Histone deacetylase expression and acetylation in the aging retinal pigment epithelium

Abstract

 

Purpose

 

Histone deacetylase (HDAC) mediated histone deacetylation is one of the major epigenetic mechanisms of regulation of gene expression. Reports on HDAC involvement in the maintenance of retinal pigment epithelium (RPE) health, led us to examine their expression and histone acetylation status in Wild-type (Wt) young/aged mouse RPE and primary human RPE isolates under oxidative stress to explore role of acetylation in aging.

 

Methods

 

RPE/choroid tissue was isolated from Wt C57BL/6J young/aged (4 weeks/18 months) mouse (n=3) eyes, processed for RNA isolation, and subjected to qPCR analysis for Class I HDAC (1/2/3/8) mRNA expression. Class I HDAC activity was measured via quantitative fluorometric HTS assay in lysates from Wt young/aged mouse RPE/choroid (n=3) and primary human RPE isolates treated with tert-butyl hydroperoxide (tBHP) or vehicle (n=3). Levels of histone H3 acetylation in Wt young/aged mouse RPE was analysed by immunofluorescence in mouse eye cryosections using anti-histone H3 antibody which detected acetyl-lysine residues 9 (K9), 14 (K14), 18 (K18), 23 (K23), and 27 (K27). Cell viability in primary human RPE treated with tBHP (50μM) or vehicle was assessed by Sytox assay.

 

Results

 

Real time PCR analysis revealed significant loss in class I HDAC (1/2/8) RNA expression in aged mouse RPE as compared to young (Figure 1), which was corroborated by reduced HDAC activity in aged mouse RPE. Tert-butyl hydroperoxide oxidative stress in primary human RPE isolates also resulted in reduced HDAC RNA expression and activity. Sytox assay showed reduced human RPE cell viability following tBHP induced oxidative stress. Increased acetylated histone H3 immunofluorescence was observed in RPE of aged mouse eye cryosections as compared to young (Figure 2).

 

Conclusions

 

Class I HDAC expression is down regulated in aging mouse RPE along with elevated acetylation of histone H3. Oxidative stress has similar effect on HDAC expression in primary human RPE isolates. Elevated histone acetylation could be due to reduced HDAC expression, which in turn may contribute to aging phenotype and age-related retinal diseases.  

 

Relative HDAC mRNA expression in young and aged mouse RPE

 

 

Acetylated histone H3 (indicated by white arrows) in young and aged mouse RPE