Translatome analysis of Tuberous Sclerosis Complex 1 patient-derived isogenic neural progenitor cells reveal rapamycin-dependent and independent alterations
Tuberous sclerosis complex (TSC) is an inherited multi-system disorder caused by mutations in the TSC1 or TSC2 gene. TSC patients are often diagnosed with a range of neurodevelopmental (ND) manifestations termed TSC-associated neuropsychiatric disorders (TAND) including autism spectrum disorder (ASD), intellectual disability (ID), anxiety and mood disorders. Hamartin (TSC1) and tuberin (TSC2) proteins form a complex inhibiting mechanistic target of rapamycin complex 1 (mTORC1) kinase signaling. Loss of TSC1 or TSC2 activates mTORC1 that, among several targets, controls protein synthesis by inhibiting translational repressor eIF4E-binding proteins. Using neural progenitor cells (NPCs) from patient-derived induced pluripotent stem cells (iPSCs), we recently reported early ND phenotypic changes, including increased cell proliferation and altered neurite outgrowth in CRISPR-modified TSC1-null NPCs, which were unaffected by mTORC1 inhibition by rapamycin,the only approved therapy for TSC1. Here, to assess TSC1-dependent gene expression programs in NPCs, we used polysome-profiling, which quantifies changes in mRNA abundance and translational efficiencies at a transcriptome-wide level. In addition to changes in mRNA abundance, this revealed numerous TSC1-dependent alterations in translational efficiencies. To assess the relevance of these gene expression alterations we performed polysome-profiling in post-mortem brains originating from ASD donors and matched controls. Strikingly, TSC1-dependent alterations in mRNA translation observed in NPCs were largely recapitulated in human brains. Furthermore, although polysome-profiling revealed a partial reversal of TSC1-associated gene expression alterations following rapamycin treatment, most genes related to neural activity/synaptic regulation or ASD that showed TSC1-dependent translation were rapamycin-insensitive. Therefore, we also examined whether early ND rapamycin-insensitive phenotypes in TSC1-null NPCs could be rescued by a third-generation bi-steric, mTORC1-selective inhibitor RMC-6272 (Revolution Medicines, Inc.). Unlike rapamycin, RMC-6272 strongly inhibited translation and reversed TSC1-associated proliferation and neurite outgrowth phenotypes. In summary, we reveal ample translational alterations in TSC1 patient-derived NPCs recapitulating human brain expression profiles and potential implications for treatment of TAND. Overall design: Analysis of total and polysomal mRNA samples of NPCs derived from a female TSC1 patient, including TSC1-null (-/-) and CRISPR-corrected TSC1-WT (+/+), treated with DMSO or rapamycin in triplicates.