|Molecular Vision 1999;
Received 27 January 1999 | Accepted 10 August 1999 | Published 11 August 1999
Lactose promotes organized photoreceptor outer segment assembly and preserves expression of photoreceptor proteins in retinal degeneration
Monica M. Jablonski,
Amira Wohabrebbi, Cynthia S. Ervin
Retinal Degeneration Research Center, Department of Ophthalmology, The University of Tennessee-Memphis, Memphis, TN
Correspondence to: Monica M. Jablonski, Ph.D., The University of Tennessee-Memphis, Department of Ophthalmology, 956 Court Avenue, Memphis, TN, 38163; Phone: (901) 448-7572, FAX: (901) 448-7193; email: email@example.com
Purpose: We have previously shown that lactose promotes the proper assembly of photoreceptor outer segments in the absence of the retinal pigment epithelium (RPE). The purpose of this study was to determine if the difference between organized and disorganized membranes was a variation in the amounts of two structural proteins, opsin and rds/peripherin.
Methods: Eye rudiments were dissected from Xenopus laevis embryos and the RPE was removed prior to culturing in the following media: Niu-Twitty medium; Niu-Twitty with mannose; Niu-Twitty with lactose. Controls included retinas that matured in vitro with an adherent RPE. Photoreceptor ultrastructure was evaluated with emphasis on outer segment membrane organization. The relative amounts of opsin and rds/peripherin, two outer segment-specific proteins, were determined, as were their immunolabeling patterns.
Results: In control retinas, outer segments were composed of stacked, flattened membranous saccules. Opsin labeling of rod outer segments was very dense, indicative of normally organized disc membranes, and rds/peripherin labeling was heavy at the outer segment disc periphery and incisures. In the absence of the RPE, a whorl-like profile of outer segments is present in what would be the sub-retinal space. Opsin immunolabeling was patchy and disorganized. Immunolabeling of rds/peripherin was present, but in a disorderly array. Mannose showed no protective effect. In contrast, lactose promoted the formation of organized outer segments and allowed for near normal expression of both photoreceptor markers. In retinas with disorganized outer segments, the expression of opsin is downregulated while the expression of rds/peripherin is maintained or upregulated.
Conclusions: Lactose protects against the retinal degeneration induced by RPE removal by preserving the outer segment structure and the photoreceptor immunolabeling patterns. It also maintains constant the relative amounts of opsin and rds/peripherin. It is possible that in degenerating retinas, photoreceptors upregulate rds/peripherin expression in attempt to provide additional support for the proper folding of nascent membranes, however this is insufficient to permit organization of the photoreceptor outer segments. Our results suggest that rescue-effect of lactose is mediated by a non-rds/peripherin related mechanism.
The photoreceptor (PR) outer segment (OS) is highly structured and is comprised of stacked, flattened membranous saccules in perfect register. This formation is thermodynamically unfavorable , suggesting that molecular forces may be needed maintain this conformation. It has been demonstrated in several laboratories that reduced levels of opsin and rds/peripherin are associated with altered OS structure and subsequent PR degeneration. In the rhodopsin knockout, mice lacking both opsin alleles fail to form rod OS and PR degenerate rapidly, while those mice with a single copy of the opsin gene elaborate OS with a disrupted morphology and have a slow PR degeneration [2,3]. These data suggest that in addition to its role in the phototransduction cascade, opsin may also play an as of yet undefined structural role [2,3].
The structural relevance of rds/peripherin has been much more widely evaluated. Experimental data suggest the existence of an adhesive force at the OS disc periphery, and support indirectly that rds/peripherin is responsible for exerting this stabilizing effect . An insertion in the rds/peripherin gene produces a truncated protein that, in the rds/rds homozygous mouse, precludes the normal development of outer segments by impairing disc folding, leading to a slowly progressive degeneration of both rod and cone PRs . The rds/+ heterozygote mouse has a much milder form of the disease in which OS are formed, yet they are disorganized, with swollen and vacuolated discs . This phenotype is most likely due to haploinsufficiency, rather than a dominant negative effect . Mutations in the orthologous human gene, peripherin/RDS, cause a wide spectrum of retinal manifestations that are inherited in an autosomal dominant fashion. Histopathology confirms the presence of shortened and disorganized OS , in close analogy to what has been observed in rds/+ mice .
