February 2007
Volume 48, Issue 2
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Biochemistry and Molecular Biology  |   February 2007
RPE65 Is Essential for the Function of Cone Photoreceptors in NRL-Deficient Mice
Author Affiliations
  • Andreas Wenzel
    From the Laboratory for Retinal Cell Biology, Eye Clinic, University Hospital Zürich, Zürich, Switzerland; the
  • Johannes von Lintig
    Institute of Biology I, Animal Physiology and Neurobiology, University of Freiburg, Freiburg, Germany; and the
  • Vitus Oberhauser
    Institute of Biology I, Animal Physiology and Neurobiology, University of Freiburg, Freiburg, Germany; and the
  • Naoyuki Tanimoto
    Retinal Diagnostics Research Group, Department of Ophthalmology II, University Eye Hospital, Tübingen, Germany.
  • Christian Grimm
    From the Laboratory for Retinal Cell Biology, Eye Clinic, University Hospital Zürich, Zürich, Switzerland; the
  • Mathias W. Seeliger
    Retinal Diagnostics Research Group, Department of Ophthalmology II, University Eye Hospital, Tübingen, Germany.
Investigative Ophthalmology & Visual Science February 2007, Vol.48, 534-542. doi:10.1167/iovs.06-0652
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      Andreas Wenzel, Johannes von Lintig, Vitus Oberhauser, Naoyuki Tanimoto, Christian Grimm, Mathias W. Seeliger; RPE65 Is Essential for the Function of Cone Photoreceptors in NRL-Deficient Mice. Invest. Ophthalmol. Vis. Sci. 2007;48(2):534-542. doi: 10.1167/iovs.06-0652.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. Phototransduction in cones is initiated by the bleaching of their visual pigment, which comprises a protein component—cone opsin—and a vitamin A derivative—11-cis retinal. Little is known about the source of 11-cis retinal for cones. In the current study, neural retina leucine zipper–deficient (Nrl −/−) and rod opsin (Rho −/−)–deficient mice were used, two mouse models that have been described as having a “cone-only” retina, to analyze the retinoid metabolism of cones. In addition, these mice were bred to retinal pigment epithelial protein 65 (Rpe65 −/−)–deficient mice to study the role of RPE65.

methods. Mice were analyzed using morphology, Western blot analysis, immunohistochemistry, electroretinography (ERG), and retinoid profiling by HPLC.

results. In comparison to wild-type mice, the retina of Nrl −/− mice contained elevated levels of RPE65 and cellular retinaldehyde-binding protein (CRALBP), suggesting a particular role of these two proteins for the retinoid metabolism of cones. In Nrl −/− mice, different retinoid species were present in proportions similar to wild type. Ablation of RPE65 in Nrl −/− and Rho −/− mice led to the absence of 11-cis retinal, but increased the total retinoid content, with retinyl esters representing the most abundant retinoid species. In the absence of RPE65, retinal sensitivity in Nrl −/− mice dropped by a factor of a thousand.

conclusions. The data show that RPE65, previously shown to be essential for rod function, is also indispensable for the production of 11-cis retinal for cones and thus for cone function.

