Identification of the first Rho–GEF inhibitor, TRIPα, which targets the RhoA-specific GEF domain of Trio

AbstractThe Rho–guanine nucleotide exchange factors (Rho–GEFs) remodel the actin cytoskeleton via their Rho–GTPase targets and affect numerous physiological processes such as transformation and cell motility. They are therefore attractive targets to design specific inhibitors that may have therapeutic applications. Trio contains two Rho–GEF domains, GEFD1 and GEFD2, which activate the Rac and RhoA pathways, respectively. Here we have used a genetic screen in yeast to select in vivo peptides coupled to thioredoxin, called aptamers, that could inhibit GEFD2 activity. One aptamer, TRIAPα (TRio Inhibitory APtamer), specifically blocks GEFD2-exchange activity on RhoA in vitro. The corresponding peptide sequence, TRIPα, inhibits TrioGEFD2-mediated activation of RhoA in intact cells and specifically reverts the neurite retraction phenotype induced by TrioGEFD2 in PC12 cells. Thus TRIPα is the first Rho–GEF inhibitor isolated so far, and represents an important step in the design of inhibitors for the expanding family of Rho–GEFs

Kidins220/ARMS regulates Rac1-dependent neurite outgrowth by direct interaction with the RhoGEF Trio

Supplementary material available online at http://jcs.biologists.org/cgi/content/full/123/12/2111/DC1Neurite extension depends on extracellular signals that lead to changes in gene expression and rearrangement of the actin cytoskeleton. A factor that might orchestrate these signalling pathways with cytoskeletal elements is the integral membrane protein Kidins220/ARMS, a downstream target of neurotrophins. Here, we identified Trio, a RhoGEF for Rac1, RhoG and RhoA, which is involved in neurite outgrowth and axon guidance, as a binding partner of Kidins220. This interaction is direct and occurs between the N-terminus of Trio and the ankyrin repeats of Kidins220. Trio and Kidins220 colocalise at the tips of neurites in NGF differentiated PC12 cells, where F-actin and Rac1 also accumulate. Expression of the ankyrin repeats of Kidins220 in PC12 cells inhibits NGF-dependent and Trio induced neurite outgrowth. Similar results are seen in primary hippocampal neurons. Our data indicate that Kidins220 might localise Trio to specific membrane sites and regulate its activity, leading to Rac1 activation and neurite outgrowth.Cancer Research UKFritz-Thyssen-StiftungCNRSANR-07-NEURO-006-01 from the Agence Nationale de la Recherch

SUMOylation regulates nucleo-cytoplasmic shuttling of Elk-1

The transcription factor Elk-1 is a nuclear target of mitogen-activated protein kinases and regulates immediate early gene activation by extracellular signals. We show that Elk-1 is also conjugated to SUMO on either lysines 230, 249, or 254. Mutation of all three sites is necessary to fully block SUMOylation in vitro and in vivo. This Elk-1 mutant, Elk-1(3R), shuttles more rapidly to nuclei of Balb/C cells fused to transfected HeLa cells. Coexpression of SUMO-1 or -2 strongly reduces shuttling by Elk-1 without affecting that of Elk-1(3R), indicating that SUMOylation regulates nuclear retention of Elk-1. Accordingly, overexpression of Elk-1(3R) in PC12 cells, where cytoplasmic relocalization of Elk-1 has been linked to differentiation, enhances neurite extension relative to Elk-1. The effect of Elk-1, but not of the 3R mutant, was blocked upon cotransfection with SUMO-1 or -2 and enhanced by coexpression with mutant Ubc-9. Thus, SUMO conjugation is a novel regulator of Elk-1 function through the control of its nuclear-cytoplasmic shuttling

Eph-Dependent Tyrosine Phosphorylation of Ephexin1 Modulates Growth Cone Collapse

SummaryEphs regulate growth cone repulsion, a process controlled by the actin cytoskeleton. The guanine nucleotide exchange factor (GEF) ephexin1 interacts with EphA4 and has been suggested to mediate the effect of EphA on the activity of Rho GTPases, key regulators of the cytoskeleton and axon guidance. Using cultured ephexin1−/− mouse neurons and RNA interference in the chick, we report that ephexin1 is required for normal axon outgrowth and ephrin-dependent axon repulsion. Ephexin1 becomes tyrosine phosphorylated in response to EphA signaling in neurons, and this phosphorylation event is required for growth cone collapse. Tyrosine phosphorylation of ephexin1 enhances ephexin1’s GEF activity toward RhoA while not altering its activity toward Rac1 or Cdc42, thus changing the balance of GTPase activities. These findings reveal that ephexin1 plays a role in axon guidance and is regulated by a switch mechanism that is specifically tailored to control Eph-mediated growth cone collapse

