Rho GTPase-activating proteins: Regulators of Rho GTPase activity in neuronal development and CNS diseases

Molecular and Cellular Neuroscience 80 (2017) 18–31

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Molecular and Cellular Neuroscience journal homepage: www.elsevier.com/locate/ymcne

Rho GTPase-activating proteins: Regulators of Rho GTPase activity in neuronal development and CNS diseases Guo-Hui Huang a, Zhao-Liang Sun a, Hong-Jiang Li a, Dong-Fu Feng a,b,⁎ a b

Department of Neurosurgery, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 201900, China Institute of Traumatic Medicine, Shanghai Jiao Tong University School of Medicine, Shanghai 201900, China

a r t i c l e

i n f o

Article history: Received 10 June 2016 Revised 6 January 2017 Accepted 29 January 2017 Available online 3 February 2017 Keywords: RhoGAPs Rho GTPase Neuronal development Neuronal morphology CNS diseases

a b s t r a c t The Rho family of small GTPases was considered as molecular switches in regulating multiple cellular events, including cytoskeleton reorganization. The Rho GTPase-activating proteins (RhoGAPs) are one of the major families of Rho GTPase regulators. RhoGAPs were initially considered negative mediators of Rho signaling pathways via their GAP domain. Recent studies have demonstrated that RhoGAPs also regulate numerous aspects of neuronal development and are related to various neurodegenerative diseases in GAP-dependent and GAP-independent manners. Moreover, RhoGAPs are regulated through various mechanisms, such as phosphorylation. To date, approximately 70 RhoGAPs have been identified; however, only a small portion has been thoroughly investigated. Thus, the characterization of important RhoGAPs in the central nervous system is crucial to understand their spatiotemporal role during different stages of neuronal development. In this review, we summarize the current knowledge of RhoGAPs in the brain with an emphasis on their molecular function, regulation mechanism and disease implications in the central nervous system. © 2017 Elsevier Inc. All rights reserved.

Contents 1. 2.

Introduction . . . . . . . . . . . . . . . . . . . Basic characteristics of RhoGAPs . . . . . . . . . . 2.1. RhoGAPs in neuronal development . . . . . . 2.1.1. srGAPs: srGAP1, srGAP2, and srGAP3 . 2.1.2. SYD-1 . . . . . . . . . . . . . . . 2.1.3. BCR and ABR. . . . . . . . . . . . 2.1.4. p250GAP . . . . . . . . . . . . . 2.1.5. TCGAP . . . . . . . . . . . . . . 2.1.6. Oligophrenin-1. . . . . . . . . . . 2.1.7. α- and β-chimaerin . . . . . . . . 2.1.8. p190RhoGAP. . . . . . . . . . . . 2.1.9. Vilse . . . . . . . . . . . . . . . 2.1.10. Nadrin . . . . . . . . . . . . . . 2.2. RhoGAP in CNS diseases . . . . . . . . . . . 2.2.1. Intellectual disability . . . . . . . . 2.2.2. Autism spectrum disorder. . . . . . 2.2.3. Alzheimer's disease. . . . . . . . . 2.2.4. Schizophrenia . . . . . . . . . . . 3. Conclusions. . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . .

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⁎ Corresponding author at: Department of Neurosurgery, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, 280 Mo-He Road, Shanghai, 201900, China. E-mail address: [email protected] (D.-F. Feng).

http://dx.doi.org/10.1016/j.mcn.2017.01.007 1044-7431/© 2017 Elsevier Inc. All rights reserved.

