INTRODUCTION: Tuberous Sclerosis Complex (TSC) is a genetic neurodevelopmental disorder caused by loss‑of‑function mutations in TSC1 or TSC2, which hyperactivate the mTORC1 pathway. One of the features of TSC is the emergence of TSC‑associated neuropsychiatric disorders (TANDs), among which anxiety and intellectual disability is prevalent.

AIM(S): We aim to uncover therapeutic targets within the Rac1 pathway to rescue disruptions in proper neurodevelopment and diminish anxiety-like behaviors in tsc2-deficient zebrafish.

METHOD(S): Using transparent, externally developing zebrafish, we can model neurodevelopmental abnormalities in vivo via plasmid electroporation and measuring GTP-bound RAC1. Lightsheet microscopy was used and samples were sent for RNA sequencing.

RESULTS: Our previous work demonstrated that tsc2‑deficient zebrafish exhibit disrupted axon bundling, a reduced width of the anterior commissure and anxiety‑like behaviors. We found elevated Rac1 levels in mutant tsc2-deficient zebrafish which correlated with observed anxiety-like behaviour. Importantly, Rac1 inhibitors reversed anxiety-like behaviour, as well as aberrant connectivity between brain hemispheres. Rac1 is a crucial factor in axon elongation during development. Transcriptomic profiling of brain samples revealed that Rac1 inhibition restored expression of the majority of differentially expressed genes in the tsc2 mutants back to wild-type levels. KEGG pathway analysis identified significant enrichment in mTOR signaling and focal adhesion, while GO terms highlighted processes including axon guidance, axogenesis and neuronal projection. Moreover, the mRNA levels of Rac1 upstream activators dock3, dock4 and elmo2 were also increased in mutant brains.

CONCLUSIONS: Collectively, these findings highlight the role of the Rac1 pathway in TSC pathology and point towards cytoskeletal dysregulation and aberrant axon development as potential mechanisms underlying TSC-associated neuropsychiatric disorders.

FINANCIAL SUPPORT: This work was supported by the OPUS Grant No. 2020/37/B/NZ3/02345, Sonata Bis grant No. 2023/50/E/NZ3/00252, both from National Science Centre, Poland