September 2024

Both GEF domains of the autism and developmental epileptic encephalopathy-associated Trio protein are required for proper tangential migration of GABAergic interneurons

Lara Eid, Ludmilla Lokmane, Praveen K. Raju, Samuel Boris Tene Tadoum, Xiao Jiang, Karolanne Toulouse, Alexis Lupien-Meilleur, François Charron-Ligez, Asmaa Toumi, Stéphanie Backer, Mathieu Lachance, Marisol Lavertu-Jolin, Marie Montseny, Jean-Claude Lacaille, Evelyne Bloch-Gallego & Elsa Rossignol

Recessive and de novo mutations in the TRIO gene are associated with intellectual deficiency (ID), autism spectrum disorder (ASD) and developmental epileptic encephalopathies (DEE). TRIO is a dual guanine nucleotide exchange factor (GEF) that activates Rac1, Cdc42 and RhoA. Trio has been extensively studied in excitatory neurons, and has recently been found to regulate the switch from tangential to radial migration in GABAergic interneurons (INs) through GEFD1-Rac1-dependent SDF1α/CXCR4 signaling. Given the central role of Rho-GTPases during neuronal migration and the implication of IN pathologies in ASD and DEE, we investigated the relative roles of both Trio’s GEF domains in regulating the dynamics of INs tangential migration. In Trio–/– mice, we observed reduced numbers of tangentially migrating INs, with intact progenitor proliferation. Further, we noted increased growth cone collapse in developing INs, suggesting altered cytoskeleton dynamics. To bypass the embryonic mortality of Trio–/– mice, we generated Dlx5/6Cre;Trioc/c conditional mutant mice (TriocKO), which develop spontaneous seizures and behavioral deficits reminiscent of ASD and ID. These phenotypes are associated with reduced cortical IN density and functional cortical inhibition. Mechanistically, this reduction of cortical IN numbers reflects a premature switch to radial migration, with an aberrant early entry in the cortical plate, as well as major deficits in cytoskeletal dynamics, including enhanced leading neurite branching and slower nucleokinesis reflecting reduced actin filament condensation and turnover as well as a loss of response to the motogenic effect of EphA4/ephrin A2 reverse signaling. Further, we show that both Trio GEFD1 and GEFD2 domains are required for proper IN migration, with a dominant role of the RhoA-activating GEFD2 domain. Altogether, our data show a critical role of the DEE/ASD-associated Trio gene in the establishment of cortical inhibition and the requirement of both GEF domains in regulating IN migration dynamics.

July 2024

The RNA-binding protein Nab2 regulates levels of the RhoGEF Trio to govern axon and dendrite morphology

Carly L. Lancaster, Pranav S. Yalamanchili, Jordan N. Goldy, Sara W. Leung, Anita H. Corbett, Kenneth H. Moberg

The Drosophila RNA-binding protein (RBP) Nab2 acts in neurons to regulate neurodevelopment and is orthologous to the human intellectual disability-linked RBP, ZC3H14. Nab2 governs axon projection in mushroom body neurons and limits dendritic arborization of class IV sensory neurons in part by regulating splicing events in ∼150 mRNAs. Analysis of the Sex-lethal (Sxl) mRNA revealed that Nab2 promotes an exon-skipping event and regulates m6A methylation on Sxl pre-mRNA by the Mettl3 methyltransferase. Mettl3 heterozygosity broadly rescues Nab2null phenotypes implying that Nab2 acts through similar mechanisms on other RNAs, including unidentified targets involved in neurodevelopment. Here, we show that Nab2 and Mettl3 regulate the removal of a 5′UTR (untranslated region) intron in the trio pre-mRNA. Trio utilizes two GEF domains to balance Rac and RhoGTPase activity. Intriguingly, an isoform of Trio containing only the RhoGEF domain, GEF2, is depleted in Nab2null nervous tissue. Expression of Trio-GEF2 rescues projection defects in Nab2null axons and dendrites, while the GEF1 Rac1-regulatory domain exacerbates these defects, suggesting Nab2-mediated regulation Trio-GEF activities. Collectively, these data indicate that Nab2-regulated processing of trio is critical for balancing Trio-GEF1 and -GEF2 activity and show that Nab2, Mettl3, and Trio function in a common pathway that shapes axon and dendrite morphology.

MARCH 2024

The RhoGEF Trio is transported by microtubules and affects microtubule stability in migrating neural crest cells

Stefanie Gossen , Sarah Gerstner, Annette Borchers

Directed cell migration requires a local fine-tuning of Rho GTPase activity to control protrusion formation, cell-cell contraction, and turnover of cellular adhesions. The Rho guanine nucleotide exchange factor (GEF) TRIO is ideally suited to control RhoGTPase activity because it combines two distinct catalytic domains to control Rac1 and RhoA activity in one molecule. However, at the cellular level, this molecular feature also requires a tight spatiotemporal control of TRIO activity. Here, we analyze the dynamic localization of Trio in Xenopus cranial neural crest (NC) cells, where we have recently shown that Trio is required for protrusion formation and migration. Using live cell imaging, we find that the GEF2 domain, but not the GEF1 domain of Trio, dynamically colocalizes with EB3 at microtubule plus-ends. Microtubule-mediated transport of Trio appears to be relevant for its function in NC migration, as a mutant GEF2 construct lacking the SxIP motif responsible for microtubule plus-end localization was significantly impaired in its ability to rescue the Trio loss-of-function phenotype compared to wild-type GEF2. Furthermore, by analyzing microtubule dynamics in migrating NC cells, we observed that loss of Trio function stabilized microtubules at cell-cell contact sites compared to controls, whereas they were destabilized at the leading edge of NC cells. Our data suggest that Trio is transported by microtubules to distinct subcellular locations where it has different functions in controlling microtubule stability, cell morphology, and cell-cell interaction during directed NC migration.

