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Protective effect of 14-3-3 antibodies on stressed neuroretinal cells via the mitochondrial apoptosis pathway.

Bell K, Wilding C, Funke S, Pfeiffer N, Grus FH - BMC Ophthalmol (2015)

Bottom Line: We found significant effects of serum antibodies on proteins of neuroretinal cells especially of the mitochondrial apoptosis pathway.The changed autoantibodies belong to the natural autoimmunity.We conclude that changes in the natural autoimmunity of patients with glaucoma can negatively impact regulatory functions.

View Article: PubMed Central - PubMed

Affiliation: Experimental Ophthalmology, Department of Ophthalmology, University Medical center of the Johannes Gutenberg University, Langenbeckstraße 1, 55131, Mainz, Germany. kbell@eye-research.org.

ABSTRACT

Background: Previous studies demonstrate changes of autoantibody concentrations against retinal and optic nerve head antigens in the serum of glaucoma patients in comparison to healthy persons. These antibodies belong to the natural autoimmunity. Previous studies showed up regulated, but also significantly down-regulated autoantibody levels. These antibodies have the ability to influence protein profiles of neuroretinal cells and possibly hold neuroprotective potential, as we have been able to demonstrate before. Aim of this study was to analyse the serum and antibody effect of glaucoma patients on neuroretinal cells in more detail and also determine the impact of antibodies found down-regulated in glaucoma patients on the pathogenesis of the neurodegenerative disease glaucoma.

Methods: Neuroretinal cells (RGC-5) were incubated with serum either from glaucoma patients or healthy controls for 24 h. Mass spectrometric analysis was performed after cell lysis. Furthermore the neuroretinal cells were preincubated with different and concentrations of 14-3-3 antibodies (0.005, 0.1, 0.5, 1, 5 and 10 μg/ml) and then stressed with H2O2, staurosporine or glutamate. Viability tests were performed with crystal violet and ROS tests with DCFH-DA. Antibody location in the cell after antibody incubation was performed with immunocytochemical methods. Additionally mass spectrometric analysis was performed with the cells after antibody incubation.

Results: Protein expression analysis with Maldi-Orbitrap MS showed changes in the expression level of regulatory proteins in cells incubated with glaucoma serum, e.g. an up-regulation of 14-3-3 and a down-regulation of Calmodulin. After preincubation of the cells with anti-14-3-3 antibody and stressing the cells, we detected an increase in viability of up to 22 % and a decrease in reactive oxygen species (ROS) of up to 31 %. Proteomic 1 analysis involvement of the mitochondrial apoptosis pathway in this protective effect and immunohistochemical analysis showed an antibody uptake in the cells.

Conclusion: We found significant effects of serum antibodies on proteins of neuroretinal cells especially of the mitochondrial apoptosis pathway. Furthermore we detected a protective potential of antibodies down-regulated in glaucoma patients. The changed autoantibodies belong to the natural autoimmunity. We conclude that changes in the natural autoimmunity of patients with glaucoma can negatively impact regulatory functions.

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Related in: MedlinePlus

14-3-3 sigma antibody uptake of RGC-5 revealed by indirect immunfluorescence. Living cells were preincubated with polyclonal rabbit anti 14-3-3 antibodies and then fixed, permeabilised, blocked and stained with Goat polyclonal secondary antibody to rabbit IgG-H&L conjugated with FITC. a: Bright light microscopy picture. b: Corresponding fluorescence micrograph merged with bright light. Red Arrows indicate vesicles, which contain 14-3-3 sigma antibodies, showing antibody uptake into the intact cell
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Fig6: 14-3-3 sigma antibody uptake of RGC-5 revealed by indirect immunfluorescence. Living cells were preincubated with polyclonal rabbit anti 14-3-3 antibodies and then fixed, permeabilised, blocked and stained with Goat polyclonal secondary antibody to rabbit IgG-H&L conjugated with FITC. a: Bright light microscopy picture. b: Corresponding fluorescence micrograph merged with bright light. Red Arrows indicate vesicles, which contain 14-3-3 sigma antibodies, showing antibody uptake into the intact cell

Mentions: To determine, whether RGC-5 cells express 14-3-3 and were the antibody binds in living cells we performed an indirect immunofluorescence staining. In permeabilised RGC-5 cells we could show binding of the 14-3-3 antibody in the cytoplasm (see Fig. 5). We further investigated the uptake of 14-3-3 antibodies in living RGC-5 cells. We were able to show the uptake of 14-3-3 antibodies in vesicles after preincubation of living RGC-5 for 3 h (Fig. 6).Fig. 5


Protective effect of 14-3-3 antibodies on stressed neuroretinal cells via the mitochondrial apoptosis pathway.

