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(The FASEB Journal. 2006;20:2036-2049.)
© 2006 FASEB

Biological characterization of gene response in Rpe65–/– mouse model of Leber’s congenital amaurosis during progression of the disease

Sandra Cottet*,1, Lydia Michaut{dagger}, Gaëlle Boisset*, Ulrich Schlecht{dagger}, Walter Gehring{dagger} and Daniel F. Schorderet*,{ddagger}

* Institute of Research in Ophthalmology, Sion, Switzerland;

{dagger} Biozentrum, University of Basel, Basel, Switzerland; and

{ddagger} University of Lausanne and Ecole Polytechnique Fédérale of Lausanne, Lausanne, Switzerland

1Correspondence: Av. Grand-Champsec 64, 1950 Sion 4, Switzerland. E-mail: sandra.cottet{at}iro.vsnet.ch


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
RPE65 is the retinal isomerase essential for conversion of all-trans-retinyl ester to 11-cis-retinol in the visual cycle. Leber’s congenital amaurosis (LCA), an autosomal recessive form of RP resulting in blindness, is commonly caused by mutations in the Rpe65 gene. Whereas the molecular mechanisms by which these mutations contribute to retinal disease remain largely unresolved, affected patients show marked RPE damage and photoreceptor degeneration. We evaluated gene expression in Rpe65–/– mouse model of LCA before and at the onset of photoreceptor cell death in 2, 4, and 6 month old animals. Microarray analysis demonstrates altered expression of genes involved in phototransduction, apoptosis regulation, cytoskeleton organization, and extracellular matrix (ECM) constituents. Cone-specific phototransduction genes are strongly decreased, reflecting early loss of cones. In addition, remaining rods show modified expression of genes encoding components of the cytoskeleton and ECM. This may affect rod physiology and interaction with the adjacent RPE and lead to loss of survival signals, as reflected by the alteration of apoptosis-related genes Together, these results suggest that RPE65 defect triggers an overall remodeling of the neurosensitive retina that may, in turn, disrupt photoreceptor homeostasis and induce apoptosis signaling cascade toward retinal cell death.—Cottet, S., Michaut, L., Boisset, G., Schlecht, U., Gehring, W., Schorderet, D. F. Biological characterization of gene response in Rpe65–/– mouse model of Leber’s congenital amaurosis during progression of the disease.


Key Words: microarray • LCA • apoptosis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
RETINITIS PIGMENTOSA (RP) is a heterogeneous group of inherited conditions involving the death of retinal photoreceptors with subsequent loss of vision. Although many different genes responsible for these diseases have now been identified, they all seem to share a common final pathway leading to progressive dysfunction of the photoreceptors and to apoptosis of both rods and cones.

RPE65, abundantly expressed in the RPE, is the retinoid isomerase responsible for the conversion of all-trans-retinyl ester to 11-cis-retinol in the visual cycle (1 , 2 , 3 , 4 , 5) . Mutations in the RPE65 protein, as several other mutations in genes related to RPE, are linked to the degeneration of photoreceptor cells. Mutations in humans are associated with several forms of inherited retinal dystrophies, such as autosomal recessive RP (6) and autosomal recessive childhood-onset severe retinal dystrophy (7) . They also account for {approx}6–15% of Leber’s congenital amaurosis (LCA), an early onset and autosomal recessive form of RP that results in blindness or severely impaired vision in children (8 , 9) . The general mechanisms by which these mutations contribute to retinal degeneration are not yet known, but affected patients show marked RPE damage, as reflected by drusen-like deposits, hypopigmentation (10) , pigment clumps (11) , and increased granularity in the epithelial monolayer (12) . Photoreceptor degeneration, characterized by congenital nystagmus, profound visual deficiency, night blindness, and reduced (along a rod-cone pattern) or nondetectable electroretinogram (ERG), is the cardinal feature observed in patients affected by RPE65 mutations. However, the relationship among genetic mutation, retinal defect, and final loss of photoreceptors remains largely unresolved.

