Recovery of Visual Functions in a Mouse Model of Leber Congenital Amaurosis

Abstract

The visual process is initiated by the photoisomerization of 11-cis-retinal to all-trans-retinal. For sustained vision the 11-cis-chromophore must be regenerated from all-trans-retinal. This requires RPE65, a dominant retinal pigment epithelium protein. Disruption of the RPE65 gene results in massive accumulation of all-trans-retinyl esters in the retinal pigment epithelium, lack of 11-cis-retinal and therefore rhodopsin, and ultimately blindness. We reported previously (Van Hooser, J. P., Aleman, T. S., He, Y. G., Cideciyan, A. V., Kuksa, V., Pittler, S. J., Stone, E. M., Jacobson, S. G., and Palczewski, K. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 8623–8628) that in Rpe65−/− mice, oral administration of 9-cis-retinal generated isorhodopsin, a rod photopigment, and restored light sensitivity to the electroretinogram. Here, we provide evidence that early intervention by 9-cis-retinal administration significantly attenuated retinal ester accumulation and supported rod retinal function for more than 6 months post-treatment. In single cell recordings rod light sensitivity was shown to be a function of the amount of regenerated isorhodopsin; high doses restored rod responses with normal sensitivity and kinetics. Highly attenuated residual rod function was observed in untreated Rpe65−/− mice. This rod function is likely a consequence of low efficiency production of 11-cis-retinal by photo-conversion of all-trans-retinal in the retina as demonstrated by retinoid analysis. These studies show that pharmacological intervention produces long lasting preservation of visual function in dark-reared Rpe65−/− mice and may be a useful therapeutic strategy in recovering vision in humans diagnosed with Leber congenital amaurosis caused by mutations in the RPE65 gene, an inherited group of early onset blinding and retinal degenerations.

Leber congenital amaurosis (LCA)¹ is a group of conditions that cause blindness or severe visual impairment from birth. All show both rod and cone dysfunction, a negligible (not recordable) electroretinogram (ERG), and nystagmus. They result in early onset retinal dystrophy (1), which over time may be accompanied by pigmentary changes in the retina, hence “amaurosis” (Greek for darken). LCA is caused by defects in at least five different genes that disrupt a variety of different cellular functions (2-6).

In ∼12% of all LCA cases the gene for a 65-kDa protein (RPE65) of retinal pigment epithelium cells (RPE) is disabled (7, 8). RPE65 is heavily expressed in RPE cells, where it plays an essential role in the retinoid cycle. This is a set of tightly interconnected events that involve both photoreceptors and RPE cells. The photoisomerization of the visual pigment chromophore (11-cis-retinal) produces all-trans-retinal, which is reduced in the photoreceptor, transferred to the RPE, converted back to 11-cis-retinal, and then transferred back to the photoreceptor to regenerate the original visual pigment (reviewed in Ref. 9). The precise function of RPE65 in retinoid processing is unknown.

Genetically engineered mice in which the gene for Rpe65 has been eliminated (Rpe65−/−) exhibit changes in retinal morphology, function, and biochemistry that closely resemble the changes seen in human LCA patients. Both rod and cone function is severely disrupted, and the ERG is severely attenuated in Rpe65−/− mice (10, 11). There is also a dramatic overaccumulation of all-trans-retinyl esters in the RPE cells in lipid-like droplets (10, 12) and degeneration of the retina. Thus, the Rpe65−/− mouse provides the opportunity to gain insight into the cellular and molecular origins and consequences of LCA as well as a means to test different therapeutic strategies.

Here we describe the results of an in-depth study of the changes in biochemistry and function that occur in Rpe65−/− mice and show how the progression of the disease can be interrupted and the functional effects reversed by providing a supply of 9-cis-retinal. The goals were to: 1) examine the beneficial effects of 9-cis-retinal treatment on the progression of the disease and on photoreceptor function; 2) evaluate using single cell electrophysiology and ERG recording how 9-cis-retinal treatment affected rod function and light-driven signals in the retina; and 3) investigate the biochemical basis for the low level of residual vision that persists in both LCA patients and Rpe65−/− mice.

We find that administration of 9-cis-retinal to Rpe65−/− mice produces and maintains rod photopigment for more than 6 months in the dark. Early intervention with 9-cis-retinal restores normal rod physiology and significantly attenuates ester accumulation in the RPE but only partially improves retinal function as measured by ERG. These studies demonstrate that pharmacological intervention produces long lasting preservation of visual function in dark-reared Rpe65−/− mice and is a potentially useful therapy for restoring vision in LCA patients.

MATERIALS AND METHODS

Animals—All of the animal experiments employed procedures approved by the University of Washington Animal Care Committee and conformed with recommendations of the American Veterinary Medical Association Panel on Euthanasia. Animals were maintained in complete darkness, and all of the manipulations were performed under dim red light employing all Kodak No. 1 Safelight filter (transmittance, >560 nm). Typically, 2–3-month-old mice were used in all of the experiments. RPE65-deficient mice were obtained from Dr. M. Redmond (NEI, National Institutes of Health) and genotyped as described previously (12, 13). Retinal G protein-coupled receptor-deficient mice were generated and genotyped as described previously (14). Double knockout Rpe65−/− Rgr−/− were generated by cross-breeding single Rpe65−/− and Rgr−/− mice to genetic homogeneity.

Analyses of Retinoids and Visual Pigments—All of the procedures were performed under dim red light as described previously (10, 15, 16). In addition to previously described methods, retinoid analysis was performed on an HP 1100 series high pressure liquid chromatograph (HPLC) equipped with a diode array detector and HP Chemstation A.07.01 software, allowing identification of retinoid isomers according to their specific retention time and absorption maxima. A normal phase column (Beckman Ultrasphere Si 5μ, 4.6 × 250 mm) and an isocratic solvent system of 0.5% ethyl acetate in hexane (v/v) for 15 min followed by 4% ethyl acetate in hexane for 60 min at a flow rate of 1.4 ml/min at 20 °C (total 75 min) with detection at 325 nm allowed the separation of 11-cis-, 13-cis-, and all-trans-retinyl esters. In addition, all of the experimental procedures related to the analysis of dissected mouse eyes, derivatization, and separation of retinoids have been described previously in detail (10). Rh and iso-Rh measurements were performed as described previously (16). Typically, two mouse eyes were used per assay, and the assays were repeated three to six times. The data are presented with S.E.M.

Light and Electron Microscopy—Eye cups were prepared by removing the anterior segment and vitreous. The eyes were collected on ice at PND 1–28 on a weekly basis. “Thin” sections (1.0 μm) were stained with Richardson's blue solution (1%) and subjected to light microscopy. “Ultrathin” sections (0.05 μm) were stained with uranyl acetate/lead citrate and subjected to electron microscopy.

