Department of Oncology-Pathology, Science for Life Laboratory, Karolinska Institute, 171 77, Stockholm, Sweden.
Center for Genomic Medicine, Department of Neurology, Massachusetts General Hospital, 185 Cambridge Street, Boston, MA, 02114, USA.
Mol Autism. 2023 Oct 25;14(1):39. doi: 10.1186/s13229-023-00572-3.
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.
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，患者常表现出神经发育（ND）表现，称为与 TSC 相关的神经精神障碍（TAND），包括自闭症谱系障碍（ASD）和智力障碍。结节蛋白（TSC1）和 tuberin（TSC2）蛋白形成复合物，抑制雷帕霉素靶蛋白复合物 1（mTORC1）信号。TSC1 或 TSC2 的缺失会激活 mTORC1，该复合物除了其他几个靶点外，还通过抑制翻译抑制剂 eIF4E 结合蛋白来控制蛋白质合成。我们最近使用 TSC1 患者来源的神经祖细胞（NPC）报告了早期 ND 表型变化，包括 TSC1 缺失 NPC 中的细胞增殖增加和神经突生长改变，而雷帕霉素（mTORC1 抑制剂）对这些变化没有影响。
在这里，我们使用核糖体图谱分析，该分析在全转录组水平上定量评估 mRNA 丰度和翻译效率的变化，比较来自一位 TSC 供体（一个克隆/基因型）的 CRISPR 编辑的 TSC1 缺失与 CRISPR 校正的 TSC1-WT NPC。为了评估鉴定的基因表达改变的相关性，我们对来自 ASD 供体和年龄匹配对照的尸检大脑进行了核糖体图谱分析。我们还比较了使用别构抑制剂雷帕霉素与第三代双特异性、mTORC1 选择性抑制剂 RMC-6272 抑制 mTORC1 后，对一组转录物的翻译影响以及在 NPC 中对早期 ND 表型的挽救。
NPC 的核糖体图谱分析显示，在 mTORC1 相关的 mRNA 翻译中存在大量 TSC1 相关改变，这些改变在人类 ASD 大脑中得到了很大程度的再现。此外，尽管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 的治疗抑制了雷帕霉素不敏感的翻译，并逆转了 TSC1 相关的早期 ND 表型，包括雷帕霉素不敏感的增殖和神经突生长。
我们的工作揭示了 TSC1 患者来源的 NPC 中存在大量的 mRNA 翻译改变，这些改变在 ASD 脑样本中得到了再现。此外，RMC-6272 抑制 TSC1 相关但雷帕霉素不敏感的翻译和 ND 表型，揭示了作为 TAND 更有效治疗策略的 mTORC1 靶向的潜在意义。
