Molecular Vision 2005; 11:152-162 <http://www.molvis.org/molvis/v11/a17/> Received 6 August 2004 | Accepted 23 February 2005 | Published 28 February 2005

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Retinal Degeneration 12 (rd12): A new, spontaneously arising mouse model for human Leber congenital amaurosis (LCA)

Ji-jing Pang,¹ Bo Chang,² Norman L. Hawes,² Ronald E. Hurd,² Muriel T. Davisson,² Jie Li,³ Syed M. Noorwez,¹ Ritu Malhotra,¹ J. Hugh McDowell,¹ Shalesh Kaushal,¹ William W. Hauswirth,¹ Steven Nusinowitz,⁴ Debra A. Thompson,⁵ John R. Heckenlively⁵
 
(The first two authors contributed equally to this publication)
 
 

Departments of ¹Ophthalmology and ³Neuroscience, College of Medicine, University of Florida, Gainesville, FL; ²The Jackson Laboratory, Bar Harbor, ME; ⁴Jules Stein Eye Institute, Harbor-UCLA Medical Center, Torrance, CA; ⁵W. K. Kellogg Eye Center, The University of Michigan, Ann Arbor, MI

Correspondence to: Bo Chang, MD, The Jackson Laboratory, 600 Main Street, Bar Harbor, ME 04609; Phone: (207) 288-6394; FAX: (207) 288-6149; email: bchang@jax.org

Abstract

Purpose: To report the phenotype and characterization of a new, naturally occurring mouse model of hereditary retinal degeneration (rd12).

Methods: The retinal phenotype of rd12 mice were studied using serial indirect ophthalmoscopy, fundus photography, electroretinography (ERG), genetic analysis including linkage studies and gene identification, immunohistochemistry, and biochemical analysis.

Results: Mice homozygous for the rd12 mutation showed small punctate white spots on fundus examination at 5 months of age. The retina in the rd12 homozygote had a normal appearance at the light microscopic level until 6 weeks of age when occasional voids appeared in the outer segments (OS) of the photoreceptor (PR) cells. The outer nuclear layer (ONL) appeared normal until 3 months of age though more obvious voids were detected in the OS. By 7 months of age, 6 to 8 layers of ONL remained in the mutant retina, and the OS were obviously shorter. The first sign of retinal degeneration was detected at the electron microscopic level around 3 weeks of age when occasional small lipid-like droplets were detected in the retinal pigment epithelium (RPE). By 3 months of age, much larger, lipid-like droplets accumulated in RPE cells accompanied by some OS degeneration. While the histology indicated a relatively slow retinal degeneration in the rd12 homozygous mutant mice, the rod ERG response was profoundly diminished even at 3 weeks of age. Genetic analysis showed that rd12 was an autosomal recessive mutation and mapped to mouse chromosome 3 closely linked to D3Mit19, a location known to be near the mouse Rpe65 gene. Sequence analysis showed that the mouse retinal degeneration is caused by a nonsense mutation in exon 3 of the Rpe65 gene, and the gene symbol for the rd12 mutation has been updated to Rpe65^rd12 to reflect this. No RPE65 expression, 11-cis retinal, or rhodopsin could be detected in retinas from rd12 homozygotes, while retinyl esters were found to accumulate in the retinal pigment epithelium (RPE).

Conclusions: Mutations in the retinal pigment epithelium gene encoding RPE65 cause an early onset autosomal recessive form of human retinitis pigmentosa, known as Leber congenital amaurosis (LCA), which results in blindness or severely impaired vision in children. A naturally arising mouse Rpe65 mutation provides a good model for studying the pathology of human RPE65 mutations and the effects of retinyl ester accumulation.

