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Comparative Study
. 2006 Jan 4;26(1):203-9.
doi: 10.1523/JNEUROSCI.3849-05.2006.

Mislocalized rhodopsin does not require activation to cause retinal degeneration and neurite outgrowth in Xenopus laevis

Beatrice M Tam  1 , Guifu Xie, Daniel D Oprian, Orson L Moritz
Affiliations
Comparative Study

Mislocalized rhodopsin does not require activation to cause retinal degeneration and neurite outgrowth in Xenopus laevis

Beatrice M Tam et al. J Neurosci. .

Abstract

Mutations in the C terminus of rhodopsin disrupt a rod outer segment localization signal, causing rhodopsin mislocalization and aggressive forms of retinitis pigmentosa (RP). Studies of cultured photoreceptors suggest that activated mislocalized rhodopsin can cause cell death via inappropriate G-protein-coupled signaling. To determine whether this pathway occurs in vivo, we developed a transgenic Xenopus laevis model of RP based on the class I rhodopsin mutation Q344Ter (Q350Ter in X. laevis). We used a second mutation, K296R, to block the ability of rhodopsin to bind chromophore and activate transducin. We compared the effects of expression of both mutants on X. laevis retinas alone and in combination. K296R did not significantly alter the cellular distribution of rhodopsin and did not induce retinal degeneration. Q350Ter caused rhodopsin mislocalization and induced an RP-like degeneration, including loss of rods and development of sprouts or neurites in some remaining rods, but did not affect the distribution of endogenous rhodopsin. The double mutant K296R/Q350Ter caused a similar degeneration and neurite outgrowth. In addition, we found no protective effects of dark rearing in these animals. Our results demonstrate that the degenerative effects of mislocalized rhodopsin are not mediated by the activated form of rhodopsin and therefore do not proceed via conventional G-protein-coupled signaling.

