Neuroprotective effects of antibodies on retinal ganglion cells in an adolescent retina organ culture.

Glaucoma, a neurodegenerative disease, is characterized by a progressive loss of retinal ganglion cells (rgc). Up- and down-regulated autoantibody immunoreactivities in glaucoma patients have been demonstrated. Previous studies showed protective effects of down-regulated antibodies [gamma (γ)-synuclein and glial fibrillary acidic protein [GFAP]) on neuroretinal cells. The aim of this study was to test these protective antibody effects on rgc in an organ culture model and to get a better understanding of cell-cell interactions of the retina in the context of the protective effect. We used an adolescent retinal organ culture (pig) with an incubation time of up to 4 days. Retinal explants were incubated with different antibodies for 24 h (anti-GFAP, anti-γ-synuclein and anti-myoglobin antibody as a control). Brn3a and TUNEL staining were performed. We also conducted glutamine synthetase staining and quantification of the retinal explants. Mass spectrometry analyses were performed as well as protein analyses via microarray. We detected a continuous decrease of rgc/mm in the retinal explants throughout the 4 days of incubation with increased TUNEL rgc staining. Immunohistochemical analyses showed a protective effect of anti-γ-synuclein (increased rgc/mm of 41%) and anti-GFAP antibodies (increased rgc/mm of 37%). Mass spectrometric, microarray and immunohistochemical analyses demonstrated Müller cell involvement and decreased endoplasmic reticulum stress response in the antibody-treated retinae. We could detect that the tested antibodies have a protective effect on rgc which seems to be the result of reduced stress levels in the retina as well as a shift of glutamine synthetase localization in the endfeet of the Müller cells towards the inner retinal layer. Loss of retinal ganglion cells (rgc) in glaucoma leads to blindness. Several antibodies are down-regulated in glaucoma patients. Our aim was to test if these antibodies have a protective effect of rgc in a retinal organ culture. This could be shown with an increase of rgc numbers. This effect results through reduced stress levels and the shift of glutamine synthetase localization.

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

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.

γ-Synuclein antibodies have neuroprotective potential on neuroretinal cells via proteins of the mitochondrial apoptosis pathway.

The family of synuclein proteins (α, ß and γ) are related to neurodegenerative disease e.g. Parkinson disease and Morbus Alzheimer. Additionally, a connection between γ-synuclein and glaucoma, a neurodegenerative disease characterized by a progressive loss of retinal ganglion cells, which finally leads to blindness, exists. The reason for the development of glaucoma is still unknown. Recent studies evaluating the participation of immunological components, demonstrate complex changed antibody reactivities in glaucoma patients in comparison to healthy people, showing not only up-regulations (e.g. alpha-fodrin antibody) but also down-regulations (e.g. γ-synuclein antibody) of antibodies in glaucoma patients. Up-regulated antibodies could be auto-aggressive, but the role of down-regulated antibodies is still unclear. Previous studies show a significant influence of the serum and the antibodies of glaucoma patients on protein expression profiles of neuroretinal cells. The aim of this study was to investigate the effect of γ-synuclein antibody on the viability and reactive oxygen species levels of a neuroretinal cell line (RGC-5) as well as their interaction with cellular proteins. We found a protective effect of γ-synuclein antibody resulting in an increased viability (up to 15%) and decreased reactive oxygen species levels (up to -12%) of glutamate and oxidative stressed RGC-5. These can be traced back to anti-apoptotic altered protein expressions in the mitochondrial apoptosis pathway indicated by mass spectrometry and validated by microarray analysis such as active caspase 3, bcl-2 associated-x-protein, S100A4, voltage-dependent anion channel, extracellular-signal-regulated-kinase (down-regulated) and baculoviral IAP repeat-containing protein 6, phosphorylated extracellular-signal-regulated-kinase (up-regulated). These changed protein expression are triggered by the γ-synuclein antibody internalization of RGC-5 we could see in immunohistochemical stainings. These findings let us assume a novel physiological function of γ-synuclein antibodies and give insights in the role of autoantibodies in glaucoma. We hypothesize that the down-regulation of autoantibodies found in glaucoma patients lead to a loss of protective autoimmunity.

Does autoimmunity play a part in the pathogenesis of glaucoma?

Glaucoma is a chronic neurodegenerative disease and one of the leading causes of blindness. Several risk factors have been described, e.g. an elevated intraocular pressure (IOP), oxidative stress or mitochondrial dysfunction. Additionally, alterations in serum antibody profiles of glaucoma patients, upregulation (e.g. anti-HSP60, anti-MBP) and downregulation (e.g. anti-14-3-3), have been described, but it still remains elusive if the autoantibodies seen in glaucoma are an epiphenomenon or causative. However, it is known that elicited autoimmunity causes retinal ganglion cell loss resulting in glaucomatous-like damage and according to the autoaggressive nature of some autoantibodies we found antibody deposits in human glaucomatous retinae in a pro-inflammatory environment. Furthermore, glaucomatous serum has the potential to influence neuroretinal cell regulatory processes. Importantly, we demonstrate that some autoantibodies hold neuroprotective potential for neuroretinal cells. The protective nature of autoantibodies and the molecular mechanisms underlying the very sensitive equilibrium between autoaggression and protection remain subject of future examinations and offer promising target sites for new therapeutic approaches. Additionally, the changes in antibody profiles could be used as highly sensitive and specific marker for diagnostics purposes. Early diagnosis and intervention in risk patients would offer the chance of early treatment and to slow down the progression of glaucoma and delay the resulting blindness.

Autoimmune biomarkers in glaucoma patients.

There is growing evidence showing an autoimmune involvement in the pathogenesis of glaucoma, and that alterations in natural occurring autoantibody levels play a key role. The upregulation of autoantibodies can be associated with fatal conditions, but several studies demonstrate that natural autoantibodies entail also protective characteristics and influence the protein expression of neuroretinal cells. A disbalance of natural occurring autoantibodies may shift the physiological equilibrium of protective immunity leading to a predisposition for developing glaucoma. This article highlights recent advances in understanding of autoimmune mechanisms in the pathogenesis of glaucoma.