Preparation of Mouse RPE Microsomes—Fresh mouse eyes were enucleated immediately after cervical dislocation or CO₂ asphyxiation. The anterior segment, vitreous, and retina were carefully removed under a microdissecting scope. Typically, 30–40 eyes were dissected for each preparation. RPE cells were separated by placing 12 dissected eyecups in 400 μl of 10 mm MOPS, pH 7.0, containing 1 μm leupeptin and 1 mm dithiothreitol and vigorously shaken for 20 min. The eyecups were then gently brushed with a fine brush to further dislodge the RPE cells. The cell suspension was removed, another aliquot of 400 μl of MOPS buffer was added, and the eyecups were shaken again for 20 min. The cell suspensions were combined and subjected to glass-glass homogenization. The homogenate was centrifuged at 10,000 × g for 10 min, and then the supernatant was centrifuged at 275,000 × g for 1 h. The pellet was then reconstituted in 200 μl of the MOPS buffer and resubjected to glass-glass homogenization. The total protein concentration (typically 0.5–1 mg/ml) was determined by the Bradford method (39).

Isomerization of All-trans-retinol to 11-cis-Retinol using Mouse RPE Microsomes—The assay used for determining isomerization to 11-cis-retinol was reported previously (17). Briefly, 20 μl of bovine serum albumin (final concentration, 1%), 125 μl of 50 mm 1,3-bis[tris(hydroxymethyl)-methylamino]propane, pH 7.5, 10 μl of ATP (1 mm final concentration), 25 μm apo-recombinant CRALBP, 40 μl of RPE microsomes (typically 25–50 μg of total protein), and 0.5 μl of 4 mm all-trans-retinol in dimethylformamide. The reactions were incubated for 2 h at 37 °C. The reaction was quenched using 300 μl of MeOH, and the retinoids were extracted with 200 μl of hexane. The mixture was shaken vigorously for 2 min and then centrifuged at 14,000 rpm for 4 min for phase separation. The upper organic layer was removed, and a 100-μl aliquot was separated and analyzed using an HP 1100 HPLC (Beckman Ultrasphere Si, 4.6 mm × 250 mm, 1.4 ml/min flow rate using 10% ethyl acetate in hexane) equipped with HP Chemstation software (version A.07.01).

Preparation of pro-S-[4-³H]NADH and pro-S-[4-³H]NADPH—Syntheses of pro-S-[4-³H]NADH and pro-S-[4-³H]NADPH were carried out with l-glutamic dehydrogenase, NAD(P), and l-[2,3-³H]glutamic acid (PerkinElmer Life Sciences), as described previously (15, 18).

RDH Assays—The assays were carried out by monitoring the production of [15-³H]retinol (reduction of retinal) using 11-cis-retinal and pro-S-[4-³H]NAD(P)H as dinucleotide substrates in the presence or absence of NADH (18).

Oral Gavage—Oral gavage was carried out as described previously (10).

Intravenous Administration of Retinoids—The chemicals were purchased from Sigma/Aldrich unless otherwise specified. Solution A contained 10 mg of 9-cis-retinal, 75 mg of Cremophor EL, 1 mg of α-tocopherol, and 0.6 mg of benzoic acid suspended in 1 ml of lactated Ringer's solution (Baxter). The mixture was vortexed for 10 min and centrifuged for 10 min at 20,000 × g, and the concentration of 9-cis-retinal (7.7 mm) was determined spectrophotometrically. Solution B contained 13 mg of 9-cis-retinal, 50 mg of Cremophor EL, 10 mg of dipalmitoylphosphatidyl choline, and 40 mg of 2-hydroxypropyl-β-cyclodextrin suspended in 1 ml of lactated Ringer's solution (Baxter). The mixture was vortexed for 10 min and centrifuged for 10 min at 20,000 × g, and the concentration of 9-cis-retinal (10 mm) was determined spectrophotometrically. Solutions A and B (typically, 100 μl) were delivered to the mouse lateral tail vein employing a 1-ml syringe equipped with a 27-gauge needle and a restraint tube.

Single Cell Recordings—Mice were dark-reared from birth and sacrificed via cervical dislocation, and the eyes were removed. The retina was isolated and stored on ice for up to 12 h in HEPES-buffered Ames' solution (10 mm HEPES, pH adjusted to 7.4 with NaOH). Isolated rods were obtained by shredding a small piece of retina (roughly 1 mm²) with fine needles in a 160-μl drop of solution. The drop was then injected into a recording chamber mounted on the stage of an inverted microscope (Nikon Eclipse) equipped with an infrared video viewing system and continuously superfused at 2–3 ml/min with bicarbonate-buffered Ames' solution warmed to 37 °C (pH 7.4 when equilibrated with 5% CO₂, 95% O₂). The entire dissection was carried out under infrared illumination using a dissecting microscope equipped with infrared-visible image converters.

An isolated rod was drawn by suction into a heat-polished, silanized borosilicate electrode with an opening 1.2–1.5 μm in diameter. The electrode was filled with HEPES-buffered Ames' solution. The electrical connections to the bath and suction electrode were made by NaCl-filled agar bridges that contacted calomel half-cells. Bath voltage was held at ground by an active clamp circuit (19). Membrane current collected by the suction electrode was amplified by an Axopatch 200A patch clamp amplifier (Axon Instruments, Foster City, CA), filtered at 30 Hz (−3 dB point) with an 8-pole Bessel low pass filter, and digitized at 1 kHz.

Light from light-emitting diodes with peak outputs at 470, 570, and 640 nm were combined using a trifurcated fiber optic and focused on the preparation using a water immersion lens in place of the microscope condensor. The light stimulus was spatially uniform and illuminated a circular area 0.57 mm in diameter centered on the recorded cell. Light intensities were measured at the preparation and converted to equivalent 500-nm photons (λ_max for rod sensitivity)) using the absorption spectrum of rhodopsin and the measured light-emitting diode spectrum. Equivalent intensities for each experiment are given in the figure legends.

Mouse Electroretinograms—Mice were dark-reared from birth and anesthetized (ketaject/xylaject, 65 mg/kg intraperitoneally), and the pupils were dilated with tropicamide (1%). A contact lens electrode was placed on the eye with a drop of methylcellulose and a ground electrode placed in the ear. ERGs were recorded and analyzed with the universal testing and electrophysiologic system 3000 (UTAS E-3000) (LKC Technologies Inc., Gaithersburg, MD). The mice were placed in a Ganzfield chamber, and flicker recordings were obtained from one eye. Flicker stimuli had a range of intensities (0.00040–41 cd·s/m²) with a fixed frequency (10 Hz).

