Involvement of ER stress in retinal cell death
Involvement of ER stress in retinal cell death
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发表时间：
2007-04
期刊：
影响因子：
2.2
通讯作者：
M. Shimazawa;Yuta Inokuchi;Yasushi Ito;Hiroshi Murata;M. Aihara;M. Miura;M. Araie;H. Hara
中科院分区：
文献类型：
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作者：
M. Shimazawa;Yuta Inokuchi;Yasushi Ito;Hiroshi Murata;M. Aihara;M. Miura;M. Araie;H. Hara
目的利用体外培养的视网膜神经节细胞（RGC-5，E1 A病毒转化的大鼠神经节细胞系）和携带人XBP 1和Venus a变体的绿色荧光蛋白（GFP）融合基因的转基因小鼠ER应激激活指示剂（ERAI），研究内质网（ER）应激是否参与视网膜细胞死亡。方法用衣霉素诱导RGC-5细胞损伤，Hoechst 33342和YO-PRO-1或碘化丙啶双染色法检测细胞活力。采用免疫印迹或免疫染色法检测衣霉素（体外或体内）或N-甲基-D-天冬氨酸（NMDA;体内）处理后葡萄糖调节蛋白78（GRP 78）/BiP、真核起始因子2α（p-eIF 2 α）的磷酸化形式和C/EBP同源（CHOP）蛋白的表达。携带F-XBP 1-DBD-venus表达基因的ERAI小鼠用于监测体内ER应激。玻璃体内注射衣霉素或NMDA后24小时，或升高眼内压（IOP）后，使用检眼镜观察麻醉动物的视网膜荧光强度，并使用激光共聚焦显微镜观察视网膜平片或横截面标本。结果衣霉素诱导RGC-5细胞凋亡，并诱导ER应激相关蛋白（BiP、eIF 2 α磷酸化形式和CHOP蛋白）的产生。在体内，衣霉素诱导视网膜神经节细胞（RGC）的损失和内丛状层变薄，玻璃体内注射后7天。在ERAI小鼠的平铺视网膜中，在衣霉素、NMDA或IOP升高后24小时，由XBP-1-venus融合蛋白产生的荧光强度增加，表明ER应激激活。在ERAI小鼠的横截面中，荧光强度首先在神经节细胞和内丛状层的细胞中增加，分别在12和24 h，NMDA注射后，并且其分别在12和24 h定位于神经节细胞和无长突细胞，并且在72 h定位于小胶质细胞。在注射NMDA后12小时，BiP和CHOP增加，并且在72小时观察期的剩余时间内持续增加。结论无论是由NMDA还是由眼压升高引起的RGC死亡，ER应激都可能起着关键作用。
Purpose To clarify whether endoplasmic reticulum (ER) stress is involved in retinal cell death, using cultured retinal ganglion cells (RGC-5, a rat ganglion cell line transformed with E1A virus), and transgenic mice ER stress-activated indicator (ERAI) mice carrying a human XBP1 and venus a variant of green fluorescent protein (GFP) fusion gene. Methods RGC-5 damage was induced by tunicamycin, and cell viability was measured by double nuclear staining (Hoechst 33342 and either YO-PRO-1 or propidium iodide). The expressions of glucose-regulated protein 78(GRP78)/BiP, the phosphorylated form of eukaryotic initiation factor 2α (p-eIF2α), and C/EBP-homologous (CHOP) protein after tunicamycin (in vitro or in vivo) or N-methyl-D-aspartate (NMDA; in vivo) treatment were measured using immunoblot or immunostaining. ERAI mice carrying the F-XBP1-DBD-venus expression gene were used to monitor ER-stress in vivo. Twenty-four hours after intravitreal injection of tunicamycin or NMDA, or after raising intraocular pressure (IOP), the retinal fluorescence intensity was visualized in anesthetized animals using an ophthalmoscope and in retinal flatmount or cross-section specimens using laser confocal microscopy. Results Treatment with tunicamycin induced apoptotic cell death in RGC-5 and also induced production of ER stress-related proteins (BiP, the phosphorylated form of eIF2α, and CHOP protein). In vivo, tunicamycin induced retinal ganglion cell (RGC) loss and thinning of the inner plexiform layer, 7 days after intravitreal injection. In flatmounted retinas of ERAI mice, the fluorescence intensity arising from the XBP-1-venus fusion protein, indicating ER-stress activation, was increased at 24 h after tunicamycin, NMDA, or IOP elevation. In transverse cross-sections from ERAI mice, the fluorescence intensity was first increased in cells of the ganglion cell and inner plexiform layers at 12 and 24 h, respectively, after NMDA injection, and it was localized to ganglion and amacrine cells at 12 and 24 h, respectively, and to microglial cells at 72 h. BiP and CHOP were increased at 12 h after NMDA injection, and the increases persisted for the remainder of the 72 h observation period. Conclusions These data indicate that ER-stress may play a pivotal role in RGC death, whether induced by NMDA or IOP elevation.