Ординатура / Офтальмология / Английские материалы / Retinal Degeneration Disease_Hollyfield, Anderson, LaVail_1999

Figure 15.3. Electron micrographs show relative normal RPE and outer segments in the 6.5-month-old r15 heterozygous control (A and C), and disorganized photoreceptor outer segments with membranous whorls (asterisks in B) and vacuoles in the RPE cells in the homozygous littermate (B and D). Scale bars: 2 mm in (A) and (B); 1 mm in (C) and (D).

Figure 15.4. Histology analysis of 1-year-old heterozygous (A and C) and homozygous (B, D, and E) r18 littermates. RPE hypertrophy and loss of outer nuclear layers are seen in homozygous r18 retina compared to the heterozygous retina. Scale bars: 50 mm in (A) and (B); 20 mm in (C) and (D); 10 mm in (E).

Figure 15.5. Electron micrographs show the apical sides of RPE cells of 2-month-old r18 heterozygous and homozygous littermates. Scale bars: 1 mm.

EM study of the r18 mutant retina reveals that substantial amount of microvilli from the apical side of RPE cells accumulate in the subretinal space in the homozygous mutation but not in the heterozygous control (Fig. 15.5).

Thus, in comparison to homozygous r15 mutation, r18 homozygous mutation represents a different phenotype in the RPE cells with a much slower degeneration of photoreceptor cells. We hypothesize that both r15 and r18 gene mutations either directly or indirectly modulate the apical microvilli to cause the degeneration of photoreceptor cells. We believe that further investigation of both mutations will lead to new knowledge for genes that play important roles in the functions of RPE cells.

3. ACKNOWLEDGEMENTS

We thank the National Eye Institute for the travel award provided to CX to attend this meeting. XG is supported by NIH grants RO1 EY12808 and RO1 EY13849.

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1995). Interestingly, this constant background activity has been proposed to lead to photoreceptor degeneration (Fain and Lisman, 1993). However, rod photoreceptors also possess a protective mechanism to prevent the activity of bleached, unliganded opsin, which is opsin phosphorylation and arrestin binding. We have shown that in the Rpe65-/- mouse retina, opsin is constitutively phosphorylated at an elevated level of ~25%. This level of phosphorylation is approximately half of the maximal light-inducible (transient) amount of phosphorylation in the wild type retina (Ablonczy et al., 2001). In addition, this constitutively phosphorylated opsin was found to bind arrestin at similar levels as bleached wild type opsin (Crouch et al., 2003). Accordingly, it was not surprising that arrestin distribution in the Rpe65-/- retina has been shown to mimic that of a light-adapted wild type retina (Mendez et al., 2003). We therefore argue that the kinetics of rod degeneration is controlled by both constitutive opsin activity and constitutive opsin phosphorylation.

2.2. Cone Responses in Rpe65-/- Mice

Rod photoreceptors appear to be more susceptible to degeneration either during aging (e.g., Gresh et al., 2003; Curcio, 2001) or as a consequence of insults (light damage; Cicerone, 1976). Thus, we chose to revisit the question of cone function and survival in the Rpe65-/- mouse. Classically, cone responses are recorded in single flash experiments in the presence of a rod-adapting background, or in flicker ERGs. However, due to the reduced light sensitivity in the Rpe65-/- rods, it is impossible to bleach them sufficiently to be certain that they no longer contribute to the elicited light response. To be able to record unequivocally from Rpe65-/- cones, we obtained the Rpe65-/-::Rho-/- cross, in which rod responses are eliminated genetically (Seeliger et al., 2001). Elimination of rhodopsin causes a delayed rod degeneration (onset ~P60), thus all experiments were performed in animals <1 month-of- age. Interestingly, if photopic ERGs were recorded using normal averaging conditions (averaging 3-5 traces) using a maximal white flash of ~2.2 ¥ 1013 photons/mm2, no ERGs could be recorded; however, if averaging was increased to 50 traces, a small but reliable cone ERG response (13.7 ± 1.87 mV; n = 9) could be recorded from these mice.

