Delayed dark adaptation in 11- cis -retinol dehydrogenase deficient mice: A role of RDH11 in visual processes in vivo

1 The oxidation of 11- cis -retinol to 11- cis -retinal in the retinal pigment epithelium (RPE) represents the final step in a metabolic cycle that culminates in visual pigment regeneration. Retinol dehydrogenase 5 (RDH5) is responsible for a majority of the 11- cis -RDH activity in the RPE, but the formation of 11- cis -retinal in rdh5 -/- mice suggests another enzyme(s) is present. We have previously shown that RDH11 is also highly expressed in RPE cells and has dual specificity for both cis - and trans- retinoid substrates. To investigate the role of RDH11 in the retinoid cycle, we generated rdh11 -/- and rdh5 -/- rdh11 -/- mice and examined their electrophysiological responses to various intensities of illumination and during dark adaptation. Retinoid profiles of dark-adapted rdh11 -/- mice did not show significant differences compared with WT mice, whereas an accumu-lation of cis -esters was detected in rdh5 -/- and rdh5 -/- rdh11 -/- mice. Following light stimulation, 73% more cis -retinyl esters were stored in rdh5 -/- rdh11 -/- mice compared with rdh5 -/- mice. Sin-gle-flash ERGs of rdh11 -/- showed normal responses under dark- and light-adapted conditions, but exhibited delayed dark adaptation following high bleaching levels. Double knockout mice also had normal ERG responses in dark- and light-adapted conditions, but had a further delay in dark adaptation relative to either rdh11 -/- or rdh5 -/- mice. Taken together, these results suggest that RDH11 has a measurable role in regenerating the visual pigment by complementing RDH5 as an 11- cis -RDH in RPE cells, and indicate that an additional unidentified enzyme(s) oxidizes 11- cis -retinol or that an alternative pathway contributes to the retinoid cycle.


ABSTRACT 1
The oxidation of 11-cis-retinol to 11-cis-retinal in the retinal pigment epithelium (RPE) represents the final step in a metabolic cycle that culminates in visual pigment regeneration. Retinol dehydrogenase 5 (RDH5) is responsible for a majority of the 11-cis-RDH activity in the RPE, but the formation of 11-cis-retinal in rdh5 -/mice suggests another enzyme(s) is present. We have previously shown that RDH11 is also highly expressed in RPE cells and has dual specificity for both cis-and trans-retinoid substrates. To investigate the role of RDH11 in the retinoid cycle, we generated rdh11 -/and rdh5 -/-rdh11 -/mice and examined their electrophysiological responses to various intensities of illumination and during dark adaptation. Retinoid profiles of dark-adapted rdh11 -/mice did not show significant differences compared with WT mice, whereas an accumulation of cis-esters was detected in rdh5 -/and rdh5 -/-rdh11 -/mice. Following light stimulation, 73% more cis-retinyl esters were stored in rdh5 -/-rdh11 -/mice compared with rdh5 -/mice. Single-flash ERGs of rdh11 -/showed normal responses under dark-and light-adapted conditions, but exhibited delayed dark adaptation following high bleaching levels. Double knockout mice also had normal ERG responses in dark-and light-adapted conditions, but had a further delay in dark adaptation relative to either rdh11 -/or rdh5 -/mice. Taken together, these results suggest that RDH11 has a measurable role in regenerating the visual pigment by complementing RDH5 as an 11-cis-RDH in RPE cells, and indicate that an additional unidentified enzyme(s) oxidizes 11-cis-retinol or that an alternative pathway contributes to the retinoid cycle.
Vision is sustained through biochemical reactions involving the regeneration of isomerized chromophores in rod and cone photoreceptors in a multi-step retinoid cycling pathway (reviewed in (5,6)). The mechanism to regain light sensitivity is not completely understood, but it has been proposed to involve two distinct pathways, one for cone and the other for rod photoreceptors (7).
Both pathways ultimately regenerate 11-cis-retinal conjugated to opsins by a retinylidene bond.
