Rac1 contributes to brain connectivity impairments and neuropsychiatric disorders in Tuberous Sclerosis Complex

Abstract Tuberous sclerosis complex (TSC) is a neurodevelopmental disorder caused by inactivating mutations in the TSC1 or TSC2 genes, leading to constitutive activation of the mTOR complex 1 pathway. Patients commonly present with epilepsy and TSC-associated neuropsychiatric disorders (TANDs), including intellectual disability and anxiety. Our previous work identified anxiety-like behavior in tsc2 vu242 zebrafish mutants, characterized by enhanced thigmotaxis. We found that anxiety-like behavior in tsc2 vu242 zebrafish mutants was rescued by Rac1 inhibitors. These findings prompted further investigation into Rac1 signaling in the context of TSC pathology. Using G-LISA and FRET live imaging, we confirmed elevated Rac1 activity in the mutant brains. Rac1 activity was reduced following NSC23766 and EHT1864 treatment. Compared to wild-type controls, Rac1 activity in mutants was elevated by 50% in untreated animals, and reduced by ∼30–40% after Rac1 inhibitors. Moreover, aberrant connectivity between brain hemispheres that correlated with anxiety before was restored after Rac1 inhibition. Transcriptomic profiling revealed 1,260 DEGs between homozygous and heterozygous tsc2 vu242 zebrafish. Rac1 inhibition restored the expression of approximately 1,000 genes toward wild-type levels. KEGG pathway analysis identified significant enrichment in mTOR signaling and focal adhesion, while GO terms highlighted processes including axon guidance, axonogenesis, and neuronal projection. Collectively, our findings support a functional role for Rac1 in TSC pathology, linking cytoskeletal dysregulation with aberrant axon development and anxiety-like behavior. Rac1 inhibition emerges as a promising therapeutic avenue for modulating both brain structure and function in TSC-associated anxiety. Highlights Rac1 activity is upregulated in tsc2 vu24/vu242 zebrafish mutants and correlates with anxiety-like behavior. Inhibition with NSC23766 and EHT1864 reduces Rac1 activity, partially normalizes mTORC1 signaling, and rescues anxiety-like behavior. Live FRET imaging and G-LISA confirm elevated Rac1 signaling and its modulation by pharmacological treatment. Transcriptomic profiling reveals 1,260 deferentially expressed genes in mutants, with restoration of ∼1,000 genes by NSC23766. Gene expression and pathway analysis link Rac1 dysregulation to altered actin dynamics, axon guidance, and focal adhesion.