If constitutive cone opsin activity were to contribute to the reduced sensitivity of the cones, one would predict that cone degeneration would likewise occur. However, as the amplification in the signal transduction cascade is lower in cones then in rods, one might predict cone degeneration to be slower than rod degeneration. However, the small cone ERG response was virtually eliminated by 4 months-of-age (unpublished results; BR 2004), suggesting that a different mechanism is responsible for the demise of the cones. Interpretation of these data is complicated by the finding that RPE65 is present in all mouse cones (Znoiko et al., 2002). The role of this protein in cones is unknown.

2.3. Rod and Cone Opsin Distribution in the Rpe65-/- Mouse Retina

Recently, Chapple and coworkers (2001) have speculated that the chromophore 11-cis retinal might act as a pharmacological chaperone that facilitates opsin folding into an appropriate conformational state that allows for its proper transport through the trans-Golgi network and integration into the photoreceptor outer membrane. Likewise, it would allow for appropriate posttranslational modifications. Wild type rhodopsin is a relatively stable protein that appears to be transported appropriately in the absence of 11-cis retinal (Figure 16.2A). However, the P23H mutation, which does not fold properly, can only be targeted

Figure 16.2. UV-cone and rod opsin distribution in C57BL/6 (wt) and Rpe65-/- mice. (Top row) UV cone opsin localization at P25 (using a polyclonal antibody against mouse UV cone opsin, generously provided by J. Chen, University of Southern California, Los Angels, CA, USA). (A) In the wt retina, cone opsin was localized predominantly to the outer segment (OS). (B) UV cone apoprotein was found to be distributed throughout the entire cone in Rpe65-/-::Rho-/- mice. [Green cone opsin (localized with a polyclonal antibody against human MWL cone opsin, generously provided by J. Nathans, Johns Hopkins University School of Medicine, Baltimore, MD, USA) showed a similar distribution profile, but is not depicted here.] (Bottom row) Rod opsin localization at P30 (using monocolonal antibody against bovine rhodopsin, generously provided by R. Molday; University of British Columbia, Vancouver, BC, Canada). Rhodopsin (C) and apoprotein (D) localization was indistinguishable between the wt and Rpe65-/- mouse retina, respectively. Scalebar: 15 mm.

appropriately in the presence of ligand (either 9-cis or 11-cis retinal; Noorwez et al., 2003). The accumulation of misfolded P23H rhodopsin apparently leads to photoreceptor degeneration (e.g., Olsson et al., 1992). Cone opsins appear to be more thermally unstable (Matsumoto et al., 1975), suggesting that they may be more dependent on a pharmacological chaperone for stabilization. We therefore tested the hypothesis that the cone opsin apoproteins are mislocalized in the cones of the Rpe65-/-::Rho-/- mice. Cone opsin in the absence of 11-cis retinal was found to be localized throughout the cones. Here we show the distribution of UV cone opsin (Figure 16.2B), a distribution profile that is recapitulated by green cone opsin (data not shown). Whether cone opsin is misfolded in the Rpe65-/-::Rho-/- retinas will be difficult to determine. One of the presumed results of misfolding is the lack of appropriate glycosylation. However, these retinas contain such small amounts of cone opsin that Western blotting to determine a difference in molecular weight will be a challenge.

2.4. Isolation of the Chromophore for Rhodopsin

Rpe65-/- mouse photoreceptors have been found to exhibit minimal levels of light sensitivity. However, even after careful pooling of retinas isolated from Rpe65-/- mice, no pigment could be measured. We recently found, however, that prolonged dark-adaptation resulted in slow accumulation of chromophore (~0.4 pmol/day), demonstrating that the process is very inefficient (Fan et al., 2003). Young adult Rpe65-/- mice were transferred into complete darkness, and retinas were collected at weekly intervals. The two retinas from each animal were pooled for pigment measurements in 1% dodecylmaltoside, determining the