Introduction

Leber congenital amaurosis (LCA) is the designation for a group of autosomal recessive blinding retinal dystrophies that represent the most common genetic causes of congenital visual impairment in infants and children. About 10% of LCA cases are caused by mutations in the gene encoding RPE65 [1,2]. RPE65 is a highly conserved 61 kDa protein that is present in the smooth endoplasmic reticulum of the retinal pigment epithelium (RPE) [3], and is essential for the conversion of vitamin A from all-trans retinol to 11-cis retinal, the chromophore of the visual pigments [4]. The Rpe65 gene has been mapped in the mouse to chromosome 3 (Chr 3) in an interspecific backcross [5]. The Rpe65 gene consists of 14 exons encoding a 533 amino acid protein [6]. To date, RPE65 studies in mouse have relied heavily on a knockout mouse reported by Redmond et al. [7]. Rpe65^-/- mice develop a slow retinal degeneration accompanied by over accumulation of all-trans-retinyl esters in the RPE, while 11-cis-retinyl esters and 11-cis retinal are absent. Concomitantly, outer segment discs of rod photoreceptors in Rpe65^-/- mice become disorganized compared with those of Rpe65^+/+ and Rpe65^+/- mice. Rpe65^-/- mice have severely depressed light and dark adapted electroretinogram (ERG) responses as a result of low levels of chromophore [7,8]. Residual responses are attributed to rod function having decreased sensitivity (similar to that of cones) [9] and proposed to be sustained by small amounts of 9-cis retinal that serves as the chromophore for opsin [10]. The associated pathogenic mechanism likely is due to increased levels of unregenerated opsin apoprotein that result in constitutive activation of the visual cascade [11].

Mouse models of spontaneous retinal degenerations have been used for many years to provide insight into the etiologies of human retinal degenerations and to provide retinal tissue to study the pathology of disease progression. Mouse models of retinal degeneration have provided good initial templates for gene and pharmacological therapies. Many of these models have come from screening mice from genetically independent mouse strains and stocks at The Jackson Laboratory (TJL) by indirect ophthalmoscopy and electroretinography (ERG) [12-16]. In the present study, we have identified a new retinal degeneration mutation, retinal degeneration 12 (rd12), which is associated with distinctive white dots on the retina that develop with age. We show that the rd12 retinal degeneration is caused by a nonsense mutation in exon 3 of the Rpe65 gene. Our functional and biochemical studies confirm that vitamin A metabolism and visual processing are disrupted in the rd12 mouse. This naturally occurring mutation (Rpe65^rd12) provides another valuable mouse model for LCA.

Methods

Animals

The mice in this study were bred and maintained in standardized conditions in the Research Animal Facility at TJL and the University of Florida. They were maintained on NIH31 6% fat chow and acidified water, with a 14 h light/10 h dark cycle in conventional facilities that were monitored regularly to maintain a pathogen free environment. All experiments were approved by the Institutional Animal Care and Use Committees and conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

Origin

rd12 was discovered in one male mouse of the B6.A-H2-T18^a/BoyEg strain at 10 months of age; at that time small, discrete white dots were present throughout the fundus (Figure 1). This male mouse was mated to a C57BL/6J female and then mated to the F1 female mice. The F1 mice had normal retinas, but some of the backcrossed mice showed a similar retinal phenotype with small, discrete dots in the fundus. These affected mice were mated with each other to produce the rd12 mouse colony. Subsequently, the rd12 stock was maintained by repeated backcrossing to C57BL/6J to make a congenic inbred strain, hereafter referred to as B6-rd12.

Clinical retinal evaluation

Twenty mice used in clinical characterization studies had pupils dilated with 1% atropine ophthalmic eye drops and were evaluated by indirect ophthalmoscopy with a 78 D lens. Signs of retinal degeneration, such as vessel attenuation, alterations in the RPE, and presence or absence of retinal dots were noted. Fundus photographs were taken with a Kowa Genesis small animal fundus camera (Torrance, CA) [17].

