Phototoxicity avoidance is a potential therapeutic approach for retinal dystrophy caused by EYS dysfunction
Yuki Otsuka, … , Akitaka Tsujikawa, Haruhisa Inoue
Yuki Otsuka, … , Akitaka Tsujikawa, Haruhisa Inoue
Published April 22, 2024
Citation Information: JCI Insight. 2024;9(8):e174179. https://doi.org/10.1172/jci.insight.174179.
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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 3

RD retinal organoids exhibited mislocalization of EYS and GRK7 in connecting cilia and outer segment.

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RD retinal organoids exhibited mislocalization of EYS and GRK7 in connec...
(A) Representative immunofluorescence images of EYS and acetylated α-tubulin (AcTub) in control and RD retinal organoids on day 180. Lower panels are higher-magnification images of the dotted boxes in the upper panels. White arrows indicate the CC and nascent OS. Scale bars: 10 μm. (B) Quantitative analysis of merged immunoreactivity of EYS and AcTub in control and RD retinal organoids. The y axis indicates the number of merged dots per field. Data represent mean ± SEM (n = 6 organoids). An unpaired, 2-tailed t test was used for statistical comparison (*P < 0.05). (C) Western blot analysis of EYS and RCVRN in control and RD retinal organoids on day 100 and day 180. Full-length pictures of the blots are presented in Supplemental Figure 8, A–F. (D) Quantification of Western blot bands in C. The y axis indicates the relative amount of intracellular EYS protein. Data represent mean ± SEM from independent experiments (n = 3). An unpaired, 2-tailed t test was used for statistical comparison (P = 0.92). (E) Representative immunofluorescence images of GRK7 and OS marker PRPH2 in control and RD 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. (F) Quantitative analysis of PRPH2 immunoreactivity in control and RD retinal organoids in E. The y axis indicates the number of reactive dots per field. Data represent mean ± SEM (n = 6 organoids). An unpaired, 2-tailed t test was used for statistical comparison (P = 0.87). (G) Quantitative analysis of merged immunoreactivity of PRPH2 and GRK7 in control and RD retinal organoids in E. The y axis indicates the number of reactive dots per field. Data represent mean ± SEM (n = 6 organoids). An unpaired, 2-tailed t test was used for statistical comparison (*P < 0.05). (H) Biochemical interactions between EYS and GRK7. 293T cell lysates were subjected to immunoprecipitation (IP) using anti-FLAG antibody and immunoblotted (IB) with antibodies against FLAG (top) or EYS (middle and bottom). A full-length picture of the blot is presented in Supplemental Figure 9. (I) Quantification of IB bands in H. The y axis indicates the relative amount of control or mutant EYS binding to GRK7. Data represent mean ± SEM from independent experiments (n = 3). An unpaired, 2-tailed t test was used for statistical comparison (P = 0.69). (J) Schematic of helper-dependent adenoviral vector containing control EYS. IRES, internal ribosome entry site; ITR, inverted terminal repeats; pac, packaging signal; stuffer, stuffer DNA. (K) Bright-field and EGFP fluorescence of RD organoids 48 hours after adenoviral infection. Scale bars: 200 μm. (L) Representative immunofluorescence images of GFP, EYS, and AcTub in RD retinal organoids after control EYS introduction with adenoviral vector. Lower panels are higher-magnification images of the dotted boxes in the upper panels. White arrows indicate the CC and nascent outer segment OS. Scale bars: 10 μm. (M) Representative immunofluorescence images of GFP, GRK7, and PRPH2 in RD retinal organoids after control EYS introduction with adenoviral vector. Lower panels are higher-magnification images of the dotted boxes in the upper panels. White arrows indicate the nascent OS. Scale bars: 10 μm.