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.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
2R01EY012017-06A1
Application #
6771363
Study Section
Special Emphasis Panel (ZRG1-AED (01))
Program Officer
Liberman, Ellen S
Project Start
1998-07-01
Project End
2008-04-30
Budget Start
2004-05-01
Budget End
2005-04-30
Support Year
6
Fiscal Year
2004
Total Cost
$577,534
Indirect Cost
Name
Washington University
Department
Ophthalmology
Type
Schools of Medicine
DUNS #
068552207
City
Saint Louis
State
MO
Country
United States
Zip Code
63130
Liu, Bin; Sun, Xiaoguang; Suyeoka, Genn et al. (2013) TGFýý signaling induces expression of Gadd45b in retinal ganglion cells. Invest Ophthalmol Vis Sci 54:1061-9
Wang, Ai Ling; Lukas, Thomas J; Yuan, Ming et al. (2010) Age-related increase in mitochondrial DNA damage and loss of DNA repair capacity in the neural retina. Neurobiol Aging 31:2002-10
Wang, Ai Ling; Lukas, Thomas J; Yuan, Ming et al. (2009) Autophagy and exosomes in the aged retinal pigment epithelium: possible relevance to drusen formation and age-related macular degeneration. PLoS One 4:e4160
Chen, Huiyi; Liu, Bin; Lukas, Thomas J et al. (2009) Changes in iron-regulatory proteins in the aged rodent neural retina. Neurobiol Aging 30:1865-76
Chen, Huiyi; Lukas, Thomas J; Du, Nga et al. (2009) Dysfunction of the retinal pigment epithelium with age: increased iron decreases phagocytosis and lysosomal activity. Invest Ophthalmol Vis Sci 50:1895-902
Liu, Bin; Suyeoka, Genn; Papa, Salvatore et al. (2009) Growth arrest and DNA damage protein 45b (Gadd45b) protects retinal ganglion cells from injuries. Neurobiol Dis 33:104-10
Chen, Huiyi; Liu, Bin; Lukas, Thomas J et al. (2008) The aged retinal pigment epithelium/choroid: a potential substratum for the pathogenesis of age-related macular degeneration. PLoS One 3:e2339
Wang, Ai Ling; Lukas, Thomas J; Yuan, Ming et al. (2008) Increased mitochondrial DNA damage and down-regulation of DNA repair enzymes in aged rodent retinal pigment epithelium and choroid. Mol Vis 14:644-51
Chen, Huiyi; Liu, Bin; Neufeld, Arthur H (2007) Epidermal growth factor receptor in adult retinal neurons of rat, mouse, and human. J Comp Neurol 500:299-310
Liu, Bin; Chen, Huiyi; Johns, Terrance G et al. (2006) Epidermal growth factor receptor activation: an upstream signal for transition of quiescent astrocytes into reactive astrocytes after neural injury. J Neurosci 26:7532-40

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