Genetically engineered mice, in which the gene for RPE65 has been disrupted, exhibit changes in retinal morphology, function, and biochemistry that closely resemble the alterations seen in human RPE-related LCA patients. Rpe65–/– mice exhibit changes in retinal physiology characterized by a slow and progressive loss of photoreceptor layers (1) , a degenerative process that seems to rely on activation of residual transduction cascade by unliganded opsin (13) . In the absence of RPE65, all-trans-retinyl esters accumulate in lipid droplets within the RPE, reflecting defects in the visual cycle in these cells. As a result of absence of chromophore biosynthesis, knock-out (ko) animals do not generate adequate levels of visual pigment and have severely depressed light- and dark-adapted ERG responses (1 , 14) . The remaining visual capacity is attributed to residual rod function mimicking cone function under normally cone-isolating lighting conditions, indicating that RPE65 deficiency may affect cones more severely than rods in mice (15) . Whereas a direct role of RPE65 in cone visual pigment regeneration remains controversial (15 , 16) , a recent study by Znoiko et al. (17) showed that the decreased expression of cone-specific opsins and transducin correlated with cone degeneration at early ages in Rpe65-deficient retinas. Swedish Briard dogs, carrying a spontaneous 4-bp deletion in RPE65 (18 , 19) , showed similar alterations and damage of the RPE displaying hypertrophied morphology, irregular apical surface (20) , and large lipoid-like inclusions (18 , 21) . Also observed is the disorganization of photoreceptor outer segments (OS), followed by degenerative changes and progressive loss of photoreceptors (21) .

The retinal pigment epithelium performs numerous functions critical for the viability and activity of the adjacent photoreceptors in the neural retina. It is notable that mutations in RPE-expressed genes encoding proteins of the visual cycle can cause diverse forms of retinal dystrophies (22) . Photoreceptor cell death can be caused by any changes that alter the composition of the signal transduction cascade, influence the energy metabolism, or disturb the structural and functional interactions between the RPE and the photoreceptors, reflecting, at least in part, the interdependence and intimate contacts of these cells. Although apoptosis is a common pathway for photoreceptor degeneration in animal models of RP, the cellular and molecular events leading to cell death in the Rpe65–/– mouse remain largely unknown.

In the present study, we sought to characterize the changes in gene expression in Rpe65–/– mouse model during disease progression. In an attempt to unravel the potential signaling pathways at the initiation of the degenerative process, we assessed differential gene expression early in the development of the disease, namely before and at the onset of photoreceptor cell death in mutant mice of 2, 4, and 6 months of age. We identified differentially expressed genes whose biological characterization indicates that they may affect the development of LCA in Rpe65–/– mice. Together, our findings indicate that in the absence of RPE65, expression of genes involved in diverse cellular pathways, including phototransduction, apoptosis regulation, cytoskeleton organization, and extracellular matrix (ECM) constituents, are altered. Furthermore, we show that, whereas cone-specific phototransduction genes are strongly decreased, reflecting early loss of cones, the remaining rods have altered expression of genes encoding components of the cytoskeleton and ECM. This may affect rod physiology and interaction with the adjacent RPE. In turn, this may lead to impaired survival signals and retinal degeneration, as shown by the alteration of apoptosis-related genes in Rpe65-deficient retina. Ultimately, these observations pave the way for putative unraveled functions of RPE65 protein in addition to its intrinsic isomerase activity and point out general mechanisms involved in retinal degeneration.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
These studies adhered to the Association for Research in Vision and Ophthalmology (ARVO) statement for the use of animals in ophthalmic and vision research and were approved by the Veterinary Service of the State of Vaud (Switzerland). Wild-type (wt) C57BL/6 mice were purchased from Charles River (Les Oncins, France); the Rpe65–/– mouse strain (on a C57BL/6 background) was a generous gift from Dr. T. M. Redmond (National Institutes of Health, Bethesda, MD). Mouse genotype was determined by polymerase chain reaction (PCR) analysis of tail DNA as described previously (1) . Animals were kept in a 12-h light/12-h dark cycle with unlimited access to food and water.

Tissue isolation and RNA preparation
Age-matched wt and Rpe65–/– animals (2, 4, and 6 months-old) were killed by cervical dislocation. Three retinas for each mouse strain and time point were dissected under a microscope to exclude extraretinal tissues and were quickly isolated in RNAlater (Ambion, Huntingdon, United Kingdom) before being transferred in TRIzol (Invitrogen, Basel, Switzerland) and stored at –80°C until RNA extraction. Total RNA was extracted according to manufacturer’s instructions. Quantity and quality of total RNA were determined by capillary electrophoresis on an RNA6000 Bioanalyzer (Agilent Technologies, Waldbronn, Germany).

Target preparation, microarray procedures, and data repository
One microgram of total RNA was used to generate double-stranded cDNA (ds-cDNA; SuperScript Double-Stranded cDNA Synthesis Kit; Invitrogen). Purified ds-cDNA was used as a template for biotinylated cRNA synthesis using Affymetrix GeneChip Expression 3'-Amplification Kit for IVT Labeling Kit. After purification on RNeasy columns (Qiagen, Basel, Switzerland), 20 µg of target cRNA were fragmented and hybridized on Affymetrix Mouse Genome 430 2.0 GeneChips for 16 h at 45°C. Washes were performed on Fluidics Station 450 (EukGE-WS2v5 procedure), and chips were scanned on an Affymetrix GeneChip Scanner 3000 using the GCOS software (Affymetrix). Data normalization was performed using the Robust Multi-Array Analysis algorithm (RMA) as implemented in the GeneSpring 7.2 software (Agilent Technologies). Triplicates were performed for each condition studied. The intensity files corresponding to our raw data are deposited in NCBI Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo).

