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Yang Liu, Abbot F Clark, Iok-Hou Pang; Phosphoproteomic changes in the retina following optic nerve crush. Invest. Ophthalmol. Vis. Sci. 2016;57(12):6004.
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© ARVO (1962-2015); The Authors (2016-present)
Phosphorylation is a major type of protein post-translational modification. The identification and characterization of protein phosphorylation changes in disease models are understudied but an effective strategy to delineate the underlying disease mechanisms. In this study, we evaluated the phosphoproteomic changes in the retina induced by optic nerve crush (ONC) in the mouse, an acute model of central nervous system (CNS) axon injury.
Intraorbital ONC was performed in adult C57BL/6J mice. Retinas were collected at 0, 6, and 12 h following optic nerve injury. Retinal proteins labeled with CyDye-C2 were subjected to 2D-PAGE. 2D gel phosphoprotein staining was performed, followed by in-gel and cross-gel image analysis. The ratio change of protein differential phosphorylation following ONC was obtained. Proteins with significant changes in phosphorylation (ratios ≥ 1.5) in retinas of the injured eyes compared to the control eyes were spot-picked, tryptic digested, and peptide fragments were analyzed by MALDI-TOF (MS) and TOF/TOF (tandem MS/MS). Proteins identity was based on 10 or more peptides. Bioinformatics analysis was performed using PATHER Classification System. Identified proteins were validated by western blotting and immunofluorescence staining in separate experiments (n ≥ 3).
Intraorbital ONC increased phosphorylation of many retinal proteins. Among them, 53 significantly phosphorylated proteins were identified. PANTHER analysis showed that these proteins fall into several specific biological themes, such as metabolic processes, cellular component organization or biogenesis, anti-apoptosis and axon guidance. Significantly phosphorylated proteins in optic nerve crushed retinas include protein kinase C alpha type, glycogen phosphorylase and tubulin-folding cofactor B. One of the identified phosphoproteins, hepatoma-derived growth factor, has been shown to be neuroprotective in rat optic nerve transection model.
This study provides new insights into mechanisms of retinal ganglion cell degeneration after optic nerve injury, as well as central nervous system (CNS) neurodegeneration, since the retina is an extension of the CNS. These new insights will lead to novel therapeutic targets for retinal and CNS neurodegeneration.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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