However, cone visual pigments are regenerated much faster than rhodopsin in rods (reviewed by McBee et al. (5)). To complete the visual cycle, photoisomerized all-trans-retinal is first reduced to all-trans-retinol within the photoreceptors. This reaction is followed by the movement of all-trans-retinol to the retinal pigment epithelium (RPE) for storage in retinosomes as all-trans-retinyl esters (8,9), which are available for isomerization to 11-cis-retinol. The final reaction in the pathway involves oxidation of 11-cis-retinol to 11-cis-retinal and movement of the chromophore back to the photoreceptors (Fig. 1).
The importance of the retinoid cycle for the development and maintenance of normal vision has prompted efforts to identify the critical enzymes and cofactors responsible for regulating each reaction of the cycle. Hereditary mutations in key regulatory enzymes involved in this pathway underlie a range of disorders from mild visual acuity problems to severe retinal dystrophies (see Retnet at www.sph.uth.tmc.edu). Although the enzymatic components of many retinoid cycle reactions are well characterized, the key mediator(s) for 11-cis-retinol oxidation to by guest on March 24, 2020 http://www.jbc.org/ Downloaded from 11-cis-retinal remains to be identified. Leading candidates include one or more of the retinol dehydrogenase (RDH) enzymes, a subfamily of the short-chain dehydrogenase/reductase (SDR) superfamily. RDH5 is expressed in RPE cells and has been shown to oxidize 11-cis-retinol in vitro.
Mutations in the RDH5 gene have been linked to the clinical diagnosis of fundus albipunctatus and are associated with delayed dark adaptation (10). However, rdh5 -/mice display no retinal degeneration and have normal dark adaptation kinetics at bleaching levels that cause a delay in patients with fundus albipunctatus (11,12). This finding indicates that there are other RDH enzymes which facilitate retinol oxidation in the RPE.
We have recently identified additional members of the RDH family that are expressed in the eye and exhibit the ability to catalyze reduction/oxidation reactions involving retinoids (13).
One of these enzymes, RDH11, was initially designated as prostate short-chain dehydrogenase reductase 1 (PSDR1) based on its hallmark SDR protein motifs and its high transcript expression level in the human prostate (14). Subsequently RDH11 was identified as a gene regulated by sterol regulatory element-binding protein (SREBP), a transcription factor that functions to coordinately regulate the expression of enzymes involved in cholesterol and fatty acid synthesis (15). SDR enzymes also utilize steroids including RDH5. RDH11, however, lacks reactivity with steroid substrates but reduces other short-chain aldehydes such as nonanal and 4-hydroxy-2-nonenal (15).
RDH5 has NADH cofactor specificity and is more efficient in oxidizing retinols rather than reducing retinals in vitro (16). In contrast, RDH11 has NADPH specificity and catalyzes the reduction of retinals ~50-fold more efficiently than it does the oxidation of retinol in vitro (17). However, the in vivo substrate of RDHs might depend upon the relative concentration of substrates in the immediate environment, and several lines of evidence indicate that RDH11 can have 11-cis-RDH by guest on March 24, 2020 http://www.jbc.org/ Downloaded from activity in the RPE. First, RDH11 is expressed in RPE cells. Second, the remaining enzymatic activity in rdh5 -/-RPE exhibited NADPH cofactor specificity and reduced all-trans-, 9-cis-, and 11-cis-retinal (11,18). Third, the residual 11-cis-RDH activity in the rdh5 -/-RPE was membrane-associated (18), a characteristic consistent with the known subcellular localization of RDH11 (19).
In this study, we examine the physiological role of RDH11 and in particular its role in the visual retinoid cycle by generating and characterizing mice with a targeted deletion of the rdh11 gene (rdh11 -/-) and combined deletions of RDH5 and RDH11 (rdh5 -/-rdh11 -/-). Electrophysiological and biochemical measurements demonstrate that RDH11 plays a minor but complementary role to RDH5 in the flow of retinoids and thus in dark adaptation. Analyses of the rdh5 -/-rdh11 -/mice reveal the existence of additional enzymes in the retina with 11-cis-RDH(s) activity, or the presence of an alternative pathway capable of generating the visual pigment.
Homologous Recombination and Genotyping Targeting vector was linearized with Sac II and electroporated into R1_129 embryonic stem (ES) cells (20), and colonies were selected in 300 µg/ml G418 for 9 days. Homologous recombination events were screened by Southern blotting and PCR using primers O7, 5'-AGGAGGGTTGCACTTTTTGCTCTCT-3' and O8, and was subsequently labeled with [α-32 P]dCTP using the random primer labeling kit (Stratagene, La Jolla, CA).
