Abstract
Purpose: :
Nell2 is a neuron-specific thrombospondin-1-like protein containing six epidermal growth factor-like domains. The aim of this study was to analyze Nell2 expression at mRNA and protein levels in the wild-type and ONT rat retinas and demonstrate its specific expression in RGCs.
Methods: :
We have identified Nell2 as a retinal ganglion cell (RGC) - specific gene by comparing expression profiles of control and RGC-deficient [2 weeks after optic nerve transaction (ONT)] rat retinas. Localization of Nell2 mRNA expression in the retina was performed with in situ hybridization with gene-specific sense and anti-sense riboprobes. Quantitative and spatial expressions of Nell2 protein were examined with western blot and immunohistochemisty, respectively. Retrograde labeling of RGCs was performed by placing a piece of Gelfoam soaked with 6% Fluorogold (FG) on the proximal cut surface of the optic nerve after ONT. Optic nerve transection (ONT) was performed on adult male Wistar rats.
Results: :
Nell2-positive in situ signals were localized predominantly to irregularly shaped cells in the retinal ganglion cell layer (GCL) and were co-localized with FG-labeled RGCs. No Nell2-positive cells were detected by in situ hybridization in 2 weeks ONT retinas characterized with approximately 90-95% RGC loss. Consistent with the in situ results, semi-quantitative RT-PCR analysis showed a dramatic decrease in the mRNA level of Nell2 mRNA after ONT compared to the controls. With respect to protein expression, staining with Nell2 antibodies was observed in the GCL. Nell2-positive cells were colocalized with FG-labeled RGCs. Immunoblot analysis of the Nell2 expression in the retina revealed the presence of two products with approximate molecular weights of 90 and 140 kDa. The 90 kDa product was more abundant than 140 kDa. Since the deduced molecular weight of Nell2 is 91,402 kDa, it is suggested that the 90 and 140 kDa bands represent non-glycosylated and glycosylated Nell2, respectively. Both, 90 and 140 kDa bands were absent in retinal protein extracts two weeks after ONT.
Conclusions: :
We have determined that Nell2 expression at both mRNA and protein levels in the retina is localized to RGCs. Since Nell2 has been reported to increase survival of neurons from the hippocampus and cerebral cortex by modulating MAPK activities, it is possible that it plays a similar function in RGCs.
Keywords: ganglion cells • gene/expression • retina