G.-H. Huang et al. / Molecular and Cellular Neuroscience 80 (2017) 18–31

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1. Introduction During brain development, each neuron typically outgrowths several neurites, which then develop into a single axon and multiple dendrites and eventually form synapses (Elston and Fujita 2014; Goldberg 2003; Kolodkin and Tessier-Lavigne 2011; McAllister 2007). To ensure precise neuronal connectivity, neurons evolve spatial and temporal coordination of multiple developmental steps, including axon specification (Cheng and Poo 2012; Tahirovic and Bradke 2009; Takano et al. 2015; Villarroel-Campos et al. 2016), axon outgrowth and branching (Kalil and Dent 2014), axon retraction/pruning (Luo and O'Leary 2005; Riccomagno and Kolodkin 2015), axon guidance and navigation (Chedotal and Richards 2010; O'Donnell et al. 2009; Quinn and Wadsworth 2008; Raper and Mason 2010), the selection of synaptic target sites, dendritic growth and branching, and synapse formation and maturation (de la Torre-Ubieta and Bonni 2011; Elston and Fujita 2014; Kolodkin and Tessier-Lavigne 2011; McAllister 2007; Williams et al. 2010). The impairment of these steps have profound effects on brain function and are linked to developmental disorders of the central nervous system (CNS), which range from intellectual disability to epilepsy, autism, and schizophrenia (Clement et al. 2012; Pavlowsky et al. 2012; Ramakers 2002; Schubert et al. 2015; Stankiewicz and Linseman 2014). To understand how the precise refinement and coordination are carried out, it is crucial to decode the role of key proteins in this process. As a result of the capability of modulating the dynamic changes and rearrangement of cytoskeletons, the Rho GTPases, in which RhoA, Cdc42 and Rac1 are best-characterized, have been highlighted as significant contributors for orchestrating neuronal development (Duman et al. 2015; Heasman and Ridley 2008; Miller et al. 2013; Quinn et al. 2008; Tolias et al. 2011; Van Aelst and Cline 2004). In CNS development and diseases, the Rho GTPases switch between two states, a GTP-bound active state and a GDP-bound inactive state, which are mediated by three regulatory family (Cherfils and Zeghouf 2013; Etienne-Manneville and Hall 2002; Hall and Lalli 2010; Heasman and Ridley 2008). Rho guanine nucleotide exchange factors (RhoGEFs), the positive regulators, promote and catalyze the release of bound GDP for GTP (Cook et al. 2014; Miller et al. 2013; Miyamoto and Yamauchi 2010); Rho GTPase-activating proteins (RhoGAPs), the negative regulators, is able to increase or stimulate the hydrolysis of Rho GTPases (Fig. 1) (Moon and Zheng 2003). The other negative regulators, Rho guanine nucleotide-dissociation inhibitors (Rho GDIs) can bind to and prevent the dissociation of GDP (Fig. 1) (Olofsson 1999). In response to upstream signaling during different developmental stages, the Rho GTPases were precisely and coordinately mediated by their regulatory proteins. Moreover, the bidirectional regulation of Rho GTPases was necessary and required for spatial and temporal signals to guide downstream biological reactions, such as axon growth or retraction, synapse maturation or elimination, during the dynamics of neuronal morphology formation (Cherfils and Zeghouf 2013; Tolias et al. 2011; Van Aelst and Cline 2004). In addition to a deactivator role, increasing evidence has demonstrated that the RhoGAPs, as signaling intermediates, are deployed downstream of key molecules and transduce or link the extracellular and transmembrane signals to the dynamic reorganization of the cytoskeleton, as well as play an irreplaceable role in the regulation of neuronal morphology via GAP-dependent and -independent mechanisms (Fig. 1) (Bacon et al. 2013; Bernards and Settleman 2005; Moon and Zheng 2003; Yang and Kazanietz 2007), including axon growth and guidance, synapse formation and plasticity (Moon and Zheng 2003; Tolias et al. 2011). Defects or mutations in RhoGAPs also cause axonal and dendritic defects (Tolias et al. 2011), which are commonly recognized as the cause of several CNS diseases. Moreover, interference with RhoGAPs may mediate axon regeneration and synaptic plasticity (Mizuno et al. 2004; Tolias et al. 2011), which may contribute to better outcomes in CNS diseases, such as autism spectrum disorder.

Fig. 1. RhoGAPs in the CNS. RhoGAPs inactivate Rho GTPases to transfer a GAP-dependent signaling. In addition, RhoGAPs may directly respond to external stimuli and trigger GAPindependent signaling pathways via downstream binding partners or effectors. Finally, RhoGAPs are involved in actin and microtubule cytoskeleton dynamics and elicit diverse biological responses.

In this review, we focus on several extensively investigated RhoGAPs that are involved in axon and spine development. We try to explore how these RhoGAPs regulate the specific Rho GTPases signals spatiotemporally by detecting their expression patterns, domain structures as well as potential protein-protein interactions. Finally, we summarize the most recent studies of RhoGAPs in CNS diseases to elucidate their functions.

2. Basic characteristics of RhoGAPs The first RhoGAP, p50RhoGAP, was discovered 27 years ago (Garrett et al. 1989) and was determined to promote the intrinsic GTPase activity of Rho GTPases. Subsequent studies have indicated that a protein sequence composed of approximately 170 amino acids was required and responsible for GAP activity and was named the RhoGAP domain. To date, several RhoGAPs have been reported. A human genome analysis indicated there are approximately 70 GAP domain containing proteins, which is far beyond the number of Rho GTPases. The over-abundance of RhoGAPs indicated that each RhoGAP may play a specialized role, and each GAP activity may be precisely regulated spatially and temporally. For example, several RhoGAPs are widely expressed; however, other RhoGAPs are specifically expressed in the brain, such as the brain-specific GAP Grit and α1-chimaerin. Moreover, an individual RhoGAP may target a specific Rho GTPase. p122RhoGAP and RARhoGAP specifically act for RhoA, whereas α1-chimaerin and ArhGAP15 are Rac1 specific (Tcherkezian and Lamarche-Vane 2007). Moreover, in addition to the GAP domain, nearly all RhoGAPs contain at least 2–3 additional domains, which may interact with different proteins and are implicated in different signaling pathways. For example, the C1 domain of α1-chimaerin may bind to phorbol esters, which, in turn, strengthen the interaction between α1-chimerin and the NMDA receptor (NMDAR) to mediate downstream signaling (Brose et al. 2004; Van de Ven et al. 2005). The Src homology 2 (SH2) or Src homology 3 (SH3) adapter domain of Rho GAPs may aid in the signal transduction of receptor tyrosine kinase pathways (Fig. 2). Thus, RhoGAPs may act as intermediator or scaffold proteins to

