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Correlation of Regenerable Opsin with Rod ERG Signal in Rpe65^-/- Mice during Development and Aging

¹From the Departments of Ophthalmology and ²Physiology and Neuroscience, Medical University of South Carolina, Charleston, South Carolina; and the ³Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, National Institutes of Health, Bethesda, Maryland.

	Abstract

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PURPOSE. RPE65 has been shown to be essential for the production of 11-cis retinal by the retinal pigment epithelium. Mutations in RPE65 are known to be associated with severe forms of early-onset retinal dystrophy. This project was designed to determine the amount of regenerable opsin in Rpe65^-/- mice during development and aging, and to examine the function of this rhodopsin by electroretinography (ERG).

METHODS. Young and aged Rpe65^-/- and wild-type (WT) mice were dark adapted. Endogenous rhodopsin and regenerable opsin were measured using absorption-difference spectrophotometry. Photoreceptor function was assessed with scotopic single-flash ERGs and photoreceptors were counted in histologic sections. Opsin’s primary structure was analyzed by mass-spectrometric mapping.

RESULTS. Unlike WT mice, amounts of regenerable opsin in Rpe65^-/- mice decreased significantly with age, which correlated with a decrease in the number of photoreceptors and a decline in ERG amplitudes. Opsin structure, however, did not change. No endogenous levels of rhodopsin were measurable in the Rpe65^-/- mice (detection limit: 0.225 pmol). 11-cis Retinal injections resulted in the regeneration of similar amounts of rhodopsin and improved rod function in a comparable way, irrespective of age.

CONCLUSIONS. In the aged Rpe65^-/- mouse, opsin levels decrease because of the loss of photoreceptors. The remaining opsin is structurally intact, and the components of the phototransduction cascade and the retinal circuitry remain functional, despite the absence of normal photoreceptor activity.

In humans, mutations in the RPE65 gene account for 10% to 15% of Leber congenital amaurosis (LCA), a severe form of autosomal recessive, childhood-onset retinal dystrophy,^{5 6} as well as some cases of recessive retinitis pigmentosa (RP).⁷ In both cases, photoreceptor degeneration occurs; however, patients with RP have good central vision during the first decade of life, whereas patients with LCA are born blind or lose vision within a few months after birth.

Thus, we set out to investigate the development and deterioration of photoreceptor structure and function in Rpe65^-/- mice during aging, with a special emphasis on the ability of the old Rpe65^-/- photoreceptors to regenerate functional photopigment. The results show that opsin levels declined during aging, in part due to the loss of photoreceptors, without, however, resulting in further deterioration of the photoresponse or alterations in opsin structure. In vivo 11-cis retinal treatment resulted in the regeneration of visual pigment and the improvement of both a- and b-waves of the scotopic ERG regardless of age, suggesting that photoreceptors in aged Rpe65^-/- mice retain their ability to regenerate rhodopsin and that the retinal circuitry remains intact, despite the degeneration that occurs in this retina.

	Material and Methods

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Animals
All experiments were in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and with approval by the University Animal Care and Use Committee. Rpe65^-/- mice were generated and genotyped as described previously and bred as homozygous animals.¹ Age-matched C57Bl/6 mice were purchased from Jackson Laboratories (Bar Harbor, ME). Animals were housed in the animal facility with a normal 12-hour light, 12-hour dark cycle and food and water provided ad libitum. For experiments requiring dark adaptation, animals were kept in complete darkness overnight, and all light-sensitive procedures were performed under dim red light. Animals were treated with 11-cis retinal (0.05 µg/g body weight) by intraperitoneal (IP) injections. 11-cis Retinal was dissolved in 10% ethanol, 10% fatty-acid–free bovine serum albumin (BSA) for stabilization,⁸ and 0.9% NaCl. After the injection, animals were kept in darkness for 24 hours.