Immunocytochemistry—Rpe65 mice were divided into five groups: Rpe65−/−, Rpe65−/− that were gavaged with 9-cis-retinal and kept in the dark, Rpe65−/− that were gavaged with 9-cis-retinal, exposed to a flash, and kept in the dark for 15 min, Rpe65+/+ that were kept in the dark, and Rpe65+/+ that were exposed to a flash and kept in the dark for 15 min. For the flash experiments, dark-adapted mice were subjected to a flash (Sunpak 433D, 1 ms) from a distance of 2 cm. The retinas were fixed in 4% paraformalydehyde in 0.13 m sodium phosphate, pH 7.4, for 15 h at 4 °C, and the tissues were transferred to 5, 10, or 15% sucrose in 0.13 m sodium phosphate, pH 7.4, for 30 min each time and stored overnight in 20% sucrose in the same buffer at 4 °C. The tissue was then transferred to optimal cutting temperature cryoembedding compound and sectioned at 10 μm. The cryosections were incubated overnight at 4 °C in mouse monoclonal anti-phosphorylated Rh A11–82P antibody diluted 1:10. Triton X-100 (0.1%) was included in all phosphate-buffered saline solutions to facilitate antibody penetration. The controls were processed by omitting primary antibodies from the incubation buffer. After incubation in primary antibodies, the sections were rinsed with phosphate-buffered saline and then incubated with indocarbocyanine (Cy3)-conjugated goat anti-mouse IgG (1:200). The sections were rinsed in phosphate-buffered saline mounted in 5% n-propylgallate in glycerol and coverslipped.

RESULTS

Early Treatment with 9-cis-Retinal Eliminates Oil-like Structures in Rpe65−/− Mice—In addition to the loss of photoreceptors, a defective interface between ROS and RPE, RPE cells of Rpe65−/− mice contained numerous lipid-like droplets (10, 12, 20). In young animals, empty vacuoles were observed in fixed electron microscopy sections of RPE from Rpe65−/− mice but not in controls (data not shown). With increasing age (>PND 21), they were filled with a diffractive material that was retained during electron microscopy section preparation. This observation correlates with the excessive accumulation of all-trans-retinyl esters in Rpe65−/− mice (Fig. 1A, open circles). Retinyl esters also accumulated with age in Rpe65+/+ mice, albeit at lower levels than for Rpe65−/− mice. By PND 21, ∼800 pmol/eye of retinyl esters accumulated compared with ∼40 pmol/eye for Rpe65+/+. For Rpe65+/+ mice, Rh levels initially exceeded the amount of retinyl esters several-fold.

Fig. 1.

Changes in retinoid levels and interface between RPE and ROS in Rpe65−/− mice gavaged with 9-cis-retinal. A, the levels of all-trans-retinyl esters (closed circles) and 11-cis-retinal (closed squares) in Rpe65+/+ compared with levels of all-trans-retinyl esters (open circles) in Rpe65−/− mice as a function of age. B, ester analysis of 9-cis-retinal-treated and untreated Rpe65−/− mice. Rpe65−/− mice were treated with 25 μg of 9-cis-retinal starting at PND 7 every other day until they were 1 month old. Note the y axis scale. C, age-related accumulation of all-trans-retinyl esters in Rpe65−/− mice (gray line with black data points) compared with the ester levels (circles) in animals treated with 9-cis-retinal starting at PND 7 (left panel) (25 μg every other day, and after PND 30 gavaged with 9-cis-retinal (250 μg) once a week) or PND 30 (right panel) gavaged with 9-cis-retinal (250 μg) once a week. The levels of iso-Rh in treated Rpe65−/− mice are indicated by triangles measured as 11-cis-retinyl oximes. D, changes in the RPE-ROS interface in Rpe65 mice treated with 9-cis-retinal. Rpe65−/− mice were treated with 9-cis-retinal (200 μg each) at PND 7, 11, and 15 and analyzed when they were PND 30 (panels c and d) and PND 90 (panels e and f). Rpe65−/− mice were treated with 9-cis-retinal (200 μg each) at PND 30 and analyzed when they were PND 120 (panels g and h). Control retina from untreated Rpe65−/− mice at PND 7 and PND 30 is shown on the top (panels a and b, respectively). Only partially filled lipid-like droplet in early treated mice (left column, red arrow), and considerably improved RPE-ROS processes (right column) in all treated mice were observed. Scale bar, 1 μm.

When PND 7 mice were treated with a 0.25-mg dose of 9-cis-retinal (10 mg/ml) every other day until they were 30 days old, a dramatic change in the ester accumulation was observed (Fig. 1B). With increasing age and continued administration of 1 dose (1.25 mg) per week, the amounts of all-trans-retinyl esters increased, similar to Rpe65+/+, but the overall amounts of esters were dramatically suppressed with concomitant formation of iso-Rh (Fig. 1C, left panel). Once deposited, the accumulated esters in the RPE were not removed if the treatment began after more than 1 month of age (Fig. 1C, right panel). When young animals or young adults were treated with 9-cis-retinal, the interface contacts between the RPE and ROS were improved (Fig. 1D, panels d, f, and h), and the vacuoles appeared to be only partially filled (Fig. 1D, panels c and e) over several months of this study. These observations suggest that formation of the regenerated pigment significantly slowed down accumulation of esters but did not promote the complete removal of the all-trans-retinyl esters that had been deposited in the eye.

Long Term Effect of 9-cis-Retinal Treatment—Treatment of mice with 9-cis-retinal produced a long lasting increase in photopigment levels and a decrease in accumulation of all-trans-retinyl esters. Rpe65−/− mice (1-month-old) were treated once (2.5 mg) with 9-cis-retinal and then kept under either a 12-h light/dark cycle, or under 24 h dark for 37 days. No appreciable depletion of retinal was observed under either set of conditions (Fig. 2A). These results suggest that a single dose of 9-cis-retinal sustains iso-Rh in these animals under normal laboratory conditions.

Fig. 2.