Electroretinography

After at least 6 h of dark adaptation, mice were anesthetized with an intraperitoneal injection of normal saline solution containing ketamine (15 mg/g) and xylazine (7 mg/g body weight). Electroretinograms (ERGs) were recorded from the corneal surface of one eye after pupil dilation (1% atropine sulfate) using a gold loop electrode referenced to a gold wire in the mouth. A needle electrode placed in the tail served as ground. A drop of methylcellulose (2.5%) was placed on the corneal surface to ensure electrical contact and to maintain corneal integrity. Body temperature was maintained at a constant temperature of 38 °C using a heated water pad. All stimuli were presented in a Ganzfeld dome (LKC Technologies, Gaithersburg, MD) whose interior surface was painted with a highly reflective white matte paint (No. 6080; Eastman Kodak, Rochester, NY). Stimuli were generated with a Grass Photic Stimulator (model PS33 Plus; Grass Instruments, Worcester, MA) affixed to the outside of the dome at 90° to the viewing porthole. Dark adapted responses were recorded to short wavelength (λ_max = 470 nm; Wratten 47A filter) flashes of light over a 4.0 log unit range of intensities (0.3 log unit steps) up to the maximum allowable by the photic stimulator. Light adapted responses were obtained with white flashes (0.3 steps) on the rod saturating background after 10 min of exposure to the background light to allow complete light adaptation. Responses were amplified (Grass CP511 AC amplifier, x10,000; 3 dB down at 2 and 10,000 Hz) and digitized using an I/O board (model PCI-1200; National Instruments, Austin, TX) in a personal computer. Signal processing was performed with custom software (LabWindows/CVI; National Instruments). Signals were sampled every 0.8 ms over a response window of 200 ms. For each stimulus condition, responses were computer averaged with up to 50 records averaged for the weakest signals. A signal rejection window could be adjusted during data collection to eliminate electrical artifacts.

Gene mapping and sequencing

To determine the chromosomal location of the rd12 gene, we mated B6-rd12 mice to CAST/EiJ mice. The F1 mice, which exhibit no retinal abnormalities, were backcrossed (BC) to B6-rd12 mice. Tail DNA was isolated as previously reported [18]. DNAs of the 92 BC offspring were genotyped using microsatellite markers to develop a structure map of the region. For PCR amplification, 25 ng DNA was used in a 10 μl volume containing 50 mM KCl, 10 mM Tris-HCl, pH 8.3, 2.5 mM MgCl₂, 0.2 mM oligonucleotides, 200 μM dNTPs, and 0.02 U AmpliTaq DNA polymerase. The reactions, were initially denatured for 3 min at 94 °C, then subjected to 40 cycles of 15 s at 94 °C, 1 min at 51 °C, 1 min at 72 °C, and then a final 7 min extension at 72 °C. PCR products were separated by electrophoresis on 3% MetaPhor (FMC, Rockland, ME) agarose gels and visualized under UV light after staining with ethidium bromide. Initially a genome scan of microsatellite (Mit) DNA markers was carried out on pooled DNA samples [19]. After detection of linkage on Chromosome 3, the microsatellite markers D3Mit19, D3Mit15, D3Mit11 were scored on individual DNA samples. To test the Rpe65 gene as a candidate, we designed four pairs of PCR primers based on mouse coding sequence from Celera mouse genomic sequence (Celera, Foster City, CA; Biosystems) to amplify overlapping cDNA fragments. The human RPE65 mRNA (NM_000329) sequence was used to blast the Celera mouse genome sequences. For direct sequencing, the PCR reaction was scaled up to 30 μl; amplification was done for 36 cycles with a 15 s denaturing step at 94 °C, a 2 min annealing step at 60 °C, and a 2 min extension step at 72 °C. PCR products were purified from agarose gels using a Qiagen kit (Qiagen Inc., Valencia, CA). Sequencing reactions were carried out with automated fluorescence tag sequencing. Total RNA was isolated from retinas of newborn mice by TRIZOL LS Reagent (Invitrogen life technologies, Carlsbad, CA) and the SuperScript^TM preamplification system (Invitrogen life technologies) was used to make first strand cDNA. Primers used in the study are shown in Table 1.

Light and transmission electron microscopy (TEM)

Both eyes from rd12 mutant mice and age matched C57BL/6J mice were enucleated and eyecups were prepared for light and electron microscopic examination with previously described procedures [20,21]. 2-6 eyes were used for each age group. Eyes were immediately removed and immersed in cold fixative, 4% glutaraldehyde in 0.1 M phosphate buffer. Corneas were removed and the eyes left in fixative for 24 h. The lens was then removed followed by dehydration with a graded series of increasing ethanol concentrations. Eyecups were embedded in Epon mixture. For each sample, 0.5 μm thin sections were stained with toluidine blue for light microscopy followed by ultra thin section preparation for TEM examination.

