Browsing by Subject "Molecular and Cellular Neuroscience"
Now showing 1 - 20 of 29
- Results Per Page
- Sort Options
Item 17 Beta-Estradiol, Integrins, and Synaptic Proteins(2009-05-01) Chandra, Manjari; Simpkins, James W.Item 17Beta-Estradiol Suppresses Hydrogen Peroxide-Induced Nuclear Factor Kappa B Activation in HT22 Cells(2008-05-01) Kim, Pil J.; Simpkins; Singh; Yang, ShaohuaKim, Pil J., 17beta-estradiol suppresses hydrogen peroxide-induced nuclear factor κappa B activation in HT22 cells. Master of Science (Biomedical Sciences), May, 2008, 78pp., 20 illustrations, 66 titles. Reactive oxygen species (ROS) are natural byproducts of normal cellular reactions. They are oxygen ions, free (non)radicals, and peroxides that are highly reactive with normal macromolecules, such as lipids, DNA, and proteins. Cells are normally able to defend against the damages of ROS via enzymes that neutralize them into water. However, when cells are not able to cope with the accumulation of ROS, distributions in signaling pathways and gene transcription will occur, which will ultimately lead to cell death. It is now widely accepted that increased oxidative stress-induced damage in the brain is a major cause of neurodegenerative diseases, such as Alzheimer’s disease (AD). Nuclear factor κappa-B (NFκB) is not only a ubiquitously expressed transcription factor but also a signaling protein that is activated by ROS-induced oxidative stress. Our laboratory has demonstrated the neuroprotective effects of 17β-estradiol (E2) are elicited via an anti-oxidant effect. The purpose of this project was to determine the role of NFκB activation in E2-mediated neuroprotection against hydrogen peroxide (H2O2)-induced oxidative stress. HT-22, a murine immortalized hippocampal neuronal cell line, was utilized to determine whether NFκB is activated by hydrogen peroxide-induced oxidative stress and whether E2 suppresses H2O2-induced NFκB activation. We observed that H2O2 activated NFκB by phosphorylation of IκBα (pIκBα), one of the NFκB inhibitor proteins, reduction of total IκBα, and induction of NFκB (p65) nuclear translocation. In contrast, E2 suppressed H2O2-induced NFκB activation by dramatic reducing pIκBα, increasing total IκBα, and inhibiting p65 nuclear translocation. Our results show that one of the mechanisms by which estrogens are neuroprotective against oxidative stress is through the attenuation of H2O2-induced NFκB activation.Item [3H] Ethynylbicycloorthobenzoate ([3H] EBOB) Binding in Native and Recombinant GABAA Receptors(2000-05-01) Yagle, Monica A.; Dillon, Glenn; Martin, Michael; de Fiebre, ChristopherYagle, Monica A., [3H] Ethynylbicycloorthobenzoate ([3H] EBOB) Binding in Native and Recombinant GABAA Receptors. Master of Science (Pharmacology), May 2000, 59 pp., 3 tables, 7 illustrations, bibliography, 75 titles. Modulation of the GABAA receptor has been studied with noncompetitive convulsant ligands such as tert-butylbicyclophosphorothionate (TBPS) and picrotoxin (PTX). EBOB is a more recently developed ligand that appears to bind in the same region of the channel at TBPS, but with a higher affinity. While only a few studies have examined the binding of EBOB to vertebrate brain tissue and insect preparations, none have examined potential subunit-dependent binding of EBOB. We have thus examined [3H] EBOB binding in rat cerebellum and HEK293 cells stably expressing human α1β2γ2, human α2β2γ2, and rat α6β2γ2 GABAA receptors. For comparison, [35S] TBPS binding was also examined in α1β2γ2 receptors. Saturation and Scatchard analyses revealed saturable [3H] EBOB binding at one site in all tissue preparations with Kd values ranging from 3 to 9nM. [3H] EBOB binding, like [35S] TBPS binding was inhibited by the CNS convulsants dieldrin, lindane, tert-butylbicyclophosphorothionate (TBOB), PTX, TBPS, and pentylenetetrazole (PTZ) at one site in a concentration dependent fashion. Affinities were in the high nM to low μM range for all compounds except PTZ (low mM range). GABA modulated [3H] EBOB binding in a biphasic manner in α1β2γ2 receptors with a 100-fold difference between stimulatory and inhibitory affinities. Inhibition of GABA-mediated current by TBOB in α1β2γ2 receptors resulted in a functional IC50 of 0.2 μM, in agreement with binding study results. Differences seen in binding between the different receptor subtypes examined suggest that some characteristics of EBOB binding are subunit dependent. In addition, we have shown that [3H] EBOB is a useful ligand in the study of recombinant GABAA receptors and that results obtained with [3H] EBOB are comparable to those obtained with [35S] TBPS.Item Alzheimer's Fibroblasts are More Susceptible to Oxidative Stress(2001-05-01) Marshall, Pamela L.; Neeraj Agarwal; Robert GracyMarshall, Pamela L., Alzheimer’s Fibroblasts Are More Susceptible to Oxidative Stress. Master’s of Science (Biomedical Sciences). May 2001. Recent evidence indicates that oxidative stress contributes to neuronal death in Alzheimer’s disease (AD). In addition, it has been suggested that AD is a systemic illness in which the development of the disease is only visible in the brain. The aim of this research is to develop experimental procedures using a simple cell model, the fibroblast, to determine if proteins derived from AD skin fibroblasts are more sensitive to oxidation by reactive oxygen species than non-AD cells, and to assess the ability of antioxidants to prevent this oxidative damage in AD fibroblasts. Preliminary findings suggest that changes in sensitivity are already detectable in fibroblasts from AD patients, probably as a consequence of genetic component as well as other risk factors. Therefore, this biochemical marker might have the potential for identifying individuals at risk for AD.Item Brain Derived Neurotrophic Factor Regulates Müller Cell Survival via MAPK and PI3K