Abstract
Purpose: :
The oxidative stress response of the RPE is thought to undergo age-related changes which may be associated with aging diseases of the posterior pole. We hypothesize that key genes for signaling proteins in this response are silenced by promoter methylation as a function of age. Each candidate gene selected was associated with cellular aging, and also possessed a CpG island near the promoter which could serve as a region for CpG methylation resulting in gene silencing.
Methods: :
C57BL/6J mice (male and female) at 6 weeks and 18 months of age were obtained from the Jackson Laboratory and the NIA, respectively. The RPE/choroid was dissected after which samples of DNA and RNA were prepared. RNA samples were quantified for gene expression using Taqman assays. The MethylCollector Kit was used to isolate methylated CpG fragments from DNA samples. Methylated DNA fractions were then labeled and used to probe an Affymetrix GeneChip Mouse Promoter 1.0R Array, or used as template for promoter PCR analysis to compare yield of methylated DNA between aged and young RPE/choroid samples. Protein expression was examined in paraffin sections from PFA fixed BALB/cBy eyes by immunohistochemistry.
Results: :
Out of a larger panel of genes which were studied, we found that levels of the p66/Shc1, 14-3-3 beta, and Pgc-1 transcripts were significantly reduced with age. Methylation analysis of these gene promoters is currently being performed.
Conclusions: :
Out of a larger panel of genes which were studied, we found that levels of the p66/Shc1, 14-3-3 beta, and Pgc-1 transcripts were significantly reduced with age. Methylation analysis of these gene promoters is currently being performed.
Keywords: aging • retinal pigment epithelium • gene/expression