The Genetic Modifier Rpe65Leu ₄₅₀ : Effect on Light Damage Susceptibility in c-Fos-Deficient Mice

Abstract

purpose. To test whether introduction of the Rpe65Leu ₄₅₀ variant can overcome protection against light-induced photoreceptor apoptosis in mice without the activator protein (AP)-1 constituent c-Fos.

methods. c-Fos-deficient mice (c-fos ^−/− ) carrying the Leu ₄₅₀ variant of RPE65 were compared with c-fos ^−/− mice with Rpe65Met ₄₅₀ . Expression of RPE65 was analyzed by Western blot analysis. Rhodopsin regeneration was determined by measuring rhodopsin after different times in darkness after bleaching. Susceptibility to light-induced damage was tested by exposure to white light and subsequent morphologic analysis. Activation of AP-1 and its complex composition was analyzed by electromobility shift assay (EMSA) and antibody interference. The contribution of AP-1 to apoptosis was tested by pharmacological inhibition of AP-1, using dexamethasone.

results. Compared with RPE65Met ₄₅₀ , introduction of the RPE65Leu ₄₅₀ variant led to increased levels of RPE65 protein, accelerated rhodopsin regeneration, loss of protection against light-induced damage, and AP-1 responsiveness to toxic light doses, despite the absence of c-Fos. c-Fos was mainly replaced by Fra-2. Application of dexamethasone restored resistance to light-induced damage.

conclusions. Increasing retinal photon catch capacity by introducing the Rpe65Leu ₄₅₀ variant overcomes light damage resistance provided by c-fos deficiency. Thus, a variation of RPE65 at position 450 is a strong genetic modifier of susceptibility to light-induced damage in mice. Under conditions of high rhodopsin availability during exposure to light, Fra-2 and, to a minor degree, FosB substitute for c-Fos and enable light-induced AP-1 activity and thus photoreceptor apoptosis. Regardless of the AP-1 complex’s composition, glucocorticoid receptor activation inhibits AP-1 and prevents apoptosis. Thus, not the absence of c-Fos per se, but rather impairment of AP-1 DNA binding is protective against light-induced damage. This impairment may result from the absence of c-Fos or glucocorticoid receptor-mediated transrepression.

Light-induced damage of photoreceptors is a model system widely used to investigate intracellular mechanisms that lead to apoptotic cell death, the common fate of photoreceptors in retinitis pigmentosa and age-related macular degeneration. ¹  

c-Fos was the first gene discovered to be essential for photoreceptor apoptosis induced by exposure to bright light. ^{2 3} Photoreceptors of c-Fos-deficient mice (c-fos ^−/− ) are fully functional, ⁴ but are highly resistant to light-induced damage. ³ Although in wild-type mice, exposure to damaging doses of light leads to a fast and sustained increase in the DNA-binding activity of AP-1, no such activation of AP-1 can be achieved by exposure to light in c-fos ^−/− mice. ³ Analysis of the composition of the dimeric AP-1 complex reveals that c-Fos is a preponderant constituent of light-activated AP-1. ⁵ Thus, the absence of c-Fos is considered to confer resistance to light-induced damage of photoreceptors by suppressing AP-1 DNA binding activity. This concept is reinforced by observations in mice with elevated corticosteroid levels. Increased activity of the glucocorticoid receptor diminishes light-induced AP-1 DNA-binding activity and counteracts light-induced apoptosis. ⁶  

Damage to photoreceptors by exposure to short periods of bright light is mediated by rhodopsin ^{7 8} in a transducin-independent manner. ⁹ In contrast to earlier observations in rodent models, the sensitivity of photoreceptors to light-induced damage in mice is modulated by the availability of rhodopsin during exposure to light: Fast rhodopsin regeneration by the visual cycle coincides with a high sensitivity, slow regeneration renders the retina more resistant, and a block of rhodopsin regeneration completely prevents light-induced damage. ^{10 11 12 13} An amino acid variation at position 450 (Leu ₄₅₀ Met) in the retinal pigment epithelial protein RPE65 has been discovered to be a genetic modifier of susceptibility to light-induced damage ¹⁴ and also modifies the speed of rhodopsin regeneration (Nusinowitz S, Nguyen LT, Farber DB, Danciger M, ARVO Abstract 3758, 2002). ¹² The methionine variant has been discovered so far only in C57BL/6 mice or mouse lines on a mixed background derived from it. All other mouse strains analyzed so far carry the leucine variant ^{12 14} (Danciger M, unpublished observation, 2003). 

