Tucker Budd A, Solivan-Timpe Frances, Roos Ben R, Anfinson Kristin R, Robin Alan L, Wiley Luke A, Mullins Robert F, Fingert John H
Department of Ophthalmology and Visual Sciences, Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA ; Stephen A. Wynn Institute for Vision Research, Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA.
Department of Ophthalmology, Johns Hopkins University, Baltimore, MD 21287, USA ; Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205, USA.
J Stem Cell Res Ther. 2014 Jan 25;3(5):161. doi: 10.4172/2157-7633.1000161.
Duplication of the gene causes normal tension glaucoma (NTG); however the mechanism by which this copy number variation leads to retinal ganglion cell death is poorly understood. The ability to use skin-derived induced pluripotent stem cells (iPSCs) to investigate the function or dysfunction of a mutant gene product in inaccessible tissues such as the retina now provides us with the ability to interrogate disease pathophysiology . iPSCs were generated from dermal fibroblasts obtained from a patient with -associated NTG, via viral transduction of the transcription factors , , , and . Retinal progenitor cells and subsequent retinal ganglion cell-like neurons were derived using our previously developed stepwise differentiation protocol. Differentiation to retinal ganglion-like cells was demonstrated via rt-PCR targeted against TUJ1, MAP2, THY1, NF200, ATOH7 and BRN3B and immunohistochemistry targeted against NF200 and ATOH7. Western blot analysis demonstrated that both fibroblasts and retinal ganglion cell-like neurons derived from NTG patients with gene duplication have increased levels of LC3-II protein (a key marker of autophagy). Duplication of has been previously shown to increase expression of and here we demonstrate that the same duplication leads to activation of LC3-II. This suggests that -associated glaucoma may be caused by dysregulation (over-activation) of this catabolic pathway.
该基因的复制会导致正常眼压性青光眼（NTG）；然而，这种拷贝数变异导致视网膜神经节细胞死亡的机制仍知之甚少。利用皮肤来源的诱导多能干细胞（iPSC）来研究突变基因产物在视网膜等难以接近的组织中的功能或功能障碍，现在为我们探究疾病病理生理学提供了可能。通过对转录因子、、、和进行病毒转导，从一名患有相关NTG的患者的真皮成纤维细胞中生成了iPSC。使用我们之前开发的逐步分化方案获得了视网膜祖细胞以及随后的视网膜神经节细胞样神经元。通过针对TUJ1、MAP2、THY1、NF200、ATOH7和BRN3B的逆转录聚合酶链反应（rt-PCR）以及针对NF200和ATOH7的免疫组织化学，证明了向视网膜神经节样细胞的分化。蛋白质免疫印迹分析表明，来自具有该基因复制的NTG患者的成纤维细胞和视网膜神经节细胞样神经元中，微管相关蛋白1轻链3-II（LC3-II）蛋白（自噬的关键标志物）水平均升高。先前已表明该基因的复制会增加的表达，在此我们证明相同的复制会导致LC3-II的激活。这表明相关青光眼可能是由这种分解代谢途径的失调（过度激活）引起的。