RNA analysis and computer-aided facial phenotyping help to classify a novel TRIO splice site variant

Sarina Schwartzmann, Max Zhao, Henrike Lisa Sczakiel, Gabriele Hildebrand, Nadja Ehmke, Denise Horn, Martin A Mensah, Felix Boschann

Pathogenic variants in TRIO, encoding the guanine nucleotide exchange factor, are associated with two distinct neurodevelopmental delay phenotypes: gain-of-function missense mutations within the spectrin repeats are causative for a severe developmental delay with macrocephaly (MIM: 618825), whereas loss-of-function missense variants in the GEF1 domain and truncating variants throughout the gene lead to a milder developmental delay and microcephaly (MIM: 617061). In three affected family members with mild intellectual disability/NDD and microcephaly, we detected a novel heterozygous TRIO variant at the last coding base of exon 31 (NM_007118.4:c.4716G>A). RNA analysis from patient-derived lymphoblastoid cells confirmed aberrant splicing resulting in the skipping of exon 31 (r.4615_4716del), leading to an in-frame deletion in the first Pleckstrin homology subdomain of the GEF1 domain: p.(Thr1539_Lys1572del). To test for a distinct gestalt, facial characteristics of the family members and 41 previously published TRIO cases were systematically evaluated via GestaltMatcher. Computational analysis of the facial gestalt suggests a distinguishable facial TRIO-phenotype not outlined in the existing literature.

JANUARY 2024

What have genetic studies of rare sequence variants taught us about the aetiology of schizophrenia?

Lea Heinzer, David Curtis

With a population prevalence of 1%, schizophrenia is widespread, yet the aetiology of this psychiatric disorder remains elusive. There is an evident genetic component of schizophrenia, with heritability estimates lying at 60%-80%. While genome-wide association studies have identified 120 gene loci associated with schizophrenia risk, these involved common variants that confer only small effects on individual risk (median odds ratio < 1.2). The recent emergence of whole exome sequencing (WES) technologies has facilitated the identification of rare sequence variants, including some protein-truncating variants that have significant effects on risk. Three key large-scale WES studies have demonstrated that rare sequence variants in the genes SETD1A, CACNA1G, CUL1, GRIA3, GRIN2A, HERC1, RB1CC1, SP4, TRIO, XPO7, and AKAP11 confer substantial risk for schizophrenia. These genes are highly expressed in central nervous system neurons and their products participate in diverse molecular functions including synaptic transmission, transcriptional regulation, and ubiquitin ligation. The understanding of these functional roles illuminates putative molecular mechanisms which may lead to schizophrenia-like phenotypes. It will also be possible to develop model systems in which the effects of impaired function of these genes can be further explored. Genetic studies of rare variants to date suggest that glutamatergic system dysregulation, chromatin modification, and the ubiquitin-proteasome system play key roles in schizophrenia aetiology.

Heterozygosity for neurodevelopmental disorder-associated TRIO variants yields distinct deficits in behavior, neuronal development, and synaptic transmission in mice

Yevheniia Ishchenko, Amanda T. Jeng, Shufang Feng, Timothy Nottoli, Cindy Manriquez-Rodriguez, Khanh Nguyen, Melissa G. Carrizales, Matthew J. Vitarelli, Ellen E. Corcoran, Charles A. Greer, Samuel A. Myers, Anthony J. Koleske

Abstract

Heterozygosity for rare genetic variants in TRIO is associated with neurodevelopmental disorders (NDDs) including schizophrenia (SCZ), autism spectrum disorder (ASD) and intellectual disability. TRIO uses its two guanine nucleotide exchange factor (GEF) domains to activate GTPases (GEF1: Rac1 and RhoG; GEF2: RhoA) that control neuronal migration, synapse development and function. It remains unclear whether and how discrete TRIO variants differentially impact these neurodevelopmental events. Here, we elucidate how heterozygosity for NDD-associated Trio variants – +/K1431M (ASD), +/K1918X (SCZ), and +/M2145T (bipolar disorder, BPD) – impact mouse behavior, brain development, and synapse structure and function. Heterozygosity for different Trio variants impacts motor, social, and cognitive behaviors in distinct ways that align with clinical phenotypes in humans. ASD- and SCZ-linked Trio variants differentially impact head and brain size with corresponding changes in dendritic arbors of motor cortex layer 5 pyramidal neurons (M1 L5 PNs). Although dendritic spine density and synaptic ultrastructure were only modestly altered in the Trio variant heterozygotes, we observe significant changes in synaptic function and plasticity including excitatory/inhibitory imbalance and long-term potentiation defects. We also identify distinct changes in glutamate synaptic release in +/K1431M and +/M2145T cortico-cortical synapses, associated with deficiencies in crucial presynaptic release regulators. While TRIO K1431M has impaired ability to promote GTP exchange on Rac1, +/K1431M mice exhibit increased Rac1 activity, suggesting possible compensation by other GEFs. Our work reveals that discrete disease-associated Trio variants yield overlapping but distinct NDD-associated phenotypes in mice and demonstrates, for the first time, an essential role for Trio in presynaptic glutamate release, underscoring the importance of studying the impact of variant heterozygosity in vivo.