Real time quantitative PCR RNA was Trizol extracted from liver and brain according to manufacturer's protocol. Five µg of RNA was used to generate the cDNA using 0.14 mM oligo-dT detector. Mouse RDH11-14 gene expression was normalized to S16 expression. PCR without cDNA templates did not produce significant amplification products. Specificity of the primers was verified by the amplification of a single PCR product, which was determined by observing a single dissociation curve from each tissue. Primers for the real-time PCR are the following: Analyses of Retinoids All experimental procedures related to extraction, derivatization, and separation of retinoids from dissected mouse eyes were carried out as described previously by guest on March 24, 2020 http://www.jbc.org/ Downloaded from (22)(23)(24). All reactions involving retinoids were carried out under dim red light. Retinoids were separated by normal phase HPLC (Beckman, Ultrasphere-Si, 4.6 µ 250 mm) with 10% ethyl acetate and 90% hexane at a flow rate of 1.4 ml/min with detection at 325 nm, using an HP1100 HPLC with a diode array detector and HP Chemstation A.03.03 software.
Electroretinograms (ERGs) Prior to recording, mice were dark-adapted overnight. Under safety light, mice were anesthetized by intraperitoneal injection using 20 µl/g body weight of 6 mg/ml ketamine and 0.44 mg/ml xylazine diluted with 10 mM sodium phosphate (pH 7.2) containing 100 mM NaCl. The pupils were dilated with 1% tropicamide. A contact lens electrode was placed on the eye, and a reference electrode and ground electrode were placed in the ear and on the tail.
ERGs were recorded with the universal testing and electrophysiologic system UTAS E-3000 (LKC Technologies, Inc. Gaithersburg, MD). The light intensity was calibrated by the manufacturer and computer-controlled. The mice were placed in a Ganzfeld chamber, and scotopic and photopic responses to flash stimuli were each obtained from both eyes simultaneously.
Single-flash recording Flash stimuli had a range of intensities (-3.7-2.8 log cd·s·m -2 ), and white light flash duration was adjusted according to intensity (from 20 µs to 1 ms). Three to five recordings were made with >10 s intervals, and for higher intensity intervals, intervals were 10 min or as indicated. There were no significant differences between the first and the fifth flash.
Light-adapted responses were examined after bleaching at 1.4 log cd·m -2 for 15 min. Typically, four to eight animals were used for the recording of each point in all conditions. Double-flash recording The protocol was followed as previously published with some modifications (25). A test flash was delivered to suppress the circulating current of the rod photoreceptors.
The recovery of this current was monitored by delivering a second flash, termed the probe flash. program. Leading edges of the ERG responses were fitted with a model of rod photoreceptor activation as previously described (23). Statistical analysis was carried out using the one-way ANOVA test.

Generation of rdh11 knockout mice
To determine the physiological role of RDH11, we generated rdh11 -/null mice by deleting exons 2 and 3 through homologous recombination ( Fig. 2A).
Exon 2 contains the NADP(H)-binding site, which is essential for enzyme activity. If the short transcript for exon 1 is stably expressed, it will produce a 22 amino acid-long peptide. Alternative splicing of exon 1 to exons 4, 5, 6, or 7 creates a frameshift resulting in a premature stop codon.
Southern blotting and PCR analysis identified embryonic stem (ES) cell clones with the proper homologous recombination event for germline transmission. Southern blotting probed with the 5'-fragment derived from the rdh11 gene labeled the anticipated DNA fragment sizes of 5.9 kb for the WT allele and 6.5 kb for the targeted allele (Fig. 2B). PCR screening with a primer outside the targeting vector arm and another within the neo gene verified the Southern results (Fig. 2C). Of three male chimeras with 80-90% agouti coat color, one had germline transmission of the mutant rdh11 allele. All the experiments in this report used mice originating from this ES cell clone.
