|Molecular Vision 2003;
Received 1 August 2003 | Accepted 25 November 2003 | Published 16 December 2003
Permissive glycan support of photoreceptor outer segment assembly occurs via a non-metabolic mechanism
Monica M. Jablonski
Retinal Degeneration Research Center, Department of Ophthalmology, The University of Tennessee, Memphis, TN
Correspondence to: Monica M. Jablonski, Ph.D, The University of Tennessee Health Science Center at Memphis, Department of Ophthalmology, 956 Court Avenue, Suite D228, Memphis, TN, 38163; Phone: (901) 448-7572; FAX: (901) 448-1299 email: firstname.lastname@example.org
Purpose: We have previously demonstrated that in RPE-deprived retinas, lactose, galactose, and structurally related glycans support the proper assembly of nascent photoreceptor outer segment membranes. Other glycans such as mannose and glucose have no effect on this process. While the ability to support outer segment assembly is highly specific, all of the permissive glycans we have tested are able to enter metabolic pathways within the retinal cells, thus the mechanism by which the glycans are functioning is still ambiguous. The present study was undertaken to determine if permissive glycan-mediated support of photoreceptor outer segment assembly occurred via a non-metabolic mechanism and if the phenomenon was reversible.
Methods: The RPE was removed from isolated Xenopus laevis embryonic eyes that were allowed to complete differentiation in (1) Niu-Twitty medium, (2) Niu-Twitty supplemented with 5x10-3 M lactose or mannose, (3) Niu-Twitty supplemented with lactose and 3H-galactose, (4) Niu-Twitty medium supplemented with isopropyl beta-D-thiogalactopyranoside (IPTG) at concentrations spanning five orders of magnitude, or (5) Niu-Twitty medium containing lactose or IPTG to which 3H-leucine was added for 2 days followed by an additional 2 days in Niu-Twitty alone. Control RPE-deprived and RPE-supported retinas were included for comparison. Under all experimental conditions, retinal photoreceptors were evaluated to determine the level of organized folding of outer segment membranes.
Results: Outer segment membranes of RPE-deprived retinas exposed to lactose were significantly more organized than both control RPE-deprived retinas and those exposed to mannose. In eyes exposed to the radiolabeled metabolizable glycan, the majority of the sugar was incorporated into Müller cells. IPTG, a non-metabolizable form of galactose, promoted the formation of organized outer segment assembly similar to lactose although at a 100 fold reduced concentration compared to metabolizable permissive glycans. Both IPTG and lactose supported outer segment assembly in a step-wise fashion with maximal support at 5x10-5 M and 5x10-3 M, respectively. Removal of the permissive glycans resulted in loss of support of outer segment assembly.
Conclusions: The ability of both lactose and IPTG to support outer segment assembly in the absence of the RPE is dose-dependent and the effect of these sugars upon membrane folding is reversible. Moreover, this effect is supported by a non-metabolic mechanism and is therefore not accounted for by simple provision of an energy source.
Photoreceptors are highly polarized and ultrastructurally unique cells that are essential for capturing photons of light and transducing them into chemical signals. The outer segment is the most highly specialized region of the photoreceptor cell. Each photoreceptor outer segment consists of up to 1000 flattened membranous discs stacked on top of one another in an orderly array [1,2]. Outer segment assembly is a dynamic process as membranes are continuously being renewed at the proximal end of the outer segment, while the distal ends of the outer segment are shed and phagocytized by the retinal pigment epithelium (RPE) .
While the nature of the molecular signals that regulate photoreceptor outer segment assembly are not fully understood, carbohydrates and their lectin receptors certainly play an important physiological role in the retina. Carbohydrates have both a metabolic and a non-metabolic role in retinal physiology. The metabolic role of carbohydrates in the retina has been studied extensively by Winkler and colleagues [4-8]. These studies have demonstrated effectively that frog retina is highly resistant to glucose deprivation and utilize an alternative endogenous carbohydrate resource in the form of stored glycogen under these circumstances  and that glucose is the preferred substrate of all retinal neurons as well as the Müller cells . Furthermore, they have demonstrated that the supply of glucose is a critical determinant of the ability of a retinal cell to withstand a metabolic stress .
