Abstract
Purpose :
Endothelin-1 (ET-1) is a member of the endothelin family of vasoactive peptides (which includes ET-1, ET-2, and ET-3) that has found to be elevated in the aqueous humor and circulation of glaucoma patients. ET-1 acts through G protein coupled receptors, namely, ETA and ETB receptors to produce neurodegenerative effects in animal models of glaucoma, however, the underlying mechanisms are not completely understood. The purpose of this study was to determine mitochondrial mechanisms by which ET-1 produces neurodegenerative effects in cultured retinal ganglion cells (RGCs).
Methods :
Primary RGCs were isolated from post-natal day 5 rat pups and treated for 1 hour with either 100 nM ET-1 or 100 nM ET-3 and reactive oxygen species were detected by treatment with the Cell Rox reagent. Another batch of RGCs was treated with ET-1 for 4 hours following which changes in mitochondrial potential was assessed by treatment with the JC-1 dye and mitochondrial bioenergetics was analyzed using the Agilent Seahorse XFe96 analyzer. IOP was elevated using the Morrison method in one eye of retired breeder Brown Norway rats and retina sections obtained were used for immunohistochemical analysis of TOM20 (an outer mitochondrial membrane protein) and LAMP-1 (a marker of lysosomes).
Results :
Primary RGCs treated with ET-1 for 1 hour showed a modest increase in reactive oxygen species, while ET-3 treatment produced an appreciable increase in reactive oxygen species. ET-1 treatment for 4 hours produced a significant (p<0.01) decrease in mitochondrial membrane potential. In the SeaHorse analysis, ET-1 treatment for 4 hours produced a significant decrease (p<0.05) in mitochondrial basal respiration, maximal respiration, ATP production and spare respiratory capacity. Following IOP elevation for 2 weeks in Brown Norway rats, an appreciable decrease in co-staining of TOM-20 and LAMP-1 was found in the retinal ganglion cells, suggestive of a decrease in transit of mitochondria to the lysosomes .
Conclusions :
Endothelins could promote glaucomatous neurodegeneration of RGCs by their damaging effects on the mitochondria and possibly leading to a decline in mitophagy.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.