Figure 16.3. Isolation of isorhodopsin from Rpe65-/- mouse retinas. Absorption spectra of endogenous pigments isolated after 2 months of dark adaptation from a wild type (black trace) and a Rpe65-/- mouse (grey trace). Nomograms were fitted to the difference spectra to determine a peak of the absorbance. The wild type spectrum could be fitted with a nomogram with a peak absorbance of 500 nm, which corresponds to rhodopsin, whereas the pigment isolated from the Rpe65-/- mouse had a blue-shifted spectrum, indicative of isorhodopsin, which is formed in the presence of 9-cis retinal. (Adapted from Fan et al., 2003; © PNAS.)

difference spectra from measurements before and after bleaching. Interestingly, the pigment isolated from the Rpe65-/- mouse retinas was found to be isorhodopsin, which is the rod pigment regenerated with 9-cis rather then 11-cis retinal (Figure 16.3). The presence of 9- cis retinal in these retinas and the RPE was confirmed by higher performance liquid chromatography (Fan et al., 2003). It is unclear at this point as to how 9-cis retinal is formed; the only definitive statements that can be made are that it is independent of light (see Figure 16.3), the light-dependent, RGR-mediated mechanism (unpublished results; J. Fan, 2003) and RPE65 (Fan et al., 2003). In addition, it is unclear whether 9-cis retinal is also the chromophore for the cones, as the amount of cone pigment generated in these retinas is too minute to be measured. Single cell spectrophotometry or electrophysiology may shed some light on this question. However, our experiments do provide indirect evidence that the proposed Müller-cell-mediated mechanisms for chromophore regeneration in cone-dominant retinas, which is independent of RPE65 (Mata et al., 2003), does not exist in the roddominant mouse retina.

3. DISCUSSION

Here we have discussed the differences in behavior between rods and cones in the absence of RPE65 and thus endogenous chromophore. We have presented evidence that both rod and cone responses can be recorded in the young Rpe65-/- mice. Rods degenerate very slowly and those kinetics are controlled by the constitutive activity of the free opsin and opsin phosphorylation. In the absence of chromophore, the apoprotein appears to be folding correctly and thus proper targeting to the rod outer segments occurs. In contrast, cones degenerate very quickly in the absence of chromophore. Cone opsin is not targeted properly to the outer segments, but is distributed throughout the entire cell membrane. We are suggesting that folding and thus targeting of the cone opsin to the cone outer segment requires a chromophore as a pharmacological chaperone.

In addition, we have demonstrated that the chromophore available for the formation of pigment in the Rpe65-/- mouse retina is 9-cis retinal. For the sake of argument, let’s assume that the cones in the Rpe65-/- mouse retina use the same chromophore. Based on the amount of chromophore generated (~0.4 pmol/day; Fan et al., 2003), the Rpe65-/- mouse retina con-

tains less then 0.2% of normal levels of chromophore. As rods outnumber cones by a factor of ~100 : 1 (Jeon et al., 1998), cone opsin has a lower affinity for chromophore than rhodopsin (Matsumoto et al., 1975) and the cone outer segments are further away from the RPE (the presumed source of the chromophore); it is, therefore, difficult to understand how any cone pigment is formed in the Rpe65-/- mouse retina. We suggest that chromophore delivery in the rods is dependent upon diffusion of IRBP-bound chromophore to the outer segments, whereas chromophore delivery to the cones is aided by the presence of the cone sheath, an extension of the RPE that ensheathes the entire cone outer segment (Fisher and Steinberg, 1982). These observed differences between rods and cones need to be addressed when considering treatment strategies for LCA patients.

4. ACKNOWLEDGEMENTS

Funding was provided by NIH grants EY-13520, EY-04939, EY-14793; Foundation Fighting Blindness; and an unrestricted grant to MUSC from Research to Prevent Blindness, Inc., New York, NY. RKC is a RPB Senior Scientific Investigator. The authors thank Jie Fan, Jian-xing Ma, Sergey Znoiko and Patrice Goletz for contributing experiments for this review; Mathias Seeliger, Michael Redmond and Peter Humphries for contributing mice, Jeannie Chen, Jeremy Nathans and Robert Molday for providing antibodies and Luanna Bartholomew for editorial assistance.

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