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Gennadiy P. Moiseyev, MD Nawajes A. Mandal, Michael H. Elliott, Anne Kasus-Jacobi, Lixin Zheng, Julie-Thu A. Tran, Hui Chen, Robert A. Floyd, Jian-xing Ma, Robert E. Anderson; PBN Protection Of Retina From Light-damage Is Through Inhibition Of Rpe65 Isomerohydrolase Activity. Invest. Ophthalmol. Vis. Sci. 2011;52(14):3641.
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PBN (alpha-phenyl-tert-butyl nitrone), a free radical spin trap, has been shown previously to protect rat retinas against light damage but the mechanism of protection is not known. The purpose of this study was to determine the mechanism underlying the PBN-mediated retinal protection by studying gene expression and the activity of the visual cycle enzymes RPE65 and retinol dehydrogenase (RDH).
For gene expression analysis, Sprague-Dawley rats raised in dim (5lux) cyclic light from birth were injected intraperitoneally with PBN or saline 0.5h before exposure to white fluorescent light intensity at 2,700 lux for 6h. Retinas were analyzed by Illumina RatRef 12 bead arrays. Effect of PBN on RPE65 isomerohydrolase activity was assayed in vitro using rat RPE microsomes. Rhodopsin recovery after bleach was measured spectrophotometrically.
PBN administered 0.5h to 12h before light stress protected against light-damage, but no protection occurred when administered after exposure. We detected a total of 86 genes significantly altered in vehicle-injected rat retinas after light damage. PBN injection 0.5h prior to light damage blocked the expression of the majorly altered genes. PBN had no effect on the activity of retinal RDH. Interestingly, PBN potently inhibited the enzymatic activity of RPE65 (IC50=100 µM) but not lecithin:retinol acyltransferase activity. PBN significantly inhibited rhodopsin recovery in the dark following light exposure.
Our results demonstrate that PBN inhibition of gene expression may contribute to the protection by up-regulating endogenous neuroprotection pathways. However, the primary mechanism of PBN protection is likely due to the inhibition of RPE65, probably by trapping the retinoid radical intermediate. This provides new insights into the mechanism for RPE65 catalytic action and indicates therapeutic potential of PBN for Stargardt’s disease and age-related macular degeneration.
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