Disruption of OS structure can also be caused by retinal pigment epithelium (RPE) dysfunction or loss. The importance of an intact and fully functional RPE on PR structure and survival has been known for many years. Retinal detachment studies indicate that separation of the neural retina from the overlying RPE layer results in rapid cone and rod OS degeneration and the degree of recovery of cell morphology and function are negatively correlated with the duration of the detachment [8,9]. In the Royal College of Surgeons rat, it has been shown that a defect in the RPE results in PR degeneration unless growth factors are injected into the subretinal space  or an RPE transplant is performed . Additionally, mutations in RPE-specific genes have been documented in human forms of retinal degenerations (e.g., RPE65 gene [12,13], cellular retinoid binding protein (CRALBP) , bestrophin , and PEDF [16-18]).
To further complicate our understanding of critical factors influencing PR health or degeneneration, a large body of work suggests that carbohydrates also are necessary for proper OS formation and organization. We have previously demonstrated that, in the absence of the RPE, OS membrane ultrastructure can be modified by adding specific sugars to the culture medium. OS membranes elaborated in the absence of the RPE, yet in the presence of permissive sugars (e.g., galactose and lactose), assemble into stacked flattened membranous saccules, very similar to those that have been maintained in the presence of the RPE. Conversely, retinas exposed to non-permissive sugars (e.g., glucose and mannose) elaborated disordered OS [19,20]. Data from other laboratories support our contention that carbohydrates are necessary for OS formation and organization. It has been demonstrated that in adult Xenopus retinas tunicamycin-induced inhibition of N-linked oligosaccharides causes nascent membranes to form tubulo-vesicular opsin-containing profiles [21-23]. A recent study in transgenic mice lacking the glycosylation site(s) of opsin corroborates the hypothesis that proper glycosylation of this protein is necessary for normal OS morphogenesis . Taken together, these data suggest that the disorganization of OS membrane saccules may be due to underglycosylation of proteins within the retina. Given the documented abundance of glycoproteins, glycolipids, glycosaminoglycans, and lectins in the retina/RPE complex it may be that sugars play a role in cellular adhesion or a more downstream role in cell signaling involving the regulation of gene transcription/translation.
While data from several laboratories suggest that opsin and rds/peripherin are involved in the proper folding of OS membranous discs, the effect of RPE dysfunction or removal upon these proteins has not yet been explored. In addition, it is unknown whether lactose-mediated rescue alters the expression of these proteins. The purpose of the present study was to combine our previously documented alterations in PR architecture with the characterization of opsin and rds/peripherin under four experimental conditions: healthy retinas, degenerating retinas, non-protected retinas exposed to mannose, and retinas that have been protected from degeneration with lactose. We evaluated the immunolocalization patterns and quantified the protein levels. The hypothesis tested was that a alteration in the amounts of opsin and rds/peripherin would be detected in retinas undergoing degeneration, while the rescuing effect of lactose may be due to a stabilizing effect upon protein expression.
Culture of Developing Retinas
The culture preparation used in these experiments has been previously described [19,20,25-27]. Xenopus laevis embryos were obtained through induced breeding of adult Xenopus by injection of human chorionic gonadotropin (Sigma Chemical Co., St. Louis, MO). Embryos were staged by external morphological criteria  after which time eye rudiments from stage 33/34 embryos were isolated. At stage 33/34, outer segments are just beginning to be elaborated, therefore all membranous disc material is produced in vitro .
Eye rudiments without a closely apposed RPE were placed into Niu-Twitty (NT) media alone , NT containing 5 mM mannose (Sigma Chemical Co., St. Louis, MO) or 5 mM lactose (Eastman Kodak Company, Rochester, NY). Controls included eye rudiments allowed to mature in vitro in the presence of an apposed RPE in NT medium alone. Cultures were maintained at 23 °C for 3 days under cyclic lighting conditions, at which time the eyes have reached approximately stage 42 of the in vivo developmental scale [19,30]. Prior to removal of the eye rudiments, embryos were maintained under identical lighting conditions. The handling of animals was in accordance with the Declaration of Helsinki and The Guiding Principles in the Care and Use of Animals (DHEW Publication NIH 80-23).
