Endoplasmic reticulum (ER) Ca2+-channel activity contributes to ER stress and cone death in cyclic nucleotide-gated channel deficiency

Abstract

Endoplasmic reticulum (ER) stress and mislocalization of improperly folded proteins have been shown to contribute to photoreceptor death in models of inherited retinal degenerative diseases. In particular, mice with cone cyclic nucleotide-gated (CNG) channel deficiency, a model for achromatopsia, display both early-onset ER stress and opsin mistrafficking. By 2 weeks of age, these mice show elevated signaling from all three arms of the ER-stress pathway, and by 1 month, cone opsin is improperly distributed away from its normal outer segment location to other retinal layers. This work investigated the role of Ca²⁺-release channels in ER stress, protein mislocalization, and cone death in a mouse model of CNG-channel deficiency. We examined whether preservation of luminal Ca²⁺ stores through pharmacological and genetic suppression of ER Ca²⁺ efflux protects cones by attenuating ER stress. We demonstrated that the inhibition of ER Ca²⁺-efflux channels reduced all three arms of ER-stress signaling while improving opsin trafficking to cone outer segments and decreasing cone death by 20-35%. Cone-specific gene deletion of the inositol-1,4,5-trisphosphate receptor type I (IP₃R1) also significantly increased cone density in the CNG-channel-deficient mice, suggesting that IP₃R1 signaling contributes to Ca²⁺ homeostasis and cone survival. Consistent with the important contribution of organellar Ca²⁺ signaling in this achromatopsia mouse model, significant differences in dynamic intraorganellar Ca²⁺ levels were detected in CNG-channel-deficient cones. These results thus identify a novel molecular link between Ca²⁺ homeostasis and cone degeneration, thereby revealing novel therapeutic targets to preserve cones in inherited retinal degenerative diseases.

Keywords: CNG channel; calcium channel; endoplasmic reticulum stress (ER stress); inositol trisphosphate receptor (InsP3R); photoreceptor; ryanodine receptor.

Figure 1.

Expression of the IP₃R1 isoform is increased in Cnga3^−/−;Nrl^−/− mice and is localized to the inner segment in cones. A, retinas prepared from Cnga3^−/−;Nrl^−/− and Nrl^−/− mice were analyzed for expression of IP₃R isoforms by qRT-PCR at P15, P30, and P60 following normalization to Hprt1 mRNA controls. B, total IP₃R1 isoform protein levels were measured at P15, P30, and P60 in Cnga3^−/−;Nrl^−/− and Nrl^−/− mice. Shown are representative Western blot images with corresponding densitometric analysis following normalization to internal loading control β-actin. C, localization of the IP₃R1 isoform was determined via immunofluorescence co-labeling with rhodopsin or Na⁺/K⁺ ATPase α1 subunit at P30 in wild-type mice. ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer. Data are presented as mean ± S.D. of at least three independent assays using retinas from at least six mice/group. Unpaired Student's t test was used to determine significance between CNG-channel-deficient and Nrl^−/− mice (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

Figure 2.

Expression of the RyR2 isoform is increased in Cnga3^−/−;Nrl^−/− mice and is localized to the inner segment in cones. A, retinas prepared from Cnga3^−/−;Nrl^−/− and Nrl^−/− mice were analyzed for expression of RyR isoforms by qRT-PCR at P15, P30, and P60 following normalization to Hprt1 mRNA controls. B, total RyR2 isoform protein levels were measured at P15, P30, and P60 in Cnga3^−/−;Nrl^−/− and Nrl^−/− mice. Shown are representative Western blot images with corresponding densitometric analysis following normalization to internal loading control β-actin. C, localization of the RyR2 isoform was determined via immunofluorescence co-labeling with rhodopsin or Na⁺/K⁺ ATPase α1 subunit at P30 in wild-type mice. ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer. Data are presented as mean ± S.D. of at least three independent assays using retinas from at least six mice/group. Unpaired Student's t test was used to determine significance between CNG-channel-deficient and Nrl^−/− mice (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

Figure 3.

Expression of phospho-IP₃R1 and phospho-RyR2 is increased and intracellular Ca²⁺ is decreased at early ages in Cnga3^−/−;Nrl^−/− mice. A, retinal protein expression levels of phospho-RyR2 and phospho-IP₃R1 at P15, P30, and P60 in Cnga3^−/−;Nrl^−/− and Nrl^−/− mice were analyzed. Shown are representative Western blot images with corresponding densitometric analysis following normalization to internal loading control β-actin. B, basal intracellular Ca²⁺levels were also measured in Cnga3^−/−;Nrl^−/− and Nrl^−/− mice at P15, P30, and P60. Data are presented as mean ± S.D. of at least three independent assays using retinas from 6–8 mice/group for immunoblotting and 15–33 photoreceptors from 3–5 mice for [Ca²⁺]_i assays. Unpaired Student's t test was used to determine significance between CNG-channel-deficient and Nrl^−/− mice (*, p < 0.05; **, p < 0.01).

Figure 4.

