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. Author manuscript; available in PMC: 2010 Feb 17.
Published in final edited form as: Eur J Neurosci. 2006 Feb;23(4):1028–1034. doi: 10.1111/j.1460-9568.2006.04639.x

Rpe65 as a modifier gene for inherited retinal degeneration

M Samardzija 1, A Wenzel 1, M Naash 2, C E Remé 1, C Grimm 1
PMCID: PMC2823586  NIHMSID: NIHMS164416  PMID: 16519667

Abstract

Light accelerates progression of retinal degeneration in many animal models of retinitis pigmentosa (RP). A sequence variant in the Rpe65 gene (Rpe65450Leu or Rpe65450Met) can act as a modulator of light-damage susceptibility in mice by influencing the kinetics of rhodopsin regeneration and thus by modulating the photon absorption. Depending on exposure duration and light intensity applied, white fluorescent light induces photoreceptor apoptosis and retinal degeneration in wild-type mice by the activation of one of two known molecular pathways. These pathways depend, respectively, on activation of the transcription factor c-Fos/AP-1 and on phototransduction activity. Here we tested Rpe65 as a genetic modifier for inherited retinal degeneration and analysed which degenerative pathway is activated in a transgenic mouse model of autosomal dominant RP. We show that retinal degeneration was reduced in mice expressing the Rpe65450Met variant and that these mice retained more visual pigment rhodopsin than did transgenic mice expressing the Rpe65450Leu variant. In addition, lack of phototransduction slowed retinal degeneration whereas ablation of c-Fos had no effect. We conclude that sequence variations in the Rpe65 gene can act as genetic modifiers in inherited retinal degeneration, presumably by regulating the daily rate of photon absorption through the modulation of rhodopsin regeneration kinetics. Increased absorption of photons and/or light sensitivity appear to accelerate retinal degeneration via an apoptotic cascade which involves phototransduction but not c-Fos.

Keywords: c-Fos/AP-1, mouse, phototransduction, retinal degeneration, Rpe65

Introduction

Age-related macular degeneration (AMD) and retinitis pigmentosa (RP) are leading causes of blindness in the Western world. Whereas AMD is a multifactorial disease including exogenous (smoking, light exposure, etc.) and endogenous (gene mutation) risk factors, RP is mostly monogenic. Mutations in genes encoding proteins involved in visual function are especially prone to cause retinal degeneration. One of the most prominent mutations is found at position 23 of rhodopsin. The exchange of proline with histidine at this position (P23H) is responsible for ~10% of autosomal dominant RP in the USA.

The molecular events during retinal degenerations remain largely unknown although it would be essential to understand the mechanisms of the disease process to develop therapeutic strategies for patients. Light exposure accelerates the disease process in several animal models and it has been suggested that light is a cofactor for AMD as well as for inherited retinal degenerations in human patients (Taylor et al., 1990; Cruickshanks et al., 1993; Simons, 1993; Cideciyan et al., 1998; Cruickshanks et al., 2001). Therefore, light-induced retinal degeneration in wild-type mice has been used to study pathophysiological mechanisms in the retina (Sanyal & Hawkins, 1986; Wang et al., 1997; Chen et al., 1999a; Chen et al., 1999b; LaVail et al., 1999; Organisciak et al., 1999; Wenzel et al., 2005a), and at least two different pathways for light-dependent photoreceptor cell death have been revealed. The first pathway, induced by short-term exposure to high light levels, depends on the activation of the transcription factor activator protein-1 (AP-1) but is independent of phototransduction. The second pathway, induced by long-term exposure to low levels of light, does not seem to depend on AP-1 but involves phototransduction (Hao et al., 2002). It has been shown that a sequence variation in the Rpe65 gene is associated with reduced light-damage susceptibility (LaVail et al., 1987a,b; Danciger et al., 2000). This variant codes for methionine instead of leucine at position 450 of the protein (RPE65450Met) causing reduced steady-state levels of RPE65, probably via a post-translational mechanism (Wenzel et al., 2001). The reduced RPE65 (‘RPE’ signifies ‘retinal pigment epithelium’) levels lead to a reduced rhodopsin regeneration rate in darkness (Wenzel et al., 2001) and during illumination (Wenzel et al., 2005b).