Effects of light exposure on iso-Rh levels in Rpe65−/− mice gavaged 9-cis-retinal and ERG responses after a long term treatment with 9-cis-retinal. A, comparison of iso-Rh levels in 1-month-old Rpe65−/− mice gavaged with a single dose of 9-cis-retinal (2.5 mg) and kept under 12 h light/dark or at constant dark for 37 days (n = 4). B, the levels of Rh or iso-Rh in 6-month-old Rpe65−/− mice. The Rh levels in wild type mice (column a) were compared with iso-Rh in Rpe65−/− mice treated twice with 9-cis-retinal (2.5 mg each time) at 1 month old with 4-day intervals (column c) and treated twice with 3-month (column d) or 4-month (column e) intervals. No Rh or iso-Rh was detected in untreated Rpe65−/− mice (column b) (n = 4). C, the intensity-dependent response of flicker ERGs in Rpe65+/+, Rpe65−/−, Rpe65−/− treated with 9-cis-retinal, and Rpe65−/− Rgr−/− mice. The flicker recordings were obtained with a range of intensities of 0.00040–41 cd·s/m² at a fixed frequency (10 Hz). Left panel, Rpe65+/+ mice; right panel, Rpe65−/− with or without treatment (open and closed circles, respectively) and Rpe65−/− Rgr−/− mice without treatment (closed triangles).

In another set of experiments, the level of Rh or iso-Rh was measured in 6-month-old Rpe65−/− mice (Fig. 2B). In these animals, the iso-Rh levels were comparable for three groups of Rpe65−/− mice: mice treated twice with 9-cis-retinal (2.5 mg/dose) at PND 30 and 34, mice treated twice at PND 30 and 120, and mice treated twice at PND 30 and 150. The 50% decrease of iso-Rh in Rpe65−/− (Fig. 2, B compared with A) matches a similar decrease in Rh in Rpe65+/+ as a function of age. The ester levels were reduced by >50% (compared with untreated animals) and were unaffected by the frequency and dose of 9-cis-retinal. No Rh or iso-Rh was detected in untreated dark-adapted Rpe65−/− mice.

9-cis-Retinal, reduced to 9-cis-retinol, can be stored in the eye and liver in the form of 9-cis-retinyl ester. When needed 9-cis-retinol would be liberated by a retinyl hydrolase. To determine how large the reservoir of 9-cis-retinoids is in the eye and liver, a group of mice were treated with 9-cis-retinal (2.5 mg) and after 48 h exposed to multiple flashes at 1-h intervals that bleached ∼30–35% of Rh/flash. Iso-Rh and 9-cis-retinyl esters were significantly depleted after more than three intense flashes (data not shown). Retinyl esters from liver and RPE were completely depleted after five flashes at 24-h intervals (data not shown). Continuous shedding and resynthesis of Rh-containing ROS discs does not affect the long term preservation of the visual pigment. Therefore, it appears that 9-cis-retinal is, in a large part, recycled from phagocytized iso-Rh to newly produced opsin molecules over an extended period of time.

Physiological effects of 9-cis-Retinal Treatment—Treatment of Rpe65−/− mice with 9-cis-retinal also provided long term improvement of retinal function. The long term physiological effect of 9-cis-retinal treatment was determined from single flash responses of different intensities (data not shown) and flicker ERG measurements on Rpe65+/+ and Rpe65−/− mice. Previous experiments showed a partial recovery of the ERG sensitivity 48 h after oral 9-cis-retinal administration (10). We found that this partial recovery persisted for more than 12 weeks in Rpe65−/− mice treated once at PND 30.

The flicker ERG in Rpe65+/+ mice reached a peak amplitude of 254.9 ± 41.5 μV at a light level of 0.015 cd·s/m² and 95.1 ± 8.9 μV at 7.5 cd·s/m² (Fig. 2C, left panel). These data resemble the rod and cone dominant ERG responses, respectively (11). In Rpe65−/− mice without treatment, the flicker ERG reached a significantly smaller peak amplitude, 76.0 ± 12.0 μV, at a light level of 7.5 cd·s/m² (Fig. 2C, right panel). Eight weeks after a single treatment with 2.5 mg of 9-cis-retinal, the flicker ERG reached peak amplitudes of 137.3 ± 24.4 μV at 0.059 cd·s/m² and 40.0 ± 7.1 μV at 13 cd·s/m² (Fig. 2C, right panel). These peaks were smaller and occurred at a higher light level than in the Rpe65+/+ mice; however, the response of treated Rpe65−/− mice was 2.1 logarithmic units more sensitive and had larger amplitude than that of untreated mice. Thus, administration of 9-cis-retinal provided a long term, partial recovery of the ERG.

Treatment with 9-cis-Retinal Eliminated Constituitive Opsin Phosphorylation—To gain additional insight into the enzymatic processes of Rpe65 mice, several direct measurements of relevant enzymatic activities were carried out. It is generally accepted that opsin has some signaling capability. Immunolabeling on retina sections from Rpe65 mice using a monoclonal antibody against phosphorylated opsin could provide a clean evaluation of this activity, whereas it would be expected that 9-cis-retinal treatment would inhibit this activity.

The retinas from Rpe65+/+ mice (Fig. 3C, panel A) and Rpe65−/− mice (Fig. 3C, panel B) were fixed in constant darkness. The ROS in Rpe65+/+ mice showed no labeling (Fig. 3C, panel A), and the ROS from untreated Rpe65−/− mice were labeled by a monoclonal antibody against phosphorylated opsin (Fig. 3C, panel B). This labeling was abolished for Rpe65−/− mice (gavaged once at PND 30 and analyzed 48 h post-treatment) treated with 9-cis-retinal (Fig. 3C, panel C). This 9-cis-treatment reduced phosphorylation of opsin to levels comparable with those in normal rods. ROS fixed in darkness at 15 min following a single flash showed immunolabeling in both Rpe65+/+ and Rpe65−/− mice treated with 9-cis-retinal (Fig. 3C, panels D and E). These data suggest that opsin is constitutively phosphorylated in Rpe65−/− mice. These experiments indicated a specific deficit in conversion of all-trans-retinol to 11-cis-retinol and constitutive opsin phosphorylation but not in oxidation of 11-cis-retinol to 11-cis-retinal. Constitutive opsin phosphorylation could be an important element in the pathogenesis of LCA.

Fig. 3.

Isomerization, dehydrogenase activity, and phosphorylation of Rh in Rpe65 mice. A, isomerization of all-trans-retinol in RPE microsomes from wild type and Rpe65−/− mice. B, 11-cis-RDH activity in RPE microsomes from wild type and Rpe65−/− mice in the presence of different combinations of dinucleotides. C, immunolabeling of the Rpe mouse retina with monoclonal antibody anti-Rh A11–82P against phosphorylated Rh. Panel A, Rpe65+/+ at constant dark. ROS showed no labeling. Panel B, Rpe65−/− at constant dark. ROS were strongly labeled. Panel C, gavage 9-cis-retinal Rpe65−/− at constant dark without labeling. Panel D, ROS of Rpe65+/+ mice 15 min after the flash showed strong labeling. Panel E, gavage 9-cis-retinal Rpe65−/− mice 15 min after the flash. Immunolabeling is heavy throughout the ROS. In all of the sections, secondary antibody used for detection of anti-phosphorylated Rh antibody recognized choroidal blood vessels and anti-phosphorylated opsin antibody-labeled neurofilaments in inner retina. Scale bar, 20 μm. OS, outer segments; IS, inner segment layer; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer.