Immunocytochemistry for Rpe65 expression

Three eyes from C57BL/6J and 3 eyes from rd12 homozygotes at 2 month of age were fixed with 4% paraformaldehyde. Eyecups were prepared as described above, and frozen with Tissue Freezing Medium (Triangle Biomedical Sciences, Durham, NC). Following permeabilization with 0.1% Triton X-100, 10 μm frozen sections were rinsed in 0.1 M PBS, blocked in 20% normal goat serum (NGS), then incubated overnight at 4 °C in rabbit polyclonal raised anti-human/bovine RPE65 antibodies (kindly provided by Dr. T. Michael Redmond, NEI, NIH), which were diluted in NGS to 1:400. After 3 rinses with 0.1 M PBS, sections were incubated in goat anti-rabbit Texas red (1:300, Molecular Probes, Eugene, OR) for 2 h followed by 3 rinses with 0.1 M PBS. Sections were then mounted with coverslips before fluorescence photography.

Purification of rhodopsin

Normal C57BL/6J and rd12 homozygotes were dark adapted overnight, euthanized, and the eyecups were prepared by removing the cornea and lens under dim red light for each experiment. Four eyes from different mice were used for each age. Retinas were then separated from the eyecups and collected individually in 1.0 ml buffer A (10 mM sodium phosphate buffer, 137 mM NaCl, 1.0% n-Dodecyl-β-D-maltopyranoside [DM] and complete protease inhibitor cocktail [Roche Diagnostics Corporation Indianapolis, IN], pH 7.0) and sonicated for 10 s. The samples were agitated gently overnight at 4 °C in the dark. The rhodopsin was purified essentially as described by Noorwez et al. [22]. Briefly, the tubes were centrifuged at 36,000 rpm for 15 min at 4 °C in a Beckman tabletop ultracentrifuge and the supernatants collected and incubated with 1D4 coupled Sepharose 4B beads (by coupling 1D4 to CNBr activated sepharose beads from Pharmacia according to their instructions) for 3.5 h at 4 °C. The beads were washed with buffer B (10 mM sodium phosphate buffer, 0.1% DM, and complete protease inhibitor cocktail, pH 6.0) and rhodopsin eluted in buffer B containing 0.1 mM synthetic peptide corresponding to the C-terminal 18 amino acids of rhodopsin for 1 h. The tubes were spun briefly, the supernatants collected. The UV/visible spectra were recorded with a Tidas II spectrophotometer (World Precision Instruments, Sarasota, FL).

Retinoid analysis

All procedures were carried out under dim red light (>660 nm). Retinoids were analyzed following slight modifications of the procedures of Groenendijk and Smith [23,24]. Freshly purified rhodopsin from mouse retina was dried under a stream of nitrogen at room temperature. A 1:1 mixture of methanol and 1 M NH₂OH (pH 6.5), 0.1 ml, was added to each sample and the samples were mixed on a vortex shaker. Methanol was added to 70% and the samples were mixed again in a 1.7 ml centrifuge tube. Water and dichloromethane were added to yield 1:1:1 ratio of water:methanol:dichloromethane. The extracts were vortexed, briefly centrifuged, and the lower organic phase collected. Addition of dichloromethane, vortexing, centrifugation and collection of lower phase was repeated twice. The collected organic phases were combined, and dried under a stream of nitrogen gas. Samples were stored in the dark at -80 °C if not analysed immediately. The residue was dissolved in 25 μl of 1% isopropyl alcohol in hexane and an aliquot was immediately injected onto an HPLC column (Luna, 5μ Silica (2) from Phenomenex Torrance, CA) and resolved according to the procedure of Smith and Goldsmith [24] using an isocratic elution with 9% ethyl acetate in hexane.