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Fig. 2. Diverse domains of different RhoGAPs.

transduce the signaling pathways between Rho GTPases and other nonRho GTPase signaling pathways. 2.1. RhoGAPs in neuronal development One of the well-known roles of RhoGAPs is to regulate the cytoskeletal organization during neuronal development. In different stages, RhoGAPs may link external stimulation to intracellular signals, which thus precisely mediates neuronal morphology. In this section, we analyze the unique role of each RhoGAP during neuronal development (Fig. 2 and Table 1). 2.1.1. srGAPs: srGAP1, srGAP2, and srGAP3 The Slit-Robo GTPase-activating proteins (srGAPs) were initially characterized as cytoplasmic effectors of Robo1, an established axonal guidance receptor. srGAP1, srGAP2 and srGAP3 have been reported to be highly expressed in various CNS regions during different developmental stages (Bacon et al. 2009; Bacon et al. 2013). All srGAPs are structurally similar and contain a Fes-Cip4 homology Bin/ Amphiphysin/Rvs (F-BAR) domain, a RhoGAP domain and an SH3 domain (Bacon et al. 2013; Tcherkezian and Lamarche-Vane 2007) (Fig. 2). srGAP1 specifically acts on RhoA and Cdc42 in vivo (Wong et al.

2001), whereas srGAP3 preferentially downregulate Rac1 and, to a lesser extent, Cdc42 in vitro (Endris et al. 2002) (Table 1). srGAP3 is also referred to as WAVE-associated Rac GTPase-activating protein (WRP) and mental disorder-associated GAP (MEGAP), based on its interactions with WAVE1 (Wasp-family verprolin-homologous protein) and its implicated role in mental disorders, respectively (Soderling et al. 2002; Westphal et al. 2000). Through the SH3 domain, srGAP1 can bind to the intracellular site of Robo, which thus converts receptor activation into intracellular signals. The binding of srGAP1 with Robo subsequently activates its GAP activity towards Cdc42, which disrupts the actin structure and inhibits neuronal migration (Wong et al. 2001). Similar to srGAP1, srGAP2 overexpression results in increasing neurite growth and branches and decreasing migration (Guerrier et al. 2009). Guerrier et al. (Guerrier et al. 2009) reported that srGAP2 can promote the induction of membrane protrusions through its F-BAR domain, resulting in increased neurite outgrowth and decreased cell migration, which is different from the mechanism of srGAP1 (Wong et al. 2001). The most recent research has indicated a potential two-component molecular mechanism that modulates specific ligand binding to srGAP2 by dramatically tightening their associations, as well as moderately auto-inhibiting and restricting binding (Guez-Haddad et al. 2015). srGAP3 has also been demonstrated

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Table 1 Roles of RhoGAPs in the CNS. Name

