2021
SELECTED PUBLICATIONS
DECEMBER 2021
In vitro fluorescence assay to measure GDP/GTP exchange of guanine nucleotide exchange factors of Rho family GTPases
Alyssa M Blaise, Ellen E Corcoran, Eve S Wattenberg, Yan-Ling Zhang, Jeffrey R Cottrell, Anthony J Koleske
Guanine nucleotide exchange factors (GEFs) are enzymes that promote the activation of GTPases through GTP loading. Whole exome sequencing has identified rare variants in GEFs that are associated with disease, demonstrating that GEFs play critical roles in human development. However, the consequences of these rare variants can only be understood through measuring their effects on cellular activity. Here, we provide a detailed user-friendly protocol for purification and fluorescence-based analysis of the two GEF domains within the protein, Trio. This analysis offers a straight-forward, quantitative tool to test the activity of GEF domains on their respective GTPases, as well as utilize high-throughput screening to identify regulators and inhibitors. This protocol can be adapted for characterization of other Rho family GEFs. Such analyses are crucial for the complete understanding of the roles of GEF genetic variants in human development and disease.
RhoGEF Trio regulates radial migration of projection neurons via its distinct domains
Chengwen Wei, Mengwen Sun, Xiaoxuan Sun, Hu Meng, Qiongwei Li, Kai Gao, Weihua Yue, Lifang Wang, Dai Zhang & Jun Li
The radial migration of cortical pyramidal neurons (PNs) during corticogenesis is necessary for establishing a multilayered cerebral cortex. Neuronal migration defects are considered a critical etiology of neurodevelopmental disorders, including autism spectrum disorders (ASDs), schizophrenia, epilepsy, and intellectual disability (ID). TRIO is a high-risk candidate gene for ASDs and ID. However, its role in embryonic radial migration and the etiology of ASDs and ID are not fully understood. In this study, we found that the in vivo conditional knockout or in utero knockout of Trio in excitatory precursors in the neocortex caused aberrant polarity and halted the migration of late-born PNs. Further investigation of the underlying mechanism revealed that the interaction of the Trio N-terminal SH3 domain with Myosin X mediated the adherence of migrating neurons to radial glial fibers through regulating the membrane location of neuronal cadherin (N-cadherin). Also, independent or synergistic overexpression of RAC1 and RHOA showed different phenotypic recoveries of the abnormal neuronal migration by affecting the morphological transition and/or the glial fiber-dependent locomotion. Taken together, our findings clarify a novel mechanism of Trio in regulating N-cadherin cell surface expression via the interaction of Myosin X with its N-terminal SH3 domain. These results suggest the vital roles of the guanine nucleotide exchange factor 1 (GEF1) and GEF2 domains in regulating radial migration by activating their Rho GTPase effectors in both distinct and cooperative manners, which might be associated with the abnormal phenotypes in neurodevelopmental disorders.
Pathophysiological Mechanisms in Neurodevelopmental Disorders Caused by Rac GTPases Dysregulation: What’s behind Neuro-RACopathies
Marcello Scala; Masashi Nishikawa, Koh-ichi Nagata and Pasquale Striano
Rho family guanosine triphosphatases (GTPases) regulate cellular signaling and cytoskeletal dynamics, playing a pivotal role in cell adhesion, migration, and cell cycle progression. The Rac subfamily of Rho GTPases consists of three highly homologous proteins, Rac 1–3. The proper function of Rac1 and Rac3, and their correct interaction with guanine nucleotide-exchange factors (GEFs) and GTPase-activating proteins (GAPs) are crucial for neural development. Pathogenic variants affecting these delicate biological processes are implicated in different medical conditions in humans, primarily neurodevelopmental disorders (NDDs). In addition to a direct deleterious effect produced by genetic variants in the RAC genes, a dysregulated GTPase activity resulting from an abnormal function of GEFs and GAPs has been involved in the pathogenesis of distinctive emerging conditions. In this study, we reviewed the current pertinent literature on Rac-related disorders with a primary neurological involvement, providing an overview of the current knowledge on the pathophysiological mechanisms involved in the neuro-RACopathies.
OCTOBER 2021
Rho GTPases in Skeletal Muscle Development and Homeostasis
Sonia Rodríguez-Fdez, Xosé R. Bustelo
Rho guanosine triphosphate hydrolases (GTPases) are molecular switches that cycle between an inactive guanosine diphosphate (GDP)-bound and an active guanosine triphosphate (GTP)-bound state during signal transduction. As such, they regulate a wide range of both cellular and physiological processes. In this review, we will summarize recent work on the role of Rho GTPase-regulated pathways in skeletal muscle development, regeneration, tissue mass homeostatic balance, and metabolism. In addition, we will present current evidence that links the dysregulation of these GTPases with diseases caused by skeletal muscle dysfunction. Overall, this information underscores the critical role of a number of members of the Rho GTPase subfamily in muscle development and the overall metabolic balance of mammalian species.
