Cone Outer Segment Morphology and Cone Function in the Rpe65 ^−/− Nrl ^−/− Mouse Retina Are Amenable to Retinoid Replacement

Abstract

purpose. RPE65, a major retinal pigment epithelium protein, is essential in generating 11-cis retinal, the chromophore for all opsins. Without chromophore, cone opsins are mislocalized and cones degenerate rapidly (e.g., Rpe65 ^−/− mouse). Function, survival, and correct targeting of opsins is increased in Rpe65 ^−/− cones on supplying 11-cis retinal. Here, we determine the consequences of 11-cis retinal withdrawal and supplementation on cone development in the all-cone Nrl ^−/− retina.

methods. Rpe65 ^−/− Nrl ^−/−, Nrl ^−/−, and wild-type mice were examined. Cone structure was analyzed by using TUNEL assay, electron microscopy, and cone-specific antibodies. Cone function was assessed with light-adapted single-flash ERGs.

results. Rpe65 ^−/− Nrl ^−/− mice had an increased number of TUNEL-positive photoreceptors during programmed cell death compared with Nrl ^−/− mice, in addition to accelerated age-related degeneration. Cone function in Rpe65 ^−/− Nrl ^−/− mice was minimal, and opsins were mislocalized. Treatment with 11-cis retinal restored cone function, promoted outer segment formation, and enabled opsin trafficking to outer segments. Eliminating Rpe65 prevented rosette formation in Nrl ^−/− retinas; supplementation of Rpe65 ^−/− Nrl ^−/− mice with 11-cis retinal resulted in their reoccurrence.

conclusions. Taken together, function and opsin trafficking in Nrl ^−/− and wild-type cones are comparable, confirming and extending our findings that cone maturation and outer segment development are dependent on the presence of chromophore. The data on age-related cone death in Rpe65 ^−/− Nrl ^−/− mice and the reintroduction of rosettes after 11-cis retinal injections confirm that outer segments, which for steric reasons appear to introduce rosettes in an all-cone retina, are essential for cell survival. These results are important for understanding and treating chromophore-related cone dystrophies.

Photoreception is supported by two types of photoreceptors: rods and cones. Rods are essential for vision under dim-light conditions and cones for vision under bright-light conditions. Despite their importance in human vision, cones are outnumbered by rods ∼20:1; however, visual acuity under photopic conditions is not impaired due to the aggregation of the cone photoreceptors in the center of the retina, the macula. This high rod-to-cone ratio is similar in most mammalian retinas, including the mouse, ¹ although no cone-rich specialization exists in that retina. In a study of the cone-rich murine retina, Mears et al. ² created a knockout mouse in which the transcription factor Nrl (essential for rod differentiation) was eliminated, creating an all-cone retina with predominantly short-wavelength opsin-positive cones. Daniele et al. ³ carefully characterized the photoreceptors in the Nrl ^−/− mouse retina, and based on 13 characteristics (e.g., ultrastructure, histology, molecular parameters, and ERG a-wave characteristics), concluded that the “Nrl ^−/− photoreceptors are cone-like,” and that “this retina provides a model for the investigation of cone function and cone-specific genetic disease.” 

We have studied cone photoreceptor development and function in the Rpe65 ^−/− mouse retina, ^{4 5} a model for Leber congenital amaurosis (LCA). ⁶ In retinas in which 11-cis retinal synthesis is disrupted, cones appear to be born in normal numbers; however, cone cell death starts at the time of eye opening, eliminating almost all cones by 1 month of age. ⁴ These cones are integrated into the retinal network such that small, but reliable cone electroretinograms (ERGs) can be recorded. ⁵ Histologically, cones in the Rpe65 ^−/− mouse retina differ from those in the wild-type retina as postnatal cone outer segment (OS) maturation fails to occur. In the Rpe65 ^−/− cones, cone opsins are mislocalized, and expression is downregulated, ⁵ as are other cone OS membrane-associated proteins. ⁷ Expression levels and mislocalization of cone opsins and cone outer-segment membrane-associated proteins are reversed by exogenous 11-cis retinal, augmenting both cone survival and function. 