Specificity

Expression

Signaling pathway and function in the CNS

srGAP1:RhoA, srGAP1: high in the brain; Binds to Robo. Increases neurite growth and srGAP2: widespread branching and decreases neuronal migration Cdc42 through its F-BAR domain via the induction of srGAP2: Rac1 filopodia-like membrane protrusions. Promotes synaptic maturation. srGAP3 Rac1Ncdc42 High in the brain Interacts with Slit/Robo. Regulated by phospholipids. in vitro Interacts with WAVE-1 and promotes Rac1-dependent neurite outgrowth. Necessary for normal spine formation and plasticity. Involved in ID. Potentially participates in schizophrenia. SYD1 Rac1 High in the brain Required for normal axon specialization. Binds to DCC and Robo and negatively regulates Rac1 activity to promote axon guidance. Recruits Trio for the positive regulation of Rac1 and is implicated in synaptic targeting. Interacts with trans-synaptic Nrx-1-Nlg1 complex to coordinate presynaptic and postsynaptic maturation. Abundantly expressed in Interacts with PSD-95 to restrict spine formation. BCR/ABR BCR:Rac1, Regulated by phosphorylation and ABR:Rac1 and the brain and dephosphorylation, intermolecular interactions and hematopoietic cells Cdc42 intramolecular interactions. Key role in cerebellar development. p250GAP Cdc42, Rac1, Rich in the brain Involved in the NGF/TrkA-p250GAP-Cdc42/RhoA and RhoA pathway to mediate axon growth. Interacts with β-catenin, N-cadherin, NMDARs and PSD-95 and is involved in NMDAR activity-dependent actin reorganization in dendritic spines. Under upstream control by tyrosine phosphorylation. Acts as a scaffold in membrane trafficking. Involved in ASD and schizophrenia. TCGAP Cdc42 High in the CNS Suppresses axon outgrowth. Necessary for normal spine formation and maturation. Regulated by phosphorylation. Interacts with SORT1 to cooperatively regulate TrkB trafficking. Associated with schizophrenia. Mediates spine length; stabilizes AMPA receptors to Broadly expressed in the Oligophrenin-1 In vitro: brain and enriched in the contribute to synapse maturation and plasticity. RhoA, Rac1 hippocampus, cortex and Involved in endocytosis of synaptic vesicles. Interacts and Cdc42 with Homer. Associated with X-linked intellectual In vivo: RhoA cerebellum disability. Mediates spine formation. Regulated through α1/α2/β2-chimaerin Rac1 α1-Chimaerin: binding of phorbol esters and DAG to C1 domain. exclusively expressed in Involved in Sema 3A and EphA4-mediated axon the brain guidance and retraction. Required for normal axon α/β2-Chimaerin: predominantly present in pruning. Mediated by intramolecular interactions in auto-inhibitory mechanism. the brain Mediated by phosphorylation. Associated with schizophrenia. p190RhoGAP RhoA High levels throughout Involved in axon outgrowth, guidance and the developing CNS fasciculation, dendritic spine maturation and dendrite stability. Controls Sema1A-mediated reverse signaling. Phosphorylation regulated by Src and Arg. Vilse Rac1NCdc42 Mainly expressed in the Downstream of Slit/Robo to inactivate Rac1/cdc42 in brain axon repulsion. Binds to CNK2 to mediate spine formation. Neuron-specific Inhibits neurite outgrowth via a Ca2+-dependent Nadrin RhoA, Rac1, and Cdc42 in exocytosis mechanism. Phosphorylation regulated vitro by Src. BAR domain-mediated auto-inhibitory mechanism. srGAP1/2

to be involved in the negative regulation of Rac1-dependent neurite outgrowth (Endris et al. 2011; Soderling et al. 2002; Zhang et al. 2014) by directly binding to WAVE-1 via its SH3 domain in vivo (Soderling et al. 2002). In addition to their roles in axon growth and guidance and neuronal migration, srGAPs, particularly srGAP3 and srGAP2, mediate cytoskeletal reorganization to regulate spine formation or synaptic maturation (Bacon et al. 2013; Charrier et al. 2012; Fossati et al. 2016; Yang et al. 2006). Fossati et al. have demonstrated that the ancestral srGAP2 (also referred to as srGAP2A) decreased the density of both excitatory and inhibitory synapses through its GAP activity (Fossati et al. 2016). srGAP2

Refs (Fossati et al. 2016; Wong et al. 2001)

(Carlson et al. 2011; Endris et al. 2002; Soderling et al. 2002; Soderling et al. 2007; Waltereit et al. 2012)

(Hallam et al. 2002; Holbrook et al. 2012; Owald et al. 2012; Xu et al. 2015; Xu and Quinn 2015)

(Kaartinen et al. 2001; Chuang et al. 1995; Mulherkar et al. 2014; Oh et al. 2010; Park et al. 2012)

(Akshoomoff et al. 2015; Chagnon et al. 2010; Long et al. 2013; Nakamura et al. 2016; Nakamura et al. 2002; Ohi et al. 2012; Okabe et al. 2003)

(Liu et al. 2006; Nakazawa et al. 2016; Rosario et al. 2007; Rosario et al. 2012; Schuster et al. 2015)

(Billuart et al. 1998; Govek et al. 2004; Khelfaoui et al. 2009; Nadif et al. 2009; Nakano-Kobayashi et al. 2009)

(Beg et al. 2007; Brown et al. 2004; Buttery et al. 2006; Iwasato et al. 2007; Kai et al. 2007; Riccomagno et al. 2012; Shi et al. 2007; Van de Ven et al. 2005; Wegmeyer et al. 2007)

(Brouns et al. 2001; Jeong et al. 2012; Sfakianos et al. 2007; Zhang and Macara 2008)

(Hu et al. 2005; Lim et al. 2014; Lundstrom et al. 2004) (Beck et al. 2014; Beck et al. 2013; Furuta et al. 2002; Harada et al. 2000)

promotes synaptic maturation via interactions with the excitatory postsynaptic scaffold Home via the F-BAR domain and the inhibitory postsynaptic scaffold Gephyrin via the SH3 domain (Fossati et al. 2016). Interestingly, srGAP2C, the human-specific paralog, inhibits all identified functions of srGAP2, thereby protracting the maturation and increasing the density of excitatory and inhibitory synapses (Fossati et al. 2016). srGAP2C has been shown to physically interact with srGAP2 (Charrier et al. 2012); however, the exact mechanism of srGAP2C in srGAP2 suppression remains unclear. Regarding the role of srGAP3 on spine or synapse formation, Waltereit et al. (Waltereit et al. 2012) reported that the dendritic spines

