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(Investigative Ophthalmology and Visual Science. 1999;40:2788-2794.)
© 1999 by The Association for Research in Vision and Ophthalmology, Inc.

Low Expression of {alpha}A-Crystallins and Rhodopsin Kinase of Photoreceptors in Retinal Dystrophy Rat

Akiko Maeda1, Hiroshi Ohguro1, Tadao Maeda1, Takashi Nakagawa1 and Yoshio Kuroki2

From the Departments of 1 Ophthalmology and 2 Biochemistry, Sapporo Medical University School of Medicine, Japan.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. The Royal College of Surgeons (RCS) rat has been extensively characterized as a model for inherited retinal dystrophy such as retinitis pigmentosa. In the present study, compositions of retinal proteins were compared between RCS (rdy-/-) and control (rdy+/+ ) rats during progression of the disease to understand the molecular pathologic course of the retinal degeneration.

METHODS. Protein mapping was performed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) or two-dimensional (2D)-PAGE using whole retinas or rod outer segments (ROS) obtained by a sucrose-density gradient centrifugation method from RCS or control rats at the age of 3 to 8 weeks.

RESULTS. 2D-PAGE showed that retinal proteins of RCS rats were generally less abundant than those of the control animals and that the difference became more evident with aging. However, no significant difference was observed in the protein-mapping patterns in 2D-PAGE between RCS and control rats in any ages tested. Analysis by SDS-PAGE of ROS proteins and by western blot using antibodies against opsin, rhodopsin kinase (RK), recoverin, or arrestin demonstrated that a 20-kDa protein and RK were selectively less abundant in RCS than in control rats. Edman sequence analysis of the proteolytic peptides obtained by in-gel digestion of the corresponding protein band using endoproteinase Lys C identified the 20-kDa protein as {alpha}A-crystallin. Reverse transcription–polymerase chain reaction confirmed selective low levels of mRNA expressions of {alpha}A-crystallins and RK in RCS rats.

CONCLUSIONS. This study demonstrates that decreased expression of {alpha}A-crystallins and RK in RCS rats, may have significant roles in the development of retinal dystrophy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The Royal College of Surgeons (RCS) rat with inherited retinal dystrophy has been widely used as a model to study human retinal degenerative diseases, such as retinitis pigmentosa (RP). It has been shown that the retinal pigment epithelium (RPE) cell is affected by the rdy- mutation and continuously expresses the rdy- phenotype.1 Histopathology has shown that retinal degeneration starts within the first month after birth and that most of the photoreceptor cells are lost in the next month.2 3 In terms of the pathologic molecular course of retinal degeneration, it has been suggested that the inability of the RPE to phagocytose shed tips of rod outer segments (ROS) debris in RCS rats is primarily involved.4 5 6 In contrast, RCS photoreceptor cells are considered to be normal in structure and functions, because RCS photoreceptor cells can survive retinal RPE transplantation.7 8 9 However, the deficit of phagocytosis in RCS RPE cells is most likely to be ROS specific, because normal capacity of the RCS RPE cells in phagocytosis of nonspecific polystyrene latex spheres has been identified.10 11 12 In addition, several studies in vitro and in vivo have shown that the ability of the RCS RPE cells to bind ROS is normal, but the ingestion of bound ROS is significantly reduced.6 13 14 15 These observations allowed us to speculate that some unknown deficit in the ROS may be involved in addition to the deficiency in phagocytosis in the RPE. In fact, it has been found that several changes occur in RCS ROS including opsin,16 arrestin,17 18 and ROS protein phosphorylation levels,19 suggesting that the quenching of the phototransduction pathway might be affected in RCS. In addition, other changes have been identified in retinal proteins including heat shock protein (hsp) 70,18 20 neurotrophic factors, fibroblast growth factors,21 and phospholipids.22 However, it is still ambiguous whether such changes are primary causes of the diseases or secondary events associated with retinal degeneration, and the relationship among these changes is unclear, because no systematic studies have been undertaken, such as the mapping of retinal proteins.

