腓骨肌萎缩症新致病基因MORC2的发病机制研究

Charcot–Marie–Tooth disease (CMT) encompasses a genetically and clinically heterogeneous group of inherited neuropathies and ranks among the most common inherited neurological disorders. The recently cloned causative gene of CMT2Z, MORC2, encoded a protein which participates in DNA Damage Response (DDR) and transcription regulations. In 2016, we found that novel mutations (p.Q338R and p.D404N) and hotspot mutation (p.S25L) of MORC2 were related to CMT2- and SMA-like phenotypes. Furthermore, Cells stably expressing the MORC2 mutants showed increased DNA damage in normal conditions, and delayed DNA damage recovery after ionizing radiation treatment. Elevated apoptosis in the early stage and increased cell death in the late stage of recovery after ionizing radiation were also detected. The mutant p.S25L exhibited the most significant effects mentioned above. We speculate that DDR dysfunction is the main pathogenesis of CMT2Z and also one of the major pathogeneses of CMT, and whether DNA damage triggers neuronal precursor cell apoptosis during development determines the phenotypic severity of CMT2Z. This project proposes to establish iPSC derived spinal motor neuron model of MORC2 mutations and normal controls, observe CMT2Z phenotypes at the cellular level by using techniques such as electron microscope and patch clamp, analyze the differences of DDR-related molecules, DNA damage and apoptosis at different stages of neuronal development by methods such as immunoblotting and Flow cytometry, analyze the changes of axonal-related molecules and pathways by transcriptional sequencing, treat the MNs using the selected drugs and observe the effects in order to elucidate the MORC2 pathogenesis, the underlying mechanism of clinical heterogeneity and to discover new therapeutic targets.

腓骨肌萎缩症（CMT）是一组最常见的周围神经单基因遗传病。新克隆的CMT2Z致病基因MORC2，其蛋白主要参与DNA损伤反应（DDR）和转录调控。我们发现新突变p.Q338R、p.D404N和热点突变p.S25L分别导致CMT2和脊肌萎缩症样表型；突变细胞系在基础状态下出现DNA损伤、电离辐射后损伤恢复延迟和细胞凋亡增加，以p.S25L最显著。我们推测DDR功能异常为CMT2Z的主要发病机制、DNA损伤是否达到触发神经元前体细胞凋亡的阈值决定CMT2Z的表型差异。本项目拟建立MORC2突变和正常对照iPSC来源的脊髓运动神经元模型；运用电镜、膜片钳等技术观察疾病表型变化；运用免疫印迹、流式细胞等技术在神经元不同分化阶段检测DDR相关分子、DNA损伤和凋亡的差异；运用转录组测序分析轴索相关分子和通路的改变；选择药物处理并观察疗效，以期阐明MORC2致病、表型差异的内在机制和发现新治疗靶点。

腓骨肌萎缩症（CMT）是一组最常见的周围神经单基因遗传病，CMT2Z致病基因MORC2的编码蛋白主要参与DNA损伤反应（DDR）和转录调控。本项目收集三名分别携带MORC2p.S87L（SMA样表型）、p.Q400R和p.D466N （CMT2表型）突变的患者和一名健康对照的尿液上皮细胞，采用重编程技术成功构建四系iPSCs细胞系；采用干细胞定向分化技术将上述四系iPSCs诱导分化为神经上皮前体细胞（NEPs）、运动神经元前体细胞（MNPs）、有丝分裂后运动神经元（HB9+MNs）和成熟运动神经元（ChAT+MNs），建立上述突变的CMT2Z iPSC-MNs细胞模型；在iPSCs和MNPs阶段，ki67免疫荧光和流式细胞术检测显示各突变组的ki67表达减低、细胞增殖受阻，以p.S87L组达最显著；在ChAT+MNs阶段，Tubulinβ3免疫染色显示各突变组均出现轴索断裂和曲张样体形成；中性彗星实验显示各突变组ChAT+MNs阶段均出现DNA损伤显著增高，而上述改变在MNPs、HB9+MNs阶段不明显；RNA测序和实时荧光定量PCR、蛋白免疫印迹验证表明，各突变组在MNPs阶段的差异基因主要富集在与增殖相关的PI3K/AKT和MAPK/ERK分子通路，以p.S87L组最显著。各突变组在HB9+MNs阶段的差异基因则主要富集于轴突终末、突触前膜和囊泡运输相关分子；对iPSCs阶段的MORC2p.S87L组运用反义寡核苷酸（ASO）靶向沉默MORC2p.S87L表达，可逆转下调的PI3K/ AKT和MAPK/ERK信号通路和提升iPSCs增殖水平，提示早期ASO靶向干预治疗有效。在HB9+MNs和ChAT+MNs阶段的各突变组开展NAR、HDAC6抑制剂等小分子药物处理，可改善神经元的轴突数量和形态。本研究在iPSC-MNs水平阐明了CMT2Z表型差异的可能机制：MORC2p.S87L主要通过导致早期神经细胞的增殖障碍而出现SMA样严重表型、MORC2 p.Q400R和p.D466N则主要通过导致成熟神经元的DNA损伤和轴索损伤而出现经典CMT2表型。本研究同时也为相应表型分别提供了具有较好临床转化能力的ASO和小分子药物靶向治疗方案。