SEPTEMBER 2021
The RhoGEF Trio: A Protein with a Wide Range of Functions in the Vascular Endothelium
Lanette Kempers , Amber J. M. Driessen, Jos van Rijssel, Martijn A. Nolte and Jaap D. van Buul
Many cellular processes are controlled by small GTPases, which can be activated by guanine nucleotide exchange factors (GEFs). The RhoGEF Trio contains two GEF domains that differentially activate the small GTPases such as Rac1/RhoG and RhoA. These small RhoGTPases are mainly involved in the remodeling of the actin cytoskeleton. In the endothelium, they regulate junctional stabilization and play a crucial role in angiogenesis and endothelial barrier integrity. Multiple extracellular signals originating from different vascular processes can influence the activity of Trio and thereby the regulation of the forementioned small GTPases and actin cytoskeleton. This review elucidates how various signals regulate Trio in a distinct manner, resulting in different functional outcomes that are crucial for endothelial cell function in response to inflammation.
August 2021
Activation of Rac1 Has an Opposing Effect on Induction and Maintenance of Long-Term Potentiation in Hippocampus by Acting on Different Kinases
Dongyang Cui, Xiaodong Jiang, Ming Chen, Huan Sheng, Da Shao, Li Yang, Xinli Guo, Yingqi Wang, Bin Lai and Ping Zheng,
Small GTPases are important signaling molecules in neurons. One of the best characterized subfamilies of the small GTPases is the Rho family, which includes Rac, Cdc42, and Rho. Among them, Rac1, a small GTPase of the Rho family, has been reported to be involved in morphological plasticity in the hippocampus.
A previous study showed that the activation of Rac1 had an opposing effect on induction and maintenance of long-term potentiation (LTP) in the hippocampus. However, the molecular mechanism underlying this opposing effect remains to be addressed. In the present work, we find that the activation of Rac1 during the induction of LTP leads to an activation of PKCι/λ by phosphatidylinositol-3-kinase (PI3K), whereas the activation of Rac1 during the maintenance of LTP leads to the inhibition of PKMζ by LIM_kinase (LIMK) in the hippocampus. This result suggests that during different stages of LTP, the activation of Rac1 can modulate different signaling pathways, which leads to an opposing effect on the induction and maintenance of LTP in the hippocampus.
Autism Spectrum Disorder/Intellectual Disability-associated mutations in Trio disrupt Neuroligin 1-mediated synaptogenesis
Chen Tian, Jeremiah D. Paskus, Erin Fingleton, Katherine W. Roche and Bruce E. Herring
We recently identified an Autism Spectrum Disorder/Intellectual Disability (ASD/ID)-related de novo mutation hotspot in the Rac1 activating GEF1 domain of the protein Trio. Trio is a Rho guanine nucleotide exchange factor (RhoGEF) that is essential for glutamatergic synapse function. An ASD/ID-related mutation identified in Trio's GEF1 domain, Trio D1368V, produces a pathological increase in glutamatergic synaptogenesis, suggesting that Trio is coupled to synaptic regulatory mechanisms that govern glutamatergic synapse formation. However, the molecular mechanisms by which Trio regulates glutamatergic synapses are largely unexplored. Here, using biochemical methods we identify an interaction between Trio and the synaptogenic protein Neuroligin 1 (NLGN1) in the brain. Molecular biological approaches were then combined with super resolution dendritic spine imaging and whole-cell voltage clamp electrophysiology in male and female rats to examine the impact ASD/ID-related Trio mutations have on NLGN1-mediated synaptogenesis. We find that an ASD/ID-related mutation in Trio's 8th spectrin repeat region, Trio N1080I, inhibits Trio's interaction with NLGN1 and prevents Trio D1368V-mediated synaptogenesis. Inhibiting Trio's interaction with NLGN1 via Trio N1080I blocked NLGN1-mediated synaptogenesis and increases in synaptic NMDA receptor function but not NLGN1-mediated increases in synaptic AMPA receptor function. Finally, we show that the aberrant synaptogenesis produced by Trio D1368V is dependent on NLGN signaling. Our findings demonstrate that ASD/ID-related mutations in Trio are able to pathologically increase as well as decrease NLGN-mediated effects on glutamatergic neurotransmission, and point to a NLGN1-Trio interaction as part of a key pathway involved in ASD/ID etiology.
