Browsing by Subject "retinal ganglion cell death"
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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 Role of Nonfeminizing Estrogen Analogues in Neuroprotection of Rat Retinal Ganglion Cells Against Glutamate-Induced Cytotoxicity(2007-05-01) Kumar, Domalapalli Maneesh; Agarwal, Neeraj; Gracy, Robert; Garner, MargaretKumar, Domalapalli Maneesh, Role of Nonfeminizing Estrogen Analogues in Neuroprotection of Rat Retinal Ganglion Cells against Glutamate-Induced Cytotoxicity, Doctor of Philosophy (Cell Biology and Genetics), May, 2007, 210 pp., 3 tables, 23 figures, bibliography, 427 titles. Retinal ganglion cell death has been determined to be the final common pathway in glaucoma. Continuous loss of retinal ganglion cells results in irreversible progressive visual field deterioration that culminates in blindness. No effective therapy is currently available to reverse retinal ganglion cell loss. Therefore, preventing the loss of retinal ganglion cells is a logical approach to maintaining vision in effected individuals. Of the methods of investigation, in vivo models of ganglion cell death provide a physiological system in which to study neuroprotective drugs and their effects, but these systems are inefficient for initial screening studies. We have addressed this by utilizing the RGC-5 clonal rat retinal ganglion cell line. Glutamate treatment of RGC-5 cells induces apoptotic death which can be attenuated by pretreatment with the anti-oxidants N-acetyl cysteine and thiourea, implicating oxidative stress as a major component of glutamate’s cytotoxicity. Also antioxidants, estrogens have been demonstrated to be potent neuroprotectants in a variety of in vitro and in vivo models of neurodegeneration. Estrogens’ antioxidant capacity has been attributed to the ability of the phenolic A ring to quench and resonance stabilize oxidative free radicals. It is also known that the estrogen A ring is responsible for binding of these hormones to estrogen receptors, producing feminizing phenotypes. The feminizing effects of estrogens narrow or preclude their use as neuroprotectants in males, and in females that may be predisposed to their deleterious effects. To address these shortcomings we screened 13, non-feminizing, non-receptor binding estrogen analogues in our glutamate-induced RGC-5 model of oxidative stress-induced cell death. The most effective of these drugs was ZYC-3. ZYC-3 was synthesized by the addition of an adamantly group to the C2 position on the A ring of estrone. This modification produced a neuroprotective compound with potency and efficacy at least equal to the prototypical estrogen, 17β-estradiol, but with no appreciable binding affinity for estrogens receptors α or β. Our preliminary findings suggest that ZYC-3 enhances glutathione synthesis and blocks mitochondrial apoptotic pathways. However, as a novel drug we are naïve to its effects on cellular physiology and as to how it affords neuroprotection. Understanding how this drug regulates cellular destructive and protective mechanisms could lead to further innovations in drug design and in methods to prevent retinal ganglion cell degeneration. In vivo studies of this drug may then form the bridge to a better clinical approach to managing ocular disorders in which ganglion cell loss is the culprit for vision loss. Although promising, evidence supporting the application of estrogen analogues in models of ocular neurodegenerative diseases are nearly non-existent. It is our objective to study the neuroprotective effects of ZYC-3 in glaucomatous models with the goal of maintaining retinal ganglion cell viability and preventing vision loss.