Pathways(2003-05-01) Taylor, Sara A.; Agarwal, Neeraj; Wordinger, Robert J.; Pang, Iok-HouTaylor, Sara A., Brain Derived Neurotrophic Factor Regulates Müller Cell Survival via MAPK and PI3K Pathways. Master of Science (Biomedical Sciences), January, 2003, 112 pp., 4 tables, 39 illustrations, bibliography, 68 titles. Purpose: Glutamate has been implicated in many pathologies affecting the Central Nervous System including those in the retina, but the exact nature of the role of glutamate in neuronal degeneration remains unclear. In the retina. Müller cells are resistant to glutamate insults that are normally toxic to other cells of the retina, however the molecular and biochemical mechanisms that control their death or survival are not well understood. We used a series of pharmacological inhibitors and molecular biology agents on cultured Müller cells to dissect two key signaling pathways normally involved in cell survival, the Mitogen Activated Protein Kinase – Extracellularly Regulated Kinase (MAPK(ERK) pathway and the Phosphatidylinositide 3 Kinase (PI3K) pathway. Since preliminary data in our laboratory showed that Müller cells upregulate their secretion of neurotrophins including Brain Derived Growth Factor (BDNF) in response to glutamate treatment, we also examined the effect of BDNF on the activation of these two signaling pathways. Methods: Early passaged Müller cells were treated with various concentrations (5 nM -50 μM) of inhibitions of the MAPK(ERK) pathway (GW5074, U0126, and PD98059) or with various concentrations (1-50 μM) of inhibitors of the PI3K pathway (LY294002 or Akt inhibitor) in the presence and absence of 50 ng/ml of BDNF for 24 hours. These experiments were repeated in Müller cells transfected with either NFκB or Bc12 DNA. Cell cultures were then analyzed for surviving cells with an MTS/PMS assay, a colorametric method for determining the number of viable cells in a proliferation assay. Results: The MAPK (ERK) inhibitors PD98059 and GW5074 both resulted in decrease in Müller cell survival. PD98059 did not decrease Müller cell survival until concentrations were high enough to suppress ERK2 phosphorylation. Müller cells transfected with NFκB or Bc12 DNA were able to resist treatment with concentrations of PD98059 that reduced cell number in untransfected cells. The PI3K inhibitor LY294002 also resulted in significant decreases in Müller cell survival in both untransfected cells and cells transfected with NFκB or Bc12 DNA. Treatment with an inhibitor farther down in the PI3K pathway, Akt inhibitor, did not significantly decrease Müller cell survival. Finally, BDNF was not able to increase cell survival in Müller cells treated with PD98059 or U0126, although it did increase the survival of cells treated wit GW5074. BDNF was also able to reverse the decrease in cell survival caused by LY294002 in both untransfected Müller cells or Müller cells transfected with NFκB or Bc12 DNA. Conclusions: Our data shows that Mitogen Activated Protein Kinase – Extracellularly Regulated Kinase (MAPK(ERK) and Phosphatidylinositide 3 Kinase (PI3K) are both essential for Müller cell survival. There is modulation between the pathways and they may interconnected far upstream at a protein previously associated with only the MAPK(ERK) pathway. These results are consistent with a role for both pathways in Müller cell survival.Item Cellular and Molecular Mechanisms that Distinguish the Effects of Progestorone and Medroxyprogesterone Acetate on Neuroprotection(2006-07-28) Kaur, Paramjit; Goldfarb, Ronald; Singh, Meharvan; Agarwal, NeerajKaur, Paramjit. Cellular and Molecular Mechanisms That Distinguish the Effects of Progesterone and Medroxyprogesterone Acetate on Neuroprotection., Doctor of Philosophy, (Pharmacology and Neuroscience), July, 2006, 203 pp., 5 illustrations, 20 figures and bibliography. Women have a higher prevalence for Alzheimer’s disease (AD) than men, suggesting that the precipitous decline in gonadal hormone levels following the menopause may contribute to the risk of developing AD. However, principal results from the Women’s Health Initiative concluded that women taking conjugated equine estrogens combined with medroxyprogesterone acetate (MPA, tradename: Prempro) incurred more harmful than beneficial outcomes versus the placebo group (Rossouw et al., 2002). This dissertation was aimed at determining if the discrepancy between basic science reports and these clinical studies could have been due to the synthetic progestin, MPA. I hypothesized that P4 and MPA differed in their ability to protect against the excitotoxic/oxidative insult, glutamate. Further, I proposed that this difference in neuroprotective potential would be reflected in the difference in the ability of these hormones to elicit key effectors of two neuroprotection-associated signaling pathways, the ERK/MAPK and P13-Kinase pathways. Finally, studies were initiated to evaluate the potential importance of BDNF (brain-derived neurotrophic factor) in mediating the protective effects of P4. I used organotypic explants of the cerebral cortex, and found that both P4 and MPA elicit the phosphorylation of ERK and Akt, two signaling pathways implicated in neuroprotection, with maximal phosphorylation occurring at a concentration of 100 nM. Interestingly, P4 protected against glutamate- induced toxicity however, while an equimolar concentration of MPA (100nM) did not. Further, P4 resulted in an increase in BDNF, while MPA did not. Our data bring into question the relevance of using MPA as a component of hormone therapies in postmenopausal women, and instead, argue that the relevant progestin for use in treating brain-related disorders is progesterone. Collectively, the data presented here suggest that P4 is protective via multiple, and potentially related mechanism, and importantly, its neurobiology is different from the