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Madeleine Kiderle, Katharina Bell, Sebastian Funke, Norbert Pfeiffer, Franz H Grus; Protein changes within the Rho pathway in neuroretinal cells after incubation with POAG serum: Cofilin, Rac1, CDC42 – causes for the effect of ROCK-inhibitors?. Invest. Ophthalmol. Vis. Sci. 2016;57(12):5635. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Animal models showed that Rho-kinase inhibitors (ROCK-inhibitors), a new group of molecules under trial for glaucoma therapy, not only lower intraocular pressure but also provide neuroprotection of ganglion cells. The pathophysiology of how Rho-kinases might initiate neuronal cell damage in glaucoma has not been demonstrated yet. In this study we performed protein analysis in a cell culture model on subcellular level by incubating neuroretinal cells with serum of glaucoma patients with primary open angle glaucoma (POAG). Protein changes might be caused by unknown substances in the POAG serum such as antibodies.
Neuroretinal cells (RGC5) were incubated with serum of POAG patients with cataract as well as cataract patients as controls (age- and sex-matched, n=6) for 24h and 48h. After subcellular protein fractionation four protein fractions (cytosolic (F1), membrane (F2), nuclear (F3), cytoskeletal (F4)) were prepared and pooled by fractions (6 samples group) before they were separated through SDS-gelelectrophoresis. Trypsin in-gel digestion was performed and proteins were analyzed using LC-ESI/MS. Evaluation was implemented with Ingenuity Pathway Analysis Software and Panther Classification System.
The Rho-kinase pathway consists of 20 proteins of which 10 proteins were identified (p<0,01) in the POAG and the control group after 24h and after 48h. Among those proteins five were expressed significantly changed (>2-fold) after 24h as well as after 48h. Cofilin showed a movement from the cytoskeleton to the nucleus (CFL1, F3 (48h): 2,254 fold up- regulated, F4 (24h): -9,894 fold down-regulated), Rac1 and CDC42 showed significantly changed expression profiles in the cytosol (RAC1, F1 (48h): -2,976; CDC42, F1 (24h): 2,772)).
Our results show changes of proteins that are part of Rho-kinase pathways. Subcellular changes in the cofilin expression profile (down-regulation in the cytoskeleton, up-regulation in the nucleus) have earlier been awarded a central role in initiating neurodegeneration and apoptosis in neurodegenerative diseases. Rac1 and CDC42 may also play significant roles in cell survival. Hence, these changes, eventually caused by antibodies of POAG serum, may contribute to the pathophysiology of POAG and may be a reason for the neuroprotective effect of ROCK-inhibitors.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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