TSC2-deficiency potentiates a catabolic signaling switch that differentiates neural and neural crest lineage development and progressive disease manifestations

inactivating mutations elicit mTORC1 hyperactivation and underlie neurological dysfunction and the development of neural and mesenchymal tumors in the monogenic disease tuberous sclerosis complex (TSC). We present a multi-lineage model of TSC2-deficiency employing CRISPR-Cas9 engineering in human pluripotent stem cells (hPSCs) and differentiation into neural and neural crest lineages, cell types predicted to drive TSC manifestations. Temporal RNA-sequencing reveals a massive proteostatic stress response underlying early neuroepithelial induction of TSC2deficient cells, which is resolved upon neural crest cell (NCC) specification but persists in neural precursor cells (NPCs). This culminates in long-term endosomal and metabolic reprogramming as cells age, and sensitivity of NPCs, but not NCCs, to death via proteasome inhibition independent of mTORC1 activity. Thus, TSC2-deficiency induces lineage-specific stress adaptations which confer differential sensitivity to a commonly targeted pathway. These results exemplify the complexity of elucidating underlying biological mechanisms and therapeutic approaches for multisystem diseases, illustrating the power of utilizing hPSC disease models with tissue-specific relevance.