Expression of ER-chaperone proteins is increased in Cnga3^−/−;Nrl^−/− mice and luminal Ca²⁺ stores undergo dynamic changes. A, retinas prepared from Cnga3^−/−;Nrl^−/− and Nrl^−/− mice were analyzed for protein expression of Grp78/BiP and calreticulin by Western blot analysis at P15, P30, and P60. Shown are representative Western blot images with corresponding densitometric analysis following normalization to internal loading control β-actin. B, intracellular Ca²⁺ levels were measured following 20 mm KCl or 10 μm ionomycin treatment in Cnga3^−/−;Nrl^−/− and Nrl^−/− mice at P15, P30, and P60 to determine relative levels of luminal Ca²⁺ stores. Data are presented as mean ± S.D. of at least three independent assays using retinas from 6–8 mice/group for immunoblotting and 13–33 photoreceptors from 3–5 mice for [Ca²⁺]_i assays. Unpaired Student's t test was used to determine significance between CNG-channel-deficient and Nrl^−/− mice (*, p < 0.05; **, p < 0.01).

Figure 5.

Inhibition of ER Ca²⁺-channel activity reduced cone apoptosis in Cnga3^−/−;Nrl^−/− mice. Cnga3^−/−;Nrl^−/− mice received IP₃R inhibitor, RyR inhibitor, or vehicle treatment for 7 days beginning on P8. At the end of the treatment, cone apoptosis was evaluated by TUNEL labeling on retinal sections. Shown are representative confocal images of TUNEL labeling of Cnga3^−/−;Nrl^−/− mice treated with IP₃R inhibitor (A), RyR inhibitor (B), or vehicle and age-matched Nrl^−/− controls and correlating quantitative analysis. ONL, outer nuclear layer; INL, inner nuclear layer. Data are represented as mean ± S.D. of at least three independent assays. Unpaired Student's t test was used to determine significance between the two groups (*, p < 0.05; ***, p < 0.001).

Figure 6.

Inhibition of IP₃R activity improved cone-opsin localization to cone outer segment and reduced ER-chaperone protein levels in CNG-channel-deficient mice. A, Cnga3^−/− mice received IP₃R inhibitor or vehicle treatment for 22 days beginning on P8. At the end of the treatment, M-opsin localization was determined via immunofluorescence on retinal sections. Shown are representative confocal images of immunofluorescence for M-opsin co-labeled with Na⁺/K⁺ ATPase α1 in IP₃R inhibitor and vehicle-treated Cnga3^−/− mice and control WT mice with quantification. ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer. B, Cnga3^−/−;Nrl^−/− mice received IP₃R inhibitor or vehicle treatment for 7 days, beginning on P8, for Western blot analysis. At the end of the treatment, the protein expression levels of Ca²⁺-sensitive ER-chaperone proteins Grp78/BiP and calreticulin were analyzed via Western blot analysis. Shown are representative Western blot images of the IP₃R inhibitor or vehicle-treated Cnga3^−/−;Nrl^−/− mice with corresponding quantitative analysis following normalization to internal loading control β-actin. Data are presented as mean ± S.D. of at least three independent assays. Unpaired Student's t test was used to determine significance between IP₃R inhibitor and vehicle-treated Cnga3^−/−;Nrl^−/− and Cnga3^−/− mice (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

Figure 7.

Inhibition of IP₃R activity reduced ER-stress marker protein levels in CNG-channel-deficient mice. Cnga3^−/−;Nrl^−/− mice received IP₃R inhibitor or vehicle treatment for 7 days beginning on P8. At the end of the treatment, the protein expression levels of ER-stress marker proteins phospho-IRE1α, cleaved ATF6, and phospho-eIF2α were analyzed in the retina via Western blot analysis. Shown are representative Western blot images of the IP₃R inhibitor or vehicle-treated Cnga3^−/−;Nrl^−/− mice and Nrl^−/− mice with corresponding quantitative analysis following normalization to internal loading control β-actin. Data are presented as mean ± S.D. of at least three independent assays using eyes from 6–8 mice/group. Unpaired Student's t test was used to determine significance between IP₃R inhibitor and vehicle-treated Cnga3^−/−;Nrl^−/− mice (*, p < 0.05; **, p < 0.01).

Figure 8.

Inhibition of RyR activity reduced ER-stress marker protein levels in CNG-channel-deficient mice. Cnga3^−/−;Nrl^−/− mice received RyR inhibitor or vehicle treatment for 7 days beginning on P8. At the end of the treatment, the protein expression levels of ER-stress marker proteins phospho-IRE1α, cleaved ATF6, and phospho-eIF2α, as well as phosphoprotein and total RyR2 protein levels, were analyzed in the retina via Western blot analysis. Shown are representative Western blot images of the RyR inhibitor or vehicle-treated Cnga3^−/−;Nrl^−/− mice and Nrl^−/− mice with corresponding quantitative analysis following normalization to internal loading control β-actin. Data are presented as mean ± S.D. of at least three independent assays using eyes from 6–8 mice/group. Unpaired Student's t test was used to determine significance between RyR inhibitor and vehicle-treated Cnga3^−/−;Nrl^−/− mice (*, p < 0.05; **, p < 0.01).

Figure 9.

Genetic deletion of IP₃R1 improved cone survival in Cnga3^−/− mice. A and B, shown are representative confocal images of immunofluorescence for peanut agglutinin (PNA) in control wild-type (WT), Itpr1^flox/flox;Hrgp-Cre,Cnga3^−/−, and Cnga3^−/−;Itpr1^flox/flox;Hrgp-Cre mice at 4 months (4M) with quantification. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. C, shown are quantifications of the scotopic and photopic ERG responses in WT and Itpr1^flox/flox;Hrgp-Cre mice at 1 month (1M) and 3–5 months (3–5M). Data are presented as mean ± S.D. using eyes from at least four mice/group. Unpaired Student's t test was used to determine significance between age-matched groups (*, p < 0.05; **, p < 0.01; ***, p < 0.001).