Crosses of heterozygous rdh11 +/mice produced offspring with genotypes in accordance with expected Mendelian ratios ( Fig. 2D and 2E). Breeding of homozygous rdh11 -/mice produced average litter sizes (~7-10 pups) and the offspring had normal survival and growth, indicating that rdh11 -/mice are fertile and healthy. Besides the retina, RDH11 is highly expressed in the liver and testes. At 2 months of age, tissues of rdh11 -/mice exhibited no histological differences grossly or upon microscopic analysis of hematoxylin-and eosin-stained tissues in comparison with organs from WT mice (data not shown).
Relative to WT mice, immunoblotting analysis demonstrated the loss or reduction of results of rdh11 mRNA expression were concordant with the protein level for RDH11 (Fig. 3B).
Rdh11 transcripts were not detected in rdh11 -/liver but were measured in liver tissue from rdh11 +/and rdh11 +/+ mice. Expression levels of rdh11 homologues, rdh12, 13, and 14, were analyzed to ensure that rdh11 on chromosome 12 was targeted and not the homologues. Rdh12, which is located ~16 kb from rdh11 on chromosome 12, has the same intron-exon genomic organization and shares 43% nucleotide identity with rdh11. Transcripts of rdh11 homologues exhibited similar expression levels in the livers of different rdh11 genotypes (Fig. 3B), indicating the correct targeted disruption of rdh11 and a lack of transcriptionally-based compensation for the loss of rdh11.
Retinoid Analysis To investigate the in vivo role of RDH11 in vertebrate retinoid cycling between photoreceptors and RPE cells, retinoid levels in the eyes of WT, rdh11 -/-, rdh5 -/-, and rdh5 -/-rdh11 -/mice were measured 48 h after dark adaptation, or 15 min after a probe flash. Typical HPLC separation profiles of retinoids are illustrated in Fig. 4. In dark-adapted conditions rdh11 -/eyes had similar levels of retinoids relative to those in WT mice. Relative to WT mice, rdh5 -/mice had elevated levels of 11/13-cis-retinyl esters, which further increased 15 min after a single light flash that photoactivated ~35% of rhodopsin (24) (Fig. 4, peaks 1, 2; Table 1), in agreement with previously published data (11,18). The separation of 13-cis-from 11-cis-retinyl esters was particularly challenging, and only partial separation of these esters was accomplished (identified by characteristic UV/visible spectra). Interestingly, after a flash probe, rdh5 -/-rdh11 -/mice had even greater amounts of 11/13-cis-retinyl esters relative to those in rdh5 -/mice (Fig. 4, peaks 1, 2).
Conversely, after the first critical 15 min of dark adaptation, the 11-cis-retinal chromophore regenerated with similar kinetics in all the mice. The ratios for all-trans-retinal/11-cis-retinal (pmol/eye) are WT 31.3 ± 3.3 %, rdh11 -/-28.3 ± 3.2 %, rdh5 -/-29.2 ± 3.1 %, and rdh5 -/-rdh11 -/-32.7 ± 3.8 %.  Fig. 6A and C, and a-and b-wave amplitude responses are plotted in Fig. 6B and D. Under dark-adapted conditions, rdh11 -/and rdh5 -/a-and b-wave amplitudes remained similar to those observed in WT mice (Fig. 6B). Rdh5 -/-rdh11 -/mice, however, displayed slightly reduced a-and b-wave amplitude responses. Under light-adapted conditions, the genetically altered mice showed no significant changes in either a-or b-wave amplitudes (Fig. 6D). The leading edges of the a-wave of the ERG responses in dark-adapted conditions were fitted with a rod phototransduction model. The maximum amplitude and sensitivity of the photoresponses were reduced from maximal responses in dark-adapted conditions, and both parameters were compared with the results in WT mice as described previously (23). The by guest on March 24, 2020 http://www.jbc.org/ Downloaded from maximal amplitude and sensitivity of the rdh11 -/-, rdh5 -/-, and rdh5 -/-rdh11 -/mouse photoresponses remained unchanged relative to WT mice (Table 1).

Retinal Morphology
Recovery function of rod photoreceptors was further studied using paired flash responses.
