Abstract
Purpose::
Glaucoma is an optic neuropathy characterized by degeneration of the optic nerve and gradual cupping of the optic disc. This results in a progressive loss of visual field, ultimately leading to blindness. Although the mechanisms of optic nerve damage in glaucoma (mechanical vs ischemic) have not been completely determined, it appears that the optic nerve head (ONH) may be the initial site of damage and the final common pathway is the apoptotic loss of retinal ganglion cells (RGC). The specific signaling mechanism(s) that lead to the observed changes in the optic nerve head leading to RGC cell death are currently not understood. The purpose of this study was to identify differentially expressed protein(s) in the optic nerve heads of glaucomatous rats.
Methods::
The RGC-5 cells were exposed to L-glutamic acid at 5 mM concentration for 24 hrs along with untreated control cells. The optic nerve heads (ONH) from the intraocular pressure induced and control rat eyes were carefully dissected and pooled from 4 individual animals. The protein from RGC-5 cells and ONHs was subjected to 2D gel electrophoresis using 200 ug of total protein. Isoelectric focusing was carried out in glass tubes using pH 3.5-10 ampholines & subjected to 2D electrophoresis. The 2D gels between glaucomatous and normal ONH and RGC-5 cells were compared with each other. The protein spots showing differences between the two were digested in situ with trypsin and the proteins identified by mass spectrometry (MALDI-TOF/MS and capillary HPLC-ESI/MS/MS, with database searching accomplished by Mascot (Matrix Science).
Results::
By mass spectrometry, we identified 5 protein spots, which were altered in glaucomatous ONH as compared to the controls. The proteins identified were transferrin, glutamate dehydrogenase-mitochondrial precursor, alpha 1-enolase, carbonic anhydrase, and alpha-crystallin-B chain. Whereas, there was an upregulation of Hsp-70, Beta 2-tubulin, alpha-1 enolase, vimentin, desmin, and prohibitin in glutamate treated RGC-5 cells as compared to controls.
Conclusions::
The identified proteins point towards possible involvement of oxidative pathway(s) in glaucomatous optic nerve head damage and RGC cell death with alpha-1 enolase being common in both insults.
Keywords: proteomics • optic nerve • oxidation/oxidative or free radical damage