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of hippocampal neurons in srGAP3 knockout (KO) mice were significantly longer, with no effect on the spine density. In contrast, Carlson et al. (Carlson et al. 2011) reported that srGAP3 conditional KO neurons exhibited a significant decrease in the spine density. The reasons for this discrepancy may be that the srGAP KO mice may induce compensatory effects on spine formation during development, whereas the conditional KO may not exhibit these effects. Moreover, the exons of the gene encoding the inverse F-BAR domain is interrupted in conditional KO mice, whereas it remains intact in srGAP3 KO mice. An increasing number of studies have demonstrated the importance of the F-BAR domain of srGAP3 in actin dynamics and spine formation. The F-BAR domain of srGAP3 has the capability of binding specifically to numerous membrane lipids, including PA, PIP2 and PIP3, which subsequently recruit srGAP3 to plasma membrane (Endris et al. 2011), thereby leading to outward filopodia-like structures in both non-neuronal cells and neurons (Coutinho-Budd et al. 2012). In addition, impaired spine formation in srGAP3 KO mice can be rescued by the F-BAR domain, which suggests that srGAP3 mediates spine formation through its F-BAR domain (Carlson et al. 2011). srGAP3 also inhibits actin dynamics via its GAP domain by inactivating Rac1 (Endris et al. 2011; Soderling et al. 2002). Moreover, Soderling et al. (Soderling et al. 2002; Soderling et al. 2007) have reported that srGAP3 may interact with WAVE1 via its SH3 domain, thereby becoming involved in the WAVE-1 complex, a key mediator in normal neuronal development. Following the generation of mutant mice of WAVE-1 without srGAP3 binding sites, Soderling et al. determined that srGAP3 anchored to WAVE-1 plays a key role in synaptic plasticity during normal neuronal development (Table. 1) (Soderling et al. 2007). Collectively, mouse models and in vitro studies have demonstrated a crucial role for srGAP3 in dendritic spine formation and activity, which may be mediated by its F-BAR domain, GAP domain and interaction with WAVE1. 2.1.2. SYD-1 SYD-1 (synapse defective protein-1), a conserved protein in C. elegans, Drosophila and mice, is a key positive regulator involved in multiple steps of neuronal development and neuronal connectivity (Holbrook et al. 2012; Patel et al. 2006; Wentzel et al. 2013; Xu and Quinn 2015). In C. elegans and Drosophila, SYD-1 is consisted of a PDZ domain, a C2 domain and a Rho GAP domain (Fig. 2). However, in mice, mSYD1A contains an N-terminal intrinsically disordered domain (IDD) as well as a C2 domain and a Rho GAP domain but without the PDZ domain (Wentzel et al. 2013). In this section, we mainly focus on SYD-1. Localized in presynaptic terminals, SYD-1 has been reported to function in defining axonal identity by sorting presynaptic components into the developing axon (Hallam et al. 2002). Loss-of-function mutations of SYD-1 impaired the normal axon component distribution, which was mis-localized to dendritic processes (Hallam et al. 2002). SYD-1 may target Liprin-α to maturing presynaptic active zones (AZs) and promote the presynaptic assembly (Owald et al. 2010; Patel et al. 2006). In addition, SYD-1 promotes the accumulation of presynaptic Nrx-1, which then recruits postsynaptic Nlg1, thereby coupling pre- to postsynaptic assembly (Owald et al. 2012). The interaction between SYD-1 and Nrx-1-Nlg1, which may also stabilize the SYD-1/Liprin-α clusters at AZs, sheds light on the role of SYD1 with respect to how the cooperation of the presynaptic scaffold protein at AZs and a pair of trans-synaptic protein module may precisely mediate synapse assembly (Owald et al. 2012). In addition, an SYD-1 mutation exhibited defects in the selection of synaptic target sites (Holbrook et al. 2012). The molecular mechanism is that in axons, SYD1 may bind to Trio (a RhoGEF protein) and promote its GEF activity, which may guide axons via Rac1 activation (Holbrook et al. 2012). Recently, SYD-1 has been determined to bind to the cytoplasmic domains of two guidance receptors, DCC and Robo, and function inter-dependently, whereas it interacts with activated Rac1 GTPase

