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 4

Generation of EYS-KO iPSCs with CRISPR/Cas9 system.

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Generation of EYS-KO iPSCs with CRISPR/Cas9 system. (A) Target sequence ...
(A) Target sequence of CRSPR/Cas9 gene editing is located in exon 4 of the EYS gene. Sanger sequencing of edited iPSCs shows single base deletion (c.621 delT). (B) Bright-field view of EYS-KO retinal organoids on day 180. Lower panels are higher-magnification images of the dotted boxes in the upper panels. Scale bars: 200 μm. (C) Western blot analysis of EYS and RCVRN in control and EYS-KO retinal organoids on day 180. Full-length pictures of the blots are presented in Supplemental Figure 8, G–L. (D) Representative immunofluorescence images of GRK7 and OS marker PRPH2 in control and EYS-KO retinal organoids on day 180. Lower panels are higher-magnification images of the dotted boxes in the upper panels. White arrows indicate the OS region. Scale bars: 10 μm. (E) Quantitative analysis of merged immunoreactivity of PRPH2 and GRK7 in control and EYS-KO retinal organoids in D. The y axis indicates the number of reactive dots per field. Data represent mean ± SEM (n = 3 organoids). An unpaired, 2-tailed t test was used for statistical comparison (*P < 0.05).