The VPP mouse is a model for autosomal dominant RP caused by the P23H mutation. It carries a rhodopsin transgene coding for a protein with three amino acid substitutions (V20G, P23H and P27L), one of which represents the P23H mutation in humans (Naash et al., 1996). Retinal degeneration in mice expressing the VPP transgene is accelerated by light (Wang et al., 1997). Genetic factors modulating disease progression have been postulated (Naash et al., 1996).

Here we used the VPP mouse to study factors known to modulate light-damage susceptibility in a model of inherited retinal degeneration. By creating several double-mutant mice, we show that Rpe65 acts as a modifier gene for retinal degeneration and that VPP-induced photoreceptor death follows the pathway of low-level long-term light exposure.

Materials and methods

Animals

All procedures concerning animals were in accordance with the regulations of the Veterinary Authority of Zurich and with the ARVO statement for the use of animals in research. Animals were kept in a 12:12-h light: dark cycle with 40–60 lux of dim white light within cages. To grow animals in constant darkness, animals were put into a wooden box at birth and kept in darkness for 6 weeks. Husbandry was carried out in dim red light with a wavelength > 650 nm. All mice used were of a mixed 129SV/C57BL/6 background. Double-transgenic mice were generated by classical breeding schemes. Genotypes were tested by polymerase chain reaction (PCR) of DNA isolated from tail tissue.

Assessment of retinal degeneration

Mice were killed with CO2 and retinal degeneration was assessed qualitatively using light microscopy on 0.5-μm sections of Epon-embedded tissues. Sections shown are from the lower temporal part of the retina. Quantitative determination of photoreceptor degeneration was carried out by measuring rhodopsin content as described previously (Kueng-Hitz et al., 2000). Statistical analyses used unpaired two-tailed t-tests.

RNA isolation and RT-PCR

Total retinal RNA and cDNA were prepared using the the RNeasy RNA isolation kit (Qiagen, Hilden, Germany) as described (Grimm et al., 2000a). cDNA was prepared using oligo(dT) and M-MLV reverse transcriptase (Promega, Madison, USA). cDNAs corresponding to 10 ng of total RNA were amplified for 23 cycles with primers specific for β-actin (up, 5′-CAA CGG CTC CGG CAT GTG C-3′; down, 5′-CTC TTG CTC TGG GCC TCG-3′) or for 25 cycles with primers specific for c-fos (up, 5′-CAA CGC CGA CTA CGA GGC GTC AT-3′; down, 5′-GTG GAG ATG GCT GTC ACC G-3′), respectively (Grimm et al., 2000a). Products were separated on a 1.5% agarose gel and visualized with ethidiumbromide staining.

Results

Rpe65 is a modifier gene for inherited retinal degeneration

We combined the VPP transgene with each of the Rpe65 variants (Rpe65450Leu and Rpe65450Met) and tested the time course of retinal degeneration qualitatively by microscopic analysis of retinal morphology and quantitatively by measuring rhodopsin content. Degeneration in both VPP;Rpe65450Leu and VPP;Rpe65450Met mice was not uniform; this is manifested in an increased severity of photoreceptor loss in the temporal retina as compared to the nasal retina (not shown). Micrographs shown in Fig. 1 (and in all other figures) were taken from the most affected region. Independent of the Rpe65 variant, VPP and wild-type mice showed a similar retinal morphology at postnatal day (PND) 11 (Fig. 1A, top panels). At PND 15, thinning of the outer nuclear layer (ONL) was observed in all VPP mice but mice with the Rpe65450Leu variant appeared to be more severely affected. At PND 18, thinning of the ONL had continued and photoreceptor outer segments were much shorter than in wild-type mice (Fig. 1A, bottom panels). At PND 21, when developmental apoptosis is completed, the number of photoreceptors in VPP mice was reduced, with the Rpe65450Leu variant slightly more affected (Fig. 1B, top panels). Photoreceptor outer segments were poorly preserved. From PND 21 to PND 56, progression of the degeneration was slow or almost nonexistent. Indeed, retinal morphology seemed to improve in the VPP;Rpe65450Met mouse strain up to PND 42. However, such a potential improvement was not represented by a statistically significant increase in the content of total retinal rhodopsin (P = 0.098, PND 21 vs. PND 42). Furthermore, expression studies of the proliferation marker ki-67 by reverse transcription (RT)-PCR and immunohistochemical detection assays of systemically applied BrdU did not show evidence of proliferative activity in the ONL of mice of the VPP;Rpe65450Met genotype (not shown). A similar biphasic degeneration as observed here (a fast phase until PND 21 and slow phase thereafter) was recently described for transgenic rats homozygous for the P23H mutation (Pinilla et al., 2005).