Cone- and rod-mediated vision is initiated by the absorption of photons by cone opsin or, in rods, rhodopsin. The activation of phototransduction involves the conversion of the visual pigment chromophore 11-cis retinal into its all-trans isomer on photon absorption. A bleached visual pigment releases all-trans retinal and, to restore light sensitivity, has to be supplied with another molecule of 11-cis retinal. 
For rods, 11-cis retinal is regenerated in a sequence of enzymatic reactions—collectively termed the visual cycle—that takes place in the rod itself and within the adjacent retinal pigment epithelium (RPE). 1 Although the mechanism of rod visual pigment regeneration has been studied in great detail using a large variety of transgenic mice (for example, Refs. 2 3 4 5 6 7 8 9 10 11 ), the rareness of cones (≤3% 12 13 ) in the rod-dominated retina of mice so far has hampered the analysis of cone visual pigment regeneration. 
However, several lines of evidence derived from other species suggest that cone visual pigment regeneration differs distinctly from the well-known visual cycle used by rods: In frogs, cones but not rods can restore light sensitivity in the absence of the RPE, suggesting mechanisms of cone opsin regeneration being completely located within the retina. 14 15 Furthermore, cones but not rods can recover light sensitivity by using 11-cis retinol. 16 In general, these differences appear to contribute to the cone’s ability to recover light sensitivity much faster than rods. In the chicken and squirrel, both having a cone-dominated retina, pathways have been proposed that involve Müller cells (retinal glia) for cone visual pigment regeneration, and that thereby accelerate regeneration 20-fold in comparison to the rod visual cycle. 17 18  
In addition, the proposed presence of retinal pigment epithelial protein 65 (RPE65)—a key enzyme of rod visual pigment regeneration—in cones, but not rods has been interpreted as evidence of an alternative cone-specific mechanism of visual pigment regeneration. 19 Whether or not RPE65 is expressed in cones is a matter for debate, with good evidence for 19 and against 20 its presence. Of note, RPE65 deficiency in mice particularly affects cone photoreceptor function and survival. 8 21 22 23  
To gain further insight into the retinoid metabolism of cones, we used two different mouse knockout models characterized as cone-only models: neural retina leucine zipper–deficient (Nrl −/−) mice 24 and rod opsin–deficient (Rho −/−) mice. 25 26 27 To study the role of RPE65 in particular, we also used either mice deficient in Nrl and Rpe65 or those deficient in Rho and Rpe65
NRL is a transcription factor essential for the expression of rod photoreceptor genes during development. Consequently, the absence of NRL in Nrl −/− mice leads to a complete absence of rods. 24 Instead, retinas of Nrl −/− mice display a dramatically increased number of photoreceptors, which predominantly express the short-wavelength cone opsin. Based on morphologic, electrophysiological, and molecular criteria, these cells have been classified as cones. 28 29 Thus, Nrl −/− mice provide a unique model to study cones without interference from rods. 
Rho −/− mice do not form any rod outer segments and completely lack any rod-mediated light responses. 26 27 Cones develop normally, however, and within a window from weeks 4 to 7 after birth, Rho −/− mice display normal cone function. 26 27  
In this study, we demonstrated that the retina of the Nrl −/− mouse contains both, increased amounts of RPE65, and elevated levels of Müller cell markers, being in line with the above-mentioned models of cone-specific retinoid metabolism. We furthermore showed that the additional knockout of Rpe65 in Nrl −/− mice led to a strongly reduced light sensitivity, lack of 11-cis retinal, and accumulation of retinyl esters. Similarly, knockout of Rpe65 in Rho −/− mice led to the absence of 11-cis retinal, accumulation of retinyl esters, and loss of function. 8 22 Collectively, these data indicate that RPE65 is essential for the production of the rod and cone visual pigment chromophore and therefore exerts its fundamental function at a level at which rod and cone pathways of retinoid regeneration have not yet separated. 
Methods
Mice
Nrl −/− mice 24 (B6 background) were purchased from the University of Michigan. Rho −/− mice (B6) were obtained from Peter Humphries (Trinity College, Dublin, Ireland). Rpe65 −/− mice 7 (129/B6) were a kind gift of T. Michael Redmond (National Eye Institute, Bethesda, MD). Wild-type mice (129/B6) were bred at the University Hospital, Zürich. Mice double deficient for Nrl and Rpe65 were bred by standard breeding schemes and genotyped as described. 24 30 Mice double deficient for Rho and Rpe65 have been described elsewhere. 8 22 All mice were homozygous for methionine at position 450 of RPE65. 31 32 All experiments involving animals were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and with the Guidelines of the Cantonal Veterinary Authorities of Zürich. 
Western Blot Analysis
Antibodies used for the detection of CRALBP and RPE65 have been described recently. 6 Anti-glial fibrillary acidic protein (GFAP, G-3893, Clone G-A-5) was purchased from Sigma-Aldrich (St. Louis, MO). Anti-glutamine synthetase (GS) was purchased from Chemicon International (MAB302; Temecula, CA). Isolation and preparation of tissues, Western blot analysis, and quantification of immunoreactivity were performed as published recently. 6 To compare the amount of immunoreactivity, equal amounts of total protein extracts from four retinas or four eye cups from different animals of each genotype were analyzed. The statistical significance of the result was tested with the Mann-Whitney test. 
Morphologic Analysis
For the morphologic analysis, eyes were fixed and embedded in Epon, as described recently. 33 Images were taken from 0.5-μm sections counterstained with methylene blue. 
Immunohistochemistry
Eyes were prepared, embedded in OCT medium, and immediately frozen in liquid nitrogen. Sixteen-micrometer sections were prepared and dried overnight at room temperature. The sections were fixed in cold acetone for 10 minutes, washed in PBS containing 0.05% Triton X-100 (PBST), blocked with 10% normal serum for 1 hour, and subsequently incubated with the primary antibodies (see Western Blot Analysis) in PBST containing 3% normal serum at 4°C overnight. After the sections were washed three times with PBST, the secondary antibody (Cy3 or Cy2 coupled; Jackson ImmunoResearch, West Chester, PA) was applied in PBST containing 3% normal serum for 1 to 2 hours at room temperature. 4′,6-Diamidino-2-phenylindol (DAPI; 3.5 μM) was applied in PBS for 2 minutes. Sections were washed two times in PBST and one time in PBS and were rinsed with water before they were covered with PBS containing 75% glycerol and 30 mg/mL diazo-bicyclo-octane. Images were then obtained with a microscope (Axiovision; Carl Zeiss Meditec, Inc., Dublin, CA) equipped with a digital camera, and merged images of Cy2 and Cy3 fluorescence were produced (Photoshop; Adobe Systems, Mountain View, CA). 
Retinoid Analysis
Tissue preparation, retinoid extraction, and HPLC were performed as described recently. 6 For dark adaptation, animals were kept in darkness overnight for 16 hours. 
ERG Analysis
ERGs were obtained in dark-adapted mice according to previously reported procedures. 8 The ERG equipment consisted of a Ganzfeld bowl, a direct current amplifier, and a PC-based control and recording unit (Multiliner Vision; VIASYS Healthcare GmbH, Höchberg, Germany). 
Results
Increased Amounts of RPE65 and Müller Cell Markers in the Retina of Nrl−/− Mice
By Western blot analysis, we compared the amount of proteins known to play important roles in the rod visual cycle in isolated eye cup or retina preparations from Nrl −/− and wild-type mice (6–8 weeks, an adult-age at which the cone function of Nrl −/− mice is still unimpaired). In comparison to the retina of wild-type mice, we found a 4.9-fold increased immunoreactivity for RPE65 and 2.2-fold increased levels of CRALBP. In the retina of Nrl −/− mice, the Müller cell markers GFAP and GS were 3.5- and 1.8-fold increased, respectively (Fig. 1)