c-Fos ^−/− mice are maintained on a mixed background of C57BL/6 and 129/Sv (see http://www.jax.org). All experiments using c-fos ^−/− mice so far ^{2 3 4 5} have been performed with mice carrying the Rpe65Met ₄₅₀ variant (fos ^−/− Rpe65M), which confers increased resistance to light-induced damage. ^{12 14} In the present study, we introduced the light-sensitive variant, Rpe65Leu ₄₅₀ , onto the c-fos ^−/− background (fos ^−/− Rpe65L), to test whether this genetic modification, known to increase susceptibility to light-induced damage in wild-type mice, would render the retinas of these animals vulnerable to light-induced damage. 

Materials and Methods

fos ^−/− Rpe65L and fos ^−/− Rpe65M Mice

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 Zurich. Pigmented c-fos heterozygous animals (mixed background B6;129, stock number 002293) were purchased from the Jackson Laboratories (Bar Harbor, ME) and reared in a 12-hour light-dark cycle with 60 to 100 lux within the cages. Screening these animals for Rpe65Leu ₄₅₀ or Met ₄₅₀ was performed as previously described. ¹² Both Rpe65 genotypes were found, indicating that these mice segregate genes of 129/Sv (embryonic stem [ES] cell donor with Rpe65Leu ₄₅₀ ) and C57BL/6 (host with Rpe65Met ₄₅₀ ). Breeding was performed to generate c-fos ^−/− mice homozygous for either Rpe65Leu ₄₅₀ (fos ^−/− Rpe65L) or Rpe65Met ₄₅₀ (fos ^−/− Rpe65M). Experiments were performed in pigmented mice aged 6 to 8 weeks. 

Western Blot Analysis for RPE65

RPE65 protein levels were determined in eyecup homogenates containing retinal pigment epithelium (RPE) and retina but not lens and vitreous. ¹² Western blot analysis was performed as described recently. ¹² Four new rabbit- and guinea pig-derived antibodies (Pin-5 to -8, Pineda Antikörper Service, Berlin, Germany) directed against amino acids 150 to 164 of mouse RPE65 (NH2-CNFITKINPETLETIK-COOH ¹⁵ ) were tested. After affinity purification, antibodies Pin-5, -6, and -8 detected a single protein of 66 kDa in mouse eyecup homogenates. No staining was obtained when extracts of Rpe65-deficient mice¹⁸ were tested (not shown). All experiments shown here were performed using the Pin-5 antibody (polyclonal rabbit anti-RPE65) at a dilution of 1:2000. Immunoreactivity was visualized using a detection kit (Western Lightning; Perkin Elmer, Boston, MA). 

Rhodopsin Regeneration

Rhodopsin content was determined spectrophotometrically in retinal extracts from both eyes. ¹² Rhodopsin was determined after overnight dark adaptation or immediately after the end of exposure to light (5000 lux of white light for 10 minutes with dilated pupils). Rhodopsin regeneration was measured after a period of 60 or 90 minutes of recovery in darkness after exposure to light. 

