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• W2989150392 abstract "Article7 November 2019free access Source DataTransparent process Cul3-Klhl18 ubiquitin ligase modulates rod transducin translocation during light-dark adaptation Taro Chaya Laboratory for Molecular and Developmental Biology, Institute for Protein Research, Osaka University, Osaka, Japan Search for more papers by this author Ryotaro Tsutsumi Laboratory for Molecular and Developmental Biology, Institute for Protein Research, Osaka University, Osaka, Japan Search for more papers by this author Leah Rie Varner Laboratory for Molecular and Developmental Biology, Institute for Protein Research, Osaka University, Osaka, Japan Search for more papers by this author Yamato Maeda Laboratory for Molecular and Developmental Biology, Institute for Protein Research, Osaka University, Osaka, Japan Search for more papers by this author Satoyo Yoshida Laboratory for Molecular and Developmental Biology, Institute for Protein Research, Osaka University, Osaka, Japan Search for more papers by this author Takahisa Furukawa Corresponding Author [email protected] orcid.org/0000-0003-4171-220X Laboratory for Molecular and Developmental Biology, Institute for Protein Research, Osaka University, Osaka, Japan Search for more papers by this author Taro Chaya Laboratory for Molecular and Developmental Biology, Institute for Protein Research, Osaka University, Osaka, Japan Search for more papers by this author Ryotaro Tsutsumi Laboratory for Molecular and Developmental Biology, Institute for Protein Research, Osaka University, Osaka, Japan Search for more papers by this author Leah Rie Varner Laboratory for Molecular and Developmental Biology, Institute for Protein Research, Osaka University, Osaka, Japan Search for more papers by this author Yamato Maeda Laboratory for Molecular and Developmental Biology, Institute for Protein Research, Osaka University, Osaka, Japan Search for more papers by this author Satoyo Yoshida Laboratory for Molecular and Developmental Biology, Institute for Protein Research, Osaka University, Osaka, Japan Search for more papers by this author Takahisa Furukawa Corresponding Author [email protected] orcid.org/0000-0003-4171-220X Laboratory for Molecular and Developmental Biology, Institute for Protein Research, Osaka University, Osaka, Japan Search for more papers by this author Author Information Taro Chaya1, Ryotaro Tsutsumi1, Leah Rie Varner1, Yamato Maeda1, Satoyo Yoshida1 and Takahisa Furukawa *,1 1Laboratory for Molecular and Developmental Biology, Institute for Protein Research, Osaka University, Osaka, Japan *Corresponding author. Tel: +81-6-6879-8631; Fax: +81-6-6879-8633; E-mail: [email protected] EMBO J (2019)38:e101409https://doi.org/10.15252/embj.2018101409 PDFDownload PDF of article text and main figures.AM PDF Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Adaptation is a general feature of sensory systems. In rod photoreceptors, light-dependent transducin translocation and Ca2+ homeostasis are involved in light/dark adaptation and prevention of cell damage by light. However, the underlying regulatory mechanisms remain unclear. Here, we identify mammalian Cul3-Klhl18 ubiquitin ligase as a transducin translocation modulator during light/dark adaptation. Under dark conditions, Klhl18−/− mice exhibited decreased rod light responses and subcellular localization of the transducin α-subunit (Tα), similar to that observed in light-adapted Klhl18+/+ mice. Cul3-Klhl18 promoted ubiquitination and degradation of Unc119, a rod Tα-interacting protein. Unc119 overexpression phenocopied Tα mislocalization observed in Klhl18−/− mice. Klhl18 weakly recognized casein kinase-2-phosphorylated Unc119 protein, which is dephosphorylated by Ca2+-dependent phosphatase calcineurin. Calcineurin inhibition increased Unc119 expression and Tα mislocalization in rods. These results suggest that Cul3-Klhl18 modulates rod Tα translocation during light/dark adaptation through Unc119 ubiquitination, which is affected by phosphorylation. Notably, inactivation of the Cul3-Klhl18 ligase and calcineurin inhibitors FK506 and cyclosporine A that are known immunosuppressant drugs repressed light-induced photoreceptor damage, suggesting