In isolated eye cups (containing the RPE) the immunoreactivity for RPE65 and CRALBP appeared slightly reduced, without gaining statistical significance (Fig. 1)
Dysmorphic Structures in the Outer Layers of the Retina of the Nrl−/− Mouse
Although the normal retina is organized in several distinct layers (Fig. 2 , left column), we found that the retina of Nrl −/− mice was heavily disorganized, in particular the outer layers, which contain the photoreceptors. The retina of adult Nrl −/− mice contained rosettes—dysmorphic structures surrounded by cones 28 29 that disrupted the normal layering throughout the entire retina (Fig. 3 , top left). A large proportion of the cones were situated in rosettes that formed between 1 and 2 weeks of age (Fig. 2) . With age, the number and density of the cones decreased and the number of rosettes declined. Nevertheless, the retina also contained disorganized areas in mice at an advanced age (Fig. 2)
Proteins of the Visual Cycle and Markers of Müller Cells in Rosettes
Previous studies have shown that cone outer segments point toward the center of the rosettes. Outer segments within a rosette contain cone opsin, suggesting that these cones are functional. 28 29 We also detected RPE65 immunoreactivity within the center of rosettes in 6- to 8-week-old Nrl −/− mice (Fig. 3) , suggesting that part of the structures within a rosette are derived from the RPE. These structures do not contain melanin, however (Fig. 3) . RPE65 immunoreactivity was only detectable in eyes of mice expressing Rpe65, while no staining was obtained in Rpe65-deficient mice (Fig. 4) . The rosettes likewise contained CRALBP, and the Müller cell markers GS and GFAP (Fig. 3) . CRALBP within the rosettes may be expressed by both Müller cells and protrusions of the RPE, as CRALBP and GFAP or GS immunoreactivities colocalized only partially—that is, within the center of rosettes GFAP, and GS staining was always colocalized with CRALBP staining, but some structures were stained by CRALBP only and may thus represent cells different from Müller glia (Fig. 3)
Our findings suggest that all outer segments of cones in Nrl −/− are in close proximity to RPE65-containing structures. In areas with apparently normal morphology, outer segments contact the regular RPE. 28 29 In rosettes, cone outer segments contact the central RPE65-containing structure (Fig. 3)
Effect of RPE65 Deficiency on the Retinal Morphology of Nrl−/− Mice
Our findings in immunohistochemical and Western blot analyses support the view that RPE65 may have an important role in the retinoid metabolism of cones. To test the role of RPE65 for cones, we mated Nrl −/− mice with Rpe65 −/− mice to obtain double-deficient animals. Surprisingly, deficiency of RPE65 dramatically changed the retinal morphology of Nrl −/− mice at all developmental stages beyond the first postnatal week. Rosettes did not form, and, despite significant loss of cones with age, retinal morphology never appeared as disorganized as in the Nrl −/− mice expressing RPE65 (Fig. 2)
RPE65 deficiency renders rod photoreceptors resistant to light damage 34 ; therefore, we wondered whether the dysmorphic appearance of the retina in Nrl −/− mice might be a consequence of light damage, which would be prevented by lack of RPE65. To test this assumption, we raised Nrl −/− mice in complete darkness from birth. However, as in Nrl −/− animals raised under cyclic light, rosettes formed between week 1 and 2 after birth. Neither onset nor severity of retinal disorganization was affected by light deprivation, excluding light damage as the reason for the observed changes (Fig. 2)
Effect of RPE65 on Retinoid Composition in Nrl−/− and Rho−/− Mice
We analyzed different retinoid species in eyes (containing retina and RPE) of Nrl −/− and Nrl/Rpe65 double-deficient mice by HPLC and used 11-cis retinal as a surrogate marker for functional cone opsins. Eyes of Nrl −/− mice dark adapted overnight contained retinoids as summarized in Table 1 . In isolated retinas of Nrl −/− mice, no retinyl esters were detectable (six mice were tested, data not shown). 
RPE65 deficiency resulted in a complete lack of 11-cis retinal, 11-cis retinol, and all-trans retinal as well as a strong accumulation of retinyl esters in the eyes of Nrl/Rpe65 double-deficient mice. Again, retinyl esters were exclusively found in isolated eye cups and not in isolated retinas (retinas and eye cups from six animals were tested, not shown), suggesting that retinyl esters accumulated in the RPE only. 9-cis Retinal was the sole vitamin A aldehyde in the retina of Nrl/Rpe65 double-deficient animals, which was not detectable in retinas of Nrl −/− mice. 
Rho −/− mice contained amounts of retinoids as listed in Table 1 . Also in these mice, retinyl esters were detectable only in the eye cup, not in the retina (n = 3, not shown). The amount of 11-cis retinal found in retinas of Rho −/− mice was strongly reduced, corresponding to approximately 3.6% of the amount of 11-cis retinal in a wild-type retina. These values and previous functional tests, 26 27 showing normal cone function in Rho −/− mice at the age analyzed in the current study, indicate that we truly measured 11-cis retinal associated with functional cones. The knockout of RPE65 in Rho −/−, as in Nrl −/− mice, led to undetectable levels of 11-cis retinoids and accumulation of retinyl esters (Table 1)
In both, Nrl −/− and Rho −/− mice, the deletion of RPE65 led to a steep increase (∼fourfold) in total retinoid content with a distinct distribution of the different retinoid species, however. Notably, retinyl esters made up more than 97% of the total in both Rpe65-deficient lines, whereas they represented only 12% in Nrl −/− and 38% in Rho −/− mice expressing RPE65 (Fig. 5)
RPE65 Dependency of Cone Function in Nrl−/− Mice
The ERG from young Nrl −/− mice completely lacks responses of the rod system, but features supranormal cone responses. At the age of 4 weeks, the maximum b-wave amplitudes for single flashes applied under photopic conditions were more than twice as high as in age-matched wild-type animals. These amplitudes showed an age-related reduction after 7 weeks of age and were substantially reduced after 5 months. The decline in amplitudes progressed up to 7 months of age to a level of one third that in a 4-week-old animal. Subsequently, no further decline of function was observed up to an age of 12 months (Fig. 6A)
Deficiency of RPE65 resulted in a 1000-fold reduction of retinal sensitivity under photopic conditions at 7 weeks of age (Fig. 6B) . At 7 months of age, no more light-evoked responses were recordable from retinas of Nrl/Rpe65 double-deficient mice (Fig. 6B)
ERGs of Rho −/− and Rho/Rpe65 double-deficient animals have been published and show that between 4 and 7 weeks after birth, the cone ERG of Rho −/− mice can be considered normal, 26 27 whereas it is virtually absent in Rho/Rpe65 double-deficient mice. 8 22  
Discussion
The results described herein for two mouse models with a “cone-only” retina, namely Nrl −/− and Rho −/− mice, point to an essential role of RPE65 in the function of cone photoreceptors, particularly in the supply of the cone visual pigment chromophore. In normal mice (i.e., those expressing NRL and rod opsin and thus having a rod-dominated retina), the absence of RPE65 results in very characteristic changes: (1) 11-cis retinal is absent, 7 (2) retinyl esters accumulate within the RPE, 7 (3) 9-cis retinal is the only detectable vitamin A aldehyde, 35 36 and (4) the retinal sensitivity to light is 1000-fold reduced. 8 We observed that the deletion of RPE65 in Nrl −/− and Rho −/− mice leads to very similar changes (Table 1 , Fig. 6B ) and therefore conclude that RPE65 is of high functional importance for the retinoid metabolism of both rods and cones. This, however, does not exclude that rods and cones in mice use different pathways for retinoid recycling below the level of RPE65. For example, RPE65 could be the sole entrance for retinoids into the visual cycle of both systems after their absorption from the blood stream and subsequent esterification by lecithin-retinol acyltransferase (LRAT). 2 In this regard, if the cone pathway proposed by Mata et al. 18 for the retina of the chicken and the squirrel would exist in a rod-dominated retina such as that of a mouse, it may be fueled by all-trans retinol derived from bleached rod visual pigments, and thus indirectly depend on the rod visual cycle and thus RPE65. 37 In the particular situation of the cone-only retinas of Nrl −/− and Rho −/− mice, this indirect link via rods would be missing, but RPE65 nevertheless seems to be essential to provide an initial accouterment of 11-cis retinal for a potential cone-specific visual cycle. 