Therefore, in the current study, to determine which molecules change during the progression of retinal degeneration, we performed protein mapping using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) or two-dimensional (2D-PAGE) of either whole retina or partially purified ROS and compared them between RCS and normal rats of different ages (3–8 weeks old). Furthermore, by western blot analysis we studied the protein changes involved in the quenching of phototransduction, including rhodopsin, RK, arrestin, and recoverin during development of retinal degeneration, because these proteins were most likely affected as described.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
All experimental procedures were designed to conform to the ARVO Statement for Use of Animals in Ophthalmic and Vision Research and our institution’s guidelines. The animals used in this study were 3- to 8-week-old inbred RCS (rdy-/-) rats and congenic control RCS (rdy+/+ ) rats originally obtained from Crea, Tokyo, Japan. Unless otherwise stated, all procedures were performed with ice-cold solutions.

Antibodies
Anti-recoverin rabbit serum or anti-arrestin rat serum was obtained by immunization of purified recoverin23 or arrestin24 from fresh bovine retinas to rabbit or rat, respectively. IgG was isolated from these sera by use of a protein G Sepharose column, according to the protocol described by the manufacturer. For affinity purification, IgG was applied to a Sepharose 6B column covalently linked with recoverin or arrestin, and IgG binding to the column was eluted by lowering the pH using 0.2 M glycine buffer (pH 2.5). An aliquot (1 ml each) was collected and mixed immediately with 0.1 ml 1 M Tris buffer (pH 8.5) to adjust the pH to 7.5. The purity and protein contents were determined by SDS-PAGE and spectrophotometry, respectively. Anti-human rhodopsin kinase (RK) monoclonal antibodies25 and anti-rhodopsin monoclonal antibodies26 were generous gifts of Krzysztof Palczewski (Department of Ophthalmology, University of Washington, Seattle) and Fumio Tokunaga (Osaka University, Japan). The specificity and titers of all antibodies were examined by western blot and enzyme-linked immunosorbent assay (ELISA), respectively.

Preparation of Rod Outer Segment Membranes
Rat ROS membranes were prepared by the method described by Papermaster,27 with some modifications using sucrose-density gradient centrifugation. Briefly, four freshly dissected rat retinas were suspended in 0.3 ml 45% (wt/vol) sucrose in ROS buffer (10 mM HEPES [ pH 7.5], 20 mM NaCl, 60 mM KCl, and 1 mM benzamidine), shaken vigorously in a tube (Eppendorf, Fremont, CA) for 20 minutes, and centrifuged for 10 minutes at 13,000 rpm. The supernatant was collected, and the pellet was again suspended in the same buffer. The sucrose floatation was repeated. The combined supernatant was diluted twice with ROS buffer, and then centrifuged at 13,000 rpm for 15 minutes. The pellet was used as a sample of ROS.

SDS-PAGE
SDS-PAGE was performed by the method of Laemmli,28 using a 12.5% SDS-PAGE laboratory gel and a minigel apparatus (Hoeffer, San Francisco, CA).

2D-PAGE
Two freshly dissected retinas were homogenized in 200 µl of solution containing 8 M urea, 2% NP40, and 2% ampholine (pH 3.5–10). The homogenate was centrifuged at 13,000 rpm for 15 minutes, and the supernatant (100 µl) was subjected to 2D-PAGE, performed by the method described by O’Farrell.29 In the first dimension, isoelectric focusing (IEF) gels were prepared in glass tubes (75 x 5 mm). The gels contained 4% acrylamide/bis-acrylamide, 8 M urea, 2% NP40, and 2% ampholine producing a pH gradient of 4.0 to 8.0. After a 2-hour prerun at 200 V, electrofocusing was performed successively for 1 hour at 200 V, 16 hours at 300 V, and 1 hour at 500 V, with 20 mM NaOH and 10 mM H3PO4 as the cathode and anode solutions, respectively. In the second dimension, IEF gels were removed from the glass tubes, incubated with solution containing 62.5 mM Tris-HCl (pH 6.8), 2% SDS and 5% ß-mercaptoethanol, and subjected to SDS-PAGE.

