Endothelin-1 Mediated Decrease in Mitochondrial Bioenergetics Involved in Neurodegeneration of Retinal Ganglion Cells

Abstract

Purpose : Endothelin-1 (ET-1) treatment has been shown to produce retinal ganglion cells (RGCs) loss, axonal injury and disruption of nerve fiber layer, however, the precise mechanisms underlying these effects are still not completely understood. The purpose of the study was to assess mitochondrial mechanisms underlying ET-1 mediated neurodegeneration of RGCs.

Methods : Primary RGCs isolated from rat pups were treated with ET-1 (100 nM) for 24h and RNA-Seq analysis was carried out. Immunocytochemical analysis of two of the key mitochondrial genes (revealed by the RNA-Seq analysis), Cytochrome C oxidase copper chaperone (COX17) and ATP synthase, H+ transporting, mitochondrial Fo Complex (ATP5H) was carried out in primary culture of RGCs treated with ET-1 (24h). 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, euthanized 24h post-injection and retina sections were analysed for expression of ATP5H and COX17. An Agilent Seahorse real-time ATP rate assay was performed to provide quantification of mitochondrial ATP production by measuring the oxygen consumption rate in control and ET-1 treated (4h and 24h) groups to assess mitochondrial function in primary RGCs. Student's t-test was used for statistical analysis.

Results : STRING network analysis revealed 156 differentially expressed genes, of which 23 genes were identified with known or predicted mitochondrial function. ET-1 treatment for 24h produced a significant (n=5, p<0.05) decrease in immunostaining for COX17 as well as an appreciable decrease in immunostaining for ATP5H in primary RGCs. Following intravitreal injection of ET-1 in rats, a significant decrease in immunostaining for ATP5H specifically was observed in ganglion cell and inner plexiform layer (n=6, p<0.01) while for COX17 decreased immunostaining was found in the ganglion cell and nerve fiber layer (n=6, p<0.05). The real time-ATP rate assay in primary RGCs revealed a significant decrease in ATP production following ET-1 treatment for 4h and 24h (n=10, p<0.05) indicating the ability of ET-1 to compromise the oxidative phosphorylation in RGCs.

Conclusions : 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 is a 2020 ARVO Annual Meeting abstract.