In addition to having metabolic functions in the retina, carbohydrates also play fundamental non-metabolic roles. Data from several labs, in addition to our own, have indicated that carbohydrates are necessary for proper photoreceptor outer segment formation and organization. Tunicamycin, an antibiotic that prevents the formation of N-linked oligosaccharides via the lipid intermediate pathway during protein glycosylation, significantly alters membrane morphogenesis in adult Xenopus retinas, suggesting that the lack of sugar moieties on glycoproteins within the retina may be responsible for the misassembly of outer segment membranes [9-11]. If post-translational trimming of oligosaccharides is inhibited with castanospermine, however, nascent disc morphology is identical to control conditions, suggesting that post-translational removal of oligosaccharides is not essential for normal disc morphogenesis . Several sub-types of lectins, including lactose-binding lectins, have been localized to the retina in many species [13-16]. In addition, many lectin-binding sites of the outer retina have been documented in detail [17-20]. In particular, a 16 kD galectin has been suggested to play a modulating role in the interactions between the RPE and the retina . Moreover, its presence throughout Müller cells points to its role in metabolic processing between Müller cells and other retinal cells . Its specific expression at the OLM level underscores the probable role of this galectin in cell-cell interactions between Müller cells and photoreceptors. In addition, galectin-1 has been proposed to be involved in regulating the adhesion of photoreceptors and outer plexiform layers based upon its localization patterns . This brief review underscores the important role of carbohydrates in retinal physiology, and emphasizes their important role in retinal development.
In our early studies, we tested a battery of 16 different glycans at concentrations over four orders of magnitude and evaluated the ability of each to support outer segment assembly in RPE-deprived retinas [23,24]. Only lactose, galactose, and substituted forms of these permissive glycans allowed outer segments to organize in the absence of the RPE. Other glycans, such as glucose, mannose, and fucose, failed to exert any organizational effect and were thus termed non-permissive glycans. We have since demonstrated that lactose also supports the expression of key photoreceptor and Müller cell proteins that are normally dysregulated in RPE-deprived retinas [25-27]. Because these glycans are able to enter the metabolic pathway within retinal cells, determination of the molecular mechanisms supporting these phenomena has been elusive. In addition, our previous experiments could not rule out the possibility that regardless of the mechanism(s), once activated, the organizational effect could remain sustained and persist also independently of further supplementation of glycans in the culture media. Herein, we show that non-metabolizable permissive glycans that are structurally similar to the permissive glycans that we have tested previously support equally well the assembly of outer segment membranes, demonstrating that their effect is not mediated by mere provision of an energy source. We further show that the organizational effect is reversible upon withdrawal of the supportive glycans.
Culture of developing retinas
The culture preparation used in these studies has been previously described . The handling of animals was in accordance with the Declaration of Helsinki, The Guiding Principles in the Care and Use of Animals (DHEW Publication NIH 80-23), and the ACUC office at the University of Tennessee Health Science Center. Human chorionic gonadotropin (Sigma Chemical Co., St Louis, MO) was used to induce adult Xenopus laevis to breed. The external staging system of Nieuwkoop and Faber  was used to determine retinal maturity as previously described. Embryos and isolated retinas were maintained under cyclic lighting conditions (12 h light:12 h dark).
In all experiments, eyes were removed from embryos at stage 33/34, just as photoreceptor outer segments are beginning to be elaborated . At this stage, the eye rudiments are not yet surrounded by the sclera, leaving the posterior segment covered only by the RPE layer. Taking advantage of this characteristic, the overlying RPE can be gently peeled away from the neuroepithelium, leaving the underlying retina exposed to the culture medium. Intact eye rudiments without an adherent RPE were cultured in Niu-Twitty medium alone , Niu-Twitty containing concentrations of lactose (Eastman Kodak Company, Rochester, NY), or isopropyl beta-D-thiogalactososide (IPTG; Sigma Chemical Company) over five orders of magnitude (5x10-6 to 5x10-2 M and 5x10-7 to 5x10-3 M, respectively). Eyes were placed cornea-side down in 35 mm culture dishes and were maintained in vitro for 3 days at 23 °C in Niu-Twitty medium . Eyes allowed to mature in vitro in the presence of an adherent RPE in Niu-Twitty medium alone were used as controls.