potential therapeutic targets. Synopsis In rod photoreceptor cells, subcellular localization of transducin changes in response to ambient light, thereby contributing to light and dark adaptation. Here, the Cul3-Klhl18 ubiquitin ligase is shown to modulate light-dependent translocation of the transducin α-subunit (Tα) to adjust light sensitivity in rod photoreceptors. Cul3-Klhl18 promotes Tα localization to the rod outer segment under dark conditions. Cul3-Klhl18 regulates Tα translocation via ubiquitination and degradation of the Tα trafficking regulator Unc119. Cul3-Klhl18-mediated Unc119 degradation is phosphorylation-dependent. Regulation of Unc119 phosphorylation by casein kinase 2 and calcineurin modulates its degradation. Inhibition of Cul3-Klhl18 or calcineurin suppresses light-induced retinal damage. Introduction Vision in vertebrates begins with light reception and conversion to electrical signals through the process of phototransduction by rod and cone photoreceptor cells (Yau & Hardie, 2009). Rod photoreceptors are sensitive to a lower range of light intensities and are responsible for low light vision, while cone photoreceptors operate at brighter intensities and are responsible for high-resolution daylight and color vision. Paradoxically, light is also harmful to these photoreceptor cells. In some animal models of retinal degenerative diseases, including retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA), photoreceptor degeneration is accelerated by light exposure and photoreceptors are protected by dark rearing (Paskowitz et al, 2006). In humans, exposure to light is a suspected risk factor for the progression of age-related macular degeneration (AMD) and RP (Parmeggiani et al, 2011; Marquioni-Ramella & Suburo, 2015; Schick et al, 2016; Mitchell et al, 2018). Adaptation, a general phenomenon observed in sensory systems, makes it possible for sensory cells to respond to ambient signals appropriately according to background levels. Rod and cone photoreceptor cells alter their photosensitivity depending on ambient light levels, enabling responses to a wide range of light intensities (Fain et al, 2001; Luo et al, 2008). In darkness, rod photoreceptors become sensitive to light and ultimately can respond even to single photons (Baylor et al, 1979; Rieke & Baylor, 1998). On the other hand, they possess the ability to reduce light sensitivity and thus avoid excessive activation, preventing saturation and protecting them from brighter light (Fain, 2006). Since rod photoreceptor degeneration leads to secondary cone photoreceptor death, rod photoreceptor protection contributes to the preservation of vision under both dim light and daylight conditions (Roska & Sahel, 2018). Accordingly, light and dark adaptation of rod photoreceptors plays a crucial role in the acquisition of proper vision and in the prevention of blindness; however, the underlying regulatory mechanisms are not fully understood. In rod photoreceptor cells, subcellular localization of transducin, a heterotrimeric G protein that is a component of the phototransduction cascade, changes in response to ambient light, thereby contributing to light and dark adaptation (Brann & Cohen, 1987; Philp et al, 1987; Whelan & McGinnis, 1988; Organisciak et al, 1991; Sokolov et al, 2002). Transducin is concentrated in the outer segment under dark-adapted conditions. After light reception, transducin is translocated from the outer segment to the inner part of the rod photoreceptor. This light- and dark-dependent transducin translocation is known to modulate photosensitivity in rod photoreceptors. For example, transgenic mice expressing a mutant form of the α-subunit of transducin (Tα), in which Tα acylation and localization to the outer segment are inhibited, show a decreased light sensitivity of rod photoreceptors (Kerov et al, 2007). In contrast, mouse lines modeling Usher syndrome, shaker1 and whirler, exhibit defective Tα translocation to the inner part and a decreased threshold of light-triggered photoreceptor degeneration (Peng et al, 2011; Tian et al, 2014). Furthermore, additional lipid modification by an amino acid substitution partially blocks light-induced Tα translocation from the outer segment to the inner