Browsing by Subject "retina"
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Item A Novel Prodrug Approach for Central Nervous System-Selective Estrogen Therapy(MDPI, 2019-11-19) Prokai-Tatrai, Katalin; Prokai, LaszloBeneficial effects of estrogens in the central nervous system (CNS) results from the synergistic combination of their well-orchestrated genomic and non-genomic actions, making them potential broad-spectrum neurotherapeutic agents. However, owing to unwanted peripheral hormonal burdens by any currently known non-invasive drug administrations, the development of estrogens as safe pharmacotherapeutic modalities cannot be realized until they are confined specifically and selectively to the site of action. We have developed small-molecule bioprecursor prodrugs carrying the para-quinol scaffold on the steroidal A-ring that are preferentially metabolized in the CNS to the corresponding estrogens. Here, we give an overview of our discovery of these prodrugs. Selected examples are shown to illustrate that, independently of the route of administrations and duration of treatments, these agents produce high concentration of estrogens only in the CNS without peripheral hormonal liability. 10beta,17beta-Dihydroxyestra-1,4-dien-3-one (DHED) has been the best-studied representative of this novel type of prodrugs for brain and retina health. Specific applications in preclinical animal models of centrally-regulated and estrogen-responsive human diseases, including neurodegeneration, menopausal symptoms, cognitive decline and depression, are discussed to demonstrate the translational potential of our prodrug approach for CNS-selective and gender-independent estrogen therapy with inherent therapeutic safety.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 CHANGES IN ENDOTHELIN RECEPTOR A EXPRESSION IN A RAT MODEL OF OCULAR HYPERTENSION(2014-03) McGrady, Nolan R.; Minton, Alena Z.; Krishnamoorthy, Raghu R.Glaucoma is the leading cause of blindness in developed countries and is the second leading cause of blindness worldwide. The most common and currently only treatable symptom associated with glaucoma is an increase in intraocular pressure (pressure inside the eye). Rodent models have been routinely used to understand the effects elevated pressure has on the eye, and this study focuses on the changes in expression of a protein molecule (endothelin A receptor) within the retina due to elevated intraocular pressure in a rat model of elevated intraocular pressure. Purpose (a): The endothelin system of peptides and their receptors have been implicated for their neurodegenerative role in glaucoma. The purpose of this study was to determine changes in ETA receptor expression within the retina in the Morrison’s elevated IOP model of glaucoma in rats. Methods (b): IOP was elevated in the left eye of adult male retired breeder Brown Norway rats using the Morrison’s model of glaucoma (by injection of hypertonic saline through episcleral veins) while the contralateral eye served as the control. The rats were maintained for two to four weeks following IOP elevation and sacrificed. Retinal sections were obtained from both control and IOP-elevated eyes, and analyzed for changes in ETA receptor expression using immunohistochemistry. ETA receptor immunostaining was co-localized with β-III-Tubulin, which is selectively expressed in retinal ganglion cells. Results (c): After two weeks, rat eyes with IOP elevation showed an increase in immunostaining for ETA receptors in several retinal layers including the inner and outer plexiform layers with a modest increase in the retinal ganglion cell layer. Following four weeks of IOP elevation, ETA receptor expression was modestly increased in the inner and outer plexiform layers of the retina, compared to that in the corresponding contralateral eyes. Conclusions (d): Elevated intraocular pressure results in a time-dependent change in ETA receptor expression. Increased ETA receptor expression is associated with neurodegenerative changes in glaucoma.Item Function of Differentially Expressed Intracellular Calcium Channels in Retinal Neurons(2008-05-01) Nixon, Everett Sheldon; Peter Koulen; Raghu Krishnamoorthy; Rong MaNixon, Everett, Function of differentially expressed intracellular calcium channels in retinal neurons. Doctor of Philosophy (Pharmacology and Neuroscience), May, 2008, pp154, 17 illustrations. The retina, a specialized part of the central nervous system (CNS) is the innermost layer of the eye responsible for capturing light and converting the light response into a signal that can be transmitted through the optic nerve and onto the brain for interpretation. The ability of the retina to perceive light is dependent on its sensory neurons and the neural circuitry present that initiate the primary stage of processing the image being visualized, which then transmits an electrical signal down the optic nerve to the brain for processing and ultimately visual perception. In the vertical pathway of the visual process that involves the photoreceptor cells, bipolar cells and the ganglion cells, glutamate is the main excitatory neurotransmitter. Communication between these cells is dependent upon the release of glutamate into the synaptic region within both the outer plexiform layer and inner plexiform layer, a process that is Ca2+ regulated. In neurons, Ca2+ regulates a plethora of processes such as gene expression, cell death, synaptic plasticity and neurotransmitter release since it serves as a critical intracellular messenger. In view of the involvement of Ca2+ in a variety of physiological processes, it is essential for the intracellular Ca2+ concentration to be tightly regulated within neuronal cell. Regulation of Ca2+ signaling within retinal neurons can occur via inositol 1,4,5-triphosphate (IP3) receptors (IP3Rs) and ryanodine receptors (RyRs). These receptors are involved in the release of Ca2+ from the intracellular stores such as the endoplasmic reticulum (ER) into the cytosol. IP3Rs and RyRs contribute substantially to cytosolic free Ca2+ concentration transients and thereby play an important role in neuronal function. The purpose of the study was to determine the role of mGluRs, IP3Rs and RyRs in increasing intracellular Ca2+ levels in retinal neurons as related to signaling and neurotransmitter release. The present study provides experimental evidence for the following mechanisms: -Activation of mGluR8 in photoreceptor cells reduced cytosolic Ca2+ concentration by inhibition of the voltage gated Ca2+ channels on the plasma membrane. –The distribution of IP3R and RyR isoforms was associated with cytosolic Ca2+ transients and the IP3R induced transients occurs by activation of group I mGluRs. –In rod bipolar cells, the main increase in cytosolic Ca2+ concentrations during depolarization is due to Ca2+ release from internal stores via activation of RyR. The results of the present study contribute to the understanding of intracellular Ca2+ signaling in retinal neurons and Ca2+ signaling mechanisms. This is of relevance for identifying mechanisms controlling neurotransmitter release and possible pharmacological targets in neurodegenerative retinal diseases characterized by Ca2+ dyshomeostasis.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 INCREASED EXPRESSION OF GLUTAREDOXIN 1 (Grx1) PROTECTS HUMAN RETINAL PIGMENT EPITHELIAL CELLS FROM OXIDATIVE DAMAGE(2014-03) Liu, Xiaobin; Jann, Jamieson; Wu, HongliOxidative stress is believed to contribute to the pathogenesis of many diseases, including age-related macular degeneration (AMD), in which retinal pigment epithelial (RPE) cells are considered as major targets. It is widely accepted that the RPE cells have enormous number of thiol-containing proteins, which can undergo modifications to change retinal protein functions. In contrast, the mechanism of thiol redox regulation in the retina and its association with AMD are still very poorly understood. In particular, the function of glutaredoxin 1 (Grx1), a thiol repair enzyme in cytosol, is virtually unknown. This project seeks to address this paucity in a comprehensive and physiological relevant fashion, and therefore is both novel and innovative. Furthermore, the ability to identify novel therapeutic targets for further research is the first critical step in finding new treatments for AMD. The overall success of this project will raise new hope that Grx1 or its mimic may be a potential therapeutic agent for AMD, and perhaps for other ocular diseases induced by oxidative stress. Purpose (a): The retina is constantly exposed to oxidative stress, which is countered by well-designed antioxidant systems present in retinal pigment epithelial (RPE) cells. Disruption of these systems may lead to the development of age-related macular degeneration (AMD). In this study, we explored the strategy of overexpressing glutaredoxin 1 (Grx1), a component of the endogenous antioxidant defense system, to combat oxidative damage in RPE cells. Methods (b): Human retinal pigment epithelial (ARPE-19) cells were transfected with either a Grx1-containing plasmid or an empty vector. Normal ARPE-19 cells and transfected cells were treated with or without 200 µM H2O2 for 24 h. Grx1 protein expression was detected by western blots and enzyme activity was measured by spectrophotometry. Cell viability was measured by a colorimetric assay with WST8. The morphology of nuclear chromatin was assessed by staining with Hoechst 33342. Apoptosis was quantitatively analyzed by flow cytometry. The level of protein glutathionylation (PSSG) was measured by immunoblotting using anti-PSSG antibody. Results (c): Grx1 protein level and enzyme activity in Grx1 transfected cells were significantly increased as compared to non-transfected and vector transfected cells. Grx1 overexpression protected ARPE-19 cells from H2O2-induced cell viability loss. Assessment of apoptosis indicated that cells transfected with Grx1 were relatively more resistant to H2O2 with fewer cells undergoing apoptosis as compared to vector control or non-transfected cells. Furthermore, PSSG accumulation was also dramatically attenuated by Grx1 overexpression. Conclusions (d): Grx1 can protect human retinal pigment epithelial cells against H2O2-induced cell death. The mechanism of this protection is likely associated with its ability to prevent lethal accumulation of PSSG.Item Mechanisms of Photoreceptor Cell Apoptosis(2000-05-01) Crawford, Matthew John; Neeraj Agarwal; Victoria Rudick; Raghu KrishnamoorthyCrawford, Matthew John, Mechanisms of Photoreceptor Cell Apoptosis. Doctor of Philosophy (Biomedical Sciences), May 2000; 168 pp; 3 tables; 23 figures; bibliography, 282 titles. Photoreceptor cell death mediated by programmed cell death pathways is responsible for many disease states of the retina, which result in vision loss. Examples of this include retinal dystrophies and age-related macular degeneration. Correspondingly, the understanding of programmed cell death, or apoptosis, in these cells is important in the formulation of preventative and treatment options. The goals of this dissertation are to characterize a suitable in vitro photoreceptor cell model and explore the molecular mechanisms resulting in apoptotic cell death secondary to oxidative cell death paradigm. Means of interrupting