The putative role of RPE65 in cones has been studied in the Nrl ^−/− mouse by Wenzel et al. ⁸ and Feathers et al. ⁹ In the retina in this mouse, with excess short-wavelength cones (S-cones), rosettes form containing cells from the photoreceptor cell layer. ² However, rosette formation is eliminated in the absence of 11-cis retinal (i.e., in the Rpe65 ^−/− Nrl ^−/− retina). ⁸ Of interest, in other models with increased density of S-cones, rosette formation has been reported. ^{10 11} Finally, electron microscopic analysis of the Rpe65 ^−/− Nrl ^−/− cone OS structure demonstrated that in the absence of RPE65, photoreceptor OS formation is initiated such that, by P14, partial OS can be observed, but cannot be maintained resulting in loss of OS and cell death. ⁹  

In the present study, we demonstrated that many features of cone development and function are dependent on the presence of 11-cis retinal, and that both wild-type and Nrl ^−/− cones behave comparably. As in the wild-type cones, cone opsin trafficking to the OS in the Nrl ^−/− cones is dependent on 11-cis retinal, and in both types of cones, wild-type and Nrl ^−/−, cone function in the absence of RPE65 can be restored by supplying exogenous 11-cis retinal. As a consequence of the lack of cone opsin trafficking, both types of cones appear to lose their OS, followed by cell death. The results are discussed in the context of finding the optimal dose of 11-cis retinal for cone opsin trafficking, the source of 11-cis retinal for cones, and potential chromophore treatment strategies for patients with chromophore-related dystrophies. 

Material and Methods

Animals

Rpe65 ^−/−, Nrl ^−/− mice were generously provided by Andreas Wenzel (University of Zurich ⁸ ) with the permission of Anand Swaroop (University of Michigan) and T. Michael Redmond (National Eye Institute); Nrl ^−/− mice were provided by Anand Swaroop (University of Michigan ² ). Both strains were bred as homozygous animals. Age-matched C57BL/6 (wild-type [WT]) mice were generated from breeding pairs obtained from Harlan Laboratories (Indianapolis, IN). To study the effect of 11-cis retinal treatments, we randomly assigned littermates to the experimental or the control groups and kept them in the dark starting at postnatal day (P)6. Once moved into the dark, the experimental animals were injected intraperitoneally with 11-cis retinal (5 mg per dose in 150 μL vehicle containing 10% ethanol, 10% bovine serum albumin, and 0.9% NaCl) on P6, P10, and P14, unless otherwise specified in the results section. As published previously, this paradigm has been shown to lead to the successful regeneration of cone opsin in the Rpe65 ^−/− retina. ⁵ Since a single injection of this dose leads to the successful regeneration of ∼10% of rhodopsin in the young Rpe65 ^−/− retina, ¹² and the adult Nrl ^−/− mouse has ∼15% of WT pigment, this dose should saturate all available cone opsin during Rpe65 ^−/− Nrl ^−/− cone development. 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. 

Anatomic Analyses

TUNEL.

Labeling was performed according to the protocol provided by the manufacturer (Roche Diagnostics, Indianapolis, IN) and published previously. ¹³ In short, eyes were fixed in 2% paraformaldehyde for 2 hours at 4°C followed by dehydration, paraffin embedding, and sectioning (7-μm sections). A TUNEL (TdT-mediated dUTP nick-end labeling) assay was performed and the DNA strand breaks were labeled with fluorescein for visualization. Slides were examined with a fluorescein isothiocyanate filter on a microscope equipped for fluorescence (Carl Zeiss Meditec, Dublin, CA). For cell counts, the number of TUNEL-positive nuclei in the outer nuclear layer (ONL) was counted in three sections per eye and were averaged to obtain a single value. For overall differences in TUNEL-positive cells between the different retinas, data were analyzed by repeated-measures ANOVA followed by the Tukey post hoc test (P < 0.05); for analysis of regional differences in cone cell counts, the one-way ANOVA followed by the pair-wise Tukey post hoc test was used (P < 0.05). 

Semithin Sections.

For semithin sections, eyes were removed, postfixed in 4% paraformaldehyde and 2% glutaraldehyde in cacodylate buffer, and bisected dorsally to ventrally through the optic nerve. Each half was embedded in an Epon-Araldite mixture, and sections were cut at 1 μm through the horizontal meridian and stained with toluidine blue. ¹⁴ Photoreceptor layers were counted in the peripheral (superior and inferior, within 350 μm of the ora serrata) and central (superior and inferior, within 350 μm of the optic nerve head) regions. Three measurements were made per field, which were averaged to provide a single value for each area, and the four area values were averaged to give a value for the entire retina. Data are expressed as mean ± SEM and analyzed by the Student t-test. 