part, leading to photoreceptor cell death (Kerov & Artemyev, 2011; Majumder et al, 2013). In the current study, we identified the cullin 3 (Cul3)–Kelch-like 18 (Klhl18) ubiquitin E3 ligase as a modulator of transducin translocation during light and dark adaptation of rod photoreceptor cells. We found that Klhl18 is predominantly expressed in retinal photoreceptor cells. Decreased light responses and Tα mislocalization from the outer segment to the inner part were observed in the rod photoreceptors of Klhl18−/− mice. Unc119, an interactor with Tα, is ubiquitinated and degraded by the Cul3–Klhl18 ligase. Overexpression of Unc119 phenocopied the Tα mislocalization observed in the Klhl18−/− retina. Unc119 expression in rod photoreceptors decreased under dark conditions in a Klhl18-dependent manner. Unc119 is phosphorylated and dephosphorylated by casein kinase 2 (CK2) and calcineurin, a Ca2+-dependent phosphatase, respectively. Unc119 degradation by Cul3–Klhl18 is suppressed by Unc119 phosphorylation. Taken together, these results suggest that Cul3–Klhl18 modulates light- and dark-dependent Tα localization changes in rod photoreceptors through Unc119 ubiquitination and degradation, which is affected by Unc119 phosphorylation and dephosphorylation. Notably, genetic or pharmacological inactivation of the Cul3–Klhl18 ligase and calcineurin inhibitors FK506 and cyclosporine A (CsA) that are known immunosuppressant drugs repressed light-induced photoreceptor damage. Our results also suggest potential therapeutic targets for photoreceptor protection. Results Klhl18 is expressed in retinal photoreceptor cells In order to identify molecules regulating retinal photoreceptor development and/or function, we searched for genes enriched in photoreceptor cells using our previously generated microarray data comparing transcripts between control and Otx2 conditional knockout retinas, in which cell fate is converted from photoreceptors to amacrine-like cells (Nishida et al, 2003; Omori et al, 2011). We focused on Klhl18, which encodes a Cul3 ubiquitin E3 ligase substrate adaptor whose in vivo function has not yet been reported (Moghe et al, 2012). To examine the tissue distribution of Klhl18 expression, we performed RT–PCR analysis using 4-week-old (4 weeks) mouse tissues. We observed Klhl18 expression in the retina but not in other tissues examined (Fig 1A). We next carried out in situ hybridization analysis using developing and mature mouse retinal sections. We observed that Klhl18 is expressed in the outer nuclear layer (ONL), where photoreceptor cells are located, at postnatal day 9 (P9) and P21 (Fig 1B, Appendix Fig S1). These results suggest that Klhl18 is predominantly expressed in maturing and mature retinal photoreceptor cells. Figure 1. Decrease in the rod light responses in Klhl18−/− mice A. RT–PCR analysis of the Klhl18 transcript in mouse tissues at 4 weeks. Klhl18 was predominantly expressed in the retina. β-Actin was used as a loading control. B. In situ hybridization analysis of Klhl18 in developing (embryonic day 17.5 (E17.5), P3, and P9) and mature (P21) mouse retinas. Klhl18 signals were detected in the ONL at P9 and P21. GCL, ganglion cell layer; NBL, neuroblastic layer; ONL, outer nuclear layer; INL, inner nuclear layer C–H. ERG analysis of Klhl18−/− mice. ERGs were recorded from Klhl18+/+ and Klhl18−/− mice at 1 month (1 M) (n = 3 per each genotype). (C) Representative scotopic ERGs elicited by four different stimulus intensities (−4.0 to 1.0 log cd s/m2) from Klhl18+/+ and Klhl18−/− mice are presented. (D, E) The scotopic amplitudes of a- (D) and b-waves (E) are shown as a function of the stimulus intensity. Data are presented as mean ± SD. n = 3 per each genotype. **P < 0.01, ***P < 0.001 (two-way repeated-measures ANOVA). (F) Representative photopic ERGs elicited by four different stimulus intensities (−0.5 to 1.0 log cd s/m2) from Klhl18+/+ and Klhl18−/− mice are presented. (G, H) The photopic amplitudes of a- (G) and b-waves (H) are shown as a function of the stimulus intensity. Data are presented as mean ± SD. n = 3 per each genotype. n.s., not significant (two-way repeated-measures ANOVA). Download figure Download PowerPoint Klhl18 deficiency decreases light response in rod