and negatively regulates Rac1 activity to promote axon guidance (Xu et al. 2015; Xu and Quinn 2015). The opposite roles of SYD-1 with respect to Rac1 activity during specific time points of developmental stages may be a result of different binding partners, such as GEF or GAP. This makes SYD-1 a spatial and temporal mediator in neuronal morphology via different protein-protein interactions (Holbrook et al. 2012; Xu et al. 2015; Xu and Quinn 2015). Together, through different partners at different developmental stages, SYD-1 has been implicated in crucial steps, including axonal specification, axon guidance, target site selection, and presynaptic and postsynaptic maturation. 2.1.3. BCR and ABR BCR (breakpoint cluster region protein) was originally recognized as BCR-Abl fusion oncogene in leukemia (Heisterkamp et al. 1985) and was subsequently identified as a RhoGAP towards Rac1 (Chuang et al. 1995; Diekmann et al. 1991). ABR (active BCR-related) was identified due to its relatedness with BCR (Tan et al. 1993). In addition to their expressions in hematopoietic cells, both BCR and ABR are enriched in brain (Oh et al. 2010; Tan et al. 1993; Tcherkezian and Lamarche-Vane 2007). BCR consists of several domains: an N-terminal coiled-coil oligomerization domain (CC domain), a serine/threonine kinase domain in the Nterminus, a DH domain (also referred to as a RhoGEF domain), a PH domain, a C2 domain, a proline-rich region and a GAP domain in the C-terminus (Fig. 2). Similar to the domains of BCR, but without the Nterminal kinase domain, ABR has been reported to negatively regulate both Rac1 and Cdc42 via its GAP activity (Fig. 2). BCR and ABR share several overlapping functions; however, they do not have identical roles in the brain during neuronal development (Um et al. 2014; Narayanan et al. 2013; Park et al. 2012; Oh et al. 2010; Vaughan et al. 2011a). Here, we mainly discuss the role of BCR. Both BCR and ABR play indispensable roles in patterning cerebellar morphology. With elevated Rac1 activity and p38 MAPK signaling, BCR and ABR KO mice exhibited cerebellar developmental defects, including ectopic neurons, defects in fissure formation and disorganized Bergmann processes and basement membrane integrity, which suggests a significant role in the control of cell morphogenesis, migration and foliation (Kaartinen et al. 2001a; Mulherkar et al. 2014). Furthermore, as a multiple domain protein, BCR may bind to Huntingtin-Associated Protein 1 or Src Homology 2 Domain Containing Protein 5 to regulate neuronal differentiation and morphology (Gray et al. 2014; Huang et al. 2015). In excitatory synapses, BCR has been reported to interact with PSD95 (postsynaptic density protein 95), an abundant postsynaptic scaffolding protein, and restrict spine formation, dendritic arborization and actin polymerization via its GAP domain (Oh et al. 2010; Park et al. 2012) (Table. 1). BCR may also interact with the Tiam1-Par complex and inhibit both Rac1 and PKCζ to control cell polarity and migration (Narayanan et al. 2013). In addition, following the formation of the GEF/GAP complex, BCR and Tiam1 together coordinately regulate Rac1-dependent spine development downstream of EphB receptors (Um et al. 2014). A GEF/GAP complex may provide an efficient mechanism for the dynamic regulation of Rac1 activity during neuronal development. It should be noted that although there is a significant difference in the spine density between BCR or ABR KO mice and wild-type mice, the increase in the spine density of BCR or ABR KO mice was small and was not accompanied by a concomitant increase in spontaneous/ evoked excitatory synaptic transmission (Oh et al. 2010). Recently, Um et al. demonstrated that striking increases in both the size and density of spines and synapses were detected in BCR and ABR double KO mice (Um et al. 2014). The more severe phenotypes observed in the double KO mice suggest the functional overlap between BCR and ABR. The BCR KO mice lack of significant synaptic defects may be a result of the compensatory mechanisms by ABR (Oh et al. 2010; Um et al. 2014), which are also present in other cell types (Cho et al. 2007; Kaartinen et al. 2001).