To directly measure the isomerase activity, RPE microsomes were isolated from Rpe65 mice using a novel procedure. In control experiments using RPE microsomes from Rpe65+/+ mice, 11-cis-retinol was produced from exogenously added all-trans-retinol only in the presence of RPE microsomes and CRALBP (Fig. 3A, upper panel). 11-cis-Retinol was absent when CRALBP was omitted, as well as when RPE microsomes or CRALBP were denatured by heat. 11-cis-Retinol was not detected in RPE microsomes from Rpe65−/− mice (Fig. 3A, lower panel).

Because 11-cis-retinol dehydrogenase (11-cis-RDH) was purified in a complex with RPE65 protein (21), oxidation of 11-cis-retinol was investigated in RPE microsomes from Rpe65 mice. Strong activity was detected in Rpe65+/+ and Rpe65−/− mice using NADPH and NADH as a dinucleotide cofactor (Fig. 3B). To distinguish NADPH-dependent activity from NADH-dependent activity, the test for dehydrogenase activity was carried out in the presence of nonradioactive NADH and [³H]NADPH. In such conditions, only NADPH-dependent dehydrogenase activity can be readily detected (Fig. 3B). The differences between Rpe65+/+ and Rpe65−/− were insignificant because this activity is much higher than required for normal flow of retinoids as determined from 11-cis-Rdh−/− mice (15). These data suggest that RPE microsomes from Rpe65−/− mice contain high NADPH-dependent and NADH-dependent dehydrogenase activities. In addition, no differences were seen in immunolocalization of 11-cis-RDH in the RPE of Rpe65 mice (data not shown).

Treatment with 9-cis-Retinal Restores Normal Rod Function—Because the ERG primarily reflects bipolar responses, the inability of 9-cis-retinal to provide complete recovery could be due to residual deficits in the photoreceptors or problems in signal transfer from rods to bipolar cells. To determine whether 9-cis-retinal treatment could restore normal photoreceptor function, we used suction electrodes to record the responses of single rods from Rpe65+/+ mice and untreated and treated Rpe65−/− mice (gavaged once at PND 30 and analyzed 48 h post-treatment).

Light-evoked changes in circulating dark current were recorded from outer segments of single rods from Rpe65+/+ mice or Rpe65−/− mice gavaged 0, 0.25, 1.25, or 2.5 mg of 9-cis-retinal (once a day for two consecutive days preceding the experiment). Retinoid analysis revealed that 300 ± 25 pmol of iso-Rh/eye was formed with a 2.5 mg dose of 9-cis-retinal, 109.8 pmol of iso-Rh/eye with a 1.25-mg dose, and 85.6 ± 6.2 of pmol/eye with a 0.25-mg dose. The nonlinear relation between the dose of 9-cis-retinal and the iso-Rh concentration presumably reflects accumulation in the liver and other tissues. All of the rod types listed supported light responses that increased with increasing flash strength to reach a maximum (saturating) amplitude when the light was bright enough to cause all of the cGMP channels to close and fully suppress the light-sensitive dark current of the cell. The response families from rods of each type are shown in Fig. 4A. They show that the amplitude of the saturating response increases with increasing doses of 9-cis-retinal. The relationship between mean dark current for each group of rods and the dose of 9-cis-retinal is plotted in Fig. 4B. Light-sensitive dark current in Rpe65−/− rods that received no supplemental 9-cis-retinal was 2.1 ± 0.3 pA, not significantly different from Rpe65−/− rods that received 0.25 mg of 9-cis-retinal (3.6 ± 0.9 pA). Rod dark current increased with larger doses of chromophore, reaching a value that was essentially the same as Rpe65+/+ when mice where given 2.5 mg of 9-cis-retinal.

Fig. 4.

Stimulus response families (A) for Rpe65+/+ and Rpe65−/− mice supplemented with 2.5, 1.25, 0.25, and 0 mg of 9-cis-retinal. Each panel is the mean family for five rods of each type. Flash (10 ms) strengths increased in two-fold steps from the dimmest intensity that was (in equivalent 500 nm photons/μm²): Rpe65+/+ (3.12), 2. 50 (10.14), 1.25 (190), 0.25 (192), and 0 (1425) mg of 9-cis-retinal. As determined by HPLC retinoid analysis, 300 ± 25 pmol iso-Rh/eye was formed with 2.5 mg, 109.8 pmol of iso-Rh/eye with 1.25 mg and 85.6 ± 6.2 pmol/eye with a 0.25-mg dose of 9-cis-retinal. B, mean light-sensitive dark current for the same sets of rods in A. The error bars are smaller than the symbols. C, mean linear range responses for same cells in A are scaled to the same peak amplitude and superimposed to illustrate differences in response kinetics.

Two other properties of the Rpe65−/− flash response varied with the amount of supplemental 9-cis-retinal, response kinetics, and light sensitivity. To illustrate the kinetic differences, the average dim flash response (in the cells linear range) was determined for each rod type. The mean responses from the five different sets of rods were scaled to the same peak amplitude and are compared in Fig. 4C. Responses recorded from Rpe65+/+ and Rpe65−/− rods from mice treated with 2.5 mg of 9-cis-retinal have essentially the same kinetics. The linear range responses are superimposed, showing that the dim flash responses of the two different rod types have the same time-to-peak and recovery times. Responses recorded from rods from Rpe65−/− mice gavaged with 1.25 or 0.25 mg of 9-cis-retinal are also essentially the same, with similar time-to-peak and recovery times; both are substantially faster than those of Rpe65+/+ (Fig. 4C, red and green traces). The dim flash kinetics of rod responses from Rpe65−/− mice that received no supplemental 9-cis-retinal (Fig. 4C, brown trace) were intermediate; they were faster than Rpe65+/+ but slower than rods from mice treated with 1.25 or 0.25 mg of 9-cis-retinal.

The differences in light sensitivity between Rpe65+/+ rods and rods from Rpe65−/− mice are shown in Fig. 5, which plots the stimulus response curves for each of the five experimental conditions (Rpe65+/+ and Rpe65−/− mice gavaged with 2.5, 1.25, 0.25, or 0 mg of 9-cis-retinal). The half-saturating flash intensity was lowest in Rpe65+/+ rods (∼30 photons/μm²) and increased by factors of 6, 66, and 131 in rods from mice gavaged with 2.5, 1.25, and 0.25 mg of 9-cis-retinal, respectively. The light sensitivity of rods from mice that did not receive 9-cis-retinal was the same as rods from mice that received the lowest dose (0.25 mg).