Mutations specific to the Rac-GEF domain of <i>TRIO</i> cause intellectual disability and microcephaly

Background: Neurodevelopmental disorders have challenged clinical genetics for decades, with over 700 genes implicated and many whose function remains unknown. The application of whole-exome sequencing is proving pivotal in closing the genotype/phenotype gap through the discovery of new genes and variants that help to unravel the pathogenic mechanisms driving neuropathogenesis. One such discovery includes TRIO, a gene recently implicated in neurodevelopmental delay. Trio is a Dbl family guanine nucleotide exchange factor (GEF) and a major regulator of neuronal development, controlling actin cytoskeleton dynamics by activating the GTPase Rac1.Methods: Whole-exome sequencing was undertaken on a family presenting with global developmental delay, microcephaly and mild dysmorphism. Father/daughter exome analysis was performed, followed by confirmatory Sanger sequencing and segregation analysis on four individuals. Three further patients were recruited through the deciphering developmental disorders (DDD) study. Functional studies were undertaken using patient-specific Trio protein mutations.Results: We identified a frameshift deletion in TRIO that segregated autosomal dominantly. By scrutinising data from DDD, we further identified three unrelated children with a similar phenotype who harboured de novo missense mutations in TRIO. Biochemical studies demonstrated that in three out of four families, the Trio mutations led to a markedly reduced Rac1 activation.Conclusions: We describe an inherited global developmental delay phenotype associated with a frameshift deletion in TRIO. Additionally, we identify pathogenic de novo missense mutations in TRIO associated with the same consistent phenotype, intellectual disability, microcephaly and dysmorphism with striking digital features. We further functionally validate the importance of the GEF domain in Trio protein function. Our study demonstrates how genomic technologies are yet again proving prolific in diagnosing and advancing the understanding of neurodevelopmental disorders.<br/

Opposite Modulation of RAC1 by Mutations in TRIO Is Associated with Distinct, Domain-Specific Neurodevelopmental Disorders

The Rho-guanine nucleotide exchange factor (RhoGEF) TRIO acts as a key regulator of neuronal migration, axonal outgrowth, axon guidance, and synaptogenesis by activating the GTPase RAC1 and modulating actin cytoskeleton remodeling. Pathogenic variants in TRIO are associated with neurodevelopmental diseases, including intellectual disability (ID) and autism spectrum disorders (ASD). Here, we report the largest international cohort of 24 individuals with confirmed pathogenic missense or nonsense variants in TRIO. The nonsense mutations are spread along the TRIO sequence, and affected individuals show variable neurodevelopmental phenotypes. In contrast, missense variants cluster into two mutational hotspots in the TRIO sequence, one in the seventh spectrin repeat and one in the RAC1-activating GEFD1. Although all individuals in this cohort present with developmental delay and a neuro-behavioral phenotype, individuals with a pathogenic variant in the seventh spectrin repeat have a more severe ID associated with macrocephaly than do most individuals with GEFD1 variants, who display milder ID and microcephaly. Functional studies show that the spectrin and GEFD1 variants cause a TRIO-mediated hyper- or hypo-activation of RAC1, respectively, and we observe a striking correlation between RAC1 activation levels and the head size of the affected individuals. In addition, truncations in TRIO GEFD1 in the vertebrate model X. tropicalis induce defects that are concordant with the human phenotype. This work demonstrates distinct clinical and molecular disorders clustering in the GEFD1 and seventh spectrin repeat domains and highlights the importance of tight control of TRIO-RAC1 signaling in neuronal development.<br/

Role de l'autophosphorylation du recepteur de l'insuline dans le controle selectif des effets metaboliques de l'hormone

SIGLECNRS T Bordereau / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Rôle du RhoGEF Trio dans la différenciation neuronale et la réponse à la nétrine-1

