TOP2基因与GGGGCC六核苷酸重复扩展突变毒性的相关性研究

GGGGCC expanded repeats in a non-coding region of C9ORF72 gene have been identified as a major genetic cause of amyotrophic lateral sclerosis (ALS). Identification of GGGGCC expanded repeats toxicity and how to modify its toxicity are the key points for finding a potential ALS drug treatment. We previously constructed GGGGCC wild type and expanded transgenic Drosophila, and showed that GGGGCC expanded repeats alone were sufficient to cause neurodegeneration in Drosophila. We then performed a crossing screen using the expanded transgenic Drosophila and found that knock-down of TOP2 using RNAi method could modulate the GGGGCC associated fly eye neurodegeneration, which were successfully replicated when using two TOP2 chemical inhibitors. Therefore, in this study, we mainly focus on three questions as following: 1) TOP2 mediate the toxicity of GGGGCC expanded repeats in Neuro-2a cells and Drosophila models; 2) Identification of pathogenic pathways and important genes, which participate in the interactions between TOP2 and GGGGCC using RNA-sequencing; 3) Whether the expression of TOP2 probably be a bio-marker of ALS. All these questions could help us find a potential ALS drug treatment.

C9ORF72基因第一外显子内部非编码区GGGGCC重复扩展突变是肌萎缩侧索硬化（ALS）的常见致病基因，明确该重复扩展突变的毒性效应并寻找其调控机制可为ALS潜在药物靶点提供重要信息。前期工作中，我们构建了GGGGCC野生型和扩展突变型转基因果蝇并证实扩展突变可导致果蝇复眼溃烂；随后，我们借助扩展突变型果蝇筛选到调控重复扩展突变毒性的关键基因—TOP2，即利用RNAi和化学抑制剂下调TOP2水平后可显著改善重复扩展突变介导的果蝇复眼神经退行性变。基于上述研究结果，本研究旨在解决：1）果蝇和细胞模型上明确TOP2表达水平对GGGGCC扩展突变毒性的调控能力；2）利用全基因组RNA测序和生物信息学分析初步明确介导TOP2和GGGGCC扩展突变相互作用的致病通路；3）探讨TOP2表达水平的变化能否作为ALS生物学标记。明确上述问题可为寻找ALS药物靶点提供重要信息。

C9ORF72基因第一外显子内部非编码区GGGGCC重复扩展突变是肌萎缩侧索硬化（ALS）的常见致病基因。前期工作中，我们借助扩展突变型果蝇筛选到调控GGGGCC重复扩展突变毒性的关键基因—TOP2。研究发现：通过化学抑制剂或siRNA下调TOP2基因表达后可显著改善GGGGCC扩展突变介导的果蝇复眼神经退行性变、孵化率降低和运动功能障碍等表型；同时，在细胞模型中进一步证实下调TOP2基因表达可营救GGGGCC扩展突变介导的细胞毒性；随后，我们发现，不仅与GGGGCC扩展突变相关的ALS患者脑组织、果蝇脑组织、Neuro-2a细胞中TOP2表达水平升高，而且在ALS常见致病基因（如SOD1、TARDBP）相关的细胞或小鼠模型中TOP2基因表达水平亦显著升高，提示TOP2表达水平升高可能参与了ALS疾病的发生，TOP2水平变化有望成为ALS疾病诊断的生物标志物；最后，我们利用全基因组RNA测序和生物信息学分析初步明确介导TOP2和GGGGCC扩展突变相互作用的致病通路及可能参与介导疾病发生的相关基因；为明确GGGGCC重复扩展突变的毒性调控机制提供重大发现，同时为ALS潜在药物靶点提供了重要信息。

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