A ‘GGGGCC’ repeat expansion in the first intron of the C9orf72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The exact mechanism resulting in these neurodegenerative diseases remains elusive, but C9 repeat RNA toxicity has been implicated as a gain-of-function mechanism. Our aim was to use a zebrafish model for C9orf72 RNA toxicity to identify modifiers of the ALS-linked phenotype. We discovered that the RNA-binding protein heterogeneous nuclear ribonucleoprotein K (HNRNPK) reverses the toxicity of both sense and antisense repeat RNA, which is dependent on its subcellular localization and RNA recognition, and not on C9orf72 repeat RNA binding. We observed HNRNPK cytoplasmic mislocalization in C9orf72 ALS patient fibroblasts, induced pluripotent stem cell (iPSC)-derived motor neurons and post-mortem motor cortex and spinal cord, in line with a disrupted HNRNPK function in C9orf72 ALS. In C9orf72 ALS/FTD patient tissue, we discovered an increased nuclear translocation, but reduced expression of ribonucleotide reductase regulatory subunit M2 (RRM2), a downstream target of HNRNPK involved in the DNA damage response. Last but not least, we showed that increasing the expression of HNRNPK or RRM2 was sufficient to mitigate DNA damage in our C9orf72 RNA toxicity zebrafish model. Overall, our study strengthens the relevance of RNA toxicity as a pathogenic mechanism in C9orf72 ALS and demonstrates its link with an aberrant DNA damage response, opening novel therapeutic avenues for C9orf72 ALS/FTD.

C9orf72基因第一个内含子中的“GGGGCC”重复扩增是肌萎缩侧索硬化症 (ALS) 和额颞叶痴呆 (FTD) 最常见的遗传原因。导致这些神经退行性疾病的确切机制仍然难以捉摸，但 C9 重复 RNA 毒性已被认为是一种功能获得机制。我们的目标是使用斑马鱼C9orf72 RNA 毒性模型来识别 ALS 相关表型的修饰因子。我们发现RNA结合蛋白异源核糖核蛋白K (HNRNPK)可逆转有义和反义重复RNA的毒性，这取决于其亚细胞定位和RNA识别，而不是C9orf72重复RNA结合。我们在C9orf72 ALS 患者成纤维细胞、诱导多能干细胞 (iPSC) 衍生的运动神经元以及死后运动皮层和脊髓中观察到 HNRNPK 细胞质错误定位，这与C9orf72 ALS 中 HNRNPK 功能破坏一致。在C9orf72 ALS/FTD 患者组织中，我们发现核易位增加，但核糖核苷酸还原酶调节亚基 M2 (RRM2) 的表达减少，RRM2 是参与 DNA 损伤反应的 HNRNPK 下游靶标。最后但并非最不重要的一点是，我们表明，在我们的C9orf72 RNA 毒性斑马鱼模型中，增加 HNRNPK 或 RRM2 的表达足以减轻 DNA 损伤。总体而言，我们的研究加强了 RNA 毒性作为C9orf72 ALS 致病机制的相关性，并证明了其与异常 DNA 损伤反应的联系，为C9orf72 ALS/FTD 开辟了新的治疗途径。