clinically used progestin, MPA.Item Characterization of the Serotonin Receptors in the Long Posterior Ciliary Artery of the Bovine Eye(2000-08-01) Landry, Theresa A.; Quist, Eugene; Martin, Michael; Pang, Iok-HouLandry, Theresa A., Characterization of the Serotonin Receptors in the Long Posterior Ciliary Artery of the Bovine Eye. Doctor of Philosophy (Biomedical Science), August 2000, 14 pp., 5 tables, 29 illustrations, bibliography, 104 titles. Vascular disease and vasospasm are implicated in the etiology of glaucoma. The long posterior ciliary (LPCA) is the major blood supply for the ciliary body including the ciliary processes that produce aqueous humor. Information about the pharmacological control of this vessel would be helpful in understanding its normal and pathologic function. Serotonin (5-HT) is a neurotransmitter that effectively constricts the LPCA. The objective of this research is to identify the serotonin receptor subtype responsible for the 5-HT induced vasoconstriction of the LPCA and to characterize the cellular mechanisms that mediate that contraction. Ring segments of the LPCA were dissected from bovine eyes and mounted on tungsten triangles attached to a force transducer. Changes in vascular tension were measured and recorded using a physiography recorder. Dose response curves with 5-HT, 5-HT 1-like agonist, 5-CT, and 5-HT2 agonist, α-methyl-5-HT, indicate that the 5-HT 1-like receptor contributed about 15.13% to the contraction and the 5-HT2 receptor contributed to 61.61%. The EC50 for the three agonists were 283 nM (5-HT), 336 nM (5-CT), and 1.7 μM (α-methyl-5-HT). Inhibition curves with selective antagonists indicate that the IC50 is (5-HT 1-like antagonist) and ketanserin (5-HT2 antagonist). Following incubation of the rings with diltiazem 10 μM or nifedipine 10μM, the response to 5-HT was reduced 65.*% and 61.7% respectively. Incubation in calcium free PB produced similar results. Ryanodine inhibited the 5-HT contraction by 58.1% and caffeine inhibited the response 100%. PKC inhibitors bisindolymaleimide II 1 μM, disindolylamalemide II 10 μM, chelerythrine 25 μM and H-7 5 μM decreased the 5-HT response by19.8%, 55.7%, 31.1% and 61.5% respectively. Incubation of the ring segments with one of three PLC antagonists, 2-NCDC 70 μM, U73122 0.5μM, or neomycin 5 mM, prior to the addition of 1 μM serotonin, significantly reduced the contraction of each vessel, p [less than] 0.0001. The 5-HT-induced vasoconstriction of the LPCA of the bovine eye is mediated through activation of both 5-HT2 and 5-HT 1-like receptors. The contraction is dependent on the mobilization of calcium and is mediated in part through PLC activated intracellular calcium release from IP3 sensitive stores.Item Corticotropin-Releasing Factor and Corticosterone Modulate the Anxiogenic-Like Effects of mCPP(1998-06-01) Jenkins, Jennifer A.; Michael Forster; Robert Luedtke; Patricia GwirtzJenkins, Jennifer A., Corticotropin-Releasing Factor and Corticosterone Modulate the Anxiogenic-Like Effects of mCPP. Doctor of Philosophy (Pharmacology), June 1998, 119 pp., 2 tables, 29 figures, bibliography, 100 titles. The administration of PTZ or mCPP produces anxiety-like behavior as measured by an increase in the percentage of entries into the open arms and the time spent on the open arms of the elevated plus maze (Prunell et al., 1994). Reportedly, PTZ and mCPP substitute for each other in the drug discrimination paradigm (Wallis and Laz, 1998). It is therefore suggested that commonality exists among anxiogenic drugs as perceived by trained animals. Andrews and Stephen (1990) suggested that this overall parallelism is an indication that anxiogenic agents may possess similar properties. Therefore, the question posed is as follows: Is there a common denominator anxiety? The global hypothesis is that the core component of anxiety produced by anxiogenic agents or processes involves stimulation of the HPA axis to release CRF, ACTH and/or CORT. Long Evans rats were trained to discriminate either mCPP (1.4 mg/kg) or PTZ (16mg/kg) from saline in a two-lever choice procedure (FR10) which is food reinforced. Animals were pretreated with CRF, α-helical CRF (a CRF antagonist), two steroid synthesis inhibitors (ketoconazole, KETZ and aminoglutethimide, AMG), CORT or underwent an adrenalectomy prior to behavioral testing in order to test the hypothesis that the release of CRF and/or CORT are components of the discriminate stimulus of the mCPP and/or PTZ. Pretreatment with CRF, KETZ, AMG and an adrenalectomy facilitated mCPP level selection. However in the absence of mCPP neither drug nor adrenalectomy produced drug lever selection. In addition CORT did not alter the mCPP dose response curve. However, CORT replacement therapy returned the does response curve to baseline in adrenalectomized animals. Alpha-helical CRF did not block mCPP discrimination. Unlike mCPP-trained animals, KETZ and AMG decreased PTZ-lever selection in PTZ-trained animals. In addition, CORT enhanced and partially substituted for the discriminative stimulus of PTZ. However, adrenalectomy completely abolished drug lever selection in PTZ animals. To compare the discriminative stimulus effects of mCPP and PTZ, PTZ-trained animals were injected with cumulative doses of mCPP. mCPP-trained animals were injected with cumulative doses of PTZ. mCPP and PTZ minimally substituted for each other. The results suggested that neither CRF nor CORT are components of the discriminative stimulus of mCCP and that the role of the HPA axis in mCPP discrimination maybe be a modulator of the stress response. However, CORT is a component of the discriminative stimulus of PTZ such that CORT is necessary for drug lever selection in PTZ trained animals.Item Effect of Progesterone on Calcium Signaling of Hippocampal Neurons(2006-05-01) Hwang, Ji-Yeon; Koulen; Singh, Meharvan; Yang, ShaohuaJi-yeon