In-Gel Digestion and Peptide Separation by High-Performance Liquid Chromatography
In-gel digestion was performed by the method described by Ohguro et al.30 Briefly, the protein band stained by Coomassie blue was excised and washed twice with 200 µl 50% acetonitrile and 0.2 M ammonium bicarbonate solution at 30°C for 20 minutes. After aspiration of the solution, the gel was incubated with 0.2 ml 100 mM Tris-HCl (pH 9.0) containing 0.1% SDS at 30°C for 1 hour. Thereafter, 1 µg endoproteinase Lys C (Boehringer Mannheim, Mannheim, Germany) was added, and the mixture was incubated at 30°C for 24 hours. The reaction was terminated by adding 20 µl 10% trifluoroacetic acid. The solution was collected, and the remaining gel was crushed and washed in 0.2 ml of the same buffer. The combined solution was then loaded onto a reversed-phase C8 column (2.1 x 250 mm; Shiseido, Tokyo, Japan) using a precolumn of DEAE-5PW (4.6 x 10 mm, Tosoh, Tokyo, Japan) to remove the SDS, and the cleaved peptides were purified by using a linear gradient of acetonitrile from 0% to 70% in 0.05% trifluoroacetic acid for 70 minutes at a flow rate of 0.3 ml/min.

Amino Acid Sequence Analysis
The peptide sequence was obtained by Edman degradations using an automated gas-phase protein sequencer (model 477; Applied Biosystems, Foster City, CA), as described by Crabb et al.31

Reverse Transcription–Polymerase Chain Reaction Analysis and Relative Amount of {alpha} Crystallins and RK
For reverse transcription–polymerase chain reaction (RT-PCR) total RNA from retinas was isolated using reagent according to the procedure recommended by the manufacturer (Isogen; Nippon Gene, Tokyo, Japan.). The cDNAs were generated from 2 µg retinal RNA in a 12-µl reaction using 1 µl oligo(dT) primer (0.5 mg/ml; Gibco Life Technologies, Rockville, MD). The reaction mix was denatured at 70°C for 10 minutes. Four microliters first-strand buffer (250 mM Tris-HCl, 375 mM KCl, 15 mM MgCl2; Superscript), 2 µl dithiothreitol (0.1 M; DTT), 1 µl dNTP (10 mM), 1 µl RNase inhibitor (40 U/µl), and 1 µl reverse transcriptase (200 U/µl; Superscript II (all agents from Gibco) were added to the mix. The incubation was performed at 42°C for 50 minutes and at 70°C for 15 minutes. The PCR amplifications were performed using 4 µl from the RT reaction, 5 µl 10x PCR buffer (200 mM Tris-HCl and 500 mM KCl), 2 µl MgCl2 (50 mM), 1 µl dNTP, 5 µl sense and antisense primers (10 pM/µl), and 0.5 µl Taq polymerase (5 U/µl; Gibco). The PCR mix was denatured at 94°C for 4 minutes and then run for 30 cycles of 94°C for 1 minute, 55°C for 1 minute, and 72°C for 2 minutes.

The primers used for RT-PCR were 5'-ATGGACGTCACCATCCAGCA-3' corresponding to bases 158 to 178 of the cDNA sequence and 5'-AGCTGGGCTTCTCCTCCCGT-3' corresponding to bases 713–732 of the cDNA sequence for {alpha}A-crystallin and {alpha}Ains-crystallin with expected PCR products of 485 bp and 574 bp, respectively; 5'-CCACATGCTGGAAACCCCAA-3' corresponding to bases 371–399 of the cDNA sequence32 and 5'-AGCTGGGCTTCTCCTCCCGT-3' corresponding to bases 713–732 of the cDNA sequence for {alpha}Ains-crystallin with an expected PCR product of 352 bp; 5'-ATGGACATAGCCATCCACCACCCCTGGAT-3' corresponding to bases 21–49 of the cDNA sequence and 5'-AATCTACTTCTTAGGGGCTGCAGTGACAGC-3' corresponding to bases 522–551 of the cDNA sequence for {alpha}B-crystallin with an expected PCR product of 531 bp; 5'-CGTCACCGTACAGCACAAGA-3' corresponding to bases 180–199 of the cDNA sequence33 and 5'-CATGAACACTGCATGCCCTC-3' corresponding to bases 541–560 of the cDNA sequence for opsin with an expected PCR product of 626 bp; 5'-AAGACCAAGGGCTATGCAGGGA-3' corresponding to bases 1226–1247 of the cDNA sequence34 and 5'-CTAGGAGATGAGACACATCCCTGA-3' corresponding to bases 1856–1879 of the cDNA sequence for RK with an expected PCR product of 654 bp; 5'-GTATGGAATCCTGTGGCATCC-3' corresponding to bases 2683–2703 of the genomic DNA sequence35 and 5'-TACGCAGCTCAGTAACAGTCC-3' corresponding to bases 3135–3155 of the genomic DNA sequence for ß-actin with an expected PCR product of 349 bp.36 The amplified PCR fragments were electrophoresed on a 1.5% agarose gel containing ethidium bromide.

Competitive PCR
We used cDNAs from rat surfactant protein A (SP-A37 ) as competitors for RK primers. Oligonucleotide primers (5'-AGACCAAGGGCTATGCAGGGATATGGCAGAAGCCACTGG-3' and 5'-CTAGGAGATGAGACACATCCCTGATGGGACACCGA-GCTACAG-3') containing sequences of both SP-A and RK were generated. After the PCR was performed using the described primers and SP-A cDNA as a template, a PCR product with the size of 1215 bp was obtained. The cDNA obtained was electrophoresed in a 1.5% agarose gel and purified (Gene Clean II; Funakoshi, Tokyo, Japan). Competitive PCR was then performed using 2.2 µl pooled cDNA derived from retinas of 5-week-old RCS rats or control rats, increasing concentrations of competitor cDNA (1–200 ng) and RK primers under the same conditions as described for RT-PCR, except the run lasted for 35 cycles.

Western Blot Analysis
Western blot analysis was performed as described previously.38 Briefly, an ROS-soluble protein sample was analyzed by SDS-PAGE using a 12.5% polyacrylamide gel. Separated proteins in the gel were electrotransferred to polyvinylidene difluoride membranes in 10 mM bis-tris-propane buffer (pH 8.4) containing 10% (wt/vol) methanol solution. After blocking nonspecific binding by 5% (wt/vol) skim milk in phosphate-buffered saline (PBS), the membrane was probed successively with antibodies and horseradish peroxidase (HRP)–labeled secondary antibodies (Funakoshi). Specific antigen and antibody binding was visualized with an enhanced chemiluminescence system (Amersham Pharmacia Biotech, Amersham, UK), according to the method described by the manufacturer.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
To understand the pathologic molecular course of retinal dystrophy in the RCS rat, retinal proteins of RCS (rdy-/-) and control rats (rdy+/+ ) were analyzed by 2D-PAGE, and the protein mapping patterns were compared during the disease progression. Retinal proteins of RCS (rdy-/-) were generally less abundant than those of the control (rdy+/+) in 3- to 8-week-old animals, and these differences became apparent with advancing age. However, the distributions of the retinal proteins were almost identical, and specific changes were not detected (data not shown). These results suggest that the amounts of major retinal protein components generally decreased during the course of retinal dystrophy, but their distributions were not significantly changed.

