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 6

Light overresponse and photoreceptor cell death after light irradiation in RD retinal organoids.

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Light overresponse and photoreceptor cell death after light irradiation ...
(A) Retinal organoids were exposed to white LED light on day 180. (B) Intracellular cGMP concentration was measured by ELISA. Percentage changes in cGMP concentration by light stimulation in control and RD retinal organoids are shown. Data represent mean ± SEM from independent experiments (n = 6). An unpaired, 2-tailed t test was used for statistical comparison (*P < 0.05). (C) Representative images of reactive oxygen species (ROS) stained with CellROX in control and RD retinal organoids. Twenty-four-hour light-stimulated (Bright) or dark-adapted (Dark) organoids on day 180 were evaluated. The lower panels are higher-magnification images of the dotted boxes in the upper panels. Scale bars: 100 μm. (D) Quantification of the data in C. The y axis indicates the ratio of CellROX-positive cells. Data represent mean ± SD from 3 retinal organoids. One-way ANOVA with Dunnett’s post hoc test was used for statistical comparison (*P < 0.05). (E) Representative immunofluorescence images of photoreceptor marker ARR1 and cleaved caspase-3 (Cl. CASP3) in retinal organoids after light exposure or dark adaptation. White arrowheads indicate cleaved caspase-3–positive cells. Scale bars: 50 μm. (F) Quantification of the data in E. The y axis indicates the number of cleaved caspase-3–positive cells per field. Data represent mean ± SEM from 3 retinal organoids. One-way ANOVA with Dunnett’s post hoc test was used for statistical comparison (*P < 0.05). (G) Representative immunofluorescence images of ARR1 and cleaved caspase-3 in control and EYS-KO retinal organoids after light exposure or dark adaptation. White arrowheads indicate cleaved caspase-3–positive cells. Scale bars: 50 μm. (H) Quantification of the data in G. The y axis indicates the number of cleaved caspase-3–positive cells per field. Data represent mean ± SEM from 3 retinal organoids. One-way ANOVA with Dunnett’s post hoc test was used for statistical comparison (*P < 0.05).