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Paulo D. Koeberle, Philippe M. D'Onofrio, Mark Magharious, Adam C. Hollander; Identification Of Protein Targets Involved In Adult Cns Neuron Apoptosis Via Quantitative Itraq Analysis Of The Injured Retina. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5439.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal ganglion cell (RGC) apoptosis is a hallmark of blinding diseases such as glaucoma. RGC apoptosis is modeled by transecting the mammalian optic nerve, which results in the death of 90% of injured RGCs by 14 days postaxotomy. The purpose of this study was to carry out a to identify retinal proteins that may be involved in key stages of RGC apoptosis after axotomy.
Optic nerve transection were performed in adult rats. At 1, 3, 4, 7, 10, 14 and 21 days following axotomy (n=8 at each timepoint) retinal lysates were labeled using iTRAQ reagents and subjected to analysis by mass spectroscopy (LC/MS/MS Analysis), in an 8-plex protocol that included a baseline group of normal retinas (n=8). After identifying hepatoma derived growth factor (HDGF) as being elevated at 3 days postaxotomy, we examined the effects of exogenous HDGF administration on RGC survival after axotomy. Axotomized animals received intraocular injections of recombinant HDGF at 3 days after axotomy, and RGC survival was quantified at 14 days, in fixed flatmounted retinas. RGCs were retrogradely labeled with Fluorogold at the time of axotomy, in order to track their survival. Western blots were used to confirm changes in HDGF expression in axotomized retinas at 4 and 7 days postaxotomy.
538 proteins were identified and quantified: 38 showed increased expression, while 29 showed decreased expression (<0.5x or >1.5x) relative to normal retinas. Cytoskeletal or intracellular (24.24% each) and microtubule or protein complex (9.09% each) represented the majority of up-regulated proteins after axotomy. Intracellular (26.32%), cytoskeletal (21.05%), microtubule (15.79%), and ribonucleoprotein complex (10.53%) classes were the most commonly down-regulated proteins. Our analysis identified several patterns of upregulation and downregulation at different timepoints after axotomy, and proteins were grouped accordingly. HDGF and HDGF-related protein 3 (HRP-3) showed an increase in expression between 3 and 4 days postaxotomy, when RGCs first begin to die by apoptosis. Administration of HDGF following axotomy resulted in a 2-fold increase in RGC survival (p<0.001) relative to controls, demonstrating that HDGF is neuroprotective for injured RGCs.
We used iTRAQ to uncover changes in protein levels at key stages of RGC apoptosis, and found 67 proteins that were differentially regulated after optic nerve transection. These proteins, as well as others that showed more modest changes in expression, represent potential targets for RGC neuroprotection.
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