Morphological Assessment and Immunocytochemistry
Prior to harvesting for morphological or biochemical analysis, each rudiment was grossly examined under a dissecting microscope for rudiment integrity and smoothness of the neuroepithelial surface to ensure that all rudiments were undamaged and intact. Any rudiments that exhibited an uneven surface or had many loose cells associated with it were discarded.
For structural analysis, eyes were fixed on ice in Tucker fix (2% glutaraldehyde and 1% osmium tetroxide), dehydrated, embedded in Araldite/EMbed812 (Electron Microscopy Sciences, Fort Washington, PA). One micron thick sections were placed on microscope slides, stained with toluidine blue O and examined on a Nikon Microphot fx microscope. Images were captured using a Dage-MTI video camera (Michigan City, IN) and NIH image version 1.61 image analysis system. For immunocytochemical localization analysis, eyes were fixed for 2 h in Davidson fixative (32% ethanol, 2% formalin, 11% acetic acid), dehydrated, embedded in Unicryl (Electron Microscopy Sciences, Fort Washington, PA), cut at one micron thickness and collected on microscope slides. The sections were incubated in 5% goat serum (Vector Laboratories, Burlingame, CA) in phosphate buffered saline (PBS), rinsed in PBS and incubated overnight in primary antibody. The following antibodies were used: B630N anti-opsin, 1:2000 dilution  which we have previously demonstrated to immunolabel the principal rods ; and xrds38 antiserum, 1:4000 [32,33], which we have previously shown to label both rods and cones . Gold-conjugated secondary antibodies followed by silver enhancement were applied to the tissue, as described by the manufacturer (Electron Microscopy Services, Fort Washington, PA).
Retinal sections were viewed on a Nikon Eclipse E400 microscope equipped with Sensys Color Camera (Photometrics, Tucson AZ) and images were collected using MetaMorph Imaging System software (Universal Imaging Corporation, West Chester, PA). Two images were collected of each retinal section: a brightfield image which shows the morphology of the tissue; and another image taken with epipolarized light which shows only the immunolabeling pattern. The epipolarized image was color enhanced and merged with the brightfield image so that the specific immunolabeling patterns could be easily distinguished.
Quantitation of outer segment proteins
Three sets of 15-20 eyes were collected, ground and solubilized with sodium cholate detergent (Sigma Chemical Co., St. Louis, MO). Extracted proteins were applied in duplicate to Hybond-P membrane (Amersham Pharmacia Biotech, Buckinghamshire, England) using a slot blot apparatus (Biorad, Hercules, CA). Blots were incubated in primary antibody overnight at 4 °C (anti-opsin; B630N  at 1:10,000 dilution and xrds38 antiserum [32,33] at 1:10,000). The ECF Western blotting kit (Amersham Pharmacia Biotech, Buckinghamshire, England) was used according to manufacturer(SGML-146)s protocols. Blots were scanned on a Storm 860 (Molecular Dynamics, Sunydale, CA) and data were quantified using ImageQuant software version 1.1 (Molecular Dynamics, Sunydale, CA). For each of the three repetitions, data were normalized to values obtained for eyes maintained with an intact RPE. The student(SGML-146)s T-test was employed to determine significant differences between eyes that were maintained with an intact RPE and any other culture condition.
Morphology and immunolocalization patterns in control retinas (3-day in vitro culture in the presence of the RPE)
In control retinas that were allowed to mature in vitro with a normally apposed RPE (Figure 1A), the OS are highly organized and tightly stacked, yielding individual profiles that are in line with individual PR inner segments. No loose whorls of membrane are seen. An amorphous material can be discerned in the interphotoreceptor space between the OS and the RPE. The opsin labeling is very heavy over the OS of the majority of photoreceptors (Figure 1B). The labeling is very dense and linear indicating organized stacked OS membranes. The immunolabeling patterns of rds/peripherin reflected the presence of this glycoprotein at the disc periphery and incisures depicted in the stripe-like patterns over the OS layer (Figure 1C).
