Abstract
Purpose::
The role of retinol dehydrogenases (RDHs) 11 and 12 is unknown but we hypothesize that they protect photoreceptors from toxic aldehydes produced during oxidative stress. Both enzymes can reduce aldehydes to alcohols, using NADPH as a coenzyme. Here we investigated their spatio-temporal expression in mouse retina during normal development. We also measured their catalytic activities and expression levels during light-induced oxidative stress.
Methods::
We performed in situ hybridization on adult wild type and RDH11 knockout retinas with an RDH11 specific probe, and immunogold staining of RDH12 on adult wild type retina. We then investigated by immunohistochemistry, quantitative RT-PCR (Q-PCR) and immunoblot the expression of RDH11 and RDH12 during post-natal development. We finally measured the catalytic activity of all RDHs in fractionated BALB/c mouse retinas during light-induced oxidative stress (3000 lux, 48h). We also quantified the expression of endogenous RDH enzymes (RDH8, RDH11, RDH12, RDH13, RDH14, and retSDR1) in these retinas.
Results::
We found the mRNA of rdh11 only in photoreceptor inner segments. Immunogold staining of RDH12 was found only in the outer nuclear layer, both inside and outside photoreceptor nuclei. Expression of RDH11 and 12 started in the retina between post-natal day 5 and 7. We found that the reducing activity towards all-trans-retinal in membrane fractions prepared from rod outer segments and from the rest of the retina was decreased by 50% after 48h of light exposure. As shown by Q-PCR, all endogenous RDHs were down-regulated during light exposure. RDH12 was the most dramatically decreased and this result was confirmed at the protein level.
Conclusions::
In mouse, the onset of expression of RDH11 and RDH12 coincides with the onset of rhodopsin expression and outer segment maturation, and is stable before eye opening. Total RDH activity is impaired during light-induced oxidative stress, which may precipitate the process of cell death if these enzymes have a protective role. The decrease of activity is probably due to the down regulation of all RDHs expression.
Keywords: retinoids/retinoid binding proteins • gene/expression • development