July 2021
The RHO Family GTPases: Mechanisms of Regulation and Signaling
Niloufar Mosaddeghzadeh and Mohammad Reza Ahmadian
Much progress has been made toward deciphering RHO GTPase functions, and many studies have convincingly demonstrated that altered signal transduction through RHO GTPases is a recurring theme in the progression of human malignancies. It seems that 20 canonical RHO GTPases are likely regulated by three GDIs, 85 GEFs, and 66 GAPs, and eventually interact with >70 downstream effectors. A recurring theme is the challenge in understanding the molecular determinants of the specificity of these four classes of interacting proteins that, irrespective of their functions, bind to common sites on the surface of RHO GTPases. Identified and structurally verified hotspots as functional determinants specific to RHO GTPase regulation by GDIs, GEFs, and GAPs as well as signaling through effectors are presented, and challenges and future perspectives are discussed.
June 2021
Neuronal Cytoskeleton in Intellectual Disability: From Systems Biology and Modeling to Therapeutic Opportunities
Carla Liaci , Mattia Camera, Giovanni Caslini, Simona Rando, Salvatore Contino, Valentino Romano and Giorgio R. Merlo
Intellectual disability (ID) is a pathological condition characterized by limited intellectual functioning and adaptive behaviors. It affects 1–3% of the worldwide population, and no pharmacological therapies are currently available. More than 1000 genes have been found mutated in ID patients pointing out that, despite the common phenotype, the genetic bases are highly heterogeneous and apparently unrelated. Bibliomic analysis reveals that ID genes converge onto a few biological modules, including cytoskeleton dynamics, whose regulation depends on Rho GTPases transduction. Genetic variants exert their effects at different levels in a hierarchical arrangement, starting from the molecular level and moving toward higher levels of organization, i.e., cell compartment and functions, circuits, cognition, and behavior. Thus, cytoskeleton alterations that have an impact on cell processes such as neuronal migration, neuritogenesis, and synaptic plasticity rebound on the overall establishment of an effective network and consequently on the cognitive phenotype. Systems biology (SB) approaches are more focused on the overall interconnected network rather than on individual genes, thus encouraging the design of therapies that aim to correct common dysregulated biological processes. This review summarizes current knowledge about cytoskeleton control in neurons and its relevance for the ID pathogenesis, exploiting in silico modeling and translating the implications of those findings into biomedical research.
MAY 2021
More evidence on TRIO missense mutations in the spectrin repeat domain causing severe developmental delay and recognizable facial dysmorphism with macrocephaly
K. Kloth, L. Graul-Neumann, K. Hermann, J. Johannsen, T. Bierhals & F. Kortüm
TRIO is a Dbl family guanine nucleotide exchange factor (GEF) and an important regulator of neuronal development. Most truncating and missense variants affecting the Dbl homology domain of TRIO are associated with a neurodevelopmental disorder with microcephaly (MIM617061). Recently, de novo missense variants affecting the spectrin repeat region of TRIO were associated with a novel phenotype comprising severe developmental delay and macrocephaly (MIM618825). Here, we provide more evidence on this new TRIO-associated phenotype by reporting two severely affected probands with de novo missense variants in TRIO affecting the spectrin repeat region upstream of the typically affected GEF1 domain of the protein.
What is the role of synaptic protein TRIO's spectrin repeats?
Ellen Corcoran, Josie Bircher, Anthony Koleske
There is a critical need to elucidate the biochemical events that control dendrite and synapse function to develop better targeted therapeutic strategies for neurodevelopmental disorders. Functionally damaging mutations in the TRIO gene are enriched in individuals with neurodevelopmental disorders. TRIO encodes a large synaptic regulatory protein with three catalytic domains and several accessory domains, including nine spectrin repeat domains. De novo missense mutations and rare damaging variants in the spectrin repeats are enriched in autism, intellectual disability, and developmental delay. However, the role of TRIO's spectrin repeats and the impact of disease-associated mutations remains completely unknown. Our goals are to identify spectrin repeat binding partners, explore the possibility of spectrin repeats intramolecularly regulating TRIO catalytic activity, and determine the impact of disease mutations on spectrin repeat function. In doing so, we will elucidate how TRIO's spectrin repeats confer proper neuronal development and determine whether and how TRIO disease-associated variants compromise signaling mechanisms.
Largest autism genetics analysis to date uncovers more high-confidence candidates
Laura Dattaro
After doubling its sample size, the largest study of genetic data from autistic people has identified 255 genes associated with the condition, an increase of more than 40 genes since the researchers’ 2019 update; 71 of the genes rise above a stringent statistical bar the team had not previously used. The new analysis also adds data from people with developmental delay or schizophrenia and considers multiple types of mutations.
There was less overlap with the Schizophrenia Exome Meta-Analysis, which had identified 10 genes strongly associated with schizophrenia. In at least one of those 10, an autism-linked gene called TRIO, one type of mutation occurred in schizophrenia and another in both autism and developmental delay, suggesting more overlap between the latter two conditions. But whether that finding will be replicated with other genes is an open question, Satterstrom said.
