Browsing by Subject "Medical Neurobiology"
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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 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 Effects of Sex Steroids on Stroke(2004-02-01) Yang, Shaohua; Simpkins, James W.Yang, Shaohua, Effects of Sex Steroids on Stroke. Doctor of Philosophy (Biomedical Science), February 2004, pp210, 5 tables, 27 illustrations, 64 titles. Estrogens and androgens are recognized as major sex steroids for females and males, respectively. However, it is clear that estrogens as well as androgens are more than gender hormones. Our data indicated that female steroids, such as 17β-estradiol (E2), exert neuroprotective effects on stroke, while male steroids, like testosterone, exert deleterious effects on stroke. The neuroprotective effects of estrogens have been very well demonstrated both in vitro as well as in vivo. Our studies indicated that neuroprotective effects of E2 are exerted both ischemic and hemorrhagic stroke. In our subarachnoid hemorrhage (SAH) model, E2 reduced secondary ischemic damage and mortality consequent to SAH. These effects were not associated with the change of the clot volume in SAH. The neuroprotective effects of estrogens were not only seen in the pre-treatment paradigms. E2 exerted neuroprotective effects even when administered after ischemia, with a therapeutic window of about 3 hours in a permanent focal cerebral ischemia model. This effect of estradiol was associated with no immediate change on blood flow, but with a delayed increasing in cerebral blood flow (CBF). Further our studies indicated that a non-estrogen receptor (ER)-binding analogue possessed both neuroprotective and vasoactive effects, which suggests that both the neuroprotective and vasoactive effects of estrogens are receptor-independent. This molecule also offers the possibility of clinical application for stroke without the side effects of estrogens. We used immunochemistry, immunoblot and mass spectrometry to demonstrate that ERβ is localized to mitochondria. Our data established this ERβ localization in a variety of cell types, suggesting that ERβ is not a nuclear receptor, which was thought to mediate the genomic function of estrogens. In contrast to estrogens, testosterone increased neuronal toxicity and exacerbated cerebral ischemia-reperfusion injury. These results suggest that sex differences in outcome after stroke may result from both the protective effects of estrogens and the damaging effects of testosterone. Further, our study indicated that stress induced testosterone reduction contributes to cerebral ischemia tolerance against ischemia reperfusion injury, providing the first in vivo evidences for a neuroendocrine mechanism for the cerebral preconditioning in males.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 Genetic Modulation of β-Amyloid Neurotoxicity and Protection by Nicotinic Agents(2007-05-01) Martin, Shelley E.; Basu, Alakananda; Forster, Michael; Singh, MeharvanMartin, Shelley E., Genetic Modulation of β-Amyloid Neurotoxicity and Protection by Nicotinic Agents. Master of Science (Pharmacology and Neuroscience), May, 2007, 53 pp., 7 figures, 2 tables, bibliography, 95 titles. Β-amyloid1-42 (Aβ42) has been implicated in the pathogenesis of Alzheimer’s disease (AD); however, the amount of this peptide in the brain does not correlate well with the presence or severity of AD. This project tested the hypothesis that individual differences exist in susceptibility to Aβ42 neurotoxicity arising from the differences in the expression of α7 nicotinic acetylcholine receptors α7 nACHRs). This hypothesis was tested in primary neuronal cultures derived from inbred mouse strains which differ in expression of α7 nAChRs. Also, the ability of nicotinic agents to modulate Aβ42 toxicity was examined. Significant strain differences in susceptibility to Aβ42 toxicity were found; however, these were not related to levels of α7 nAChRs. Additionally, strain differences were found in the ability of α7-selective partial agonist, an α7-selective antagonist and a α4β2 nAChR-selective antagonist to protect against this toxicity. Inbred strains of mice may be useful in uncovering the pathophysiology of AD.