A test flash was used to desensitize rod photoreceptors, and then a second probe flash at various times following the initial flash was used to examine the recovery of dark adaptation by monitoring a-wave amplitude responses (Fig. 7, left panel). Recovery of the a-wave in rdh11 -/-, rdh5 -/-, and rdh5 -/-rdh11 -/mice was normalized to a-wave amplitude responses in WT mice. Dark adaptation rates in rdh11 -/mice and rdh5 -/mice showed no significant difference, whereas photoreceptors in rdh5 -/-rdh11 -/mice resensitized significantly more slowly compared with WT (P < 0.02; rdh5 -/-rdh11 -/-, 1230.3 ± 121.3 ms; WT, 893.3 ± 47.8 ms) after the test flash (Fig. 7, right panel; Table 2). As mentioned above, other parameters of rod photoreceptor function ( Table 2) showed no significant differences between mice with different genotypes. Taken together these results suggest that the photoexcitation function of rod photoreceptors is not affected by the loss of either rdh11 or rdh5 alone but is slightly reduced when both genes are disrupted, and that both genes must be functional in order to maintain normal dark adaptation kinetics following illumination.
These differences might be due to a slower replacement of the chromophore once the flash is applied. To further evaluate the mild delay in dark adaptation, we determined if a more dramatic phenotype could be elicited by stressing the mice with an intense bleaching condition (500 cd·m -2 for 3 min) prior to monitoring the recovery of a-wave amplitude. Typical a-wave traces in the recovery phase following the bleach for WT, rdh11 -/-, rdh5 -/and rdh5 -/-rdh11 -/mice are shown in

DISCUSSION
Retinoids play an essential role in vertebrate development, differentiation, and reproduction. In vitro studies have demonstrated the ability of RDH11 to catalyze the reduction/oxidation of cis-and trans-retinals/retinols (13,17), thereby suggesting that this enzyme can play a role in retinoid homeostasis. Rdh11 is expressed during murine embryonic development, and levels gradually increase from embryonic day 7 to day 17 (26). Although RDH11 utilizes retinoid substrates and is expressed during embryogenesis, the studies reported here analyzing rdh11 -/mice did not identify any consistent abnormalities in development, post-natal survival, or fertility. The lack of a developmental phenotype in rdh11 -/mice can be due to the overlapping expression of RDH11 homologues (13) or other enzymes compensating for the loss of rdh11. In addition, RDH 11 was hypothesized to play a protective role in cells by converting highly reactive and toxic short chain aldehyde byproducts of unsaturated fatty acid oxidation to non-reactive alcohols. Since SREBP regulates fatty acid and cholesterol synthesis as well as rdh11 expression, we examined the liver for a possible phenotype. Up to 6 months of age, no histological difference in the liver of rdh11 -/compared to that of the rdh11 +/+ mice was observed, suggesting that either a liver phenotype can have a late onset, RDH11 might not play a role in preventing oxidative damage, or the mice need to be put on a special diet in order to manifest a liver phenotype. Another dehydrogenase, RDH5, is also expressed during embryogenesis (27) and catalyzes the oxidation of cis-retinol. Interestingly, mice with both rdh11 and rdh5 genes disrupted also appear healthy with no defects in development and fertility, a finding that further supports the functional redundancy of the retinoid metabolic pathway.
Maintaining the retinoid cycle in the retina is also essential for vision. In dark-adapted by guest on March 24, 2020 http://www.jbc.org/ Downloaded from conditions the retinoid levels in eyes of rdh11 -/mice were similar to measurements in eyes from WT mice, whereas rdh5 -/-rdh11 -/mice had elevated levels of cis-retinol and cis-retinyl esters that were greater than in rdh5 -/eyes ( Table 1). The reason for elevated cis-retinol levels in dark-adapted conditions is unclear, because the rate of 11-cis-retinal formation remained unchanged. A possible explanation is that the slower rate of 11-cis-retinol oxidation did not disrupt the 11-cis-retinal supply because the oxidation reaction rate was still comparable to the rate-limiting step in the retinoid cycling process, which is the reduction of all-trans-retinal (28,29).
One hypothesis could be that RDH5 might act as a modulator of RPE65-mediated isomerase activity such that the complex of RDH5 and RPE65 with a yet unidentified enzyme has lower isomerase activity compared with RPE65 free of RDH5 (16). Thus, the conversion of all-trans-retinyl ester to cis-retinol by the RPE65-mediated process (Fig. 1, d) would accelerate in the absence of RDH5, whereas the subsequent oxidation of cis-retinol (Fig. 1, e) by the remaining enzymes would not be affected by the loss of RDH5. This would result in increased levels of 11-cis-retinol, and thus cis-retinyl esters due to lecithin:retinol acyl transferase (LRAT; Fig. 1 (30)).