Dysfunction of Trio GEF1 involves in excitatory/inhibitory imbalance and autism-like behaviors through regulation of interneuron migration
Xiaoxuan Sun, Lifang Wang, Chengwen Wei, Mengwen Sun, Qiongwei Li, Hu Meng, Weihua Yue, Dai Zhang & Jun Li
Autism spectrum disorders (ASDs) are a group of highly inheritable neurodevelopmental disorders. Functional mutations in TRIO, especially in the GEF1 domain, are strongly implicated in ASDs, whereas the underlying neurobiological pathogenesis and molecular mechanisms remain to be clarified. Here we characterize the abnormal morphology and behavior of embryonic migratory interneurons (INs) upon Trio deficiency or GEF1 mutation in mice, which are mediated by the Trio GEF1-Rac1 activation and involved in SDF1α/CXCR4 signaling. In addition, the migration deficits are specifically associated with altered neural microcircuit, decreased inhibitory neurotransmission, and autism-like behaviors, which are reminiscent of some features observed in patients with ASDs. Furthermore, restoring the excitatory/inhibitory (E/I) imbalance via activation of GABA signaling rescues autism-like deficits. Our findings demonstrate a critical role of Trio GEF1 mediated signaling in IN migration and E/I balance, which are related to autism-related behavioral phenotypes.
March 2021
Targeted Sequencing Detects Variants That May Contribute to the Risk of Neuropsychiatric Disorders
Jayant Mahadevan, Reeteka Sud, Ravi Kumar Nadella, Pulaparambil Vani, Anand G. Subramaniam, Pradip Paul, Aparna Ganapathy, Ashraf U. Mannan, Vijay Chandru, Biju Viswanath, Meera Purushottam; Meera Purushottam, Sanjeev Jain
Genetic variants identified with next-generation sequencing (NGS) could help in accurate diagnosis, provide insights into aspects of disease biology, and, thus, lead to improved care. Psychiatric symptoms and syndromes overlap with rare genetic disorders associated with intellectual disability (ID) and autism spectrum disorders (ASDs) at one end and with neurodegenerative disease at the other end. These rare variants may be shared within family members. Hence, the study of rare variants of moderate-to-large effects becomes important, as they may highlight the dysfunction of disease-associated pathways.
Rare variants associated with disease syndromes may arise de novo and may be related to founder effects, population bottlenecks, genetic drift, or natural selection pressures. Therefore, they also offer insights into the inherent mutability of genes, genetic repair mechanisms, and population diversity.
We reviewed the records of 11 individuals presenting to the psychiatric services in our hospital, who also underwent targeted sequencing through a genetic analysis service, after informed consent. In general, genetic analysis was requested because of clinical suspicion based on rapid disease progression, atypical disease course, younger age of onset, or significant family history. Specific indications for each case are mentioned in the respective sections.
FEBRUARY 2021
Trio cooperates with Myh9 to regulate neural crest-derived craniofacial development
Shuyu Guo, Li Meng, Haojie Liu, Lichan Yuan, Na Zhao, Jieli Ni, Yang Zhang, Jingjing Ben, Yi-Ping Li, and Junqing Ma
Trio is a unique member of the Rho-GEF family that has three catalytic domains and is vital for various cellular processes in both physiological and developmental settings. TRIO mutations in humans are involved in craniofacial abnormalities, in which patients present with mandibular retrusion. However, little is known about the molecular mechanisms of Trio in neural crest cell (NCC)-derived craniofacial development, and there is still a lack of direct evidence to assign a functional role to Trio in NCC-induced craniofacial abnormalities.
Trio family proteins as regulators of cell migration and morphogenesis in development and disease – mechanisms and cellular contexts
Josie E. Bircher , Anthony J. Koleske
Trio family proteins are key regulators of cell motility and morphogenesis, tissue development, and protein trafficking and secretion in numerous biological contexts, including their prominent roles in developing nervous systems. These diverse roles are achieved through Trio protein interactions with membrane receptors, cytoskeleton-interacting proteins, lipids, endocytic machinery, kinases and Rho family GTPases in the cell. Recent studies have linked mutations in the human genes TRIO and kalirin (KALRN) to neurological diseases (Paskus et al., 2020) and cancers (Schmidt and Debant, 2014), highlighting the need to understand the primary functions of Trio family proteins and underscoring the outstanding questions in the field. How are the different catalytic activities balanced within Trio proteins? How do the accessory domains in Trio proteins contribute to Trio function, and how does the primary function of Trio proteins differ based on its interactions with cellular binding partners?
January 2021
TRIO-related intellectual disability with microcephaly: a case report of a patient with novel clinical findings
Bevilacqua, Florenciaa; Alberto, Guillermoa; Duarte, Santiago Pabloa; Serra, Marinaa; Basterra, Julietaa; Espeche, Lucíab; Cerretini, Roxana Inésb; Solari, Andrea Paulaa