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Importantly, the GAP activity of BCR was regulated via means of conversions between the intra- and inter-molecular interactions through phosphorylation and dephosphorylation (Lee 2015; Park et al. 2012). PTPRT, a member of the receptor-type protein tyrosine phosphatase family, may dephosphorylate BCR on the N-terminus tyrosine phosphorylation sites and decrease the intermolecular interaction (multimerization) of BCR, which leads to the increase of the intramolecular interaction between the BCR N- and C-termini (Lee 2015; Park et al. 2012). As a result, the C-terminal GAP domain cannot be released from the N-terminus, and the GAP activity is suppressed (Park et al. 2012). Thus, the reduced GAP activity of BCR increased the dendritic arborization, branching and spine formation (Lee 2015; Park et al. 2012). Furthermore, the deletion of the CC domain modulated the dendritic arborization by increasing the GAP activity, which indicates that the GAP activity may also be regulated by the CC domain (Park et al. 2012). The CC domain of BCR-Abl has been reported to control the Abl activity by inducing protein multimerization (Beissert et al. 2003; He et al. 2002). Thus, the mechanism of CC-mediated GAP activity is obscure; however, it appears that the CC domain may enhance the Rac1 GAP activity via the multimerization of BCR. Interestingly, both BCR and ABR contain a DH domain (also referred to as a RhoGEF domain), which exhibits the GEF activity towards RhoA, Rac1 and Cdc42 in vitro (Chuang et al. 1995; Dubash et al. 2013; Sahay et al. 2008; Vaughan et al. 2011). As reported, BCR may interact with and serve as a substrate of the protein-tyrosine kinase c-Fes to function coordinately in regulating Rac1 and Cdc42 activation, resulting in neuritogenesis (Laurent and Smithgall 2004). The GEF and GAP domains of BCR and ABR may interact with Rho GTPases in a non-competitive way, indicating that they may coordinately and precisely regulate Rho GTPase following different stimuli (Laurent and Smithgall 2004; Chuang et al. 1995; Peck et al. 2002). 2.1.4. p250GAP p250GAP is also referred to as Grit (GTPase regulator interacting with TrkA), RICS (RhoGAP involved in β-catenin-N-cadherin and NMDAR signaling), p200RhoGAP and GC-GAP (Hayashi et al. 2007; Moon et al. 2003; Nakamura et al. 2002; Okabe et al. 2003; Taniguchi et al. 2003; Zhao et al. 2003). Researchers have reported that p250GAP stimulates GTP hydrolysis equally in Cdc42, Rac1, and RhoA (Nakamura et al. 2002), whereas other researchers have reported that p250GAP prefers Cdc42/Rac1 or only RhoA as a substrate (Impey et al. 2010; Long et al. 2013; Simo and Cooper 2012; Zhao et al. 2003). p250GAP has a unique phospholipid binding domain, the Phox-homology (PX) domain, potential protein kinase phosphorylation sites, a proline-rich sequence and a β-catenin binding site, in addition to the GAP domain (Fig. 2). p250GAP is exclusively and richly expressed in the brain, particularly in neurons (Moon et al. 2003; Nakazawa et al. 2003) (Table. 1). In axonal development, p250GAP may directly interact with the upstream protein TrkA to mediate axon growth via the inactivation of RhoA and Cdc42, rather than Rac1 (Nakamura et al. 2002). Expression of the TrkA binding region or the RhoGAP domain of p250GAP strongly blocked neurite extension induced by NGF, which suggests that p250GAP is one significant component in NGF-induced neurite outgrowth (Nakamura et al. 2002). In vivo, Yukiko reported that p250GAP regulates neurite outgrowth as a RhoGAP toward Cdc42, and the β-catenin-binding site may be vital for its GAP activity (NasuNishimura et al. 2006). In addition to the regulation of axonal development, p250GAP has been implicated in the regulation of spine morphogenesis. p250GAP may interact with β-catenin, N-cadherin, NMDARs and PSD95, and cluster together in the postsynaptic density (Nakazawa et al. 2003; Okabe et al. 2003) (Table. 1). Both Long et al. (Long et al. 2013) and Nakazawa et al. (Nakazawa et al. 2008) have demonstrated that p250GAP, an NMDAR-associated RhoGAP, regulated spine morphogenesis via the modulation of RhoA activity. The knockdown of p250GAP significantly

