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Corina Wilding, Katharina Bell, Sebastian Funke, Sabine Beck, Norbert Pfeiffer, Franz Grus; Protective effect of GFAP antibody on RGC5 cells via actin cytoskeleton pathways. Invest. Ophthalmol. Vis. Sci. 2013;54(15):432. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
The pathogenesis of glaucoma still is unknown. Next to increased reactive oxygen species (ROS) levels, recent studies show the participation of immunological components. Previous studies demonstrate significant changes in the pattern of autoantibodies against ocular antigens in the serum of glaucoma patients in comparison to healthy controls. GFAP antibody (ab) is found in a lower concentration in glaucoma patients. Our aim was to analyse the effect of GFAP ab on neuroretinal cells (RGC5), those cells affected by glaucoma and which pathways are triggered in RGC5, when they are incubated with GFAP ab.
RGC5 were preincubated with different GFAP ab concentrations (0.05, 0.01, 0.5,1,5, 10, 20 μg/ml) for 3h and stressed with 50µM H2O2 (1h) to induce ROS. Controls were incubated without ab. Cell viability was measured with crystal violet and ROS with DCFH-DA. We also performed proteomic analysis of cells incubated with GFAP ab. Analysis of peptides was performed using a capillary LC-ESI-MS system. The obtained mass spectra were identified and quantified using MaxQuant and pathway analysis were performed with Ingenuity Pathway Analysis (IPA).
A significant increase of viability and decrease of ROS levels of cells stressed with H2O2 and preincubated with different ab levels (0.05, 0.1, 0.5 and 1 µg/ml) was in both detected (9%, p<0.05). Based on IPA we were able to indicate significant changes in actin cytoskeleton pathways, where we could find 6 significant changed proteins in RGC5 after GFAP ab treatment such as Vinculin (2.187), Talin1 (2.157), Profilin1 (-4.399), Cofilin (-3.735), Actin beta (2.036),actin regulated protein 2/3 (-2.285).
We detected significant neuroprotective effects especially of the lower GFAP ab concentrations on the cells stressed with H2O2, e.g. raised viability and decreased ROS level. Actin and actin regulated proteins, which we found differently regulated, participated in the mitochondrial apoptosis pathway and could play a key role in the neuroprotective effect of GFAP ab. During the induction of apoptosis e.g. dephosphorylated cofilin gets translocated from the cytosol to mitochondria and is able to induce cytochrom c release. Therefore we hypothesize that the changes of the actin cytoskeleton proteins, found in GFAP Ab treated RGC5, have a pro survival potential against oxidative stress.
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