Activation of Bax in Three Models of Retinitis Pigmentosa
Activation of Bax in Three Models of Retinitis Pigmentosa
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DOI：
10.1167/iovs.14-13917
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发表时间：
2014-06-01
影响因子：
4.4
通讯作者：
Marigo, Valeria
中科院分区：
文献类型：
--
作者：
Comitato, Antonella;Sanges, Daniela;Marigo, Valeria
目的.视网膜色素变性感光细胞死亡的过程仍然没有得到很好的表征，识别共同的机制将有助于治疗策略的发展。在此，我们研究了在rd1、P23 H转基因和Rho敲除视网膜中Bax的激活。Bax活化通过使用抗活化的Bax特异性抗体的免疫荧光和通过线粒体蛋白提取物上的Western印迹进行评价。敲低组织蛋白酶D，钙蛋白酶1，钙蛋白酶2实现了短发夹RNA（shRNA）交付rd1突变体光感受器细胞分化的视网膜神经球。通过玻璃体内注射钙蛋白酶抑制剂，在体内评估Bax通过钙蛋白酶激活的机制。我们定义了视网膜色素变性的三种模型中Bax的激活和线粒体定位以及钙蛋白酶和组织蛋白酶D的激活。利用rd1突变型光感受器的体外培养系统，我们解开了Bax激活的机制。我们证明，钙蛋白酶1和组织蛋白酶D有助于激活Bax和凋亡诱导因子（AIF）核转位。在体内干扰钙蛋白酶的活性阻断Bax的激活在rd1和Rho敲除视网膜和减少激活在P23 H转基因视网膜。在所有三种视网膜色素变性模型中均观察到Bax的激活，并通过定位于视网膜而导致神经损伤。我们的数据表明，Bax可以设想作为一个有前途的目标分子，抑制感光细胞变性。
PURPOSE. The process of photoreceptor cell death in retinitis pigmentosa is still not well characterized, and identification of common mechanisms will be instrumental for development of therapeutic strategies. Here we investigated activation of Bax in rd1, P23H transgenic, and Rho knockout retinas.METHODS. Bax activation was evaluated by immunofluorescence using anti-activated Bax-specific antibodies and by Western blotting on mitochondrial protein extracts. Knockdown of cathepsin D, calpain 1, and calpain 2 was achieved by short hairpin RNA (shRNA) delivery in rd1 mutant photoreceptors cells differentiated from retinal neurospheres. The mechanism of Bax activation through calpains was evaluated in vivo by intravitreal injection of calpastatin.RESULTS. We defined activation and mitochondrial localization of Bax as well as activation of calpains and cathepsin D in the three models of retinitis pigmentosa. Taking advantage of an in vitro culture system for rd1 mutant photoreceptors, we unraveled the mechanism of Bax activation. We demonstrated that calpain 1 and cathepsin D contributed to activation of Bax and to apoptosis-inducing factor (Aif) nuclear translocation. In vivo interference with calpain activity blocks Bax activation in the rd1 and Rho knockout retinas and reduces activation in the P23H transgenic retina.CONCLUSIONS. Activation of Bax was observed in all three models of retinitis pigmentosa and leads to neurodamage by localization at the mitochondrion. Our data suggest that Bax can be envisaged as one of the promising target molecules for restraining photoreceptor degeneration.