TSC patient-derived isogenic neural progenitor cells reveal altered early neurodevelopmental phenotypes and rapamycin-induced MNK-eIF4E signaling
TSC patient-derived isogenic neural progenitor cells reveal altered early neurodevelopmental phenotypes and rapamycin-induced MNK-eIF4E signaling
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DOI：
10.1186/s13229-019-0311-3
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发表时间：
2020-01-06
期刊：
影响因子：
6.2
通讯作者：
Ramesh, Vijaya
中科院分区：
文献类型：
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作者：
Martin, Pauline;Wagh, Vilas;Ramesh, Vijaya
研究背景脑硬化综合征（TSC）是一种神经发育障碍性疾病，常见于癫痫、自闭症谱系障碍（ASD）、智力残疾（ID）和多器官肿瘤。TSC中mTORC 1的异常激活导致使用mTORC 1抑制剂雷帕霉素作为肿瘤的终身治疗，但TSC相关的神经认知表现不受雷帕霉素的影响。在这里，我们从TSC患者中产生了患者特异性的诱导多能干细胞（iPSC），该患者在TSC 1的外显子15中具有杂合，种系，无义突变，并使用CRISPR/Cas9介导的基因编辑建立了杂合（Het），无效和校正野生型（Corr-WT）iPSC的同基因组。我们将这些iPSC分化为神经祖细胞（NPC），并通过RNA-seq检查神经发育表型，信号传导和基因表达的变化。结果TSC 1-Het和TSC 2-Het NPCs的细胞体积增大，与mTORC 1的激活一致。TSC 1-Het和TSC 2-NPCs也显示了增强的增殖和以基因型依赖的方式改变的神经突生长，这并不被雷帕霉素逆转。TSC 1-NPC的转录组分析揭示了显示基因型依赖性线性反应的差异表达基因，即，在Het中上调/下调的基因在Het中进一步增加/减少。特别是，与ASD，癫痫和ID相关的基因显着上调或下调，以进一步研究。在TSC 1-Het和TSC 2-NPCs中，我们还观察到ERK 1/2的基础激活，其在雷帕霉素处理后进一步激活。雷帕霉素还增加了TSC 1缺陷型NPC中的MNK 1/2-eIF 4 E信号传导。结论MEK-ERK和MNK-eIF 4 E通路调节蛋白质翻译，提示TSC 1/2缺陷的NPC中存在的异常翻译可能在神经发育缺陷中发挥作用。我们的数据显示雷帕霉素上调这些信号通路，支持联合收割机组合MEK或MNK抑制剂与雷帕霉素的策略，其对于TSC相关的CNS缺陷可能是上级的。重要的是，我们从TSC患者中产生的NPC的等基因组为translatome和大规模药物筛选研究提供了有价值的平台。总的来说，我们的研究进一步支持了这样的观点，即早期发育事件，如NPC增殖和初始过程形成，如神经元分化前发生的神经突数量和长度，代表了神经发生中对神经发育障碍如ASD的疾病发病机制至关重要的主要事件。
Background Tuberous sclerosis complex (TSC) is a neurodevelopmental disorder with frequent occurrence of epilepsy, autism spectrum disorder (ASD), intellectual disability (ID), and tumors in multiple organs. The aberrant activation of mTORC1 in TSC has led to treatment with mTORC1 inhibitor rapamycin as a lifelong therapy for tumors, but TSC-associated neurocognitive manifestations remain unaffected by rapamycin. Methods Here, we generated patient-specific, induced pluripotent stem cells (iPSCs) from a TSC patient with a heterozygous, germline, nonsense mutation in exon 15 of TSC1 and established an isogenic set of heterozygous (Het), null and corrected wildtype (Corr-WT) iPSCs using CRISPR/Cas9-mediated gene editing. We differentiated these iPSCs into neural progenitor cells (NPCs) and examined neurodevelopmental phenotypes, signaling and changes in gene expression by RNA-seq. Results Differentiated NPCs revealed enlarged cell size in TSC1-Het and Null NPCs, consistent with mTORC1 activation. TSC1-Het and Null NPCs also revealed enhanced proliferation and altered neurite outgrowth in a genotype-dependent manner, which was not reversed by rapamycin. Transcriptome analyses of TSC1-NPCs revealed differentially expressed genes that display a genotype-dependent linear response, i.e., genes upregulated/downregulated in Het were further increased/decreased in Null. In particular, genes linked to ASD, epilepsy, and ID were significantly upregulated or downregulated warranting further investigation. In TSC1-Het and Null NPCs, we also observed basal activation of ERK1/2, which was further activated upon rapamycin treatment. Rapamycin also increased MNK1/2-eIF4E signaling in TSC1-deficient NPCs. Conclusion MEK-ERK and MNK-eIF4E pathways regulate protein translation, and our results suggest that aberrant translation distinct in TSC1/2-deficient NPCs could play a role in neurodevelopmental defects. Our data showing upregulation of these signaling pathways by rapamycin support a strategy to combine a MEK or a MNK inhibitor with rapamycin that may be superior for TSC-associated CNS defects. Importantly, our generation of isogenic sets of NPCs from TSC patients provides a valuable platform for translatome and large-scale drug screening studies. Overall, our studies further support the notion that early developmental events such as NPC proliferation and initial process formation, such as neurite number and length that occur prior to neuronal differentiation, represent primary events in neurogenesis critical to disease pathogenesis of neurodevelopmental disorders such as ASD.