Parmar Tanu, Parmar Vipul M, Arai Eisuke, Sahu Bhubanananda, Perusek Lindsay, Maeda Akiko
Department of Ophthalmology and Visual Sciences, Case Western Reserve University, Cleveland, Ohio, United States.
Department of Ophthalmology and Visual Sciences, Case Western Reserve University, Cleveland, Ohio, United States 2Department of Pharmacology, Case Western Reserve University, Cleveland, Ohio, United States.
Invest Ophthalmol Vis Sci. 2016 Jun 1;57(7):3257-67. doi: 10.1167/iovs.15-18993.
Mice lacking ATP-binding cassette transporter 4 (ABCA4) and retinol dehydrogenase 8 (RDH8) mimic features of human Stargardt disease and age-related macular degeneration. RNA-sequencing of whole eyes was done to study early gene expression changes in Abca4-/-Rdh8-/- mice.
Abca4-/-Rdh8-/- mice at 4 weeks of age were exposed to intense light. Total RNA was extracted from whole eyes and used to generate RNA libraries that were paired-end sequenced on the Illumina HiSeq 2500 device. Differentially expressed genes were annotated using Gene set enrichment analysis (GSEA). Selected genes in enriched pathways exhibiting differential expression were validated using quantitative qRT-PCR and ELISA.
Transcriptome analysis of the whole eye identified 200 genes that were differentially expressed 24 hours after light exposure compared to no light in Abca4-/-Rdh8-/- mice. Expression of several visual cycle and photoreceptor genes were decreased, indicative of photoreceptor/RPE cell death. Gene categories of early stress response genes, inflammatory cytokines, immune factors, and JAK STAT components were upregulated. Lipocalin 2 (Lcn2) was the most upregulated early stress response gene identified. Protein LCN2 was produced by RPE cells and the neural retina after intense light exposure as well as in cultured RPE cells from mice and humans incubated with lipopolysaccharide or photoreceptor outer segments.
Identification of important mediators involved in the crosstalk between the acute stress response and immune activation in RPE cells and the neural retina, such as LCN2, provide novel molecular targets for reducing cellular stress during retinal degeneration.
缺乏ATP结合盒转运蛋白4（ABCA4）和视黄醇脱氢酶8（RDH8）的小鼠可模拟人类斯塔加特病和年龄相关性黄斑变性的特征。对全眼进行RNA测序以研究Abca4-/-Rdh8-/-小鼠早期的基因表达变化。
4周龄的Abca4-/-Rdh8-/-小鼠暴露于强光下。从全眼中提取总RNA，并用于构建RNA文库，该文库在Illumina HiSeq 2500设备上进行双端测序。使用基因集富集分析（GSEA）对差异表达基因进行注释。使用定量qRT-PCR和ELISA对富集途径中表现出差异表达的选定基因进行验证。
对全眼的转录组分析确定，与未暴露于光的Abca4-/-Rdh8-/-小鼠相比，在强光暴露24小时后有200个基因差异表达。几个视觉循环和光感受器基因的表达下降，表明光感受器/RPE细胞死亡。早期应激反应基因、炎性细胞因子、免疫因子和JAK STAT成分的基因类别上调。脂质运载蛋白2（Lcn2）是鉴定出的上调最明显的早期应激反应基因。蛋白质LCN2在强光暴露后由RPE细胞和神经视网膜产生，在来自小鼠和人类的培养RPE细胞中，用脂多糖或光感受器外段孵育后也会产生。
鉴定出参与RPE细胞和神经视网膜中急性应激反应与免疫激活之间相互作用的重要介质，如LCN2，为在视网膜变性期间减轻细胞应激提供了新的分子靶点。
