Phototoxicity avoidance is a potential therapeutic approach for retinal dystrophy caused by EYS dysfunction
Yuki Otsuka, Keiko Imamura, Akio Oishi, Kazuhide Asakawa, Takayuki Kondo, Risako Nakai, Mika Suga, Ikuyo Inoue, Yukako Sagara, Kayoko Tsukita, Kaori Teranaka, Yu Nishimura, Akira Watanabe, Kazuhiro Umeyama, Nanako Okushima, Kohnosuke Mitani, Hiroshi Nagashima, Koichi Kawakami, Keiko Muguruma, Akitaka Tsujikawa, Haruhisa Inoue
Yuki Otsuka, Keiko Imamura, Akio Oishi, Kazuhide Asakawa, Takayuki Kondo, Risako Nakai, Mika Suga, Ikuyo Inoue, Yukako Sagara, Kayoko Tsukita, Kaori Teranaka, Yu Nishimura, Akira Watanabe, Kazuhiro Umeyama, Nanako Okushima, Kohnosuke Mitani, Hiroshi Nagashima, Koichi Kawakami, Keiko Muguruma, Akitaka Tsujikawa, Haruhisa Inoue
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Research Article Ophthalmology Stem cells

Phototoxicity avoidance is a potential therapeutic approach for retinal dystrophy caused by EYS dysfunction

Abstract

Inherited retinal dystrophies (IRDs) are progressive diseases leading to vision loss. Mutation in the eyes shut homolog (EYS) gene is one of the most frequent causes of IRD. However, the mechanism of photoreceptor cell degeneration by mutant EYS has not been fully elucidated. Here, we generated retinal organoids from induced pluripotent stem cells (iPSCs) derived from patients with EYS-associated retinal dystrophy (EYS-RD). In photoreceptor cells of RD organoids, both EYS and G protein–coupled receptor kinase 7 (GRK7), one of the proteins handling phototoxicity, were not in the outer segment, where they are physiologically present. Furthermore, photoreceptor cells in RD organoids were vulnerable to light stimuli, and especially to blue light. Mislocalization of GRK7, which was also observed in eys-knockout zebrafish, was reversed by delivering control EYS into photoreceptor cells of RD organoids. These findings suggest that avoiding phototoxicity would be a potential therapeutic approach for EYS-RD.

Authors

Yuki Otsuka, Keiko Imamura, Akio Oishi, Kazuhide Asakawa, Takayuki Kondo, Risako Nakai, Mika Suga, Ikuyo Inoue, Yukako Sagara, Kayoko Tsukita, Kaori Teranaka, Yu Nishimura, Akira Watanabe, Kazuhiro Umeyama, Nanako Okushima, Kohnosuke Mitani, Hiroshi Nagashima, Koichi Kawakami, Keiko Muguruma, Akitaka Tsujikawa, Haruhisa Inoue

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Figure 5

eys-KO zebrafish exhibited GRK7 mislocalization in outer segment and light-induced photoreceptor cell death.

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eys-KO zebrafish exhibited GRK7 mislocalization in outer segment and li...
(A) Generation of eys-KO zebrafish with CRISPR/Cas9 technology. The zebrafish eys genomic structure is shown. The protospacer-adjacent motifs (PAMs) and crRNA targeting sequences in exon 2 are underlined. Sanger sequencing identified a 2–base pair insertion in eys-KO zebrafish. (B) Representative Eys immunostaining images of WT and eys-KO zebrafish. No punctate staining in photoreceptor cells was observed in eys-KO zebrafish. CC-C, cone connecting cilium; CC-R, rod connecting cilium; ONL-C, cone outer nuclear layer; ONL-R, rod outer nuclear layer; OS-DC, double cone outer segment; OS-LSC, long single cone outer segment; OS-R, rod outer segment. Scale bars: 20 μm. (C) Images of WT and eys-KO zebrafish immunostained for Grk7 and OS marker Gnat2. Lower panels are higher-magnification images of the dotted boxes in the upper panels. White arrowheads indicate the CC, and white arrows indicate the OS. OS-C, cone outer segment. Scale bars: 10 μm. (D) Three-month-postfertilization WT and eys-KO (heterozygous and homozygous) zebrafish were exposed to white LED light. (E) Retinal sections of WT and eys-KO (heterozygous and homozygous) zebrafish after light stimulation (Bright) or dark adaptation (Dark) were stained with hematoxylin and eosin. Scale bars: 50 μm. (F) Representative immunohistochemistry of light-stimulated or dark-adapted WT and eys-KO (heterozygous and homozygous) zebrafish. Rhodopsin and Gnat2 identify rod OS and cone OS, respectively. Scale bars: 20 μm. (G) Method for counting the number of rod or cone nuclei in light-stimulated or dark-adapted zebrafish. Dotted boxes show areas with a width of 100 μm. Dorsal and ventral areas 150 or 300 μm from the center of optic nerve (ON) were evaluated. Scale bar: 200 μm. (H) Quantification of rod or cone cell numbers by nuclei counting in light-stimulated or dark-adapted WT and eys-KO (heterozygous and homozygous) zebrafish. The x axis indicates the distance from the ON. The y axis indicates the number of nuclei within a range of 100 μm. One-way ANOVA with Dunnett’s post hoc test was used for statistical comparison (*P < 0.05). Data represent mean ± SEM (n = 3). (I) Representative TUNEL staining images of WT and eys-KO zebrafish after light stimulation or dark adaptation. White arrowheads indicate TUNEL-positive cells. Scale bars: 100 μm. (J) The number of TUNEL-positive cells was quantified in each zebrafish after light exposure or dark adaptation. The y axis indicates the number of TUNEL-positive cells per eye. Data represent mean ± SEM (n = 3). One-way ANOVA with Dunnett’s post hoc test was used for statistical comparison (*P < 0.05). (K) Representative transmission electron microscopy images of photoreceptor cells in WT and eys-KO (heterozygous and homozygous) zebrafish. Right panels are higher-magnification images of the OS in the dotted boxes in the left panels. Scale bars: 500 nm.