Project description:Tuberous Sclerosis Complex (TSC), an autosomal dominant condition, is engendered by heterozygous mutations in either TSC1 or TSC2 genes, manifesting in systemic growth of benign tumors. In addition to brain lesions, neurologic sequelae represent the greatest morbidity in TSC patients. Investigations utilizing TSC1/2-knockout animal or human stem cell models suggest that TSC deficiency-causing hyper-activation of mTOR signaling might precipitate anomalous neurodevelopmental processes. However, how the pathogenic variants of TSC1/2 genes identified in TSC patients affect the trajectory of human brain development and how they contribute to the neurological manifestations in TSC remain largely unexplored. Here, we employed 3-dimensional cortical organoids derived from induced pluripotent stem cells (iPSCs) from TSC patients harboring TSC2 mutations, alongside organoids from age- and sex-matched healthy individuals as controls. Through comprehensively longitudinal molecular and cellular analysis of TSC organoids, including transcriptomics and single cell transcriptomics, we found that TSC2 pathogenic variants led to dysregulated neurogenesis, synaptogenesis, and gliogenesis, particularly for reactive astrogliosis. The altered developmental trajectory of TSC organoids significantly resembles the molecular signatures of neuropsychiatric disorders, including autism spectrum disorders, epilepsy, and intellectual disability. Through cell-cell communication analysis at the single cell level, we identified that TSC2 pathogenic variants disrupted the cell-cell communications, in particular, neuron-astrocyte interactions within the NLGN-NRXN signaling network. Furthermore, cellular and electrophysiological assessments of TSC cortical organoids, along with proteomics and phosphoproteomics analyses of synaptosomes, revealed that pathogenic TSC2 variants precipitate perturbations in mitochondrial translational integrity, neurofilament formation, synaptic transmission, and neuronal network activity. Intriguingly, the increased neu

Project description:Tuberous sclerosis complex (TSC) is an inherited neurodevelopmental (ND) disorder with frequent manifestations of epilepsy and autism spectrum disorder (ASD) caused by mutations in TSC1 or TSC2 genes. TSC1 and TSC2 form a complex inhibiting mechanistic target of rapamycin complex-1 (mTORC1) signaling, leading to hyperactive mTORC1 signaling upon TSC1/2 loss of function. Although rapalogs, which are FDA-approved allosteric mTORC1-selective inhibitors, are used to treat TSC-associated hamartomas, they are not effective for treating ND manifestations. mTORC1 signaling controls protein synthesis by regulating formation of the eIF4F complex, whose activity is further modulated by the MNK1/2 kinases via phosphorylation of the eIF4F subunit eIF4E. While both these pathways modulate translation in transcript-selective fashion depending on features in target mRNAs’ 5’ untranslated regions (5’UTRs), their effect on transcriptome-wide patterns of mRNA translation has not been compared. Here, employing CRISPR-modified, isogenic TSC2 patient-derived neural progenitor cells (NPCs), we examined how loss of TSC2 affects gene expression via changes in mRNA abundance and translation at a transcriptome-wide scale. This revealed abundant mRNA translation alterations in TSC2-Null NPCs overlapping with those we previously observed in TSC1-Null NPCs. Surprisingly, numerous non-monogenic ASD- and NDD-associated genes, identified in patients harboring putative loss-of-function mutations, were selectively translationally suppressed in TSC2-Null NPCs consistent with their distinct repertoire of 5’UTR features. Importantly, translation of these ASD- and NDD-associated genes was reversed upon inhibition of either mTORC1 or MNK1/2 signaling using RMC-6272 or eFT-508, respectively. This study thereby establishes mTORC1-eIF4F and MNK-eIF4E-sensitive mRNA translation as key components in TSC, ASD and other neurodevelopmental disorders; and lay the groundwork for evaluating drugs in clinical development that target these pathways as a treatment strategy for TSC as well as ASD/NDD.

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Convergent and Divergent Molecular Pathways in FMR1, TSC2, and double Knockout iPSC-Derived Neurons

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