Morphology and immunolocalization patterns in degenerating retinas (absence of the RPE and also with the non-permissive sugar, mannose)
In retinas that matured in the absence of the RPE, PR-OS membrane structure was markedly disorganized, with little evidence of normal disc stacking (Figure 2A). A whorl-like arrangement of OS membranes was present, rather than compact individual OS structures as seen in Figure 1A. The same array of OS abnormalities was observed when retinas were cultured in the presence of mannose (Figure 3A), a sugar we have previously shown to be non-permissive in allowing OS organization in the absence of the RPE [20,25]. In these degenerating retinas the inner segment structure was also missing from some photoreceptors, however, the outer nuclear layer appeared to be intact (Figure 2A and Figure 3A).
In parallel to these structural changes, the immunolabeling and localization patterns were dramatically altered in retinas undergoing degeneration induced by RPE removal. Opsin immunolabeling appeared reduced and altered in conformation (Figure 2B). Individual OS profiles could not be distinguished. Rather heavy patches of label alternating with complete lack of label and still other areas of a non-uniform irregularly shaped profiles of immunoreactivity were present distal to the inner segments, similar to the OS contours seen in Figure 2A. Some areas of immunolabelling are out of the plane of focus, reflecting in three dimensions the whorls of membrane rather than tight cylinders of OS evident in the presence of the RPE. Opsin trafficking appears to be normal in that no opsin label is detected in other areas of the cell including the synaptic terminus. Also reflecting the altered OS conformation, rds/peripherin labeling was present in a random pattern in degenerating retinas (Figure 2C and Figure 3C), rather than in the orderly linear pattern notable in control retinas (see Figure 1C). Identical labeling patterns were observed in retinas that matured in the presence of mannose (Figure 3B and Figure 3C).
Morphology and immunolocalization patterns in retinas cultured in the absence of the RPE and the permissive sugar, lactose
In the absence of the RPE, lactose promoted the formation of well-organized OS segment membranes (Figure 4A), and allowed for normal expression of both PR-specific proteins (Figure 4B,C). The structure of the OS in lactose-protected retinas (Figure 4A) resembled very closely the organization of membranes under control conditions (Figure 1A). Individual OS were in line with inner segments with no whorl-like arrays, as those seen in degenerating retinas (Figure 2A and Figure 3A). In protected retinas, opsin labeling was very similar to those of control retinas (Figure 4B). Individual OS profiles with linear labeling patterns were evident, consistent with the organized OS membrane disc lamellae observed morphologically. Similar to control conditions, rds/peripherin labeling was found in linear arrays over the OS periphery (Figure 4C).
Quantitation of opsin and rds/peripherin
In eyes undergoing degeneration induced by RPE removal, there is a significant decrease in opsin, with no change in the level of rds/peripherin. A similar pattern is noted in retinas maintained in the presence of mannose: opsin expression is downregulated with a slight elevation in level of rds/peripherin. Only in retinas rescued by lactose was the level of opsin maintained compared to controls with an intact retina-RPE complex. In the lactose-rescued eyes, an increase in the amount of rds/peripherin also was measured relative to controls (Figure 5).
Our results indicate that RPE-deprived retinas undergo PR degeneration demonstrated by a derangement of OS membrane structure, altered immunolocalization patterns of opsin and rds/peripherin and loss of inner segments. These degenerating retinas also downregulate opsin expression, while maintaining constant rds/peripherin levels. Mannose, a non-permissive sugar [20,25], improved neither OS morphology, nor immunolocalization patterns. It also lacked a positive effect upon the expression of opsin. Lactose, a permissive sugar [20,25], exerted a neuroprotective action on the retinal degeneration induced by RPE removal as evidenced by the preservation of the expression and localization patterns of opsin and rds/peripherin, as well as both outer and inner segment structure. In addition, it maintained constant the relative amount of opsin while increasing rds/peripherin compared to control non-degenerating retinas with an adherent RPE.
Both opsin [34,35] and rds/peripherin  are intramembranous glycoproteins with distinct functions and expression patterns in the PR-OS. The apoprotein, opsin, is the most abundant protein of the PR-OS, comprising approximately 80-90% of the rod OS protein . Opsin is localized on the lamellar portion of OS discs and the surrounding plasma membrane. Although opsin coupled with its chromophore initiates the phototransduction cascade, evidence suggests that it also plays structural role that is yet undefined [2,3,37]. The data presented herein provide further evidence that a decrease in the amount of opsin (Figure 5) is associated with a degenerative retinal condition.
