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Invest Ophthalmol Vis Sci. 2016 Apr;57(4):1553-62. doi: 10.1167/iovs.15-18551.

Interphotoreceptor Retinoid-Binding Protein Mitigates Cellular Oxidative Stress and Mitochondrial Dysfunction Induced by All-trans-Retinal.

Abstract

PURPOSE:

Point and null mutations in interphotoreceptor retinoid-binding protein (IRBP) cause retinal dystrophy in affected patients and IRBP-deficient mice with unknown mechanism. This study investigated whether IRBP protects cells from damages induced by all-trans-retinal (atRAL), which was increased in the Irbp(-/-) retina.

METHODS:

Wild-type and Irbp(-/-) mice retinal explants in buffer with or without purified IBRP were exposed to 800 lux light for different times and subjected to retinoid analysis by high-performance liquid chromatography. Purity of IRBP was determined by Coomassie Brilliant Blue staining and immunoblot analysis. Cellular damages induced by atRAL in the presence or absence of IRBP were evaluated in the mouse photoreceptor-derived 661W cells. Cell viability and death were measured by 3-(4,5-dimethyl-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) and TUNEL assays. Expression and modification levels of retinal proteins were determined by immunoblot analysis. Intracellular reactive oxygen species (ROS) and nitric oxide (NO) were detected with fluorogenic dyes and confocal microscopy. Mitochondrial membrane potential was analyzed by using JC-1 fluorescent probe and a flow cytometer.

RESULTS:

Content of atRAL in Irbp(-/-) retinal explants exposed to light for 40 minutes was significantly higher than that in wild-type retinas under the same light conditions. All-trans-retinal caused increase in cell death, tumor necrosis factor activation, and Adam17 upregulation in 661W cells. NADPH oxidase-1 (NOX1) upregulation, ROS generation, NO-mediated protein S-nitrosylation, and mitochondrial dysfunction were also observed in 661W cells treated with atRAL. These cytotoxic effects were significantly attenuated in the presence of IRBP.

CONCLUSIONS:

Interphotoreceptor retinoid-binding protein is required for preventing accumulation of retinal atRAL, which causes inflammation, oxidative stress, and mitochondrial dysfunction of the cells.

PMID:
27046120
PMCID:
PMC4824376
DOI:
10.1167/iovs.15-18551
[Indexed for MEDLINE]
Free PMC Article

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