Lack of RPE65 led to deregulation of the total vitamin A content in both lines of mice (Table 1) , as has been observed in Rpe65 −/− mice. 7 In Nrl −/− and Rho −/− mice, the total amount of retinoids largely correlated with the amount of functional opsin (i.e., 11-cis retinal: Nrl −/− mice had approximately 10% of the amount of 11-cis retinal of a wild-type animal and the total of all retinoids corresponded to 12% of that found in wild-type eyes). Rho −/− mice had approximately 4% of the 11-cis retinal content of wild-type eyes and 7% of their total retinoid content. In both lines of mice, RPE65 deficiency and thus 11-cis retinal starvation led to a distinct accumulation of retinyl esters, increasing the amount of total retinoids dramatically. It might be speculated that a retina deprived of 11-cis retinal signals its demand for chromophore to the RPE, facilitating the entrance of vitamin A, which subsequently is converted into retinyl esters by LRAT. As RPE65 apparently is the sole isomerohydrolase able to use retinyl esters as a substrate, its absence causes excessive accumulation of retinyl esters, and apparently there is no product inhibition exerted on LRAT. What might be the nature of this signal? Activation of phototransduction may indicate an increased demand of retinoids. As opsin is constitutively active in photoreceptors of RPE65-deficient mice, 38 it may permanently stimulate the influx of vitamin A into the RPE. However, when constitutive activation of phototransduction by unliganded opsin is prohibited by deletion of transducin, retinyl esters nevertheless accumulate. 38 Thus, unliganded opsin possibly generates a signal independent of phototransduction that leads to increased uptake of retinoids into the RPE. Of interest, exogenous application of 9-cis retinal, which is an inverse agonist for opsin, leads to a decrease in retinyl esters in the RPE of Rpe65 −/− mice. 39  
Alternatively, RPE65 may control the function of LRAT, possibly by protein–protein interaction (Crabb JS et al., IOVS 2006;47:ARVO E-Abstract 2039). In this case, lack of RPE65 could lead to uncontrolled buildup of retinyl esters. RPE65’s activity in turn may be controlled by the availability of free opsin, which indirectly may constitute a sink for the product of Rpe65. Thus, opsin, RPE65, and LRAT may be connected in a regulatory network controlling the uptake of vitamin A in which lack of RPE65 leads to an increased retinoid content, lack of opsin to a decreased retinoid content, and lack of LRAT to complete absence of retinoids. 2  
Nrl −/− mice do not develop rod photoreceptors, but instead have an increased number of cones and are thus considered a unique model to study cone function in a mouse. 24 28 29 However, we observed that the retina of Nrl −/− mice not only undergoes developmental disorganization but also progressive degeneration in adulthood (Fig. 2) . These degenerative changes lead to a dramatic loss of photoreceptor function, as indicated by the loss of b-wave amplitudes recorded under photopic conditions. Being supranormal at 1 month of age, the amplitudes decreased to less than one third of the maximum between approximately 2 and 7 months of age (Fig. 6A)
All cones in the Nrl −/− mouse have their outer segments organized in a way that they may gain access to RPE65-containing structures (this work and Ref. 29 ). For those cones in areas with largely normal morphology, RPE65 is provided in a regularly structured RPE. 29 For cones located in rosettes, which are physically separated from the normal RPE, RPE65 is provided by structures most likely originating from the subretinal space and reaching into the center of the rosettes (Fig. 3) . These structures do not contain melanin, but appear to be connected to the RPE. They express RPE65 and CRALBP and may thus in fact be extensions of the RPE. It appears that the dysmorphic development in Nrl −/− mice ensures a close proximity of cone outer segments with the RPE and RPE65. In Nrl −/− mice, the RPE65-containing structures in the rosettes account for the increased immunoreactivity for RPE65 in isolated retinas, as observed in Western blot analysis (Fig. 1) . In wild-type mice, we found that the amount of RPE65 in the retina corresponds to 0.3% of the amount found in the eye cup (when equal amounts of total protein are analyzed); in Nrl −/− mice the amount of RPE65 in the retina corresponded to 3% of the amount found in the eye cup (n = 4 mice each; not shown). Whether RPE65 within rosettes is operative as an isomerohydrolase converting all-trans retinyl esters into 11-cis retinol, 40 41 42 appears questionable, as the substrate for this reaction—all-trans retinyl esters—was undetectable in the isolated retina of Nrl −/− mice. 
ONL dysmorphogenesis (e.g., rosette formation) appears to depend on the presence of RPE65; no rosettes formed in Nrl −/− mice in the absence of RPE65 (Fig. 2) . It is tempting to speculate that lack of 11-cis retinal caused by absence of RPE65 may be responsible for this phenomenon: Rpe65 −/− mice have dramatically reduced expression of both cone opsins 23 43 and this reduction can largely be corrected by Rpe65 gene-replacement 43 or by treating the mice with 11-cis retinal. 22 Although the effects of reduced opsin expression on cone outer segments are not known, reduced opsin expression in rods results in shortened outer segments. 25 Thus, a decrease in the expression of cone opsin may alter the morphology of cone outer segments, which in turn may influence rosette formation. In this regard, Nrl −/− mice deficient for RDS show altered cone outer segment morphology, reduced levels of key visual cycle proteins, a corresponding decrease in retinoid levels and—in comparison to Nrl −/− mice—very few rosettes. 44  
Apparently, not only RPE protrusions but also Müller cell–derived structures are present in the centers of the rosettes as indicated by GFAP, GS, and CRALBP immunoreactivity (Fig. 3) . The increase in total GFAP immunoreactivity in isolated retinas of Nrl −/− mice (observed in the present study and in the initial study of Nrl −/− mice 24 ) may be due to a mixture of two effects. Whereas GFAP expression in normal mouse retinas is not associated with the photoreceptor layer, degenerative changes of photoreceptors in Nrl −/− mice may increase the expression of GFAP in their vicinity. Also, the reduced amount of outer segment proteins in the Nrl −/− mouse retina may lead to a proportional increase of proteins from nonphotoreceptor cells, among them Müller glia. The latter explanation may also hold true for the increased immunoreactivity of GS in preparations of the Nrl −/− mouse retina. The suspicious proximity of cone outer segments and Müller cells—not only within the rosettes—will be analyzed in more detail in future experiments to reveal whether it is caused by the degenerative process and/or is an indication of a Müller cell-cone interaction for visual pigment regeneration as proposed by Mata et al. 18  
To sum up, in Rho −/− and Nrl −/− mice, both representing retinas with exclusively cone function, deletion of RPE65 abolishes this function due to lack of 11-cis retinal production. In the retinas of Rho −/−, Nrl −/− and wild-type mice, changes in retinoid metabolism due to deletion of RPE65 are qualitatively comparable. Thus, RPE65 and therefore the retinal pigment epithelium play a central role in rod and cone function in mice, and postulated cone-specific mechanisms of 11-cis retinal degeneration are either specific for cone-dominant retinas 18 or depend on RPE65 and the retinal pigment epithelium. 
 
