The Role of Histone Deacetylases in Age-related Macular Degeneration

By 2020, nearly 200 million individuals in the world will be affected by age-related macular degeneration (AMD) [1]. This blinding disease is hallmarked by the death of retinal pigment epithelium (RPE), choroidal neovascularization, and loss of the overlying photoreceptors in the central retina. Several recent spectacular scientific advances have revealed an array of molecular mechanisms that result in RPE atrophy, yet there is still no effective therapy to halt dry AMD progression. New insights into epigenetic derangements and chromatin remodeling have garnered great interest as a powerful approach to alter genome-wide transcription and reprogram cell signaling pathways. Chromatin regulation of gene expression occurs through multiple mechanisms that affect higher-order DNA structure including direct conjugation of methyl groups to DNA and post-translational modification of histones including methylation, acetylation, ubiquitination and phosphorylation. Histone deacetylases (HDACs) serve as a unique control of the chromatin remodeling process through regulation of post-translational histone acetylation to activate or silence gene expression. There are 18 human HDACs classified depending on homology to their yeast counterparts; classes I, II, and IV are zinc-dependent enzymes, while class III are NAD+ dependent. Numerous chemical inhibitors of specific HDACs (HDACi) have now been engineered and are currently being evaluated in a multitude of clinical studies (138 open studies on clinicaltrials.gov website accessed 2/15/2016) including a trial of valproic acid, a Class I/II/IV HDACi, for the treatment of retinitis pigmentosa. Interestingly, HDACi is cytoprotective in multiple neurodegeneration models, but, paradoxically, induces apoptosis in numerous cancer cell types as well as microglia. In the retina, HDACi is reported to improve inner retinal function after ischemic insult and suppresses choroidal neovascularization in mouse models. However, the biologic effects of pharmacologic manipulation of histone acetylation in the RPE remain unclear. My preliminary data strongly suggest that the expression and function of specific HDAC classes may be altered in the RPE in dry AMD. Importantly, new data in this revised R01 submission indicate that recapitulating HDAC dysfunction observed in AMD induced both inflammatory and cell death responses in the RPE in experimental models in vivo and in vitro. The proposed research approach will provide critical insights on the biologic intersection of epigenetic acetylation pathways with inflammation and apoptosis of the RPE to identify key signaling mechanisms in the pathogenesis and therapeutic targeting of dry AMD. The central hypotheses of this proposal are that selective HDAC dysfunction and altered expression is present in AMD, pathogenetic chemokine induction in AMD is secondary to histone hyperacetylation, and disruption of specific HDAC enzymes in animal and cell culture models will result in RPE degeneration with cardinal features of dry AMD. Preliminary data demonstrate significant down-regulation of Class I/II/IV HDAC expression in the RPE of eyes with dry AMD. I also observed RPE cytotoxicity and patterned inflammatory gene expression after inhibition of Class I/II/IV HDACs in vitro and in vivo. This mounting evidence serves as a strong foundation to rigorously pursue further investigation of the role of HDAC and acetylation biology in AMD pathogenesis and RPE degeneration. In order to study these important biological questions, the following independent but synergistic Specific Aims are proposed: Aim 1. Dissect the role of HDACs in models of RPE gene expression, function, and histone acetylation. The degenerative and pro-apoptotic effects of selective or combinatorial inhibition of HDACs will be examined utilizing small molecule and gene targeted approaches. Detailed gene expression and functional analyses will be conducted in novel animal and cell-culture models to gain critical insight on the role of histone acetylation in inflammation and cell death in the RPE. Advanced techniques in quantitative proteomics including stable isotope labeling will be utilized to measure specific acetyl-lysine residues that are changed in these models. These data will be subjected to bioinformatics pipelines to chart histone acetylation signatures in RPE degeneration which will serve as a novel epigenetic roadmap for AMD. Aim 2. Analyze the RPE transcriptome and gene specific acetyl-histone marks in dry AMD. The expression and localization of acetyl-lysine regulatory components including HDACs, HATs and acetylated residues in AMD and normal age-matched control eyes will be examined using established techniques in massively parallel RNA sequencing, chromatin immuno-precipitation, DNA sequencing, immunofluorescence, in situ hybridization, mass-spectrometry and Western blotting. These Specific Aims will provide critical and novel data on the significance of HDAC function and histone acetylation in the pathogenesis of dry AMD and RPE degeneration. Given the expanding use of HDACi as a potential therapeutic modality, the clinical and scientific community will greatly benefit from these studies, as they will offer valuable insight into cell and HDAC class dependent effects of this potent epigenetic pathway.