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Raymond A. Enke, Karl J. Wahlin, Donald J. Zack, Shannath L. Merbs; Characterizing the Role of DNA Methylation in Retinal Neurons. Invest. Ophthalmol. Vis. Sci. 2012;53(14):5125.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal cells undergo programmed cell death (PCD) during normal development as well as in response to injury and disease. We hypothesize that epigenetic mechanisms may regulate aspects of apoptosis of retinal neurons in the developing and diseased retina. As a prelude to studying the role of DNA methylation in the onset and progression of glaucoma and photoreceptor disease, we set out to determine the normal expression pattern and functional role of DNA methyltransferase (Dnmt) enzymes in the vertebrate retina and mouse models of glaucoma and retinal degeneration.
Eyes from human donor globes, adult wild type mice, and aging DBA/2J mice were fixed in 4% PFA and cryopreserved. Retinal cross sections were cut and used for immunohistochemical (IHC) analysis of Dnmt localization. Additionally, eyes from the rd1 mouse were studied by IHC analysis using an antibody against 5-methyl cytosine (5-meC) as well as markers of PCD. Primary cultured retinal ganglion cells (RGCs) were immunopanned from dissociated retina using an antibody against Thy1. Newborn wild type and transgenic mice with a targeted mutation in the Dnmt1 gene were studied. Cultured RGCs grown in the presence of Dnmt inhibitors as well as from Dnmt1 mutant mice were assayed for survival using a CellTiter-Glo viability assay.
IHC analysis of the human and mouse retina demonstrated that Dnmt1 and Dnmt3b are expressed in the nuclei of adult and aging ganglion cell layer (GCL) cells. Additionally, TUNEL positive neurons in the degenerating retina of adult rd1 mice demonstrated enhanced staining for 5-meC. Although cleaved caspase-3 (cCaspase-3) positive retinal neurons were present at similar periods of development there was little overlap with 5-meC positive cells suggesting that they are expressed at different stages of PCD. We are currently evaluating the viability of primary cultured RGCs with crippled Dnmt activity.
Vertebrate retinal neurons in the late stages of PCD demonstrated DNA hypermethylation by IHC, suggesting that epigenetic mechanisms might play a role in apoptosis of neurons during retinal development and degeneration. Likewise, nuclear localization of Dnmt1 and Dnmt3b in GCL cells from aging human and rodent retina indicates that DNA methylation may be involved in RGC homeostasis. Further investigation is needed to determine if these enzymes play a role in RGC survival.
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