Figure 1.
 
Quantitative analysis of protein expression in the eyes of Nrl −/− mice. Protein levels of RPE65, CRALBP, GFAP, and GS are presented as percentage of wild-type levels. (□) Protein levels in isolated retinas; (▪) protein levels in eye cups containing the RPE but no retina. Statistical significance was found for the increased expression of RPE65, CRALBP, GFAP, and GS in the retina (P = 0.0286). The decrease in RPE65 and CRALBP in the eye cup was not statistically significant. For the comparison, equal amounts of retinal or eye cup extract from four mice each of the Nrl −/− or wild-type background were loaded. Quantitation was performed as described elsewhere. 6 Numbers above the bars represent the average percentage relative to levels in wild-type mice.
Figure 1.
 
Quantitative analysis of protein expression in the eyes of Nrl −/− mice. Protein levels of RPE65, CRALBP, GFAP, and GS are presented as percentage of wild-type levels. (□) Protein levels in isolated retinas; (▪) protein levels in eye cups containing the RPE but no retina. Statistical significance was found for the increased expression of RPE65, CRALBP, GFAP, and GS in the retina (P = 0.0286). The decrease in RPE65 and CRALBP in the eye cup was not statistically significant. For the comparison, equal amounts of retinal or eye cup extract from four mice each of the Nrl −/− or wild-type background were loaded. Quantitation was performed as described elsewhere. 6 Numbers above the bars represent the average percentage relative to levels in wild-type mice.
Figure 2.
 
Retinal morphology of wild-type, Nrl −/−, Nrl/Rpe65 −/− and dark-reared Nrl −/− mice. Retinas were prepared at 1, 2, 4, and 6 weeks and 2, 3, and 6 months after birth. Images were taken from a central retinal area. Nrl −/− mice develop rosettes between weeks 1 and 2 after birth. With age, the number of photoreceptors decreased and, concomitantly, the rosettes disappeared. No rosettes developed in Nrl/Rpe65 −/− mice at any age. Photoreceptor loss also in these mice was progressive. Dark-rearing did not prevent or slow rosette-formation in Nrl −/− mice.
Figure 2.
 
Retinal morphology of wild-type, Nrl −/−, Nrl/Rpe65 −/− and dark-reared Nrl −/− mice. Retinas were prepared at 1, 2, 4, and 6 weeks and 2, 3, and 6 months after birth. Images were taken from a central retinal area. Nrl −/− mice develop rosettes between weeks 1 and 2 after birth. With age, the number of photoreceptors decreased and, concomitantly, the rosettes disappeared. No rosettes developed in Nrl/Rpe65 −/− mice at any age. Photoreceptor loss also in these mice was progressive. Dark-rearing did not prevent or slow rosette-formation in Nrl −/− mice.
Figure 3.
 
Expression of RPE65 and glial markers in rosettes. First row, left: rosettes were widely distributed across the retina; right: higher magnification of the rosette in the boxed area at left. There was a continuum from the center of the rosette toward the RPE. Second row: sections of Nrl −/− mouse eyes were stained with antibodies directed against RPE65 (left) and counterstained with DAPI (middle) to reveal cell nuclei. The center of rosettes, previously shown to contain the outer segments of cones, 28 29 contained RPE65. The center was connected to the RPE as indicated by the continuous staining of RPE65 from the regular RPE into the center of the rosette; right: merger of the left and the middle images. Third row: sections of Nrl −/− mouse eyes were stained with antibodies directed against CRALBP (left) and GS (middle). The RPE and some structures within the center of the rosette were exclusively stained with the CRALBP antibody. Other areas of the center and the outside of the rosette were immunoreactive for both CRALBP and GS, as indicated by yellow areas in the merged image (right). Fourth row: sections of Nrl −/− mouse eyes were stained with antibodies directed against CRALBP (left) and GFAP (middle left). The RPE and some structures within the center of the rosette were exclusively stained with the CRALBP antibody. Other areas of the center and the outside of the rosette were immunoreactive for both CRALBP and GFAP, as indicated by the yellow in the merged image shown in the middle right image. Right: GFAP was virtually absent from the photoreceptor layer in a wild-type mouse, indicating the closer proximity of Müller cells and photoreceptors in the Nrl −/− mouse.
Figure 3.
 
