MODELING MTOR PATHWAY DYSREGULATION IN PATIENT-DERIVED NEURONS

The MTOR gene encodes a central kinase regulating cellular growth, protein synthesis, and survival. Pathogenic MTOR variants, frequently arising as somatic mosaicism, cause pathway hyperactivation and are associated with epileptogenic malformations of cortical development (MCDs). We previously identified the MTOR p.Thr1977Ile variant in a patient with megalencephaly, polymicrogyria, and drug-resistant focal epilepsy. While rodent studies support its pathogenicity, a patient-derived in vitro model is required to investigate precision therapeutic approaches. We first assessed the functional impact of the variant in patient-derived fibroblasts by measuring P-RPS6 levels and responses to mTOR pathway inhibitors. We then reprogrammed fibroblasts into induced pluripotent stem cells (iPSCs), differentiated iPSCs into neural progenitor cells (NPCs), and further NPCs into mature neurons. Across differentiation stages, we evaluated mTOR activation, NPC proliferation and apoptosis, and neuronal intrinsic excitability and network synchrony. Patient-derived fibroblasts showed marked mTOR pathway hyperactivation, which was reversed by everolimus or metformin. In the neural model, mTOR hyperactivation and cytomegaly were most pronounced at the NPC stage. Mutant NPCs displayed increased proliferation and reduced apoptotic markers, consistent with the patient’s megalencephaly. Nutrient starvation assays revealed a mutant NPC subpopulation maintaining elevated P-RPS6 expression and abnormal nuclear morphology despite metabolic stress, indicating autonomous mTOR signaling that bypasses physiological growth arrest. This iPSC-based neural model demonstrates how the MTOR p.Thr1977Ile variant disrupts early neurodevelopment and metabolic responses. It also provides a valuable tool for screening targeted therapies and advancing precision treatment strategies for refractory epilepsy in mTOR-related neurodevelopmental disorder.