Supporting online database
The Eyebase database (www.eyebase.unibas.ch) was set up to facilitate visualization of our data. Eyebase is a searchable database displaying the graphical view of the intensity signal for probe sets (PS) after normalization. Each gene described in this report can be queried using gene names, symbols, synonyms, and GeneID or Affymetrix PS identifiers. When a gene is represented by several PS, the signals of all PS can be displayed on the same page for comparison. The information for all PS was retrieved from NetAffx Analysis Center (https://www.affymetrix.com/analysis/index.affx; tsv files as released on 21.06.2005). For each gene, links to relevant external databases are also provided, such as the Mouse Retina SAGE Library, which displays in situ hybridization data for over a 1,000 retina enriched transcripts (23) .

Gene ontology annotations
Gene ontology (GO) annotations provide controlled vocabularies for the description of the molecular function (MF; the biochemical activity of the gene product); biological process (BP; the biological objective to which the gene product contributes); and cellular component (CP; the place in the cell in which a gene product is active) of the gene product. The annotations for the mouse genome and the relation scheme between the GO terms were obtained at the EBI’s Gene Consortium site (www.ebi.ac.uk/GOA/) from the QuickGO database. A statistical test available at the Database for Annotation, Visualization and Integrated Discovery (DAVID) from the National Institutes of Health (www.david.niaid.nih.gov/david/; refs 24 , 25 ) was used to identify GO terms in which modulated genes were over-represented when compared with their frequency in the entire mouse genome.

Quantitative reverse transcription-PCR
The same RNA samples that were used to generate the biotinylated cRNA were also used for real-time PCR; 500 ng of total RNA in a 50 µl reaction were used for cDNA synthesis using oligo (dT)18 according to the manufacturer’s procedure (StrataScript Reverse Transcriptase; Stratagene).

The equivalent of 40 ng original total RNA was used for quantitative PCR amplification using the 2x brilliant SYBR Green QPCR Master Mix (Stratagene) and 1.5 µM forward and reverse primer pair, designed to span an intron of the target gene. Real-time PCR was performed in triplicate in a Mx3000PTM system (Stratagene) with the following cycling conditions: 50 cycles of denaturation at 95°C for 30 s, annealing either at 60°C (Bmf and Bcl-2) or 55°C (Bad and Bax) for 30 s, and extension at 72°C for 30 s. Quantitative values were obtained by the cycle number (Ct value), reflecting the point at which fluorescence starts to increase above background at a fixed threshold level.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
To assess the signaling events that may be at the initiation of retinal cell death, we generated global gene expression profiling of the retina of control and Rpe65–/– mice during the early stages of the disease. Three time points ranging from the very early disorganization of the photoreceptor OS to the onset of photoreceptor loss were investigated: 2 month-old retina, characterized by the initial disorganization of the photoreceptor OS without apparent outer nuclear layer (ONL) loss, 4 month-old retina where ONL decrease becomes slightly apparent, and 6 month-old retina showing significant photoreceptor cell death accompanied by a highly impaired OS organization (1 , 13) .

Identification of differentially expressed genes in Rp65–/– retina
We identified 300 PS down-regulated at least 2-fold (P value<0.05) in Rpe65 ko retina in at least one of the three stages studied (2, 4, and 6 months; Supplementary Table 1A). Among those, 246 could be associated to a unique gene, with 23 genes represented by more than 1 PS. Altogether, they represent 213 nonredundant genes down-regulated in Rpe65-deficient retinas (Supplementary Table 1B). Among them, 130 are known genes and 83 are cDNAs only showing homology to sequences in the expressed sequence tag (EST) or genomic databases. Table 1 displays the top 30 down-regulated known genes and 10 unknown cDNAs. Among the genes showing decreased expression, the great majority (180 out of 213, i.e., 84.5%) are down-regulated in all three stages (Fig. 1 A and Supplementary Tables 1A–B). However, two clusters of genes show specific decreased expression only at 4 and 6 months of age (Fig. 1A ). The first group (9 genes and 6 unknown cDNAs) encompasses the transcription factor c-Fos and Mapk1/Erk-2, a gene related to MAP kinase signal transduction (Fig. 1B and Supplementary Table 1C). Another transcription factor, Egr-1, is down-regulated at 4 and 6 months. Growth hormone-induced expression of c-Fos and Egr-1 through activation of ERK-1/-2 has been reported previously (26) . The second cluster that comprises {alpha}-, ß-, and {gamma}-crystallin genes, belonging to the superfamily of crystallin proteins, is reduced in 4 and 6 month-old mutant retinas (Figs. 1B and 3) . As expected, RPE65 mRNA is absent from mutant retina, whereas variable transcript levels can be detected in wt samples. This may come from the fact that, due to technical limitations, it is not always possible to completely remove all the RPE cells attached to the retina during tissue isolation.