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 Identifying Unique Therapeutic Targets To Rescue Retinal Ganglion Cells From Degeneration After Optic Nerve Crush(2014-05-01) Sharma, Tasneem P.; Clark, Abbot F.Central nervous system (CNS) trauma and neurodegenerative disorders trigger a cascade of cellular and molecular events resulting in neuronal apoptosis and regenerative failure. The pathogenic mechanisms and gene expression changes associated with these detrimental events can be effectively studied using a rodent optic nerve crush (ONC) model. The purpose of this study was to use a mouse ONC model to: (a) evaluate changes in retina and ON gene expression, (b) identify neurodegenerative pathogenic pathways, (c) discover potential new therapeutic targets, and (d) evaluate the neuroprotective and axogenic properties of one selected therapeutic target on axotomized RGCs in vitro and the optic nerve crush (ONC) mouse model in vivo. Meta-analysis of altered gene expression (≥1.5 changes and ≤1.5 changes, p [less than] 0.05 demonstrated 29 up- and 20 downregulated retinal gene clusters and 82 up- and 42 down-regulated optic nerve clusters. Regulated gene clusters included regenerative change, synaptic plasticity, axonogenesis, neuron projection, and neuron differentiation related genes. Expression of selected genes (Vsnl1, Syt1, Synpr and Nrn1) from retinal and ON neuronal clusters was qualitatively and quantitatively examined for their relation to axonal neurodegeneration by immunohistochemistry and qRT-PCR. Axotomized RGCs treated with recombinant hNrn1 (selected target) significantly increased survival of RGCs by 29% (n=8, p [less than] 0.01) and neurite outgrowth of cultured neurons by 261% compared to controls in cultured neurons (n=5-7, p [less than] 0.05). RGC transduction with AAV2-CAG-hNRN1 prior to ONC promoted RGC survival (42%, n=5-8, p [less than] 0.05) and significantly preserved ERG RGC function by 41% until 28 dpc (n=6, p [less than] 0.05) compared to the control AAV2-CAG-GFP transduction group. These ONC induced neuronal loss and regenerative failure associated clusters can be extrapolated to changes occurring in other forms of CNS trauma or in clinical neurodegenerative pathological settings. In conclusion, this study identified potential therapeutic targets to address two key mechanisms of CNS trauma and neurodegeneration: neuronal loss and regenerative failure and presented Nrn1 as a potential therapeutic target for CNS neurodegenerative diseases.Item Mechanisms of Chemoreflex Control of Muscle Sympathetic Nerve Activity and Blood Pressure in Humans(2004-05-01) Hardisty, Janelle M.; Smith, Michael; Shi, Xiangrong; Clark, MichaelHardisty, Janelle M., Mechanisms of Chemoreflex Control of Muscle Sympathetic Nerve Activity and Blood Pressure in Humans. Doctor of Philosophy (Integrative Physiology), May 2004. The mechanisms linking obstructive sleep apnea (OSA) and cardiovascular disease are not fully understood; however, studies report patients with OSA exhibit chronic elevations in muscle sympathetic nerve activity (MSNA). This appears to be due to altered chemoreflex control of MSNA, mediated primarily by hypoxia. Yet, a correlation between degree of hypoxia and chemoreflex control of MSNA is unknown. Therefore, it was evaluated whether degree of hypoxia occurring during apnea determines the sympathoexcitatory and blood pressure responses, and whether these responses are augmented in OSA patients. Additionally, it was studied whether altered chemoreflex function in OSA patients is predictive of blood pressure response to apnea. In a clinical setting, the blood pressure response to voluntary apnea was determined to evaluate whether this could be used as a non-invasive measure of chemoreflex gain in OSA. Finally, the effect of hyperoxia on MSNA was studied to determine whether 15 min of hyperoxia, following intermittent hypoxic apnea, reverses the elevation of MSNA and altered chemoreflex control of MSNA. Consistent with the hypotheses, a relationship between MSNA responses, blood pressure response and level of hypoxia were determined. MSNA and peak systolic pressure responses were augmented in OSA subjects (p≤0.05 and p≤0.05, respectively), as well as, chemoreflex gain (p≤0.05). Clinically, peak systolic pressure responses to apnea were augmented in OSA patients (p˂0.001). Finally, basal MSNA and chemoreflex control of MSNA, following hyperoxia, was not different from baseline through 180 min of recovery (p=0.940 and p=0.278, respectively). These data support the hypotheses that chemoreflex gain