Abstract
Purpose :
Endothelin-1 (ET-1), a potent vasoactive peptide has been shown to produce degeneration of the optic nerve and retinal ganglion cell loss in animal models of glaucoma. However, the precise mechanisms underlying these neurodegenerative effects are still not completely understood. The purpose of the study was to assess mitochondrial bioenergetics and also determine if a decrease in mitophagy accompanied ET-1 mediated neurodegeneration of RGCs.
Methods :
Primary RGCs isolated from post-natal day 4–6 rat pups were treated with either vehicle or ET-1 (100nM) for 24h in trophic factor free medium. A Seahorse Mito Stress kit was used to determine the effects of ET-1 on mitochondrial respiration in primary RGCs using the Agilent Seahorse XFe96 analyzer. In another set of experiments, GFP-LC3 mice were intravitreally injected in one eye with 2μl of 500μM ET-1 while the contralateral eye was injected with the vehicle. Mice were euthanized 24hours after injection and retinal sections obtained were analyzed for expression of LC3B (a marker of autophagosomes) and TOM20 (mitochondrial marker).
Results :
ET-1 treatment of primary RGCs for 24 h produced a significant change in several parameters of mitochondrial bioenergetics. RGCs treated with ET-1 showed a decrease in basal respiration by 30% from 31.99 ± 2.38 pmoles/min to 22.29 ± 3.5 pmoles/min (p=0.03), maximal respiration by 28% from 48.65 ± 3.23 pmoles/min to 34.79 ± 3.5 pmoles/min (p=0.01), ATP-linked respiration by 45% from 23.25 ± 1.6 pmoles/min to 12.84 ± 0.72 pmoles/min (p<0.0001) and an appreciable decrease of 25% in the spare respiratory capacity from 16.66 ± 3.8 pmoles/min to 12.51 ± 2.3 pmoles/min (not statistically significant), compared to vehicle treated RGCs. In the GFP-LC3 mice, a decreased co-localization of TOM20 and LC3B from 0.52 ± 0.02 to 0.45 ± 0.01 (p=0.01, n=3) was found in RGC layer following intravitreal administration of ET-1, compared to the vehicle injected eye, indicative of decreased autophagosome formation.
Conclusions :
Treatment of primary RGCs with ET-1 produced a decline in mitochondrial bioenergetics which may be an indication of mitochondrial damage. ET-1 mediated decrease in autophagosome formation could compromise mitophagy in RGCs and result in accumulation of defective mitochondria which could be one potential mechanism contributing to neurodegeneration.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.