Molecular mechanisms of C9orf72 and Matrin 3 interaction in ALS

摘要 GGGGCC六核苷酸重复序列扩增(G4C2)，在C9orf72基因的非编码区 染色体9p21是肌萎缩侧索硬化症和额颞叶痴呆最常见的原因。 C9orf72中的G4C2扩展导致核膜完整性丧失，进而导致定位错误 C9 ALS患者脊髓中核TDP-43(另一种ALS致病蛋白)的表达。最近，扩大了 在诱导多能性分化的神经元中发现了六核苷酸重复序列结合蛋白 C9-ALS患者的干细胞和运动皮质组织。Matrin-3(MATR3)与六核苷酸相互作用 在两个独立的研究中重复。MATR3是一种与RNA结合的核基质蛋白，它的突变是已知的 肌萎缩侧索硬化症的遗传病因。MATR3与核基质蛋白如Lamin A相互作用提示其可能的作用 MATR3在调节核基质结构和功能中的作用。MATR3阳性胞浆内含物 在一名已知携带C9orf72重复扩增的ALS患者中被发现，提示可能存在联系 在这两种形式的肌萎缩侧索硬化症之间。MATR3与C9orf72中的G4C2结合，并识别 已发现MATR3在散发性ALS患者的选择性剪接盒外显子中富含。然而， MATR3和扩展的G4C2重复之间相互作用的生理后果，以及 这种相互作用与肌萎缩侧索硬化症发病机制的相关性尚不清楚。 我们发现野生型(WT)MATR3的异位表达强烈抑制了神经退行性变的表型 在果蝇模型中突变的C9ORF72六核苷酸重复序列扩展(HNE)。我们观察到 去除WT-MATR3中的RNA结合结构域可阻断其在体内抑制C9-HNE毒性的能力， 提示救援表型依赖于HNE RNA转录产物与WT-MATR3的结合。在这里，我们 建议研究WT-MATR3和C9ORF72-HNE之间的这种新的相互作用及其对 C9ORF72-HNE神经退行性表型。在这份R21提案中，我们将彻底研究 用生化、遗传学和分子生物学方法研究哺乳动物细胞中MATR3和C9的关系 C9orf72介导的C9患者和果蝇模型多能干细胞的培养和诱导 神经退行性变。

Abstract GGGGCC hexanucleotide repeat expansions (G4C2), in the non-coding region of the C9orf72 gene on chromosome 9p21, are the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia. G4C2 expansions in C9orf72 cause a loss of nuclear membrane integrity which in turn leads to mislocalization of nuclear TDP-43 (another ALS causing protein) in the spinal cords of C9 ALS patients. Recently, expanded hexanucleotide repeat binding proteins have been identified in neurons differentiated from induced pluripotent stem cells and motor cortex tissue from C9-ALS patients. Matrin-3 (MATR3) interacts with hexanucleotide repeats in two independent studies. Mutations in MATR3, an RNA binding nuclear matrix protein, are a known genetic cause of ALS. MATR3 interacts with nuclear matrix proteins such as Lamin A suggesting potential role of MATR3 in regulating nuclear matrix architecture and function. MATR3 positive cytoplasmic inclusions have been identified in an ALS patient known to carry the C9orf72 repeat expansion suggesting a potential link between these two forms of ALS. MATR3 binds to the G4C2 in C9orf72 and the binding motif recognized by MATR3 has been found to be enriched in sporadic ALS patients’ alternative splicing cassette exons. However, the physiological consequences of the interaction between MATR3 and expanded G4C2 repeats, as well as the relevance of this interaction to ALS pathogenesis, are not yet known. We found that ectopic expression of wild type (WT) MATR3 strongly suppresses neurodegenerative phenotype in a Drosophila model of mutant C9ORF72 hexanucleotide repeat expansions (HNE). We observed that ablating the RNA binding domain in WT-MATR3 blocks its ability to suppress C9-HNE toxicity in vivo, suggesting that the rescue phenotype depends on HNE RNA transcripts binding to WT-MATR3. Here, we propose to investigate this novel interaction between WT-MATR3 and C9ORF72-HNE and its impact on C9ORF72-HNE neurodegenerative phenotype. In this R21 proposal, we will thoroughly examine the relationship between MATR3 and C9 using biochemical, genetic and molecular approaches in mammalian cell cultures, induced pluripotent stem cells from C9 patients and Drosophila models of C9orf72-mediated neurodegeneration.