Electron Microscopy.

The same blocks that were used for semithin sections were trimmed for ultrathin sections. Ultrathin sections were stained with uranium and lead salts as described elsewhere. ¹⁵ Sections from three mice per treatment (11-cis retinal versus vehicle, injected on P6, P10, and P14) were analyzed. 

Immunohistochemistry.

For immunohistochemical analysis, eyes were fixed in 4% paraformaldehyde, rinsed, cryoprotected in 30% sucrose overnight, frozen in optimum cutting temperature compound (TissueTek O.C.T.; Fisher Scientific, Suwanee, GA), and cut into 14-μm cryostat sections. Immunohistochemistry was performed as described previously, ⁵ using rabbit polyclonal antibodies against mouse cone opsins (UV- and MWL-cone opsin; a generous gift of Jeannie Chen, University of Southern California, Los Angeles, CA). 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 fluorescence microscopy (Carl Zeiss Meditec, Inc.), with identical exposure settings used for all images. 

ERG Analysis

Photopic full-field ERGs were recorded as described previously, with minor modifications. ⁵ 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 quartz halogen lamp and could be controlled with mechanical shutters, manually operated neutral density, and a 500-nm band-pass filter. The two pathways were combined and the light beam focused to the end of the light guide. Light intensity per 10 ms provided in the stimulus path could be varied in steps of 0.3 log units from 2.1 × 10⁷ to 2.2 × 10¹³ photons/mm² using white-light conditions, whereas the adaptation beam provided a continuous flux of 5.5 × 10⁸ photons/mm²/s 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 both mouse mid-wavelength (MWL) cones (λmax, 508 nm) and short wavelength (UV) cones (λmax, 355 nm). ¹⁶ Photopic ERGs were recorded in response to single-flash stimuli of increasing light intensities, averaging three to five responses. Peak b-wave amplitude was measured from the trough of the a-wave to the peak of the positive b-wave after applying a high-pass filter to eliminate oscillatory potentials. ¹⁷ ERG recordings were stored, displayed, and analyzed with a PC interface (Pclamp; Axon Instruments, Burlingame, CA) and software (Origin; OriginLab, Northampton, MA). Data are expressed as the mean ± SEM and analyzed with Student t-test. 

Results

RPE65 and Cone Cell Death

RPE65 protein in the rodent eye cup can be detected by P4, which is concomitant with the detection of 11-cis retinyl esters by P6 to P7. ¹⁸ The timing of this onset of 11-cis retinal production correlates with the onset of cone OS elongation. We investigated whether the lack of 11-cis retinal correlates with an increase in cone cell loss. Two phases of cone cell loss were investigated: programmed cell death early during postnatal development and age-related loss. 

Programmed cell death in the ONL has been shown in the wild-type C57BL/6 mouse retina to occur between P2 and P12. ¹⁹ Analysis of photoreceptor cell death in C57BL/6 mice was repeated to establish a temporal profile for normal programmed cell death in the central retina (Fig. 1A) , confirming that TUNEL-positive cells can be detected as early as P2, with a peak by P10, and diminishing by P12. This time course was compared with that of the Nrl ^−/− and the Rpe65 ^−/− Nrl ^−/− mouse. Using a repeated-measure ANOVA, we identified a genotype (P < 0.0001) and genotype by time-course–specific effect (P < 0.0001). In the Nrl ^−/− mouse retina, which contains only 60% of the number of photoreceptors when compared to the C57BL/6 mouse, ³ programmed cell death occurs with the same temporal profile as in the control mouse (Fig. 1A) , but to a lesser degree; but which did not reach statistical significance when the Tukey post hoc test was used (P > 0.05). However, when the number of TUNEL-positive cells in the Nrl ^−/− was compared with those in the Rpe65 ^−/− Nrl ^−/− mouse, a significant genotypic effect was established. The period of programmed cell death was prolonged, lasting from P2 through P19, whereas no TUNEL-positive cells were detected in the Nrl ^−/− retina beyond P12 (Fig. 1A) . In addition, at any given time point, significantly more TUNEL-positive cells were identified (Tukey post hoc test; P < 0.05). To determine whether counts of TUNEL-positive cells at day 10 differ in C57BL/6, Nrl ^−/−, and Rpe65 ^−/− Nrl ^−/− mice, a pair-wise Tukey post hoc analysis was undertaken, which revealed that all pair-wise comparisons between the three genotypes were statistically significant (P < 0.05). If the effect of Rpe65 on cell death were due to the lack of 11-cis retinal, treatment of the Rpe65 ^−/− Nrl ^−/− mice with exogenous 11-cis retinal should reduce the number of TUNEL-positive cells. 11-cis Retinal was injected intraperitoneally on two days (P5 and P8), using lipid-free bovine serum albumin as a carrier ¹² and retinas analyzed on P10. Addition of 11-cis retinal reduced the number of TUNEL-positive cells by 30% (Tukey post hoc test; P < 0.05; Fig. 1B ). 