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  		Table 1. Top 30 down-regulated known genes and 10 unknown genes/ESTs in Rpe65–/– retina


Figure 1
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  		Figure 1. Identification of differentially expressed genes during progression of the disease. The 534 probe sets modulated at least 2-fold (P value<0.05) in Rpe65 ko vs. wt retina in at least 1 of the 3 stages studied (2, 4, and 6 mo) were clustered using the Pearson similarity measure as implemented in the GeneSpring7.2 software. Resulting gene tree (A) shows that the great majority of genes falls into 2 main clusters. Clusters (1) and (2) encompass down-regulated and up-regulated genes in all 3 stages of the disease, respectively. B) Cluster (3) contains PS whose expression decreases only at 4 and 6 mo but remains constant at 2 mo; C) cluster (5) increased genes at same period. Down-regulated genes in cluster (4) are depicted in Fig. 3 . Color bar on left indicates relative levels of expression: red for highly expressed genes and green for nonexpressed ones.


Figure 2
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  		Figure 2. Biological characterization of the retina expression profiles in Rpe65-deficient animals. Enriched functional categories identified among down-regulated (A) and up-regulated (B) genes in Rpe65–/– retina. Modulated genes during disease progression were annotated according to the approved GO vocabulary. Differentially expressed genes that are over-represented when compared with the entire mouse genome were classified into the following functional groups: Biological Process (BP) and Molecular Function (MF). PIR, functional group related to the UniProt Knowledgebase Keyword (based on UniProtKB/Swiss-Prot entries).


Figure 3
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  		Figure 3. Altered expression of genes encoding superfamily of crystallin proteins. A) Down-regulated expression in 4 and 6 month-old mutant mice of crystallins {alpha}A (Cryaa), ßA1 (Cryba1), ßB3 (Crybb3), {gamma}B (Crygb), and {gamma}S (Crygs) in the enriched GO category related to eye lens proteins (cluster 4 from Fig. 1A ). B) Other crystallin genes show similar decreased expression levels in 4 and 6 month-old Rpe65–/– as compared with wt mice, although with a P value > 0.05.

From a total of 234 PS up-regulated in Rpe65–/– retina (Supplementary Table 2A), 159 nonredundant genes (32 genes being represented by more than one PS) showed at least a 2-fold increase (P value<0.05) in one of the three stages studied (Supplementary Table 2B). Among them, 117 are known genes and 42 are cDNAs only showing homology to sequences in the EST or genomic databases. Table 2 shows the top 30 up-regulated known genes and 10 unknown genes/ESTs. As for the down-regulated genes, most of the genes that show significant increased expression in the early stages of the disease remain modulated the same way later on, with 127 out of 159 (79.9%) up-regulated in the three stages (Fig. 1A and Supplementary Table 2). One cluster comprising 45 PS corresponding to 24 known genes and 6 unknown cDNAs escapes this trend in being up-regulated only at the later stages of the disease (4 and 6 months; Fig. 1C and Supplementary Table 2C).


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  		Table 2. Top 30 up-regulated known genes and 10 unknown genes/ESTs in Rpe65–/– retina

Biological characterization of the overall retinal expression profile in Rpe65-deficient animals
To assess the biological relevance of our study, we annotated the modulated genes in all three stages of the disease with the approved vocabulary provided by the Gene Ontology Consortium (27) . Functional groups of differentially expressed genes that were over-represented in this subset of genes when compared with their frequency in the entire mouse array were identified using DAVID informatic ressource and tested for significant P value.

The following functional categories have been observed to be over-represented in the down-regulated genes: visual perception (8 genes), eye lens proteins (6 genes), cytoplasm organization, and biogenesis (9 genes), as well as cytoskeletal protein binding (7 genes) and DNA repair (4 genes; Fig. 2 A and Table 3 ). The enriched functional group corresponding to actin binding (Myo7a, Mtmr7, Fmn2, Sntg2, and Dixdc1) is included in cytoskeletal protein binding, whereas genes over-represented in microtubule-based process (Cryaa, Kif1b, Kif5b, and Mtap2) are comprised of cytoplasm organization and biogenesis (Table 3) . A prominent set of genes diminished in all the stages of the disease and linked to visual perception is indeed the cone-specific phototransduction genes, with a strong decrease of cone phosphodiesterases (Pde6c and Pde6h), SWL (UV-sensitive), and MWL (green-sensitive) cone opsins, and cone transducin (Gnat2), as well as cone arrestin (arr3; Fig. 2A and Table 3 ). These data correlate well with a recent study by Znoiko et al. (17) describing down-regulated expression of cone-specific opsins and transducin very early in 2 and 3 wk-old Rpe65–/– mice. On the contrary, rod-specific genes remain unchanged at this stage of the disease, as reflected by the similar level of expression of rod phosphodiesterases (Pde6a, Pde6b, Pde6d, and Pde6g), rhodopsin, and rod arrestin (Gnat1) in Rpe65-deficient retina as compared with control retina. However, the rod-specific rhodopsin kinase (Grk1) is significantly up-regulated at 2 months of age and remains increased ({approx}4-fold) with time (Table 2) . Over-represented genes related to eye lens proteins are the crystallins, with components of the three major classes of this protein family {alpha}, ß, and {gamma} being down-regulated (Fig. 2A and Table 3 ). The same proteins are also part of the functional group related to peripheral nervous system development (Table 3) . Interestingly, down-regulated genes related to the two functional groups cytoplasm organization/biogenesis and cytoskeletal protein binding comprise proteins associated with microtubule activity, such as kinesin family member 1B and 5B, and microtubule-associated protein 2, as well as Ca2+-regulated myosin light chain kinase and myosin VIIa, which are actin-associated molecules (Table 3) . In retina, the latter is speculated to be involved in trafficking of ribbon-synaptic vesicle complexes and renewal of the outer photoreceptor disks (28) . Atm and Pttg1 genes, showing decreased expression in mutant retina, are related to DNA repair. Whereas Atm, belonging to PI3/PI4-kinase family, binds and phosphorylates p53, Pttg1 plays a central role in p53 pathway and negatively regulates transcriptional and related apoptosis activity of p53.


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  		Table 3. Enriched functional categories among genes down-regulated in mutant retina.

Regarding the genes whose expression is increased in Rpe65-deficient retina, several of them are significantly over-represented in the functional categories including protein kinase activity (11 genes) and ECM structural constituents and collagen (4 genes; Fig. 2B and Table 4 ). Several proteins of the collagen family are specifically increased in Rpe65-deficient retina, namely collagens IV {alpha}3 (col4a3) and {alpha}4 (col4a4) and collagen VIII {alpha}1 (col8a1), which also belong to ECM structural constituents. The following proteins are among the enriched genes encoding kinase activity: tyrosine kinases ephrin type-A receptor 4 (Epha4) and tyrosine kinase 9 (Ptk9), and the c-jun N-terminal kinase 1 (JNK1 /mapk8; Table 4 ; Fig. 2B ). The latter is known to respond to environmental stress by phosphorylating a number of transcription factors such as the activating protein (AP)-1 constituent c-Jun, whereas Ptk9 is an actin binding protein that regulates cytoskeletal dynamics by preventing actin filament assembly. Likewise, functional categories related to glycoprotein (21 genes) and structural molecule activity (8 genes) are enriched in Rpe65-knockout retina (Fig. 2B and Table 4 ). Lrat, Rdh5, and retinal G protein coupled receptor (Rgr), three genes related to visual perception, are also significantly increased at 4 and 6 months of age.


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  		Table 4. Enriched functional categories among genes up-regulated in mutant retina