ERG Recordings
Mice were anesthetized with ketamine (100 mg/kg) and xylazine (25 mg/kg) and their pupils dilated with 1% atropine and 2.5% phenylepinephrine. The animals were kept on a heating pad held at 37°C. Full-field ERGs were recorded, using a device adapted from Lyubarsky and Pugh,⁹ as described in detail.¹⁰ The optical bench was modified to include two pathways for light stimulation and background light. The optical pathways were controlled by mechanical shutters and manually operated neutral-density and bandpass filters. A filter cube combined the two pathways, and lenses focused the light beam to the end of the light guide that delivered the light stimulus to the eye. Scotopic ERGs were recorded in response to 10-ms light flashes of increasing intensity.

Microspectrophotometry
Retinas from dark-adapted mice were collected under dim red light. The samples were prepared according to previously published methods, with some modifications.¹ An aliquot of 0.5 mL of 10 mM Tris-HCl and 1 mM EDTA (pH 7.5) along with 1 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (AEBSF; Roche Molecular Biochemicals, Indianapolis, IN), a protease inhibitor cocktail tablet (Complete Mini; Roche Molecular Biochemicals), and 10 µg DNase I (Sigma, St. Louis, MO) were added to samples (n = 2–4 retinas) which were homogenized by a series of syringe triturations by using increasingly smaller needle sizes (18–26 gauge). Samples were spun at 27,000g for 15 minutes, and the resultant pellet resuspended in 100 µL of 1% n-dodecyl-ß-D-maltoside (ULTROL grade; Calbiochem, La Jolla, CA) in 0.1 M sodium phosphate buffer (pH 7.4) for solubilization (2 hours at 4°C on a rotator). Unsolubilized material was removed by centrifugation (100,000g for 15 minutes), and the supernatant was analyzed on a microspectrophotometer (SpectroPette; World Precision Instruments, Sarasota, FL). To obtain difference spectra, we bleached the samples for 30 seconds in the presence of hydroxylamine hydrochloride (pH 6.0–7.0, 20 mM final concentration). Five spectra were summed for each sample. For regeneration, 11-cis retinal (5x excess, 14 nM) was incubated with the solubilized sample overnight at 4°C on a rotator. Data were exported into a computer for difference spectra calculations (KaleidaGraph software; Synergy Software, Reading, PA).

Histology
Eyes were enucleated and immersion fixed in 4% paraformaldehyde in phosphate-buffered saline (pH 7.4) overnight at 4°C, after which they were dehydrated over several hours and embedded in paraffin in transverse orientation. Eyes were sectioned at 7 µm and mounted on poly-L-lysine–coated slides. The sections were stained with 1% toluidine blue in deionized water, dehydrated, coverslipped with mounting medium (Permount; Fisher Scientific, Fair Lawn, NJ), and photographed. The thickness of individual retinal layers was measured in a central area of the retina within 100 to 300 µm of the optic nerve, on computer with the measuring tool of a software program (Spot; Diagnostic Instruments, Sterling Heights, MI). Because retinal thicknesses are affected by extensive tissue shrinkage during the dehydration and wax infiltration process, there is an underestimation of retinal thickness by a factor of approximately 2.5 when compared with in vivo measurements.¹¹

Mass Spectrometry
Rod outer segments of dark-adapted young (3 months) and old (19–22 months) Rpe65^-/- (n = 9–13) or WT mice (n = 8–10) mice were isolated, digested, and analyzed by mass spectrometry (MS) as described previously.^{12 13}

Data Analysis
Experiments were performed on 3 to 10 animals (ERG, histology) or three to four samples (microspectrophotometry) per group, which was found to be appropriate to generate statistically reliable results for all the methods used. Data were expressed as mean ± SEM. For statistical purposes, a standard t-test was used, with a significance level of P < 0.05.