Slow photoreceptor degeneration occurs in the wild-type C57BL/6 mouse retina with aging. ²⁰ We compared cell counts in the Nrl ^−/− and Rpe65 ^−/− Nrl ^−/− mice at 5 months-of-age in the inferior and superior retina. As shown by Feathers et al., ⁹ although both genotypes lose photoreceptors with aging, overall the adult Rpe65 ^−/− Nrl ^−/− mice had fewer rows of photoreceptor nuclei, with cell loss in the superior central being more severe than the inferior central retina (Fig. 2A) . At 5 months of age, the inferior retinas (inferior periphery and inferior central combined) of the Rpe65 ^−/− Nrl ^−/− mice contained 3.22 ± 0.39 rows compared with 4.66 ± 0.19 in the Nrl ^−/− mice (Tukey post hoc test; P < 0.05), whereas the photoreceptors in the superior retina had almost completely degenerated (Rpe65 ^−/− Nrl ^−/− 0.76 ± 0.11 vs. Nrl ^−/− 4.66 ± 0.22; Tukey post hoc test; P < 0.05; Fig. 2B ). Taken together, programmed cell death was augmented in the absence of Rpe65, as was photoreceptor cell loss during aging. 

Rpe65 and Cone Function

Previously, we have shown that a minute cone ERG can be elicited from wild-type cones in the juvenile Rpe65 ^−/− mouse retina. ⁵ Similarly, small but reliable cone ERGs were recorded from the Rpe65 ^−/− Nrl ^−/− mice, resulting in maximum b-waves of 10.8 ± 1.93 μV at light levels calibrated to be ∼2.2 × 10¹³ photons/mm² (Fig. 3B) . The chromophore is presumed to be 9-cis retinal, which is the sole chromophore that can be identified in these mice ⁸ and which we have shown to be the chromophore used for the formation of the rod pigment in the Rpe65 ^−/− mouse. ²¹ To investigate whether function can be restored in Rpe65 ^−/− Nrl ^−/− mouse cones using chromophore injections, 11-cis retinal was injected intraperitoneally, using lipid-free bovine serum albumin as a carrier starting at P6 (start of OS elongation in wild-type cones at ∼P4) followed by two additional treatments at P10 and P14. ERG analysis was performed at P17, using photopic single-flash ERGs. Cone ERG amplitudes were compared to those elicited from age-matched Nrl ^−/− mice (Figs. 3A 3B 3C) . Single-flash cone ERG amplitudes in the P17 Rpe65 ^−/− Nrl ^−/− mice treated with 11-cis retinal reached maximum amplitudes of 158.2 ± 22.8 μV, which was not significantly different from those elicited from the Nrl ^−/− mice (194.7 ± 38.0 μV; P = 0.3; Fig. 3D ). Thus, Rpe65 ^−/− Nrl ^−/− photoreceptor function can be restored on the application of exogenous chromophore. 