Fig. 5.

Mean stimulus response curves (n = 5) of Rpe65+/+ (squares) and Rpe65−/− mice treated with 2.5 (filled circles), 1.25 (open circles), 0.25 (filled triangles), and 0 (brown circles) mg of 9-cis-retinal. The differences in light sensitivity were evaluated by comparing the half-saturating flash intensity (I₀) obtained from fitting the mean data with an equation for exponential saturation (38).

$$ECA=A\cdot QE\cdot (1-{10}^{\alpha l})$$ (Eq 1)

where R is the peak amplitude of the response, R_max is the amplitude of the maximum response, and i is the flash strength in photons/μm². The solid lines are the exponential saturation function (Equation 3) fitted to data with I₀ (equivalent 500 nm photons/μm²): 25 (Rpe65+/+), 164 (2.5), 1995 (1.25), 3929 (0.25), and 3714 (0 mg of 9-cis-retinal). Inset, the kinetics of responses adapted by similar amounts (∼4-fold) by steady background illumination (336 equivalent 500-nm photons/μm²/s, black traces) in a Rpe65+/+ rod and by dark light (free opsin) in rod from Rpe65−/− mouse treated with 1.25 mg of 9-cis-retinal. Each trace is from a single rod and is the mean of 10–20 flashes either 6.25 (wild type) or 910 (Rpe65−/− 1.25 mg of 9-cis-retinal (500 nm photon/μm²/flash).

In the Absence of 9-cis-Retinal Treatment, 11-cis-Retinal Is Produced in Rpe65−/− Mice by Photoisomerization— Rpe65−/− mice that were never exposed to light have 11-cis-retinal (identified as oximes) below detection level in conventional microanalysis of retinoids (10). However, these mice respond to intense illumination in ERG experiments (10, 11) and in single cell recordings (current study). To identify whether 11-cis-retinal is produced by exposure to bright light, four or eight eyes were used for retinoid analysis instead of two eyes. For Rpe65−/−, no significant amounts of 11-cis-retinal were detected for dark-adapted animals (Fig. 6). When more eyes were used for analysis, less than 0.2 pmol/eye of 11-cis-retinal oximes were detected in a typical chromatogram. All-trans-retinal (4.2 ± 1.1 pmol/eye, n = 8) was present, and an intense flash converted this aldehyde to 2.1 ± 0.6 pmol/eye of 11-cis-retinal (Fig. 6A). The retinoids were identified by the retention time with authentic standards, and their UV spectra were measured during the chromatography. Next, it was important to determine whether photoisomerization resulted from the action of the “photo-isomerase” retinal G protein-coupled receptor protein. Double knockout Rpe65−/− Rgr−/− mice were generated, and retinoid analyses were carried out. A significant reduction in free all-trans-retinal was observed (2.2 ± 0.2 pmol/eye), but light flash photo-converted a similar fraction (∼50%) to 11-cis-retinal (Fig. 6). To identify where in the RPE or in the retina these retinals are present, retina and RPE were separated and analyzed individually (note that eight eyes were used). Clearly, the majority of all-trans-retinal was observed in the retina, whereas 11-cis-retinal was present mostly in the RPE. Bleaching converted all-trans-retinal to 11-cis-retinal that also resided in the retina (Fig. 6B). Once 11-cis-retinal is formed, its level does not change after 15, 30, or 120 min in the dark (data not shown).

Fig. 6.

Photoisomerization of all-trans-retinal in the eyes of Rpe65−/− and Rpe65−/− Rgr−/− mice. A, Rpe65−/− and Rpe65−/− Rgr−/− mice were exposed to a flash that bleached ∼30–35% of Rh in Rpe65+/+ mice. Light-dependent isomerization that resulted in the production of 11-cis-retinal was observed. Four eyes were analyzed. Note that light converts ∼50% of all-trans-retinal to 11-cis-retinal, where the smaller differences in the chromatogram are a result of higher absorption coefficient for all-trans-retinal compared with 11-cis-retinal. B, identification of retinals in Rpe65−/− mice in retina and RPE layers before and after flash. Eight eyes were analyzed. syn-13-cis-Retinal oxime is indicated by an asterisk. The experiments were done using mice, and the tissue was dissected under dim red illumination.

The ERG analyses of Rpe65−/− Rgr−/− mice were not qualitatively different from the responses obtained from Rpe65−/− mice (Fig. 2C, right panel). Together, these results indicate that there is a retinal photoisomerization pathway that produces 11-cis-retinal and regenerates Rh in prior bleached animals.

Different Methods of 9-cis-Retinal Delivery—An important point was to compare different ways to deliver 9-cis-retinoids with a goal to not only regenerate iso-Rh but to also build up reservoirs of cis-retinoids. Two methods² were tested: gavage (as described previously (10)) and intravenous injections. Intravenous injection is an efficient way of delivering retinoids, and there were no major differences between aldehyde and alcohol forms or their isomeric compositions (11-cis versus 9-cis)of cis-retinoids (data not shown). Intravenous injection of 9-cis-retinal produced iso-Rh when delivered with and without cyclodextrins (data not shown). Retinal was cleared out rapidly from the blood but could be stabilized in the circulation for a longer time in the presence of cyclodextrins (t_½ = 12 h versus 23 h) (data not shown). The addition of cyclodextrin, possibly by extending the time of circulation, also led to higher accumulation of 9-cis-retinyl esters in the liver or RPE (data not shown). A rapid clearance of 9-cis-retinal from the bloodstream makes it necessary to give multiple intravenous injections to fully regenerate iso-Rh. This is not the case with gavage, in which the presence of retinal in the bloodstream lasts for >48 h. Together, gavage and intravenous injections were effective in producing iso-Rh in Rpe65−/− mice. The advantages and disadvantages of both methods are described under “Discussion.”

DISCUSSION

The Role of RPE65 and LCA—Although the sequence of events that lead to the diseased state in Rpe65−/− mice, the animal model of LCA, has not been established, it is likely that the primary defect is an interruption of the retinoid cycle. This cycle is responsible for regenerating the visual pigment through the enzymatic conversion of all-trans-retinal to 11-cis-retinal in the RPE and its return to the photoreceptor cell. Disruption of the normal retinoid flow between the RPE and photoreceptor can explain the overaccumulation of retinal esters in the RPE. Furthermore, the failure to regenerate rhodopsin can account for diminished rod and cone light sensitivity (23, 24). The absence of 11-cis-retinal also increases free opsin in the photoreceptor. A high level of free opsin produces substantial activation of the phototransduction cascade (Figs. 3C, 4, and 5), mimicking the effects of continuous light exposure. This ongoing activity may cause the reduction in the thickness of the ROS layer and photoreceptor degeneration, effects also produced in animals exposed to continuous light (25-28). This sequence of events may be further aggravated by the phosphorylation of free opsin, which has been shown in other studies to lead to retinal degeneration (29, 30).