is predicative of the blood pressure response; and furthermore, the MSNA and blood pressure responses to hypoxic apnea are augmented in OSA. Additionally, peak systolic pressure responses to voluntary apnea are augmented in OSA. Additionally, peak systolic pressure responses to voluntary apnea are augmented in OSA patients and could possibly be used as a marker of chemoreflex gain. Moreover, these data support the hypothesis that hyperoxia can reverse basal sympathoexcitation and augmented chemoreflex control of MSNA, associated with hypoxic apnea, supporting that elevations in MSNA are hypoxia mediated.Item Myocilin Regulation by Brain-Derived Neurotophic Factor and Transforming Growth Factor-Beta2 in Normal and Glaucomatous Human Trabecular Meshwork Cells(2003-05-01) Liu, Xiaochun; Wordinger, Robert J.; Rudick, Victoria; Clark, Abbot F.Liu, Xiaochun, Myocilin Regulation by Brain-derived Neurotrophic Factor and Transforming Growth Factor beta2 in Normal and Glaucomatous Human Trabecular Meshwork. Doctor of Philosophy (Biomedical Sciences), May 2003, 119 pp., 3 tables, 26 illustrations, bibliography, 188 titles. Glaucoma, of which primary open-angle glaucoma (POAG) is the most common form, is the second leading cause of irreversible blindness in the world. Ocular hypertension is an important risk factor in the development of POAG. The human trabecular meshwork (HTM) is the major regulation site for aqueous humor outflow thus controlling intraocular pressure. In POAG, there are specific morphological and pathological changes in the HTM, including an increase in extracellular matrix components and a decrease in the number of HTM cells. Myocilin (also known as GLC1A or TIGR) is associated with hypertensive POAG by both genetic linkage analysis and glucocorticoid induction studies. Brain-derived neurotrophic factor (BDNF) and transforming growth factor-beta isoforms (TGFβ1-3) have been shown to be present both in normal cultured HTM cells and aqueous humor. In addition, biologically active TGFβ2 levels are increased in the aqueous humor of POAG patients. Mechanical stretch, an important factor in HTM during intraocular hypertension, may up-regulate the expression of BDNF in the HTM cells. Therefore, BDNF and TGFβ2 may be modulators of extracellular proteins in response to the hypertensive glaucomatous injury. However, the regulation of myocilin expression by these growth factors in the HTM has not been studied. Moreover, HTM cells may signal each other via paracrine and autocrine pathways involving BDNF and TGFβ2. In this study, HTM cells were isolated and cultured in vitro. Myocilin gene expression and protein secretion by normal and glaucomatous HTM cells were compared. The regulatory effects of BDNF and/or TGFβ2 or myocilin gene expression and protein secretion by normal and glaucomatous HTM cells were also examined, as well as the reciprocal induction between BDNF and TGFβ2 gene expression and protein secretion. The interdependence between BDNF and TGFβ2 in regulating myocilin expression was determined. The results of the study established the regulatory effects of BDNF and TGFβ2 on myocilin expression as well as on each other. It is possible that both BDNF and TGFβ2 interact with each other in response to an increase of intraocular pressure through paracrine/autocrine mechanisms, resulting in differential gene expression of myocilin.Item NEUROPROTECTIVE PROPERTIES OF SIGMA-1 RECEPTOR IN GLAUCOMA(2014-05-01) Mueller, Brett H.; Thomas YorioGlaucoma is an optic neuropathy commonly associated with elevated intraocular pressure (IOP) that affects over 70 million individuals worldwide. Glaucoma pathology is manifested as cupping of the optic disk, damage to the nerve fiber layer, and visual field deficits. The final pathological step of this disease contributing to visual field loss is the apoptosis of retinal ganglion cells (RGCs). Currently, the only therapeutic agents that are used to treat glaucoma are IOP lowering drugs. However, even when IOP is brought within normal range, a significant number of patients still have progression of visual deficits. Currently, there are no treatment options that have the ability to sustain the viability of RGCs during the disease process of glaucoma. Therefore, neuroprotective drugs that protect RGCs need to be developed as adjunct therapeutic agents to IOP lowering drugs. The sigma-1 receptor (σ-1r) is a non-opioid