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K. Torres, M. Assadian, S. L. Merbs; DNA Methylation Changes Associated With Ganglion Cell Apoptosis in a Rat Model of Glaucoma. Invest. Ophthalmol. Vis. Sci. 2010;51(13):1689.
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© ARVO (1962-2015); The Authors (2016-present)
Previously published analyses of animal glaucoma models have demonstrated that the induction of retinal ganglion cell injury and subsequent apoptosis leads to complex changes in gene expression. In recent years, there has been growing interest in the role of epigenetic mechanisms that regulate gene expression because they appear to provide an important dimension to transcriptional regulation. To correlate DNA methylation changes with gene expression changes in an experimental model of glaucoma, bisulfite sequencing was used to determine the DNA methylation status of the region around the transcription start site of genes known to change expression with experimentally induced elevation of IOP.
Five genes whose expression was increased by elevated IOP as determined by both array analysis and QPCR (Ednrb, Edn2, Tnfrsf1a, Timp1, and Anxa3) and five genes whose expression was decreased by elevated IOP (Sncg, Nefl, Nrn1, Cplx1, and Sorl1) were bisulfite-sequenced in rat retina, kidney and testes. Then 16 rats underwent laser treatment in one eye, and half of these underwent as second laser treatment one week later. A second set of 16 rats had corresponding sham treatments. All rats were sacrificed 1 week after their final laser treatment. The eye cups from the experimental and sham treated eyes were cryoprotected, 10-um sections were thaw-mounted onto PEN foil slides, and laser capture microdissection was used to isolate cells from the ganglion cell layer (GCL). Isolated genomic DNA was bisulfite-modified and the above genes sequenced.
Some of the genes (ex. Sorl1) had DNA methylation patterns that were unique in each of the tissues/cells tested (kidney/testes, retina, and cells from the GCL). Other genes had similar or identical DNA methylation patterns in all tissues (ex. all CpG sites around the TSS of Ednrb were methylated in all tissues/cells). The DNA methylation patterns of the genes in GCL cells from the laser-treated rats are being compared the DNA methylation patterns from the sham-treated rats.
In normal rats, we have demonstrated GCL-specific DNA methylation patterns of genes known to change expression levels in a rat model of glaucoma, and we are currently correlating these patterns in the same cell population from rats with elevated IOP. Characterizing and understanding the role that DNA methylation might play in the apoptosis of ganglion cells in glaucoma will provide necessary fundamental knowledge and set the stage for future mechanistic studies exploring the role of epigenetic mechanisms in glaucoma.
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