School of Optometry and Vision Science Program, University of California, Berkeley, Berkeley, CA, USA.
School of Optometry and Vision Science Program, University of California, Berkeley, Berkeley, CA, USA.
Exp Eye Res. 2018 Nov;176:29-39. doi: 10.1016/j.exer.2018.06.026. Epub 2018 Jun 26.
We studied the role of sodium/proton exchanger 8 (NHE8) in retinal pigment epithelium (RPE) and photoreceptor cells of adult mouse retina by using the clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease (Cas)9 from Neisseria meningitidis (Nm). Specific single guide RNAs (sgRNAs) were designed to knockdown the Slc9a8 gene, which encodes the NHE8. Nuclease null NmCas9 and sgRNAs were packaged respectively using adeno-associated viral vector (AAV), and delivered into mouse eyes in vivo by subretinal injection on wild-type mice of about four-week-old when mouse retina is fully developed. Eye samples were collected four weeks after injection for phenotype examination. Real-time PCR analysis demonstrated ∼38% reduction of NHE8 transcripts in retinas injected with AAV-knockdown sgRNA and AAV-Cas9. Loss of photoreceptor cells was found in eyes injected with AAV-knockdown sgRNA and AAV-Cas9 under either the human rhodopsin promoter or the minimal chicken β-actin promoter, while normal morphology was observed in control eyes injected with AAV-Cas9 and AAV-control sgRNA; immunostaining data showed degenerating photoreceptor cells and RPE cells in eyes injected with knockdown sgRNA and Cas9 AAVs. We further determined that mutant M120K-NHE8 displayed altered intracellular pH regulation in human RPE and primary mouse RPE cells using genetically encoded pH sensor pHluorin and that primary cultured NHE8 mutant RPE cells showed different pH titration curves. These results indicate that NHE8 plays essential function in both RPE and photoreceptor cells. NHE8 dysfunction either in photoreceptor or RPE is sufficient to cause retinal degeneration in adult mice at any age.
我们使用奈瑟氏脑膜炎球菌（Neisseria meningitidis）的 CRISPR 相关内切酶（Cas）9 研究了钠/质子交换器 8（NHE8）在成年小鼠视网膜的视网膜色素上皮（RPE）和光感受器细胞中的作用。设计了特定的单引导 RNA（sgRNA）来敲低编码 NHE8 的 Slc9a8 基因。分别使用腺相关病毒（AAV）包装无核酸酶的 NmCas9 和 sgRNA，并在大约四周大的野生型小鼠的视网膜完全发育时通过视网膜下注射将其递送至活体小鼠眼内。在注射后四周收集眼样本进行表型检查。实时 PCR 分析表明，在注射了 AAV 敲低 sgRNA 和 AAV-Cas9 的视网膜中，NHE8 转录本减少了约 38%。在注射了 AAV 敲低 sgRNA 和 AAV-Cas9 的眼中，无论使用人视紫红质启动子还是最小鸡 β-肌动蛋白启动子，光感受器细胞都丢失了，而在注射了 AAV-Cas9 和 AAV 对照 sgRNA 的对照眼中观察到正常形态；免疫染色数据显示，在注射了敲低 sgRNA 和 Cas9 AAV 的眼中，光感受器细胞和 RPE 细胞退化。我们进一步确定，使用遗传编码的 pH 传感器 pHluorin，突变体 M120K-NHE8 显示出人 RPE 和原代小鼠 RPE 细胞中细胞内 pH 调节的改变，并且原代培养的 NHE8 突变体 RPE 细胞显示出不同的 pH 滴定曲线。这些结果表明 NHE8 在 RPE 和光感受器细胞中均发挥重要功能。NHE8 在光感受器或 RPE 中的功能障碍足以在任何年龄的成年小鼠中引起视网膜变性。
