Targeted Disruption of the Murine Retinal Dehydrogenase Gene Rdh12 Does Not Limit Visual Cycle Function

Abstract

RDH12 codes for a member of the family of short-chain alcohol dehydrogenases/reductases proposed to function in the visual cycle that supplies the chromophore 11-cis retinal to photoreceptor cells. Mutations in RDH12 cause severe and progressive childhood onset autosomal-recessive retinal dystrophy, including Leber congenital amaurosis. We generated Rdh12 knockout mice, which exhibited grossly normal retinal histology at 10 months of age. Levels of all-trans and 11-cis retinoids in dark-and light-adapted animals and scotopic and photopic electroretinogram (ERG) responses were similar to those for the wild type, as was recovery of the ERG response following bleaching, for animals matched for an Rpe65 polymorphism (p.L450M). Lipid peroxidation products and other measures of oxidative stress did not appear to be elevated in Rdh12 ؊/؊ animals. RDH12 was localized to photoreceptor inner segments and the outer nuclear layer in both mouse and human retinas by immunohistochemistry. The present findings, together with those of earlier studies showing only minor functional deficits in mice deficient for Rdh5, Rdh8, or Rdh11, suggest that the activity of any one isoform is not rate limiting in the visual response.Retinoid dehydrogenases/reductases (RDHs) perform critical oxidation-reduction reactions in the visual cycle mechanism that converts vitamin A (all-trans retinol) to 11-cis retinal, the lightabsorbing chromophore of the visual pigments in photoreceptor cells (Fig. 1) (14). One of these reactions involves the oxidation of 11-cis retinol produced by retinoid isomerohydrolase activity in the retinal pigment epithelium (RPE), resulting in formation of 11-cis retinal that is delivered to the photoreceptor cell outer segments. The second reaction involves reduction of all-trans retinal released from rhodopsin and cone opsins upon bleaching, resulting in formation of all-trans retinol that is returned to the RPE for trans-to-cis isomerization. A number of RDH isoforms are expressed in the RPE and/or the neuroretina that differ in terms of substrate specificity and sites of expression.Within the RPE, RDH5 is thought to be the key enzyme involved in converting 11-cis retinol to 11-cis retinal (25). Mutations in RDH5 in humans result in fundus albipunctatus, a form of congenital stationary night blindness (31). Since Rdh5 knockout mice exhibit very mild visual disturbances (7), it has been suggested that Rdh5 activity may be redundant with that of other isoforms. Consistent with this notion, mice deficient for Rdh11 and for prRdh, two isoforms expressed in the photoreceptors, exhibit a mild phenotype without signs of retinal degeneration (15,16). Functional interaction of RDH5 with RDH11 has been proposed (16), but studies showing that RDH11 is expressed in the photoreceptor cell inner segment complicate this interpretation (15). RDH10 is expressed in both the RPE and Müller cells and is specific for the oxidation of all-trans retinol (30).

Reduction of All-trans-retinal in Vertebrate Rod Photoreceptors Requires the Combined Action of RDH8 and RDH12

“…The results obtained in our study are consistent with the previous localization of Rdh8 in the outer segment (23) and Rdh12 in the inner segment (24,38) of rod photoreceptors cells. We find that Rdh8 activity is essential for reducing the majority of the retinal generated in the outer segment.…”

“…To investigate the functional roles of RDH8 and RDH12 in photoreceptor physiology, previous studies have developed knock-out mice lacking either one or both of these enzymes (23)(24)(25). Analysis of retinoid metabolism and visual performance in these animals showed that chromophore production necessary for continuous visual cycle activity does not appear to depend on the rate of reduction of all-trans-retinal in the rod outer segments.…”

Rdh12 Activity and Effects on Retinoid Processing in the Murine Retina

“…In studies employing retinoid extraction and HPLC analysis, we showed previously that steady-state levels of visual cycle retinoids were similar in the eyes of Rdh12-deficient and wild-type mice heterozygous for the Rpe65-L450M (L/M) polymorphism that impacts rates of chromophore synthesis (15). We have now used these methods to evaluate retinoid processing during dark adaptation in Rdh12-deficient mice homozygous for the Rpe65-Leu 450 (L/L) variant associated with high visual cycle throughput (38).…”

“…Previous studies of the retinal phenotype of Rdh12-deficient mice did not detect marked changes in retinoid processing or visual cycle function (15,16). In one study, delayed recovery of the electroretinogram a-wave after bleaching was observed as well as increased levels of 11-cis-retinal, but no decrease in alltrans-retinal reductase activity was detected in vitro (16).…”

“…Subsequent in vitro studies showed that RDH12 can also act upon C9 aldehydes resulting from lipid photo-oxidation (11)(12)(13), as well as certain steroid substrates (14), suggesting that it contributes to additional pathways important for photoreceptor function. In previous studies, we generated Rdh12-deficient mice and evaluated their retinal phenotype, finding grossly normal retinal histology, visual responses, retinoid content, and assays of oxidative stress (15). We also showed that RDH12 localizes to photoreceptor inner segments in humans and mice, distant from the phototransduction reactions occurring in the outer segment disc membranes.…”