Convergent and Divergent Molecular Pathways in FMR1-, TSC2- and FMR1/TSC2 Knockout Neurons.

Fragile X syndrome (FXS) and tuberous sclerosis complex (TSC) are common monogenic causes of autism spectrum disorder (ASD). FXS arises from FMR1 silencing, while TSC results from mutations in TSC1 or TSC2, both converging on dysregulated ERK and mTORC1 signaling. Animal knockout models suggest opposing effects on synaptic plasticity, with reciprocal compensation in double knockouts (dKO). However, clinical case with dual mutations shows severe neurodevelopmental deficits; here, we explored a human cellular model to dissect the shared and divergent mechanisms. We generated isogenic human pluripotent stem cell (hPSC)-derived models of FMR1KO, TSC2KO, and FMR1/TSC2 dKO neurons. Neuronal transcriptomes were profiled by RNA-seq, integrating ERK, mTOR, FMRP targets, and ASD risk genes. Validation via qPCR of key genes, protein synthesis, proliferation assays, and microelectrode array was performed. The FMR1/TSC2 dKO neural progenitor cells (NPCs) demonstrated high DNA damage response but normalized proliferation. Convergent transcriptomic pathways across FMR1KO, TSC2KO, and dKO neurons included upregulated extracellular matrix and stress responses, and downregulated synaptic and neurotransmission-related pathways. TSC2KO and dKO neurons showed greater similarity transcriptionally and functionally. Translational pathways and global protein synthesis were oppositely regulated in TSC2KO and dKO versus FMR1KO neurons. The dKO neurons showed hyperexcitable network activity, mTOR hyperactivation, with distinct dysregulated FMRP targets and ASD risk gene expression. Unlike mouse models, FMR1/TSC2 dKO hPSC-derived neurons did not show rescue of synaptic gene expression. Rather, dKO neurons predominantly resembled TSC2KO neurons with translational, synaptic, and neurotransmission abnormalities. These findings highlight complex interplay between FMRP and TSC, providing a foundation for future studies of ASD-relevant mechanisms.