Department of Ophthalmology, State Key Laboratory of Medical Neurobiology and MOE Frontiers Center for Brain Science, Institutes of Brain Science, Zhongshan Hospital, Fudan University, Shanghai, 200032, China.
Department of Ophthalmology and Visual Science, Eye & ENT Hospital, Shanghai Key Laboratory of Visual Impairment and Restoration, State Key Laboratory of Medical Neurobiology and MOE Frontiers Center for Brain Science, Institutes of Brain Science, Fudan University, Shanghai, 200031, China.
Neuropharmacology. 2020 Nov 1;178:108228. doi: 10.1016/j.neuropharm.2020.108228. Epub 2020 Aug 1.
Previous studies have demonstrated that EphA4 participates in neuronal injury, and there is a strong interaction between ephrinA3 and EphA4. In this study, we showed that in a rat chronic ocular hypertension (COH) experimental glaucoma model, expression of EphA4 and ephrinA3 proteins was increased in retinal cells, including retinal ganglion cells (RGCs) and Müller cells, which may result in ephrinA3/EphA4 forward signaling activation on RGCs, as evidenced by increased p-EphA4/EphA4 ratio. Intravitreal injection of ephrinA3-Fc, an activator of EphA4, mimicked the effect of COH on p-EphA4/EphA4 and induced an increase in TUNEL-positive signals in normal retinas, which was accompanied by dendritic spine retraction and thinner dendrites in RGCs. Furthermore, Intravitreal injection of ephrinA3-Fc increased the levels of phosphorylated src and GluA2 (p-src and p-GluA2). Co-immunoprecipitation assay demonstrated interactions between EphA4, p-src and GluA2. Intravitreal injection of ephrinA3-Fc reduced the expression of GluA2 proteins on the surface of normal retinal cells, which was prevented by intravitreal injection of PP2, an inhibitor of src-family tyrosine kinases. Pre-injection of PP2 or the Ca-permeable GluA2-lacking AMPA receptor inhibitor Naspm significantly and partially reduced the number of TUNEL-positive RGCs in the ephrinA3-Fc-injected and COH retinas. Our results suggest that activated ephrinA3/EphA4 forward signaling promoted GluA2 endocytosis, then resulted in dendritic spine retraction of RGCs, thus contributing to RGC apoptosis in COH rats. Attenuation of the strength of ephrinA/EphA signaling in an appropriate manner may be an effective way for preventing the loss of RGCs in glaucoma.
先前的研究表明 EphA4 参与神经元损伤，并且 EphrinA3 和 EphA4 之间存在强烈的相互作用。在这项研究中，我们表明在大鼠慢性高眼压 (COH) 实验性青光眼模型中，视网膜细胞（包括视网膜神经节细胞 [RGC] 和 Müller 细胞）中 EphA4 和 EphrinA3 蛋白的表达增加，这可能导致 EphrinA3/EphA4 正向信号在 RGC 上激活，正如增加的 p-EphA4/EphA4 比值所证明的那样。 EphrinA3-Fc（EphA4 的激活剂）的玻璃体内注射模拟了 COH 对 p-EphA4/EphA4 的影响，并在正常视网膜中诱导 TUNEL 阳性信号增加，同时伴有 RGC 树突棘回缩和树突变细。此外，玻璃体内注射 EphrinA3-Fc 增加了磷酸化 src 和 GluA2（p-src 和 p-GluA2）的水平。共免疫沉淀实验表明 EphA4、p-src 和 GluA2 之间存在相互作用。玻璃体内注射 EphrinA3-Fc 减少了正常视网膜细胞表面 GluA2 蛋白的表达，这一现象被 src 家族酪氨酸激酶抑制剂 PP2 的玻璃体内注射所阻止。在注射 EphrinA3-Fc 之前注射 PP2 或钙通透性 GluA2 缺失型 AMPA 受体抑制剂 Naspm 可显著减少 EphrinA3-Fc 注射和 COH 视网膜中 TUNEL 阳性 RGC 的数量。我们的研究结果表明，激活的 EphrinA3/EphA4 正向信号促进了 GluA2 内吞作用，从而导致 RGC 树突棘回缩，从而导致 COH 大鼠中 RGC 凋亡。以适当的方式减弱 EphrinA/EphA 信号的强度可能是防止青光眼 RGC 损失的有效方法。
