C9orf72六核苷酸重复形序列 G4C2 G-四链体引起的肌萎缩侧索硬化和额颞叶痴症的分子机制研究

肌萎缩侧索硬化症（ALS）和额颞叶痴呆（FTD）是致命性的神经系统疾病。位于C9orf72基因非编码区六核苷酸重复序列(GGGGCC)n的异常扩增是ALS/FTD最常见的遗传诱因。我们已发表的结果表明该序列可折叠成不同构象的G-四链体。同时发现核仁蛋白以及异构核糖核蛋白均可特异性结合反向平行椅式构象的C9orf72 G-四链体。本项目中，我们将对该序列形成的G-四链体及其结合蛋白形成的复合物进行系统的生化和结构表征并确定它们的三维结构。并设计研发针对这些G-四链体具有高特异性和亲和力的小分子化合物以及类肽化合物，进行化学修饰优化其特异性和亲和力，使用相应的细胞模型测定这些化合物的生物活性。所提议的研究成果不仅可以深入了解G-四链体的生物学意义并帮助我们阐明ALS/FTD的发病机制。而且发现的新化合物将促进针对致命神经退行性疾病的治疗。

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are devastating neurological diseases. Abnormal expansion of a hexanucleotide repeat, (GGGGCC)n, is the most common genetic cause of familial and sporadic forms of ALS and FTD. This nucleotide repeat element is located in the first non-coding region of the C9orf72 gene. Previous studies suggested that the C9orf72 G4C2 repeats, either as DNA or the transcribed RNA, can fold into G-quadruplexes with distinct structures. Scientists have identified several proteins that interact with the C9orf72 G4C2 repeats. These include nucleolin (NCL) and heterogeneous nuclear ribonuleoprotein F (hnRNP F) that specifically binds to the C9orf72 G4C2 G-quadruplexes. Despite the critical connection of C9orf72 G4C2 G-quadruplexes and their complexes with nucleoproteins to the pathogenic mechanism of C9orf72 G4C2-linked ALS/FTD, the molecular basis of G-quadruplex formation by C9orf72 G4C2 repeats and the interactions between C9orf72 G4C2 G-quadruplexes and the protein partners remains elusive...In this project, we plan to perform systematic biochemical and structural characterizations for C9orf72 G4C2 G-quadruplexes and their complexes with NCL and hnRNP F respectively. First, we will determine the 3D structure of G-quadruplex formed by the C9orf72 G4C2 DNA. In our preliminary study, we successfully obtained a homogenous DNA sample, d(GGGGCC)4 and d(GGGGCC)2. We have determined the topology of this G-quadruplex DNA and will further resolve its detailed structure using NMR and X-ray techniques. We have also successfully obtained a homogeneous C9orf72 G4C2 RNA sample with sufficient quality for NMR/X-ray structure determination. We will perform more NMR/X-ray studies to obtain its 3D structure. The structural information of C9orf72 G4C2 DNA/RNA to be obtained in the current project will provide valuable insights into the molecular mechanism of abortive transcripts formation by C9orf72 G4C2 repeats and RNA foci appearance in ALS/FTD patients. Meanwhile, we will aim to discover small compounds with therapeutic potential by targeting both DNA and RNA G4C2 quadruplexes with high specificity and affinity (<50nM) to block G4C2 RNA transcription and translation into toxic dipeptide repeat proteins （DPRs）. Small compound libraries will be screened to select candidate compounds together with the published inhibitors for further study using biophysical and biochemical methods. Subsequently, chemical modifications will seek to optimize their specificity and affinity. Finally, the lead compounds will be assayed in vitro and in vivo using model systems...The results from this proposed study will help to enhance our understanding of DNA/RNA G-quadruplex biology and elucidate the etiology of ALS/FTD. The small compounds discovered will promote the development therapy to treat these deadly neurodegenerative diseases.