Abstract
Purpose:
Several studies show an autoimmune component for the disease glaucoma, showing not only up-regulated autoantibodies, but also down-regulated antibodies. One of these antibodies (ab) is targeted against gamma- synuclein and is down-regulated in glaucoma patients. We were able to demonstrate a protective effect of ɤ- synuclein antibodies on RGC5 cells, after stressing these with glutamate or H2O2. The next step was to look at the effect of the antibody on retinal ganglion cells (RGC) in organ culture.
Methods:
The retina along with the RPE was isolated from a porcine eye derived from 3-6 month old pigs. The RPE was placed on nitrocellulose and the retina was placed on the RPE, RGC facing upwards. Using Transwell inserts, the retinae were either incubated with medium containing 10% FBS, or medium containing 10% FBS and additionally 0.5µg/ml ɤ- synuclein ab (n= 4 in each group). After 24 h in culture, the retinae were removed from the insert and fixed in paraffin. The slices were stained for BRN3a, with a tunel assay and DAPI. Retinal ganglion Cell counts were performed for 6 slices per retina. Also proteomic measurements of the retina incubated with the antibody as well as the control retina were performed.
Results:
We were able to detect an increase in RGC/mm of 41 % in the retinal organ culture incubated with 0.5µg/ml ɤ- synuclein ab (p < 0.01; Standard deviation 2.3). Tunel staining was equal in both groups. The proteomics analysis found 406 proteins in total and showed a significant increase of glutamine synthetase expression of 4.5 fold in the retina incubated with ɤ- synuclein ab as well as significantly increased CDC 42 (9.5 fold) and decreased reticulon 4 (-2.8 fold), both involved in endoplasmic reticulum stress.
Conclusions:
We were able to show that ɤ- synuclein ab has a protective effect on retinal ganglion cell in retina organ culture. The increased glutamine synthetase expression shows that Mueller cells are also affected by the antibodies and are involved in the protective mechanisms. Additionally, endoplasmic reticulum stress proteins such as CDC 42 and reticulon 4 seem to play a role in the protective mechanism of the antibody.