Rpe65 and Cone Structure

The absence of Rpe65 has been shown to affect cone morphology. Lack of 11-cis retinal results in cone OS membrane protein mislocalization ^{5 7 22} and downregulation of cone opsins ⁵ and hence is predicted to reduce OS stability. In addition, a lack of Rpe65, typically associated with S-cone syndrome, ¹¹ has been shown to prevent rosette formation in the Nrl ^−/− ONL. ⁸  

Immunohistochemistry using antibodies specific for mouse ultraviolet (UV) wavelength and mid-wavelength (MWL) cone opsins showed that opsins are restricted to the cone OS in the Nrl ^−/− both in the rosettes and the remainder of the retina. On the other hand, cone opsins were mislocalized in the Rpe65 ^−/− Nrl ^−/− retina, which was partially corrected in the 11-cis retinal-treated animals (Fig. 4A) . Relative distribution profiles were used to determine the percentage of incorrect trafficking to the cell body, axon, and pedicle in both control and 11-cis retinal-treated animals. For UV opsin, ∼48% of the label was mislocalized in the Rpe65 ^−/− Nrl ^−/− retina compared with ∼6% after 11-cis retinal treatment; for MWL opsin, the mislocalized opsin was reduced from ∼48% to ∼27% (data not shown). Thus, 11-cis retinal-dependent cone opsin trafficking occurs both in true cones, as shown for cones in the Rpe65 ^−/− Rho ^−/− retina, ⁵ and in cone-like cells. 

Rosette formation was initiated around P14 in the Nrl ^−/− mouse retina, ⁸ resulting in a mixture of rosettes, with some being clearly demarcated and others just starting to form. Rosette formation is eliminated in the Rpe65 ^−/− Nrl ^−/− retina; however, whereas the photoreceptors in the wild-type mouse form a sharp border, the transition from the ONL to photoreceptor IS and OS is wavy in the P14 Rpe65 ^−/− Nrl ^−/− retina. ⁸ We tested whether exogenous 11-cis retinal treatment would result in the reoccurrence of photoreceptor rosettes and other disturbances in the Rpe65 ^−/− Nrl ^−/− retina. The same regimen that resulted in improved cone opsin trafficking and cone function also triggered some photoreceptor rosette formation when the retinas were analyzed by P17. The area highlighted in Figure 4Bshows one mature rosette (arrow) and three rosettes that are beginning to form (asterisk). 

Finally, electron microscopy analysis was used to evaluate OS structure in Rpe65 ^−/− Nrl ^−/− retina treated with 11-cis retinal when compared with that in untreated animals (Fig. 5) . Untreated Rpe65 ^−/− Nrl ^−/− photoreceptors were almost devoid of OS, and only a few misaligned discs were observed (Fig. 5A) . Rpe65 ^−/− Nrl ^−/− retinas treated with 11-cis retinal exhibited morphologic features similar to those of the Nrl ^−/− mouse. ² Both the Nrl ^−/− (see Fig. 3in Ref. ² ) and the 11-cis retinal-treated Rpe65 ^−/− Nrl ^−/− photoreceptors had short OS with abnormal disc morphology. Although some OS were found to show the same stacked array of OS discs as wild-type photoreceptors, many of the OS discs were misaligned (Fig. 5B) . 

Thus, the three identified aspects of cone morphology introduced into the Nrl ^−/− retina by the elimination of the RPE65 protein were reversed by administering exogenous 11-cis retinal. 

Discussion

The main results of the present study are early postnatal Rpe65 ^−/− Nrl ^−/− cones undergo enhanced programmed cell death, and adult cones degenerate rapidly, in particular in the superior retina; Rpe65 ^−/− Nrl ^−/− cone function is impaired; the two cone opsins fail to traffic properly to the OS, destabilizing the OS; and in Rpe65 ^−/− Nrl ^−/− mice, early treatment with 11-cis-retinal reverses the anatomic and physiological defects associated with the lack of Rpe65. Programmed cell death is reduced, cone opsin trafficking is partially restored, resulting in stabilization of OS and normal levels of cone function, with the exception of the partial reintroduction of the photoreceptor rosettes. These results confirm and extent our previous findings in wild-type cones that early supplementation with chromophore can stabilize cone OS and reemphasize the importance of early intervention in cone dystrophies associated with chromophore loss. 

