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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: Previous studies showed up regulated, but also significantly down-regulated autoantibody levels.We found significant effects of serum antibodies on proteins of neuroretinal cells especially of the mitochondrial apoptosis pathway.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

Experimental setup for RGC-5 cells incubated with 14-3-3 ab. The cells were seeded in 24 well plates and were let to rest for 24 h. Then antibody preincubation of the cells with different antibody concentrations was performed for 3 h. The control cells not incubated with the antibody were incubated with normal medium for the same amount of time. After 3 h the medium containing the antibodies or the control medium was replaced with medium containing one of the stress factors (glutamate, staurosporine or H2O2). Depending on the stress factor, the incubation time varied. After stressing the cells, viability tests with crystal violet staining was performed with all cells, and ROS level measurements were performed in the cells stressed with H2O2
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Fig1: Experimental setup for RGC-5 cells incubated with 14-3-3 ab. The cells were seeded in 24 well plates and were let to rest for 24 h. Then antibody preincubation of the cells with different antibody concentrations was performed for 3 h. The control cells not incubated with the antibody were incubated with normal medium for the same amount of time. After 3 h the medium containing the antibodies or the control medium was replaced with medium containing one of the stress factors (glutamate, staurosporine or H2O2). Depending on the stress factor, the incubation time varied. After stressing the cells, viability tests with crystal violet staining was performed with all cells, and ROS level measurements were performed in the cells stressed with H2O2

Mentions: RGC-5 cells were seeded in 24 well plates. Depending on the stress factor and therefore the overall incubation time, the cells were either seeded with 45000 cells per well (for the experiments with H2O2 and staurosporine) or 40000 cells per well (experiments with the stress factor glutamate). The cells were preincubated with different concentrations of chicken polyclonal anti 14-3-3 sigma antibodies (0.005, 0.1, 0.5, 1, 5 and 10 μg/ml) for 3 h. As an additional control group the cells were incubated with a non-retina specific antibody against myoglobin. To induce apoptosis the cells were stressed with staurosporine (1.5 μM for 5 h) or glutamate (20 mM for 24 h). Furthermore oxidative stress was induced by incubating the cells with 50 μM H2O2 for 1 h (n in each experimental group = 4). We used this amount of H2O2 because test showed that we were able to detect a rise in ROS without a loss of viability of the cells using this concentration. Subsequently cell viability tests and ROS measurements were performed. Figure 1 shows an overview of the experimental setup.Fig. 1


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)

Experimental setup for RGC-5 cells incubated with 14-3-3 ab. The cells were seeded in 24 well plates and were let to rest for 24 h. Then antibody preincubation of the cells with different antibody concentrations was performed for 3 h. The control cells not incubated with the antibody were incubated with normal medium for the same amount of time. After 3 h the medium containing the antibodies or the control medium was replaced with medium containing one of the stress factors (glutamate, staurosporine or H2O2). Depending on the stress factor, the incubation time varied. After stressing the cells, viability tests with crystal violet staining was performed with all cells, and ROS level measurements were performed in the cells stressed with H2O2
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig1: Experimental setup for RGC-5 cells incubated with 14-3-3 ab. The cells were seeded in 24 well plates and were let to rest for 24 h. Then antibody preincubation of the cells with different antibody concentrations was performed for 3 h. The control cells not incubated with the antibody were incubated with normal medium for the same amount of time. After 3 h the medium containing the antibodies or the control medium was replaced with medium containing one of the stress factors (glutamate, staurosporine or H2O2). Depending on the stress factor, the incubation time varied. After stressing the cells, viability tests with crystal violet staining was performed with all cells, and ROS level measurements were performed in the cells stressed with H2O2
Mentions: RGC-5 cells were seeded in 24 well plates. Depending on the stress factor and therefore the overall incubation time, the cells were either seeded with 45000 cells per well (for the experiments with H2O2 and staurosporine) or 40000 cells per well (experiments with the stress factor glutamate). The cells were preincubated with different concentrations of chicken polyclonal anti 14-3-3 sigma antibodies (0.005, 0.1, 0.5, 1, 5 and 10 μg/ml) for 3 h. As an additional control group the cells were incubated with a non-retina specific antibody against myoglobin. To induce apoptosis the cells were stressed with staurosporine (1.5 μM for 5 h) or glutamate (20 mM for 24 h). Furthermore oxidative stress was induced by incubating the cells with 50 μM H2O2 for 1 h (n in each experimental group = 4). We used this amount of H2O2 because test showed that we were able to detect a rise in ROS without a loss of viability of the cells using this concentration. Subsequently cell viability tests and ROS measurements were performed. Figure 1 shows an overview of the experimental setup.Fig. 1

Bottom Line: Previous studies showed up regulated, but also significantly down-regulated autoantibody levels.We found significant effects of serum antibodies on proteins of neuroretinal cells especially of the mitochondrial apoptosis pathway.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