Au cours de la différenciation neuronale, les GTPases de la famille Rho contrôlent divers processus dont la migration cellulaire, la dynamique du cône de croissance, et l'extension et le guidage de l'axone. Les Rho GTPases sont activées par des facteurs d'échange (GEFs). Trio est un GEF pour les GTPases Rac et RhoG impliqué dans le guidage axonal chez les invertébrés et dans la croissance neuritique chez les Vertébrés. Au cours de ma thèse, j'ai caractérisé le rôle de Trio dans le développement neuronal. En utilisant des souris knock-out pour le gène trio, j'ai montré que Trio est important pour la croissance des axones de neurones corticaux in vitro. Je me suis intéressée au rôle de Trio dans la réponse à la nétrine-1, un facteur de guidage axonal capable d'induire la croissance de l'axone via l'activation de Rac. Le GEF responsable de l'activation de Rac au cours de la réponse à la nétrine n'était cependant pas identifié. J'ai pu mettre en évidence que Trio est essentiel pour la croissance axonale en réponse à la nétrine-1 dans des neurones corticaux. Nous avons également montré que Trio est impliqué dans la réponse à la nétrine d'explants de neurones commissuraux in vitro, et dans le guidage de ces neurones in vivo. Enfin, je me suis intéressée aux partenaires protéiques de Trio, et nous avons montré que Trio se trouvait dans un complexe comprenant le récepteur à la nétrine, DCC, ainsi que plusieurs protéines de signalisation comme Nck, FAK et PAK. D'un point de vue fonctionnel, nous avons observé que Trio permet l'activation de Rac en réponse à la nétrine dans les neurones, cette activation de Rac pouvant être à l'origine du remodelage du cytosquelette et donc de la croissance axonale. Ces travaux suggèrent donc que Trio soit un membre important de la voie de signalisation en aval de la nétrine-1 au cours du développement du système nerveux.During neuronal differentiation, RhoGTPases determine numerous processes such as cell migration, growth cone dynamics or axon outgrowth and guidance. RhoGTPases are activated by exchange factors (GEFs). Trio is a Rac- and RhoG-specific GEF involved in axon guidance in invertebrates and in neurite outgrowth in Vertebrates. My PhD theme was to characterize Trio's role in neuronal development. In vitro studies using cortical neurons from Trio knock-out mice proved that Trio is a major element in axon outgrowth. We then addressed the role of Trio in netrin-1 signalisation. Netrin-1 is an attractive guidance cue inducing axon outgrowth through a Rac-dependent mechanism, but the GEF responsible for Rac activation was unknown. We showed that Trio is necessary for netrin-induced axon outgrowth in cortical neurons and in commissural neurons explants in vitro. Trio is also involved in commissural axon guidance in vivo. Furthermore, we showed that Trio is present in a signalling complex including DCC (the netrin-1 receptor), Nck, FAK and PAK. Trio is also necessary for netrin-1-induced Rac activation in neurons, which can lead to actin remodelling and axon outgrowth. We therefore suggest that Trio is involved in netrin-1 signalling pathway through its ability to activate Rac.MONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF

Etude du Rho-GEF Trio dans la morphologie neuronale et caractérisation des partenaires de son domaine SH3-1

MONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF

Fonction de la signalisation des Rho GTPases au cours du développement du cervelet

La cellule de Purkinje (PC) est l'élément central du réseau neuronal du cortex cérébelleux et possède un arbre dendritique très développé qui se développe au cours des trois premières semaines post-natales chez la souris. Cette arborisation nécessite de nombreux réarrangement du cytosquelette, un processus contrôlé par les GTPases et leurs régulateurs, les GEFs et les GAPs, dans de nombreux types cellulaires. Au cours de ma thèse, j'ai étudié l'implication de la signalisation des RhoGTPases dans le développement post-natal du cervelet, et plus particulièrement des PCs chez la souris. Afin d'identifier de nouveaux acteurs de la signalisation des RhoGTPases impliqués dans la différenciation des PCs, nous avons établi le profil d'expression de toutes les GTPases et des GEFs de la famille DOCK à différents stades de développement de ces cellules (P3, P7, P15, P20) par Q-PCR en temps réel. Cette approche globale nous a permis d'identifier une GTPase, RhoQ, et un GEF, DOCK10, dont l'expression est très fortement augmentée au cours du développement des PCs. Nous avons montré que l'extinction de leur expression par infection lentivirale dans un modèle de coupes organotypiques de cervelet ou dans des neurones d'hippocampe entraine une très forte diminution du nombre d'épines dendritiques, révélant un rôle crucial de ces protéines dans la différenciation des PCs.Purkinje cell (PC) occupy a central and integrative position in the synaptic network of the cerebellum and have the most elaborate dendritic tree among CNS neurons, which develops remarkably in the first three postnatal weeks in mice. This arborization requires intensive actin cytoskeleton remodeling, a process known in many cell types to be controlled by Rho GTPases and their regulators, GEFs and GAPs. During my thesis, I investigated the importance of Rho signaling during postnatal mouse cerebellar development, focusing on PC differentiation.In order to identify novel regulators of PC differentiation among members of the Rho signaling pathway, I undertook a global approach, comparing gene expression profiles of all mammalian Rho GTPases and all GEFs of the DOCK family at various stages of postnatal PC differentiation (P3, P7, P15 and P20) using real-time quantitative PCR. My global approach has allowed the identification of two Rho signaling actors, the GTPase RhoQ and the RhoGEF DOCK10, whose expressions increase dramatically during cerebellar development. Lentiviral shRNA-mediated knock down of their expression in organotypic cerebellar cultures and in hippocampal neurons showed strong dendritic spine defects, revealing an essential role for these proteins in PC differentiation.MONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF