State Key Laboratory of Medical Neurobiology and MOE Frontiers Center for Brain Science, Institutes of Brain Science, Fudan University, Shanghai, China.
Institutes of Neuroscience and Third Affiliated Hospital, Henan Provincial People's Hospital, Henan Eye Institute, Henan Eye Hospital, People's Hospital of Zhengzhou University, Zhengzhou University, Zhengzhou, China.
Glia. 2023 Mar;71(3):720-741. doi: 10.1002/glia.24307. Epub 2022 Nov 23.
Deficiency of glutamate transporter GLAST in Müller cells may be culpable for excessive extracellular glutamate, which involves in retinal ganglion cell (RGC) damage in glaucoma. We elucidated how GLAST was regulated in rat chronic ocular hypertension (COH) model. Western blot and whole-cell patch-clamp recordings showed that GLAST proteins and GLAST-mediated current densities in Müller cells were downregulated at the early stages of COH. In normal rats, intravitreal injection of the ephrinA3 activator EphA4-Fc mimicked the changes of GLAST in COH retinas. In purified cultured Müller cells, EphA4-Fc treatment reduced GLAST expression at mRNA and protein levels, which was reversed by the tyrosine kinase inhibitor PP2 or transfection with ephrinA3-siRNA (Si-EFNA3), suggesting that EphA4/ephrinA3 reverse signaling mediated GLAST downregulation. EphA4/ephrinA3 reverse signaling-induced GLAST downregulation was mediated by inhibiting PI3K/Akt/NF-κB pathways since EphA4-Fc treatment of cultured Müller cells reduced the levels of p-Akt/Akt and NF-κB p65, which were reversed by transfecting Si-EFNA3. In Müller cells with ephrinA3 knockdown, the PI3K inhibitor LY294002 still decreased the protein levels of NF-κB p65 in the presence of EphA4-Fc, and the mRNA levels of GLAST were reduced by LY294002 and the NF-κB inhibitor SN50, respectively. Pre-injection of the PI3K/Akt pathway activator 740 Y-P reversed the GLAST downregulation in COH retinas. Western blot and TUNEL staining showed that transfecting of Si-EFNA3 reduced Müller cell gliosis and RGC apoptosis in COH retinas. Our results suggest that activated EphA4/ephrinA3 reverse signaling induces GLAST downregulation in Müller cells via inhibiting PI3K/Akt/NF-κB pathways, thus contributing to RGC damage in glaucoma.
谷氨酸转运体 GLAST 在 Müller 细胞中的缺乏可能导致细胞外谷氨酸的过度积累，从而参与青光眼的视网膜神经节细胞（RGC）损伤。我们阐明了在大鼠慢性高眼压（COH）模型中 GLAST 是如何被调节的。Western blot 和全细胞膜片钳记录显示，在 COH 的早期阶段，Müller 细胞中的 GLAST 蛋白和 GLAST 介导的电流密度下调。在正常大鼠中，玻璃体内注射 EphrinA3 激动剂 EphA4-Fc 可模拟 COH 视网膜中 GLAST 的变化。在纯化培养的 Müller 细胞中，EphA4-Fc 处理降低了 GLAST 在 mRNA 和蛋白水平上的表达，这可以被酪氨酸激酶抑制剂 PP2 或 EphrinA3-siRNA（Si-EFNA3）转染逆转，表明 EphA4/ephrinA3 反向信号介导 GLAST 下调。EphA4/ephrinA3 反向信号诱导的 GLAST 下调是通过抑制 PI3K/Akt/NF-κB 通路介导的，因为 EphA4-Fc 处理培养的 Müller 细胞降低了 p-Akt/Akt 和 NF-κB p65 的水平，而 Si-EFNA3 的转染则逆转了这一过程。在 EphrinA3 敲低的 Müller 细胞中，PI3K 抑制剂 LY294002 仍能在 EphA4-Fc 存在的情况下降低 NF-κB p65 的蛋白水平，而 LY294002 和 NF-κB 抑制剂 SN50 分别降低了 GLAST 的 mRNA 水平。预先注射 PI3K/Akt 通路激活剂 740 Y-P 可逆转 COH 视网膜中的 GLAST 下调。Western blot 和 TUNEL 染色显示，转染 Si-EFNA3 可减少 COH 视网膜中的 Müller 细胞胶质增生和 RGC 凋亡。我们的结果表明，激活的 EphA4/ephrinA3 反向信号通过抑制 PI3K/Akt/NF-κB 通路诱导 Müller 细胞中的 GLAST 下调，从而导致青光眼的 RGC 损伤。
