STUDYING THE EFFECT OF NOONAN SYNDROME-ASSOCIATED GENE <EM>LZTR1</EM> ON BRAIN DEVELOPMENT USING IPSC-DERIVED MODELS

The RAS/MAPK is a crucial signaling pathway regulating cell proliferation, differentiation, and survival during development. LZTR1, an adaptor protein linking the CUL3 ubiquitin ligase to RAS GTPases, mediates their degradation and prevents RAS/MAPK hyperactivation. LZTR1 loss-of-function mutations cause Noonan syndrome (NS), where excessive RAS signaling manifests as cardiac defects and a spectrum of developmental delays, intellectual disabilities, and various sensory issues, including long-term neuropathic pain. We aim to investigate the molecular mechanisms underlying these nervous system manifestations in NS patients with mutations in LZTR1 using iPSC-derived models. Proteomic analysis of LZTR1^KO sensory neurons revealed upregulation of RAS GTPases (MRAS, RIT1, HRAS), sensory lineage markers (BRN3A, TrkA, TrkB), and early neuronal marker βIII-tubulin, alongside decreased CHCHD2 and subsequent upregulation of multiple COX subunits, suggesting altered sensory neuronal differentiation and an adaptive mitochondrial response to impaired complex IV function. To examine cortical abnormalities, we used LZTR1^KO iPSCs to generate 2D NGN2 iNeurons and 3D brain organoids. In NGN2 iNeurons, MRAS and RIT1 RAS GTPases and the pERK/ERK ratio were increased, consistent with RAS/MAPK hyperactivation. In 3D organoids, flow cytometry at day 60 revealed premature differentiation with elevated βIII-tubulin⁺ early neurons and fewer progenitors, while MAP2⁺ expression remained stable. Mitochondrial analyses showed higher mitochondrial mass and membrane potential, suggesting compensatory bioenergetic changes. LZTR1 loss-of-function in NS hyperactivates RAS/MAPK signaling, driving premature neuronal differentiation and mitochondrial adaptations, potentially contributing to neurodevelopmental deficits. Ongoing single-cell RNA sequencing and high-density MEA recordings will link molecular to functional outcomes, informing RAS-targeted therapies for NS patients.