Hwang, Effects of Progesterone on Calcium Signaling of Hippocampal Neurons. Master of Science (Pharmacology and Neuroscience), May 2006, 74 pp., 18 Figures. Progesterone (P4) is one of the steroid hormones responsible for female sexual behavior. It has been recently show that P4 plays also multiple roles in the central nervous system (CNS) including neuroprotection. Calcium (Ca2+) is involved in numerous cellular processes in nerve cells such as neurotransmitter release and cell death. In the present studies, we present evidence that P4 increases the activity of IP3R-mediated Ca2+ release within nerve cells leading to cell survival and neuroprotection. The purpose of the present study is to identify the subcellular distribution of all IP3Rs and other signaling proteins including Akt and phosphor-Akt, in the primary hippocampal neuron and to test the hypothesis that P4 controls the gain of IP3R-mediated intracellular Ca2+ signaling in neurons. We observed that primary hippocampal neurons express predominantly IP3R type 1, 2, and 3. The cellular distribution of all IP3R isoforms as well as Akt and phospho-Akt was increased in primary hippocampal neurons by P4 treatment. In addition, phospho-Akt was translocated to nucleus in response to P4. P4-pretreated neurons showed potentiated IP3R-mediated intracellular Ca2+ responses. Acute application of P4 resulted in transient elevations of intracellular Ca2+ concentrations. Our results will contribute to establishing potential pharmacological approaches for the treatment of pathological conditions characterized by a dysregulation of cellular Ca2+ concentrations such as Alzheimer’s disease.Item Elucidation of Mechanism and Molecular Determinants Important in Picrotoxin Action in the 5-Hydroxytryptamine Type 3 Receptor(2003-09-01) Das, Paromita; Basu, Alakananda; Forster, Michael J.; Luedtke, Robert R.Das, Paromita, Elucidation of mechanism and molecular determinants important in picrotoxin action in the 5-hydroxytryptamin type 3 receptor. Doctor of Philosophy (Pharmacology and Neuroscience), September 2003, pp. 192, 3 tables, 26 illustrations, 67 titles. The 5-HT3 receptor belongs to the superfamily of ligand-gated ion channels (LGIC), which mediate fast neurotransmission. Till date, only two subtypes of the receptor i.e. 5-HT3A and 5-HT3B have been investigated. The GABAA receptor antagonist picrotoxin inhibits other anion-selective members of the LGIC. Whether PTX inhibits the cation-selective 5-HT3 receptors was previously unknown. Thus, the primary goal of this study was to elucidate the mechanism of action of PTX and identify the amino acids involved in the action of PTX in 5-HT3 receptors. The overall hypothesis tested was that PTX inhibits the 5-HT3 receptor by interacting in the ion channel. PTX-mediated blockade of the 5-HT3A receptors was non-competitive and use-facilitated similar to GABAA receptors suggesting a conserved site of action of these ligands. The inhibitory effect of PTX was reduced drastically in heteromeric 5-HT3A/3B receptors, compared to homomeric 5-HT3A receptors. Picrotoxin should prove to be a useful probe for determining the presence of homomeric vs. heteromeric 5-HT3 receptors in native tissue and recombinant receptor preparations. In anion-selective ion channels, the 2’, 3’ and 6’resides in cytoplasmic aspect of TM2 are known to modulate PTX sensitivity. While mutation of 2’ and 3’ residues in 5-dramatic loss of sensitivity to PTX in 5-HT3A receptors. A converse mutation at 6’ residue in the 5-HT3B subunit caused gain of sensitivity to PTX, suggesting that 6’ is a key determinant of PTX sensitivity. A novel finding was the involvement of 7’ residue in increasing PTX sensitivity in 5-HT3A but not the 5-HT3B subunit. The lack of specific binding by radioligand [3H]EBOB in 5-HT3A receptors suggested that the site of action of convulsants may be different from that anion-selective receptors. The overall results suggest that PTX interacts from that in the anion-selective receptors. The overall results suggest that PTX interact in the ion channel in the 5-HT3 receptors but also underscores the complexity of its interaction with LGICs.Item Endothelin-1-Induced Signaling Involved in Extracellular Matrix Remodeling(2006-12-01) He, Shaoqing; Thomas Yorio; Neeraj Agarwal; Peter KoulenET-1-Induced Signaling in ECM Remodeling in Astrocytes. Shaoqing He, Department of Pharmacology & Neuroscience, University of North Texas Health Science Center, Fort Worth, TX 76107. ET-1 levels are elevated under pathophysiological conditions, including glaucoma, however, ET-1’s ocular functions are not fully documented. Therefore, ET-1-induced signaling and ECM remodeling in astrocytes and at the optic nerve head were determined in this study. Three signaling pathways, including ERK1/2, PKC, and P13 kinase, were involved in ET-1-medicated cell proliferation of U373MG astrocytoma cells. Blocking one of these pathways completely abolished cell proliferation. It appeared that ERK1/2 activation was involved, but was independent of PKC and P13 kinase activation by ET-1. It was also determined that the ETB receptor was the dominant receptor involved in ERK1/2 phosphorylation and cell proliferation. In addition, ERK1/2 phosphorylation was not transactivated by the EGF receptor by ET-1. The studies also indicated that there was no activation of c/nPKC, although PKC was involved in cell proliferation. In U373MG astrocytoma cells, MAPK-ERK, PKC and P13K pathways appear to exert their roles in parallel without a direct, apparent “cross-talk”. Based on the signaling pathways obtained from U373MG astrocytoma cells, the regulation of MMPs/TIMPs and fibronectin in ET-1-activated human optic nerve head astroctyes (hONAs) was also determined. ET-1 not only induced rapid phosphorylation of ERK1/2 and PKC βI/ βII/δ but also increased the