Title: LLGL2 protein participates in the brain development and cognitive function, likely through regulation of glutamate neurotransmission
LLGL2 (MIM: 618483) encodes the Lethal giant larvae 2 protein, a key regulator of cell polarity, asymmetric division, and migration. In this study, we identified eight affected individuals from four unrelated families harboring one splicing and three missense variants in LLGL2 that segregate with neurodevelopmental disorder (NDD), epilepsy, and microcephaly. Overexpression of constructs carrying NDD-associated variants altered the subcellular localization of LLGL2 and disrupted cytoskeletal organization. Using homology-directed repair (HDR)-mediated CRISPR-Cas9 genome editing, we introduced the c.1456G>A variant into HEK293T cells. The knock-in cells exhibited pronounced mitotic defects, including aberrant centrosome assembly, multipolar spindle formation, increased multinucleation, and abnormal cytokinesis, along with altered centrosome number and diameter. These cells also displayed a significant increase in γ-H2AX foci, indicative of elevated DNA damage, and a reduced proliferation rate. Flow cytometric profiling revealed marked perturbations across all phases of the cell cycle.
Bulk mRNA sequencing of knock-in cells demonstrated significant dysregulation of the glutamatergic pathway and ERK1/2 regulatory networks—key modulators of excitatory neurotransmission and higher-order brain functions such as cognition and behavior. Further, to investigate LLGL2 function in vivo, we generated llgl2 knockdown zebrafish larvae. Consistent with the human phenotype, llgl2 morphants exhibited developmental abnormalities and reduced locomotor activity, both of which were rescued by co-injection of human wild-type LLGL2 mRNA but not mRNAs harboring NDD variants, confirming their pathogenicity. Parallel studies using c.1456G>A knock-in human induced pluripotent stem cells (iPSCs) revealed altered neural progenitor cell (NPC) morphology, including changes in embryoid body diameter and shape, and an accelerated differentiation potential relative to controls. Ongoing work using cortical neurons derived from these NPCs aims to delineate the impact of LLGL2 mutation on neuronal morphology and glutamatergic function. In summary, our findings establish LLGL2 as a critical regulator of cell division, genomic stability, and glutamatergic signaling, and highlight its essential role in human neurodevelopment and cognitive function.