	Results

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Reduction of Opsin in Aged Rpe65^-/- Mice
Retinas from dark-adapted, age-matched C57Bl/6 and Rpe65^-/- mice were homogenized and regenerated with 11-cis retinal. The dodecyl-maltoside extracts were measured for rhodopsin levels by absorption-difference spectrophotometry. The total regenerable rhodopsin levels (rho_max) remained constant for the first year in WT mice at 0.154 ± 0.005 to 0.175 ± 0.023 nmol/retina and decreased slightly but significantly at 18 months to 0.131 ± 0.004 nmol/retina (P = 0.001; Fig. 1A , C57B1/6). During the same time frame, the length of the photoreceptors and the thickness of the photoreceptor nuclear layer (ONL) decreased slightly but insignificantly (P = 0.3; Table 1 ). In comparison, rho_max in Rpe65^-/- mice remained constant for the first 6 months (range, 0.098 ± 0.003 to 0.1063 ± 0.003 nmol/retina). By 12 months of age rho_max had decreased to 0.064 ± 0.014 nmol/retina, and by 18 months of age, rho_max was reduced approximately 80% of the 1-month level to 0.0197 ± 0.0036 nmol/retina (P < 0.001; Fig. 1A , gray bars).

View larger version (14K): [in this window] [in a new window]   FIGURE 1. Total regenerable opsin in WT and Rpe65-/- mice. Retinas from age-matched, dark-adapted WT and Rpe65-/- mice were regenerated with excess 11-cis retinal, solubilized in dodecyl-maltoside, and measured for rhodopsin concentration by difference-absorption spectrophotometry. (A) Rhodopsin content per retina and (B) percentage of rhodopsin regenerated in Rpe65-/- compared with WT mice.

View larger version (95K): [in this window] [in a new window]   FIGURE 2. Retina structure in young and aged Rpe65-/- mice. Eyes from 1-month-old (left) and 18-month-old (right) Rpe65-/- mice were sectioned in transverse orientation, to compare thicknesses of the retinal layers. INL, inner nuclear layer; IPL, inner plexiform layer; IS, inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer; OS, outer segments; RGC, retinal ganglion cell layer; RPE, retinal pigment epithelium. Scale bar, 25 µm.

Opsin Primary Structure up to 22 Months
The complete mass spectrometric mapping of mouse rhodopsin was performed by digesting the protein into 16 peptide fragments of various lengths.¹⁴ As reported previously, no differences were found between the opsins of 3-month-old WT and Rpe65^-/- mice (data not shown; for additional information, please refer to Table 1 in Ref. ¹³ ). We extended these observations by mapping opsin from 19- to 22-month-old WT and Rpe65^-/- mice and found no differences in the retention times and relative abundance of these peptide fragments during HPLC separation. The identity of the 16 peptides was confirmed by tandem mass spectrometric sequencing, and no differences were observed between the opsin fragments isolated from young or aged animals in either WT or Rpe65^-/- animals. Both the sequences and the types of detected posttranslational modifications were the same. However, as reported previously for young Rpe65^-/- mice,¹³ the mass spectrometric analysis of opsin from Rpe65^-/- mice revealed unusually high levels of phosphorylation in dark-adapted mice, which was confirmed in the aged Rpe65^-/- animals (data not shown).

Residual Rod Function up to 18 Months
To determine whether the rods are functional in the Rpe65^-/- mouse retina during development and aging, we recorded single-flash ERGs from animals in the same age range as those used for opsin measurements. At the maximal light intensities tested, the Rpe65^-/- photoreceptor responses elicited only a small b-wave and no a-wave, and the b-wave amplitudes remained constant, regardless of the age of the Rpe65^-/- mice (Fig. 3) . When WT mice were tested at the same age points, it was demonstrated that both a- and b-waves declined in amplitude in animals aged from 1 to 6 months, but remained unchanged during further aging (data not shown).

