrate linked to a streptavidin-binding domain that is initially held within an inactive conformation. An in vitro selection strategy optimized the linkage so that the streptavidin-binding domain is only activated by an editing-induced conformational change. The reporter RNA is labeled with a ruthenium complex, and an electrochemiluminescent signal results from the ruthenium label when the reporter is bound to the bottom of a streptavidin-coated microtiter plate where it can be stimulated by a carbon electrode. Chemical probing, mutagenesis and binding affinity measurements were used to characterize the reporter. This highly sensitive assay was optimized and validated for use in high-throughput screening, and a pilot screen of a 1280 compound library identified compounds that are the first specific inhibitors of the editing reaction. Some of the identified inhibitors will have value as probes of the editing reaction and have already provided insights into possible regulatory mechanisms. The identification of novel drugs through screens of large chemical libraries is now possible with the new assay.Item Investigation of proteins that interact with NAADP-Gated two-pore channels.(2012-02) Moshier, Yaping LinAll living organisms respond to environmental stimuli by eliciting a sequence of signaling cascades, many of which converge in regulating [Ca2+]cyt via intracellular Ca2+ stores. Three agonist-mediated second messengers have been identified, including inositol 1,4,5-trisphosphate (IP3), cyclic ADP ribose (cADPR), and nicotinic acid adenine dinucleotide phosphate (NAADP). NAADP is the most potent calcium mobilizer identified to date, and unlike IP3 and cADPR that target ER Ca2+ stores, NAADP-mediated Ca2+ response is restricted to acidic Ca2+ stores. Several candidate Ca2+ channels expressed in the endolysosomal system have been proposed to be gated by NAADP, with recently two-pore channels (TPCs) emerging as NAADP targets. My research project utilized a radioactive photoactivable NAADP analogue, 32P-5 azido-NAADP (32P-5N3-NAADP), to perform an unbiased assay in a variety of model systems, attempting to verify NAADP targets via a direct crosslinking approach. My results revealed that 5N3-NAADP labeled protein candidate(s) were significantly smaller than predicted sizes of TPC proteins (85-100kDa) in all systems examined (22/23kDa doublet in mammals, 41kDa in sea urchin). Further, the labeling pattern and intensity of the NAADP-targeted doublet remained unchanged in TPC-overexpressing cells. Surprisingly, photoaffinity labeling (PAL) of the high affinity NAADP targets was preserved in TPC-knockout pancreatic samples, further suggesting that NAADP binding and Ca2+ release are mediated by distinct protein identities. My data contradicts current models that TPCs are direct NAADP receptors, implying an alternative cellular binding partner for NAADP on endolysosomes that then serves to indirectly regulate TPC activity. This is an important revision of current dogma, and crucial for rational design of drugs that may modulate NAADP activity. Such therapeutics may be important in disorders (diabetes, lysosomal storage disorders, and neuronal excitotoxicity) where NAADP signaling is pathologically perturbed.Item Mechanisms of HIV-associated neurotoxicity: Tat-induced synapse loss and recovery(2013-05) Brunner, Angela HaijungHIV infection is a worldwide pandemic. A debilitating neurological consequence of HIV infection is progressive cognitive decline, known as HIV-associated neurocognitive disorders (HAND). HAND afflicts up to 50% of all HIV patients to varying degrees, and as survival of HIV patients improves with current antiretroviral therapies, the prevalence of HAND is also increasing. This, coupled with the lack of current effective HAND therapies, creates a dire need to understand the mechanisms underlying the cognitive decline associated with HIV. HAND symptoms correlate closely with processes of neuronal injury, which are early events that precede overt neuronal death. One such injurious process is synapse loss. The HIV protein transactivator of transcription (Tat) is a neurotoxic viral protein released from infected cells into the central nervous system. Tat contributes to the pathologies seen in HAND patients, and induces loss of excitatory synapses between rat hippocampal neurons in culture. Using an innovative live cell imaging assay, our laboratory has previously shown that Tat induces reversible synapse loss via a pathway that is distinct from cell death. In this dissertation, I outline three studies that stem from the current knowledge involving Tat-induced synapse loss. These studies elucidated important information regarding the mechanisms by which HIV Tat exerts its neurotoxic effects, emphasizing the importance of subunit composition when determining toxic or beneficial effects of NMDA receptor activation as well as unmasking the importance of the postsynaptic density as the central target of Tat's effects. Furthermore, these studies highlight the reversibility of synapse loss and uncover a new role for the canonical NO/cGMP/PKG pathway in modulating synapse recovery downstream of GluN2B-containing NMDA receptors. Tat-induced synapse loss and subsequent recovery can correlate to symptoms of cognitive decline seen in HAND. Targeting these mechanisms can shed new light on therapeutic strategies to treat HAND patients.Item Modulation of hippocampal endocannabinoid plasticity by homer proteins and Delta(9)-tetrahydrocannabinol.