As the next step in the investigation, we isolated rod outer segments (ROS) of 3- to 8-week-old animals, and their protein components were analyzed by SDS-PAGE. As shown in Figure 1 (upper panel), ROS protein components of 3-week-old RCS rats, in which retinal dystrophy was not apparent, were almost identical with those of control rats. However, the amount of each ROS protein component in 7-week-old RCS rats, in which retinal dystrophy had become obvious, was generally decreased compared with those of the control when equal volumes of ROS were applied. In addition, it was noted that the 67-kDa protein, 23-kDa protein and 20-kDa protein bands, designated with arrowheads and an arrow, were significantly less abundant than the other proteins. In the case of overloaded SDS-PAGE gel, the most significant difference was noted in the corresponding 20-kDa band (Fig. 1 , lower panel).



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  		Figure 1. Analysis of ROS proteins in RCS and control rats by SDS-PAGE. ROS was isolated from four retinas of 3-week-old or 7-week-old RCS (rdy-/-) and control (rdy+/+) rats by a sucrose-density gradient centrifugation method. ROS pellets were each suspended in 100 µl 10 mM HEPES buffer (pH 7.5) containing 100 mM NaCl. An aliquot (10 µl) was mixed with the sample buffer (10 µl) and loaded on an SDS-PAGE gel (top). An aliquot (50 µl) from 7-week-old animals was lyophilized, dissolved in the sample buffer (10 µl), and loaded on an SDS-PAGE gel (bottom). Significant differences between RCS and control are noted in 67-kDa and 23-kDa bands designated by arrowheads and a 20-kDa band designated by an arrow.

 
To identify the 20-kDa protein, the corresponding band in the gel was cut out and subjected to in-gel digestion with endoproteinase Lys C. The resultant peptides were purified on a reversed-phase high-performance liquid chromatography C8 column using a linear gradient of acetonitrile from 0% to 70% during 70 minutes (Fig. 2) . The six major peak fractions were then subjected to the Edman sequence analysis. As shown in Figure 3 , sequence analysis of the proteolytic peptides identified the 20-kDa protein as {alpha}A-crystallin.



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  		Figure 2. High-performance liquid chromatography (HPLC) separation of proteolytic peptides of 20-kDa protein after in-gel digestion by endoproteinase Lys C. In-gel digestion of 20-kDa proteins from control rats (rdy+/+) and separation of the proteolytic peptides by reversed-phase HPLC C8 column were performed as described in the Materials and Methods section. Major peaks (designated 1–6) were collected and subjected to Edman sequencing analysis.

 


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  		Figure 3. Amino acid sequence of 20-kDa proteolytic peptides and their homology with {alpha}A-crystallin. The amino acid sequences of six proteolytic peptides from 20-kDa protein obtained by Edman sequencing are indicated (bold letters with underlines) in the rat {alpha}A-crystallin sequence. The peptide designations (P1–P6) correspond to those in Figure 2 .

 
In the previous reports, changes in opsin,16 arrestin,17 18 and ROS protein phosphorylation levels19 have been identified. Our present study identified that the 67-kDa and 23-kDa bands, which have molecular masses similar to those of RK and recoverin, respectively, were significantly less abundant in RCS ROS (Fig. 1 , upper panel). These observations suggested that the quenching of the phototransduction pathway might be impaired in RCS. To study changes of the protein components in the process of quenching of the phototransduction pathway during the development of retinal dystrophy in RCS rats, western blot analysis was performed using antibodies against opsin, RK, arrestin, and recoverin. As shown in Figure 4 , immunoreactive bands were detected in 4- and 6-week-old rats that corresponded to recoverin, arrestin, and opsin, but those in 8-week-old rats were almost missing. The development of the destruction of ROS components at this age may have been the cause of these findings. In contrast, a trace of a band of RK was found in 4-week-old rats, and no band of RK was detected in 6- and 8-wee-old rats. These results indicate that the expressions of {alpha}A-crystallins and RK in RCS rat were already low at the beginning stage of retinal dystrophy, and they were specifically less abundant among proteins in ROS. The following two possibilities were considered as a possible molecular mechanism of these changes in {alpha}A-crystallins and RK: mRNA expressions of {alpha}A-crystallins and RK are low in RCS rat, and {alpha}A-crystallin and RK are specifically proteolyzed.



