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1From the Departments of Ophthalmology and 2Physiology and Neuroscience, Medical University of South Carolina, Charleston, South Carolina; the 3Ocular Genetics Unit, Department of Genetics, Trinity College, Dublin, Ireland; the 4National Eye Institute, National Institutes of Health, Bethesda, Maryland; and the 5Retinal Electrodiagnostics Research Group, Department of Ophthalmology II, Eberhard-Karls University, Tübingen, Germany.
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METHODS. Rpe65–/–::Rho–/– mice were used to remove any interference of rods and compared with wild-type (wt) mice. Pups were injected intraperitoneally with 11-cis retinal, starting at postnatal day (P)10, and were maintained in complete darkness. At P25, cone function was assessed with photopic single-flash and flicker ERGs. Cone survival was determined immunohistochemically with cone-specific antibodies, and cone opsin levels were obtained by quantitative RT-PCR.
RESULTS. At P25, cone density and transcript levels of cone opsins were drastically reduced, but a minute cone electroretinogram was detected, indicating that the cones were functional. Confocal microscopy revealed that the cone opsins were mislocalized, suggesting that their transport to the outer segments was impaired. Intraperitoneal administrations of 11-cis retinal before P25 led to increased transport of cone opsins to the outer segments and preserved cones anatomically and functionally.
CONCLUSIONS. The results suggest that the ligand is required during cone opsin synthesis for successful opsin trafficking and that without 11-cis retinal, cones may degenerate because of opsin mislocalization. These results may have important consequences for the treatment of cone dystrophies.
Leber’s congenital amaurosis (LCA) is an autosomal recessive retinal dystrophy, for which mutations have been identified in approximately 50% of patients. Of these, approximately 6% were in the protein RPE65.11 Because of the abundant and selective expression of the Rpe65 gene in the RPE, this protein is a promising candidate for human gene therapy (e.g., see Refs. 12 ,13 ). It is therefore critical to understand the pathophysiology of the Rpe65–/– phenotype, particularly with respect to the cones, as cones are the predominant photoreceptor type used for human high-acuity visual performance.
In the Rpe65–/– mouse, a small visual response remains, even though no 11-cis retinal can be detected.1 This response has been shown to correlate with the generation of a small amount of 9-cis retinal, forming the photosensitive isorhodopsin pigment in the rods.14 Seeliger et al.15 have reported that the remaining photoresponse is due to a rod response, based on their failure to elicit a response from a 4- to 5-week-old Rpe65–/–::Rho–/– mouse using normal ERG protocols.
The purpose of this study was to determine the fate of the cones in the absence of RPE-generated 11-cis retinal. We further sought to determine whether administration of 11-cis retinal can restore function to the cones as has been shown for the rod photoreceptors. The Rpe65–/–::Rho–/– mouse cross was used to eliminate the huge pool of rod opsin that could obscure the uptake of exogenously applied ligand. Furthermore, the cone responses in the Rpe65–/– retina could be masked by desensitized rod responses with altered kinetics.7 15 In this double-knockout model, we report that cone responses are unequivocally identified, so cones are functional at early ages. We further show that cones were lost rapidly in the Rpe65–/–::Rho–/– mouse retina, with the remaining cones containing mislocalized opsin. Finally, treating the animals with 11-cis retinal starting at postnatal day (P)10 resulted in increased cone survival, properly localized cone opsin, and increased cone responses.
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10% of rhodopsin in the Rpe65–/– retina.7 All experiments were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Medical University of South Carolina (MUSC) Animal Care and Use Committee.
ERG Analysis
Full-field ERGs were recorded as described previously,17 with minor modifications.7 Briefly, the setup was modified to include a dual-channel optical bench for light stimulation, providing stimulus and background light. Both optical pathways were driven by a single 250-W halogen lamp and controlled with mechanical shutters, manually operated neutral-density filters, and a 500-nm band-pass filter. The two pathways were combined and the light beam focused on the end of the light guide. Light intensity per 10 ms provided in the stimulus path was be varied in steps of 0.3 log units from 2.1 x 107 to 2.2 x 1013 photons/mm2, in white-light conditions, whereas the adaptation beam provided a continuous flux of 5.5 x 108 photons/mm2 per second at 510 nm. Based on the characteristics of the Halogen lamp, which has a continuous spectral output from 300 to 2000 nm and the transmittance of the optical components of 350 to 2100 nm, we stimulated predominantly mouse midwavelength (MWL) cones (
max 508 nm) and, to a lesser degree, mouse short-wavelength (SWL) cones (max 355 nm).18 Photopic electroretinograms were recorded by two protocols: (1) single-flash ERGs, and (2) 10-Hz flicker ERG of increasing light intensities, averaging three to five responses for single-flash and 10 responses for flicker ERGs. Peak b-wave amplitude was measured from the trough of the a-wave to the peak of the positive b-wave after a high-pass filter was applied, to eliminate oscillatory potentials.18 ERG recordings were stored, displayed, and analyzed with a computer interface and PClamp (Axon Instruments, Burlingame, CA) and commercial software (Origin; Microcal, Northampton, MA). ERG recordings were performed on Rpe65–/–::Rho–/– mice (n = 21) and 11-cis retinal-injected Rpe65–/–::Rho–/– mice (n = 31; 
6 for each treatment paradigm). In previous experiments, such group sizes were found to be appropriate for generating statistically reliable results.7 Data are expressed as the mean ± SEM and were analyzed by Student’s t-test.
