Abstract
Purpose :
The objective of this study is to determine the role of DNA methylation in RGC death during glaucoma pathology. We administered a selective DNA methylation inhibitor (Decitabine) or delta-opioid receptor agonist (SNC-121) to regulate the DNA methylation and methyltransferases activities in a rat glaucoma model and measured changes in biochemical and functional outcomes.
Methods :
We used male and female Brown Norway rats (3-4 months old) to elevate intraocular pressure (IOP) by 2.0 M hypertonic saline injections in limbal veins. The changes in global DNA methylation were measured using the ELISA-based 5-methylcytosine (5mC) DNA Quantification Kit (# P-1030, EpiGenetek, Farmingdale, NY) according to manufacturer instructions. The activity of DNA methyltransferases (DNMTs) were measured by Enzyme Assay Kit from EpiGentek (Cat # P-3139, Farmingdale, NY), and changes in the expression pattern of DNMT-1, -3a, and -3b were determined by RT-PCR, WB, and immunohistochemistry.
Results :
A significant RGC neuroprotection was offered by Decitabine and SNC-121 treatment at day 42, post glaucomatous injury as measured by Pattern ERGs. We also find an increase in genome-wide DNA methylation in the retina samples of ocular hypertensive animals at day 42, post glaucomatous injury, which was significantly inhibited with SNC-121 treatment. Additionally, we found an overall increase in the DNA methyltransferases (DNMTs) activity, mRNA, and protein expression of DNMT1, DNMT3a, and DNMT3b in the retina sections of ocular hypertensive animals at day 7 and 42, post glaucomatous injury.
Conclusions :
Our novel data demonstrate that mRNA and protein expression of DNA methyltransferases (DNMTs) and global DNA methylation were increased in the retina of ocular hypertensive animals. Based on these findings it appears that Inhibition of DNA methylation may provide RGC neuroprotection in glaucoma.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.