Light-Induced Damage

Before exposure to light, animals were dark-adapted for 16 hours overnight. The pupils of the animals were dilated under dim red light (Cyclogyl 1%; Alcon, Cham, Switzerland; and phenylephrine 5%, Ciba Vision, Niederwangen, Switzerland) and the mice were exposed to diffuse white fluorescent light (TLD-36 W/965 tubes; Philips, Hamburg, Germany; UV-impermeable diffuser) for 2 and 6 hours (lights on at 10 AM) with an intensity of 15,000 lux in cages with a reflective interior. ¹²  

AP-1 Electromobility Shift Assay and Antibody Interference

Preparation of nuclear extracts, electromobility shift assay (EMSA) and antibody interference were performed as described previously. ^{3 5} Briefly, one retina was homogenized in 400 μL 10 mM HEPES-KOH (pH 7.9), 1 mM β-mercaptoethanol, and 1 mM dithiothreitol (DTT) in the presence of protease inhibitors. After incubation on ice for 10 minutes, the homogenate was vortexed for 10 seconds and centrifuged. The pellet was resuspended in 50 μL 20 mM HEPES-KOH (pH 7.9), 25% glycerol, 420 mM NaCl, 1.5 mM MgCl₂, 0.2 mM EDTA, 1 mM β-mercaptoethanol, and 1 mM DTT in the presence of protease inhibitors and incubated on ice for 10 minutes. Cellular debris was removed by centrifugation at 23,000g for 30 minutes at 4°C. Protein concentrations were determined using the Bradford protein assay (Bio-Rad, Hercules, CA) with BSA as a standard. Nuclear extracts (5 μg) were incubated with 1 μL oligonucleotides coding for an AP-1-specific (5′-AAG CAT GAG TCA GAC AC-3′) DNA-binding sequence (TPA response element, TRE) labeled with ³²P-γ-adenosine triphosphate (ATP) (Hartmann Analytic GmbH, Braunschweig, Germany) on ice for 20 minutes with 19 μL 5 mM MgCl₂, 0.1 mM EDTA, 0.75 mM DTT, 7.5% glycerol, 0.05% NP-40 containing 24 μg BSA and 2 μg poly d(I-C) (Roche Molecular Chemicals, Mannheim, Germany). Protein/DNA complexes were resolved at 150 V on a 1.5-mm 6% polyacrylamide gel using 0.25× tris borat EDTA (TBE) buffer and visualized on x-ray film. 

For antibody interference, the following antibodies from Santa Cruz (Santa Cruz Biotechnology, Santa Cruz, CA) were preincubated with the nuclear extracts: anti-c-Fos (catalog number sc-052), anti-Fra-1 (catalog number sc-183), anti-Fra-2 (catalog number sc-604), anti-FosB (catalog number sc-048), and a mixture (pan-Jun), of anti-c-Jun (catalog number sc-045), anti-JunB (catalog number sc-046), and anti-JunD (catalog number sc-074). 

Dexamethasone Treatment

Mice received an intraperitoneal injection of dexamethasone (52 mg/kg body weight) 2 minutes before exposure to light, as described recently. ^{6 17} Control animals received an injection of an equal volume of saline. 

Morphology

For histologic sections, enucleated eyes were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.3) at 4°C overnight. After fixation, the inferior central retina adjacent to the optic nerve of each eye was trimmed, washed in cacodylate buffer and embedded in Epon 812. Sections (0.5 μm) were stained with methylene blue and analyzed by light microscopy. ³  

Results

RPE65 Protein Levels in fos ^−/− Rpe65L and fos ^−/− Rpe65M Mice

Western blot analysis of eyecup homogenates of fos ^−/− Rpe65L and fos ^−/− Rpe65M mice using an RPE65 specific antibody revealed significantly higher levels of RPE65 in fos ^−/− Rpe65L mice (Fig. 1) . 

Rhodopsin Regeneration Kinetics in fos ^−/− Rpe65L and fos ^−/− Rpe65M Mice

After dark adaptation overnight, retinas of fos ^−/− Rpe65L mice contained 0.45 ± 0.04 nmol rhodopsin (n = 3) and those of fos ^−/− Rpe65M mice 0.48 ± 0.04 nmol (n = 3). Exposure of mice with dilated pupils to white light of 5,000 lux for 10 minutes bleached more than 90% of rhodopsin in both lines of mice (Fig. 2) . Regeneration in darkness was two times faster in fos ^−/− Rpe65L (Fig. 2) . Although 90 minutes of dark adaptation was sufficient to regenerate maximal rhodopsin levels in fos ^−/− Rpe65L, fos ^−/− Rpe65M mice regenerated only 50% during the same period. 