Altered expression of genes involved in regulation of apoptosis in mutant retina
We observed that several genes related to apoptotic functions are down-regulated in the retina of mice lacking RPE65. Among them is the family of crystallin proteins that has been shown to play a role in preventing physiological stress and apoptosis induced by various environmental and metabolic factors. We indeed observed that expression of crystallins {alpha}A, ßA1, ßB3, {gamma}B, and {gamma}S, present in the category related to eye lens proteins, is decreased in 4 and 6 month-old animals (Fig. 3 A and Table 3 ). Although not significantly enriched in the latter functional group, other {alpha}-({alpha}B), ß-(ßA2, ßA4, ßB1, and ßB2), and {gamma}-({gamma}A, {gamma}F, and {gamma}N) crystallins also showed decreased raw expression level during disease progression, which is reflected by the appearance in the gene tree of a predominant green color in knock-out samples (Fig. 3B ). Crystallins have been shown to be expressed in the adult mouse retina, where they are suggested to play crucial functions in protecting retinal neurons from metabolic stress and/or environmental damage (29) . Several genes of the Bcl-2 protein family, known for their role in apoptosis regulation, are also altered in Rpe65-deficient retina. As shown in the microarray data, the expression level of the gene encoding antiapoptotic Bcl-2 was reduced, whereas Bax, Bad, and Bmf proapoptotic genes were increased. These results were confirmed by real-time PCR analysis. Moreover, to assess persistent modulation of these apoptotic genes during progression of photoreceptor degeneration, qPCR was also performed on 12 month-old animals (Fig. 4 ). A 2-fold decrease in Bcl-2 mRNA was observed since 4 months of age in mutant retina (Fig. 4A ). On the opposite, Bax showed increased expression at 2 months of age and remained constantly up-regulated later on (Fig. 4B ), whereas Bmf increase was more pronounced at 12 months (Fig. 4C ). The gene encoding Bad behaved similarly, although with a different kinetics, being up-regulated only at later stage of disease progression, namely at 6 and 12 months (Fig. 4D ).


Figure 4
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  		Figure 4. Altered expression of Bcl-2 family members is confirmed by real-time PCR analysis. A) Decreased expression of antiapoptotic Bcl-2 in Rpe65–/– as compared with wt retinas, whereas expression levels of proapoptotic Bax (B), Bmf (C), and Bad (D) are increased at different ages (2–12m, 2–12 months).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The sequential microarray analysis of Rpe65–/– LCA mouse model reveals broad changes in retinal gene expression during the early stages of the disease, both toward increased as well as decreased expression. Phototransduction, cytoskeleton organization, and apoptosis regulation are the pathways showing the strongest variations.

Among the genes with decreased expression in all three stages, a cluster of genes related to visual perception including cone-specific Pde6c, Pde6h, Opn1sw, Opn1mw, Gnat2, and arr3 is of particular interest. Rod and cone functions have been shown to be severely disrupted in Rpe65–/– mice. Strongly decreased ERG response, only reflecting residual rod but no cone response, was recorded in these animals (14 , 15 , 30) . Recently, Znoiko et al. (17) observed a massive degeneration of cone inner and outer segments and cone cell death in the early life of Rpe65-deficient mice. Similarly, it has been suggested that retinal detachment has a greater morphological impact on cones than on rods (31) , and clinical studies have demonstrated that cone photopigments show a slow recovery after reattachment (32 , 33) . The fact that cone loss seems to precede degeneration of rod photoreceptors suggests that cones are more susceptible to direct or indirect effect of the absence of RPE65 protein. It remains controversial whether RPE65 is expressed not only in RPE but also in cones (15 , 16) . Lack of this protein in cones might be a contributing factor to early cone degeneration; however, the putative function of RPE65 in cones is still not understood. As it has been demonstrated, cone visual function does not seem to rely exclusively on retinoid synthesis from the RPE, but Müller cells may also specifically provide cones with 11-cis-retinol (34) . This leads to the suggestion that early cone-specific degeneration might be dependent, at least in part, on other unraveled function of RPE65. During development, specific and strong increased level of RPE65 transcript coincides with the extension of RPE microvilli and growth of photoreceptor outer segments, suggesting that this visual protein may play a role in the development and structure of the whole retina (35) . Furthermore, extensive embryonic retinal degeneration in LCA2 mutations (RPE65 mutations) is characterized by impaired ocular cellular interactions at the RPE and neuroretina levels, together with choriocapillaris and Bruch’s membrane defects. This suggests that RPE65 function may be critical not only for photoreceptor maintenance but also for early retinal and ocular development and connectivity (36) .