receptor that has been shown to have the ability to bind to benzomorphans, steroids, and psychotropic drugs. This receptor is ubiquitously expressed throughout the entire body; however, the endogenous ligand and function of σ-1r is not yet known. Several in vitro and in vivo studies have demonstrated the neuroprotective effects of σ-1r stimulation in several models of retinal neurodegenerative diseases including glaucoma and diabetic retinopathy. Numerous studies have linked the neuroprotective effects of σ-1r to its ability to block cytotoxic calcium ion influx through ligand gated and voltage gated ion channels, modulation of ER stress, maintenance of mitochondrial homeostasis, and stimulation of pro-survival intracellular signaling pathways. However in primary RGCs, there have been no studies demonstrating σ-1 receptor mechanism of action. The only proposed neuroprotective mechanism of action of σ-1r that has been performed in retinal flat mounts is blockage of calcium ion influx through activated NMDA receptors. This present research project investigated the mechanism of neuroprotective effects of σ-1rs in primary RGCs, particularly involving L-type voltage gated calcium channels (VGCCs) and activation of extracellular-signal-regulated kinases (ERK 1/2). We demonstrated that VGCCs were activated using KCl (20mM). Pre-treatment with a known L-type VGCC blocker produced a 57% decrease in calcium ion influx through activated VGCCs (following depolarization by KCl). In addition, calcium imaging showed that σ-1r agonists, (+)-N-allylnormetazocine hydrochloride [(+)-SKF10047] and (+)-Pentazocine, inhibited calcium ion influx through activated VGCCs. Treatment with a σ-1r antagonist, BD1047, produced a potentiation of calcium ion influx through activated VGCCs and abolished all inhibitory effects of the σ-1r agonists on VGCCs. This confirms that these ligands were acting through the σ-1r. An L-type VGCC blocker (Verapamil) also inhibited KCl activated VGCCs and when combined with the σ-1r agonists there was not a further decline in calcium entry suggesting similar mechanisms of action of both these agents. Lastly, co-localization studies demonstrated that σ-1rs and L-type VGCCs are co-localized in primary RGCs. Taken together, these results indicated that σ-1r agonists can inhibit KCl induced calcium ion influx through activated L-type VGCCs in primary RGCs. This is the first report of attenuation of L-type VGCC signaling through the activation of σ-1rs in primary RGCs. The ability of σ-1rs to co-localize with L-type VGCCs in primary RGCs implies that these two proteins are in close proximity to each other and that such interactions regulate L-type VGCCs Another signaling pathway that was studied as a potential target of σ-1r mediated neuroprotection was the MAP kinase pathway, in particular, ERK phosphorylation as an index of cell survival. RGCs subjected to oxygen and glucose deprivation (OGD) for 6 hours induced 50% cell death in primary RGCs and inhibited pERK1/2 expression by 65%. Cell death was attenuated when RGCs were treated with pentazocine under OGD and pERK1/2 expression was increased by 1.6 fold compared to OGD treated RGCs without pentazocine treatment. The co-treatment of with an ERK1/2 inhibitor PD098059 with pentazocine significantly abolished the protective effects of pentazocine on the RGCs during this OGD insult. These results established a link between σ-1 receptor stimulation and the neuroprotective effects of the ERK1/2 pathway in purified RGCs subjected to OGD. In conclusion, we have established two novel mechanisms underlying σ-1 receptor mediated neuroprotection in primary RGCs. These findings suggest that activation of the σ-1 receptor in RGCs has a role in calcium regulation and the activation of the ERK1/2 pathway. In addition, this study also demonstrates the robust neuroprotective effects of σ-1 receptor in RGCs when subjected to OGD. These data also provide evidence suggesting that σ-1 receptor may be a therapeutic target to protect RGCs during ocular neurodegenerative diseases like glaucoma.Item Opioid Receptors in Aging and Oxidative Stress(2007-01-01) Raut, Atul M.; Ratka, Anna; Simpkins, James W.; Dillon, GlennRaut, Atul M., Opioid Receptors in Aging and Oxidative Stress. Doctor of Philosophy (Pharmacology and Neuroscience), January 2007, 181 pp, 4 illustrations, 21 figures, 159 titles. Oxidative stress has been implicated