the cell death process were also investigated. An immortalized clonal mouse retinal cell line was shown to express photoreceptor-specific genes and proteins by RT-PCR amplification, Western blot analysis, and immunocytochemical localization. Exposing these cultured cells to visible light resulted in oxidative stress, as exhibited by elevated malonyldialdehyde and reduced gluthathione levels, as well light exposure-dependent apoptosis was shown using multiple techniques which identified fragmentation of chromosomal DNA, a key finding in the apoptotic cell death process. Molecular regulators of apoptotic cell death, including bcl-2 family proto-oncogenes and the nuclear transcription factor NF-kB, were found to be important in oxidative stress-induced pathogenesis of 661 W photoreceptor cells. mRNA and protein levels of the anti-apoptotic proto-oncogene bcl-2 declined following oxidative stress disturbing the balance proto-oncogene regulators and initiating the apoptotic pathway. The nuclear transcription factor NF-kB was found to be constitutionally expressed in the photoreceptor cells with its down-regulation during apoptosis. Permanent transfection of the photoreceptor cells with bcl-2 gene imparted protection from apoptosis and sustained NF-kB levels. The results presented in this dissertation help define the molecular mechanisms which occur during apoptosis of photoreceptor cells. Photo-oxidative stress results in programmed cell death mediated through changes in NF-kB binding activity and bcl-2 family genes. The involvement of caspase-1 in the degradation of NF-kB and the execution of apoptosis is also demonstrated. Over-expression of the proto-oncogene bcl-2 interrupts the apoptotic events, protecting against down-modulation of NF-kb binding activity and cell death. Our proposed mechanism for apoptosis in photoreceptor cells provides several points at which targeted gene expression (bcl-2 or NF-kB), or pharmaceuticals (anti-oxidants, caspase inhibitors, or calcium channel blockers) may prevent apoptotic cell death.Item Regulation of Endothelin-1 (ET-1) Synthesis and Secretion at the Outer Blood Retinal Barrier(2003-08-28) Narayan, Santosh; Thomas Yorio; Glenn Dillon; Michael W. MartinRegulation of Endothelin-1 (ET-1) Synthesis and Secretion at the Outer Blood-Retinal Barrier. Santosh Narayan, Department of Pharmacology & Neuroscience, University of North Texas Health Science Center Fort Worth, TX 76102. Summary The retinal pigment epithelium (RPE) constitutes the outer blood retinal barrier at the posterior segment of the eye. The RPE provides metabolic support to the photoreceptors in the neural retina. A breakdown in the barrier supported by RPE is a hallmark in several retinopathies including proliferative vitreoretinopathy, choroidal neovascularization and macular edema. Characteristic to all epithelial cells, mature RPE cells display a polarized phenotype both in culture (ARPE-19 cells) and in vivo, with specific apical and basolateral domains. This provides a testable model to study the RPE in vitro. The purpose of this study was to characterize the RPE as a source for endothelin-1, using both in vitro and in situ models. Endothelins (ET-1,-2, and -3) are known regulators of vascular tone, that are produced at sites close to their target, ET-1, being a potent vasoconstrictor may be involved in regulating blood supply to the choroid and the neural retina. We identified the RPE to be a major source for endothelin-1 (ET-1) in situ in the human retina as well as in pigmented and albino rat retinas. Additionally, using a cell-culture model of mature polarized ARPE-19 cells, we studied the synthesis and expression of ET-1 in response to muscarinic receptor stimulation, TNF-α and more recently to thrombin. We have identified other components involved in the synthesis and turnover of ET-1 in ARPE-19 cells including the proprotein convertase-furin, endothelin-converting enzyme-1 and its isoforms and the endothelin receptor B subtype. ARPE-19 cells grown on collagen filters helped determine if secretion of ET-1 was polarized or discriminative towards either the apical or basolateral surface. We consistently observed changes in cell shape and tight junction disassembly in ARPE-19 cells following TNF-α and thrombin addition. Additionally, thrombin caused an increase in preproET-1 mRNA at earlier time points that was dependent on the rhokinase (ROCK1/2) pathway. We report a novel signaling mechanism for regulating preproET-1 mRNA and mature ET-1 secretion in ARPE-19 cells that involves the thrombin receptor (protease activated receptor-1/PAR-1) dependent activation of the rho/ROCK1/2 signaling pathway that may also be involved in thrombin induced changes in the cytoskeleton. In conclusion, the RPE may be an important source for ET-1 at the posterior segment of the eye, secretion of which is greatly enhanced by substances that promote breakdown of blood retinal barriers, inflammation and changes in the RPE cytoskeleton. In conclusion, the RPE may be an important source for ET-1 at the posterior segment of the eye, secretion of which is greatly enhanced by substances that promote breakdown of blood retinal barriers, inflammation and changes in the RPE cytoskeleton. ET-1 secreted by the RPE, under physiological conditions may provide an autoregulatory mechanism for controlling blood flow at the outer blood retinal barrier. Excessive ET-1 secretion following breakdown of the barrier may either promote wound repair or may mediate further damage to the retina, the substrates of which are presently unknown. Future experimental approaches are planned to address these possibilities.