Fig. 1.

Fig. 1

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Time course of VPP-mediated retinal degeneration was dependent on the Rpe65 sequence. (A) Retinal morphology of VPP;Rpe65450Met (left column), VPP;Rpe65450Leu (middle column) and wt;Rpe65450Leu (right column) mice at PND 11 (top row), 15 (middle row) and 18 (bottom row). Shown are representative sections of the lower temporal part of at three or more independent mice. (B) Retinal morphology of VPP;Rpe65450Met (left column), VPP;Rpe65450Leu (middle column) and Rpe65−/− (right column) mice at PND 21 (top row), 28 (second row), 42 (third row) and 56 (bottom row). The Rpe65450Leu variant accelerated the VPP-mediated retinal degeneration leading to a more pronounced thinning of the ONL and less preserved photoreceptor segments. The Rpe65 knockout only partially rescued the degeneration. Shown are representative sections of the lower temporal part of at least three independent mice. (C) Rhodopsin per retina expressed in nmol. Values are given as means + SD. Open bars, VPP;Rpe65450Met; solid bars, VPP;Rpe65450Leu. Numbers of mice are indicated. *P = 0.0046, **P < 0.0001. (D) Representative section through the lower temporal part of the retina of an Rpe65−/− mouse at PND 56. PS, photoreceptor segment; ONL, outer nuclear layer; INL, inner nuclear layer.

Nevertheless, at PND 28 (P = 0.0046) and PND 42 (P < 0.0001), retinas of VPP mice expressing the RPE65450Met variant had significantly higher rhodopsin values than retinas of VPP mice expressing the RPE65450Leu variant (Fig. 1C). This suggests that the two Rpe65 sequence variants have different influences on the morphology and rhodopsin content of the retinas in the degenerative process caused by the VPP transgene.

Complete removal of RPE65 by crossing the VPP transgene onto an Rpe65-knockout background slightly delayed the degeneration but did not protect the retina to the same extent as the Rpe65450Met variant. Of note, deletion of Rpe65 alone causes a late onset and slowly progressing retinal degeneration (Rohrer et al., 2003; Woodruff et al., 2003), which proceeds much more slowly than observed here for Rpe65-deficient VPP mice (Fig. 1B and D).

The progression of VPP-mediated retinal degeneration is not only dependent on the genetic background but is also accelerated by light (Naash et al., 1996; Wang et al., 1997). The two variants of the Rpe65 gene influence rhodopsin regeneration in the visual cycle (Wenzel et al., 2001) and therefore modulate the photon absorption capacity by the visual pigment during light exposure (Wenzel et al., 2005b). To eliminate the influence of light, VPP;Rpe65450Leu mice were raised in darkness. This treatment partly protected retinal morphology at PND 42 as compared to animals of the same genotype reared in cyclic light (Fig. 2A). The morphological observation was supported by rhodopsin values which were approximately two-fold higher when the animals were reared in darkness (P = 0.0164). In fact, dark-rearing of VPP;Rpe65450Leu mice resulted in rhodopsin levels that were no longer statistically significantly different (P = 0.304) from those of mice with the VPP;Rpe65450Met genotype reared in cyclic light (Fig. 2B).

Fig. 2.