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  		Figure 4. Analysis of ROS proteins in RCS and control rats by western blot analysis using antibody against rhodopsin, RK, arrestin, or recoverin. ROS was isolated from four retinas of 4-week-old, 6-week-old, or 8-week-old RCS rats (rdy-/-) or from 8-week-old control rat (rdy+/+ ) by a sucrose-density gradient centrifugation method. ROS pellets were each suspended in 100 µl 10 mM HEPES buffer (pH 7.5) containing 100 mM NaCl. An aliquot (10 µl) was mixed with the sample buffer (10 µl), loaded on an SDS-PAGE gel, and then electrotransferred to a polyvinylidene difluoride membrane. Western blot analysis was performed using either anti-rhodopsin monoclonal antibody (mAb) (1:2 dilution), anti-RK mAb (1:3000 dilution), affinity purified anti-arrestin polyclonal Ab (1:2000 dilution), or anti-recoverin polyclonal Ab (1:2000 dilution). The details of the western blot are described in the Materials and Methods section.

 
To answer these questions, mRNA expressions of {alpha}A-crystallins and RK were examined by RT-PCR during the development of retinal dystrophy. As shown in Figure 5 , mRNA expressions of {alpha}B-crystallin and opsin were comparable between RCS and control rats at 4, 5, 6, and 8 weeks of age. mRNA levels of {alpha}A-crystallin and its splice variant form ({alpha}Ains-crystallin) were significantly less and those of RK were relatively less in RCS rats at all ages examined than in control rats. Lower mRNA expression of RK in RCS rats was confirmed by competitive quantitative PCR, as shown in Figure 6 .



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  		Figure 5. Expressions of mRNA for {alpha}A-crystallins, {alpha}B-crystallin, RK, opsin, and ß-actin in RCS and control rats. Two micrograms RNA from retinas were reverse transcribed to generate a cDNA pool. Pooled cDNA (2.2 µl from a total pool of 22 µl) was used for PCR with specific primers as described in the Materials and Methods section. PCR products were evaluated by agarose gel electrophoresis and ethidium bromide staining. Two close PCR products of 485 bp and 574 bp indicated by white arrows are from {alpha}A-crystallin and {alpha}Ains-crystallin, respectively.

 


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  		Figure 6. Competitive PCR for RK. Competitive PCR was performed using 2.2 µl pooled cDNA from retinas of 5-week-old RCS and control rats, increasing concentrations of competitor cDNA (1–200 ng) and RK primers, as described in the Materials and Methods section. PCR products were evaluated by agarose gel electrophoresis and ethidium bromide staining.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The RCS rat (rdy-/-) is the most extensively studied as a model of inherited retinal dystrophy such as RP. In this strain, it is thought that a primary genetic defect in the RPE causes a deficit in phagocytosis of shed ROS materials by the RPE.4 5 6 Accumulation of the ROS debris between photoreceptor and RPE causes unknown processes and finally results in photoreceptor degeneration, but the process has not been clarified. The molecular defect that causes this rdy-/- mutation remains to be identified, although it has been reported that several proteins including opsin,16 arrestin,17 18 hsp 70,18 20 neurotrophic factors21 including basic fibroblast growth factor, phospholipids,22 and others are changed quantitatively by aging in RCS rats.

In the present study, we performed protein mapping of either whole retina or ROS by electrophoresis and western blot analysis and found that {alpha}A-crystallins and RK in ROS of RCS rat were selectively expressed at lower levels than in control rats. It is unclear whether these changes are the causes or the consequence of retinal degeneration. However, it is speculated that these changes are not simply consequences of photoreceptor destruction but may be involved in the pathogenesis of retinal degeneration because of the following reasons: expression of {alpha}A-crystallins and RK were already low before retinal destruction was apparent in RCS rat; abundance of other ROS proteins, such as opsin, recoverin, and arrestin also decreased during the development of the photoreceptor destruction, but their decreases were slower in RCS rat than in control; and mRNA expressions of {alpha}B-crystallin and opsin in RCS rats were comparable to those in control rats.