Other down-regulated genes identified during the progression of the disease are involved in biological processes related to cytoskeleton/cytoplasm organization and biogenesis. These results suggest cell remodeling in Rpe65–/– retina, as strengthened by the altered expression of genes encoding structural proteins (Myo7a, Kif1B, Kif5B, Mtap2, and Mtmr7). Cytoskeletal systems comprising microtubules proteins closely associated with microtubules and cytoplasmic proteins serve many functions. They are known to be involved in the ontogenetic outgrowth of the OS, in their maintenance during renewal, and in phototransduction (37) . In photoreceptor cells, the molecular motor proteins myosin VIIa and kinesin II have been identified that function in the transport of the phototransductive opsin and arrestin proteins (38 , 39) . Other phototransduction molecules such as RDS/peripherin, retinitis pigmentosa GTPase regulator (RPGR), and ATP-binding cassette transporter retina (ABCR), whose defects have been implicated in RP, are associated with the cytoskeleton in photoreceptor OS (37) . These data strongly suggest that disturbance in photoreceptor cytoskeletal architecture may lead to disease phenotype and retinal degeneration. Myosin VIIa, a protein associated to actin filament, was found in this study to be decreased in mutant mice. Mutations in Myo7a gene are the cause of Usher syndrome 1B, a severe deafness-blindness disorder with patients becoming progressively blind as a result of retinal degeneration (40) . As well, the kinesin protein family, whose expression of Kif1B and 5B members is decreased in mice lacking RPE65, is comprised of motor proteins closely associated with microtubules in the photoreceptor OS that may participate in transport of materials along the microtubules and/or connect these with each other or with other components (41) . It appears that microtubule-dependent processes are critical to the function and morphogenesis of the photoreceptor and RPE cells in the retina. Indeed, loss of the kinesin II subunit Kif3a in photoreceptors caused large accumulation of opsin, arrestin, and membranes within their inner segments (38) . This was associated with apoptotic cell death, suggesting that kinesins are not only implicated in transport from photoreceptor inner to outer segments but also play a crucial role in retinal cell survival. As molecular motor proteins are responsible with scaffolding proteins for intracellular spatial organization and correct localization of signal transduction molecules, down-regulation of myosin- and kinesin-related proteins may result in impaired signaling network and affect photoreceptor function and viability. Furthermore, highly polarized and compartmentalized photoreceptors are susceptible to any perturbation in cytoplasm organization that may be crucial for the proper function and maintenance of these cells and the entire retina.

Through the interphotoreceptor matrix (IPM), the retina and the photoreceptor OS are in close contact with the underlying RPE. Modification of the IPM constituents may influence the ability of RPE cells to interact with their environment and with the maintenance of photoreceptors. This may also be linked to the altered expression of structural genes that we observed in this study. We also identified increased expression of IMPG1, a chondroitin sulfate proteoglycan bearing hyaluronan-binding motif (42) in our model. This gene is mapped to chromosome 6q13-q15, where genes of several cone-rod macular dystrophies overlapped (43 , 44) , but so far, it has not been associated to any RP. It is known that up-regulated expression of ECM genes, including chondroitin sulfate proteoglycans, occurs in injured neural tissues (45) , as well as in injured optic nerve (46 , 47) and induced glaucoma (48 , 49) . Hylauronan, a high molecular mass glycosaminoglycan, is also distributed in the IPM. Through its interaction with hyaluronan-binding motif of IMPG1, it participates in the macromolecular scaffold within the IPM (50) . Although IPM glycoconjugates mediate physical attachment of the neural retina to the RPE, the underlying molecular mechanisms have not been elucidated, but chondroitin sulfate proteoglycans appear to be crucial in this function. Thus, IMPG1 as well as other IPM constituents may affect retinal adhesion and maintenance of photoreceptor cells.

We thus postulate that in addition to its role in chromophore synthesis in the RPE, RPE65 insufficiency may impair retinal cell homeostasis and function through alteration of the structural architecture of the neuroretina. Whether this is a primary cause of RPE65 deficiency or a secondary consequence remains to be elucidated.

Apoptosis is mediated by a series of positive and negative regulators. Among these factors, members of the Bcl-2 family and the small heat shock proteins, which include the {alpha}-crystallins, play an important role.

In Rpe65–/– mice, a marked decrease of several genes encoding members of {alpha}-({alpha}A and {alpha}B), ß-(ßA1, ßA2, ßA4, ßB1, ßB2, and ßB3), and {gamma}-({gamma}A, {gamma}B, {gamma}F, {gamma}N, and S) crystallin proteins was obvious from 4 months of age. Although initially found in lens fiber cells, previous works confirmed that many crystallins are expressed in nonlens tissues such as the retina (51 , 52 , 53 , 54 , 55 , 56) . This has led Piatigorsky and colleagues (57) to propose their gene sharing theory in which proteins may be recruited not only for their enzymatic activity but also for their pure physical properties. A recent study revealed that not less than 20 different members, including {alpha}-, ß-, and {gamma}-crystallins, are found in the adult mouse retina, mainly in the ONL, inner nuclear (INL), and ganglion cell layers (GCL; 29 ). Under stress conditions, the expression level of crystallins is susceptible to modulation. Increased expression of {alpha}-, ß-, and {gamma}-crystallin transcripts has been described in the rat retina after ischemia-reperfusion injury (58) , on exposure to light injury (59) , as well as in RCS rat during retinal degeneration (60) . In the rd1 mouse, apoptosis of rod cells paralleled increased expression of crystallins in surrounding cells, suggesting that they may exert a neuroprotective effect on the remaining cones (61) . Decreased protein content and truncation of {alpha}A-crystallin have also been observed in the aged rat retina (62) .

