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

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.

Fig. 1

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

Fig. 2

Changes of proteins in RGC-5 cells incubated with POAG serum versus healthy serum. Proteomic measurements of the pooled samples of the cells incubated with POAG serum or healthy serum were performed. Each pooled sample contained protein from each of the 8 samples of either the POAG or the healthy group. After identification of the proteins a quantification of the proteins of the cells incubated with POAG serum in comparison to healthy serum was performed. The graph shows the changes in %. The most up-regulated protein in the cells incubated with POAG serum was 14-3-3 eta. But also proteins such as Calmodulin (CALM) or zink-finger protein (CNBP) were significantly differently regulated. Proteins up- or down-regulated more than 2 fold (100 %) were considered to be statistically significant

Fig. 3

Protective effects of 14-3-3 antibody on RGC-5 stressed with H₂O₂. RGC-5 cells were preincubated with different concentration of 14-3-3 antibodies for 3 h and additionally stressed with 50 μM H₂O₂ for 1 h. This amount of H₂O₂ was used, as we did not want to produce a loss of viability through H₂O₂ our aim was to just increase ROS in the cells. The graph shows the ROS levels in % in the cells (blue line) as well as the viability (red line) of the cells after pre-incubation with different 14-3-3 antibody concentrations and stress with 50 μM H₂O₂ for 1 h. N in each experimental group is 4. Significantly decreased ROS levels (−31 %) were measured in the cells incubated with 10 μg/ml 14-3-3 antibodies. ROS-production was measured using DCFH-DA and expressed as percent of the control cells, which were only treated with H₂O_2. Significantly increased viability (+22 %) was measured for the cells incubated with 10 μg/ml 14-3-3 antibodies. Viability was measured using crystal violet and expressed as percent of the control cells additionally incubated with H₂O₂ (* = p < 0.05; **p < 0.01)

Fig. 4

Viability of glutamate and staurosporine stressed RGC-5 upon 14-3-3 sigma antibody treatment. RGC-5 were preincubated with different 14-3-3 antibody concentrations and additionally stressed with 20 mM glutamate for 24 h, or 1.5 μM stauorsporine for 5 h. Cell viability was determined using crystal violet and expressed as percent of the control cells + the stress factor (glutamate or staurosporine) (* = p < 0.05; **p < 0.01). N in each experimental group is 4. a: Increased highly significant and significant cell viability of up to 7 % was demonstrated after the cells were preincubated with 0.5, 1 μg/ml 14-3-3 sigma antibodies and additionally stressed with staurosporine. b: Increased cell viability in a range of 0.05-5 μg/ml 14-3-3 antibodies were obtained after the cells were stressed with glutamate, whereby the result of 0.5 μg/ml is highly significant and of 1.5 μg/ml is significant. We were able to detect an increase of viability of up to 12 %

Fig. 5

Expression of 14-3-3 sigma in RGC-5 revealed by indirect immunfluorescence. RGC-5 cells were fixed, permeabilised, blocked and incubated with rabbit polyclonal anti 14-3-3 antibodies. Subsequently the cells were incubated with Goat polyclonal secondary antibody to rabbit IgG-H&L conjugated with FITC. The cells were visualized with a fluorescence microscope. The pictures show that 14-3-3 was expressed in all cells and it seems to be distributed in the cytoplasm

Fig. 6

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

Fig. 7

Changed mitochondrial apoptosis pathway in RGC-5 conditioned of treatment with 14-3-3 sigma antibodies. a: This graph shows several of the significantly differently regulated proteins in the cells incubated with 14-3-3 antibodies in comparison to control cells. The proteins listed here in some way all are involved in the mitochondrial apoptosis pathway. The changes of the proteins are shown in percent. b: This graph schematically visualizes the significantly changed proteins of the mitochondrial apoptosis pathway. Proteins highlighted in green were found to be significantly down-regulated) and proteins highlighted in red significantly up-regulated. The protein 14-3-3 interacts with p53, which is also shown in the graph. It is conceivable that the modulation of 14-3-3 through 14-3-3 sigma antibodies leads to a changed interaction of p53 and thereby to changed expression of p53 target genes such as PRAF2, S100A4 and BAX, which are significantly changed in the cells incubated with 14-3-3 abs. BAX plays a role in releasing cytochrome c from the mitochondrion. Cytochrome C interacts with caspase3, which triggers apoptosis of the cells. It also is conceivable that the interaction of 14-3-3 and STAT3 is altered, which comes to the up-regulation of BIRC6. Another protein which plays also a role in mitochondrial apoptosis is the anti-apoptotic ERK1, which is up-regulated in 14-3-3 sigma treated RGC-5 as well the down-regulation of VDAC 1/2/3