The severe autosomal dominant retinitis pigmentosa rhodopsin mutant Ter349Glu mislocalizes and induces rapid rod cell death

Abstract

Mutations in the rhodopsin gene cause approximately one-tenth of retinitis pigmentosa cases worldwide, and most result in endoplasmic reticulum retention and apoptosis. Other rhodopsin mutations cause receptor mislocalization, diminished/constitutive activity, or faulty protein-protein interactions. The purpose of this study was to test for mechanisms by which the autosomal dominant rhodopsin mutation Ter349Glu causes an early, rapid retinal degeneration in patients. The mutation adds an additional 51 amino acids to the C terminus of the protein. Folding and ligand interaction of Ter349Glu rhodopsin were tested by ultraviolet-visible (UV-visible) spectrophotometry. The ability of the mutant to initiate phototransduction was tested using a radioactive filter binding assay. Photoreceptor localization was assessed both in vitro and in vivo utilizing fluorescent immunochemistry on transfected cells, transgenic Xenopus laevis, and knock-in mice. Photoreceptor ultrastructure was observed by transmission electron microscopy. Spectrally, Ter349Glu rhodopsin behaves similarly to wild-type rhodopsin, absorbing maximally at 500 nm. The mutant protein also displays in vitro G protein activation similar to that of WT. In cultured cells, mislocalization was observed at high expression levels whereas ciliary localization occurred at low expression levels. Similarly, transgenic X. laevis expressing Ter349Glu rhodopsin exhibited partial mislocalization. Analysis of the Ter349Glu rhodopsin knock-in mouse showed a rapid, early onset degeneration in homozygotes with a loss of proper rod outer segment development and improper disc formation. Together, the data show that both mislocalization and rod outer segment morphogenesis are likely associated with the human phenotype.

Keywords: Cilia; G Protein-coupled Receptors (GPCRs); Neurodegeneration; Neurodegenerative Diseases; Photoreceptors; Phototransduction; Retinal Degeneration; Rhodopsin; Ter349Glu; Vision.

FIGURE 1.

Analyses of proper folding and GPCR functional activity of Ter349Glu rhodopsin. A, schematic diagram of the Ter349Glu rhodopsin mutant (left). The sequence of the additional 51 amino acids is shown in red (right). B,Ter349Glu-transfected COS cells harvested, reconstituted with 11-cis-retinal, and solubilized. Absorbance spectra were taken before and after exposure to saturating white light, and a difference spectrum was charted (solid line). WT difference spectrum (dashed line) was taken from purified rhodopsin from bovine ROS. Spectra are as indicated and normalized to 0.1. C, ability of WT (squares) and Ter349Glu (triangles) rhodopsin in COS cell membranes to activate transducin. Activity was assayed as detailed under “Experimental Procedures.” Dark, filled symbols; light, outlined symbols; light exposure, hν. Error bars are S.D., n = 3. Data were analyzed using a two-tailed t test. p value = 0.229. D, immunocytochemically labeled COS cells expressing WT, Ter349Glu, P23H, or Gln344Ter rhodopsins. Arrows show areas of plasma membrane localization; asterisks (*) show areas of perinuclear localization. Scale bar, 10 μm.

FIGURE 2.

Localization of Ter349Glu opsin in high and low expressing IMCD cells. IMCD cells were transfected with rhodopsin constructs in high or low expression vectors and immunocytochemically labeled. A, all rhodopsins in high expression transfections were labeled using B6-30N antibody and a goat anti-mouse IgG₁ secondary antibody conjugated to Alexa Fluor 488 (green). Cilia were labeled using anti-Arl13b antibody followed by an anti-mouse IgG_2a secondary conjugated to Alexa Fluor 568 (red, arrows). Tight junctions were labeled using ZO-1 primary antibody followed by a goat anti-rat IgG secondary conjugated to Alexa Fluor 647 (purple). Rhodopsins are as indicated. B, quantitation of apical (superior to ZO-1) and basolateral (inferior to ZO-1) localization was measured using ImageJ. Data were analyzed using a two-tailed t test and are expressed as the mean ± S.D. *, p < 0.01; **, p < 0.001. C, rhodopsins in low expression transfections were labeled using K62-82 antibody and a goat anti-mouse IgG₃ secondary conjugated to Alexa Fluor 488 (green). Cilia were labeled similarly to high expression IMCDs. Basal body was labeled using anti-γ-tubulin antibody and goat anti-mouse IgG₁ secondary conjugated to Alexa Fluor 647 (purple). All nuclei were labeled with DAPI (blue). Rhodopsins are as indicated. Scale bars, 10 μm.

FIGURE 3.

Localization of Ter349Glu rhodopsin in transgenic X. laevis. Using a modified Amaya and Kroll method of transgenesis, WT and Ter349Glu rhodopsin-expressing tadpoles were generated and euthanized at 2 weeks of age, processed and cryosectioned, and labeled using immunohistochemistry. WT rhodopsin was labeled using B6-30N primary antibody. Ter349Glu rhodopsin was labeled using mammalian rhodopsin-specific primary antibody A5-3-12. Both were labeled with anti-mouse IgG secondary antibody conjugated to Alexa Fluor 488 (green). Nuclei were labeled with DAPI (blue). OPL, outer plexiform layer. Arrows, normally developed outer segments; arrowheads (>), inner segment mislocalization. Asterisks, synaptic mislocalization. Scale bar, 20 μm.

FIGURE 4.

Examining the Ter349Glu rhodopsin mouse retina for rhodopsin localization, apoptosis, and degeneration. A and B, eyes from WT (+/+), Ter349Glu heterozygote (Ter349Glu/+), and Ter349Glu homozygote (Ter349Glu/Ter349Glu) mice were fixed, cryosectioned, and processed by immunohistochemistry. In +/+ and Ter349Glu/Ter349Glu mice, rhodopsin was labeled using B6-30N primary antibody; Ter349Glu/+ was labeled with 1D4 (WT) and K62-82 (Ter349Glu). WT rhodopsin is shown in red; Ter349Glu rhodopsin is shown in green. Nuclei labeled with DAPI are shown in blue. ONL counts from three animals are expressed as the mean ± S.D. Scale bar, 20 μm. C and D, apoptotic nuclei labeled using ApopTag Kit. Fluorescently tagged nuclei (purple) were counted from 5–8 sections per genotype per slide. Data were analyzed using a two-tailed t test and are expressed as the mean ± S.D. (error bars). **, p < 0.001. Scale bar, 20 μm.

FIGURE 5.

Assessing the Ter349Glu rhodopsin mouse retina for photoreceptor ultrastructural morphology. Eyes from WT (+/+), Ter349Glu heterozygote (Ter349Glu/+), Ter349Glu homozygote (Ter349Glu/Ter349Glu), and heterozygote rhodopsin null (+/−) mice were fixed. Ultra-thin sections were taken and processed for transmission electron microscopy. Arrowheads indicate extracellular vesicles from rod photoreceptors. Diamonds denote basal bodies. Arrow shows ROS containing abnormally oriented discs. Stars represent membranous whirls, indicative of malformed disc membranes. Scale bars: A, D, and F, 2 μm; B, C, E, G, and H, 0.5 μm.