PDF(Pubmed)
Abstract：
Autosomal dominant optic atrophy (ADOA) is a rare progressive disease mainly caused by mutations in OPA1, a nuclear gene encoding for a mitochondrial protein that plays an essential role in mitochondrial dynamics, cell survival, oxidative phosphorylation, and mtDNA maintenance. ADOA is characterized by the degeneration of retinal ganglion cells (RGCs). This causes visual loss, which can lead to legal blindness in many cases. Nowadays, there is no effective treatment for ADOA. In this article, we have established an isogenic human RGC model for ADOA using iPSC technology and the genome editing tool CRISPR/Cas9 from a previously generated iPSC line of an ADOA plus patient harboring the pathogenic variant NM_015560.3: c.1861C>T (p.Gln621Ter) in heterozygosis in OPA1. To this end, a protocol based on supplementing the iPSC culture media with several small molecules and defined factors trying to mimic embryonic development has been employed. Subsequently, the created model was validated, confirming the presence of a defect of intergenomic communication, impaired mitochondrial respiration, and an increase in apoptosis and ROS generation. Finally, we propose the analysis of OPA1 expression by qPCR as an easy read-out method to carry out future drug screening studies using the created RGC model. In summary, this model provides a useful platform for further investigation of the underlying pathophysiological mechanisms of ADOA plus and for testing compounds with potential pharmacological action.
摘要：
常染色体显性视神经萎缩（ADOA）是一种罕见的进行性疾病，主要由OPA1突变引起，OPA1是一种编码线粒体蛋白的核基因，在线粒体动力学中起着至关重要的作用。细胞存活,氧化磷酸化，和mtDNA维护。ADOA的特征在于视网膜神经节细胞(RGCs)的变性。这会导致视力丧失，这在许多情况下可能导致法律失明。如今,ADOA没有有效的治疗方法。在这篇文章中,我们使用iPSC技术和基因组编辑工具CRISPR/Cas9从先前生成的具有致病性变异NM_015560.3：c.1861C>T的ADOA加患者的iPSC细胞系中建立了ADOA的等基因人类RGC模型（p.Gln621Ter)在OPA1中的杂合。为此,已经采用了基于向iPSC培养基补充几种小分子和试图模拟胚胎发育的定义因子的方案。随后，创建的模型经过验证，确认基因组间通信缺陷的存在，线粒体呼吸受损，以及细胞凋亡和ROS产生的增加。最后,我们建议通过qPCR分析OPA1表达作为一种简单的读出方法，用于使用创建的RGC模型进行未来的药物筛选研究。总之,该模型为进一步研究ADOAplus的潜在病理生理机制以及测试具有潜在药理作用的化合物提供了有用的平台.