activity of MMP-2 and the expression of TIMP=1 and 2. The activity of MMP-2 was enhanced in the presence of inhibitors of MAPK or PKC in hONAs, whereas the expression of TIMP-1 and 2 was abolished. ET-1 increased the soluble fibronectin (FN) expression as well as FN matrix formation, however, the expression and deposition of FN were MAPK- and PKC-independent, whereas expression and activity of MMps and TIMPs were MAPK- and PKC-dependent. Therefore, ET-1 shifted the balance of MMPs/TIMPs and substrates that altered the ECM composition and subsequently let to ECM remodeling in activated hONA cells. ET-1’s effects on ECM remodeling at the optic nerve head were also examined following intravitreal administration of ET-1 in rats. The increased expression of MMP-9 and collagen VI was detected in both ETB deficient rats and wildtype Wistar rats post ET-1 intravitreal injection for 2 and 14 days, whereas the deposition of FN and collagen IV was unchanged. There was no significant difference in staining of MMP-9 and collagen VI between ETB deficient rats and wildtype Wistar rats. In this study, ECM remodeling was demonstrated in rats injected with ET-1 into the vitreous. Such changes in the ECM seen in the current study provide additional insight into the mechanisms that might explain the glaucomatous changes observed in ET-1-injection or perfusion models. In summary, ET-1 not only activated several signaling pathways in cell proliferation of astrocytes, but also modulated the expression of ECM molecules in vitro and in vivo, indicating that ET-1 plays a regulatory role in ECM remodeling. These effects coupled with observations that ET-1 levels are elevated in glaucoma patients, suggests that ET-1 may be involved in glaucomatous optic neuropathy.Item Evaluation of NK Cell – Astrocyte Interactions: Potential Role in HIV-Associated Neurocognitive Disorders and HIV- Associated Dementia(2015-05-01) Bowen, Kelly E.; Mathew, Porunelloor A.; Mathew, Stephen O.; Hodge, Lisa M.NK cells play important roles in immunity against pathogens and cancer. NK cell functions are regulated by inhibitory and activating receptors binding corresponding ligands on the surface of target cells. During pathological conditions, NK cells were shown to be recruited to the CNS and could impact CNS physiology by killing glial cells and by secreting IFN-g. Astrocytes are intimately involved in immunological and inflammatory events occurring in the CNS and reactive astrogliosis is a key feature in HIV-associated neurocognitive disorders (HAND). There is little data on NK cell-astrocyte interactions and ligands expressed on astrocytes that could impact NK cell function. This study aimed to identify NK-associated ligands expressed by human astrocytes that confer this NK-directed cytotoxicity of astrocytes and assay the cytotoxicity differences in presence and absence of HIV 3S peptide. Using a fusion protein consisting of the extracellular domain of NKp44 fused to Fc portion of human IgG, we determined the expression of a novel ligand for NKp44 (NKp44L) on astrocytes. Incubation of astrocytes with 3S peptide downregulated NKp44L expression on astrocytes implicating protection from NK mediated killing. Thus, our study demonstrated that NKp44 has a protective effect on astrocytes from NK cell mediated killing during HIV infection. Astrocytes could also secrete cytokines that affect the expression of NK receptors on NK cells. We evaluated the expression of receptors on NK cells after co-culture with astrocytes. CD38 expression was increased on primary NK cells after incubation with astrocytes. CD38 is expressed on both NK cells and astrocytes and has an important implication in HIV-1 infection. Blocking CD38 signaling in our studies decreased astrocyte lysis, suggesting CD38 signaling has important implications in NK-astrocyte interactions. Future studies providing novel insights into the role of NK cells in the pathogenesis of HAND and other brain disorders might result in the development of NK cell based therapies for brain pathologies.Item Extracellular PACE4 is increased following transient oxygen glucose deprivation in Optic Nerve Astrocytes(2008-05-01) Fuller, John Anthony; Wordinger, Robert J.; Clark, Abbot F.; Krishnamoorthy, Raghu R.Fuller, John Anthony Extracellular PACE4 is increased following transient oxygen glucose deprivation in Optic Nerve Astrocytes. Doctor of Philosophy (Biomedical Sciences), May, 2008, 140 pp., 2 tables, 25 illustrations, bibliography, 218 titles. Primary Open Angle Glaucoma (POAG) is a family of heterogeneous optic neuropathies characterized by progressive retinal ganglion cell (RGC) death that leads to peripheral vision loss and eventually blindness. Various risk factors are associated with glaucoma, however the molecular mechanisms leading to RGC cell death remain unknown. The optic nerve serves as the conduit for the transmission of retinal ganglion action potentials to the brain. The cells that compromise the optic nerve form a scaffold that forms a physical support for the RGC axons. One cell type found throughout the optic nerve and associated with the RGC axon is the optic nerve astrocyte (ONA). Astrocytes are a predominant cell throughout the CNS and are believed to play crucial roles in metabolic, growth factor, and structural support, and respond to protect neurons during injury. The neuronal-glial interface in the optic nerve is poorly understood and believed to plan an important role in POAG pathophysiology, as unmyelenated RGC axons have direct contact with astrocyte processes. IN this study, the subtilisin-like Proprotein Convertases, (SPC) a family of proteases responsible for cleaving a wide variety of protein substrates, were examined in the retina and optic nerve head. PACE4, an SPC found to be secreted and active in the extracellular matrix was found to be highly expressed