Early treatment of Rpe65−/− mice with 9-cis-retinal inhibited the accumulation of all-trans-retinal, improved the attachment contacts between RPE processes and ROS, led to dephosphorylation of opsin (Figs. 1D and 3C), and prevented the further progression of retinal degeneration. These observations suggest that ester accumulation in the RPE and the presence of high levels of active opsin in the photoreceptor may be the principle causes of retinal degeneration in the Rpe65−/− mouse.

Rescued Rod Function—The light sensitivity of rods from Rpe65−/− mice was restored in a dose-dependent manner by dietary supplemental 9-cis-retinal. The highest dose supported rod responses with normal sensitivity and kinetics (Fig. 4). Treatment with lower doses of 9-cis-retinal gave rise to rod responses that were desensitized and had faster kinetics, closely resembling the changes in sensitivity and kinetics that occur during steady background illumination in wild type rods. The changes in the light sensitivity of rod responses recorded from mice treated with the lower amounts of 9-cis-retinal could be accounted for by a combination of two factors. One source of desensitization was a decrease in the effective collecting area of the rod because of a reduction in both the amount of visual pigment and its quantum efficiency; the quantum efficiency of iso-Rh is about one-third that of Rh. The remaining reduction in sensitivity could be explained by steady activation of the transduction cascade by free opsin, producing an effect equivalent to that caused by steady background illumination in wild type rods.

Rods from Rpe65−/− mice that were not treated with 9-cis-retinal also generated light responses that were strongly desensitized. The presence of residual rod responses in untreated Rpe65−/− mice is consistent with previous reports of reduced but present light responses in children with LCA. Our results indicate that under these conditions the generation of light responses by flashes of intense light is most likely due to the production of 11-cis-retinal from the photoconversion of all-trans-retinal in the retina (Fig. 6). It is open to speculation whether all-trans-retinal is free or coupled (either covalently or noncovalently) to opsin. The preassociation of the chromophore and opsin would make the formation of the light-sensitive 11-cis-retinal complex (i.e. Rh) fast enough for it to be subsequently photoisomerized and transduction-triggered within the period of a brief (10 ms) flash of light.

Phototransduction in Rods of Rpe65 Mice—The shifts in light sensitivity rods from treated and untreated Rpe65 mice can be attributed to a decrease in the effective collecting area of the rod acting either alone (2.5 mg of 9-cis-retinal) or in addition to desensitization by an “equivalent background” (31) because of a low level of steady activation of the transduction cascade by free opsin (32).

The effective collecting area (ECA) depends on the geometric collecting area of the rod (A), the quantum efficiency of the pigment (QE), and the pigment density (α).

$$S_{\mathrm{f}}∕S_{\mathrm{f}}^{\mathrm{d}}=1∕1+I_{\mathrm{b}}∕I{\mid }_{\mathrm{b}}^{\mathrm{o}}$$ (Eq. 2)

where l is the path length. The pigment regenerated using 9-cis-retinal is iso-Rh, which has about one-third of the quantum efficiency of Rh (0.22 versus 0.67). The biochemical measurements indicate that in mice gavaged with 2.5 mg of 9-cis-retinal, all of the pigment is iso-Rh (no free opsin ± 10%) and is about 57% of the amount of rhodopsin in Rpe65+/+ rods (i.e. 300 pm iso-Rh versus 525 pm Rh). The decreases in quantum efficiency and axial pigment density would be expected to cause ∼5-fold decrease in effective collecting area of rods from mice fed with 2.5 mg of 9-cis-retinal. This is in agreement with the 6-fold increase in half-saturating flash strength in rods from Rpe65−/− mice gavaged with 2.5 mg of 9-cis-retinal, compared with rods of Rpe65+/+. In rods from mice treated with 1.25 and 0.25 mg of 9-cis-retinal, the axial densities of iso-Rh were 21 and 16%, respectively, of the amount of Rh in Rpe65+/+ rods. By the same reasoning as above, these changes would be expected to increase the half-saturating flash strength by 14.5- and 19-fold compared with Rpe65+/+. This is not enough to account for the observed shifts in sensitivity; rods from mice gavaged with 1.25 and 0.25 mg of 9-cis-retinal are further desensitized by factors of 4.5- and 6.8-fold, respectively.

The additional desensitization could be attributed to an equivalent background (31) that acts like “dark light” to cause steady activation of the cascade. In separate experiments on Rpe65+/+ rods the change in flash sensitivity by background illumination was described by the Weber-Fechner relationship.

$$R∕R_{\max }=1-{\exp }^{\ln 2\cdot i{I}_o}$$ (Eq. 3)

where S_f is the flash sensitivity in steady light, $S_{\mathrm{f}}^{\mathrm{d}}$ is flash sensitivity in darkness, I_b is the background light intensity, and $I_{\mathrm{b}}^{\mathrm{o}}$ is the background intensity (10⁸ photons/μm²/s) that reduces the flash sensitivity by half its dark value. Hence, background intensities of 378 and 648 photons/μm²/s would be expected to cause 4.5- and 7-fold changes in flash sensitivity. With an effective collecting area of 0.5 μm² and an integration time of 0.3 s, these background intensities correspond to equivalent activation in Rpe65+/+ rods of 57 and 97 Rh*/s.