Immunohistochemistry
For immunohistochemical analysis, eyes were fixed in 4% paraformaldehyde, rinsed, cryoprotected in 30% sucrose overnight, frozen in optimal cutting temperature (OCT) compound (TissueTek; Sakura Finetek, Torrance CA) and cut into 14-µm cryostat sections. Immunohistochemistry was performed as described previously,17 with the following primary antibodies: rabbit anti-mouse SWL-cone opsin19 (a generous gift from Jeanny Chen, University of Southern California, Los Angeles, CA) and rabbit anti-human MWL-cone opsin20 (JH 492; a generous gift from Jeremy Nathans, Johns Hopkins University School of Medicine, Baltimore, MD). For visualization, fluorescently labeled secondary antibodies (Alexa 488; Molecular Probes) were used. Each staining was performed on slides from at least three animals per condition. Sections were examined by confocal microscopy (Leica, Deerfield, IL), and the images were pseudo-colored (Photoshop; Adobe Systems, Mountain View, CA). To determine cone opsin distribution, images were binarized and thresholded, to set the background to zero, and distribution profiles were analyzed in Image J (NIH; available by ftp at zippy.nimh.nih.gov/ or at http://rsb.info.nih.gov/nih-image; developed by Wayne Rasband, National Institutes of Health, Bethesda, MD). In sections in which cone spacing allowed imaging of individual cones (i.e., Rpe65–/–::Rho–/– mice), single cone profiles were analyzed. In all other cases, a group of photoreceptors (20–30) were measured together. Cone lengths (OS to synaptic pedicle) and distribution profiles were normalized to 100%, and data points were binned based on the smallest image of the samples to be compared (Matlab, ver. 6.5; The MathWorks, Natick, MA). The profiles of the mean ± SD opsin distribution were plotted (Origin software; Microcal). Opsin distribution profiles were compared by averaging over the three regions of interest (0%–20%, OS; 20%–90%, inner segment cell body, and axon; and 90%–100%, synaptic pedicle relative lengths), with Student’s t-test used to determine statistical significance.
Real-Time PCR
Real-time PCR was performed as described previously.9 Briefly, littermates (treated and untreated) were killed by decapitation, and retinas were quickly removed. Total RNA was isolated (TRIzol; Ambion, Austin, TX) and equal amounts of RNA were used in reverse transcription reactions (Invitrogen, Carlsbad, CA). PCR amplifications were conducted using a kit (QuantiTect SYBR Green; Qiagen, Valencia, CA), according to the manufacturer’s instructions, with 0.2 µM forward and reverse primers and equal amounts of complimentary DNA (1 µL of 1:10 dilutions): ß-actin, forward GCT ACA GCT TCA CCA CCA CA, reverse TCT CCA GGG AGG AAG AGG AT, 123 bp; SWL-opsin, forward TTG GGC TCT GTA GCA GGT CT, reverse CAA GTA GCC AGG ACC ACC AT, 137 bp; and MWL-opsin, forward CTC TGC TAC CTC CAA GTG TGG, reverse AAG TAT AGG GTC CCC AGC AGA, 148 bp. Reactions were treated with 0.01 U/µL Uracil N-glycosylase (AmpErase; Applied Biosystems, Inc. [ABI], Foster City, CA) to prevent carryover contamination. Real-time PCR was performed in triplicate in a sequence detection system (GeneAmp 5700; ABI) using the after cycling conditions: 50°C for 2 minutes, 94°C for 15 minutes, 40 cycles of 94°C for 15 seconds, and 58°C for 1 minute. Quantitative values were obtained from the cycle number (Ct), which is inversely proportional to the amount of a specific mRNA species in the tissue sample from which the cDNA was derived. Relative gene expression levels were calculated with the equation y = (1 + AE)
Ct, where AE is the amplification efficiency of the target gene (set at 1.0 for all calculations), and Ct is the difference between the mean experimental and control Ct. Ct is the difference between the Ct for a retina-associated gene and the ß-actin internal reference control gene.
