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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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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
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Fig7: 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

Mentions: To further investigate the effect of 14-3-3 abs on the protein expression of RGC-5, proteomic analyses were performed. Using a pooled sample, we could identify 1204 proteins of which 225 were significantly differently regulated in cells incubated with 14-3-3 abs (>2 fold increased or < 2 fold decreased) (see Additional file 4: Table S1). The pathway analysis (performed with IPA) showed that many of the differently regulated proteins belong to apoptosis signalling pathways of the cells. We could indicate several changed proteins, such as BAX, BIRC6, PRFA2, S100A4, VDAC 1/2/3 and ERK1, which are involved in the regulation of the mitochondrial apoptosis pathways. BAX, PRFA1, VDAC 1/2/3 and S100A4 were significant down-regulated and BIRC6 and ERK1 were significant up-regulated in cells treated with 14-3-3 abs in comparison to untreated cells (Fig. 7a).Fig. 7


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)

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
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig7: 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
Mentions: To further investigate the effect of 14-3-3 abs on the protein expression of RGC-5, proteomic analyses were performed. Using a pooled sample, we could identify 1204 proteins of which 225 were significantly differently regulated in cells incubated with 14-3-3 abs (>2 fold increased or < 2 fold decreased) (see Additional file 4: Table S1). The pathway analysis (performed with IPA) showed that many of the differently regulated proteins belong to apoptosis signalling pathways of the cells. We could indicate several changed proteins, such as BAX, BIRC6, PRFA2, S100A4, VDAC 1/2/3 and ERK1, which are involved in the regulation of the mitochondrial apoptosis pathways. BAX, PRFA1, VDAC 1/2/3 and S100A4 were significant down-regulated and BIRC6 and ERK1 were significant up-regulated in cells treated with 14-3-3 abs in comparison to untreated cells (Fig. 7a).Fig. 7

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