Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, USA.
J Biol Chem. 2012 May 25;287(22):18018-29. doi: 10.1074/jbc.M112.342220. Epub 2012 Apr 9.
Cyclic nucleotide-gated (CNG) channels play a pivotal role in phototransduction. Mutations in the cone CNG channel subunits CNGA3 and CNGB3 account for >70% of all known cases of achromatopsia. Cones degenerate in achromatopsia patients and in CNGA3(-/-) and CNGB3(-/-) mice. This work investigates the molecular basis of cone degeneration in CNG channel deficiency. As cones comprise only 2-3% of the total photoreceptor population in the wild-type mouse retina, we generated mouse lines with CNG channel deficiency on a cone-dominant background, i.e. CNGA3(-/-)/Nrl(-/-) and CNGB3(-/-)/Nrl(-/-) mice. The retinal phenotype and potential cell death pathways were examined by functional, biochemical, and immunohistochemical approaches. CNGA3(-/-)/Nrl(-/-) and CNGB3(-/-)/Nrl(-/-) mice showed impaired cone function, opsin mislocalization, and cone degeneration similar to that in the single knock-out mice. The endoplasmic reticulum stress marker proteins, including Grp78/Bip, phospho-eIF2α, phospho-IP(3)R, and CCAAT/enhancer-binding protein homologous protein, were elevated significantly in CNGA3(-/-)/Nrl(-/-) and CNGB3(-/-)/Nrl(-/-) retinas, compared with the age-matched (postnatal 30 days) Nrl(-/-) controls. Along with these, up-regulation of the cysteine protease calpains and cleavage of caspase-12 and caspase-7 were found in the channel-deficient retinas, suggesting an endoplasmic reticulum stress-associated apoptosis. In addition, we observed a nuclear translocation of apoptosis-inducing factor (AIF) and endonuclease G in CNGA3(-/-)/Nrl(-/-) and CNGB3(-/-)/Nrl(-/-) retinas, implying a mitochondrial insult in the endoplasmic reticulum stress-activated cell death process. Taken together, our findings suggest a crucial role of endoplasmic reticulum stress in cone degeneration associated with CNG channel deficiency.
环核苷酸门控（CNG）通道在光转导中起着关键作用。在所有已知的色盲病例中，圆锥细胞 CNG 通道亚基 CNGA3 和 CNGB3 的突变占比超过 70%。在色盲患者和 CNGA3(-/-)和 CNGB3(-/-)小鼠中，圆锥细胞会退化。这项工作研究了 CNG 通道缺陷导致的圆锥细胞退化的分子基础。由于在野生型小鼠视网膜中，圆锥细胞仅占总光感受器群体的 2-3%，我们在以圆锥细胞为主的背景下生成了 CNG 通道缺陷的小鼠模型，即 CNGA3(-/-)/Nrl(-/-)和 CNGB3(-/-)/Nrl(-/-)小鼠。通过功能、生化和免疫组织化学方法研究了视网膜表型和潜在的细胞死亡途径。CNGA3(-/-)/Nrl(-/-)和 CNGB3(-/-)/Nrl(-/-)小鼠表现出圆锥细胞功能受损、视蛋白定位错误和与单敲除小鼠相似的圆锥细胞退化。内质网应激标记蛋白，包括 Grp78/Bip、磷酸化 eIF2α、磷酸化 IP(3)R 和 CCAAT/增强子结合蛋白同源蛋白，在 CNGA3(-/-)/Nrl(-/-)和 CNGB3(-/-)/Nrl(-/-)视网膜中显著升高，与年龄匹配的（出生后 30 天）Nrl(-/-)对照相比。此外，在通道缺陷的视网膜中发现半胱氨酸蛋白酶钙蛋白酶的上调和 caspase-12 和 caspase-7 的切割，表明存在内质网应激相关的细胞凋亡。此外，我们观察到凋亡诱导因子（AIF）和内切核酸酶 G 在 CNGA3(-/-)/Nrl(-/-)和 CNGB3(-/-)/Nrl(-/-)视网膜中的核转位，暗示在内质网应激激活的细胞死亡过程中存在线粒体损伤。总之，我们的研究结果表明，内质网应激在 CNG 通道缺陷相关的圆锥细胞退化中起着关键作用。
