title: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Experimental Ophthalmology, Department of Ophthalmology, University Medical center of the Johannes Gutenberg University, Langenbeckstraße 1, 55131, Mainz, Germany. kbell@eye-research.org.
authors.forename: Katharina
authors.initials: K
authors.lastname: Bell
authors.affiliation: Experimental Ophthalmology, Department of Ophthalmology, University Medical center of the Johannes Gutenberg University, Langenbeckstraße 1, 55131, Mainz, Germany. cwilding@eye-research.org.
authors.forename: Corina
authors.initials: C
authors.lastname: Wilding
authors.affiliation: Experimental Ophthalmology, Department of Ophthalmology, University Medical center of the Johannes Gutenberg University, Langenbeckstraße 1, 55131, Mainz, Germany. sfunke@eye-research.org.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Funke
authors.affiliation: Experimental Ophthalmology, Department of Ophthalmology, University Medical center of the Johannes Gutenberg University, Langenbeckstraße 1, 55131, Mainz, Germany. norbert.pfeiffer@unimedizin-mainz.de.
authors.forename: Norbert
authors.initials: N
authors.lastname: Pfeiffer
authors.affiliation: Experimental Ophthalmology, Department of Ophthalmology, University Medical center of the Johannes Gutenberg University, Langenbeckstraße 1, 55131, Mainz, Germany. grus@eye-research.org.
authors.forename: Franz H
authors.initials: FH
authors.lastname: Grus
mesh_terms: D048948:14-3-3 Proteins / Q000276:immunology*; D017209:Apoptosis / Q000502:physiology*; D001323:Autoantibodies / Q000502:physiology*; D002460:Cell Line; D002470:Cell Survival; D004591:Electrophoresis, Polyacrylamide Gel; D019084:Fluorescent Antibody Technique, Indirect; D005902:Glaucoma, Open-Angle / Q000097:blood*; D018698:Glutamic Acid / Q000494:pharmacology; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008928:Mitochondria / Q000378:metabolism*; D018384:Oxidative Stress; D047428:Protein Kinase Inhibitors / Q000276:immunology*; D040901:Proteomics; D017382:Reactive Oxygen Species / Q000378:metabolism; D012165:Retinal Ganglion Cells / Q000166:cytology* / Q000187:drug effects; D015398:Signal Transduction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D019311:Staurosporine / Q000494:pharmacology
chemical_list: D048948:14-3-3 Proteins; D001323:Autoantibodies; D047428:Protein Kinase Inhibitors; D017382:Reactive Oxygen Species; D018698:Glutamic Acid; D006861:Hydrogen Peroxide; D019311:Staurosporine