in aging and neurodegenerative disorders. Pain sensitivity and responses to opioids change with aging. The effect of aging and oxidative stress on opioid receptor system is not yet well understood. To study the effects of aging on pain sensitivity and opioid-induced antinociception, and to determine the possible association of oxidative stress with these pain parameters, in vivo studies were conducted. To further elucidate the effects of oxidative stress on opioid receptor proteins and their function, in vitro studies were carried out. The effects of aging on pain sensitivity and opioid-induced antinociception were studied in male Fischer 344 rats. Oxidative stress markers in cerebral cortex, hippocampus, striatum and midbrain of these rats were estimated. It was concluded that sensitivity to nociceptive stimulus increases and responses to opioids decrease with aging and age-related oxidative damage is negatively correlated with opioid-induced antinociception. To characterize the effects of oxidative stress on function of opioid receptors, changes in intracellular cyclic adenosine monophosphate (cAMP) was measured in human SK-N-SH neuronal cells under oxidative stress conditions. It was found that oxidative stress decreased the function of mu opioid receptor (MOR) but not that of delta or kappa opioid receptors (DOR and KOR respectively). Antioxidant intervention preserved the function of MOR. Western immunoassays revealed that MOR but not DOR and KOR proteins were decreased under oxidative stress conditions. Thus, these findings show a selective impairment of the MOR function and reduction in MOR protein under conditions of oxidative stress. The results from the in vivo and in vitro projects demonstrate the involvement of aging and oxidative stress in modulation of pass sensitivity, opioid-induced antinociception and opioid receptor function and expression.Item Presenilins Modulate Cellular Activity of Ryanodine Receptors(2012-12-01) Payne, Andrew J.; Peter KoulenPayne, Andrew J., Presenilins Modulate Cellular Activity of Ryanodine Receptors. Doctor of Philosophy (Biomedical Sciences), December, 2012, 160 pp., 7 tables, 39 figures, bibliography 241 titles. Ryanodine Receptors (RyRs) are large, endoplasmic reticulum (ER) intracellular calcium channels in excitable cells. RyRs are major cellular mediators of calcium-induced calcium release and crucial regulators of intracellular calcium homeostasis. Disruption of RyR function has been described in pathologies of dysregulated calcium such as Alzheimer’s disease (AD). Presenilins (PS1 and PS2) are ER transmembrane proteins expressed in the central nervous system mediating calcium homeostasis and Notch signaling, and act as the proteolytic core of γ-secretase in amyloid cleavage. Previous single channel electrophysiology studies described a direct interaction between RyR and the N-termini of presenilin 1 (PS1NTF) and presenilin 2 (PS2NTF) that resulted in differential modulation of the RyR open probability and mean Ca2+ current at the RyR single channel level. We herein tested the hypothesis that PS1NTF and PS2NTF functionally modulate RyRs in a physiologically relevant in vitro model resulting in changes to RyR-mediated intracellular calcium release. Confocal microscopy, microfluorimetry, and coimmunoprecipitation studies confirmed a physical interaction between RyRs and PS-NTFs in human neuroblastoma SH-SY5Y cells, an in vitro AD model. Live cell fluorescent calcium imaging was used to quantify the effects of overexpression of PS1NTF or PS2NTF on Ca2+ release from RyR mediated stores. PS1NTF was found to increase RyR gating to the full open state at physiologically normal calcium concentrations. Verifying the previous electrophysiology data, PS2NTF had no effect on RyR at physiological calcium concentrations. Mutagenesis of critical cysteine residues on the PSNTFs was applied to determine the effect of specific structure-function differences between PS1 and PS2 molecules that underlie the isoform specific modulation of RyR calcium release. Mutations to PS2NTF removing disulfide bridging cysteines recapitulated PS1NTF-like regulation of RyR Ca2+ release. Our findings indicate that PS1NTF and PS2NTF bind RyRs differentially. Our results indicate a novel mechanism of intracellular calcium regulation by the PS-RyR interaction and a novel target for the treatment of AD, neurodegenerative disorders, and diseases controlled by RyR and PS functions.