After a continuous 3 min bleach, cis-retinal levels decreased in rdh5 -/and rdh5 -/-rdh11 -/mice, which can explain the delayed dark adaptation kinetics. After prolonged illumination, enzymes responsible for steps b, c, and d in Fig. 1 are capable of handling the increase in substrate concentrations because of photoisomerization of 11-cis-retinal to all-trans-retinal, but a bottleneck effect occurs at step e because of the slower oxidation rate of the compensating enzymes. Thus, 11-cis-retinal demand following mild perturbations of ~30% bleach can be met by the remaining dehydrogenase activity, but these enzymes are insufficient to rapidly replenish large depletions of by guest on March 24, 2020 http://www.jbc.org/ Downloaded from 11-cis-retinal chromophore following higher bleaches. Changes in retinoid levels in rdh5 -/-rdh11 -/mice compared with rdh5 -/mice also provide evidence that RDH11 plays a role in the normal flow of retinoids.
Both rdh11 -/and rdh5 -/-rdh11 -/mice have normal photopic and scotopic ERG kinetics (Fig. 6, Table 2), indicating that these mice are capable of regenerating the visual chromophore and have normal rod and cone photoreceptor phototransduction signaling. However, after prolonged intense illumination conditions delayed dark adaptation kinetics were observed in the single and double knockout mice. The severity of the attenuated dark adaptation (Fig. 8) appeared to correlate with decreasing 11-cis-retinal concentrations observed in rdh11 -/-, rdh5 -/and rdh5 -/-rdh11 -/mice relative to WT (Fig. 9). Photoreceptors in both rdh11 -/and rdh5 -/mice displayed biphasic recovery kinetics: a rapid initial phase followed by a slower period (Fig. 8). This biphasic recovery has been observed in patients exhibiting clinical fundus albipunctatus due to an Arg257Trp mutation in RDH5 (31). After a 0.5% bleach, patients have normal rod recovery, but following intermediate bleaches in the 2-12% range, a rapid partial recovery is followed by a transitory plateau. A mild visual phenotype in rdh5 -/-rdh11 -/mice suggests that alternative enzymes can oxidize 11-cis-retinol to 11-cis-retinal in RPE cells. Likely candidates might be Dhrs9 (retsdr8), an oxidoreductase, which has dual substrate specificity for both 11-cis and all-trans-retinol with NADPH specificity (21) or RDH10, a SDR that catalyzes retinol oxidation with NADP specificity (32). Rather than a microsomal enzyme, perhaps the compensating enzyme can be the plasma membrane-associated 11-cis-RDH found in the RPE (33). Another possibility is that the remaining 11-cis-RDH activity observed in rdh5 -/-RPE cells results from a NAD + -dependent enzyme (18).
Candidate NADH-dependent enzymes might be amongst the eight SDR genes present as a cluster on mouse chromosome 10D3. Rdh5 is also located on chromosome 10D3 but it is separated from the other SDRs by ~1 Mb. RDH1, RDH5, RDH6 (CRAD1), RDH7 (CRAD2), and RDH9 (CRAD3) (34-37) catalyze retinol oxidation with NAD + specificity, suggesting that these SDRs most likely originated through local intra-chromosomal duplication events. If these enzymes are expressed in the RPE as demonstrated for RDH6 and RDH7, and if indeed RDH1, RDH7, and RDH9 lack a human orthologue, one or more of these enzymes can catalyze 11-cis-retinol oxidation and contribute to the more efficient dark adaptation mechanism in mice.
Biochemical and genetic studies indicate that alcohol dehydrogenase (ADH) metabolizes retinol to retinal (38). Database searches have identified adh1 expressed sequence tags (EST) in mouse RPE and choroid libraries and adh4 ESTs in mouse and human RPE and choroid libraries.