{alpha}-Crystallins belong to the small heat shock protein family of molecular chaperones. In addition to their chaperone-like activity (63 , 64) , {alpha}A and {alpha}B-crystallins interact with cytoskeleton (65) and protect cells from metabolic stress (66) . These stress-responsive proteins can prevent apoptosis induced by a large number of stress factors including staurosporine (67 , 68) , TNF (68 , 69) , and hydrogen peroxide (70) . Recently, Mao and colleagues (71) provided evidence about their capacity to block translocation of proapoptotic proteins of the Bcl-2 family into mitochondria during staurosporine-induced apoptosis. They also have the ability to prevent apoptosis by inhibiting caspases (72 , 73) . On the other hand, much less is known about the role of the members of the ß/{gamma}-superfamily, including ß- and {gamma}-crystallins, which are related to microbial proteins induced by physiological stress (74 , 75) . The fact that ß/{gamma}-crystallins also have nonlens expression and therefore probably nonstructural roles may suggest a function analogous to the antistress properties established for {alpha}-crystallins. Furthermore, several members of the ß/{gamma}-superfamily have plausible connections with cytoarchitecture, leading us to imagine that ß- and {gamma}-crystallins may possibly play a role associated with control of cell morphology, perhaps involving assembly or protection of the cytoskeleton (54 , 56) .

These overall data indicate that crystallins, in conjunction with their possible functions as chaperones and stress-responsive proteins, have more general physiological functions than only to augment the refractive power of the transparent lens tissue and may thus constitute a family of defensive proteins. Although their specific role in the retina remains unclear, expression of crystallins in the retinal layers is consistent with their proposed role in cell survival. In this sense, it suggests that the marked decrease of several members of crystallin transcripts observed in our study might somehow affect survival signals and promote photoreceptor cell death. However, the precise function of crystallins in retinal disease and photoreceptor degenerescence requires further study.

We also showed that regulation of genes of the Bcl-2 family was altered in Rpe65-deficient retina. Decreased expression of antiapoptotic Bcl-2 was paralleled with up-regulated levels of the proapoptotic genes Bax, Bad, and Bmf. Bcl-2 promotes cell survival by binding and inhibiting proapoptotic Bcl-2 proteins such as the BH-multidomain proteins Bax and Bak and the BH3-only proteins Bad, Bik, Bid, and Bim (76 , 77) . The latter proapoptotic proteins act in concert to promote apoptosis by altering mitochondrial functions and activating effector caspases (78) . Bax oligomerizes and translocates to the mitochondrial membranes, triggering disruption of the mitochondria and signaling irreversible commitment to apoptotic fate. Bad proapoptotic effect relies on its ability to bind Bcl-2 and block its prosurvival activity. Signaling pathways related to cell death promoted by disruption of ECM contact and by loss of cytoskeletal architecture may affect the levels and posttranslational modifications of Bcl-2 proteins, which may play a direct role as sensors of cytoskeletal integrity. Bmf was shown to be released from its cytoskeletal localization and moves to mitochondria where it associates with Bcl-2 on cell detachment and actin cytoskeleton disruption (79) . Modulation of two MAP kinases as shown in our experiments, increased JNK1 and decreased ERK-2 expression, might also have a direct affect Bcl-2 protein activity and affect its antiapoptotic function. Indeed, JNK1, through specific phosphorylation, has been shown to antagonize the protective function of Bcl-2 (80 , 81 , 82) . ERK-2 activation has been reported to induce phosphorylation of Bcl-2 at specific sites, preventing its ubiquitin-mediated destruction (83) . Thus, altered ratio of the different Bcl-2 members may finally influence the balance between anti- and pro-apoptotic signals to determine the life or death of the retinal cells (84) .

In summary, we demonstrate that the main cone-specific phototransduction genes are strongly decreased in Rpe65–/– mouse model of LCA, reflecting the early loss of cone photoreceptors. In the remaining retinal cells, altered expression of ECM constituents and cytoskeletal proteins may impair retinal cell homeostasis through defect of RPE-photoreceptor cell interaction. Finally, disruption of ECM-cell contact and cytoskeletal function may favor the balance toward proapoptotic intracellular cascades as reflected by the inhibition of antiapoptotic proteins in Rpe65–/– retina.


   ACKNOWLEDGMENTS
 
We thank Dr. T. M. Redmond for the Rpe65–/– mice, Francis Munier and Yvan Arsenijevic for housing some of the mice, and T. Favez for technical assistance.

Received for publication March 27, 2006. Accepted for publication May 15, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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