photoreceptor cells To investigate roles of the Cul3–Klhl18 ligase in retinal photoreceptor development and/or function, we generated Klhl18 flox mice by targeted gene disruption (Fig EV1A). We mated the Klhl18 flox mice with CAG-Cre mice, in which Cre recombinase is expressed in female germ cells (Sakai & Miyazaki, 1997), and generated conventional Klhl18-deficient (Klhl18−/−) mice (Fig EV1A and B). In the Klhl18−/− retina, no Klhl18 mRNA or protein expression was detected by RT–PCR and Western blot with an anti-Klhl18 antibody that we generated (Fig EV1C and D). We observed substantial Klhl18 immunofluorescence signals in the inner segment (IS) and ONL in the Klhl18+/+ retina but not in the Klhl18−/− retina (Fig EV1E). Klhl18−/− mice were viable and fertile, and exhibited no gross morphological abnormalities. Click here to expand this figure. Figure EV1. Generation of the Klhl18−/− mice Schematic representation of the wild-type allele, targeting vector, Klhl18 recombinant allele, and Cre recombinant allele. Red arrows indicate primers to detect the Cre recombinant allele. Removal of exon 6 by Cre-mediated recombination is predicted to result in a translational frameshift and loss of Klhl18 function. Ex, exon. PCR products of 163 and 544 bp were amplified from the wild-type and Cre recombinant allele, respectively. RT–PCR analysis of the Klhl18 transcript in Klhl18+/− and Klhl18−/− retinas. The primers were designed within exon 6 and exon 10 of Klhl18. No Klhl18 transcript was detected in the Klhl18−/− retina. β-Actin was used as a loading control. Western blot analysis of the Klhl18 protein in Klhl18+/+ and Klhl18−/− retinas. No Klhl18 band was detected in the Klhl18−/− retina. α-Tubulin was used as a loading control. An asterisk indicates non-specific bands. Immunostaining of retinas from Klhl18+/+ and Klhl18−/− mice at P14 with an anti-Klhl18 antibody (green). Klhl18 signals were detected in the IS and ONL of the Klhl18+/+ retina. No significant Klhl18 signal was observed in the IS and ONL of the Klhl18−/− retina. IS, inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer. The scotopic b/a-wave ratio of Klhl18+/+ and Klhl18−/− mice. The scotopic amplitudes of a- and b-waves at the stimulus intensity of 1.0 log cd s/m2 were used for calculation. Data are presented as mean ± SD. n.s., not significant (unpaired t-test). Source data are available online for this figure. Download figure Download PowerPoint To examine the electrophysiological properties of the Klhl18−/− retina, we measured the electroretinograms (ERGs) of Klhl18−/− mice under dark-adapted (scotopic) and light-adapted (photopic) conditions. Under scotopic conditions, the amplitude of a-waves and b-waves, originating mainly from the population activity of rod photoreceptor cells (a-waves) and of rod bipolar cells (b-waves), significantly decreased in Klhl18−/− mice compared with that of Klhl18+/+ mice (Fig 1C–E). To evaluate synaptic transmission from rod photoreceptors to rod bipolar cells, we calculated the scotopic b/a-wave ratio. We did not observe a significant difference in the scotopic b/a-wave ratio between Klhl18+/+ and Klhl18−/− mice (Fig EV1F). In contrast, under photopic conditions, the amplitude of a-waves and b-waves, mainly reflecting the population activity of cone photoreceptor cells (a-waves) and of cone ON bipolar cells (b-waves), was comparable between Klhl18+/+ and Klhl18−/− mice (Fig 1F–H). These results suggest that rod photoreceptor function is compromised by Klhl18 deficiency, but not cone photoreceptor function. To investigate how the scotopic ERG amplitude decreased in Klhl18−/− mice, we performed histological analyses using retinal sections. Toluidine blue staining showed that the ONL thickness was comparable between Klhl18+/+ and Klhl18−/− retinas (Fig 2A and B). Immunohistochemical examination using marker antibodies against rhodopsin (rod outer segments), Rom1 (rod outer segments), S-opsin (S-cone outer segments), and M-opsin (M-cone outer segments) showed no substantial differences between Klhl18+/+ and Klhl18−/− rod and cone outer segments (Fig EV2A–C). We also performed immunohistochemical analysis using antibodies against Chx10 (a marker for bipolar cells), Pax6 (a marker for amacrine and