View larger version (12K): [in this window] [in a new window]   FIGURE 3. Scotopic single-flash ERGs of Rpe65-/- mice, with and without 11-cis retinal treatment during aging. Scotopic flash ERGs were recorded at maximum light intensity (3.1 x 1011 photons/mm2) before 11-cis retinal treatment (smaller responses per panel) at 1 month (A), 6 months (B) and 18 months of age (C), and after 11-cis retinal treatment (larger responses at each age). Regardless of age, 11-cis retinal improved photoreceptor function.

View larger version (18K): [in this window] [in a new window]   FIGURE 4. ERG analysis of 1-, 6-, and 18-month-old Rpe65-/- mice. The a- and b-wave amplitudes were measured in response to single-flash stimuli with increasing flash intensities at 1 (squares), 6 (circles), and 18 (triangles) months. (A) No a-waves were recorded at any age without 11-cis retinal treatment (filled symbols), whereas in all age groups, 11-cis retinal treatment regenerated enough rhodopsin that the rod response resulted in a recordable a-wave (open symbols). (B) The b-waves improved significantly in mice with 11-cis retinal treatment (open symbols) when compared with their untreated littermates (filled symbols).

View larger version (16K): [in this window] [in a new window]   FIGURE 5. Exogenous 11-cis retinal remained available for pigment regeneration after bleaching. (A) Rpe65-/- animals (1 month of age) were treated with a single dose of 11-cis retinal and tested for successful regeneration (prebleach). All rhodopsin was bleached in vivo with a strong adaptation light (postbleach) and allowed to recover for 10 days, after which animals were retested (recovery). (B) The a- and b-wave amplitudes recovered significantly, demonstrating that 11-cis retinal remained available for at least 2 weeks. All ERGs were recorded in response to a flash of 3.11 x 1011 photons/mm2.

	Discussion

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A steady decline in the amount of regenerable rhodopsin in the Rpe65^-/- mouse was observed (Fig. 1) that was paralleled by a loss of photoreceptors and a shortening of the remaining outer segments (Table 1) . These results are consistent with the 35% loss of photoreceptor nuclei in the ONL of 1-year-old Rpe65^-/- mice reported by Katz and Redmond.⁴ Despite the loss of photoreceptors, the remaining b-waves stayed constant in amplitude at approximately 50 to 65 µV during aging, suggesting that the remaining photoreceptors are structurally intact (Fig. 3B) .

In the WT animals, rhodopsin levels remained constant during the first 12 months, whereas photoreceptor sensitivity, as measured by a-wave amplitude, peaked at 1 month of age and subsequently declined to an adult level. Similar observations were made by Fulton et al.¹⁹ who attributed this effect to possible differences in physical events during development or changes in extracellular resistance with age. An age-related decline in responsiveness has also been reported in humans²⁰ and mice.²¹ In the mouse, Li et al.²¹ demonstrated that the age-related loss in both a- and b-waves between 2 and 12 months of age could not be attributed to a change in cellular density in rod photoreceptors and thus suggested that it could be due to changes in ocular resistance. Our data on rhodopsin concentration support these hypotheses. No significant changes in retinal thickness were observed in the WT mice during aging, which is in support of data reported by Trachimowicz et al.,²² also in C57Bl/6 mice.

Correlation of Regeneration of Rhodopsin with Age
The photoreceptors in the aged Rpe65^-/- mice remained functionally intact and integrated into the retinal circuitry. The latter point is of particular importance, because it has been noted that in the rd mouse²³ as well as in other models of photoreceptor dystrophies (Jones et al., ARVO Abstract, 1885, 2002), cells in the inner retina undergo dramatic morphologic modifications accompanying the photoreceptor loss, in particular in aged animals.

