Functional analysis of the role of EZH2 in the progression of autosomal recessive retinitis pigmentosa

Abstract

The retina is a photosensitive neuroepithelium in the inner region of the eye and is an important part of the central nervous system (CNS). A physiological imbalance or changes in the number of functional photoreceptors can lead to important visual deficits. A disease that can lead to almost total vision loss is retinitis pigmentosa (RP). Among the causes of autosomal recessive RP include mutations in the PDE6B gene, culminating in the initial death of rod-type photoreceptors and subsequent degradation of cones. However, the processes underlying the primary rod death are not fully known, although studies point to mechanisms involved in inflammatory response, oxidative and metabolic stress, and epigenetic factors. Among the latter, the polycomb complex II (PRC2), which was recently associated with neurodegenerative processes. In animal models for autosomal recessive RP, PRC2 has already been studied in contexts of hypermethylation and hypoacetylation of DNA, with influence on degeneration in these animals when blockade of the catalytic subunit of PRC2, the zeste 2 homologous enhancer (EZH2). Furthermore, PRC2 has been associated with the regulation of two major intracellular signaling pathways, the mammalian rapamycin targeting pathway (mTOR) and nuclear factor kappa beta (NF-kB), both important in cell survival, inflammation, and metabolism. Given this information, the aim of this project is to analyze the effects of pharmacological blockade of EZH2 in an animal model of autosomal recessive RP, focusing on analyses of morphometric aspects, inflammatory and regulation of the aforementioned pathways. The pharmacological blockade will be performed with the use of the drug GSK343 by subretinal injection in neonate animals. To analyze the effects caused by the interventions, immunofluorescence techniques will be used for analysis of protein localization, accompanied by the analysis of protein levels and gene expression by western blot and rt-qPCR, respectively. In addition, we will use the multiplex technique will be used to analyze the inflammatory response in conjunction with specific cellular markers. Finally, we will analyze cell death by apoptosis, using the TUNEL assay. With this study we intend to further evaluate the role of the EZH2 protein in the progression of RP, and may open way for therapies focused on epigenetic processes.