(2009-10) Roloff, Alan MathhewThe endocannabinoid (eCB) system comprised of lipophylic signaling molecules, postsynaptic production enzymes, and presynaptic G-protein coupled receptors (GPCR) has recently emerged as a key mediator in a broad range of neuronal plasticity. Initial interest in the system stemmed from, delta(9)-tetrahydrocannabinol (THC) the main psychoactive ingredient in the illicit drug marijuana which elicits various effects on the central nervous system (CNS) through interactions with the eCB system. Further study described a nearly ubiquitous yet tunable system that may underlie many CNS functions. Studying the eCB system offers insight into the basic science of synaptic transmission but more importantly the mechanism by which postsynaptic determinants may influence their synaptic inputs to adapt to changing CNS conditions. The objective of these studies was to determine the properties of THC that dictate its relationship to the intact eCB system and to characterize how specific members of the homer family of postsynaptic scaffolding proteins impact differing forms of eCB-mediated plasticity. Activation of the cannabinoid receptor-1 (CB1R) by THC or eCBs leads to several presynaptic consequences, but most importantly to inhibition of voltage-gated calcium channels (VGCC) and thus a reduction of action potential induced neurotransmitter release. In the first study presented here, we described a voltage-mediated switch by which THC acts as an agonist of CB1R or as an antagonist to effects at CB1R mediated by eCBs. Using patch clamp electrophysiology to study excitatory synaptic transmission we discovered that the rate at which the presynaptic neuron is depolarized has drastic effects on THC mediated inhibition. Excitatory postsynaptic currents (EPSCs) evoked at 0.1 Hz were suppressed by THC, however THC did not effect EPSCs evoke at 0.5 Hz. Experiments using other CB1R agonists Win55212,2 (Win-2) and 2-Arachydonylglycerol (2-AG) did not demonstrate sensitivity to stimulus rate. THC application at 0.5 Hz stimulation antagonized the inhibition produced by Win-2. THC but not Win-2 inhibited influx of calcium through VGCCs in a rate dependent manner. Win-2 but not THC shifted the voltage-dependent activation of tail currents. THC but not Win-2 mediated inhibition of VGCCs displayed mild prepulse facilitation. THC antagonized depolarization-induced suppression of excitation (DSE). These findings illustrate the complex interplay between THC and the eCB system and suggest that behavioral responses to THC may stem from a combination of both CB1R activation and inhibition of eCB action at CB1R. Postsynaptic induction of the eCB system and subsequent production of eCBs may be achieved via several interrelated molecular mechanisms that focus around glutamatergic ionotropic and metabotropic signaling and postsynaptic intercellular Ca2+ concentration ([Ca2+]i). Homer, a family of postsynaptic scaffolding proteins, is present in the hippocampus and has been found to organize many of the structural and mechanistic proteins necessary for several forms of synaptic plasticity. In the second study we describe a homer isoform-specific switch in eCB plasticity that primes neurons for ionotropic mediated production of eCBs but shifts them away from metabotropic eCB production. Transfection of hippocampal cultures with homer 1a (H1a) enhances DSE. Transfection of cultures with H1a inhibits metabotropic suppression of excitation (MSE). In combination experiments on the same neuron with DSE followed by MSE, DSE is enhanced and MSE is inhibited in H1a expressing neurons. Brain derived neurotrophic factor (BDNF) induces formation of H1a mRNA and leads to functional expression of H1a which dissociates homer 1c-GFP puncta. BDNF mediates an enhancement of DSE and inhibition of MSE similar to that found in H1a transfected neurons. These findings identify H1a as a crucial mediator of eCB signaling that may be induced by BDNF and can lead to an enhancement of depolarization mediated eCB production. The second study delineated how postsynaptic homer proteins can influence differential paths to activating eCB mediated synaptic plasticity. The physiological manifestations of eCB signaling have tentacles that extend from neurotransmitter release to higher brain function through the regulation of Ca2+ channels and induction of synaptic plasticity. These studies are directed toward understanding the way in which THC interacts with the eCB system, and the postsynaptic scaffolding protein complement which induces changes to eCB-mediated plasticity. Delineating these concepts may lead to better understanding of higher brain function and will offer new targets for therapeutic development.