Recently, ADH4 has been immunolocalized to the RPE and shown to have significant enzymatic activity toward cis-retinoids (39). Although soluble ADHs catalyze retinal/retinol redox reactions, the soluble protein fraction of bovine RPE cells contained low amounts of retinol oxidative activity, which is insufficient to explain the remaining alternative enzyme(s) activity in rdh5 -/-RPE. This indicates that soluble enzymes do not play a major role in the visual retinoid cycle (18). Thus, ADH1 and ADH4, although expressed in the RPE, most likely contribute a minor amount to redox activity toward cis-retinoids in RPE cells.
Factors that determine whether an enzyme catalyzes the reduction or oxidation of a substrate include the equilibrium constant, the reduced/oxidized cofactor, and substrate/product ratios.
The pH independent equilibrium constant is 3.3 x10 -9 for the oxidation of retinol (40). It was reported that RDH11 displayed ~50-fold more efficient activity for the NADPH-dependent reduction of all-trans-retinal than for the oxidation of all-trans-retinol (17). However, in the retina, the ratio of NADP to NAPDH is between 4 to 1 or 1.5 to 1 (41), suggesting that NADPH-dependent enzymes such as RDH11 and RDH5 have the ability to catalyze both oxidative and reductive reactions depending on the substrate concentrations. Rdh -/-, rdh11 -/and Rdh -/-rdh11 -/mice have elevated levels of cis-retinol, which is stored in the form of cis-retinyl esters, suggesting that RDH11 and RDH5 catalyze the oxidation of retinol to retinal. It should be considered that the oxidation reaction is preferred by the means of sequestering the 11-cis-retinal product by CRALBP.
In other tissues where RDH11 is expressed at different substrate/product concentrations, it is conceivable that RDH11 might catalyze the reductive reaction.
EM results revealed no retinal degeneration in rdh11 -/-, rdh5 -/or rdh5 -/-rdh11 -/retina, which is concordant with normal ERG kinetics. Contact between photoreceptor outer segments and RPE cells remained intact in all mice with different genotypes, suggesting that normal phagocytosis was occurring. Not surprisingly, disrupting both rdh11 and rdh5 genes did not elicit the white punctata in the fundus that is observed in patients with RDH5 mutations (data not shown). by guest on March 24, 2020 http://www.jbc.org/ Downloaded from A number of mice models are providing evidence that disruption of genes known to play a role in the retinoid cycle exhibit a more severe degenerative retinal pathology in humans compared with mice, such as abcr (42,43), cralbp (44,45), and rgr (24,46) mutations. These mouse models exhibit delayed dark adaptation kinetics at higher bleaching intensities but normal ERG kinetics under dark-adapted conditions. In contrast, mice models of other genes involved in the retinoid cycle such as lrat (30) and rpe65 (47) more closely mimic human retinal dystrophies. Mutations in LRAT cause early childhood onset retinal dystrophy (CSRD), and RPE65 has been identified as one of the disease-causing genes for Leber's Congenital Amaurosis (LCA), also an early onset form of retinal dystrophy. Thus, genes associated with later onset of retinal degeneration in humans produce a mild phenotype in mice, whereas genes causing early childhood onset retinal dystrophies produce a severe phenotype.
Mutations in RDH12 were linked with progressive rod-cone dystrophy in a subset of LCA patients (50,51). In humans, RDH12 is located on chromosome 14, ~27 kb apart from its homologue RDH11. Both RDH11 and RDH12 have the same substrate specificity and similar enzymatic properties, but RDH11 is localized to the RPE and Müller cells, whereas RDH12 is localized to the photoreceptor cell layer. Both groups screened their cohort of patients for polymorphisms in the RDH11 gene, but neither group found an association between the disease and RDH11. A lack of a severe phenotype in rdh11 -/mice provides additional evidence that loss of RDH11 function might not manifest in LCA. If this is indeed true, a defect in the rate-limiting step of all-trans-retinal reduction in photoreceptors appears to be the critical step in the retinoid cycle. RDH12 must be the by guest on March 24, 2020 http://www.jbc.org/ Downloaded from dominant enzyme, given the redundancy of all-trans-retinal reducing enzymes such as retSDR1 (52) and prRDH (53) in photoreceptors. It will be interesting to examine whether rdh12 -/mice display a similar retinal electrophysiological and pathological phenotype as seen in LCA patients.