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.Item Role of catecholaminergic A2 neurons of nucleus of the solitary tract(NTS) in cardiovascular and respiratory adaptations to chronic intermittent hypoxia (CIH) in rats(2014-05-01) Bathina, Chandra Sekhar; Steve MifflinThis study examined the role played by the catecholaminergic A2 neurons of the nucleus of the solitary tract (NTS) of adult male Sprague- Dawley rats in the increased mean arterial pressure (MAP) noticed following exposure to chronic intermittent hypoxia (CIH), a rodent model to simulate arterial hypoxemic conditions occurring in humans suffering from sleep apnea. In one study, we tested the hypothesis that tyrosine hydroxylase (TH) knockdown in NTS reduces the sustained elevation in MAP noticed in the rats exposed to CIH. Adult male Sprague-Dawley rats were implanted with radiotelemetry transmitters and adeno-associated viral constructs with a GFP reporter having either short hairpin RNA for TH (shRNA) or scrambled virus (scrambled) were injected into caudal NTS. shRNA through formation of RNA-induced silencing complex reduced the amount of TH levels in the NTS. Virus injected rats were exposed to 7 days CIH (alternating 6 min periods of 10% O2 and 4 min of 21% O2 from 8am to 4pm; from 4pm to 8am rats were exposed to 21% O2). CIH increased MAP and HR during the day in both the scrambled (n= 14, p Experiments were also conducted to understand the molecular level changes occurring in the A2 neurons, following CIH exposure. mRNA expression changes occurring in the A2 neurons were analyzed by novel technique of laser capture microdissection (LCM) by labeling the A2 neurons using adeno-associated virus with TH promoter attached to green fluorescent protein (GFP). A2 neurons are found to express mRNA of angiotensin receptor subtypes AT1a and AT1b. Moreover, excitatory amino acids (EAAs) like glutamate released from chemoreceptor afferents during chronic intermittent hypoxia (CIH) are found to modulate the activity of the neurons in the region of NTS. The aim of this study was to assess the effect of CIH on the mRNA expression levels of AT1a, AT1b and EAAs receptor subunits in the A2 neurons. We utilized commercially available adeno associated virus (AAV) vector mediated delivery of green fluorescent protein (GFP) labeled tyrosine hydroxylase promoter (AAV-GFP-TH), which will incorporate into the TH genome and express GFP with the TH expression to label the A2 neurons. 7 virus injected rats were exposed to 7 days CIH (alternating 6 min periods of 10% O2 and 4 min of 21% O2 from 8am to 4pm; from 4pm to 8am rats were exposed to 21% O2). Laser capture microdissection was performed to capture the A2 neurons from caudal NTS. Total RNA from these neurons was extracted and the gene expression for different genes were assessed by quantitative real time reverse transcription polymerase chain reaction and compared between the control and CIH rats using 2-ΔΔct method. CIH is found to decrease AT1a (p=0.002; control - 1.08 ± 0.13, n=7; CIH – 0.48 ± 0.07, n= 6) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor (AMPA) receptor subunit GluR2 (p=0.03; control - 1.11 ± 0.24, n=7; CIH- 0.52 ± 0.12, n= 6) and increase transcription factor FosB (p=0.03; control - 1.14 ± 0.25, n=7; CIH- 1.97 ± 0.25, n= 5) mRNA expression levels in the A2 neurons. These results suggests that there is increase in activity of these neurons following CIH and a possibility of these neurons becoming more calcium permeable as GluR2 is found to resist calcium permeability. Western blot studies were also conducted from the whole NTS punches, to study the changes in protein levels of the genes studied using LCM. The changes in TH protein levels were not significant in both caudal and sub-postremal NTS (P [greater than] 0.05). GluR1 and GluR2 protein level changes were not significant in the caudal NTS, however, there was a significant decrease (P As the mRNA analysis of A2 neurons suggested, there might be changes occurring in the calcium permeability of A2 neurons following CIH, attempts were made to do calcium imaging studies on the A2 neurons. There was difficulty in the colocalization of GFP with the fura-2AM, the calcium imaging dye. So, calcium imaging was conducted on the NTS neurons of sham Sprague-Dawley rats and CIH exposed rats. 30 μM AMPA application caused a 340/380 ratio change of 