Retinyl ester quantification and analysis

At least 3 animals (6 eyes) were used for analysis at each age. From each mouse the two eyecups with their RPE cell layer intact were homogenized in 200 μl PBS. The homogenate was then extracted with 300 μl cold methanol and 300 μl hexane or 300 μl of dichloromethane and centrifuged at 10,000 x g for 5 min. The organic phase was collected and the aqueous phase was extracted twice more. The organic phase was dried under a stream of nitrogen, dissolved in 25 μl of 1% isopropyl alcohol and typically 20 μl was used to analyze for retinyl ester content by HPLC. The analysis was performed using a Luna, 5μ Silica (2) HPLC column from Phenomenex. The analysis was performed as described by Maeda et al. [25] with slight modifications to the conditions focusing on the retinyl ester elution region. After injection, the column was eluted with 1% ethyl acetate in hexane for 15 min. The column was then washed with 8% ethyl acetate in hexane for at least 5 min and then equilibrated with 1% ethyl acetate in hexane for at least 5 min.

Results

Clinical phenotype

Mice homozygous for rd12 showed small evenly spaced white dots throughout their retinas (Figure 1). These small white dots became apparent upon ophthalmoscopic examination by 5 months of age. They are under retinal vessels (that is, vessels pass over the white dots). The fundus developed a mildly pigmented granular and mottled appearance by 15 months of age; individual spots could still be seen, although less frequently, in mice up to about 2 years of age (data not shown).

ERG phenotype

ERG records obtained from rd12 mice at 1 and 8 months of age are shown in Figure 2. Recordings obtained from a 1 month old C57BL/6J mouse are shown for comparison. Under dark adapted conditions, homozygous rd12 mice exhibited recordable, but severely attenuated, ERG signals to the brightest stimuli, even at the earliest age tested (Figure 2B). A comparison of response amplitudes at 1 and 8 months of age relative to a normal control mouse is shown in Figure 2D. Under light adapted conditions, ERG responses were significantly more robust, with response amplitudes at or near the normal range (Figure 2H). However, the timing of peak components was delayed with respect to normal at all ages with progressive delays and amplitude loss with age (data not shown).

Genetic analysis

Genetic analysis showed that rd12 is an autosomal recessive mutation that maps to mouse Chromosome 3. It is closely linked to D3Mit19 (no crossover was found between rd12 and D3Mit19 in 92 linkage samples), suggesting that the human homolog might be on Chromosome 1p31 where the human RPE65 gene is located (Figure 3A,B). Sequence analysis of Rpe65 cDNA from rd12 homozygotes showed a single base substitution at position 130 (C to T) in exon 3, which leads to a stop codon at amino acid position 44 (CGA to TGA, Figure 3C). This single base substitution was confirmed by sequencing four more rd12 homozygotes. There were no other changes in the Rpe65 gene sequence compared to C57BL/6J (wildtype). We have deposited the C57BL/6J cDNA sequence of the Rpe65 gene in GenBank (AF410461). Since the retinal degeneration in rd12 homozygotes was caused by a nonsense mutation in exon 3 of the Rpe65 gene, the gene symbol for the rd12 mutation has been changed to Rpe65^rd12.

Morphological phenotype

By light microscopy, there was little change in photoreceptor cells at 6 weeks except occasional voids in the rod outer segments (OS) of rd12 homozygotes (Figure 4A). Such voids increased progressively in frequency and size and became very obvious by 3 months of age (Figure 4B). However, the thicknesses of the OS layer and the outer nuclear layer (ONL) were maintained (Figure 4B). By 7 months of age (Figure 4C), both OS and ONL became shortened. The ONL in rd12 homozygotes at 7 months contained 6 to 7 layers of nuclei compared with 10 to 11 layers in normal C57BL/6J mice. By 27 months (Figure 4D), the OS of rods were nearly absent while the ONL was decreased to 3-4 layers in rd12 homozygotes. RPE cells at this age became atrophied and hypopigmented.