ganglion cells), calbindin (a marker for horizontal cells and a part of amacrine cells), Brn3a (a marker for ganglion cells), and S100β (a marker for Müller glial cells) and found no substantial differences between the Klhl18+/+ and Klhl18−/− retinas (Fig EV2D–G). To observe whether photoreceptor degeneration occurs in the Klhl18−/− retina, we measured the ONL thickness in the Klhl18−/− retina at 6 months. No significant change was observed in the ONL thickness between Klhl18+/+ and Klhl18−/− retinas (Fig EV2H and I). Figure 2. Rod Tα mislocalization in the Klhl18−/− mouse retina A, B. Toluidine blue staining of retinas from Klhl18+/+ and Klhl18−/− mice at 1 M. The ONL thickness was measured. Data are presented as mean ± SD. n.s., not significant (unpaired t-test), n = 3 mice per genotype. C–F. Subcellular localization of Tα and Tγ in photoreceptor cells of the Klhl18−/− retina. Retinal sections obtained from dark- or light- (˜ 1,000 lx) adapted (4 h) Klhl18+/+ and Klhl18−/− mice at 1 M were immunostained with an anti-Tα (C) or anti-Tγ (E) antibody. The immunofluorescence signals of Tα (D) and Tγ (F) detected in the inner part of photoreceptors were quantified using ImageJ software. The measured signals of Tα or Tγ in the inner part of photoreceptors were expressed as a proportion of the total (OS + the inner part) signals of Tα or Tγ in photoreceptors, respectively. Data are presented as mean ± SD. n = 4, 4, 3, and 4 mice (Klhl18+/+; dark, Klhl18−/−; dark, Klhl18+/+; light, and Klhl18−/−; light, respectively). *P < 0.05, **P < 0.01, n.s., not significant (unpaired t-test). Data information: GCL, ganglion cell layer; ONL, outer nuclear layer; INL, inner nuclear layer; OS, outer segment. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Histological analysis of the Klhl18−/− retina A–G. Immunohistochemical analysis of Klhl18+/+ and Klhl18−/− retinas at 1 M using marker antibodies as follows: Rhodopsin (rod outer segments, A), Rom1 (rod outer segments, A), S-opsin (S-cone outer segments, B), M-opsin (M-cone outer segments, C), Chx10 (bipolar cells, D), Pax6 (amacrine and ganglion cells, D), Calbindin (horizontal cells and a subset of amacrine cells, E), Brn3a (ganglion cells, F), and S100β (Müller glial cells, G). Nuclei were stained with DAPI (blue). No obvious difference was observed between Klhl18+/+ and Klhl18−/− retinas. H, I. Toluidine blue staining of retinas from Klhl18+/+ and Klhl18−/− mice at 6 M. The ONL thickness was measured. Data are presented as mean ± SD. n.s., not significant (unpaired t-test). J, K. Subcellular localization of visual arrestin in photoreceptor cells of the Klhl18−/− retina. Retinal sections obtained from dark- or light- (˜ 1,000 lx) adapted (4 h) Klhl18+/+ and Klhl18−/− mice at 1 M were immunostained with an anti-visual arrestin antibody (J). The immunofluorescence signals of visual arrestin detected in the inner part of photoreceptors were quantified using ImageJ software. The visual arrestin signal measurements were expressed as a proportion of the total (OS + the inner part) signal of visual arrestin in photoreceptors (K). Data are presented as mean ± SD. n.s., not significant (unpaired t-test), n = 3 mice each. Data information: OS, outer segment; ONL, outer nuclear layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Download figure Download PowerPoint Inhibition of the Cul3–Klhl18 ligase activity causes mislocalization of rod Tα It was previously reported that mice having a substitution of aspartic acid for glycine at residue 90 of mouse Rhodopsin, a G protein-coupled receptor, show constitutive light adaptation of rod photoreceptor cells (Sieving et al, 2001). These mice exhibit decreased rod light sensitivity without any changes in rod photoreceptor cell number and outer segment formation, resembling Klhl18−/− mice. Thus, we hypothesized that Klhl18−/− mice present constitutive light adaptation of rod photoreceptors and then observed the subcellular distribution of transducin in rod photoreceptor cells, which depends on ambient light conditions (Calvert et al, 2006). In the Klhl18+/+ retina, rod Tα was localized mainly in the outer segment in the dark-adapted state, whereas Tα was translocated to the inner part of the photoreceptors in the