in the optic nerve, and colocalized to Mϋller cells in the retina and astrocytes in the optic nerve. Exposure of primary optic nerve astrocytes to oxygen-glucose deprivation (OGD) induces an increase in PACE4 mRNA. Furthermore, protein levels of extracellular, processed PACE4 increase following transient ODG, whereas the pro form of the molecule is degraded, and is believed to be chaperoned by the cleaved cysteine rich domain, a product found at high levels in the optic nerve in situ and the ONA in vitro. Due to the extracellular activity of PACE4, we hypothesized that it may regulate the bioactivity of TGF-β2, a growth factor believed to be involved in glaucoma-associated ONH remodeling by inducing the production of extracellular matrix (ECM). When PACE4 is inhibited via siRNA-mediated knockdown, as well as extracellular inactivation, TGF-β2 levels decrease. In addition, fibronectin, a major component of the ECM, is decreased. Furthermore, there is an increase in latent TGF-β2 secreted from the cell. It is therefore possible that PACE4 plays an active role in extracellular growth factor maturation, and may be a central mediator for growth factor bioactivity in the glaucomatous ONA.Item Identification of Actin Binding Proteins Associated with Cross-Linked Actin Networks(2006-12-01) Mills, Christy E.; Clark, Abbot F.; Yorio, Thomas; Wordinger, Robert J.Mills, Christy E., Identification of Actin Binding Proteins Associated with Cross-Lined Actin Networks. Master of Science (Pharmacology and Neuroscience), December 2006, 95 pp., 9 tables, 16 figures, references, 122 titles. Glucocorticoid therapy can leady to ocular hypertension and glaucoma. The purpose of this study is to examine mechanisms contributing to increased intraocular pressure using tissue culture models of steroid-induced ocular hypertension through identification of specific actin-binding proteins associated with cross-linked actin network (CLANs). Human trabecular meshwork ™ cells were cultured to confluence and treated with dexamethasone or vehicle for 14 days. Total RNA was extracted for gene expression analysis to confirm steroid-induced expression of actin binding proteins in human TM cells. Western blots confirmed expression of actin binding proteins and demonstrated the specificity of selected antibodies. Fluorescence microscopy of treated TM cells showed cytoskeleton rearrangements from linear actin stress fibers to cross-linked actin networks and the position of candidate proteins in relation to CLANs. Dexamethasone treatment of TM cells altered the expression of actin-associated proteins that may be important in the formation of CLANs and increased outflow resistance.Item Identification of Oxidized Proteins in Alzheimer's Disease(2002-08-01) Choi, Joungil; Gracy, Robert R.; Harris, B.; Lacko, Andras G.Joungil Choi, Identification of Oxidized Proteins in Alzheimer’s Disease. Doctor of Philosophy (Molecular Biology and Immunology). August, 2002. Pages-110. Tables 8. Figures 24. Oxidative modification of specific proteins is central to the pathology of Alzheimer’s disease (AD). The purpose of this study was to identify the oxidation-sensitive proteins in neuronal cells, fibroblasts from AD subjects, and in the blood of AD patients. In all cases, age-matched non-Alzheimer’s samples were used as controls. Proteomic methods were used to isolate and characterize the oxidized proteins. These included two-dimensional gel electrophoresis, immunolocalization of oxidized proteins and identification by MALDI-TOF mass spectroscopic methods. It was hypothesized that knowledge of these critical oxidation-sensitive proteins would shed light on the underlying mechanism of the disease. In addition, it was postulated that these proteins might prove to be biomarkers for early detection and monitoring the progress of the disease. The results show that two different oxidative stressors (H2O2 generated enzymatically, or the amyloid beta peptide, AB25-35) induce apoptotic cell death and oxidation of specific proteins (heat shock protein 60 and vimentin) in skin fibroblasts from AD subjects and in neuronal cells. In addition, the results indicate that susceptibility of these two proteins to oxidative stress is increased in fibroblasts from AD patients, compared to non-AD controls. Pretreatment with antioxidants (e.g., vitamin E or flavonoids) protect these proteins from oxidative damage. Both heat shock protein 60 and vimentin, have been suggested to function as antiapoptotic proteins. Thus, their oxidative damage could lead to the apoptotic neuronal cell death in Alzheimer’s disease. In the blood plasma of AD subjects, isoforms of fibrinogen gamma chain and alpha-1 antitrypsin were found to be oxidized. These proteins exhibited to a two- to six-fold greater specific oxidation index in plasma from AD subjects when compared to controls. Both of these proteins have been suggested to be implicated in oxidation-mediated damage of inflammation in the AD brain.Item Met-Enkephalin-Arg-Phe (MERF) and Metabolism of MERF Across the Canine Heart Vascular Bed(2000-08-01) Pearlman, Eric Brian; Barbara Barron; Patricia A. Gwirtz; Michael L. SmithPearlman, Eric B., Met-Enkephalin-Arg-Phe (MERF) and Metabolism of MERF Across the Canine Heart Vascular Bed. Master of Science (Biomedical Science), August, 2000, 37 pp., 3 tables, 11 figures, references, 20 titles. Methionine enkephalin arginine phenylalanine (MERF) has been shown to be co-stored with catecholamines in vesicles. The catecholamines appear to decrease the degradation rate of 3H-MERF in vitro. The aim of this study is to investigate the spillover and metabolism of MERF across the canine heart vascular bed. I hypothesize that 3H-MERF is either degraded in the plasma or taken up and degraded by the heart. I further hypothesize that the exogenous catecholamine, isoproterenol, inhibits or reduces