Wang Juan, Chen Xin, Wang Fang, Zhang Jieping, Li Peng, Li Zongyi, Xu Jingying, Gao Furong, Jin Caixia, Tian Haibin, Zhang Jingfa, Li Weiye, Lu Lixia, Xu Guo-Tong
Department of Ophthalmology of Shanghai Tenth People's Hospital, and Tongji Eye Institute, Tongji University School of Medicine, Shanghai, China.
Department of Regenerative Medicine and Stem Cell Research Center, Tongji University School of Medicine, Shanghai, China.
PLoS One. 2016 May 19;11(5):e0155860. doi: 10.1371/journal.pone.0155860. eCollection 2016.
Ofd1 is a newly identified causative gene for Retinitis pigmentosa (RP), a photoreceptor degenerative disease. This study aimed to examine Ofd1 localization in retina and further to investigate its function in photoreceptor degeneration models. Ofd1 localization in rat retina was examined using immunofluorescence. N-methyl-N-nitrosourea (MNU)-induced rats and Royal College of Surgeons (RCS) rats were used as photoreceptor degeneration models. The expression pattern of Ofd1, other ciliary associated genes and Wnt signaling pathway genes were examined in rat models. Furthermore, pEGFP-Ofd1-CDS and pSUPER-Ofd1-shRNA were constructed to overexpress and knockdown the expression level in 661W and R28 cells. MNU was also used to induce cell death. Cilia formation was observed using immunocytochemistry (ICC). Reactive oxygen species (ROS) were detected using the 2', 7'-Dichlorofluorescin diacetate (DCFH-DA) assay. Apoptosis genes expression was examined using qRT-PCR, Western blotting and fluorescence-activated cell sorting (FACS). Ofd1 localized to outer segments of rat retina photoreceptors. Ofd1 and other ciliary proteins expression levels increased from the 1st and 4th postnatal weeks and decreased until the 6th week in the RCS rats, while their expression consistently decreased from the 1st and 7th day in the MNU rats. Moreover, Wnt signaling pathway proteins expression was significantly up-regulated in both rat models. Knockdown of Ofd1 expression resulted in a smaller population, shorter length of cell cilia, and lower cell viability. Ofd1 overexpression partially attenuated MNU toxic effects by reducing ROS levels and mitigating apoptosis. To the best of our knowledge, this is the first study demonstrating Ofd1 localization and its function in rat retina and in retinal degeneration rat models. Ofd1 plays a role in controlling photoreceptor cilium length and number. Importantly, it demonstrates a neuroprotective function by protecting the photoreceptor from oxidative stress and apoptosis. These data have expanded our understanding of Ofd1 function beyond cilia, and we concluded that ofd1 neuroprotection could be a potential treatment strategy in retina degeneration models.
Ofd1是一种新发现的导致色素性视网膜炎（RP）的致病基因，RP是一种光感受器退行性疾病。本研究旨在检测Ofd1在视网膜中的定位，并进一步研究其在光感受器退行性变模型中的功能。采用免疫荧光法检测Ofd1在大鼠视网膜中的定位。以N-甲基-N-亚硝基脲（MNU）诱导的大鼠和皇家外科学院（RCS）大鼠作为光感受器退行性变模型。检测大鼠模型中Ofd1、其他纤毛相关基因和Wnt信号通路基因的表达模式。此外，构建了pEGFP-Ofd1-CDS和pSUPER-Ofd1-shRNA以在661W和R28细胞中过表达和敲低表达水平。MNU也用于诱导细胞死亡。采用免疫细胞化学（ICC）观察纤毛形成。使用2',7'-二氯荧光素二乙酸酯（DCFH-DA）检测法检测活性氧（ROS）。采用qRT-PCR、蛋白质免疫印迹法和荧光激活细胞分选（FACS）检测凋亡基因的表达。Ofd1定位于大鼠视网膜光感受器的外段。在RCS大鼠中，Ofd1和其他纤毛蛋白的表达水平从出生后第1周和第4周开始升高，直到第6周下降，而在MNU大鼠中，它们的表达从第1天和第7天开始持续下降。此外，在两种大鼠模型中，Wnt信号通路蛋白的表达均显著上调。敲低Ofd1的表达导致细胞数量减少、细胞纤毛长度缩短和细胞活力降低。Ofd1过表达通过降低ROS水平和减轻细胞凋亡，部分减轻了MNU的毒性作用。据我们所知，这是第一项证明Ofd1在大鼠视网膜和视网膜退行性变大鼠模型中的定位及其功能的研究。Ofd1在控制光感受器纤毛的长度和数量方面发挥作用。重要的是，它通过保护光感受器免受氧化应激和细胞凋亡，表现出神经保护功能。这些数据扩展了我们对Ofd1在纤毛之外功能的理解，我们得出结论，Ofd1神经保护可能是视网膜退行性变模型中的一种潜在治疗策略。
