Tuberous sclerosis complex (TSC) is an inherited neurocutaneous disorder caused by mutations in the TSC1 or TSC2 genes, with patients often exhibiting neurodevelopmental (ND) manifestations termed TSC-associated neuropsychiatric disorders (TAND) including autism spectrum disorder (ASD) and intellectual disability. Hamartin (TSC1) and tuberin (TSC2) proteins form a complex inhibiting mechanistic target of rapamycin complex 1 (mTORC1) signaling. Loss of TSC1 or TSC2 activates mTORC1 that, among several targets, controls protein synthesis by inhibiting translational repressor eIF4E-binding proteins. Using TSC1 patient-derived neural progenitor cells (NPCs), we recently reported early ND phenotypic changes, including increased cell proliferation and altered neurite outgrowth in TSC1-null NPCs, which were unaffected by the mTORC1 inhibitor rapamycin. Here, we used polysome profiling, which quantifies changes in mRNA abundance and translational efficiencies at a transcriptome-wide level, to compare CRISPR-edited TSC1-null with CRISPR-corrected TSC1-WT NPCs generated from one TSC donor (one clone/genotype). To assess the relevance of identified gene expression alterations, we performed polysome profiling in postmortem brains from ASD donors and age-matched controls. We further compared effects on translation of a subset of transcripts and rescue of early ND phenotypes in NPCs following inhibition of mTORC1 using the allosteric inhibitor rapamycin versus a third-generation bi-steric, mTORC1-selective inhibitor RMC-6272. Polysome profiling of NPCs revealed numerous TSC1-associated alterations in mRNA translation that were largely recapitulated in human ASD brains. Moreover, although rapamycin treatment partially reversed the TSC1-associated alterations in mRNA translation, most genes related to neural activity/synaptic regulation or ASD were rapamycin-insensitive. In contrast, treatment with RMC-6272 inhibited rapamycin-insensitive translation and reversed TSC1-associated early ND phenotypes including proliferation and neurite outgrowth that were unaffected by rapamycin. Our work reveals ample mRNA translation alterations in TSC1 patient-derived NPCs that recapitulate mRNA translation in ASD brain samples. Further, suppression of TSC1-associated but rapamycin-insensitive translation and ND phenotypes by RMC-6272 unveils potential implications for more efficient targeting of mTORC1 as a superior treatment strategy for TAND.

中文翻译：

---

结节性硬化症复合体 1 患者来源的神经祖细胞的翻译组分析揭示了雷帕霉素依赖性和独立性改变

结节性硬化症 (TSC) 是一种由 TSC1 或 TSC2 基因突变引起的遗传性神经皮肤疾病，患者经常表现出称为 TSC 相关神经精神疾病 (TAND) 的神经发育 (ND) 表现，包括自闭症谱系障碍 (ASD) 和智力障碍。Hamartin (TSC1) 和 tuberin (TSC2) 蛋白形成复合物，抑制雷帕霉素复合物 1 (mTORC1) 信号传导的机制靶点。TSC1 或 TSC2 的缺失会激活 mTORC1，mTORC1 在多个靶标中通过抑制翻译阻遏物 eIF4E 结合蛋白来控制蛋白质合成。使用 TSC1 患者来源的神经祖细胞 (NPC)，我们最近报告了早期 ND 表型变化，包括 TSC1 缺失 NPC 中细胞增殖增加和神经突生长改变，而这些变化不受 mTORC1 抑制剂雷帕霉素的影响。在这里，我们使用多核糖体分析（在转录组范围内量化 mRNA 丰度和翻译效率的变化）来比较 CRISPR 编辑的 TSC1-null 与由一个 TSC 供体（一个克隆/基因型）生成的 CRISPR 校正的 TSC1-WT NPC。 。为了评估已识别基因表达改变的相关性，我们对 ASD 供体和年龄匹配对照的死后大脑进行了多核糖体分析。我们进一步比较了使用变构抑制剂雷帕霉素与第三代双位阻 mTORC1 选择性抑制剂 RMC-6272 抑制 mTORC1 后对转录本子集的翻译和挽救 NPC 早期 ND 表型的影响。NPC 的多核糖体分析揭示了许多与 TSC1 相关的 mRNA 翻译改变，这些改变在人类 ASD 大脑中得到了重现。此外，虽然雷帕霉素治疗部分逆转了 TSC1 相关的 mRNA 翻译改变，但大多数与神经活动/突触调节或 ASD 相关的基因对雷帕霉素不敏感。相比之下，RMC-6272 治疗可抑制雷帕霉素不敏感的翻译，并逆转 TSC1 相关的早期 ND 表型，包括不受雷帕霉素影响的增殖和神经突生长。我们的工作揭示了 TSC1 患者来源的 NPC 中大量 mRNA 翻译的改变，这些改变概括了 ASD 大脑样本中的 mRNA 翻译。此外，RMC-6272 对 TSC1 相关但雷帕霉素不敏感的翻译和 ND 表型的抑制揭示了更有效地靶向 mTORC1 作为 TAND 的优越治疗策略的潜在影响。