Expression of RPE65 and glial markers in rosettes. First row, left: rosettes were widely distributed across the retina; right: higher magnification of the rosette in the boxed area at left. There was a continuum from the center of the rosette toward the RPE. Second row: sections of Nrl −/− mouse eyes were stained with antibodies directed against RPE65 (left) and counterstained with DAPI (middle) to reveal cell nuclei. The center of rosettes, previously shown to contain the outer segments of cones, 28 29 contained RPE65. The center was connected to the RPE as indicated by the continuous staining of RPE65 from the regular RPE into the center of the rosette; right: merger of the left and the middle images. Third row: sections of Nrl −/− mouse eyes were stained with antibodies directed against CRALBP (left) and GS (middle). The RPE and some structures within the center of the rosette were exclusively stained with the CRALBP antibody. Other areas of the center and the outside of the rosette were immunoreactive for both CRALBP and GS, as indicated by yellow areas in the merged image (right). Fourth row: sections of Nrl −/− mouse eyes were stained with antibodies directed against CRALBP (left) and GFAP (middle left). The RPE and some structures within the center of the rosette were exclusively stained with the CRALBP antibody. Other areas of the center and the outside of the rosette were immunoreactive for both CRALBP and GFAP, as indicated by the yellow in the merged image shown in the middle right image. Right: GFAP was virtually absent from the photoreceptor layer in a wild-type mouse, indicating the closer proximity of Müller cells and photoreceptors in the Nrl −/− mouse.
Figure 4.
 
Specificity of the RPE65 antibody. Sections from wild type, Nrl −/− and Nrl/Rpe65 double-deficient mice were incubated with (+) or without (−) RPE65 antibody. Images were taken with an acquisition time of 50 ms for wild-type and Nrl −/− with primary antibody. In wild type and Nrl −/− sections incubated without primary antibody and the Nrl/Rpe65 double-deficient sections, an acquisition time of 200 ms did not result in signals above background.
Figure 4.
 
Specificity of the RPE65 antibody. Sections from wild type, Nrl −/− and Nrl/Rpe65 double-deficient mice were incubated with (+) or without (−) RPE65 antibody. Images were taken with an acquisition time of 50 ms for wild-type and Nrl −/− with primary antibody. In wild type and Nrl −/− sections incubated without primary antibody and the Nrl/Rpe65 double-deficient sections, an acquisition time of 200 ms did not result in signals above background.
Table 1.
 
Retinoid Composition in Eyes of Mice with Different Gene Deletions
Table 1.
 
Retinoid Composition in Eyes of Mice with Different Gene Deletions
Genotype 11-cis retinal 11-cis Retinol all-trans Retinal 9-cis Retinal all-trans Retinol Retinyl Ester Total Retinoid Content Eyes (n)
Wild type 414.1 ± 73.2 9.4 ± 1.7 90.5 ± 5.7 13.8 ± 3.0 65.1 ± 5.6 605.8 12
Nrl −/− 42.0 ± 5.9 4.1 ± 2.4 12.8 ± 2.9 3.2 ± 0.5 8.8 ± 1.6 70.9 6
Rho −/− 15.1 ± 2.9 4.5 ± 1.5 5.5 ± 1.6 0.6 ± 0.1 1.5 ± 0.9 16.7 ± 5.2 43.9 13
Nrl −/− 1.4 ± 0.4 1.0 ± 0.3 295.7 ± 3.7 298.1 8
Rpe65 −/−
Rho −/− 0.82 ± 0.1 4.7 ± 0.6 167.9 ± 16.3 173.4 8
Rpe65 −/−
Figure 5.
 
Proportional retinoid composition of wild-type, Nrl −/−, Rho −/−, Nrl/Rpe65 −/−, and Rho/Rpe65 −/− mice. After dark adaptation, the preponderant retinoid in eyes (combined eye cup and retina) of wild-type and Nrl −/− mice was 11-cis retinal, corresponding to 69% and 60% of the total retinoids; esters made up 11% and 12%, respectively. In Rho −/− mice, 11-cis retinal represented 34% of the total, and esters amounted to 38%. In all three lines, all-trans retinal represented 13% to 18% of the total. Rpe65 deficiency dramatically altered the proportional composition of retinoids. In Nrl/Rpe65 −/− and Rho/Rpe65 −/− mice retinyl esters represented more than 97% of the total, whereas 11-cis retinal and all-trans retinal were not detectable. The total amount of measured retinoids was set to 100%; bars, fraction of a particular retinoid in the percentage of the total.
Figure 5.
 
Proportional retinoid composition of wild-type, Nrl −/−, Rho −/−, Nrl/Rpe65 −/−, and Rho/Rpe65 −/− mice. After dark adaptation, the preponderant retinoid in eyes (combined eye cup and retina) of wild-type and Nrl −/− mice was 11-cis retinal, corresponding to 69% and 60% of the total retinoids; esters made up 11% and 12%, respectively. In Rho −/− mice, 11-cis retinal represented 34% of the total, and esters amounted to 38%. In all three lines, all-trans retinal represented 13% to 18% of the total. Rpe65 deficiency dramatically altered the proportional composition of retinoids. In Nrl/Rpe65 −/− and Rho/Rpe65 −/− mice retinyl esters represented more than 97% of the total, whereas 11-cis retinal and all-trans retinal were not detectable. The total amount of measured retinoids was set to 100%; bars, fraction of a particular retinoid in the percentage of the total.
Figure 6.
 
ERG age series in Nrl −/− mice and the effect of additional RPE65 deficiency. Starting out supranormal, cone photoreceptor function in Nrl −/− mice progressively decreased with age; the absence of RPE65 led to an additional severe loss of function. (A) ERGs were recorded under photopic conditions from Nrl −/− mice 4 and 7 weeks and 5, 7, and 12 months after birth. In comparison to a 4-week-old wild-type mouse, the photopic ERG responses of Nrl −/− mice were substantially larger in amplitude, which is in agreement with the fact that there are many more cones present than normal. Up to an age of approximately 7 weeks, ERG amplitudes remained largely unaltered. Thereafter, amplitudes decreased until a plateau was reached at and beyond 7 months of age. (B) The additional knockout of Rpe65 in Nrl −/− mice led to a dramatic desensitization of the photoreceptors. Roughly 1000-fold more light was necessary to trigger a threshold response in 7-week-old animals. At 7 months of age, no detectable light-evoked responses were recordable in double-knockout mice.
Figure 6.
 
