Rao Narsing A, Saraswathy Sindhu, Wu Guey Shuang, Katselis George S, Wawrousek Eric F, Bhat Suraj
Doheny Eye Institute, Keck School of Mewdicine, University of Southern California, Los Angeles, CA 90033, USA.
Invest Ophthalmol Vis Sci. 2008 Mar;49(3):1161-71. doi: 10.1167/iovs.07-1259.
During the early phase of experimental autoimmune uveitis (EAU), before macrophages infiltrate the retina and uvea, photoreceptor mitochondrial oxidative stress, nitration of photoreceptor mitochondrial proteins, and release of cytochrome c have been observed. However, no apoptosis has been detected during this phase. In this study, alphaA-crystallin upregulation in the retina and its antiapoptotic protective role were evaluated in early EAU.
Gene microarrays were first used to identify upregulated genes in retinas with early EAU. Among highly upregulated crystallins, alphaA was confirmed by real-time polymerase chain reaction and Western blot, and the site of upregulation was localized by immunohistochemistry. The association of alphaA-crystallin to nitrated cytochrome c and interaction with a procaspase-3 subunit was assayed. Photoreceptor apoptosis in alphaA knockout mice was compared with that in wild-type animals with EAU, by using the terminal transferase dUTP nick-end labeling assay and polymerase chain reaction.
In early EAU, alphaA-crystallin was increased 33-fold, and the site of increase was localized to the photoreceptor inner segments. This crystallin suppressed apoptosis by associating with the nitrated cytochrome c and p24. The association with nitrated cytochrome c, in particular, appeared to be restricted to nitrated cytochrome c, and thus, no association of non-nitrated cytochrome c was detected. The knockout mice showed signs of EAU development early and showed apoptosis in the retina; no such changes were seen in the wild-type control animals.
alphaA-Crystallin is highly upregulated in the retina during early EAU. This upregulation is localized primarily in the photoreceptor inner segments, the site of mitochondrial oxidative stress. Further, in early EAU, the photoreceptors preferentially use alphaA-crystallin to suppress mitochondrial oxidative stress-mediated apoptosis.
在实验性自身免疫性葡萄膜炎（EAU）的早期阶段，在巨噬细胞浸润视网膜和葡萄膜之前，已观察到光感受器线粒体氧化应激、光感受器线粒体蛋白硝化以及细胞色素c的释放。然而，在此阶段未检测到细胞凋亡。在本研究中，评估了EAU早期视网膜中αA-晶体蛋白的上调及其抗凋亡保护作用。
首先使用基因微阵列来鉴定早期EAU视网膜中上调的基因。在高度上调的晶体蛋白中，通过实时聚合酶链反应和蛋白质印迹法确认了αA，并用免疫组织化学法定位上调的部位。检测了αA-晶体蛋白与硝化细胞色素c的关联以及与半胱天冬酶-3前体亚基的相互作用。通过末端脱氧核苷酸转移酶dUTP缺口末端标记法和聚合酶链反应，比较了αA基因敲除小鼠与患有EAU的野生型动物的光感受器细胞凋亡情况。
在早期EAU中，αA-晶体蛋白增加了33倍，增加部位定位于光感受器内节。这种晶体蛋白通过与硝化细胞色素c和p24结合来抑制细胞凋亡。特别是与硝化细胞色素c的结合似乎仅限于硝化细胞色素c，因此未检测到与未硝化细胞色素c的结合。基因敲除小鼠早期出现EAU发展迹象，并在视网膜中出现细胞凋亡；野生型对照动物未出现此类变化。
在EAU早期，视网膜中αA-晶体蛋白高度上调。这种上调主要定位于光感受器内节，即线粒体氧化应激部位。此外，在EAU早期，光感受器优先利用αA-晶体蛋白来抑制线粒体氧化应激介导的细胞凋亡。