By TEM, RPE cells appeared normal except occasional small atypical lipid-like droplets at the age of 3 weeks in rd12 homozygotes (data not shown). The frequency and size of lipid-like droplets increased slowly with age. By 3 months, relative to the normal C57BL/6J mice (Figure 5A), there was significant accumulation of such droplets in the RPE cytoplasm of rd12, with more degenerating OS, perhaps corresponding to OS voids in light microscopic picture at the same age (Figure 5B). Lipid-like droplets became progressively more frequent and larger in RPE cells with an accompanying OS shortening with age (data not shown). In contrast, there were no comparable droplets detected at any age in the RPE of C57BL/6J mice.

Biochemical phenotype

Studies of RPE65 expression using immunohistochemical analysis of retinal sections with an anti-RPE65 antibody showed that RPE65 protein was mainly localized to RPE cells in C57BL/6J mice, but was undetectable in rd12 homozygotes (Figure 6). Quantitation of rhodopsin visual pigment, formed when opsin apoprotein combines with the chromophore 11-cis retinal, showed the typical UV/visible spectrum with an absorption maximum at 500 nm in C57BL/6J mice. The absorption spectra from different experiments varied by less than 5%. A representative spectrum is shown in Figure 7. No rhodopsin absorbance was detected in rd12 homozygotes at a variety of ages from 2 weeks to 5 months, while rhodopsin had reached nearly its adult level by 5 weeks in normal C57BL/6J retina (Figure 7). In addition, the typical purified opsin protein peak at 280 nm, detectable in C57BL/6J mouse retinas as early as 8 days after birth (data not shown), was much lower in rd12 homozygotes (Figure 7). This lower level of opsin expression in rd12 homozygotes was confirmed by western blot analysis (data not shown). Levels of 11-cis retinal extracted from purified rhodopsin preparations and quantitated by HPLC analysis were undectable in the retinas of rd12 homozygotes (data not shown). Levels of retinyl esters in normal C57BL/6J and rd12 mice could be detected at about 3 weeks of age. In normal C57BL/6J mice, the levels remained low and stable thereafter (Figure 8). In contrast, retinyl esters in rd12 homozygotes were detected at levels similar to normal at 3 weeks of age but increased dramatically with increasing age. While the analyses were quite variable, from 6 weeks of age there was a clear over accumulation of retinyl esters relative to normal controls. Compared to C57BL/6J mice, retinal esters in rd12 homozygotes were approximately 10 fold higher by 5 months of age when the typical retinal white dots first appeared in the fundus of rd12 homozygotes.

Discussion

With the model reported in this paper, there are now two mouse models for the human disease resulting from mutations in RPE65. The first model was created by homologous recombination involving replacement of the first three exons and intervening introns of Rpe65 by a neomycin resistance cassette [7]. No fundus abnormalities in this Rpe65 knockout mouse model were reported. This is in contrast to the rd12 mice, which have small white dots throughout their retinas. These are most easily seen at 6 to 9 months of age. It is not clear that such late stages were examined in the Rpe65 knockout mouse. In the model we now describe, a naturally occurring homozygous 130 C to T transition creates a premature stop codon, R44X, in exon 3 that is predicted to result in loss of function due to severe truncation of the protein and nonsense mediated mRNA degradation. At least five other nonsense mutations are known among the more than 60 RPE65 mutations found in retinal dystrophy patients, with over half of all reported mutations predicting premature stop codons due to splice site defects, point mutations, and small rearrangements [26]. The retinas of some of these Rpe65 lesion patients (RP20) have little white dots [27]. RPE65 loss of function due to the R44X mutation in rd12 homozygotes was confirmed in our studies by analysis of retinal function, protein expression, and vitamin A metabolism. A complementation test between Rpe65^rd12 and Rpe65 knockout was not done; however, phenotypic rescue is seen upon gene replacement therapy (data to be published).