To test further whether the photoreceptors in the aged Rpe65^-/- mice remain fully functional, we injected animals in all age groups with a constant amount of 11-cis retinal. If the photoreceptors from an aged animal (in a retina with substantial degeneration) were as healthy as those in a young animal (in a retina in which no significant degeneration has yet occurred), similar amounts of rhodopsin regeneration and similar improvement in the ERG parameters would be expected. Regardless of age, photoreceptors in all three age groups tested (1, 6, and 18 months of age) regenerated functional rhodopsin, resulting in ERGs with increased sensitivity. Thus, all the transport and uptake mechanisms for 11-cis retinal that are present in the retina of a young animal appear to remain active in the aged animal, and so does the signal transduction cascade and the postsynaptic machinery. However, only one dose of 11-cis retinal was tested in this study (50 ng/g body weight). The levels of rhodopsin formed certainly were not maximal, indicating that the transport and uptake systems are the limiting factors. However, these are only inferences from the ERG recordings, and more detailed biochemical and histologic analyses are necessary.

Although IP injections are a successful way to deliver 11-cis retinal to the photoreceptors, it was of interest to analyze whether 11-cis retinal stabilized by BSA can provide a reservior over time. Our experiments in which the newly regenerated as well as the residual rhodopsin was completely bleached and allowed to regenerate, demonstrate, that there appears to be sufficient 11-cis retinal available to regenerate approximately 30% of the prebleaching level. We suggest that the 11-cis retinal used to regenerate rhodopsin after bleaching was stored in a pool (most likely in the retina or RPE) inaccessible to light and stabilized by either BSA or a retinoid-binding protein. This hypothesis is based on the observation by Van Hooser et al.²⁴ who demonstrated that 9-cis retinal is not stored in an alcohol or retinyl ester form in the eye or liver, but rather is preserved as 9-cis retinal in the eye for the long term, if the animals are kept in darkness. They also suggest that "it appears that 9-cis retinal is, in a large part, recycled from phagocytized iso-Rho(dopsin) to newly produced opsin molecules over an extended period."²⁴ The experiment discussed herein showed that the 11-cis retinal available for regeneration must come from another source, as the rods were completely bleached, and therefore the 11-cis retinal in the rhodopsin was completely converted to all-trans retinol. Additional experiments are needed to identify the source of this 11-cis retinal.

	Summary

Top Abstract Material and Methods Results Discussion Summary References

 
Photoreceptors in the Rpe65^-/- mice are almost completely devoid of 11-cis retinal, due to a missing crucial step in the retinoid pathway. We determined the long-term consequences of the absence of chromophore on photoreceptor survival and activity. The maximum amount of regenerable rhodopsin in vitro declined steadily with age, which is reflected in the loss of cells in the ONL and a reduction in OS length. Despite the loss of photoreceptors, the Rpe65^-/- mice retained minimal light responsiveness that did not decline significantly with age. The remaining photoreceptors were structurally intact and functionally integrated into the retinal circuitry, as all age groups responded to exogenous 11-cis retinal treatment with an increase in photoreceptor sensitivity and an increase in b-wave amplitude. This finding is partly explained by the fact that the structure of the residual opsin stayed intact up to 22 months of age and was indistinguishable from the WT opsin. Although it is unclear what causes photoreceptor degeneration in the Rpe65^-/- mice, our results demonstrate that the remaining photoreceptors are functionally intact.

	Acknowledgements

 
The authors thank Yumei Chen for genotyping and Greg Beall for assistance with dissections.

	Footnotes

 
The mass spectrometry was performed in the Medical University of South Carolina’s Mass Spectrometry Institutional Research Resource Facility.

Supported by National Eye Institute Grants EY13520, EY04939, and EY12231; the Foundation Fighting Blindness; and an unrestricted grant to the Medical University of South Carolina from Research to Prevent Blindness, Inc.

Submitted for publication June 11, 2002; revised August 13, 2002; accepted August 17, 2002.

Disclosure: B. Rohrer, None; P. Goletz, None; S. Znoiko, None; Z. Ablonczy, None; J. Ma, None; T.M. Redmond, 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, Charleston, SC 29425; rohrer{at}musc.edu.

	References

Top Abstract Material and Methods Results Discussion Summary References

 

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