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Previous results have demonstrated that cones are lost rapidly in the central portion of the Rpe65–/– mouse retina, whereas peripheral cones are more resilient.9 To determine whether these remaining cones are functional, photopic ERG responses were recorded from dark-adapted P25 Rpe65–/–::Rho–/– mice by using white light at maximum flash intensity (2.2 x 1013 photons/mm2). As reported previously,15 when normal averaging procedures (i.e., three to five times) were used, no ERG was elicited from 4- to 5-week-old animals. However, minute cone responses (13.7 ± 1.87 µV; n = 9) were revealed if 50 traces were averaged in 3.5-week-old animals (Fig. 1) , indicating that both the cone opsins and the cone transduction pathways are functional in the absence of RPE65. In rods, the chromophore of the small amount of active pigment has been shown to be 9-cis retinal.14 The ligand of the pigment(s) responsible for the minute cone response in the Rpe65–/– mouse retina has not yet been identified.
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3.5 - 4-fold when compared with those in wt mice (Fig. 2) . A similar downregulation of cone opsins was observed in the P28 Rpe65–/– mouse,9 although the MWL cone opsin showed a somewhat less dramatic decrease. These results show that cone opsins in a <1-month-old mouse are not adversely affected by the loss of the rhodopsin gene and thus by the lack of the rod OS.
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Effect of Chromophore on Cone Opsin Localization
Cone opsin localization was examined by confocal microscopy in radial sections by using antibodies specific to SWL-19 and MWL cone opsins.20 Because the remaining cones in the Rpe65–/– and the Rpe65–/–::Rho–/– mouse retinas are localized almost exclusively in the peripheral retina at P25, the analyses for all animals was restricted to those areas. As expected, both cone opsins were localized to the OS in the P25 wt mouse retina (Figs. 4A 4D) . However, in the Rpe65–/–::Rho–/– cones, both the SWL and the MWL opsins appeared to be mislocalized. A significant amount of the protein was found to be localized in the cell membrane of the inner segment, cell body, axon, and synaptic pedicle (Figs. 4B 4E) . The antibody staining was found to be more pronounced for the SWL- than for the MWL-cone opsin, which may be due to differences in the binding affinity of the primary antibodies and/or differences in overall opsin levels. The wt mice have an SWL:MWL ratio of 3:1, and there was mixing of the cone opsins within single photoreceptors.22 11-cis Retinal injections, which increased cone opsin transcripts (Fig. 2) and cone function (Fig. 3) , resulted in the expected higher density of cones stained for cone opsins (Figs. 4C 4F) . In addition, significantly more of the generated cone opsin was localized appropriately to the OS (Figs. 4C 4F) .
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50 times) is necessary to uncover the response from the background activity. In the Rpe65–/– mouse, the level of rod opsin remains stable for approximately 6 months, and rhodopsin can be regenerated from opsin in vitro from animals 18 month of age.5 However, the cone photoreceptors die very quickly in this mouse model.9 Rod opsin in the absence of chromophore has been shown to be constitutively active.24 25 26 Rod degeneration in the Rpe65–/– mouse is proposed to result from opsin activity.6 This rod degeneration is slow, possibly because of the phosphorylation of opsin.7 Although constitutive activity of cone opsins may also be involved in cone degeneration in the Rpe65–/– mouse retina, the rapid time course of cone loss suggests the involvement of additional factors. Indeed, in this model, cone opsins were found to be mislocalized, resulting in apoprotein localization to the cell membrane, in particular in the axon and the synaptic pedicle, concomitant with rapid cone loss. Cone opsin distribution resembling that in the wt retina could be produced by injection of 11-cis retinal, resulting in significant cone survival and almost normal levels of cone opsin mRNA. The prevention of cone loss and of opsin mislocalization by exogenous 11-cis retinal suggests these defects were caused by the lack of ligand, rather then indirectly by the lack of rod OS, the slow accumulation of OS debris, or the potential lack of the rod-derived cone survival factor.27 28
Cone and rod opsins are similar at the genetic level, suggesting that they evolved from a common precursor. They are integral membrane proteins of the OS membrane or disc membrane and function as G-protein-coupled receptors in light. Transport of rhodopsin to the OS and correct insertion into the disc membranes appears to be dependent on protein structure and folding. It is proposed that the vesicular transport of the immature and unfolded rhodopsin from the endoplasmic reticulum (ER) through the Golgi apparatus to the OS discs is aided by two chaperones: NinaA and 
