Glycine-alanine dipeptide repeat protein contributes to toxicity in a zebrafish model of C9orf72 associated neurodegeneration.
Glycine-alanine dipeptide repeat protein contributes to toxicity in a zebrafish model of C9orf72 associated neurodegeneration.
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DOI：
10.1186/s13024-016-0146-8
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发表时间：
2017-01-14
影响因子：
15.1
通讯作者：
Schmid B
中科院分区：
文献类型：
--
作者：
Ohki Y;Wenninger-Weinzierl A;Hruscha A;Asakawa K;Kawakami K;Haass C;Edbauer D;Schmid B
额颞叶变性 (FTLD) 和肌萎缩侧索硬化症 (ALS) 最常见的遗传原因是 9 号染色体开放阅读框 72 (C9orf72) 位点非编码区中 GGGGCC 六核苷酸重复的扩展。在 C9orf72 重复扩增载体中观察到的病理特征是 RNA 焦点的形成和源自重复相关非 ATG (RAN) 翻译的二肽重复 (DPR) 蛋白的沉积。目前，尚不清楚 RNA 灶的形成、DPR 翻译产物或 C9orf72 的部分缺失是否主要驱动体内神经毒性。通过在斑马鱼中使用转基因方法，我们解决了人类 ALS/FTLD 大脑中最常见的 DPR 聚甘氨酸 (聚 GA) 蛋白在体内是否有毒的问题。我们生成了几个转基因 UAS 应答系，这些系单独表达 80 个重复的 GGGGCC，或者与翻译起始 ATG 密码子一起表达，在与 Gal4 驱动程序交叉时强制 GA80-GFP 蛋白的翻译。将 GGGGCC 重复序列和 GA80 与缺乏起始密码子的绿色荧光蛋白 (GFP) 融合，以通过 GFP 荧光监测蛋白质翻译。缺乏 ATG 密码子的 GGGGCC 重复转基因斑马鱼在缺乏聚 GA 的情况下表现出非常轻微的毒性。然而，在 ATG 启动聚 GA 表达后，会产生强烈的毒性，通过注射反义吗啉干扰起始密码子依赖性聚 GA 翻译可以挽救这种毒性。这种吗啉代仅干扰GA80-GFP翻译而不影响重复转录，表明毒性来自GA80-GFP。这些新型转基因 C9orf72 相关重复斑马鱼模型证明了多聚 GA 对斑马鱼的毒性。聚 GA 蛋白的减少可挽救毒性，验证了这种治疗 C9orf72 重复扩增载体的治疗方法。这些新颖的动物模型为药物发现提供了一个有价值的工具，以减少 C9orf72 重复扩增的 ALS/FTLD 患者中与 DPR 相关的毒性。本文的在线版本 (doi:10.1186/s13024-016-0146-8) 包含补充材料，可供授权用户使用。
The most frequent genetic cause of frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS) is the expansion of a GGGGCC hexanucleotide repeat in a non-coding region of the chromosome 9 open reading frame 72 (C9orf72) locus. The pathological hallmarks observed in C9orf72 repeat expansion carriers are the formation of RNA foci and deposition of dipeptide repeat (DPR) proteins derived from repeat associated non-ATG (RAN) translation. Currently, it is unclear whether formation of RNA foci, DPR translation products, or partial loss of C9orf72 predominantly drive neurotoxicity in vivo. By using a transgenic approach in zebrafish we address if the most frequently found DPR in human ALS/FTLD brain, the poly-Gly-Ala (poly-GA) protein, is toxic in vivo. We generated several transgenic UAS responder lines that express either 80 repeats of GGGGCC alone, or together with a translation initiation ATG codon forcing the translation of GA80-GFP protein upon crossing to a Gal4 driver. The GGGGCC repeat and GA80 were fused to green fluorescent protein (GFP) lacking a start codon to monitor protein translation by GFP fluorescence. Zebrafish transgenic for the GGGGCC repeat lacking an ATG codon showed very mild toxicity in the absence of poly-GA. However, strong toxicity was induced upon ATG initiated expression of poly-GA, which was rescued by injection of an antisense morpholino interfering with start codon dependent poly-GA translation. This morpholino only interferes with GA80-GFP translation without affecting repeat transcription, indicating that the toxicity is derived from GA80-GFP. These novel transgenic C9orf72 associated repeat zebrafish models demonstrate poly-GA toxicity in zebrafish. Reduction of poly-GA protein rescues toxicity validating this therapeutic approach to treat C9orf72 repeat expansion carriers. These novel animal models provide a valuable tool for drug discovery to reduce DPR associated toxicity in ALS/FTLD patients with C9orf72 repeat expansions. The online version of this article (doi:10.1186/s13024-016-0146-8) contains supplementary material, which is available to authorized users.