light-adapted state. Contrastingly, in the Klhl18−/− retina, we found increased Tα localization to the inner part of photoreceptors in the dark-adapted state, as observed in light-adapted Klhl18+/+ mice. In the light-adapted state, we also observed increased Tα localization to the inner part of photoreceptors in the Klhl18−/− retina compared with that in the Klhl18+/+ retina (Fig 2C and D). On the other hand, we did not find significant differences in the transducin γ-subunit (Tγ) localization in the inner part of photoreceptors between Klhl18+/+ and Klhl18−/− retinas under dark or light conditions (Fig 2E and F). In rod photoreceptors, the subcellular distribution of visual arrestin also changes in response to ambient light. After light reception, visual arrestin translocates from the inner part to the outer segment (Broekhuyse et al, 1985; Philp et al, 1987; Whelan & McGinnis, 1988). To investigate the subcellular localization of visual arrestin in Klhl18+/+ and Klhl18−/− retinas under dark- and light-adapted conditions, we performed immunohistochemical analysis. While we observed light-triggered translocation of visual arrestin to the outer segment of rod photoreceptors, we did not find significant differences in the localization of visual arrestin in the inner part of photoreceptors between Klhl18+/+ and Klhl18−/− retinas under dark- and light-adapted conditions (Fig EV2J and K). To further investigate the role of Cul3–Klhl18 in subcellular localization of transducin in rod photoreceptor cells, we injected MLN4924, a small molecule inhibitor of the NEDD8-activating enzyme (NAE), into wild-type mice (Soucy et al, 2009). Covalent attachment of the ubiquitin-like protein Nedd8 to cullin family proteins is required for the function of the cullin-based ubiquitin E3 ligases (Pan et al, 2004). We observed increased Tα localization in the inner part of photoreceptors in the retina of MLN4924-treated mice compared with that of DMSO-treated mice under dark conditions (Fig EV3A and B). In contrast to Tα, Tγ localization to the inner part of retinal photoreceptors was not increased in MLN4924-treated mice (Fig EV3A and B). These results suggest that Cul3–Klhl18 is involved in the regulation of dark- and light-dependent Tα but not Tγ translocation in rod photoreceptor cells. Click here to expand this figure. Figure EV3. Rod Tα mislocalization in the MLN4924-injected mice, and phenotypic analyses of Klhl18−/− and MLN4924-injected mice after light exposure A, B. Subcellular localization of Tα and Tγ in retinal photoreceptor cells of MLN4924-injected mice. (A) MLN4924 was subcutaneously injected into wild-type mice every day for 4 days. Retinal sections obtained from dark-adapted (4 h) mice treated with MLN4924 were immunostained using an anti-Tα or anti-Tγ antibody. (B) The immunofluorescence signals of Tα and Tγ detected in the inner part of photoreceptors were quantified using ImageJ software. The measured signals of Tα or Tγ in the inner part of photoreceptors were expressed as a proportion to the total (OS + the inner part) signals of Tα or Tγ in photoreceptors, respectively. The Tα signals in the inner part of photoreceptors increased in the retina from the MLN4924-treated mice. Data are presented as mean ± SD. *P < 0.05 (unpaired t-test), n = 4 mice each. OS, outer segment; ONL, outer nuclear layer. C. The length of rod outer segments stained with an anti-Rhodopsin antibody was measured in Klhl18+/+ and Klhl18−/− mice after LED light exposure. The length of rod outer segments in the Klhl18+/+ retina significantly decreased compared with that in the Klhl18−/− retina. Data are presented as mean ± SD. **P < 0.01 (unpaired t-test), n = 5 mice per genotype. D, E. ERG analysis of Klhl18+/+ and Klhl18−/− mice after LED light exposure. The scotopic (D) and photopic (E) a-wave amplitudes are shown as a function of the stimulus intensity. Data are presented as mean ± SD. n.s., not significant (two-way repeated-measures ANOVA), n = 4 and 5 mice (Klhl18+/+ and Klhl18−/−, respectively). F. The length of rod outer segments stained with an anti-Rhodopsin antibody was measured in MLN4924-treated mice after LED light exposure. The length of rod outer segments in DMSO-treated mice significantly decreased compared