Fig. 2

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Dark rearing partially rescued retinal degeneration in VPP;RPE65450Leu mice. (A) Representative sections of the lower temporal retina of mice reared in cyclic light (left) or reared in darkness (right) for 42 days. Mice were of the Rpe65450Leu genotype. (B) Rhodopsin per retina [nmol] in VPP;Rpe65450Leu and VPP;RPE65450Met mice reared in cyclic light and in dark-reared VPP;Rpe65450Leu mice as indicated. Values are given as means + SD. Numbers of retinas are indicated. *P = 0.0164. Rhodopsin values in dark-reared VPP;Rpe65450Leu mice were not significantly different (P = 0.304) from rhodopsin values in cyclic-reared VPP;RPE65450Met mice.

Retinal degeneration in the VPP mice involves phototransduction

Depending on the intensity and duration, respectively, of exposure, light-induced retinal degeneration depends on phototransduction and activation of c-Fos/AP-1 (Hao et al., 2002). To test the potential involvement of c-Fos/AP-1 in the degenerative process in the VPP mice, we determined the relative expression levels of c-fos mRNA by RT-PCR during postnatal development and degeneration in retinas of wild-type and transgenic animals. The c-fos mRNA expression showed a similar initial increase in both strains of mice and reached high levels especially in retinas of adult VPP mice during the degenerative period (Fig. 3A). Although mRNA expression may not always reflect protein levels, this result indicates a potential role for c-Fos in retinal degeneration in the VPP mouse. To test this hypothesis, we crossed the VPP transgene onto the c-fos-null background. As judged by morphological appearance at PND 28, lack of c-Fos did not rescue retinal degeneration in the VPP mouse (Fig. 3B). When the global degeneration was assessed by the measurment of rhodopsin in whole retinas, the two types of mice had similar levels of the visual pigment. In wild-type mice, lack of c-Fos leads to a reduction in the rhodopsin content by 20% (Kueng-Hitz et al., 2000). A similar reduction in retinal rhodopsin levels was evident when VPP mice were compared to VPP;c-fos−/− mice (Fig. 3C).

Fig. 3.

Fig. 3

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VPP-mediated retinal degeneration was independent of c-Fos. (A) c-fos gene expression in retinas isolated from wt or VPP mice at the PNDs indicated. Total RNA from three retinas of three mice were pooled for RT-PCR analysis at each timepoint; M, 100-bp DNA marker. (B) Representative sections of the lower temporal retina of PND 28 VPP or VPP;c-fos−/− mice as indicated. All mice were of the Rpe65450Met genotype. (C) Rhodopsin per retina expressed in nmol. Values are given as means + SD. Numbers of retinas are indicated. *P < 0.0001.

To test the potential involvement of phototransduction in the degenerative process, we ablated transducin by combining the VPP transgene with the guanine nucleotide-binding protein G(t), alpha-1 subunit, gene knockout (Gnat1α−/−). This genetic block of rod phototransduction resulted in protection of photoreceptors at least up to PND 42 (Fig. 4A). In addition, photoreceptor outer segments were much longer but preservation of the outer segment structure was poor. Outer segments showed large vesiculations and condensations. Nevertheless, retinas from VPP;Gnat1a−/− mice had significantly higher rhodopsin contents than VPP mice at PND 28 (P = 0.0166) and at PND 42 (P = 0.0053; Fig. 4B).

Fig. 4.

Fig. 4

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VPP-mediated retinal degeneration was influenced by phototransduction. (A) Representative sections of the lower temporal retina of VPP and of VPP;Gnat−/− mice raised in cyclic light. Lack of functional rod transducin improved retinal morphology in VPP;Rpe65450Leu mice: an increased number of photoreceptor nuclei in the ONL is apparent and photoreceptor segments are better preserved. All mice were of the Rpe65450Leu genotype and were taken at the PNDs indicated. (B) Rhodopsin per retina expressed in nmol. Values are given as means + SD. Solid bars, VPP;Rpe65450Leu; open bars, VPP;Gnat-1−/−;Rpe65450Met. Numbers of retinas are indicated. *P = 0.0166, **P = 0.0053. Abbreviations as in Fig. 1.