Since the original observation that cone opsins are mislocalized in the absence of Rpe65, ⁵ we have repeated these results in another model of Leber congenital amaurosis, the Lrat ^−/− mouse to confirm that the cone opsin mislocalization is due to the lack of 11-cis retinal and not some unknown function of Rpe65. ²² In the wild-type mouse retina, mislocalized apoprotein throughout the entire cell in rods and cones is a hallmark of development, and in all species examined, cell membrane labeling disappears around the onset of vision. ^{23 24 25 26} In unpublished results (Rohrer B, 2007) we have shown that this restriction of cone opsin to the OS by the time of eye opening does not occur in the absence of 11-cis retinal, arguing for a trafficking defect in these cells, rather than a mislocalization due to cell damage (e.g., retinal detachment ²⁷ ). Similar to the wild-type cones, it is thought that in Nrl ^−/− cones 11-cis retinal is necessary for opsin stabilization and trafficking to the OS, where it is essential for disc formation and stabilization. This hypothesis is supported by the data of Feathers et al. ⁹ who have shown with electron microscopy that the Rpe65 ^−/− Nrl ^−/− cones form the initial OS structures by eye opening, which are then disassembled rapidly, and by our own data (Fig. 5)showing that supplementing Rpe65 ^−/− Nrl ^−/− cones with 11-cis retinal resulted in OS that were similar in structure to the Nrl ^−/− OS. ² In this and previous publications ^{5 7} we have shown that supplementing the retina with exogenous 11-cis retinal leads to improvement of OS membrane protein trafficking and hence improved OS stability, functional recovery, and photoreceptor cell survival. 

Thus, an important, but unsolved question is how much chromophore is needed to support both the development and the maintenance of the cone OS. Based on the availability of knockout and transgenic animals, we can bracket the required level. Two isoforms exist for Rpe65, the leucine and the methionine 450 variant. The two variants differ in the rate with which they regenerate 11-cis retinal, such that at steady state, C57BL/6 retinas (Met450) contain only ∼40% of the amount of 11-cis retinal when compared to BALB/c retinas (Leu450). ²⁸ Mice heterozygous for Rpe65 ^Met450/−, which have approximately half of the C57BL/6 levels of 11-cis retinal based on rhodopsin measurements ⁶ (i.e., 20%), have normal localization of cone opsins. The R91W Rpe65 knockin mouse, which expresses a relatively inefficient human mutation of Rpe65 that results in the generation of less than one tenth the amount of chromophore present in the C57BL/6 eye (∼25 pM 11-cis retinal/eye plus ∼8 pmol 9-cis retinal/eye; i.e., 2%), ²⁹ has mislocalized cone opsin. ³⁰ Hence, the 11-cis retinal threshold for cone opsin trafficking lies between 2% and 20% of the maximum 11-cis retinal, if rods, a major sink for the chromophore, are present. Although supplementation experiments have been shown to be useful in confirming the role of 11-cis retinal in opsin trafficking, the results are more difficult to interpret with respect to the amount of chromophore available to the cones. Although we have shown that almost normal cone function can be attained by supplying excess amounts of exogenous chromophore by intraperitoneal injection, it is unclear what levels of chromophore are available for opsin trafficking and regeneration. It may be necessary for chromophore to be delivered to the cone photoreceptors via a specific pathway that cannot be mimicked correctly by systemic delivery. This latter point is of particular importance, given the recent discovery of a cone-specific retinoid pathway ^{31 32} in the cone-dominant retina. This mechanism involves the Müller cells that reisomerize all-trans retinol released from the photoreceptors into 11-cis retinol that is then taken up by the cones presumably through the inner segment, ³³ where it is reduced to 11-cis retinal. Evidence suggests that two pathways also exist to provide chromophore to cones in the rod-dominant retina, one involving the RPE, a second one the Müller cells (Kefalov VJ, et al. IOVS 2008;49:ARVO E-Abstract 1661). However, it is unclear how the retina-mediated cycle is fed its substrate, all-trans retinol. Qtaishat et al. ³⁴ have shown that the inward flux of all-trans retinol from the circulation into the RPE is preserved in Rpe65 ^−/− mice. However, this all-trans retinol does not seem to be accessible to the Müller cells, as no 11-cis retinal is generated for the Rpe65 ^−/−, Lrat ^−/−, Rpe65 ^−/− Rho ^−/−, or Rpe65 ^−/− Nrl ^−/− cones. Thus, the Müller cell retinoid cycle appears to be dependent on a functioning RPE cell cycle and hence requires functional RPE65. 

The Rpe65 ^−/− Nrl ^−/− retina has provided us with a tool to study conelike photoreceptors in the mouse retina. Our TUNAL assay results in the Nrl ^−/− retina demonstrated that cones, like rods, undergo programmed cell death during early postnatal development. Of interest, cell death was increased during this phase in the absence of Rpe65 and was ameliorated in the Rpe65 ^−/− Nrl ^−/− retina by exogenous 11-cis retinal (Fig. 1) . However, it is unclear how 11-cis retinal or a downstream product could increase cone survival during programmed cell death. Our results showing long-term survival in Nrl ^−/− and Rpe65 ^−/− Nrl ^−/− retinas confirm and extend the results reported by Feathers et al. ⁹ that although rosette formation is eliminated by the lack of Rpe65, cell death during aging is accelerated in the Rpe65 ^−/− Nrl ^−/− retinas when compared with those in the Nrl ^−/− mouse. Rosette formation is thought to be eliminated in the Rpe65 ^−/− Nrl ^−/− retina due to the lack of OS. However, the lack of OS is also the presumed reason that cells die more rapidly in these retinas. Rosette formation is reintroduced in the 11-cis retinal-treated Rpe65 ^−/− Nrl ^−/− retina although not to the same extent as in the Nrl ^−/− retina (Grimm C, personal communication, 2009), suggesting that 11-cis retinal may be required continuously for this effect. Another contributing factor for cone degeneration is thought to be the mislocalization of cone opsins. ^{5 7} Of note, the superior retina is more susceptible to cell death than is the inferior retina—opposite to the findings in wild-type cones, which die more rapidly in the inferior and central retina, ⁴ but similar to the results reported for bright-light damage, where rod degeneration is more pronounced in the superior retina. ³⁵ Finally, restoration of cone function can be studied more readily in the Rpe65 ^−/− Nrl ^−/− retina than in the Rpe65 ^−/−, as the Nrl ^−/− retina is an all-cone retina. We found that early (starting at P6) and frequent (every 4 days) intervention with chromophore is essential for functional cone recovery, which may be the crucial protocol difference explaining why Feathers et al. ⁹ were unable to improve cone structure and function in Rpe65 ^−/− Nrl ^−/− mice. 

In summary, our results show that many aspects of cone development, maturation, and function are dependent on sufficient and constant chromophore supply. These results still must be verified in other models such as the Briard dog or in Rpe65-mediated LCA in humans, to assure that this is not a species-associated phenomenon. However, our ligand replacement experiments suggest that ligand can be used to promote cone survival. This aspect is particularly important in the context of developing therapies (gene therapy or pharmacologic manipulation) for patients with retinoid dysfunction. In those patients, cone and rod dysfunctions have been reported. ³⁶ Of interest, Jacobson et al., ³⁷ have shown that in patients with Rpe65-mutations, the photoreceptor layer thickness in the central retina is actually much greater than one would have predicted based on the amount of cone vision loss, suggesting that there is a window of opportunity during which cone-based vision may be recoverable and implying that implementation of an early intervention in these patients may be critical. Thus, understanding the role of retinoids in cone development, function, and cone dystrophies is of significant clinical importance. 

 Supported by National Institutes of Health Grants EY13520 (BR) and EY04939 (RKC), Vision Core Grant EY014793, and an unrestricted grant to MUSC from Research to Prevent Blindness (RPB), Inc., New York, NY. BR is an RPB Olga Keith Weiss Scholar and RKC is an RPB Senior Scientific Investigator. The animal studies were conducted in a facility constructed with support from the National Institutes of Health, Grant C06 RR015455 from the Extramural Research Facilities Program of the National Center for Research Resources.

 Submitted for publication October 14, 2008; revised April 1 and 20, 2009; accepted July 6, 2009.

 Disclosure: K. Kunchithapautham, None; B. Coughlin, None; R.K. Crouch, None; B. Rohrer, 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.

 

Figure 1.

 

Programmed photoreceptor cell death was augmented in the absence of Rpe65. Photoreceptor cell death was documented by counting positive cells per retina section (i.e., circumference) in a TUNEL assay. (A) The temporal profile of programmed cell death (PCD) in C57BL/6 mice placed the peak at P10. In the Nrl ^−/− and the Rpe65 ^−/− Nrl ^−/− mouse, the peak of PCD overlapped that of the C57BL/6 mouse, but the period of PCD was shorter in the Nrl ^−/− retina and prolonged in the Rpe65 ^−/− Nrl ^−/− retina. (B) At postnatal day 10, the Rpe65 ^−/− Nrl ^−/− mice had a higher number of TUNEL-positive cells than did the Nrl ^−/− mice (*P < 0.05), a difference that was reduced significantly by the addition of 11-cis retinal (*P < 0.05).

