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Sushil Kumar Dubey, Dingyuan Lou, Jacob Roney, Kyung Jung, Jennifer Brown, Kabhilan Mohan, Mark Ellsworth Kleinman; Epigenetic regulation of autophagy by histone deacetylases in retinal pigment epithelium. Invest. Ophthalmol. Vis. Sci. 2017;58(8):5253. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Autophagy in retinal pigment epithelium (RPE) plays a critical role in regulating retinal homeostasis through the degradation of malfunctioning proteins and organelles. Impaired autophagy in the RPE contribute to the progression of age-related macular degeneration (AMD). However, the underlying mechanism causing impaired autophagy in RPE remains unclear. In this study, we show the altered expression signatures of specific histone deacetylases (HDACs) and the resulting autophagy impairment in the RPE due to the altered HDAC function.
Gene expression analysis of HDAC isoforms was performed on human macular RPE/choroid samples from dry AMD and normal age-matched eyes (n= 6-8 per group) by qPCR. ARPE19 (ATCC) and low passage (<P4) primary hRPE cell isolates (three independent biological replicates) were treated with class I (MS-275, 10µM; MGCD0103, 10µM) and class III (Sirtinol, 25µM; EX527, 20µM) HDAC inhibitors (HDACi). Quantitative PCR of autophagy related genes (ATG7, LAMP1, LC3a, LC3b and p62) was performed (n=3, 24 hrs), and HDACi effect on basal autophagic flux was further evaluated via Western blot for LC3-II/LC3-I, and immunofluorescence assays for GFP-LC3 puncta and P62 levels (n=3, 5/12/24 hrs). Statistical significance was determined with Student's unpaired two-tailed t-test to compare the means of the two groups (p < 0.05, GraphPad Software).
Gene expression data demonstrated significant loss of HDAC1/2/6/7/11 isoforms (*P<0.05) in human macula RPE/choroid tissues in advanced dry AMD. Inhibiting HDAC function in vitro with class I and class III HDACi showed upregulation of autophagy genes (ATG7, LAMP1, LC3a, LC3b and p62 transcripts with class I and LC3a, LC3b and p62 with class III HDACi). Evaluation of autophagic flux showed induction of autophagy after treatment with either class I or class III HDACi.
These observations clearly suggest that HDACs play a critical role in autophagy regulation and that altered expression of these regulators disrupt the normal autophagy process in the RPE. These autophagy disruptions may lead to retinal phenotypes associated with AMD.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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