At the end of the culture protocol, each intact eye was grossly examined under a dissecting microscope for integrity and smoothness of the neuroretinal surface. Any rudiment that exhibited an uneven surface or had many loose cells associated with it was discarded. For an evaluation of photoreceptor outer segment organization, eyes were fixed in Tucker fix and processed as previously described . To ensure that photoreceptors of equivalent stages of maturation were compared, all structural analyses were performed exclusively on tissue sections taken from the posterior pole region of the retina. Three individual eyes from each concentration from three replicate experiments were evaluated in all studies.
To determine the cellular fate of a permissive glycan, RPE-deprived retinas were removed from stage 33/34 tadpoles were cultured in Niu-Twitty medium containing 5x10-3 M lactose supplemented with 0.10 mCi/ml 3H-galactose (specific activity 29.5 Ci/mmol, NEN, Boston, MA). The lighting and temperature regulation was identical to that described above. After a total of three days in vitro, eyes were fixed in Tucker fix and embedded in Araldite/EMbed812 (Electron Microscopy Sciences, Fort Washington, PA), as previously described .
To determine if the supportive effect of permissive glycans upon photoreceptor outer segment assembly was reversible, RPE-deprived retinas removed from stage 33/34 tadpoles were cultured in Niu-Twitty medium containing either lactose or IPTG at the concentration at which it bests supports outer segment membrane assembly (5x10-3 M lactose or 5x10-5 M IPTG) supplemented with 0.10 mCi/ml 3H-leucine (specific activity 171 Ci/mmol; ICN, Irvine, CA) for a period of two days, followed by an additional two days in non-supplemented culture medium containing neither the permissive glycan nor the 3H-leucine. After a total of four days in vitro, eyes were fixed in Tucker fix and embedded in Araldite/EMbed812 (Electron Microscopy Sciences), as previously described .
For both autoradiographic studies, 1 μm thick sections were cut through the posterior pole region of the retina followed by drying onto microscope slides. Slides were dipped in liquid emulsion (NTB-2; NEN, Boston, MA) and allowed to develop for 2 weeks at 4 °C and 0% humidity. After developing with Kodak developer and fixer (Eastman Kodak Co.) as recommended by manufacturer, the slides were lightly stained with Toluidine blue-O and examined with bright- and epi-illuminated microscopy, as previously described [25,26,32-34]. Silver grains are colorized red prior to overlying with the brightfield image. Using this methodology to demonstrate the reversibility of the ability of the permissive glycans to support outer segment assembly, only those outer segments that were assembled in the presence of the permissive sugar and 3H-leucine will have silver grains over them. Those membranes that were produced during the second half of the culture protocol during which eyes were cultured in Niu-Twitty medium only will not present with silver grains.
Grading of photoreceptor outer segment assembly
The grading of photoreceptor outer segment assembly was evaluated using our previously described grading scale . The key criterion of this grading system is the amount of outer segment membrane organization, i.e., the amount of stacked and organized photoreceptor outer segment membrane that was associated with each of the evaluated cells. In brief, each step in grade represented a linear progression by an approximate 25% interval, ranging from 100% organization as seen in retinas with an adherent RPE (grade 4) to complete absence of organization despite the presence of whorls of membranous outer segment material attributable to the underlying photoreceptor cell (grade 0). The grading also included a sixth level (grade -1) to account for the complete absence of the photoreceptor outer segments . For each experimental condition, eight contiguous photoreceptors from three individual retinas (n=24) were evaluated using this scale. The grader was blinded to the experimental condition under which each graded micrograph was obtained. For the autoradiographic study to determine the reversibility of the ability of the glycan to support outer segment assembly, each photoreceptor outer segment was divided into two parts, those with overlying silver grains and those without silver grains, which were graded independently.
