C9orf72 多聚重复蛋白对miRNA生成和功能影响及其在ALS/FTD发病机制中的作用研究

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are two common neurodegenerative diseases with a high incidence and so far there is no effective drug. A large GGGGCC hexanucleotide repeat expansion within a noncoding region of the gene C9orf72 is the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia. Yet the mechanism underlying the pathogenesis of C9orf72-ALS /FTD is still unclear.It has been previously reported that through unconventional repeat-associated non-ATG (RAN) translation, sense and antisense transcripts of those repeats produce dipeptide-repeat proteins (referred to as RAN proteins). And in the previous published study, we found that all five types of RAN proteins are not prone to aggregate and the turnover of these RAN proteins was not regulated by the ubiquitin–proteasome system or autophagy. In addition, only poly-GR and poly-PR located in the nucleolus causing critical nucleoprotein B23 (nucleophosmin) transported from the nucleolus to nucleoplasm, impairing stress granule formation, leading to nucleolar stress and cell death. In this project, we will explore the effects of these dipeptide repeat proteins, especially the poly-GR and poly-PR containing arginine repeat proteins on the biogenesis of miRNA, and further explore the underlying molecular mechanism through deep sequencing, qRT-PCR, immunofluorescence, Western blot and other molecular biology, cell biology and biochemistry methods. This promising study to explore the relationship between these dipeptide repeat proteins and miRNA will be helpful to further understanding the basic molecular mechanism underlying the pathogenesis of C9orf72-ALS /FTD，which provides a new perspective for the therapy of ALS/FTD.

肌萎缩性侧索硬化症（ALS）和额颞叶痴呆（FTD）是两种常见的神经退行性疾病，发病率高，至今尚无有效治疗药物。新发现的C9orf72基因内含子上的GGGGCC六碱基高度重复序列突变与ALS和FTD发病相关，但是其详细的致病机制仍不明确。项目申请人先前发表研究结果表明GGGGCC不依赖ATG翻译的含精氨酸的多聚重复蛋白poly-GR和poly-PR定位于核仁导致核仁应激引起细胞死亡，同时其介导的核功能缺陷还可抑制核糖体RNA及应激颗粒形成。本项目利用各种细胞模型，通过qRT-PCR，miRNA深度测序，免疫荧光，免疫印迹等实验方法探索多聚重复蛋白尤其是poly-GR 和poly-PR对miRNA生成和功能的影响，并探索其内在的分子机制。这有助于进一步阐明C9orf72突变相关的ALS/FTD发病机制，为ALS/FTD的治疗提供新的视点。

肌萎缩性侧索硬化症（ALS）和额颞叶痴呆（FTD）是两种常见的神经退行性疾病，发病率高，至今尚无有效治疗药物。近年来发现的C9orf72基因内含子上的GGGGCC六碱基高度重复序列突变与ALS和FTD发病相关。这种内含子上的重复序列不依赖于ATG翻译出五种多聚重复蛋白poly-GA，poly-GP，poly-GR，poly-PA和poly-PR。虽然提出了许多的致病理论，但是其详细的致病机制仍不明确。项目申请人通过研究不同类型不同重复数目的的五种多聚重复蛋白，发现随着重复数目的增加这些多聚重复蛋白均能诱导更严重的细胞毒性，且poly-PR更为显著。通过小RNA深度测序以及qRT-PCR验证发现特异性的只有poly-PR能够显著引起miRNA表达紊乱。其中丰度较高的miR-30b-5p显著下调，而miR-30b-5p又能够在细胞核中抑制关键的溶酶体转录因子TFEB活性而影响自噬。进一步的分子机制表明poly-PR并不影响miRNA 的剪切，而是通过影响关键的miRNA成熟蛋白YBX3，poly-PR通过结合YBX3将其从细胞质招募只细胞核中从而影响到miRNA的剪切成熟。本研究有助于进一步阐明C9orf72突变相关的ALS/FTD发病机制，为ALS/FTD的治疗提供新的理论依据。

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