Figure 2.

 

Age-related photoreceptor cell death was augmented in the absence of RPE65. Age-related photoreceptor degeneration was determined at 5 months of age, comparing cell counts in four different quadrants of the retina. (A) Toluidine-stained 1-μm plastic-mounted sections obtained from Nrl ^−/− and Rpe65 ^−/− Nrl ^−/− retinas were compared. (B) Whereas retinas from young adult 4-week-old Nrl ^−/− and Rpe65 ^−/− Nrl ^−/− mice had approximately seven to eight rows of photoreceptor nuclei (data not shown), by 5 months of age, both genotypes had lost ∼50% of their photoreceptor nuclei in the inferior retina. In the superior retina, the Nrl ^−/− retina also retained ∼50% of photoreceptor nuclei, whereas the Rpe65 ^−/− Nrl ^−/− had almost completely degenerated. *P < 0.05; Tukey post hoc analysis.

Figure 3.

 

Functional recovery of Rpe65 ^−/− Nrl ^−/− cone ERG after administration of 11-cis retinal. (A–C) Family of photopic cone ERGs in response to increasing light intensity were recorded in steps of 0.3 log units (maximum light intensity, ∼2.2 × 10¹³ photons/mm²) at P17. ERG amplitudes and sensitivity were compared between Nrl ^−/− (A), vehicle (B), and 11-cis-retinal-treated Rpe65 ^−/− Nrl ^−/− mice (C). Small but reliable b-waves were recorded in vehicle-treated Rpe65 ^−/− Nrl ^−/− mice. After three injections of 11-cis retinal, light sensitivity and photopic ERG amplitudes of Rpe65 ^−/− Nrl ^−/− mice are indistinguishable from those of Nrl ^−/− mice. (D) Summary of maximum photopic ERG amplitudes of Nrl ^−/−, vehicle, and 11-cis-retinal-treated Rpe65 ^−/− Nrl ^−/− mice.

Figure 4.

 

Structural changes in Rpe65 ^−/− Nrl ^−/− cones on 11-cis retinal administration. (A) Cone opsin localization was compared in Nrl ^−/−, vehicle, and 11-cis-retinal-treated Rpe65 ^−/− Nrl ^−/− mice, with antibodies specific for mouse UV and MWL cone opsins. In Nrl ^−/− mice, the opsins were restricted to the cone OS. In the absence of 11-cis retinal, the opsins were mislocalized, which was partially reversed by 11-cis retinal treatment. (B) Rosette formation, which started at the time of cone OS elongation in the Nrl ^−/− mouse retina (data not shown), was eliminated in the Rpe65 ^−/− Nrl ^−/− retina (control), but reoccurred in 11-cis retinal-treated Rpe65 ^−/− Nrl ^−/− eyes when analyzed after three injections at P17 (11-cis retinal). INL, inner nuclear layer; IPL, inner plexiform layer; ONL, outer nuclear layer; OPL, outer plexiform layer; OS, outer segment; RGC, retinal ganglion cells; RPE, retinal pigment epithelium. Scale bar, 50 μm.

Figure 5.

 

OS analysis in Rpe65^−/−Nrl^−/− cones on 11-cis retinal administration. Electron micrographs of Rpe65^−/−Nrl^−/− retinas in the absence (Aa) and presence (Bb) of 11-cis retinal. (Aa) Cone OS in untreated animals were almost completely devoid of disc structures (black arrow), although cilia formation was occurring (white arrow). (Bb) 11-cis-Retinal-treated Rpe65^−/−Nrl^−/− photoreceptors significantly increased the number of OS discs. Although some OS exhibited normal disc structure, the majority of the discs are misaligned ( ) with respect to both the photoreceptor axis and the RPE. Qualitatively, these results are indistinguishable from those reported for the Nrl^−/− mouse.²IS, inner segment; ONL, outer nuclear layer; OS, outer segment; RPE, retinal pigment epithelium. Magnification: (A, B) ×5,000; (Aa, Bb) ×25,000.

The authors thank Christian Grimm, Andreas Wenzel, Debra Thompson, Anand Swaroop, and Muna Naash for helpful discussions; Luanna Bartholomew for critical review; and Patrice Goletz and Carol Moskos (MUSC Electron Microscopy Facility) for excellent technical assistance.