We determined the equivalent background of residual free opsin in the treated Rpe65−/− rods by combining biochemical measurements of the free opsin concentration with physiological estimates of desensitization. The number of Rh molecules in a Rpe65+/+ rod is estimated to be about 2 × 10⁷ (i.e. 3 mm Rh in 0.02 pl). Biochemical measurements on rods from Rpe65−/− mice indicate that they make ∼40% less pigment than Rpe65+/+ 48 h after treatment. Thus, the number of iso-Rh molecules in rods from Rpe65−/− mice gavaged with 2.5 mg of 9-cis-retinal would be about 1.2 × 10⁷. Smaller doses of 9-cis-retinal do not regenerate all of the available pigment to form iso-Rh, causing there to be a pool of free opsin. The retinoid analysis suggests that the amount of free opsin in rods from Rpe65−/− mice gavaged with 1.25 and 0.25 mg of 9-cis-retinal would be 63 and 72% of the total amount of available pigment (i.e. 7.5–8.6 × 10⁶ molecules). For this amount of free opsin to cause desensitization in the rods from Rpe65−/− mice that is equivalent to the desensitization in Rpe65+/+ rods caused by a steady light that bleaches 57 and 97 Rh*/s, about 1 × 10⁵ opsin would have to activate the cascade as well as 1 Rh* (1.3–0.9 × 10⁵ opsin: Rh*). This value is broadly consistent with previous estimates of activation ratio of free opsin: Rh* (i.e. 10⁶:1) (33). The inset in Fig. 5 shows that background light adaptation and adaptation by an equivalent (free opsin) background that desensitized the flash response by similar amounts had similar effects on the kinetics of the dim flash response. This is also in general agreement with previous studies (32) that showed the adaptational changes in the kinetics of the dim flash response were similar, whether adaptation was due to background light or the equivalent background associated with dark adaptation.

The highly desensitized rod responses recorded from untreated Rpe65−/− mice did not show the acceleration in response kinetics seen in rods from treated mice (Fig. 4C). There are several possible explanations for this difference. One possibility is that the activity of free opsin is less in rods from untreated Rpe65−/− mice than in those from treated mice, perhaps because of phosphorylation of the opsin in untreated rods. This explanation would require that treatment with a low dose of 9-cis-retinal converts most or all of the remaining free opsin to a state of higher activity, perhaps through dephosphorylation. Another possibility is that the activation and deactivation of the photopigment are altered in the untreated mice. For example, it is not clear that the photopigment created by photoconversion is identical to normal rhodopsin; for example, the opsin may still be phosphorylated. Further studies are required to distinguish these possibilities.

The complete or nearly complete rescue of normal rod function after treatment with 9-cis-retinal contrasted with the partial rescue of the sensitivity of the electroretinogram. Because the electroretinogram primarily reflects activity of bipolar cells, this difference indicates that responses in the rods are not properly transmitted across the rod-bipolar synapse. It is possible this synapse does not develop properly in Rpe65−/− mice because of a lack of visual signals. Continuous treatment with 9-cis-retinal from birth may help remedy this problem.

Advantages and Disadvantages of 9-cis-Retinal Treatments—Retinals can be delivered to the eye effectively by one (or a combination) of two methods: gavage (10) and intravenous injection (current study). The most effective delivery system is gavage, which restores visual pigment in 1–2 days and also produces accumulation of 9-cis-retinyl esters in the liver and RPE microsomes. It is a highly reproducible procedure. There is a transient elevation of retinoids in the blood for 48 h that is followed by recovery to the normal level. The only noticeable drawback is that much of the retinoid is secreted rather than stored, requiring a higher dose than other delivery methods.

Intravenous injection is also an effective method for retinoid delivery to the eye, but it has the disadvantage of the retinoids being rapidly eliminated from the bloodstream by the kidneys. This can be prevented to some degree by “caging” retinal in a cyclodextrin net. For full regeneration, multiple or large doses must be injected, causing potential problems with local infection. To lower the amounts of circulating all-trans-retinoids, it would be helpful to inhibit liver carboxylesterase to prevent all-trans-retinal from being released to the bloodstream. Such inhibitors, if they are potent, are highly toxic, because they inhibit other processes that require hydrolase activity. General and mild inhibitors, such as vitamins K₁ and E (34), are effective to some degree,³ but more specific inhibitors would be useful to enhance the level of cis-retinoids in the bloodstream. Finally, intraocular injection (22) is an option in same cases. It requires the smallest amounts of material and is useful for specialized retinoids, but this is not a readily accessible or routine procedure, and repeated intraocular injections are associated with the formation of cataracts (20–40%).

There is not a large reservoir of cis-retinoids in the liver and RPE, most likely because of nonenzymatic conversion of free retinal or retinol to the all-trans isomer (22). However, the efficiency of mammalian vision is remarkable and worth consideration in light of potential cis-retinoid therapy. For example, the mammalian retina contains ∼10⁸ photoreceptors. If each photoreceptor absorbs on average 1–2 × 10³ photons/s, with a quantum yield of 0.65 (or 0.3 for 9-cis-retinal), the daily requirement of 11-cis-retinal is only <1 μg, an amount that could be easily delivered by dietary supplement even if the majority of retinoids are retained in liver or secreted. The recommendations for vitamin A intake is 0.8 mg/day for men and 0.7 mg/day for women, with the upper safety limit of 3 mg/day is only an estimate, because of lack of data (35).

Multiple gavages do not increase the amount of retinyl esters in the eye. In contrast, early intervention significantly lowers the accumulation of all-trans-retinyl esters (Fig. 1). This could be one of the prerequisites of successful cis-retinoid therapy for retinal diseases. The level of all-trans-retinyl esters in the RPE is predetermined by the time of the intervention. If the treatment is initiated very early in life, the esters only gradually increase with age, as in wild type mice. The treatment does not remove the esters from the eye but prevents accumulation of the esters. One possible explanation is that the retina sends a signal that opsin is not regenerated, and this causes retinol capture from the blood circulation and retention as retinyl ester in RPE. When retinyl esters cannot be converted to 11-cis-retinal, and the “opsin signal” is on, these two factors ultimately lead to ester accumulation. The mechanism of such communication is unknown on a molecular level.

In all of the experiments described here, we did not observe any adverse effects of 9-cis-retinal. However, in applying these results to other species, it is important to note that retinoid flow in the mouse may be substantially different from in other animals. For example, dogs have very high levels of retinoids in the bloodstream (36, 37) that will need to be combated to efficiently deliver cis-retinoids to the eye via gavage.

In summary, we provide evidence that administration of 9-cis-retinal restores rod photopigment and rod retinal function for more than 6 months and that early intervention significantly attenuates the ester accumulation. Opsin in Rpe65−/− mice is constitutively phosphorylated in rods of Rpe65−/− mice, and this modification of the visual pigment could be involved in the pathophysiology of LCA; fortunately, after 9-cis-retinal-treatment, opsin is dephosphorylated. We also provide evidence that the source of 11-cis-retinal in Rpe65−/− mice results from photoisomerization of all-trans-retinal present in the retina and that other mechanisms in addition to photoisomerase retinal G protein-coupled receptor are involved in this process, as shown in double Rpe65−/− Rgr−/− knockout mice. Electrophysiological data using single cell recordings suggest that 11-cis-retinal is formed in situ in rod outer segments. These studies provide information about the etiology of LCA on a molecular level and demonstrate that pharmacological intervention produces long lasting preservation of the visual function in dark-reared Rpe65−/− mice.