Mice homozygous for rd12 have small, evenly spaced yellowish white dots throughout their retinas. These small white dots become apparent upon ophthalmoscopic examination by 5 months of age. The clinical phenotype of retinal spots and flecks associated with retinal degeneration in rd12 homozygotes is similar to human fundus albipunctatus (FA) caused by biochemical defects in the RDH5 gene encoding 11-cis retinol dehydrogenase [28]. The Rdh5 gene maps to distal mouse Chromosome 10 [29,30], whereas the rd12 mutation is located on distal Chromosome 3 where the Rpe65 gene is located. Vitamin A deficiency also can be associated with white dots evenly distributed throughout the fundus and such a phenotype can be difficult to distinguish from FA. However, with vitamin A therapy, spots correlated with the deficient state rapidly disappear [31]. We therefore suggest that the clinical phenotype of the rd12/rd12 mouse retina further supports the proposed role of RPE65 in disease and in vitamin A metabolism in the visual cycle.

Our biochemical studies of rd12 homozygotes are consistent with previous findings that RPE65 is required for the conversion of vitamin A to 11-cis retinal by the RPE [4]. The ERG phenotype in the rd12 mouse is similar to that previously reported in RPE65 deficient animal models in which ERG responses were essentially undetectable to all but the brightest stimuli. A recent study investigating the origin of residual function in the RPE65 -/- mouse suggests that a desensitized rod system is responsible for the detectable signals at the highest intensities, even under light adapted conditions [9]. We find no detectable 11-cis retinal in the opsin purified from the rd12 mouse, and a steady accumulation of retinyl esters in the RPE cells with age. In the absence of RPE65, all-trans retinyl esters are expected to accumulate in droplets within RPE cells, consistent with a block in 11-cis retinal synthesis following esterification of vitamin A to membrane phospholipids before the isomerization reaction. Although the details of the role of RPE65 in the isomerase reaction have not been fully elucidated, recent studies suggest that RPE65 is a retinyl ester binding protein that may function to present all-trans retinyl esters to the isomerase [32-34]. The requirement for RPE65 in the synthesis of 11-cis retinal has been further substantiated by the finding that addition of recombinant RPE65 to RPE microsomal membranes prepared from Rpe65 knockout mice restores retinoid isomerase activity to the preparations [35,36]. Accordingly, adding 11-cis retinal to retinal organ cultures or homogenized retinas from young rd12 mice leads to formation of rhodopsin (data not shown).

As a result of decreased 11-cis retinal synthesis, rd12 homozygotes do not generate normal levels of visual pigments and have severely depressed dark adapted ERG responses. A similar phenotype is also present in a strain of Swedish Briard dogs carrying a functionally null allele of canine Rpe65 [37,38] that are afflicted with progressive retinal dystrophy [39,40]. Affected Briards were recently used in the first successful gene replacement therapy experiments in a large animal model of retinal degeneration [41]. Similar treatment has been attempted in the rd12 mouse with a significant fraction of the visual function restoration [42], but in the Rpe65 knockout mouse with limited success [43]. In other studies, administration of oral retinoids to Rpe65 knockout mice resulted in partial restoration of visual pigment and function [8,44]. The same feeding experiment has not yet been done in our laboratory, but rd12 mice are available from TJL. The identification of rd12 as a spontaneously arising mouse model, which has similar RPE65 mutations to those found in human patients now makes available a powerful new independent model for studying the pathogenesis and progression of LCA. One of the most exciting future uses is likely to involve the study of therapies for LCA and autosomal recessive retinitis pigmentosa in humans.

Acknowledgements

We thank The Jackson Laboratory Microchemistry Service for DNA sequencing. These data were generated through the use of GenBank, Celera Discovery System, and Celera Genomics' associated databases. We also thank Dr. T. Michael Redmond (NEI, NIH) for the RPE65 antibody and Denifield W. Player, Dept. of Anatomy and Cell Biology at University of Florida for the EM pictures. This study was supported by National Institutes of Health Grants EY07758, EY11123, EY11996, EY13729, RR01183, and CA34196 and Research to Prevent Blindness, Inc. Parts of this work were presented in abstract form at the annual meeting of The Association for Research in Vision and Ophthalmology, May 2002, Ft. Lauderdale, FL.

W. W. Hauswirth has a financial interest in the use of AAV vectors for treating retinal diseases associated with his involvement with Applied Genetic Technologies Corporation (Alachua, FL).

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