Characterisation of novel therapies to mitigate inflammation in retinal degenerations
Creators: name: Saxena, Kartik
Publisher: name: The Australian National University
Year: 2015
Subjects: Inflammation
retinal degeneration
microRNA
therapeutics

description: Inflammation is established as a key factor in mediating the progression of a number of retinal degenerations, including both wet and dry age-related macular degeneration (AMD) [1-3]. MicroRNAs (miRNAs) are a class of endogenously occurring non-coding RNA (ncRNA) molecules that are gaining momentum as therapeutic targets for treating a number of human conditions [4-6] and have been identified to modulate inflammation [7]. The purpose of this study is to investigate the modulation of miRNAs in a model of retinal degeneration, the light damage model. MiRNA and their potential roles in mitigating retinal inflammation will also be investigated in animals treated with 670nm red light therapy. Albino rats raised in dim cyclic light conditions (5lux; 12hr on, 12hr off; controls) were exposed to bright continuous light (1000lux) for 24 hours and returned to dim light conditions for 0, 3 or 7 days. At each timepoint animals were culled and their eyes removed and processed either for histological analyses or RNA based analyses. For histology eyes were fixed in 4% paraformaldehyde, cryoprotected and sectioned to determine the photoreceptor cell death using TUNEL or perform immunohistochemistry experiments using Vimentin and IBA1 or in situ hybridisation for Ccl2 and miR-124-3p. RNA was extracted from dissected retinas, reverse transcribed and used for low density array and qPCR analysis to determine the expression changes of genes and miRNAs. Immortalised cell lines were also used for performing cell transfections and similar RNA based analyses as above. Intense light exposure for 24 hours led to differential expression of a number of miRNAs, 37 of which were significantly modulated by 2-fold or more. Of those, 19 may potentially regulate the inflammatory immune response observed in the model. MicroRNAs -125-3p, -155, -207, -347, -449a, -351, and -542-3p are all upregulated at 24 hours of exposure along with peak photoreceptor cell death. The miRNAs -542-3p and -351 reached maximum expression at 7 days after exposure, while -125-3p, -155, -207, -347, and -449 reached a peak expression at 3 days. MiR-124-3p expression showed significant downregulation post intense (1000lux) light induced damage to the rat retina. Its expression was localized predominantly to the Müller glia and showed co-localisation with expression of a target gene, Ccl2 (which is a potent chemoattractant molecule responsible for targeted monocyte recruitment in the retina). Luciferase assays in MIO-M1 and HeLa cells confirmed direct binding between miR-124-3p and the CCL2-3’UTR. Additionally, in vitro overexpression of miR-124-3p in Müller cells using miRNA mimics significantly inhibited CCL2 upregulation post stimulation with inflammatory cues. Post mortem human tissue showed a similar expression profile for miR-124-3p in the retina. Changes is miRNA expression were also seen in 670nm light treated retinas with or without bright light induced damage, along with the modulation of chemokine gene expression in the pre-treated retinas. Differential expression of miR-351 and miR-155 was confirmed using qPCR, both of which are predicted to target inflammation related genes. The data indicate that miRNAs are involved in modulating the inflammatory immune response elicited during retinal degeneration. Indeed we found a number of potential candidates, including miR-124-3p, which could prove to be novel therapeutic interventions in mitigating retinal inflammation and the consequent photoreceptor death. 670nm light therapy mitigated retinal inflammation including the modulation of specific miRNAs in the retina.