Bell K, Wilding C, Funke S, Pfeiffer N, Grus FH - BMC Ophthalmol (2015)

14-3-3 sigma antibody uptake of RGC-5 revealed by indirect immunfluorescence. Living cells were preincubated with polyclonal rabbit anti 14-3-3 antibodies and then fixed, permeabilised, blocked and stained with Goat polyclonal secondary antibody to rabbit IgG-H&L conjugated with FITC. a: Bright light microscopy picture. b: Corresponding fluorescence micrograph merged with bright light. Red Arrows indicate vesicles, which contain 14-3-3 sigma antibodies, showing antibody uptake into the intact cell
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4482181&req=5

Fig6: 14-3-3 sigma antibody uptake of RGC-5 revealed by indirect immunfluorescence. Living cells were preincubated with polyclonal rabbit anti 14-3-3 antibodies and then fixed, permeabilised, blocked and stained with Goat polyclonal secondary antibody to rabbit IgG-H&L conjugated with FITC. a: Bright light microscopy picture. b: Corresponding fluorescence micrograph merged with bright light. Red Arrows indicate vesicles, which contain 14-3-3 sigma antibodies, showing antibody uptake into the intact cell
Mentions: To determine, whether RGC-5 cells express 14-3-3 and were the antibody binds in living cells we performed an indirect immunofluorescence staining. In permeabilised RGC-5 cells we could show binding of the 14-3-3 antibody in the cytoplasm (see Fig. 5). We further investigated the uptake of 14-3-3 antibodies in living RGC-5 cells. We were able to show the uptake of 14-3-3 antibodies in vesicles after preincubation of living RGC-5 for 3 h (Fig. 6).Fig. 5

Bottom Line: We found significant effects of serum antibodies on proteins of neuroretinal cells especially of the mitochondrial apoptosis pathway.The changed autoantibodies belong to the natural autoimmunity.We conclude that changes in the natural autoimmunity of patients with glaucoma can negatively impact regulatory functions.

View Article: PubMed Central - PubMed

Affiliation: Experimental Ophthalmology, Department of Ophthalmology, University Medical center of the Johannes Gutenberg University, Langenbeckstraße 1, 55131, Mainz, Germany. kbell@eye-research.org.

ABSTRACT

Background: Previous studies demonstrate changes of autoantibody concentrations against retinal and optic nerve head antigens in the serum of glaucoma patients in comparison to healthy persons. These antibodies belong to the natural autoimmunity. Previous studies showed up regulated, but also significantly down-regulated autoantibody levels. These antibodies have the ability to influence protein profiles of neuroretinal cells and possibly hold neuroprotective potential, as we have been able to demonstrate before. Aim of this study was to analyse the serum and antibody effect of glaucoma patients on neuroretinal cells in more detail and also determine the impact of antibodies found down-regulated in glaucoma patients on the pathogenesis of the neurodegenerative disease glaucoma.

Methods: Neuroretinal cells (RGC-5) were incubated with serum either from glaucoma patients or healthy controls for 24 h. Mass spectrometric analysis was performed after cell lysis. Furthermore the neuroretinal cells were preincubated with different and concentrations of 14-3-3 antibodies (0.005, 0.1, 0.5, 1, 5 and 10 μg/ml) and then stressed with H2O2, staurosporine or glutamate. Viability tests were performed with crystal violet and ROS tests with DCFH-DA. Antibody location in the cell after antibody incubation was performed with immunocytochemical methods. Additionally mass spectrometric analysis was performed with the cells after antibody incubation.

Results: Protein expression analysis with Maldi-Orbitrap MS showed changes in the expression level of regulatory proteins in cells incubated with glaucoma serum, e.g. an up-regulation of 14-3-3 and a down-regulation of Calmodulin. After preincubation of the cells with anti-14-3-3 antibody and stressing the cells, we detected an increase in viability of up to 22 % and a decrease in reactive oxygen species (ROS) of up to 31 %. Proteomic 1 analysis involvement of the mitochondrial apoptosis pathway in this protective effect and immunohistochemical analysis showed an antibody uptake in the cells.

Conclusion: We found significant effects of serum antibodies on proteins of neuroretinal cells especially of the mitochondrial apoptosis pathway. Furthermore we detected a protective potential of antibodies down-regulated in glaucoma patients. The changed autoantibodies belong to the natural autoimmunity. We conclude that changes in the natural autoimmunity of patients with glaucoma can negatively impact regulatory functions.

Show MeSH
Related in: MedlinePlus