0.17 ± 0.01 (n=5) in control rats and this change was significantly higher 0.55 ± 0.13 in CIH rats. The probability of neurons responding to AMPA application was considerably higher in CIH rats. CNQX treatment of the slices abolished the changes in intracellular calcium in neurons from both control and CIH rats, demonstrating that the responses noticed after AMPA application were AMPA receptor mediated. Increases in intracellular calcium levels following 500 μM potassium chloride applications validate the fact that the neurons were viable. Further studies on quantifying the phosphorylated GluR1 and GluR2, subunits of AMPA receptors are required to explain the driving force behind this uniform increase in intracellular calcium levels of NTS neurons after CIH. We conclude that the sustained hypertension observed during CIH can be prevented by TH knockdown and this mechanism might involve paraventricular nucleus (PVN) of forebrain, hypothalamo-pituitary adrenal axis (HPA axis) or intermediolateral cell column (IML) of spinal cord. A2 neurons also undergo molecular alterations that might increase their calcium influx in to the neuron and vise-versa.Item Studies of Protein F1 (GAP-43) Expression and Function in Spinal Neuronal Cultures(1994-08-01) El-Badawy, Hassan M.E. Azzazy; Ming-Chi Wu; Guenter W. Gross; Scott NortonEl-Badawy, Hassan M. E. Azzazy, Studies of Protein F1 (GAP-43) Expression and Function in Spinal Neuronal Cultures. Doctor of Philosophy (Biochemistry and Molecular Biology), August 1994, 167 pp., 32 illustrations, References, 194 titles. Protein F1 (GAP-43, B-50, neuromodulin) is a membrane-bound phosphoprotein that has been studied mainly in neurons and is implicated in synaptic plasticity, axonal growth and regeneration, and neurotransmitter release. In this study, a 21 amino acid polypeptide that corresponds to the C-terminus sequence of protein F1 and contains a potential PKC phosphorylation sequence (SXR) was synthesized. The synthetic peptide was phosphorylated by rat PKC in a concentration-dependent manner suggesting that this site in the intact protein may be phosphorylated by PKC in vivo. Polyclonal antibodies against the peptide were produced in a rabbit and used to: (i) recognize native non-phosphorylated protein F1 purified from rat brain, (ii) immunoprecipitate phosphorylated protein F1, and (iii) stain the cell bodies and neuritis of cultured neurons. Electron microscopic studies revealed intracellular protein F1 immunoreactivity but no specific subcellular association of the gold label could be demonstrated. The antibodies were also used to compare protein F1 levels during the development of spinal neurons in culture and in vivo. The highest levels of protein F1 were detected by ELISA, at 2 days in culture. These results are in accordance with previous reports that correlate high expression of protein F1 to neurite outgrowth. In vivo, however, protein F1 reached maximal level at one day after parturition. Two approaches were utilized to investigate the potential physiological functions of protein F1 in spinal neurons networks. First, interaction of positively charged, rhodamine-labeled liposomes with spinal neurons was characterized by fluorescence microscopy and electrophysiological recording. Uniform, non-toxic, and preferential interaction of liposomes with spinal neurons over glia was established. These liposomes were used to deliver anti-protein F1 antibodies into spinal neurons but did not affect neurite formation by these cells. Second, antisense oligodeoxynucleotides internalized into spinal neurons in order to interfere with protein F1 expression had no effect on the development of these cells in culture. Data from this study suggest that Ser-210 at the C-terminus of protein F1 may be a substrate for PKC phosphorylation in vivo. Antibodies raised against F1 peptide revealed protein F1 immunoreactivity that outlined cell bodies and neuritis of cultured spinal neurons. Positively charged liposomes were characterized as a potential delivery system for macromolecules into spinal neurons. Protein F1 levels were shown to be developmentally regulated in mouse spinal neurons in culture and in vivo. Finally, the use of antisense oligodeoxynucleotides against protein F1 mRNA revealed that protein F1 may not be essential for neurite outgrowth of mouse spinal neurons in culture.Item Synergy 2007: Annual Research Report(2007-01-01)