Dong Ling-Dan, Gao Feng, Wang Xiao-Han, Miao Yanying, Wang Shu-Yue, Wu Yi, Li Fang, Wu Jihong, Cheng Xiang-Lin, Sun Xing-Huai, Yang Xiong-Li, Wang Zhongfeng
Institutes of Brain Science, Institute of Neurobiology, State Key Laboratory of Medical Neurobiology, Collaborative Innovation Center for Brain Science, Fudan University, Shanghai 200032, People's Republic of China, and.
Institutes of Brain Science, Eye & ENT Hospital, State Key Laboratory of Medical Neurobiology, Shanghai Key Laboratory of Visual Impairment and Restoration, and Collaborative Innovation Center for Brain Science, Fudan University, Shanghai 200032, People's Republic of China, and.
J Neurosci. 2015 Apr 1;35(13):5409-21. doi: 10.1523/JNEUROSCI.4376-14.2015.
EphB1, expressed in Müller cells, and ephrinB2, expressed in both Müller cells and retinal ganglion cells (RGCs), constitute an EphB/ephrinB reverse signaling in RGCs. Whether and how this reverse signaling is involved in RGC apoptosis in a rat chronic ocular hypertension (COH) model was investigated. In the COH model, both EphB1 and ephrinB2 were significantly increased and the reverse signaling was activated, which was accompanied by increased protein levels of phosphorylated (p) src, GluA2, and p-GluA2. Intravitreal injection of EphB2-Fc, an activator of ephrinB2, induced an increase in TUNEL-positive signals in normal retinae. A coimmunoprecipitation assay demonstrated direct interactions among ephrinB2, p-src, and GluA2. Moreover, in COH rats the expression of GluA2 proteins on the surface of retinal cells was decreased. Such GluA2 endocytosis could be prevented by preoperational intravitreal injection of 4-amino-3-(4-chlorophenyl)-1-(t-butyl)-1H-pyrazolo [3,4-d] pyrimidine (PP2), an inhibitor of src family tyrosine kinases, and possibly involved the protein interacting with C kinase 1 and phosphorylation of GluA2. In normal rats, intravitreal injection of EphB2-Fc caused changes in these protein levels similar to those observed in COH rats, which all could be avoided by preinjection of PP2. Patch-clamp experiments further showed that the current-voltage relationship of AMPA receptor-mediated EPSCs of RGCs exhibited stronger inward rectification in EphB2-Fc-injected rats. Furthermore, preinjection of PP2 or N-[3-[[4-[(3-aminopropyl)amino]butyl]amino]propyl]-1-naphthaleneacetamide trihydrochloride) (Naspm), a Ca(2+)-permeable GluA2-lacking AMPA receptor inhibitor, remarkably inhibited RGC apoptosis in either EphB2-Fc-injected or COH rats. Together, elevated GluA2 trafficking induced by activated EphB2/ephrinB2 reverse signaling likely contributes to RGC apoptosis in COH rats.
在穆勒细胞中表达的EphB1和在穆勒细胞及视网膜神经节细胞（RGCs）中均表达的ephrinB2，在RGCs中构成EphB/ephrinB反向信号通路。本研究探讨了该反向信号通路是否以及如何参与大鼠慢性高眼压（COH）模型中的RGC凋亡。在COH模型中，EphB1和ephrinB2均显著增加，反向信号通路被激活，同时磷酸化（p）src、GluA2和p-GluA2的蛋白水平升高。玻璃体内注射ephrinB2激活剂EphB2-Fc可诱导正常视网膜中TUNEL阳性信号增加。免疫共沉淀试验证明ephrinB2、p-src和GluA2之间存在直接相互作用。此外，在COH大鼠中，视网膜细胞表面GluA2蛋白的表达降低。玻璃体内预先注射src家族酪氨酸激酶抑制剂4-氨基-3-（4-氯苯基）-1-（叔丁基）-1H-吡唑并[3,4-d]嘧啶（PP2）可防止这种GluA2内吞作用，这可能涉及与C激酶1相互作用的蛋白和GluA2的磷酸化。在正常大鼠中，玻璃体内注射EphB2-Fc导致这些蛋白水平发生与COH大鼠中观察到的类似变化，而预先注射PP2可避免所有这些变化。膜片钳实验进一步表明，在注射EphB2-Fc的大鼠中，RGCs的AMPA受体介导的兴奋性突触后电流（EPSCs）的电流-电压关系表现出更强的内向整流。此外，预先注射PP2或N-[3-[[4-[(3-氨基丙基)氨基]丁基]氨基]丙基]-1-萘乙酰胺三盐酸盐（Naspm，一种缺乏Ca(2+)通透性的GluA2的AMPA受体抑制剂）可显著抑制注射EphB2-Fc的大鼠或COH大鼠中的RGC凋亡。总之，由激活的EphB2/ephrinB2反向信号通路诱导的GluA2转运增加可能促成了COH大鼠中的RGC凋亡。
