A human iPSC-derived microglia model of tuberous sclerosis complex exhibits LPL-driven increased lipid metabolism affecting neuronal hyperexcitability

Tuberous sclerosis complex (TSC) is an autosomal dominant genetic disorder caused by mutations in either TSC1 or TSC2, presenting with systemic growth of benign tumors. In addition to the brain lesions, neurologic impairment causes the greatest morbidity in TSC patients. Up to 50% of TSC patients are affected with autism spectrum disorders (ASDs), including a wide range of autistic symptoms overlapping with that in idiopathic ASD patients. Examination of postmortem tissue uncovered activated microglia and neuroinflammation in the brains of TSC patients. Microglia are the resident immune cells of the central nervous system. Studies from animal or human stem cell models with TSC1/2 knockout suggest that TSC1/2 deficiency can cause hyper-activation of the mammalian target of rapamycin complex 1 (mTORC1) signaling, which may result in abnormal neurodevelopment. However, how the causal variants of TSC1/2 genes identified in TSC patients affect human microglia and how they contribute to the neurological manifestations in TSC remain largely unexplored. To address these questions, in this study, we generated human induced pluripotent stem cells-derived microglia (hiPSC-microglia) from TSC patients carrying TSC2 mutations as well as age- and sex-matched healthy controls for the first time. Bulk RNA sequencing (bulk-RNAseq), proteome, and phosphoproteome analyses of TSC microglia revealed that TSC2 mutations not only hyperactivate mTORC1 signaling pathway, but also alter lipid metabolism through the peroxisome proliferator-activated receptor (PPAR) signaling pathway driven by lipoprotein lipase (LPL) gene. This leads to dysregulated phagocytic capacities and pro-inflammatory responses, which further impair neuronal function including altered complexity of neuronal dendrites, dysregulated formation of morphological excitatory synapses and neuronal hyperexcitability. Additionally, unbiased lipidomics revealed alterations of glycerophosphocholines (GPC) and fatty acyls (FA) in TSC microglia. The developmental abnormalities i To determine the causal role of LPL by knocking-down LPL in TSC microglia and overexpressing LPL in control microglia, respectively.