Phosphoproteomic changes in the retina following optic nerve crush

Purpose: Phosphorylation is a major type of protein post-translational modification. The identification and characterization of protein phosphorylation changes in disease models is 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. Methods: Intraorbital ONC was performed in adult C57BL6/J 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 immunostaining was performed, followed by in-gel image analysis. Proteins with significant changes in phosphorylation 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). Identified proteins were validated by Western blotting and immunofluorescence staining. Results: Intraorbital ONC increased phosphorylation of many retinal proteins. Among them, 96 were spot-picked and identified. An initial DAVID analysis showed that these proteins fall into several specific biological themes, such as apoptosis, survival, and regeneration of neurons, as well as glial activation. One of the identified phosphoproteins, PEA-15, has been confirmed by Western blot analysis; ONC increased phosphorylation of this protein without affecting its expression level. Immunofluorescence staining using phospho-PEA-15-specific antibody demonstrated that increased phosphorylated PEA-15 co-localized with GFAP, a marker for Müller cells and astroglia in the retina. Conclusions: 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.