Original research
Distinct functions of Trio GEF domains in axon outgrowth of cerebellar granule neurons

https://doi.org/10.1016/j.jgg.2019.02.003Get rights and content

Abstract

As a critical guanine nucleotide exchange factor (GEF) regulating neurite outgrowth, Trio coordinates multiple processes of cytoskeletal dynamics through activating Rac1, Cdc42 and RhoA small GTPases by two GEF domains, but the in vivo roles of these GEF domains and corresponding downstream effectors have not been determined yet. We established multiple lines of knockout mice and assessed the respective roles of Trio GEF domains and Rac1 in axon outgrowth. Knockout of total Trio in cerebellar granule neurons (CGNs) led to an impaired F-actin rearrangement of growth cone and hence a retarded neurite outgrowth. Such a retardation was reproduced by inhibition of GEF1 domain or knockdown of Cdc42 and restored apparently by introduction of active Cdc42. As Rac1 deficiency did not affect the neurite outgrowth of CGNs, we suggested that Trio GEF1-mediated Cdc42 activation was required for neurite outgrowth. We established a GEF2-knockout line with deletion of all Trio isoforms except a cerebella-specific Trio8, a short isoform of Trio without GEF2 domain, and used this line as a GEF2-deficient animal model. The GEF2-deficient CGNs had a normal neurite outgrowth but abolished Netrin-1-promoted growth, without affecting Netrin-1 induced Rac1 activation. We thus suggested that Trio GEF1-mediated Cdc42 activation rather than Rac1 activation drives the F-actin dynamics necessary for neurite outgrowth, while GEF2 functions in Netrin-1-promoted neurite elongation. Our results delineated the distinct roles of Trio GEF domains in neurite outgrowth, which is instructive to understand the pathogenesis of clinical Trio-related neurodevelopmental disorders.

Introduction

Neurodevelopmental disorders, including schizophrenia, autism, bipolar disorder, and intellectual disability, are complex diseases affecting approximately 1%–3% of population and associated with hundreds of genes (Chelly et al., 2006; van Bokhoven, 2011). Many of the genes functionally converge onto common cellular processes including neurogenesis, neuronal migration and morphogenesis, and synaptic formation (van Bokhoven, 2011). It is well known that actin-enriched neuronal growth cone is fundamental for the neuronal migration, morphogenesis and axon guidance. As Rho family GTPases are the potent effectors regulating F-actin dynamics, these small GTPases are considered as the key regulators involved in the pathogenesis of the neurodevelopmental disorders (Nadif Kasri and Van Aelst, 2008; Hall and Lalli, 2010; Ba et al., 2013).

Among the Rho GTPases, Rac1, Cdc42 and RhoA are the most extensively studied members in neuronal development (Luo, 2000; Dickson, 2001). Rac1 and Cdc42 are believed to be required for neurite elongation while RhoA is involved in neurite retraction (Govek et al., 2005; Garvalov et al., 2007; Tahirovic et al., 2010). The activities of Rho GTPases are determined by cycles between GTP- and GDP-bound states, which are tightly regulated by guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs), respectively (Jaffe and Hall, 2005). GEFs accelerate the exchange of GDP for GTP to activate Rho GTPases, while GAPs activate the intrinsic GTPase activity to hydrolysis GTP to GDP to inactivate them (Rossman et al., 2005). In mammals, 85 GEFs and 80 GAPs regulate the activities of a much smaller number of Rho GTPases (Jaffe and Hall, 2005), and thus GEFs and GAPs are thought to determine the signal specificity of Rho GTPase-mediated extracellular response.

As a key GEF regulating all of the three Rho GTPases (Debant et al., 1996), Trio was recently identified as an important player in various neurodevelopmental disorders (Ba et al., 2016; Pengelly et al., 2016; S Katrancha et al., 2017; Sadybekov et al., 2017). Trio contains two GEF domains, GEF1 (the N-terminal GEF domain) and GEF2 (the C-terminal GEF domain), which respectively activate Rac1/RhoG and RhoA, and the activated RhoG further activates Cdc42 (Blangy et al., 2000; Bateman and Van Vactor, 2001). Trio plays diverse roles in multiple processes of neuronal development, including neurite outgrowth, axon guidance, and migration (Briancon-Marjollet et al., 2008; Peng et al., 2010). Trio was also implicated in learning and memory (Zong et al., 2015), and synaptic transmission and long-term potentiation (LTP) (Herring and Nicoll, 2016). Although previous in vitro experiments suggested a role of Trio GEF2 domain in regulation of RhoA, its in vivo role had yet to be determined, particularly in neurite outgrowth. Trio GEF2 domain-induced RhoA activation was implicated in apical constriction during early eye development (Plageman et al., 2011), the neuronal death induced by enterovirus infection (Lee et al., 2012) and bipolar disorder by M2145T mutation in humans (Katrancha et al., 2017). Therefore, elucidating the regulatory scenario of Trio GEF domains in vivo will lead to significantly understanding the mechanism underlying these disorders.

In this study, cerebellar granule neurons (CGNs) with deletion of specific Trio GEF domains were generated to address the in vivo roles of Trio GEF domains in neurite outgrowth, as well as their response to guidance cues. We found that Trio GEF1-mediated Cdc42 activation was required for outgrowth of CGN neurites, and Trio GEF2 was required for Netrin1-mediated neurite elongation but not required for intrinsic neurite elongation. Our results uncover the in vivo roles of Trio GEF domains in neurite outgrowth, which may lead to significantly understanding the pathogenesis of Trio-related neuronal disorders.

Section snippets

Trio is required for cytoskeletal dynamics during CGN neurite outgrowth

Considering that Trio deletion leads to neurite growth inhibition (Peng et al., 2010), and GEF1 and GEF2 of Trio respectively activate Rac1/Cdc42 and RhoA which are able to regulate F-actin turnover (Hall, 1998), we speculated that Trio regulates neurite growth through F-actin turnover. As Cytochalasin D (CytoD) is a widely used reagent for increasing actin dynamics and promoting axon development (Bradke and Dotti, 1999), we bath-applied CytoD to cultured Trio-deficient CGNs derived from TrioNKO

Discussion

In this study, we genetically dissected the functions of Trio's two GEF domains in neurite outgrowth. Our results revealed that Trio functioned essentially in maintaining the neuronal growth cone morphology by regulating cytoskeletal dynamics. Trio GEF1 domain regulated cytoskeleton reorganization of neurite outgrowth via activation of Cdc42 rather than Rac1. We generated a mouse line that only expressed a shorter Trio isoform lacking GEF2 domain to explore the role of Trio GEF2 domain. GEF2

Plasmids

The Cdc42 CDS was PCR amplified from mouse cerebellar cDNA and cloned into the pMD19T vector (TaKaRa, Japan). The Cdc42(G12V) point mutation was then generated using a Mut Express II Fast Mutagenesis Kit (C212, Vazyme, China) and recombined with the P2A-EGFP sequence into the pcDNA3.1(+) backbone to construct pcDNA-Cdc42(G12V)-P2A-EGFP using a ClonExpress MultiS One Step Cloning Kit (C113, Vazyme). A control plasmid, pcDNA-EGFP, was also constructed by inserting the EGFP sequence into the same

Acknowledgments

We thank other members of Dr. Zhu's lab for their critical discussion and comments on the manuscript. This work was supported by grants from the National Natural Science Foundation of China (31272311 and 31330034) to M.S.Z.

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