Discussion

Exposure to light above a given threshold induces retinal degeneration in wild-type animals. Similarly, environmental light accelerates photoreceptor apoptosis in many models of inherited retinal degeneration. Here we show that these events share some common molecular mechanisms. The degree of damage or the rate of disease progression can be modulated by regulating the availability of rhodopsin and the activity of the phototransduction cascade. It has been suggested that the genetic background influences progression of inherited retinal degeneration. We have identified RPE65 as one of the proposed genetic modifiers. Our findings indicate that sequence variants in RPE65 differentially influence progression of inherited retinal degeneration through the modulation of light sensitivity (the rate of photon absorption) and the subsequent initiation of phototransduction by activated rhodopsin.

RPE65 is the isomerohydrolase essential for the generation of 11-cis-retinal in the visual cycle (Jin et al., 2005; Moiseyev et al., 2005; Redmond et al., 2005). Lack of functional RPE65 results in the almost complete absence of rhodopsin and the accumulation of retinylester in the RPE (Redmond et al., 1998; Seeliger et al., 2001; Gollapalli et al., 2003). The recently identified RPE65 variant 450Met causes a 13-fold reduction in the steady-state level of RPE65 protein in the RPE (Lyubarsky et al., 2005), presumably by a post-translational process (Wenzel et al., 2001). This reduces rhodopsin regeneration kinetics after bleaching more than three-fold (Lyubarsky et al., 2005). As rhodopsin is essential for light damage (Grimm et al., 2000b), pharmacological (Keller et al., 2001; Sieving et al., 2001) or genetic (Wenzel et al., 2001) interference with efficient rhodopsin regeneration leads to a reduced light-damage sensitivity. Similarly, introducing the Rpe65450Met variant attenuated progression of photoreceptor cell loss in the inherited retinal degeneration mediated by the VPP transgene. This indicates that the Rpe65 gene can act as a genetic modifier for inherited retinal dgenerations, presumably through the modulation of the rate of photon absorption by rhodopsin.

Naash and coworkers showed that VPP-mediated degeneration depends cumulatively on the genetic background and light exposure (Naash et al., 1996). The influence of the genetic background was tested by comparing VPP-induced retinal degeneration in albino and pigmented mice. The Rpe65450Leu variant is found in most mouse strains including FVB (M. Danciger, personal communication) from which the albino mice used in the Naash studies were derived. It is therefore probable that the albino VPP mice carried the Rpe65450Leu variant and that the pigmented animals (which were derived from the Rpe65450Met-expressing C57BL/6 strain) used for comparison carried the Rpe65450Met variant. When Naash and coworkers raised albino and pigmented VPP mice in cyclic light, the rhodopsin content in pigmented animals was roughly three times as much as in the albino VPP mice. We found a very similar difference between VPP;Rpe65450Leu and VPP;Rpe65450Met mice at PND 42 (Fig. 1). Furthermore, the increase in rhodopsin content in the VPP;Rpe65450Leu mouse after dark-rearing closely resembles the increase in the dark-reared albino VPP mice reported by Naash and colleagues. This suggests that the genetic component Naash et al. (1996) proposed may be found in the Rpe65 gene. It was also reported that the sequence variants of Rpe65 can influence disease progression in a model of Stargardt disease: in the ABCR−/− mouse, the Rpe65450Met variant decreased accumulation of A2E in the RPE, presumably by modulating photon absorption and rhodopsin bleaching (Kim et al., 2004).

However, the difference in photon absorption capacity between mice with the methionine and the leucine variants may not fully account for the different degeneration rates observed in the different VPP mice. Otherwise, dark-reared VPP;Rpe65450Leu mice should show higher rhodopsin levels than VPP;Rpe65450Met mice reared in cyclic light. Thus, we postulate additional genetic factor(s) which may cosegregate with Rpe65. New quantitative trait loci influencing retinal damage in models of light- and age-related degeneration have recently been identified on several mouse chromosomes. However, none of these loci map to distal chromosome 3, the genetic location of Rpe65 (Danciger et al., 2003; Danciger et al., 2004; Danciger et al., 2005). It will nevertheless be of importance to identify these factors and test them in combination with models of inherited retinal degeneration.