with that in MLN4924-treated mice. Data are presented as mean ± SD. *P < 0.05 (unpaired t-test), n = 4 mice each. G, H. ERG analysis of MLN4924-treated mice after LED light exposure. The scotopic (G) and photopic (H) a-wave amplitudes are shown as a function of the stimulus intensity. Data are presented as mean ± SD. *P < 0.05, (two-way repeated-measures ANOVA), n = 4 mice each. n.s., not significant. Download figure Download PowerPoint Light-induced retinal damage is suppressed by inhibiting the Cul3–Klhl18 ligase activity It was previously reported that fixation of Tα to the outer segment increases light-triggered photoreceptor cell death (Majumder et al, 2013), suggesting that Tα translocation to the inner part decreases photoreceptor damage caused by light. To examine whether inhibition of Cul3–Klhl18 suppresses photoreceptor damage by light, Klhl18+/+ and Klhl18−/− mice were exposed to light-emitting diode (LED) light and their retinal damage was compared (Fig 3A). We observed decreased ONL thickness in the Klhl18+/+ retina compared with that in the Klhl18−/− retina (Fig 3B). To analyze light-induced retinal damage in more detail, we immunostained retinal sections using antibodies against Rhodopsin, S-opsin, M-opsin, and IbaI (a marker for macrophage and microglia). We observed that rod and cone outer segments were disorganized in the Klhl18+/+ retina compared with those in the Klhl18−/− retina. The number of macrophages and/or microglia in the ONL increased in the Klhl18+/+ retina compared with those in the Klhl18−/− retina (Figs 3C and EV3C). We also performed an ERG analysis and found that scotopic and photopic b-wave amplitudes in Klhl18−/− mice are higher than those in Klhl18+/+ mice, although no significant differences in scotopic and photopic a-wave amplitudes were detected between Klhl18+/+ and Klhl18−/− mice (Figs 3D and E, and EV3D and E). The scotopic a-wave amplitude was significantly higher in Klhl18+/+ mice compared with that in Klhl18−/− mice (Fig 1C and D), and this result shows that LED light exposure decreased the scotopic a-wave amplitude in Klhl18+/+ mice more than in Klhl18−/− mice, suggesting that light-induced photoreceptor damage is suppressed in Klhl18−/− mice compared with that in Klhl18+/+ mice. Figure 3. Klhl18 deficiency represses light-induced retinal damage A. Experimental design for exposure of Klhl18+/+ and Klhl18−/−mice to LED light. B. Retinal sections from Klhl18+/+ and Klhl18−/− mice after light exposure were stained with DAPI, and then, the ONL thickness was measured. Data are presented as mean ± SD. **P < 0.01 (two-way repeated-measures ANOVA), n = 5 mice per genotype. C. Immunohistochemical analysis of retinas from Klhl18+/+ and Klhl18−/− mice after light exposure using marker antibodies as follows: Rhodopsin (rod outer segments), S-opsin (S-cone outer segments), M-opsin (M-cone outer segments), and IbaI (microglia or macrophages). Nuclei were stained with DAPI. Rod and cone outer segments were severely disorganized in the Klhl18+/+ retina. D, E. ERG analysis of Klhl18+/+ and Klhl18−/− mice after LED light exposure. Scotopic (D) and photopic (E) amplitudes of b-waves are shown as a function of the stimulus intensity. Data are presented as mean ± SD. *P < 0.05 (two-way repeated-measures ANOVA), **P < 0.01 (two-way repeated-measures ANOVA followed by Sidak's multiple comparisons test), n = 4 and 5 mice (Klhl18+/+ and Klhl18−/−, respectively). F. Experimental design for exposure of mice treated with MLN4924, an NAE inhibitor, to LED light. MLN4924 was injected into mice every day for 3 days. G. Retinal sections from MLN4924-treated mice after light exposure were stained with DAPI, and the ONL thickness was measured. Data are presented as mean ± SD. ***P < 0.001 (two-way repeated-measures ANOVA), n = 4 mice each. H. Immunohistochemical analysis of retinas from MLN4924-treated mice after light exposure using marker antibo" @default.
• W2989150392 created "2019-11-22" @default.
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• W2989150392 date "2019-11-07" @default.
• W2989150392 modified "2023-10-15" @default.
• W2989150392 title "Cul3‐Klhl18 ubiquitin ligase modulates rod transducin translocation during light‐dark adaptation" @default.
