Only through a better understanding of the pathogenic mechanisms of glaucoma will improved treatments evolve. Ongoing studies during the original grant period have supported the involvement of tumor necrosis factor-alpha (TNF-a) signaling in glaucoma and illuminated many aspects of the TNF-a-mediated death of retinal ganglion cells (RGCs) during glaucomatous neurodegeneration. The proposed experiments in this renewal application are based on the hypothesis that an innovative analytical approach using proteomics technology can further elucidate TNF-a signaling in glaucoma by identifying time-dependent alterations in the protein complement.
The specific aims that will address this hypothesis are: (1) To comparatively identify proteomic alterations in RGCs and glia exposed to TNF-a, in vitro; and (2) To identify proteomic alterations associated with TNF-a signaling in RGCs during the course of glaucomatous neurodegeneration, in vivo. For in vitro experiments, RGCs and glial cells will be isolated from adult tissues. For large-scale identification of TNF-a-induced alterations in the protein complement, differential proteomics will utilize protein lysates obtained from cultured cells. Time-dependent alterations in protein expression will be quantitatively evaluated by comparing the proteomic datasets obtained from RGCs and glial cells incubated in the presence and absence of TNF-a. Complementary approaches will be utilized to increase the sensitivity of protein identification. In addition, phosphorylated proteins will be identified through targeted proteomics using tandem mass spectrometry. Findings of the proposed comparative analyses should provide comprehensive information about differential responses of RGCs and glia to TNF-a at the protein level. A better understanding of the cellular mechanisms associated with the relative protection of glial cells against glaucomatous injury can facilitate efforts to similarly improve RGC survival in glaucoma. The proposed in vivo experiments will utilize an experimental rat model of glaucoma in which intraocular pressure (IOP) elevation will be unilaterally induced by hypertonic saline injections into limbal veins. IOP exposure and axon loss will be determined for each rat sacrificed at different time points during a follow-up period of up to 12 weeks. RGC protein samples will be pooled from rat eyes matched for IOP exposure and axon loss. Interacting and phosphorylated RGC proteins in enriched multi-protein complexes will be identified using targeted proteomic approaches. The signaling complexes studied will include the TNF-a/TNF receptor complex and those associated with mitogen-activated protein kinase and nuclear factor-kappaB pathways (which are involved in TNF-a signaling and glaucomatous neurodegeneration). Due to diverse bioactivities of TNF-a, which promote both cell death and survival signals, specific inhibition of cell death signaling and/or the amplification of survival signaling (rather than the inhibition of receptor binding), should accomplish neuroprotection against TNF-a-mediated RGC death. An improved understanding of TNF-a signaling during glaucomatous neurodegeneration in a proteome-wide scale should therefore provide new and specific treatment targets for effective neuroprotective interventions in glaucoma, a leading cause of blindness. ? ? ?

National Institute of Health (NIH)
National Eye Institute (NEI)
Research Project (R01)
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Anterior Eye Disease Study Section (AED)
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Agarwal, Neeraj
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University of Louisville
Schools of Medicine
United States
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Tezel, Gülgün (2014) A decade of proteomics studies of glaucomatous neurodegeneration. Proteomics Clin Appl 8:154-67
Križaj, David; Ryskamp, Daniel A; Tian, Ning et al. (2014) From mechanosensitivity to inflammatory responses: new players in the pathology of glaucoma. Curr Eye Res 39:105-19
Tezel, Gulgun (2013) Immune regulation toward immunomodulation for neuroprotection in glaucoma. Curr Opin Pharmacol 13:23-31
Tezel, Gulgun (2013) A proteomics view of the molecular mechanisms and biomarkers of glaucomatous neurodegeneration. Prog Retin Eye Res 35:18-43
Tezel, Gulgun; Yang, Xiangjun; Luo, Cheng et al. (2012) An astrocyte-specific proteomic approach to inflammatory responses in experimental rat glaucoma. Invest Ophthalmol Vis Sci 53:4220-33
Tezel, Gulgun; Thornton, Ivey L; Tong, Melissa G et al. (2012) Immunoproteomic analysis of potential serum biomarker candidates in human glaucoma. Invest Ophthalmol Vis Sci 53:8222-31
Tezel, Gulgun (2011) The immune response in glaucoma: a perspective on the roles of oxidative stress. Exp Eye Res 93:178-86
Yang, Xiangjun; Luo, Cheng; Cai, Jian et al. (2011) Neurodegenerative and inflammatory pathway components linked to TNF-?/TNFR1 signaling in the glaucomatous human retina. Invest Ophthalmol Vis Sci 52:8442-54
Tezel, Gülgün; Yang, Xiangjun; Luo, Cheng et al. (2010) Hemoglobin expression and regulation in glaucoma: insights into retinal ganglion cell oxygenation. Invest Ophthalmol Vis Sci 51:907-19
Luo, Cheng; Yang, Xiangjun; Kain, Angela D et al. (2010) Glaucomatous tissue stress and the regulation of immune response through glial Toll-like receptor signaling. Invest Ophthalmol Vis Sci 51:5697-707

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