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Natik Piri, Jacky Man Kwong Kwong, Haksu Kyung, Joseph Caprioli, Yasunari Munemasa; The Neuronal EGF-related Gene Nell2 Supports Survival Of Retinal Ganglion Cells After Optic Nerve Axotomy. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3480.
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Nell2, a neuron-specific protein containing six epidermal growth factor-like domains, has been identified as a retinal ganglion cell (RGC)-expressed gene by comparing mRNA profiles of control and RGC-deficient rat retinas. The aim of this study was to evaluate the role of Nell2 in RGCs.
Proteome analysis was performed by matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MS). EGFP-fused Nell2 expression construct was delivered to RGCs by electroporation-mediated cell transfection. The optic nerve transection (ONT) model was generated in adult male Wistar rats. Retrograde labeling to estimate the number of RGCs was performed by application of the Fluorogold to the proximal cut surface of the optic nerve at the time of optic nerve axotomy.
Microtubule-actin crosslinking factor 1 (Macf1) was identified by MS as Nell2 interacting protein in the retina in three consecutive experiments. The results obtained with MS were confirmed by reverse immunoprecipitation with a Macf1 antibody and immunoblot with Nell2 antibody. The expression pattern of Macf1 in the retina was determined by immunohistochemistry. Strong Macf1 expression in the GCL and inner plexiform layer was observed. Macf1 expression in RGCs was co-localized with Thy-1 staining, a commonly used marker for RGCs. The effect of Nell2 overexpression on RGC survival was evaluated 2 weeks after ONT. RGCs in the upper nasal retina were consistently more efficiently transfected than in other areas. Approximately 49% of RGCs in the upper nasal area were transfected whereas the transfection efficiency for the entire retina was only ~13% (n=5, p<0.05). In non-transfected ONT retinas the loss of RGCs was approximately 95% compared to the untreated control. In the upper nasal region, Nell2 transfection led to the preservation of approximately 58% more cells damaged by axotomy compared to untransfected (n=5, p<0.01) or pEGFP-transfected controls (n=5, p<0.01).
The results of MS that were further corroborated with immunoprecipitation/immunoblot and immunohistochemistry data provide strong evidence for the Nell2 interaction with Macf1 in RGCs. Furthermore, we evaluated the cell-protective effect of Nell2 after ONT and demonstrated an increase in RGC survival by approximately 58% compared to controls.
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