Photoreceptor outer segment grading data were statistically analyzed by one-way analysis of variance (ANOVA) using SAS statistical software (SAS Institute, Inc. Cary, NC). Observations within each eye could not be considered independent, because graded photoreceptors were analyzed in groups of eight from three separate eyes under each experimental condition. To account for this, the average grading for each eye was calculated, and comparisons among groups were conducted only on the averages of each of the eyes that were analyzed. P values less than 0.05 were considered statistically significant.
Metabolizable permissive glycan supports outer segment assembly: quantification of effect
Figure 1 illustrates examples of intact retinas that were removed from stage 33/34 Xenopus laevis tadpoles and placed into culture in Niu-Twitty medium for three days. Using this paradigm, all outer segment material is elaborated while in culture . In retinas that were maintained with a normally apposed RPE, the outer segments are tightly stacked, properly folded and contain discs of equal diameter (Figure 1A). This morphology is identical to retinas maturing in vivo . In RPE-deprived retinas that were otherwise similarly maintained, photoreceptor outer segment membranes are markedly disorganized, with little evidence of normal disc stacking (Figure 1B). The addition of 5x10-3 M mannose did not influence favorably the folding of outer segments in RPE-deprived retinas (Figure 1C), whereas the addition of 5x10-3 M lactose (Figure 1D) supported nicely the formation of nascent outer segments (membranes into organized assemblies) in the absence of the RPE.
Application of our six step classification scheme of photoreceptor outer segment morphology  revealed significant differences in the organization of outer segments under the aforementioned culture protocols. By one way ANOVA, the overall F-test for differences among the four groups was highly significant (F=42.10; p<0.0001). In retinas that completed morphogenesis with an adherent RPE, the vast majority of photoreceptor outer segments were highly structured, properly folded and contained discs of equal diameter. Utilization of our grading scale yielded a grade of 3.71±0.09 with a grade of 4 representing the highest level of organization. In the absence of the RPE, the average grade of photoreceptor outer segment organization decreased to 0.58±0.24 (Figure 1E; p<0.0001 compared to control RPE-supported retinas and lactose-exposed retinas), indicating that, on average, less than 25% of the outer segment material was organized into stacked flattened membranous saccules. The addition of 5x10-3 M mannose to the medium did not change the level of organization of the outer segments in RPE-deprived retinas (0.54±0.22, p=0.90; Figure 1E). In lactose-exposed retinas, the average organizational grade was 2.50±0.28 (Figure 1E), indicating that between 50 to 75% of the outer segment membranes were highly structured. Although the average value from lactose-supported retinas was lower than the control values from RPE-supported retinas (p=0.0009), it was significantly greater than the organizational grade of both RPE-deprived retinas and those that were exposed to mannose (p<0.0001).
Fate of metabolizable permissive gycans
While our data indicate that lactose, an example of a permissive glycan, is able to support photoreceptor outer segment membrane assembly to a level that is significantly greater than in its absence, they do not determine the mechanism by which permissive glycans are functioning within the retina. Because they are sugars, they may exert their organizational effect by simply providing an energy source (e.g., by feeding into the Krebs' cycle), by permitting the glycosylation of some essential proteins, or both. To answer this question, we performed experiments with tritiated (3H)-galactose (i.e., an alternative permissive glycan that was preferred over 3H-lactose because the former is much less expensive than the latter) to follow the destiny of these sugars during each of the three days of culture.
In these experiments, the distribution of silver grains is indicative of the cell type in which the 3H-galactose was incorporated. Our results indicate that relatively few silver grains are localized the photoreceptor region. A radial line of silver grains, however, is localized to the intercellular space between photoreceptors at a periodicity of every 2-4 cells, which continues throughout the retina to the ganglion cell layer (Figure 2). This radial pattern is present throughout the entire thickness of the retina with two exceptions: (1) silver grains are grouped in clusters in the outer portion of the inner nuclear layer, corresponding to the location of Müller cell bodies, and (2) there is also a higher density of grains that are present in the inner plexiform layer, although even here a radial pattern can be appreciated. The overall pattern of silver grain distribution is very similar to that which we see in immunocytochemical analysis of Müller cell-specific markers [25,32,34].
