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Peroxynitrite Mediates Retinal Neurodegeneration by Inhibiting Nerve Growth Factor Survival Signaling in Experimental and Human Diabetes

¹ Program in Clinical and Experimental Therapeutics, University of Georgia, Augusta, Georgia
² Department of Pharmacology and Toxicology, Augusta, Georgia
³ Department of Ophthalmology, Medical College of Georgia, Augusta, Georgia
⁴ VA Medical Center, Augusta, Georgia

Address correspondence and reprint requests to A.B. El-Remessy, PhD, RPh, Program in Clinical and Experimental Therapeutics, College of Pharmacy, University of Georgia, Augusta, GA 30912. E-mail: aelremessy{at}mcg.edu

Key Words: 4-HNE, 4-hydroxynonenal • bFGF, basic fibroblast growth factor • DR, diabetic retinopathy • ELISA, enzyme-linked immunosorbent assay • MAPK, mitogen-activated protein kinase • NGF, nerve growth factor • PI3K, phosphatidylinositol 3-kinase • RGC, retinal ganglion cells • ROD, relative optical density • PN, peroxynitrite • TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling • VEGF, vascular endothelial growth factor

OBJECTIVE—Recently we have shown that diabetes-induced retinal neurodegeneration positively correlates with oxidative stress and peroxynitrite. Studies also show that peroxynitrite impairs nerve growth factor (NGF) survival signaling in sensory neurons. However, the causal role of peroxynitrite and the impact of tyrosine nitration on diabetes-induced retinal neurodegeneration and NGF survival signaling have not been elucidated.

RESEARCH DESIGN AND METHODS—Expression of NGF and its receptors was examined in retinas from human and streptozotocin-induced diabetic rats and retinal ganglion cells (RGCs). Diabetic animals were treated with FeTPPS (15 mg · kg^–1 · day^–1 ip), which catalytically decomposes peroxynitrite to nitrate. After 4 weeks of diabetes, retinal cell death was determined by TUNEL assay. Lipid peroxidation and nitrotyrosine were determined using MDA assay, immunofluorescence, and Slot-Blot analysis. Expression of NGF and its receptors was determined by enzyme-linked immunosorbent assay (ELISA), real-time PCR, immunoprecipitation, and Western blot analyses.

RESULTS—Analyses of retinal neuronal death and NGF showed ninefold and twofold increases, respectively, in diabetic retinas compared with controls. Diabetes also induced increases in lipid peroxidation, nitrotyrosine, and the pro-apoptotic p75^NTR receptor in human and rat retinas. These effects were associated with tyrosine nitration of the pro-survival TrkA receptor, resulting in diminished phosphorylation of TrkA and its downstream target, Akt. Furthermore, peroxynitrite induced neuronal death, TrkA nitration, and activation of p38 mitogen-activated protein kinase (MAPK) in RGCs, even in the presence of exogenous NGF. FeTPPS prevented tyrosine nitration, restored NGF survival signal, and prevented neuronal death in vitro and in vivo.

CONCLUSIONS—Together, these data suggest that diabetes-induced peroxynitrite impairs NGF neuronal survival by nitrating TrkA receptor and enhancing p75^NTR expression.

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