rds/Peripherin is localized exclusively at the disc rims and incisures of both rod and cone outer segments [31,38-40]. It is postulated that rds/peripherin belongs to a group of adhesion molecules that stabilize the folding of the OS disc into its proper conformation [36,41,42]. We have recently isolated and characterized three Xenopus homologs, termed xrds, of mammalian rds/peripherin. In both photoreceptor types, xrds is hypothesized to form heterotetrameric or higher-order rds/peripherin complexes that stabilize disc rims by bridging the intradiscal space in the terminal loop region of OS discs. In the rds/+ heterozygote mouse, in which an insertion in one allele of the rds/peripherin gene produces a truncated protein, outer segments are formed, but they are disorganized, with swollen and vacuolated discs , similar to what we have shown in the retinal degeneration induced by RPE removal (Figure 2A).
The disorderly array of OS membranes in our degenerating retinas cannot be ascribed to haploinsufficiency of rds/peripherin because the levels of this protein are maintained or slightly elevated, despite a downregulation of opsin (Figure 5A). This results in a relative decrease in the amount of opsin while maintaining the amount of rds/peripherin, suggesting that these two glycoproteins are differentially regulated and that this form of degeneration has minimal effect upon rds/peripherin transcription or translation. Another possible explanation is that in response to the downregulation of opsin, photoreceptors upregulate the relative amount of rds/peripherin in attempt to assist the nascent membranes into an organized, properly folded array. Our results suggest that an increase in the amount of rds/peripherin relative to opsin is insufficient to explain the rescue of photoreceptors from degeneration in this model and that the lactose-mediated rescue must occur by a yet unknown mechanism.
Although many of the permissive sugars (e.g., lactose and galactose), as well as many of the non-permissive sugars we previously tested (e.g., mannose and glucose) can readily participate in glycolysis, they differ in their respective abilities to exert an organizational effect upon OS assembly [20,25]. This demonstration provides evidence that the structural manifestations provided by lactose and the other permissive sugars we previously described are not accounted for by providing an energy source via the glycolytic pathway. It is possible, however, that mannose and other non-permissive sugars are unable to fulfill a particular functional role or participate in glycosylation in the same manner as lactose and other permissive sugars. It has been documented that opsin is transiently hyperglycosylated with galactose during disc morphogenesis and that it is removed upon assembly of nascent discs . If indeed, galactose is temporarily required for proper membrane assembly, it is plausible that in our system, exogenous lactose participates in this assembly process, after which time it is removed and secreted into the media. Mannose, the predominant sugar residue of native opsin , may have no role in the assembly, but rather serve a separate function. For this reason, exogenous mannose may not stimulate proper assembly. In addition, although both mannose-binding and lactose-binding lectins [44-52] are present in the retina-RPE complex, the downstream effects of sugar-lectin binding are unknown.
In summary, our results indicate that removal of the RPE dramatically altered PR structure, as well as the immunolabeling patterns and relative amounts of opsin and rds/peripherin. A decreased opsin content is associated with the degenerating phenotype, while an elevation in rds/peripherin is not sufficient to permit outer segments to fold with the proper structural conformation. Lactose exerted a neuroprotective action on the retinal degeneration induced by RPE removal by preserving the expression and localization patterns of both PR-specific proteins. This effect was not accounted for by nonspecific glycosylation or by providing an energy source, since mannose did not exert a rescuing effect. These data suggest that lactose-mediated rescue of PR degeneration occurs via a non-rds/peripherin related mechanism. Other possible modes of rescue are currently being evaluated.
The authors gratefully acknowledge Alessandro Iannaccone, M.D. for his insights into the clinical relevance of our experimental findings and for his critical review of this manuscript. We also acknowledge Andre DeBellevué and Mike Davis from Advanced Scientific Inc., Meraux, LA for their assistance with collecting the images presented in this manuscript. The following have generously provided antibodies for our use: Dr. Paul Hargrave, University of Florida, Gainesville, FL and Dr. Gabriel Travis, University of Texas Southwestern. This study was supported by a National Eye Institute grant EY10853 (MMJ), a pre-doctoral fellowship from the Center for Neuroscience at the University of Tennessee (AW), an unrestricted departmental grant from Research to Prevent Blindness (RPB), New York. MMJ is an RPB William and Mary Greve Special Scholar.
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