Support of outer segment assembly by a non-metabolizable glycan
To separate out the metabolizable properties of permissive glycans, we performed experiments in which we exposed intact RPE-deprived retinas from Xenopus laevis tadpoles to concentrations over five orders of magnitude of isopropyl beta-D-thiogalactopyranoside (IPTG), a non-metabolizable form of galactose . By one way ANOVA, the overall F test for differences among the four groups was significant (F=6.15, p=0.0002). We found a step-wise improvement in photoreceptor outer segment organization with increasing concentrations of IPTG up to a maximum effect (i.e., 5x10-5 M; Figure 3A-C), after which we observe a reduction in the ability of IPTG to support proper outer segment assembly. The maximum response was obtained with 5x10-5 M with an average grade of 2.61±0.37. This value is significantly greater than that of RPE-deprived eyes, but it is remarkably similar to that obtained in eye exposed to 5x10-3 M lactose (compare to Figure 1). It is also remarkably similar to that obtained in eye exposed to 5x10-3 M lactose. At concentrations greater than 5x10-5 M (Figure 3D-E), the reduced supportive effect may be due to toxicity, an effect similar to that which we have seen with pigment epithelium-derived factor .
Removal of permissive glycans eliminates the stimulus to support outer segment membrane assembly
To test the hypothesis that the organizational effect permissive glycans upon outer segment assembly was reversible, we exposed RPE-deprived retinas to the permissive sugars at the optimal concentrations (5x10-3 M lactose or 5x10-5 M IPTG) supplemented with 3H-leucine for a period of two days, followed by an additional two days in non-supplemented culture medium. Our data demonstrate that the supportive effect upon outer segment assembly is indeed reversible. Illustrated in Figure 4A is an example of an RPE-deprived retina that was exposed to IPTG using the above outlined culture paradigm. In the majority of the photoreceptors, the outer segments that are most displaced from the inner segment portion of the photoreceptor, and therefore were assembled during the early part of the culture paradigm when eyes were exposed to IPTG and 3H-leucine, are properly assembled. The average grade of these membranes was 2.88±0.37 (Figure 4B), which is not statistically different from that of RPE-deprived eyes exposed to lactose or IPTG. On the contrary, outer segment membranes that are close proximity to the inner segments, and therefore were assembled most recently when eyes were exposed to non-supplemented medium, were significantly more disorganized. These membranes had an average grade of 1.21±0.31 (p=0.0014; Figure 4B). Even though this value is significantly less than that obtained from the early part of the culture paradigm, it is significantly greater than that obtained from RPE-deprived eyes (p=0.043). This suggests two significant findings: (1) that proper outer segment assembly is responsive to stimulation and that withdrawal of the permissive glycan ligand removes the support for proper folding of outer segment membranes; and (2) that the intracellular pathways by which the permissive glycans support outer segment membrane assembly are not completely shutdown immediately upon removal of the stimulus and therefore the membranes assembled in the two-days immediately following glycan removal have an intermediate level of organization. Eyes exposed to lactose in a similar protocol demonstrated an identical trend (data not shown).
We have previously shown that lactose and substituted forms of this glycan exert an organizational effect on photoreceptor outer segment membranes in the absence of the RPE [23,24,26,37]. The utilization of IPTG as a substitute for the metabolizable permissive glycans allowed us to separate out the non-metabolic aspects of permissive glycan function. With IPTG, not only do we observed an effect upon outer segment assembly in RPE-deprived retinas that is similar in all ways to what we described previously for lactose [23,24,26,37], but this was also observed at 100 fold lower concentrations. Specifically, IPTG and lactose stimulate maximal support of photoreceptor outer segment assembly at 5x10-5 M and 5x10-3 M, respectively. Since IPTG is non-metabolizable , demonstration that IPTG supports outer segment assembly in RPE-deprived retinas as effectively as lactose yet at 100 fold lower concentrations offers evidence that, although the majority of the metabolizable permissive glycans are likely taken up for metabolic purposes, they exert their organizational effect on outer segments via a non-metabolic pathway.