Susceptibility to Light-Induced Damage in fos ^−/− Rpe65L and fos ^−/− Rpe65M Mice

Exposure of fos ^−/− Rpe65M mice to 15,000 lux of white light for 2 or 6 hours did not cause any signs of irreversible damage (Figs. 3A 3B 3C) . Ten days after exposure to light, retinas of fos ^−/− Rpe65M mice appeared morphologically normal and outer nuclear layer (ONL) thickness in the central inferior area amounted to 10 to 11 rows of photoreceptor nuclei. In contrast, severe loss of photoreceptors was caused by 2 hours of exposure in fos ^−/− Rpe65L mice. Large central areas of fos ^−/− Rpe65L mice displayed a reduction in ONL thickness to three to four rows of nuclei (Figs. 3D 3E) . Many of the remaining nuclei still contained condensed chromatin, indicating ongoing apoptosis. Rod outer segments (ROS) and rod inner segments (RIS) were completely disintegrated, and the RPE was swollen. 

AP-1 DNA-Binding Activity in fos ^−/− Rpe65L and fos ^−/− Rpe65M Mice

As determined by EMSA, no AP-1 DNA-binding activity was induced by exposure to 15,000 lux for 2 hours in fos ^−/− Rpe65M mice (Fig. 4A) , which is in line with our previous results. ³ However, in fos ^−/− Rpe65L mice, AP-1 DNA-binding activity showed a similar increase as described for wild-type mice, ^{2 3} notably, despite absence of c-Fos (Fig. 4A) . 

AP-1 Complex Composition in fos ^−/− Rpe65L Mice

Antibody interference with AP-1 DNA-binding activity was strongest when retinal nuclear extracts were incubated with antibodies directed against Fra-2 or a mixture of Jun-specific antibodies (Fig. 4B) . Little interference (super-shift) was observed on incubation of FosB antibodies. AP-1 DNA-binding activity was only very slightly impaired by addition of an anti-Fra-1 antibody (Fig. 4B) . The reduction in AP-1 DNA binding after addition of anti-c-Fos antibodies may have been due to unspecific action of the antibody, because extracts were derived from c-fos ^−/− mice. 

Prevention of Photoreceptor Apoptosis by Application of Dexamethasone

Pretreatment of fos ^−/− Rpe65L mice with dexamethasone (52 mg/kg, intraperitoneal) restored retinal resistance against exposure to 15,000 lux of light for 2 hours. Three days after exposure to light, saline-injected animals displayed severe light-induced damage affecting RPE, ROS, and RIS morphology. Furthermore, ONL thickness was reduced in large central areas of the retina (Fig. 5) . In contrast, only a few scattered apoptotic nuclei of photoreceptors and a small number of condensed RIS and ROS resulted from exposure to light in dexamethasone-treated animals (Fig. 5) . 

Discussion

Replacing the Met ₄₅₀ variant of RPE65 by the Leu ₄₅₀ variant in c-fos ^−/− mice rendered the retina of these animals susceptible to light-induced apoptosis of photoreceptors. Introducing Rpe65Leu ₄₅₀ increased the amount of RPE65 protein present in the RPE and accelerated regeneration of rhodopsin after strong bleach. Faster kinetics of rhodopsin regeneration increases the photon catch capacity of photoreceptors. ¹² Thus, in a given period of exposure to light, photoreceptors of mice carrying the Rpe65Leu ₄₅₀ variant absorb more photons than those of mice carrying the Rpe65Met ₄₅₀ variant. c-Fos ^−/− mice with Rpe65Met ₄₅₀ resisted exposure to white light of 15,000 lux for up to 6 hours, while fos ^−/− Rpe65L mice showed severe irreversible loss of photoreceptors after exposure for only 2 hours. These results demonstrate the strong influence of the different RPE65 variants and show that the Rpe65Leu ₄₅₀ genetic modification is sufficient to overcome the protection against light-induced damage conferred by absence of c-Fos. ^{2 3}  

