The long-term objective of our research is to identify classes of compounds that protect against the neurodestructive activity of inducible nitric oxide synthase (NOS-2) and that have the potential to be developed as treatments for glaucoma. Our overall hypothesis is that excessive nitric oxide, synthesized by NOS-2 in astrocytes of the optic nerve head, is neurotoxic to the axons of the retinal ganglion cells in glaucoma. The proposed experiments in this renewal application are focused on identifying the signal transduction pathways required for the induction of this neurotoxic enzyme in human optic nerve head astrocytes and to test compounds for their ability to inhibit the induction of NOS-2 in a model of glaucoma. Specifically, we will determine the molecular mechanisms by which activation of epidermal growth factor receptor (EGFR) tyrosine kinase acts as a signal transduction pathway regulating the induction of NOS-2 in response to elevated hydrostatic pressure. We will determine which ligands of EGFR are present in human and rat glaucomatous optic nerve head tissues by immunohistochemistry and RT-PCR. Using specific inhibitors, we will determine the intracellular mediator pathways that act downstream and/or in parallel to activated EGFR to induce NOS-2. We will define additional signal transduction pathways that differentially regulate the induction of expression of the NOS-2 gene in response to elevated hydrostatic pressure versus cytokines. We will identify pharmacological compounds that block key signal transduction pathways leading to the induction of NOS-2 and test these compounds as neuroprotective agents against loss of retinal ganglion cells using the rat glaucoma model and retrograde labeling with Fluoro-Gold. Using human and rat optic nerve head astrocytes in vitro, we will characterize ligand binding to EGFR. Once putative sites in the promoter region of the NOS-2 gene are identified for EGFR- or pressure-stimulated induction of NOS-2, we will use transfection, mutagenesis and electrophoretic mobility shift analysis to characterize the sites. We will determine whether translocation of the ligand-receptor complex to the nucleus and binding to the promoter region of the NOS-2 gene is necessary for induction. This research will demonstrate pharmacological compounds that may be useful for accomplishing neuroprotection for the treatment of glaucoma.
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