Interestingly, complete absence of RPE65 function, a condition dramatically reducing the photon catch capacity of the retina (Seeliger et al., 2001), did not notably reduce the rate of retinal degeneration in VPP mice. Lack of RPE65 on its own causes an early cone degeneration (Znoiko et al., 2005) followed by a slowly progressing rod cell death. Thus, by combining the VPP transgene with a Rpe65 deletion, two effects may sum and cause the degeneration observed. An alternative explanation is provided by recent findings made in cell-based or purified in vitro systems: Noorwez et al. (2004) found that opsin with the P23H mutation incorporated 11-cis-retinal more efficiently when the chromophore was present during synthesis of the protein. Complexation of P23H opsin with 11-cis-retinal stabilized the mutant rhodopsin facilitating its cellular folding and transport (Noorwez et al., 2004). In the absence of RPE65 protein and therefore of a functional visual cycle, no or only minimal amounts of 11-cis-retinal is present to assist biogenesis of mutant rhodopsin. This may lead to an increased abundance of un- or misfolded and/or of mislocalized proteins. As a consequence, large aggregates of mutant opsin may form and impair the ubiquitin proteasome system, resulting in cytotoxic effects and cell death (Illing et al., 2002; Saliba et al., 2002). Accumulation of high amounts of unfolded proteins may additionally elicit a cytotoxic effect by affecting the unfolded protein response (Bucciantini et al., 2002; Forman et al., 2003; Forman et al., 2004). Intriguingly, such mechanisms can also be found in other neurodegenerative diseases such as Alzheimer’s disease. An additional explanation for the lack of rescue in the VPP;Rpe65−/− mice argues for increased signalling of unliganded opsin. It has been shown that the degeneration observed in the Rp65−/− mouse retina can be blocked by the genetic ablation of phototransduction (Woodruff et al., 2003). To understand the pathophysiological signals leading to photoreceptor cell death in inherited retinal degenerations, it will be of crucial importance to elucidate the molecular mechanisms of such light-independent degenerative processes.

At least two different apoptotic pathways downstream of photon absorption and rhodopsin activation exist and these mediate light-induced photoreceptor degeneration in wild-type mice: short exposure to high-level light depends on c-Fos/AP-1 and is independent of transducin (and therefore phototransduction), while long exposure to low-level light is independent of c-Fos and dependent on transducin (Hao et al., 2002). To discriminate between these two pathways for the deleterious effect of light in the VPP mouse, we analysed retinal degeneration in VPP mice deficient for c-Fos and VPP mice deficient for transducin, respectively. Although c-fos gene expression was strongly induced in VPP mice (Fig. 4A), ablation of c-Fos did not alter the degenerative process (Fig. 4B and C). In contrast, lack of transducin considerably preserved the number of photoreceptor nuclei. This strongly suggests that VPP-mediated retinal degeneration follows a pathway similar to that induced in wild-type mice by long-term low-level light exposure.

Collectively, our data reinforce the observation that progression of RP can be enhanced by light acting directly on the diseased photoreceptor. Thus, reducing light exposure is advisable for patients suffering from RP. However, it is similarly important to identify and understand the molecular mechanisms enforced by modifier genes. For a successful pharmacological intervention in the disease process, it thus may be necessary not only to target the consequences of the primary genetic defect but also to pay close attention to the function of potential modifier genes.

Acknowledgments

We thank C. Imsand, G. Hoegger and H. Wariwoda for expert technical assistance. This work was supported by the Swiss National Science Foundation, the VELUX Foundation (Glarus, Switzerland), the H. Messerli Fonds, Grant EY-10609 from the National Eye Institute, a Core Grant for Vision Research at the University of Oklahoma (EY12190) and a National Institutes of Health Grant P20 RR 017703 from the Center of Excellence in Biomedical Research Program of the National Center for Research Resources to the University of Oklahoma.

Abbreviations

AMD

age-related macular degeneration

AP-1

activator protein-1

ONL

outer nuclear layer

PCR

polymerase chain reaction

PND

postnatal day

RP

retinitis pigmentosa

RPE

retinal pigment epithelium

RT

reverse transcription

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