Application of dexamethasone, a synthetic glucocorticoid agonist, restored resistance against light-induced damage in fos ^−/− Rpe65L mice. Activation of the glucocorticoid receptor interferes with AP-1 DNA-binding activity. ^{6 17} Similarly, the absence of c-Fos in fos ^−/− Rpe65M mice disables light-induced AP-1 activity. ³ Under extreme situations such as those present in fos ^−/− Rpe65L mice, in which photon catch capacity is dramatically increased, absent c-Fos may be replaced. Antibody interference indicates that among the Fos family members mainly Fra-2 and to a minor extent FosB replaced c-Fos under these conditions. Both, Fra-2 and FosB are not involved in light-induced photoreceptor apoptosis in wild-type animals. ⁵ Recent experiments using mice in which the coding sequence of c-fos was replaced by the coding sequence of Fra-1 ¹⁷ indicate that the regulatory sequences of the c-fos gene are particularly responsive to light-induced damage. Although Fra-1 under control of its own regulatory sequences is unresponsive to exposure to light, ⁵ it can very well substitute for c-Fos when controlled by the c-fos regulatory sequences. Under these circumstances, Fra-1 is a constituent of light-activated AP-1, and photoreceptor apoptosis occurs despite the absence of c-Fos. ¹⁷ Fra-2 and FosB proteins normally may not be involved in light-induced photoreceptor apoptosis, because the c-fos gene is more readily activated by light treatment and promotes the apoptotic cascade before Fra-2 and FosB can contribute. In addition, both proteins may be activated only beyond a certain level of proapoptotic stimulation that is surpassed under extreme conditions such as a combination of increased photon catch (due to fast regeneration), extreme light intensity, and long duration of exposure. In the absence of c-Fos, introduction of RPE65Leu ₄₅₀ may produce this kind of extreme situation, enabling Fra-2 and FosB to contribute to AP-1-mediated apoptotic signaling. 

Nevertheless, AP-1 whether containing Fra-1, ¹⁷ Fra-2, or FosB remained amenable to glucocorticoid receptor-mediated inhibition, indicating that AP-1 is the essential mediator of photoreceptor apoptosis. c-Fos is an important constituent of AP-1, but its absence can be overcome depending on the strength of the proapoptotic stimulus. 

These data emphasize that for any analysis of light-induced retinal degeneration it is essential to know the genetic background of the mice under investigation. This includes not only the Rpe65 genotype affecting rhodopsin regeneration, but also other genetic modifiers of yet unknown function. ¹⁴  

²  Contributed equally to the work and therefore should be considered equivalent authors.

 Supported by the Swiss National Science Foundation; the Velux Foundation, Glarus, Switzerland; and the German Research Council.

 Submitted for publication November 6, 2002; revised January 8, 2003; accepted January 27, 2003.

 Disclosure: A. Wenzel, None; C. Grimm, None; M. Samardzija, None; C.E. Remé, 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.

 

Different RPE65 protein expression levels due to different Rpe65 genotype. c-Fos ^−/− mice homozygous for Rpe65Leu ₄₅₀ had significantly higher steady state levels of RPE65 in the pigment epithelium compared with fos ^−/− Rpe65M mice. Eyecup homogenate (10 μg) from three animals of each genotype was subjected to 10% SDS-PAGE followed by Western blot analysis with affinity-purified Pin-5 antibody at a dilution of 1:2000.

Figure 2.

 

Rhodopsin regeneration is accelerated in fos ^−/− Rpe65L mice. After overnight dark adaptation, rhodopsin in both types of mice was bleached by exposing animals with dilated pupils to 5000-lux white light for 10 minutes. This exposure reduced dark-adapted levels of rhodopsin by more than 90% (t = 0 minutes) in fos ^−/− Rpe65L and fos ^−/− Rpe65M. Recovery of rhodopsin in darkness was measured 60 and 90 minutes after the end of exposure to light in two to three animals. A trend line was generated and tested by the R ² test. fos ^−/− Rpe65L mice regenerated rhodopsin two times faster than fos ^−/− Rpe65M mice.