the rate of MERF degradation. Mongrel dogs were anesthetized and instrumented to record cardiovascular parameters, infuse 3H-MERF, and obtain blood samples across the heart. Blood samples were taken before and after stopping 3H-MERF infusion to evaluate kinetics, show steady state, and test the effect of treatments. Steady state concentration of 3H-MERF was observed after 30 min of infusion. Chromatography separated intact from degraded 3H-MERF. Three experimental groups were used: control, propranolol plus isoproterenol, and propranolol only. Blockade of β-receptors was necessary to prevent changes in coronary blood flow. Propranolol bolus (0.2 mg/kg) was administered IV at 50 min. 3 μg/min isoproterenol or 0.5 ml/min normal saline was infused starting at 70 min until the end of sample collection. The 3H-MERF venous-arterial (V-A) difference prior to treatment was negative, indicating degradation in the plasma or uptake and degradation by the heart. The 75 min V-A difference was used to calculate the effect of the infusions on the degradation or uptake of the 3H-MERF; this value was unchanged by any treatment. Spillover of 3H-MERF was significantly lower in the propranolol + isoproterenol dogs (p [less than] 0.05) compared to propranolol only treatment at 75 min. Heart rate was significantly lower for the propranolol only group compared to control. Blood pressure and change in coronary flow were unchanged. In conclusion, isoproterenol does not affect the metabolism of 3H-MERF across the canine heart vascular bed. Propranolol, however, does increase the intact 3H-MERF in the plasma, but additional β adrenergic blockade agents need to be investigated to determine the mechanism by which this takes place.Item N-Acylethanolamine Signaling in Neurons(2008-12-01) Duncan, Raymond Scott; Koulen, Peter; Simpkins, James; Forster, MichaelDuncan, Raymond S., N-acylethanolamine signaling in neurons. Doctor of Philosophy (Biomedical Sciences), December 2008, 356 pp., 1 table, 70 illustrations, bibliography, 576 titles. Neurodegenerative diseases including Alzheimer’s disease are and will continue to be significant health problems as the aging population increases. The maintenance of neuronal calcium homeostasis has been a focus in degenerative disease research for many years. Within the last several years, lipids that activate cannabinoid receptors, and thus called cannabinoids, have gained recognition as neuroprotectants in models of neurodegenerative diseases. A subset of these cannabinoids, the N-acylethanolamines (NAEs), includes the well characterized neuroprotective lipid, arachidonylethanolamine. Other NAEs, such as palmitoylethanolamine (PEA), are more abundant in neurons and do not activate cannabinoid receptors, suggesting other targets for these lipids exist. Since non-cannabinoid NAEs rapidly accumulate after neuronal injury, it is likely they play a role in cellular responses to injury. Interestingly, some NAEs can alter intracellular Ca2+ signaling, but the underlying mechanism of action remains unclear. I hypothesized that the non-cannabinoid NAEs, such as PEA, protect the hippocampal cell line, HT22, from oxidative stress in part by reducing intracellular calcium release. I determined that HT22 cells and cultured mouse cortical neurons express proteins involved in NAE signaling, thus warranting the use of pharmacological inhibitors of these proteins in subsequent neuroprotection studies. Using HT22 cells, I determined that PEA exhibitis antiproliferative effects and neuroprotects against oxidative stress. In addition, I determined that PEA facilitates the nuclear translocation of putative protective proteins that can be regulated by Ca2+ through a mechanism not involving cannabinoid receptor activation. These findings led me to hypothesize that PEA alters release of Ca2+ from intracellular stores. To test this hypothesis, I determined that our cell models express inositol 1,4,5-trisphosphate receptors (IP3Rs) and ryanodine receptors (RyRs) both of which are intracellular Ca2+ channels elevated in response to oxidative stress. I determined that treatment of HT22 cells with PEA reduced intracellular Ca2+ release elicited by chemical depolarization with KCI. My results suggest that non-cannabinoid NAEs, such as PEA, protect the hippocampal cell line, HT22, from oxidative stress in part by activating putative neuroprotective signaling proteins and by reducing intracellular calcium release.Item Oxidative Stress Alters IP3 Receptor Function in the Neuronal Cell Line HT22(2008-05-01) Longoria, Sandra; Peter Koulen; Kati Prokai; Tina MachuSandra Longoria., Oxidative Stress Alters IP3 Receptor Function in the Neuronal Cell Line HT22, Master of Science (Biomedical Sciences), May 2008, 72 pp., 25 Figures. Oxidative stress contributes to the genesis of several neurodegenerative disorders such as Alzheimer’s Disease (AD). Oxidants such as, tert-butyl hydrogen peroxide (tBHP), have been used in in vitro models of neurodegeneration to induce oxidative stress. Small changes in the regulation of the intracellular calcium (Ca2+) concentration can contribute to brain aging and increase vulnerability of neurons to cellular and functional damage in neurodegenerative diseases. In neurons, inositol 1, 4, 5-trisphosphate (IP3) is a second messenger that is generated through receptor activity at the plasma membrane. IP3 receptors (IP3R) are located on endoplasmic reticulum (ER) membranes and are intracellular calcium channels (ICC) that release Ca2+ into the cytoplasm in response to activation by their ligand IP3. The goal of the present study was to measure the contribution of ICCs to Ca2+ dysregulation in neurons experiencing oxidative stress. I tested the hypothesis that oxidative stress induced with tBHP causes increased intracellular Ca2+ release via activation of IP3 receptors. I used the murine