Downregulation of ATP synthase subunit-6, cytochrome c oxidase-III, and NADH dehydrogenase-3 by bright cyclic light in the rat retina
Downregulation of ATP synthase subunit-6, cytochrome c oxidase-III, and NADH dehydrogenase-3 by bright cyclic light in the rat retina
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DOI：
10.1167/iovs.03-1081
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发表时间：
2004-08-01
影响因子：
4.4
通讯作者：
Anderson, RE
中科院分区：
文献类型：
--
作者：
Huang, H;Li, F;Anderson, RE
目的.在明亮的周期性光（300-800勒克斯）下出生和长大的白化病大鼠的视网膜比在昏暗的周期性光（5勒克斯）下出生和长大的动物的视网膜对光诱导的细胞凋亡更不敏感。本研究旨在通过差异显示（DD）-PCR技术筛选差异表达的基因，研究周期性光照对大鼠视网膜神经保护作用的机制。白化病大鼠出生并在5或400勒克斯循环光照（12小时开/关）下饲养。在6至8周龄时，将动物处死以收获视网膜或暴露于1700勒克斯照明12或24小时。通过DNA片段化和定量组织学方法评价400勒克斯周期性光照饲养的神经保护作用。DD-PCR鉴定差异表达的候选基因。北方印迹分析用于定量所选基因的差异表达。Western blot和酶活性分析确定蛋白质的差异表达。转录本的细胞定位通过原位杂交确定。DNA片段化和定量组织学结果表明，400勒克斯的周期性光饲养保护视网膜光诱导的细胞凋亡相比，5勒克斯的周期性光饲养。DD-PCR分析表明，一个283 bp的表达序列标签（EST）在大鼠视网膜从出生到400勒克斯循环光下调。经BLAST检索，该EST被鉴定为大肠杆菌编码的NADH脱氢酶亚基3（ND-3）的3 '末端序列。北方印迹分析表明，EST与两个mRNA转录本杂交，其中较大的一个转录本包含三磷酸腺苷（ATP）合成酶亚基6（ATPase-6）、细胞色素c氧化酶亚基III（CO-III）、ND-3和tRNA-Gly。北方印迹分析表明，CO-III和ATP酶-6下调1.8-和2.3-倍，由400-lux的循环光相比，5-lux的循环光，分别，但是，没有变化，细胞色素c氧化酶亚基I和II（CO-I和-II）或12 S核糖体RNA（12 S rRNA），线粒体管家基因。用抗CO-III抗体进行的Western blot分析显示，暗光饲养大鼠视网膜线粒体中CO-III蛋白表达增加。在暗光饲养的大鼠视网膜匀浆中，CO的酶活性是强光饲养的大鼠的2倍。用35 S标记的CO-III探针进行的原位杂交显示，CO-III转录本在强光饲养动物的视网膜层中存在并下调。饲养在400勒克斯循环光下调ATP酶-6，CO-III和ND-3的表达相比，饲养在5勒克斯循环光。作者假设，这些变化是对光应激的适应性反应，通过升高应激相关因子的水平和降低氧化细胞色素c的水平来为视网膜细胞提供神经保护，氧化细胞色素c是激活细胞死亡的凋亡级联的形式。
PURPOSE. Retinas of albino rats born and raised in bright cyclic light (300-800 lux) are less susceptible to light-induced apoptosis than retinas of animals born and raised in dim cyclic light (5 lux). In this study, the objective was to study the mechanisms of neuroprotection in the bright cyclic light-reared retina by identification of differentially expressed genes with differential display (DD)-PCR.METHODS. Albino rats were born and raised in 5- or 400-lux cyclic light (12 hours on/off). At 6 to 8 weeks of age, animals were either killed to harvest retinas or exposed to 1700 lux illumination for 12 or 24 hours. The neuroprotection of 400-lux cyclic light rearing was evaluated by DNA fragmentation and quantitative histology. The differentially expressed candidate genes were identified by DD-PCR. Northern blot analysis was used to quantitate differential expression of selected genes. Differential expression of protein was determined by Western blot and enzyme activity analysis. Cellular localization of transcripts was determined by in situ hybridization.RESULTS. DNA fragmentation and quantitative histology results indicated that 400-lux cyclic light rearing protected the retina from light-induced apoptosis compared with 5-lux cyclic light rearing. DD-PCR analysis showed that a 283-bp expressed sequence tag (EST) was downregulated in retinas of rats raised from birth in 400-lux cyclic light. A BLAST search identified the EST as the 3'-terminal sequence of mitochondria-encoded NADH dehydrogenase subunit 3 (ND-3). Northern blot analysis showed that the EST hybridized to two mRNA transcripts, the larger of which was confirmed to encompass the adenosine triphosphate (ATP) synthase subunit 6 (ATPase-6), cytochrome c oxidase subunit III (CO-III), ND-3, and tRNA-Gly. Northern blot analysis demonstrated that CO-III and ATPase-6 were downregulated 1.8- and 2.3-fold by 400-lux cyclic light compared with 5-lux cyclic light, respectively; however, there was no change in cytochrome c oxidase subunit I and II (CO-I and -II) or in 12S ribosomal RNA (12S rRNA), a mitochondrial housekeeping gene. Western blot analysis using anti-CO-III antibody showed more CO-III protein in retinal mitochondria from dim-light-raised rats. The enzyme activity of CO was two times higher in retinal homogenates from dim-light-raised rats than those from bright-light-raised rats. In situ hybridization using a (35)S-labeled CO-III probe showed that the CO-III transcript was present and downregulated in most of the retinal layers of bright-light-reared animals.CONCLUSIONS. Rearing in cyclic light at 400-lux downregulates the expression of ATPase-6, CO-III, and ND-3 compared with rearing in 5-lux cyclic light. The authors hypothesize that these changes are adaptive responses to light stress that provide neuroprotection to retinal cells by elevating the level of stress-related factors and reducing the level of oxidized cytochrome c, the form that activates the apoptotic cascade of cell death.