Abstract
The RhoGEF TRIO is known to play a major role in neuronal development by controlling actin cytoskeleton remodeling, primarily through the activation of the RAC1 GTPase. Numerous de novo mutations in the TRIO gene have been identified in individuals with neurodevelopmental disorders (NDDs). We have previously established the first phenotype/genotype correlation in TRIO-associated diseases, with striking correlation between the clinical features of the individuals and the opposite modulation of RAC1 activity by TRIO variants targeting different domains. The mutations hyperactivating RAC1 are of particular interest, as they are recurrently found in patients and are associated with a severe form of NDD and macrocephaly, indicating their importance in the etiology of the disease. Yet, it remains unknown how these pathogenic TRIO variants disrupt TRIO activity at a molecular level and how they affect neurodevelopmental processes such as axon outgrowth or guidance. Here we report an additional cohort of individuals carrying a pathogenic TRIO variant that reinforces our initial phenotype/genotype correlation. More importantly, by performing conformation predictions coupled to biochemical validation, we propose a model whereby TRIO is inhibited by an intramolecular fold and NDD-associated variants relieve this inhibition, leading to RAC1 hyperactivation. Moreover, we show that in cultured primary neurons and in the zebrafish developmental model, these gain-of-function variants differentially affect axon outgrowth and branching in vitro and in vivo, as compared to loss-of-function TRIO variants. In summary, by combining clinical, molecular, cellular and in vivo data, we provide compelling new evidence for the pathogenicity of novel genetic variants targeting the TRIO gene in NDDs. We report a novel mechanism whereby the fine-tuned regulation of TRIO activity is critical for proper neuronal development and is disrupted by pathogenic mutations.
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Acknowledgements
We are grateful to all the patients and their families for their participation in this study, and in particular to Candice Williams. We also acknowledge Eva-Lena Stattin (Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden) and Philip Wyatt (Department of Obstetrics and Gynecology, York Central Hospital, Toronto, Ontario, Canada) for their patient case contribution.
We thank all members of the Debant team as well as Xavier Nicol (Institut de la Vision, Paris) for helpful discussions, and Damien Laouteouet and Jean-Christophe Perez for generating and performing initial experiments with the artificial spectrin mutants. This work was supported by grants from the Agence Nationale de la Recherche to AD (ANR-2019 TRIOTISM) and to CF (ANR-20-CE16-0019) and from The Fondation pour la Recherche Médicale (program Equipes FRM2016, DEQ20160334942) to AD. MaxB and MarB are recipients of a PhD fellowship from the Ministère de l’Enseignement Supérieur et de la Recherche (MESR). DB is generously supported by a National Institute for Health and Care Research research professorship RP-2016-07-011. We acknowledge the imaging facility MRI, and in particular Volker Bäcker, part of the national France-BioImaging infrastructure supported by the French National Research Agency (ANR-10-INBS-04, “Investments for the future”).
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MaxB, AD, CF and SS conceived research and designed research experiments; DB, GG, DH, MPR, AC, DS, MM, FV and AC conducted subject recruitment and consented assessment; DB and GG interpreted subjects phenotypes; MaxB, CFK, IT, SS, FC and SB performed site-directed mutagenesis, cloning of plasmids, Western Blot and pulldown experiments; MaxB and SB prepared neuronal cultures; MaxB performed immunofluorescence and microscopy studies, with the help of MarB; MaxB and NN performed image analyses and statistical analyses. FR and CF performed the zebrafish experiments; MaxB, FR and CF analyzed and quantified images from zebrafish motoneurons and made statistics; AVK performed molecular modeling and suggested artificial TRIO mutants; SS and AD supervised the project and coordinated the study, in collaboration with DB; SS, AD, MaxB and CF wrote the manuscript. All authors reviewed and approved the manuscript.
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Bonnet, M., Roche, F., Fagotto-Kaufmann, C. et al. Pathogenic TRIO variants associated with neurodevelopmental disorders perturb the molecular regulation of TRIO and axon pathfinding in vivo. Mol Psychiatry 28, 1527–1544 (2023). https://doi.org/10.1038/s41380-023-01963-x
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DOI: https://doi.org/10.1038/s41380-023-01963-x
