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Corina Wilding, Katharina Bell, Sebastian Funke, Norbert Pfeiffer, Franz H Grus; Participation of ERP57 in the pro-survival potential of GFAP abs on RGC5. Invest. Ophthalmol. Vis. Sci. 2014;55(13):2438. doi: https://doi.org/.
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Recent studies show significant up- and down regulated autoantibodies against retinal antigens in the serum of glaucoma patients in comparison to healthy controls. One down-regulated antibody (ab) in glaucoma patients is targeted against GFAP. Since we were able to detect a protective effect of GFAP abs on H2O2 stressed neuroretinal cells (RGC5), the aim of this study was to analyze which mechanisms possibly lead to the protective effect of GFAP abs on RGC5.
A proteomic analysis of cells incubated with GFAP abs using a capillary LC-ESI-MS system was performed. Pathway analysis was implemented with Ingenuity Pathway Analysis program (IPA). Furthermore, possible interaction partners of GFAP abs in RGC5 were identified by western blot, mass spectrometry analysis and indirect immunofluorescence staining.
The mass spectrometric analysis showed significant changes in the actin cytoskeleton pathway. We found 6 proteins significantly (more than 2 fold increased or decreased) changed belonging to this pathway 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). Western blot and mass spectrometry analyses could detect ERP57 as a possible interaction partner of GFAP ab. The binding of GFAP abs on ERP57 on the cell membrane of RGC5 could be confirmed with immunofluorescence staining.
Recent studies could detect significant neuroprotective effects especially of lower GFAP ab concentrations on neuroretinal cells stressed with H2O2. Actin and actin regulated proteins, which we found differently regulated in this study, can influence the mitochondrial apoptosis pathway and therefore could play a role in the neuroprotective effect of GFAP abs. This could be mediated via the binding of GFAP abs to ERP57, a protein disulfide isomerase participating in protein folding, signal transduction from the cell membrane into the nucleus.
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