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Abdallah Hamieh, Nawel Hadjout, Géraldine Millet-puel, Thierry D. Leveillard, Emeline F Nandrot; Implication of various oxidative stress pathways in the aging phenotype of Prpf31-mutant mice. Invest. Ophthalmol. Vis. Sci. 2018;59(9):6074.
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© ARVO (1962-2015); The Authors (2016-present)
Mutations in the ubiquitous Pre-mRNA Processing Factors 3, 8, and 31 (PRPF3, 8 and 31) constitute the second most prominent cause of autosomic dominant retinitis pigmentosa. Human ARPE-19 cells down-regulated for PRPF31 and Prpf31-mutant primary RPE cells display a 40% decrease of RPE phagocytosis, suggesting similar pathological processes occur in both species. With age, Prpf31-mutant RPE cells display abnormal apical and basement membrane structures and accumulation of vacuoles. We thus investigated RPE-related stress pathways and lipids accumulation at different ages in Prpf31-mutant mice.
Studies were conducted on 3 to 18-month-old animals in order to dissect the full series of molecular events. Gene and protein expression levels for the mitochondrial respiratory chain, detoxifying enzymes and the endoplasmic reticulum (ER) unfolded protein response (UPR) pathways were analyzed by qPCR and immunoblots. RPE lipids accumulation with age was quantified on histological sections by Oil-Red-O and Bodipy stainings.
In the mitochondrial respiratory chain, complexes IV and V seemed affected in PRPF31-mutant RPE/Choroid samples with increased CoxIV gene expression at ages 6-18 months, while CoxIV protein and ATP synthase gene/protein expressions were decreased between 3-12 and 6-18 months, respectively. Surprisingly, expression of the mitochondrial superoxide dismutase SOD2 was decreased from 6 months of age in contrast to other non-mitochondrial detoxifying enzymes such as SOD1 and catalase. Several UPR pathways were activated leading to increases in Bip, Atf4 and Atf6 at 12 months, suggesting an increased ER stress at this age. Mitochondrial, ER and detoxification pathway perturbations were also observed in retinal extracts, showing that oxidative processes are not limited to the RPE. Prpf31-mutant RPE cells gradually accumulate on average twice as much lipid droplets in number and in size than wildtype littermates as soon as 3 months of age.
Taken together our results suggest that oxidative imbalance and associated stress take place at different levels in Prpf31-mutant RPE cells. Functional evaluation of mitochondrial activity using the Seahorse technology is under way to confirm these results. In addition, nature and amounts of lipids is currently assessed by thin-layer chromatography analysis.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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