Abstract
Purpose: :
The pathogenesis of glaucoma still is unknown. Next to elevated glutamate levels, recent studies show the participation of immunological components. Previous studies demonstrate significant changes in the pattern of autoantibodies against ocular antigens in the serum of glaucoma patients in comparison to healthy controls. γ-synuclein antibody (ab) is found in a lower concentration in glaucoma patients. Our aim was to analyse the effect of γ-synuclein ab on rgc’s, those cells affected by glaucoma and which pathways are triggered in RGC5, when they are incubated with γ-synuclein ab.
Methods: :
The retinal ganglion cell line RGC5 was cultured in 24 well plates. The cells were preincubated with different IgG γ-Synuclein ab (Santa Cruz) concentrations (0.005, 0.05, 0.1, 0.5,1,5 μg/ml) for 3h and stressed with 20mM glutamate (24h) to induce apoptosis. Controls were incubated without ab. Cell viability was measured with crystal violet. We also performed proteomic analyses of cells incubated with γ-synuclein ab. After lysis of the experimental cells the proteins were separated in a denaturing gel electrophoresis and the proteins were subsequently digested. Analysis of peptides was performed by use of a capillary LC-ESI-MS system. The obtained mass spectra were identified and quantified using MaxQuant and pathway analyses were performed with Ingenuity Pathway Analysis (IPA).
Results: :
A significant increase of viability of cells stressed with glutamate and preincubated with different antibody levels (0.1 and 1 µg/ml) was detected (15%, p<0.05). Overall we could identify 1110 proteins and 201 significant protein changes in RGC5, which were incubated with γ-synuclein ab more than 2 fold increased or decreased. Based on IPA we were able to indicate significant changes in mitochondrial apoptosis, where we could find 6 significant changed proteins in RGC5 after γ-Synuclein ab treatment such as BAX (-2,337), BIRC6 (+2.931), S100A4 (-4.193), and VDAC1 (-2.345), VDAC2 (-2.64), VDAC3 (-4.483).
Conclusions: :
We detected significant neuroprotective effects especially of the lower γ-synuclein ab concentrations on the cells stressed with glutamate, e.g. raised viability. Mitochondrial apoptotic pathways might play a key role in this effect. For example glutamate-induced apoptotic cell death is associated with the up regulation of BAX, which leads to cytochrome c release and apoptosis. The down regulation of BAX in RGC5 incubated with γ-synuclein ab could lead to a protection against glutamate induced apoptosis.
Keywords: cell survival • ganglion cells • autoimmune disease