hippocampal cell line HT22, as a model for neuronal oxidative stress. Immunocytochemistry and Ca2+ imaging experiments were performed to identify areas of altered IP3R expression and activity under normal conditions and induced oxidative stress. tBHP treatment increased expression and Ca2+ release activity of neuronal IP3 receptors. My findings support that oxidative stress as seen in a number of neurodegenerative diseases negatively affects regulation of Ca2+ release through increased expression and activity of IP3 receptors.Item Pharmacological Assessment of Novel Phenylacetamide as a Sigma 1 Receptor Ligand(2014-05-01) Malik, Maninder; Robert LuedtkeThe symptoms of psychosis have been categorized as positive, negative and cognitive. Traditionally drug discovery in psychiatric disorders has focused on positive symptoms of the disease. However, cognitive impairment is equally prevalent and represents a major impediment to the recovery of patients. Hence, research on the drug discovery and development that can improve overall quality of life of patients with neuropsychiatric conditions is important. The main aim of this project was to evaluate a selective and potent sigma 1 receptor phenylacetamide (LS-1-137) as a potential pharmacotherapeutic agent for treating neuropsychiatric disorders and associated cognitive impairment. The sigma 1 receptor is an endoplasmic reticulum (ER) resident protein located on the interface of ER and mitochondria. The sigma 1 receptor is a 25 KDa protein that shares no amino acid sequence homology with any other known mammalian proteins. The research being done on these novel receptors suggests that rather than being a classical receptor-signaling unit sigma 1 receptors act as a molecular chaperone. Several studies suggest sigma 1 receptor ligands modulate abnormal neurotransmission that contributes to the pathogenesis of several CNS disorders. In recent studies by our laboratory, it was found that our novel sigma 1 receptor ligand, LS-1-137 (developed by our collaborators Mach and colleagues) exhibit neuroprotection both in vitro and in vivo. In this project we further characterized LS-1-137 as a potential treatment option for cognitive and neuropsychiatric disorders. Our fundamental hypothesis is that sigma 1 receptor selective compounds represent a potential neuroprotective pharmacotherapy for treating psychosis and cognitive deficits associated with neuropsychiatric disorders. LS-1-137 was evaluated in a 2, 5-dimethoxy-4-iodoamphetamine (DOI) induced head twitch response (HTR) model and a scopolamine-induced cognition impairment model. Our findings suggest that LS-1-137 attenuates the DOI-induced HTR and alleviates scopolamine-induced impairment in learning. Our in vitro data suggest that LS-1-137 is an agonist at sigma 1 receptors and triggers the release of BDNF from rat astrocytes. Furthermore, rotarod and swim test studies indicated that unlike currently prescribed neuroleptics, LS-1-137 does not compromise the agility or muscular coordination of animals. Therefore, in this dissertation we have assessed a novel pharmacotherapeutic agent that may treat the psychosis and cognitive dysfunction associated with neuropsychiatric disorders.Item Regulation of intracellular calcium channels by their associated proteins homer 1 and presenilin 1(2006-05-01) Hwang, Sung-Yong; Koulen, Peter; Dillon, Glenn; Singh, MeharvanSung-Yong, Hwang, Regulation of intracellular calcium channels by their associated proteins homer 1 and presenilin 1. Doctor of Philosophy (Pharmacology and Neuroscience), May, 2006, 184 pp., 4 tables, 20 illustrations, 74 titles. In neurons, Calcium (CA2+) serves as a critical intracellular messenger that regulates a variety of cellular processes such as gene expression, neurotransmitter release, cell death, and synaptic plasticity. Therefore, it is essential for neurons to control their Ca2+ levels tightly. Ca2+ is released within the cell from intracellular stores such as the endoplasmic reticulum by activation of intracellular Ca2+ channels (ICCs) such as the inositol 1,4,5-triphosphate (IP3) receptors (IP3Rs) and ryanodine receptors (RyRs). Each of these two groups of ICC has three isoforms. A number of associated proteins of these two ICCs that were shown to modulate activity of the respective channel have been identified. Homer 1, a synaptic scaffolding protein not only physically associated with IP3R type1 (IP3R1), but also changes the activity of IP3R1, suggesting that Homer 1 is involved in intracellular Ca2+ signaling. Based on the similarity in amino acid sequence and molecular and physiological properties among IP3R isoforms and the fact that IP3R type 3 (IP3R3) contains the proline-rich motif (PPxxFr) that is required for the interaction with Homer, it was hypothesized that Homer 1 associates with IP3R3, leading to changes in the channel activity. Presenilin 1 (PS1) is a transmembrane protein, being expressed in cell body, dendrites, and axon in the neuron. Mutations in PS1 account for most cases of early-onset familial Alzheimer’s disease (AD). PS1 was shown to associate with RyRs and to modulate their channel activity. Therefore, it was hypothesized that specific regions of PS-1 bind to RyR type 2 (RyR2), a major isoform in the brain, resulting in changes in the channel activity. Homer 1c was shown to associate with IP3R3, leading to a decrease in channel activity. A specific region of PS1 that interacts with RyR2 was identified to increase the channel activity of RyR2. Results of the present study contributed to the understanding of the nature of intracellular Ca2+ signaling as well as the mechanisms of action by which ICCs are regulated by their associated proteins. These findings provide the rationale for novel strategies to study neurological disorders including AD and epilepsy that are mediated by Ca2+ dysregulation.