Effect of IVNS1ABP mutation, knockout on gene expression during iPSCs differentiation to Neural Progenitors

We identified a new disease characterized by severe neurological deficits in addition to progeria symptoms. It is caused by IVNSABP gene mutation. The association between IVNS1ABP and aging has never been reported before. By generating isogenic iPSCs from the patients' fibroblasts and differentiating the iPSCs into neural progenitor cells (NPCs), we found that mutant IVNS1ABP fibroblasts, iPSCs, and NPCs exhibited disrupted cytokinesis, DNA damage, and cellular senescence. Transcription analysis of isogenic iPSCs and iPSCs derived neural progenitors (NPCs) also showed impaired cytokinesis and senescence alteration. Correspondingly, cerebral organoids displayed premature differentiation of NPCs to neurons. Molecular profiling as well as biochemical and cellular analysis revealed altered binding of mutant IVNS1ABP to actin and actin-associated proteins and dysregulated actin dynamics during cytokinesis. Taken together, we propose that IVNS1ABP mutation dysregulates actin polymerization and organization which is at least partly responsible for the cellular senescence phenotypes in this undiagnosed disease. Overall design: To get insight into the gene's affection in CNS (central nerve system), we corrected one patient iPSC line, and generated the mutation corrected isogenic iPSC line and KO iPSC line by CRISPR/Cas9. These iPSCs lines were further differentiated to NPCs. We then performed gene expression profiling analysis using data obtained from RNA-seq of the isogenic cell lines (CON, MT and KO), at two tome points (iPSCs and NPCs)我们发现了一种兼具严重神经功能缺损与早衰症症状的新型疾病，该疾病由IVNSABP基因突变引发。此前从未有过IVNS1ABP与衰老相关的研究报道。 我们通过从患者成纤维细胞中诱导生成同基因诱导多能干细胞（iPSCs），并将iPSCs分化为神经前体细胞（NPCs），发现携带突变IVNSABP的成纤维细胞、iPSCs及NPCs均出现胞质分裂紊乱、DNA损伤及细胞衰老现象。对同基因iPSCs及其分化所得神经前体细胞的转录组分析，同样证实了胞质分裂受损与衰老状态异常。 相应地，大脑类器官也表现出神经前体细胞向神经元的过早分化。 分子谱分析结合生化与细胞实验分析显示，突变型IVNSABP与肌动蛋白及肌动蛋白结合蛋白的结合能力发生改变，且胞质分裂过程中的肌动蛋白动力学失调。 综合以上结果，我们提出：IVNSABP基因突变通过失调肌动蛋白聚合与组织架构，至少部分介导了这一未确诊疾病中的细胞衰老表型。 整体实验设计：为探究该基因对中枢神经系统（central nerve system, CNS）的影响，我们对1株患者来源iPSCs系进行基因校正，通过CRISPR/Cas9技术构建了携带校正后基因型的同基因iPSCs系及基因敲除（KO）iPSCs系。随后将上述iPSCs系均分化为神经前体细胞。我们分别在两个时间节点（iPSCs阶段与神经前体细胞阶段），对同基因细胞系（野生型对照CON、突变型MT及敲除型KO）进行RNA测序，以此开展基因表达谱分析。