{alpha}-Crystallins are known to be major components of lens proteins. It has been shown that {alpha}-crystallins are expressed in many extralenticular tissues, including retina, heart, lung, spinal cord, skin, muscle, brain, and kidney.39 40 41 42 The proteins belong to a family of hsps, which are involved as molecular chaperons in the biologic protection to suppress protein aggregation, denaturation, and misfolding under stress conditions.43 44 45 Among these chaperon functions, {alpha}-crystallins have been shown to bind specifically to post-Golgi membranes and to be involved in the transportation of newly synthesized rhodopsin.42 In fact, Nir et al.16 have reported that opsin abnormally accumulates within the inner segment plasma membranes of photoreceptors of RCS rat. These observations suggest that {alpha}-crystallins may participate in the renewal of photoreceptor outer segment membrane and that immature ROS may not be properly phagocytized by RPE cells because of the absence of {alpha}-crystallins. Alternatively, it is speculated that {alpha}-crystallins may directly affect phagocytotic processes, because they exist in RPE cells.46 Recent observations have also shown that {alpha}-crystallins are involved in other degenerative diseases. Vicart et al.47 reported that a missense mutation in the {alpha}B-crystallin gene causes a desmin-related myopathy. Van Noort et al.48 reported that {alpha}B-crystallin is a candidate for an autoantigen in multiple sclerosis. Tezel et al.49 found serum autoantibodies against {alpha}-crystallins in patients with normal-tension glaucoma and suggested that autoimmunity toward {alpha}-crystallins may be related to the pathogenesis of the glaucomatous optic neuropathy.

RK is a member of the G-protein–coupled receptor kinase family and plays a pivotal role in desensitization of phototransduction by phosphorylating rhodopsin in a light-dependent manner.50 51 In terms of the relationship with retinal diseases, a mutation of RK has been identified as a cause of Oguchi’s disease, a retinal degenerative disease that involves congenital stationary night blindness.52 53 54 It has been suggested that absence of or abnormally high levels of rhodopsin phosphorylation are possible mechanisms of retinal degeneration in RP.55 56 57 58 In cancer-associated retinopathy, which is caused by an autoimmune reaction toward retinal antigens,59 it has been suggested that an autoantibody against recoverin may block its function of regulating rhodopsin phosphorylation in a Ca2+-dependent manner60 61 and that this is a possible mechanism of retinal degeneration.62 63 Taken together, these observations indicate that abnormal regulation of rhodopsin phosphorylation may commonly be involved in the pathogenesis of photoreceptor degeneration. We do not know why RK expression is low in RCS rats, in addition to the low expression of {alpha}-crystallins, compared with that in control. However, if {alpha}-crystallins are really involved in the transportation of newly synthesized rhodopsin, low expressions of {alpha}-crystallins and RK may both be involved synergistically in the pathogenesis of the retinal degeneration.

In conclusion, we have found selectively low expressions of {alpha}-crystallins and RK in ROS of RCS rat, which we believe may be related to the pathogenesis of retinal dystrophy. Further study is required to clarify the mechanism by which these changes of {alpha}-crystallins and RK cause retinal degeneration.


   Acknowledgements
 
The authors thank Hitomi Sano, Department of Biochemistry, Sapporo Medical University School of Medicine for generous support of the study.


   Footnotes
 
Supported by grants from the Japanese Ministry of Health, Naito Memorial Foundation, Ciba–Geigy Foundation for the Promotion of Science, The Mochida Memorial Foundation for Medical and Pharmaceutical Research, Uehara Memorial Foundation, and Japanese Retinitis Pigmentosa Society Research Foundation.

Submitted for publication March 31, 1999; revised July 9, 1999; accepted July 19, 1999.

Commercial relationships policy: N.

Corresponding author: Hiroshi Ohguro, Department of Ophthalmology, Sapporo Medical University, School of Medicine, S-1, W-16, Chuo-ku Sapporo 060-8543, Japan. E-mail: ooguro{at}sapmed.ac.jp


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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