Abstract
Autism spectrum disorder (ASD) is a neurodevelopmental condition characterized by impairments in social interaction, challenges with communication, and repetitive behaviors. Genetic mutations associated with ASD can either activate or inactivate the responsible proteins, affecting neuronal morphogenesis and contributing to the disorder’s hallmark features. However, the molecular mechanisms driving these changes remain incompletely understood. Here, we report for the first time that the small GTP/GDP-binding protein Arf6 and FE65, which act together with the genetically conserved engulfment and cell motility 2 (ELMO2) signalosome to control Rac1, underlie the excessive neuronal process elongation phenotype associated with the ASD-linked semaphorin-5 A (Sema5A) Arg676-to-Cys protein (p.Arg676Cys). Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas13-mediated knockdown of Arf6 or FE65 reversed the excessively elongated processes in primary cortical neurons. Similar results were obtained in the N1E-115 cell line, a model capable of neuronal morphological differentiation. Moreover, expression of the ELMO2-binding domain of FE65 restored Rac1 activity required for process elongation, recapitulating the effects seen in the knockdown experiments. These findings suggest that signaling through FE65 specifically couples Sema5A p.Arg676Cy to the ELMO2 signalosome molecule, driving excessively elongated processes with elevated Rac1 activity. One or more of these molecules may provide possible therapeutic targets for correcting the cellular phenotypes associated with the Sema5A p.Arg676Cys mutation in ASD.
Data availability
The datasets used and/or analyzed for the current study are available from the corresponding author upon reasonable request.
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Acknowledgements
We thank Drs. Takako Morimoto and Yoichi Seki (School of Life Sciences, Tokyo University of Pharmacy and Life Sciences) for the insightful comments they provided throughout this study.
Funding
This work was supported by the Core Research for Evolutional Science and Technology (CREST) program of the Japan Science and Technology Agency (JST). Additional support was provided by Grants-in-Aid for Scientific Research from the Japanese Ministry of Education, Culture, Sports, Science and Technology (MEXT) and Grants-in-Aid for Medical Scientific Research from the Japanese Ministry of Health, Labour and Welfare (MHLW). We also received funding from the Daiichi Sankyo Science Foundation, Japan Foundation for Pediatric Research, Mishima Kaiun Memorial Foundation, Mitsubishi Tanabe Science Foundation, Otsuka Science Foundation, and Takeda Science Foundation.
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Junji Yamauchi designed and organized the study. Yuki Miyamoto and Junji Yamauchi wrote and edited the manuscript. Mikito Takahashi and Hideji Yako performed experiments. Mikito Takahashi and Hideji Yako performed statistical analyses. Hideji Yako, Mutsuko Kukimoto-Niino, and Mikako Shirouzu evaluated experimental and statistical data.
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Takahashi, M., Yako, H., Miyamoto, Y. et al. Autism spectrum disorder-associated Sema5A p.Arg676Cys drives Arf6/FE65 signaling and aberrant cell morphogenesis. Sci Rep (2026). https://doi.org/10.1038/s41598-026-39722-x
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DOI: https://doi.org/10.1038/s41598-026-39722-x
