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Katharina Bell, Corina Wilding, Norbert Pfeiffer, Franz H. Grus; Down Regulation Of 14-3-3 Ab In Glaucoma Patients Could Lead To Loss Of Protective Effects. Invest. Ophthalmol. Vis. Sci. 2012;53(14):6592.
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Studies show changed autoantibody levels against retinal antigens in the serum of glaucoma patients in comparison to healthy people, such as a significant down regulation of 14-3-3 Ab. Furthermore studies showed that retinal ganglion cells (rgc) react with different protein expressions, such as an up regulation of 14-3-3 protein, when incubated with serum of glaucoma patients, and that this effect could at least partly be caused by the antibodies (Ab) in the serum of glaucoma patients. Aim of this study was to detect if 14-3-3 Ab has an influence on stressed rgc and which pathway could be involved.
RGC5 cells were incubated with different concentrations of chicken polyclonal anti 14-3-3 sigma Ab (Abcam) (0.005, 0.1, 0.5, 1, 5 µg/ml) for 3 hours. In order to induce oxidative stress or apoptosis the cells then were stressed with different stress factors (H2O2, Staurosporine, glutamate). Viability and reactive oxygen species (ROS) then were measured. Furthermore a proteomic analysis of the cells after incubation with 14-3-3 Ab was performed with ESI- Orbitrap MS. The significantly changed proteins in the cells incubated with 14-3-3 Ab in comparison to cells without 14-3-3 Ab incubation were further analyzed using the Ingenuity software. Ab uptake into the cells was confirmed with immunohistochemical staining.
We detected significant protective effects of the 14-3-3 Ab against different stress factors, e.g. up to 31% higher viability and up to 32% decreased ROS level. Furthermore our proteomic analysis was able to measure 1204 proteins from which 225 were significantly differently regulated in cells incubated with 14-3-3 Ab. The functional analysis by Ingenuity showed that many of these proteins belonged to cellular apoptosis pathways such as the mitochondrial apoptosis pathway (e.g. BAX and ERK1).
We were able to show that the Ab againt 14-3-3, which is down regulated in glaucoma patients, has a protective effect on rgc. Possibly the protective effect can be explained through the influence of 14-3-3 Ab on the mitochondrial apoptosis pathway. This leads to the conclusion that natural Ab’s have an impact on the intracellular protein homeostasis which could be in a misbalance in patients suffering from glaucoma, eventually leading to apoptosis.
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