ERG age series in Nrl −/− mice and the effect of additional RPE65 deficiency. Starting out supranormal, cone photoreceptor function in Nrl −/− mice progressively decreased with age; the absence of RPE65 led to an additional severe loss of function. (A) ERGs were recorded under photopic conditions from Nrl −/− mice 4 and 7 weeks and 5, 7, and 12 months after birth. In comparison to a 4-week-old wild-type mouse, the photopic ERG responses of Nrl −/− mice were substantially larger in amplitude, which is in agreement with the fact that there are many more cones present than normal. Up to an age of approximately 7 weeks, ERG amplitudes remained largely unaltered. Thereafter, amplitudes decreased until a plateau was reached at and beyond 7 months of age. (B) The additional knockout of Rpe65 in Nrl −/− mice led to a dramatic desensitization of the photoreceptors. Roughly 1000-fold more light was necessary to trigger a threshold response in 7-week-old animals. At 7 months of age, no detectable light-evoked responses were recordable in double-knockout mice.
The authors thank Gabi Hoegger, Coni Imsand, and Hedi Wariwoda for technical assistance and John C. Saari (University of Washington, Seattle, WA) for the CRALBP antibody. 
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Figure 1.
 
Quantitative analysis of protein expression in the eyes of Nrl −/− mice. Protein levels of RPE65, CRALBP, GFAP, and GS are presented as percentage of wild-type levels. (□) Protein levels in isolated retinas; (▪) protein levels in eye cups containing the RPE but no retina. Statistical significance was found for the increased expression of RPE65, CRALBP, GFAP, and GS in the retina (P = 0.0286). The decrease in RPE65 and CRALBP in the eye cup was not statistically significant. For the comparison, equal amounts of retinal or eye cup extract from four mice each of the Nrl −/− or wild-type background were loaded. Quantitation was performed as described elsewhere. 6 Numbers above the bars represent the average percentage relative to levels in wild-type mice.
Figure 1.
 
Quantitative analysis of protein expression in the eyes of Nrl −/− mice. Protein levels of RPE65, CRALBP, GFAP, and GS are presented as percentage of wild-type levels. (□) Protein levels in isolated retinas; (▪) protein levels in eye cups containing the RPE but no retina. Statistical significance was found for the increased expression of RPE65, CRALBP, GFAP, and GS in the retina (P = 0.0286). The decrease in RPE65 and CRALBP in the eye cup was not statistically significant. For the comparison, equal amounts of retinal or eye cup extract from four mice each of the Nrl −/− or wild-type background were loaded. Quantitation was performed as described elsewhere. 6 Numbers above the bars represent the average percentage relative to levels in wild-type mice.
Figure 2.
 
Retinal morphology of wild-type, Nrl −/−, Nrl/Rpe65 −/− and dark-reared Nrl −/− mice. Retinas were prepared at 1, 2, 4, and 6 weeks and 2, 3, and 6 months after birth. Images were taken from a central retinal area. Nrl −/− mice develop rosettes between weeks 1 and 2 after birth. With age, the number of photoreceptors decreased and, concomitantly, the rosettes disappeared. No rosettes developed in Nrl/Rpe65 −/− mice at any age. Photoreceptor loss also in these mice was progressive. Dark-rearing did not prevent or slow rosette-formation in Nrl −/− mice.
Figure 2.
 
Retinal morphology of wild-type, Nrl −/−, Nrl/Rpe65 −/− and dark-reared Nrl −/− mice. Retinas were prepared at 1, 2, 4, and 6 weeks and 2, 3, and 6 months after birth. Images were taken from a central retinal area. Nrl −/− mice develop rosettes between weeks 1 and 2 after birth. With age, the number of photoreceptors decreased and, concomitantly, the rosettes disappeared. No rosettes developed in Nrl/Rpe65 −/− mice at any age. Photoreceptor loss also in these mice was progressive. Dark-rearing did not prevent or slow rosette-formation in Nrl −/− mice.
Figure 3.
 
Expression of RPE65 and glial markers in rosettes. First row, left: rosettes were widely distributed across the retina; right: higher magnification of the rosette in the boxed area at left. There was a continuum from the center of the rosette toward the RPE. Second row: sections of Nrl −/− mouse eyes were stained with antibodies directed against RPE65 (left) and counterstained with DAPI (middle) to reveal cell nuclei. The center of rosettes, previously shown to contain the outer segments of cones, 28 29 contained RPE65. The center was connected to the RPE as indicated by the continuous staining of RPE65 from the regular RPE into the center of the rosette; right: merger of the left and the middle images. Third row: sections of Nrl −/− mouse eyes were stained with antibodies directed against CRALBP (left) and GS (middle). The RPE and some structures within the center of the rosette were exclusively stained with the CRALBP antibody. Other areas of the center and the outside of the rosette were immunoreactive for both CRALBP and GS, as indicated by yellow areas in the merged image (right). Fourth row: sections of Nrl −/− mouse eyes were stained with antibodies directed against CRALBP (left) and GFAP (middle left). The RPE and some structures within the center of the rosette were exclusively stained with the CRALBP antibody. Other areas of the center and the outside of the rosette were immunoreactive for both CRALBP and GFAP, as indicated by the yellow in the merged image shown in the middle right image. Right: GFAP was virtually absent from the photoreceptor layer in a wild-type mouse, indicating the closer proximity of Müller cells and photoreceptors in the Nrl −/− mouse.
Figure 3.
 
Expression of RPE65 and glial markers in rosettes. First row, left: rosettes were widely distributed across the retina; right: higher magnification of the rosette in the boxed area at left. There was a continuum from the center of the rosette toward the RPE. Second row: sections of Nrl −/− mouse eyes were stained with antibodies directed against RPE65 (left) and counterstained with DAPI (middle) to reveal cell nuclei. The center of rosettes, previously shown to contain the outer segments of cones, 28 29 contained RPE65. The center was connected to the RPE as indicated by the continuous staining of RPE65 from the regular RPE into the center of the rosette; right: merger of the left and the middle images. Third row: sections of Nrl −/− mouse eyes were stained with antibodies directed against CRALBP (left) and GS (middle). The RPE and some structures within the center of the rosette were exclusively stained with the CRALBP antibody. Other areas of the center and the outside of the rosette were immunoreactive for both CRALBP and GS, as indicated by yellow areas in the merged image (right). Fourth row: sections of Nrl −/− mouse eyes were stained with antibodies directed against CRALBP (left) and GFAP (middle left). The RPE and some structures within the center of the rosette were exclusively stained with the CRALBP antibody. Other areas of the center and the outside of the rosette were immunoreactive for both CRALBP and GFAP, as indicated by the yellow in the merged image shown in the middle right image. Right: GFAP was virtually absent from the photoreceptor layer in a wild-type mouse, indicating the closer proximity of Müller cells and photoreceptors in the Nrl −/− mouse.
Figure 4.
 
