Department of Psychiatry & Behavioral Sciences, Center for Therapeutic Innovation, University of Miami Miller School of Medicine, 1501 NW 10th Ave, Biomedical Research Building Room 413, Florida, Miami, 33136, USA.
Department of Biochemistry and Molecular Biology, University of Miami Miller School of Medicine, 1600 NW 10th Ave, Miami, FL, 33136, USA.
Mol Neurodegener. 2020 Feb 24;15(1):13. doi: 10.1186/s13024-020-00365-9.
The C9ORF72 hexanucleotide repeat expansion is the most common known genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two fatal age-related neurodegenerative diseases. The C9ORF72 expansion encodes five dipeptide repeat proteins (DPRs) that are produced through a non-canonical translation mechanism. Among the DPRs, proline-arginine (PR), glycine-arginine (GR), and glycine-alanine (GA) are the most neurotoxic and increase the frequency of DNA double strand breaks (DSBs). While the accumulation of these genotoxic lesions is increasingly recognized as a feature of disease, the mechanism(s) of DPR-mediated DNA damage are ill-defined and the effect of DPRs on the efficiency of each DNA DSB repair pathways has not been previously evaluated.
Using DNA DSB repair assays, we evaluated the efficiency of specific repair pathways, and found that PR, GR and GA decrease the efficiency of non-homologous end joining (NHEJ), single strand annealing (SSA), and microhomology-mediated end joining (MMEJ), but not homologous recombination (HR). We found that PR inhibits DNA DSB repair, in part, by binding to the nucleolar protein nucleophosmin (NPM1). Depletion of NPM1 inhibited NHEJ and SSA, suggesting that NPM1 loss-of-function in PR expressing cells leads to impediments of both non-homologous and homology-directed DNA DSB repair pathways. By deleting NPM1 sub-cellular localization signals, we found that PR binds NPM1 regardless of the cellular compartment to which NPM1 was directed. Deletion of the NPM1 acidic loop motif, known to engage other arginine-rich proteins, abrogated PR and NPM1 binding. Using confocal and super-resolution immunofluorescence microscopy, we found that levels of RAD52, a component of the SSA repair machinery, were significantly increased iPSC neurons relative to isogenic controls in which the C9ORF72 expansion had been deleted using CRISPR/Cas9 genome editing. Western analysis of post-mortem brain tissues confirmed that RAD52 immunoreactivity is significantly increased in C9ALS/FTD samples as compared to controls.
Collectively, we characterized the inhibitory effects of DPRs on key DNA DSB repair pathways, identified NPM1 as a facilitator of DNA repair that is inhibited by PR, and revealed deficits in homology-directed DNA DSB repair pathways as a novel feature of C9ORF72-related disease.
C9ORF72 六核苷酸重复扩展是肌萎缩侧索硬化症（ALS）和额颞叶痴呆（FTD）这两种致命的年龄相关性神经退行性疾病最常见的已知遗传原因。C9ORF72 扩展编码五种二肽重复蛋白（DPR），这些蛋白通过非典型翻译机制产生。在 DPR 中，脯氨酸-精氨酸（PR）、甘氨酸-精氨酸（GR）和甘氨酸-丙氨酸（GA）是最具神经毒性的，并且会增加 DNA 双链断裂（DSB）的频率。虽然这些遗传毒性损伤的积累越来越被认为是疾病的一个特征，但 DPR 介导的 DNA 损伤的机制尚不清楚，并且 DPR 对每种 DNA DSB 修复途径的效率的影响以前尚未评估。
使用 DNA DSB 修复测定法，我们评估了特定修复途径的效率，并发现 PR、GR 和 GA 降低了非同源末端连接（NHEJ）、单链退火（SSA）和微同源介导的末端连接（MMEJ）的效率，但不降低同源重组（HR）的效率。我们发现 PR 通过与核仁蛋白核磷蛋白（NPM1）结合，部分抑制 DNA DSB 修复。NPM1 的缺失抑制了 NHEJ 和 SSA，表明在表达 PR 的细胞中 NPM1 的功能丧失会导致非同源和同源定向 DNA DSB 修复途径的障碍。通过删除 NPM1 亚细胞定位信号，我们发现 PR 与 NPM1 结合，而不管 NPM1 被导向哪个细胞区室。删除已知与其他富含精氨酸的蛋白质结合的 NPM1 酸性环基序，阻断了 PR 和 NPM1 的结合。使用共聚焦和超分辨率免疫荧光显微镜，我们发现 SSA 修复机制的组成部分 RAD52 的水平在源自诱导多能干细胞（iPSC）的神经元中相对于使用 CRISPR/Cas9 基因组编辑删除 C9ORF72 扩展的同基因对照显著增加。对死后脑组织的 Western 分析证实，与对照相比，C9ALS/FTD 样本中的 RAD52 免疫反应性显著增加。
总的来说，我们描述了 DPR 对关键 DNA DSB 修复途径的抑制作用，确定 NPM1 为促进 DNA 修复的辅助因子，而 PR 会抑制其活性，并揭示了同源定向 DNA DSB 修复途径的缺陷是 C9ORF72 相关疾病的一个新特征。
