Abstract
Purpose :
R Retinitis Pigmentosa (RP) is the most common form of inherited retinal degeneration. The PRPF family of splicing factors represents the second most frequent cause of autosomic dominant RP (adRP). A surprising feature of these ubiquitous factors is that outcomes of their alterations are restricted to the retina. With age, Prpf31-mutant RPE cells display abnormal apical and basement membrane structures and accumulation of vacuoles. Moreover PRPF31 mutations or downregulation lead to loss in RPE phagocytic activity. We previously showed that various stress pathways were activated in RPE of Prpf31-mutant mice as early as 3 months of age. We now investigated further oxidative damages, mitochondrial activity and lipid droplets accumulation.
Methods :
Studies were conducted on 3- to 18-month-old animals in order to dissect the full series of molecular events. RPE lipids accumulation with age was quantified on histological sections and their identification by thin-layer chromatography plates is ongoing. We identified activation of different stress pathways by qPCR, western blot, immunofluorescence and phagocytosis assays. Functional evaluation of mitochondrial activity was done using the Seahorse technology on freshly-isolated RPE/choroid fractions.
Results :
Prpf31-mutant RPE cells gradually accumulate in the cytoplasm on average twice as much lipid droplets in number and in size than wildtype cells. We detected increased signals in RPE cells and the retina of 4-HNE and 8-oxoguanine, and to a lesser extent nitrotyrosine, all markers of oxidative stress. Stress inducers such as tunicamycin and DTT affected RPE phagocytosis capabilities only when Prpf31 was downregulated. Analysis of the mitochondrial respiration and oxygen consumption show a marked decrease in ATP production and non-mitochondrial respiration.
Conclusions :
Taken together our results suggest that Prpf31-mutant RPE undergo oxidative and endoplasmic reticulum stresses. As well, addition of stress inducers worsens the phagocytic loss phenotype of RPE cells. Moreover, our results suggest that other energy production sources could also be affected in Prpf31-mutant RPE. Overall, oxidative imbalance and associated stress take place at different levels in Prpf31-mutant RPE cells that may directly affect the retinal homeostasis.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.