Specificity of the RPE65 antibody. Sections from wild type, Nrl −/− and Nrl/Rpe65 double-deficient mice were incubated with (+) or without (−) RPE65 antibody. Images were taken with an acquisition time of 50 ms for wild-type and Nrl −/− with primary antibody. In wild type and Nrl −/− sections incubated without primary antibody and the Nrl/Rpe65 double-deficient sections, an acquisition time of 200 ms did not result in signals above background.
Figure 4.
 
Specificity of the RPE65 antibody. Sections from wild type, Nrl −/− and Nrl/Rpe65 double-deficient mice were incubated with (+) or without (−) RPE65 antibody. Images were taken with an acquisition time of 50 ms for wild-type and Nrl −/− with primary antibody. In wild type and Nrl −/− sections incubated without primary antibody and the Nrl/Rpe65 double-deficient sections, an acquisition time of 200 ms did not result in signals above background.
Figure 5.
 
Proportional retinoid composition of wild-type, Nrl −/−, Rho −/−, Nrl/Rpe65 −/−, and Rho/Rpe65 −/− mice. After dark adaptation, the preponderant retinoid in eyes (combined eye cup and retina) of wild-type and Nrl −/− mice was 11-cis retinal, corresponding to 69% and 60% of the total retinoids; esters made up 11% and 12%, respectively. In Rho −/− mice, 11-cis retinal represented 34% of the total, and esters amounted to 38%. In all three lines, all-trans retinal represented 13% to 18% of the total. Rpe65 deficiency dramatically altered the proportional composition of retinoids. In Nrl/Rpe65 −/− and Rho/Rpe65 −/− mice retinyl esters represented more than 97% of the total, whereas 11-cis retinal and all-trans retinal were not detectable. The total amount of measured retinoids was set to 100%; bars, fraction of a particular retinoid in the percentage of the total.
Figure 5.
 
Proportional retinoid composition of wild-type, Nrl −/−, Rho −/−, Nrl/Rpe65 −/−, and Rho/Rpe65 −/− mice. After dark adaptation, the preponderant retinoid in eyes (combined eye cup and retina) of wild-type and Nrl −/− mice was 11-cis retinal, corresponding to 69% and 60% of the total retinoids; esters made up 11% and 12%, respectively. In Rho −/− mice, 11-cis retinal represented 34% of the total, and esters amounted to 38%. In all three lines, all-trans retinal represented 13% to 18% of the total. Rpe65 deficiency dramatically altered the proportional composition of retinoids. In Nrl/Rpe65 −/− and Rho/Rpe65 −/− mice retinyl esters represented more than 97% of the total, whereas 11-cis retinal and all-trans retinal were not detectable. The total amount of measured retinoids was set to 100%; bars, fraction of a particular retinoid in the percentage of the total.
Figure 6.
 
ERG age series in Nrl −/− mice and the effect of additional RPE65 deficiency. Starting out supranormal, cone photoreceptor function in Nrl −/− mice progressively decreased with age; the absence of RPE65 led to an additional severe loss of function. (A) ERGs were recorded under photopic conditions from Nrl −/− mice 4 and 7 weeks and 5, 7, and 12 months after birth. In comparison to a 4-week-old wild-type mouse, the photopic ERG responses of Nrl −/− mice were substantially larger in amplitude, which is in agreement with the fact that there are many more cones present than normal. Up to an age of approximately 7 weeks, ERG amplitudes remained largely unaltered. Thereafter, amplitudes decreased until a plateau was reached at and beyond 7 months of age. (B) The additional knockout of Rpe65 in Nrl −/− mice led to a dramatic desensitization of the photoreceptors. Roughly 1000-fold more light was necessary to trigger a threshold response in 7-week-old animals. At 7 months of age, no detectable light-evoked responses were recordable in double-knockout mice.
Figure 6.
 
ERG age series in Nrl −/− mice and the effect of additional RPE65 deficiency. Starting out supranormal, cone photoreceptor function in Nrl −/− mice progressively decreased with age; the absence of RPE65 led to an additional severe loss of function. (A) ERGs were recorded under photopic conditions from Nrl −/− mice 4 and 7 weeks and 5, 7, and 12 months after birth. In comparison to a 4-week-old wild-type mouse, the photopic ERG responses of Nrl −/− mice were substantially larger in amplitude, which is in agreement with the fact that there are many more cones present than normal. Up to an age of approximately 7 weeks, ERG amplitudes remained largely unaltered. Thereafter, amplitudes decreased until a plateau was reached at and beyond 7 months of age. (B) The additional knockout of Rpe65 in Nrl −/− mice led to a dramatic desensitization of the photoreceptors. Roughly 1000-fold more light was necessary to trigger a threshold response in 7-week-old animals. At 7 months of age, no detectable light-evoked responses were recordable in double-knockout mice.
Table 1.
 
Retinoid Composition in Eyes of Mice with Different Gene Deletions
Table 1.
 
Retinoid Composition in Eyes of Mice with Different Gene Deletions
Genotype 11-cis retinal 11-cis Retinol all-trans Retinal 9-cis Retinal all-trans Retinol Retinyl Ester Total Retinoid Content Eyes (n)
Wild type 414.1 ± 73.2 9.4 ± 1.7 90.5 ± 5.7 13.8 ± 3.0 65.1 ± 5.6 605.8 12
Nrl −/− 42.0 ± 5.9 4.1 ± 2.4 12.8 ± 2.9 3.2 ± 0.5 8.8 ± 1.6 70.9 6
Rho −/− 15.1 ± 2.9 4.5 ± 1.5 5.5 ± 1.6 0.6 ± 0.1 1.5 ± 0.9 16.7 ± 5.2 43.9 13
Nrl −/− 1.4 ± 0.4 1.0 ± 0.3 295.7 ± 3.7 298.1 8
Rpe65 −/−
Rho −/− 0.82 ± 0.1 4.7 ± 0.6 167.9 ± 16.3 173.4 8
Rpe65 −/−
×
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