• W2989150392 cites W1509709236 @default.
• W2989150392 cites W1525882285 @default.
• W2989150392 cites W1590951841 @default.
• W2989150392 cites W1602968125 @default.
• W2989150392 cites W1719249858 @default.
• W2989150392 cites W1788141166 @default.
• W2989150392 cites W1820330395 @default.
• W2989150392 cites W1968686595 @default.
• W2989150392 cites W1968800607 @default.
• W2989150392 cites W1969259847 @default.
• W2989150392 cites W1973383826 @default.
• W2989150392 cites W1973796123 @default.
• W2989150392 cites W1973875627 @default.
• W2989150392 cites W1977361305 @default.
• W2989150392 cites W1977571897 @default.
• W2989150392 cites W1978295625 @default.
• W2989150392 cites W1982036931 @default.
• W2989150392 cites W1983698424 @default.
• W2989150392 cites W1984641428 @default.
• W2989150392 cites W1984754431 @default.
• W2989150392 cites W1987018613 @default.
• W2989150392 cites W1989088998 @default.
• W2989150392 cites W1989433038 @default.
• W2989150392 cites W1991163286 @default.
• W2989150392 cites W1994120696 @default.
• W2989150392 cites W1994152829 @default.
• W2989150392 cites W1999179844 @default.
• W2989150392 cites W1999354994 @default.
• W2989150392 cites W2001040160 @default.
• W2989150392 cites W2002098580 @default.
• W2989150392 cites W2006459253 @default.
• W2989150392 cites W2010538044 @default.
• W2989150392 cites W2014899116 @default.
• W2989150392 cites W2017487928 @default.
• W2989150392 cites W2020295187 @default.
• W2989150392 cites W2027937017 @default.
• W2989150392 cites W2028135910 @default.
• W2989150392 cites W2028426734 @default.
• W2989150392 cites W2029224830 @default.
• W2989150392 cites W2038163496 @default.
• W2989150392 cites W2039109528 @default.
• W2989150392 cites W2039596772 @default.
• W2989150392 cites W2043274355 @default.
• W2989150392 cites W2045519476 @default.
• W2989150392 cites W2048058414 @default.
• W2989150392 cites W2052149638 @default.
• W2989150392 cites W2052547930 @default.
• W2989150392 cites W2052626303 @default.
• W2989150392 cites W2052899636 @default.
• W2989150392 cites W2057688490 @default.
• W2989150392 cites W2058709834 @default.
• W2989150392 cites W2060526583 @default.
• W2989150392 cites W2065909084 @default.
• W2989150392 cites W2066359202 @default.
• W2989150392 cites W2066962733 @default.
• W2989150392 cites W2073681930 @default.
• W2989150392 cites W2075735241 @default.
• W2989150392 cites W2076194625 @default.
• W2989150392 cites W2080904282 @default.
• W2989150392 cites W2088938650 @default.
• W2989150392 cites W2092019931 @default.
• W2989150392 cites W2093475470 @default.
• W2989150392 cites W2093867927 @default.
• W2989150392 cites W2100104320 @default.
• W2989150392 cites W2101436175 @default.
• W2989150392 cites W2104301336 @default.
• W2989150392 cites W2105129317 @default.
• W2989150392 cites W2113585002 @default.
• W2989150392 cites W2117193130 @default.
• W2989150392 cites W2119324374 @default.
• W2989150392 cites W2129618668 @default.
• W2989150392 cites W2134281520 @default.
• W2989150392 cites W2134685947 @default.
• W2989150392 cites W2134844061 @default.
• W2989150392 cites W2142913230 @default.
• W2989150392 cites W2144704200 @default.
• W2989150392 cites W2148504678 @default.
• W2989150392 cites W2151900188 @default.
• W2989150392 cites W2155376306 @default.
• W2989150392 cites W2161318689 @default.
• W2989150392 cites W2164105351 @default.
• W2989150392 cites W2167700121 @default.
• W2989150392 cites W2167745656 @default.
• W2989150392 cites W2168006411 @default.
• W2989150392 cites W2286751849 @default.
• W2989150392 cites W2322029944 @default.
• W2989150392 cites W2529546502 @default.
• W2989150392 cites W2548499955 @default.
• W2989150392 cites W2581258821 @default.

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