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Renuka Chaphalkar, Dorota L Stankowska, Shaoqing He, Bindu Kodati, Raghu R Krishnamoorthy; Endothelin-1 Mediated Decrease in Expression of Mitochondrial Proteins ATP5H and COX17 in Retinal Ganglion Cells.. Invest. Ophthalmol. Vis. Sci. 2019;60(9):24.
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© ARVO (1962-2015); The Authors (2016-present)
Endothelin-1 (ET-1) treatment has been shown to promote apoptosis of retinal ganglion cells (RGCs), however, the precise mechanisms underlying these effects are still unknown. The purpose of the study was to assess the changes in gene expression at the level of the translatome, occurring during ET-1 mediated neurodegeneration of RGCs.
Primary RGCs isolated from post-natal day 5 rat pups were treated with ET-1 (100 nM) for 24 h in trophic factor-free medium. Polysomal RNA was isolated and libraries for RNA-Seq were prepared. Trimmed mean of M-values (TMM) was used to normalize the gene expression. Genes with expression changes more than 1.5 fold with p < 0.05 were considered differentially expressed. Two of the key mitochondrial genes, Cytochrome C Oxidase Copper Chaperone (COX17) and ATP Synthase, H+ Transporting, Mitochondrial Fo Complex (ATP5H) involved in oxidative phosphorylation were tested for their protein expression in primary culture of RGCs treated with ET-1. Following ET-1 treatment for 24 hours, primary RGCs were fixed with 4% PFA and immunocytochemical analysis was performed using specific antibodies to COX17 and ATP5H. To confirm these findings in vivo, retired breeder Brown Norway rats were intravitreally injected in one eye with either 2 nmole of ET-1 or vehicle. The animals were euthanized 24 hours post-injection and retina sections obtained were analysed for expression of ATP5H and COX17.
STRING network analysis revealed 156 differentially expressed genes, of which 23 genes were identified with known or predicted mitochondrial function. Immunostaining of primary RGCs showed an appreciable decline in expression of COX17, while ATP5H expression was modestly decreased. A decreasing trend (three out of four rats) in immunostaining for ATP5H as well as COX17 was found in retinas of rats intravitreally injected with ET-1 (n=4).
ET-1 treatment produced a decrease in expression of key components of mitochondrial electron transport chain. A compromise in bioenergetics could be one mechanism by which ET-1 promotes neurodegeneration of RGCs in glaucoma.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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