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An Optic Nerve Crush Injury Murine Model to Study Retinal Ganglion Cell Survival
Published on: April 25, 2011
Early phosphoproteomic changes in the retina following optic nerve crush
Yang Liu1, Huahong Zhong2, Emily L Bussan3
1Department of Pharmacology & Neuroscience, North Texas Eye Research Institute, University of North Texas Health Science Center, Fort Worth, TX, USA.
Abstract:
Retinal ganglion cell (RGC) death causes irreversible blindness in adult mammals. Death of RGC occurs in diseases including glaucoma or injuries to the optic nerve (ON). To investigate mechanisms involved in RGC degeneration, we evaluated the phosphoproteomic changes in the retina induced by ON injury. Intraorbital optic nerve crush (ONC) was performed in adult C57BL/6J mice. Retinas were collected at 0, 6, and 12 h following ONC. Retinal proteins labeled with CyDye-C2 were subject to 2D-PAGE, followed by phosphoprotein staining and in-gel/cross-gel image analysis. Proteins with significant changes in phosphorylation (ratios ≥1.2) 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). Intraorbital ONC increased phosphorylation of many retinal proteins. Among them, 29 significantly phosphorylated proteins were identified. PANTHER analysis showed that these proteins are associated with a variety of protein classes, cellular components, biological processes and signaling pathways. One of the identified proteins, phosphoprotein enriched in astrocytes 15 (PEA15), was further validated by western blotting and immunofluorescence staining. Functions of PEA15 were determined in cultured astrocytes. PEA15 knockdown reduced astrocyte phagocytic activity but promoted cell migration. Long term PEA15 knockdown also decreased astrocyte ATP level. This study provides new insights into mechanisms of RGC degeneration after ON 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.
Insights
Optic nerve injury triggers significant retinal protein phosphorylation changes, impacting astrocyte function and offering new therapeutic targets for blindness and neurodegeneration.
Area of Science:
- Neuroscience
- Proteomics
- Ophthalmology
Background:
- Retinal ganglion cell (RGC) death leads to irreversible blindness.
- RGC degeneration is implicated in glaucoma and optic nerve (ON) injuries.
- Understanding RGC degeneration mechanisms is crucial for developing treatments.
Purpose of the Study:
- To investigate the phosphoproteomic changes in the retina following optic nerve injury.
- To identify key proteins and pathways involved in RGC degeneration.
- To explore the role of phosphoprotein enriched in astrocytes 15 (PEA15) in astrocyte function.
Main Methods:
- Optic nerve crush (ONC) model in adult mice.
- Phosphoproteomic analysis using 2D-PAGE, phosphoprotein staining, and MALDI-TOF/TOF MS.
- Western blotting and immunofluorescence staining for PEA15 validation.
- Functional assays in cultured astrocytes with PEA15 knockdown.
Main Results:
- ONC induced significant phosphorylation changes in numerous retinal proteins.
- 29 significantly phosphorylated proteins were identified, associated with diverse biological functions.
- PEA15 knockdown altered astrocyte phagocytosis, migration, and ATP levels.
- PEA15 was identified as a key player in the retinal response to injury.
Conclusions:
- Optic nerve injury triggers complex phosphoproteomic alterations in the retina.
- PEA15 plays a critical role in astrocyte function, influencing their response to injury.
- These findings provide novel insights into RGC degeneration and potential therapeutic targets for neurodegenerative diseases affecting the central nervous system (CNS).
