Wang X et al. (2010), Cellular retinol binding protein 1 modulates ph...

XB-ART-44277

Dev Neurobiol 2010 Aug 01;709:623-35. doi: 10.1002/dneu.20798.

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Cellular retinol binding protein 1 modulates photoreceptor outer segment folding in the isolated eye.

Wang X , Tong Y , Giorgianni F , Beranova-Giorgianni S , Penn JS , Jablonski MM .

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In a previous study, we used differential proteomics to identify retinal proteins whose steady-state levels were altered in an experimental system in which photoreceptor outer segments were improperly folded. We determined that the steady-state level of cellular retinol binding protein 1 (CRBP1) was downregulated in eyes lacking organized outer segments. The purpose of this study was to determine if CRBP1 is a plausible candidate for regulating outer segment assembly. We used Morpholinos to directly test the hypothesis that a decreased level of CRBP1 protein was associated with the misfolding of outer segments. Results from these studies indicate that downregulation of CRBP1 protein resulted in aberrant assembly of outer segments. Because CRBP1 plays a dual role in the retina-retinal recycling and generation of retinoic acid-we evaluated both possibilities. Our data demonstrate that outer segment folding was not modified by 11-cis retinal supplementation, suggesting that CRBP1 influences outer segment assembly through a mechanism unrelated to rhodopsin regeneration. In contrast, retinoic acid is required for the proper organization of nascent outer segment membranes. The localization of CRBP1 within Muller cells and the RPE and its demonstrated role in modulating the proper folding of nascent outer segment membranes through retinoic acid further elucidates the role of these cells in directly influencing photoreceptor physiology.

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Species referenced: Xenopus laevis
Genes referenced: actl6a glul mag myh3 rbp1 rho rpe
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	Figure 1 Representative analysis of spot 22 from our previous investigation (Wang et al., 2009). A: The Cy3 (RPE-supported; top) and Cy5 (RPE-deprived; bottom) images of the gel area surrounding the spot are indicated by black circles. The spot is located on the gel at a relative molecular weight of 16 kD and pI of 5.4. B: The relative abundance levels for each biological replicate are shown graphically. For spot 22, the levels of protein are consistently higher in samples derived from RPE-supported retinas. This difference is significant at p ¼ 0.039. C: Semi-quantitative Western blot analysis performed on another biological replicate confirms the findings of the 2D DIGE analysis. Steady-state CRBP1 levels were normalized to actin levels to account for possible variations in the amount of protein loaded in each lane.
	Figure 2 A: Base peak chromatogram for the LC-MS/MS analysis of spot 22. (B) through (D) MS/MS spectra for the three peptides ions from the LC-MS/MS analysis that matched to Xenopus laevis hypothetical protein MGC81232. The peptide ions (doubly charged, [M+2H]2+, with precursor ion masses 567.25, 705.81, and 630.29) were matched to sequences NDQLVC#EQK (Xcorr 2.98; C# denotes carbamidomethyl-cysteine), EFEEDLSGVDDR (Xcorr 3.92), and NYIM* EFDVGR (Xcorr 3.02; M* denotes oxidized methionine), respectively.
	Figure 3 A: Protein sequence of Xenopus laevis hypothetical protein MGC81232. The peptides from our MS/MS analysis are shown in gray. A total of three peptides were matched for 24% sequence coverage throughout the length of the protein. B: BLAST analysis of the protein sequence of Xenopus laevis hypothetical protein MGC81232 revealed that the Xenopus protein was 77.6% identical to mouse CRBP1. Identical matches between the two sequences are marked with an asterisk.
	Figure 4 A–D: Localization of CRBP1 protein in the RPE and Mu¨ ller cells using immunohistochemistry. Immunopositive labeling is shown in RPE-supported retinas in A and B and in RPEdeprived retinas in C and D. Red ¼ CRBP1; green ¼ glutamine synthetase; blue ¼ nuclei; and yellow ¼ co-localization of CRBP1 and glutamine synthetase in Mu¨ller cells. Labeling of CRBP1 protein is shown individually in A and C and overlapped with nuclei in B and D. (E–G) Localization of rbp1 mRNA in the RPE and Mu¨ ller cells using in situ hybridization. Green ¼ rbp1 mRNA; and red ¼ nuclei. Labeling of rbp1 mRNA is shown individually in E and overlapped with nuclei in F. G is a sense control. RPE, retinal pigment epithelium; OS, outer segments; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Mag bar ¼ 10 lm. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]
	Figure 5 A: Graphic representation of outer segment organization (gray bars) and normalized CRBP1 protein levels (black bars) in retinas exposed to Morpholinos. Exposure of retinas to endoporter, which facilitates entry of the Morpholino into the cytoplasmic compartment, is included as a negative control. By one-way ANOVA, the overall Ftest for differences among the groups was highly significant (F ¼ 23.02; p value <0.0001 for outer segment organization; F ¼ 82.76; p value <0.001 for CRBP1 protein levels). * ¼ p value <0.05 vs. RPE-supported retinas; ^ ¼ p value <0.05 vs. RPE-deprived retinas. B: Representative image of RPE-supported eye exposed to control Morpholino. C: Representative image of RPE-supported eye exposed to CRBP1 Morpholino. RPE, retinal pigment epithelium; OS, outer segments; IS, inner segments; ONL, outer nuclear layer. Mag bar ¼ 10 lm.
	Figure 6 A: Graphic representation of the degree of organization of the outer segments of retinas exposed to 11- cis retinal. By one-way ANOVA, the overall F-test for differences among the groups was highly significant (F ¼ 21.38; p value <0.0001 for outer segment organization). * ¼ p value <0.05 vs. RPE-supported retinas; ^ ¼ p value <0.05 vs. RPE-deprived retinas. B: Representative image from a retina exposed to 11-cis retinal. C: Representative image from a retina exposed to Morpholino and 11-cis retinal. D: Representative image from a retina exposed to alltrans retinal. OS, outer segments; IS, inner segments; ONL, outer nuclear layer. Mag bar ¼ 10 lm.
	Figure 7 Localization of retinoic acid in photoreceptors, the outer plexiform layer and Mu¨ller cells using immunohistochemistry. Immunopositive labeling is shown in RPE-supported retinas in A and B and in RPE-deprived retinas in C and D. Green ¼ retinoic acid; and blue ¼ ToPro III iodide labeling of nuclei. Labeling of retinoic acid is shown individually in right-hand panels and overlapped with nuclear stain in left-hand panels. RPE, retinal pigment epithelium; IS, inner segments; OPL, outer plexiform layer; INL, inner nuclear layer; GCL, ganglion cell layer. Mag bar ¼ 10 lm. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]
	Figure 8 A: Graphic representation of the degree of organization of the outer segments of retinas exposed to retinoic acid. By one-way ANOVA, the overall F-test for differences among the groups was highly significant (F ¼ 21.52; p value <0.0001). * ¼ p value <0.05 vs. RPE-supported retinas; ^ ¼ p value <0.05 vs. RPE-deprived retinas. B: Representative image from an RPE-deprived retina exposed to 0.5 lM retinoic acid. C: Representative image from an RPE-supported retina exposed to Morpholino and retinoic acid. D: Representative image from an RPE-supported retina exposed to citral. E: Representative image from an RPE-supported retina exposed to citral and 0.5 lM retinoic acid. OS, outer segments; IS, inner segments; ONL, outer nuclear layer. Mag bar ¼ 10 lm.

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