In the majority of the photoreceptors, the outer segments that incorporated the 3H-leucine and therefore were assembled during the early part of the culture paradigm when eyes were exposed to lactose or IPTG, were properly organized. Vice versa, outer segment membranes that were not labeled by 3H and therefore were assembled during the latter part of the experiments when eyes were exposed to non-supplemented medium, were more disorganized. These results strongly suggest that proper outer segment assembly is directly responsive to stimulation by lactose or IPTG and that removal of these glycans results in disruption of outer segment assembly. Therefore, these results provide evidence for a reversibility of the observed organizational effect.
It has been previously shown  that Müller cells incorporate galactose and store it as glycogen, which serves as an energy source. Consistent with this observation, our results suggest that, indeed, the permissive metabolizable glycan, galactose, is taken up by Müller cells. Therefore, to this end, these glycans may be providing an energy source for Müller cells, assuming the fate of all metabolizable permissive glycans is equivalent to that of galactose. However, since the organizational effect was observed also with IPTG, which is non-metabolizable, the mechanism(s) underlying this specific effect must be other than metabolic ones. It must be noted, though, that the fact that the majority of the metabolizable glycans were taken up by Müller cells does not allow us to rule out that Müller cells were implicated in mediating the organizational effect upon photoreceptor outer segments via alternative and entirely separate mechanism(s). In fact, preliminary data indicates that this may well be the case. When cultured in the presence of a Müller cell inhibitor, α-aminoadipic acid, permissive glycans fail to exert the typically observed organizational effect (Jablonski, 2003, unpublished observation). These findings corroborate our previous results, suggesting that the organizational effect of permissive glycans may have been exerted not by a direct effect upon photoreceptors, but indirectly through Müller cells [23-26,37]. Moreover, while a significant body of evidence indicates that factors supplied by the RPE are critical for the development and survival of photoreceptors, data in the scientific literature demonstrate that photoreceptors themselves most often do not directly respond to the stimulus. Rather, it is the Müller cell population that responds by upregulating c-fos, c-jun and/or the mitogen-activated protein (MAP) kinase pathways [39-41], thus further suggesting that Müller cells are able to support photoreceptor health and integrity, although the mechanisms governing this phenomenon have not been fully elucidated.
These findings represent important steps forward in narrowing down the plausible mechanisms by which these permissive glycans promote and regulate outer segment assembly. Specifically, several aspects of this phenomenon point to the involvement of a receptor. First, the ability to induce a physiologic effect (i.e., support of outer segment assembly) is very specific; only glycans structurally related to lactose and galactose elicit this response. Second, both metabolizable (e.g., lactose) and non-metabolizable (e.g., IPTG) permissive glycans stimulate outer segment assembly in a stepwise dose-dependent fashion with maximum permissive effects at 5x10-3 and 5x10-5 M, respectively. Third, the effect is reversible; removal of the stimulus eliminates the effect. Last, the concentration at which IPTG is most effective (10-5 M) is on the high end of the typical range that is documented for receptor-ligand interactions , which might suggest a low affinity interaction between a putative receptor and its ligand, IPTG.
In conclusion, our results demonstrate that permissive glycans exert their organizational effect via a non-metabolic mechanism at very low concentrations, that this effect is reversible, and possibly mediated by Müller cells. Collectively, these results are consistent with the exciting possibility that a receptor for these glycans may exist in the retina, and that its stimulation results in support of outer segment assembly. Studies are currently underway to isolate and identify this putative receptor using structurally-related multivalent ligands.
Supported by NEI grant EY10853 (MMJ), Fight for Sight Grant-in Aid number GA02046 (MMJ), KTEF grant (XFW), NEI core grant EY013080 to the University of Tennessee Health Science Center at Memphis, and an unrestricted grant to the Department of Ophthalmology from Research to Prevent Blindness, Inc., New York. MMJ is the recipient of a Research to Prevent Blindness William and Mary Greve Special Scholar Award. AI is the recipient of a Research to Prevent Blindness Career Development Award. The authors gratefully acknowledge the technical assistance Sharon Frase of The Integrated Microscopy Center at the University of Memphis and Amira Wohabrebbi of the University of Tennessee Health Science Center at Memphis.
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