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Figures

Figure 1.
Figure 1.
Effects of M13F on antibody cross-reactivity. Western blots of extracts of COS cells transfected with plasmids designed to express GFP or rhodopsin N1-38–GFP fusion proteins or mock-transfected cells (control) are shown. Blots were probed with antibodies B630N, 514-18, or anti-GFP. Mr, Relative mobility.
Figure 2.
Figure 2.
Characterization of the effects of rhodopsin mutations on chromophore formation and transducin activation. A, Absorption spectra for mutant rhodopsins purified from COS cell detergent extracts. Spectra are for the mutants eRho (1) and eRhoK296R (2). B, Effect of rhodopsin mutations on ability to activate transducin. Filled symbols, Dark; open symbols, after exposure to light (arrow). Circles, eRho; triangles, eRhoQ350Ter; squares, eRhoK296R. ERhoK296R membranes that were not treated with retinal were also assayed for ability to activate transducin but showed no activity (data not shown).
Figure 3.
Figure 3.
Dot blot analysis of the effects of rhodopsin expression on total rhodopsin content of transgenic eyes. Dot blots of total eye extract from 22 transgenic animals of each type probed with B630N (total rhodopsin) (A) and 514-18 (eRho rhodopsin) (B) are shown. The positions of four control samples of wt and eRho rhodopsin are indicated on each blot. The ratio of 514-18 signal to B630N signal was used to calculate the percentage of transgenic rhodopsin in each eye. C, Plots of expression level versus total rhodopsin. The log scale data for eRhoQ350Ter and eRhoK296R/Q350Ter were fit to dose–response curves, and eRho and eRhoK296R data were fit to straight lines.
Figure 4.
Figure 4.
Effect of dark rearing on total rhodopsin content of eRhoQ350Ter transgenic eyes. Dot blots of total eye extract from 33 animals of each type were probed with B630N (total rhodopsin) and 514-18 (transgenic rhodopsin) as in Figure 3, and plots of expression level versus total rhodopsin were similarly derived from the dot blots and fit to dose–response curves. Note that the greatest difference in rhodopsin content occurs at the lowest expression levels.
Figure 5.
Figure 5.
Effects of transgene expression on rod and cone photoreceptor density. A–D, Confocal micrographs of retinas expressing high levels of eRho (A), eRhoK296R (B), eRhoQ350Ter (C), and eRhoK296R/Q350Ter (D) labeled with 2B2 anti-eRho rhodopsin (green), wheat germ agglutinin (red), and Hoechst 33342 nuclear stain (blue). Note the gaps in the photoreceptor layer in C and D. E, Section of retina expressing eRho, with bright-field image superimposed on 2B2 label. F, Similar section expressing eRhoQ350Ter. Note the absence of rods relative to E. Cones are present in the spaces between rods (arrowheads). Mislocalized rhodopsin is present in the rod synapses in C, D, and F (compare regions marked by white arrows to similar regions marked in A, B, and E). Scale bars: (in A) A–D, 100 μm; (in E) E, F, 10 μm. ONL, Outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer.
Figure 6.
Figure 6.
Localization of transgene products in rod photoreceptors. Retinas were labeled with 2B2 anti-eRho rhodopsin (green), wheat germ agglutinin (red), and Hoechst 33342 nuclear stain (blue) Rods are expressing eRho (A), eRhoK296R (B), eRhoQ350Ter (C), or eRhoK296R/Q350Ter (D). Note the strong 2B2 signal from IS plasma membranes in C and D relative to A and B (arrowheads). The asymmetric labeling of OS is an artifact caused by poor penetration of the densely packed disk membranes in fixed tissue. Scale bar, 10 μm. N, Nucleus; S, synapse.
Figure 7.
Figure 7.
Expression of mislocalized rhodopsin mutants does not cause mislocalization of endogenous rhodopsin and induces formation of sprouts. Retinas were labeled with 2B2 anti-eRho rhodopsin (green) wheat germ agglutinin (red), and Hoechst 33342 nuclear stain (blue). A, B, Retina expressing eRhoK296R/Q350Ter with very nonuniform expression labeled with 2B2 (A) and 11D5 (B). The 11D5 IS label is uniform and relatively faint, with very little label in the synaptic region (compare white arrows in A and B). C, 2B2-labeled section from the same retina shown in B, with horizontal and vertical processes indicated by arrowheads. D, E, ERhoQ350Ter (D) and eRhoK296R/Q350Ter (E) retinas labeled with 2B2 showing several processes (arrowheads). C, D, and E are two-dimensional projections constructed from Z-series of ∼24 confocal images taken at 0.5 μm intervals. Gamma was adjusted in all three channels of C, D, and E to maximize image detail. Scale bars: A, B, 50μm; C–E, 10 μm. RPE, Retinal pigment epithelium; ONL, outer nuclear layer; OPL, outerplexiformlayer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer.
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References

    1. Adamus G, Zam ZS, Arendt A, Palczewski K, McDowell JH, Hargrave PA (1991) Anti-rhodopsin monoclonal antibodies of defined specificity: characterization and application. Vision Res 31: 17–31. - PubMed
    1. Alfinito PD, Townes-Anderson E (2002) Activation of mislocalized opsin kills rod cells: a novel mechanism for rod cell death in retinal disease. Proc Natl Acad Sci USA 99: 5655–5660. - PMC - PubMed
    1. Batni S, Scalzetti L, Moody SA, Knox BE (1996) Characterization of the Xenopus rhodopsin gene. J Biol Chem 271: 3179–3186. - PubMed
    1. Berson EL, Rosner B, Weigel-DiFranco C, Dryja TP, Sandberg MA (2002) Disease progression in patients with dominant retinitis pigmentosa and rhodopsin mutations. Invest Ophthalmol Vis Sci 43: 3027–3036. - PubMed
    1. Besharse JC, Hollyfield JG, Rayborn ME (1977) Turnover of rod photoreceptor outer segments. II. Membrane addition and loss in relationship to light. J Cell Biol 75: 507–527. - PMC - PubMed

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