-crystallin.29 Wild-type rhodopsin is core glycosylated in the ER, followed by glycan group modifications in the Golgi apparatus.30 These modifications appear to be sufficient for proper folding and protein stability, as transport of the apoprotein to the OS and insertion into the disc membrane appear to be normal in the Rpe65–/– mouse retina.1 However, in the case of the thermally unstable P23H rhodopsin mutation, chromophore binding to the opsin in the ER has been found to improve folding, posttranslational modifications of the protein, and subsequent transport to the cell membrane in HEK293 cells.31 Although the two mouse cone opsins have sites for potential glycosylation, glycosylation has been reported not to be required for the formation of functional bovine green cone (MWL) pigment.32
Misfolded proteins are selectively destroyed in a ubiquitin-dependent process by the large adenosine triphosphate (ATP)-dependent proteolytic machine, the 26S proteasome.33 However, under conditions when the normal proteolytic machinery gets saturated (i.e., in the presence of constitutive production of misfolded proteins such as in cystic fibrosis and in the major neurodegenerative diseases), aggresomes or ubiquitinated inclusions start to form.34 The formation of aggresomes appears to lead to the disruption of the Golgi and intermediate filament networks and involves the upregulation of several cellular chaperones,34 35 all of which may ultimately lead to cell death by apoptosis. One proposed mechanism by which photoreceptors may reduce the formation of stress-induced aggregates of protein(s) is by increasing the production of molecular chaperones such as -crystallin.36 37
Little is known about the retinoid cycling mechanisms in cones. Mata et al.38 have proposed from studies in the cone-dominant ground squirrel retina that a mechanism involving the Müller cells is operational for the generation of 11-cis retinal for the cones. It is of interest to note the virtual absence of a cone response in a mouse that lacks Rpe65, suggesting that this pathway may be restricted to cone-dominant retinas or that it may involve RPE65, which we have found in cones of several species, including the mouse.2 Further evidence for a variance in retinoid processing between cones and rods comes from the work of Jin et al.,39 who found that whereas isolated bleached salamander rods can only incorporate retinal through the OS, bleached cones can incorporate 11-cis retinal through either the inner or OS, suggesting that binding to the opsin occurs in the inner segment. Although the role of the RPE65 protein in cones is unknown, its absence apparently does not prevent uptake of exogenous 11-cis retinal by the cones.
The results described herein provide evidence that ligand binding to opsin is necessary for protein stability and transport to the OS in cones. In its absence, both MWL- and SWL-cone opsins are mislocalized in part to the membrane of the pedicle and the axon, presumably resulting in rapid loss of cone OS and cone degeneration. On supplying 11-cis retinal, significantly more cone opsin is transported to the cone OS, where it can participate in light absorption and signal transduction. Concurrently, cones degenerate more slowly. Of note, the correction of this opsin mislocalization is more striking with the MWL opsin than the SWL opsin, for reasons that deserve further study. Whether the formation of aggresomes is involved in the degenerative process of the Rpe65–/–cones is currently unclear. However, in gene array experiments, crystallins have been found to be upregulated (more than twofold) in the Rpe65–/– retina (Rohrer B, unpublished data, 2004), whereas genes encoding for cone proteins are downregulated.9
In summary, our results show that if sufficient chromophore is supplied to the cones during OS development, cone opsin synthesis and transport can progress normally, preventing cone degeneration. These results need to be verified in other models, such as the Briard dog,13 to assure that this is not a species-specific phenomenon. However, our results suggest that if gene therapy were to be adopted for Rpe65 LCA patient treatment with the goal of recovering both rod and cone function, an intermediate strategy should be adopted to prevent missorting of cone opsins and subsequent cone degeneration. Treatment frequency and ligand dose must be identified, and a mode of delivery that specifically targets cones must be designed for this approach to be useful for cone survival. More generally, the systemic supplementation of 11-cis retinal may be useful in improving vision and ameliorating cone degeneration in a variety of disorders in which the chromophore supply to cones is primarily or secondarily impaired.
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Submitted for publication April 28, 2005; revised June 7, 2005; accepted August 16, 2005.
Disclosure: B. Rohrer, None; H.R. Lohr, None; P. Humphries, None; T.M. Redmond, None; M.W. Seeliger, None; R.K. Crouch, None
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. 
1734 solely to indicate this fact.
Corresponding author: Baerbel Rohrer, Department of Ophthalmology, Medical University of South Carolina, 167 Ashley Avenue, Room 511, Charleston, SC 29425; rohrer{at}musc.edu.
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