Phosphoproteomic changes in the retina following optic nerve crush

Purpose: Phosphorylation is a major type of protein post-translational modification. 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) axonal injury. The functional role of an identified major phosphoprotein was further studied. Methods: 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. Identified proteins were validated by western blotting and immunofluorescence staining in separate experiments (n ≥ 3). Cell migration assay and flow cytometry-based phagocytosis assay were performed using primary cultured mouse optic nerve astrocytes. Results: Intraorbital ONC increased phosphorylation of many retinal proteins. Among them, 53 significantly phosphorylated proteins were identified. Significantly phosphorylated proteins in optic nerve crushed retinas include protein kinase C alpha, glycogen phosphorylase, tubulin-folding cofactor B, among others. One of the identified phosphoproteins, PEA-15, was confirmed by western blot analysis; ONC increased phosphorylation of this protein without affecting its basal protein 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 and optic nerve. PEA-15 knockdown significantly promoted optic nerve astrocyte migration and suppressed phagocytosis. Conclusions: Our novel approach identified specific proteins whose phosphorylation was increased by ONC. One of these proteins, PEA-15, mediates major optic nerve astrocytic functions, which likely affect retinal neuronal survival and regeneration after injury. These new insights will lead to novel therapeutic targets for retinal and CNS neurodegeneration.