Figure 3.

 

Rpe65Leu ₄₅₀ rendered the retina of c-fos ^−/− susceptible for light-induced damage. (A–C) Morphology of fos ^−/− Rpe65M and (D, E) of fos ^−/− Rpe65L mice. (A) Morphology of the inferior central retina of a fos ^−/− Rpe65M mouse 24 hours after 2 hours of exposure to 15,000 lux. Apart from slightly disorganized ROS tips and a few scattered apoptotic bodies (white arrowhead), no morphologic signs of light-induced damage were observed. (B) Ten days after the same treatment retinal morphology appeared entirely normal in fos ^−/− Rpe65M mice. (C) Even after 6 hours of exposure to 15,000 lux, no damage was visible in fos ^−/− Rpe65M mice 10 days later. (D) fos ^−/− Rpe65L mice displayed severe retinal damage 24 hours after exposure to 15,000 lux for 2 hours. In large central areas, the RPE was swollen, ROS and RIS were disintegrated, and most of the photoreceptor nuclei in the ONL contained condensed chromatin (arrowheads), indicative of apoptosis. (E) Ten days after the same treatment, ONL thickness was reduced to three to four rows of photoreceptor nuclei, with many of them being pyknotic (arrowheads). The RPE showed cystic spaces in the cytoplasm (arrow); RIS and ROS were completely destroyed. RPE, retinal pigment epithelium; ROS, rod outer segments; RIS, rod inner segments; ONL, outer nuclear layer; INL, inner nuclear layer.

Figure 4.

 

Light-induced AP-1 activity in fos ^−/− Rpe65L mice. EMSA was performed with 5 μg of retinal nuclear extracts. (A) Lanes 2 to 4: AP-1 DNA-binding activity in light-damage susceptible wild-type mice before (lane 2), immediately after (lane 3), and 6 hours after (lane 4) exposure to 15,000 lux for 2 hours. Lanes 5 to 7: AP-1 DNA-binding activity in fos ^−/− Rpe65L mice before (lane 5), immediately after (lane 6), and 6 hours after (lane 7) exposure to 15,000 lux for 2 hours. Lanes 8 and 9: AP-1 DNA-binding activity in fos ^−/− Rpe65M mice before (lane 8) and 6 hours after (lane 9) exposure to 15,000 lux for 2 hours. Lane 1: Control, no nuclear extract added. (B) Antibody interference analysis with nuclear extracts of fos ^−/− Rpe65L mice 6 hours after exposure to 15,000 lux for 2 hours. No antibody (lane 1), anti-c-Fos (lane 2), anti-Fra-1 (lane 3), anti-Fra-2 (lane 4), anti-FosB (lane 5), or a mixture of anti-c-Jun, -JunB, and -JunD (lane 6) antibodies were added to nuclear extracts before incubation with the radiolabeled AP-1 probe. The strongest interference with AP-1 DNA binding was observed for Fra-2 and the mixture of Jun antibodies. Minor interference, resulting in a supershifted complex, resulted from incubation with FosB antibodies Arrow: super-shifted complex consisting of anti-FosB antibody and AP-1. (✶)f Position of AP-1 DNA complexes.

Figure 5.

 

Dexamethasone restored light-induced damage resistance in fos ^−/− Rpe65L mice. fos ^−/− Rpe65L mice were exposed to 15,000-lux white light for 2 hours and analyzed 72 hours later. Mice that received a saline injection (No Dex) showed severe retinal degeneration, including RPE edema (arrow), disintegration of ROS and RIS, and reduction of ONL thickness. Mice pretreated with a single dose of dexamethasone (52 mg/kg, + Dex) were highly resistant, with only a few scattered pyknotic photoreceptor nuclei present in the ONL (arrowheads). Abbreviations are as in Figure 3 .

The authors thank Dora Greuter, Gabi Hoegger, and Coni Imsand for excellent technical assistance.