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decreased the dendritic spine length and branching and increased the endogenous RhoA activity. This phenotype may be rescued by the overexpression of dominant negative mutants of ROCK or RhoA or the inhibition of ROCK activity (Long et al. 2013; Nakazawa et al. 2008), which indicates that p250GAP is a RhoAGAP essential for the regulation of RhoA/ROCK signaling to control dendritic spine formation. NMDAR activation leads to the redistribution of p250GAP (Nakazawa et al. 2008), which suggests that p250GAP is involved in the NMDAR-mediated regulation of RhoA activity in spine morphological plasticity. Furthermore, via its phosphorylation sites, p250GAP is controlled by tyrosine kinases, such as TrkA (Nakamura et al. 2002), the Src-family kinase Fyn (Taniguchi et al. 2003), other Src kinases (Moon et al. 2003), Ca2+/calmodulin-dependent protein kinase II (CaMKII) (Okabe et al. 2003; Simo and Cooper 2012), and receptor tyrosine phosphatase sigma (RPTPσ) (Chagnon et al. 2010) (Table. 1). The GAP activity of p250GAP was reportedly inhibited by the phosphorylation by CaMKII (Okabe et al. 2003). Moreover, the RPTPσ can increase the p250GAP activity to control axonal outgrowth (Chagnon et al. 2010). The opposite pattern of p250GAP activity may be caused by different influences of phosphorylation by distinct kinases. An increasing number of evidence suggest that the PX domain of p250GAP participates in several aspects of membrane trafficking (Nakamura et al. 2010; Nakamura et al. 2008). In a recently published paper, Nakamura reported that the multiple-domain-containing protein p250GAP plays a pivotal role as a linker in the assembly of the constituents of the trafficking complex (Nakamura et al. 2016). In mouse cortical neurons, p250GAP formed an adaptor complex with other proteins and subsequently interconnected GABAAR and dynein/dynactin, which contributes to the GABAAR trafficking and surface expression (Nakamura et al. 2016). These findings suggest that the p250GAP may be involved in the delivery of numerous membrane-related proteins to mediate synapse formation and synaptic transmission (Nakamura et al. 2016), as well as indicate another uncovered role for p250GAP in neuronal morphology. p250GAP interacts with more than 60 proteins, many of which have potential functions on developmental stages of neurons, such as neurite extension and Rho GTPase pathway regulation. Clearly, more investigations are needed to determine their roles in CNS development. Despite its role as a protein binding partner, p250RhoGAP was also mediated by small RNAs in the regulation of neuronal morphogenesis. Many assays have indicated that p250GAP may act as a target of miR132, which thus regulates Rac1 to exert its function on neuronal morphogenesis (Impey et al. 2010; Marler et al. 2014; Vo et al. 2005; Wayman et al. 2008). Together, through diverse binding partners, signaling pathways, and effectors (Rac1, RhoA, and Cdc42), p250GAP is involved in each stage of brain development and fine-tunes neural circuits at various time points. 2.1.5. TCGAP TCGAP (Tc10/Cdc42 GTPase-activating protein) has been extensively investigated under various names, including NOMA-GAP/ARHGAP33/ SNX26/TCGAP (Simo and Cooper 2012). Similar to p250GAP, it contains an N-terminal PX domain, an N-terminal RhoGAP domain, several proline-rich sequences, and an SH3 domain, and it has been identified as a Cdc42-specific GAP (Rosario et al. 2012) (Fig. 2 and Table 1). The function of TCGAP has been thoroughly investigated in axon extension because of its high expression in the CNS. TCGAP overexpression suppressed neurite outgrowth, whereas a GAP mutant TCGAP or TCGAP knockdown enhanced axon outgrowth (Liu et al. 2006; Rosario et al. 2007). Similar to p250GAP, the function of TCGAP in neuronal morphology is under upstream control by tyrosine phosphorylation. TCGAP may be phosphorylated in at least two regions, namely, the GAP domain by Fyn (Liu et al. 2006) and the C-terminal tail by TrkA (Liu et al. 2006; Rosario et al. 2007). In NGF-stimulated PC12 cells, Fyn-dependent phosphorylation of TCGAP inhibited the GAP activity and promoted neurite outgrowth in a Cdc42-dependent manner (Liu et al. 2006). NGF also

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stimulated TrkA-dependent phosphorylation of the C-terminal tail of TCGAP, which recruited and activated downstream effectors, such as Shc, Grb2 and Shp2, to mediate the extension of neuronal processes via the SHP2/ERK5 pathway (Rosario et al. 2007). Thus, TCGAP, via its dual function as a multi-adaptor and a RhoGAP protein, plays an essential role downstream of NGF in the promotion of neurite outgrowth and extension in vitro (Rosario et al. 2007). All axon data were collected in vitro until a recent in vivo study using TCGAP-deficient mice (Rosario et al. 2012). However, the TCGAP-deficient mice did not exhibit defects in axon pathological findings or changes in cortico–cortico or cortico– thalamic projections (Rosario et al. 2012). These data suggest that in vivo, TCGAP does not play a significant role in cortical axonal outgrowth and pathfinding or other molecules may compensate for TCGAP function in these events. In the spine, TCGAP may positively regulate dendritic spine formation (Shen et al. 2011). Knockdown of TCGAP resulted in a decreased dendrite length and spine density (Shen et al. 2011). Similar phenotype, a simpler dendritic tree, was also detected in TCGAP KO mice, exhibiting lower spine density and less mature spines (Nakazawa et al. 2016; Rosario et al. 2012); these findings indicate the necessity of this protein for proper dendritic development in the neocortex (Rosario et al. 2012; Simo and Cooper 2012). In contrast, in another line of research, Kim determined that shRNAmediated knockdown showed the same result, including an increased total spine, but a reduction in the proportion of mature mushroomshaped spines (Kim et al. 2013). This phenotype was further validated by a recent study (Schuster et al. 2015) in which the spine density increased, whereas the density of PSD95-containing synapses was significantly decreased in cultured and in vivo neurons of TCGAP KO mice. These two independent studies indicated that the loss of TCGAP interferes with dendritic spine development and results in an increase in total dendritic spines at the expense of mature spines (Kim et al. 2013; Schuster et al. 2015). Contradictory data exist among different studies regarding TCGAP in spine formation and maturation. This contradiction may be a result of several differences, such as in vivo vs. vitro assays, differences in the time point during spine development, and discrepancies in gene KO strategies or the KO mouse strain. However, despite the discrepancies regarding the total spine density, the common feature is that a lack of TCGAP leads to disrupted normal spine formation and maturation, particularly decreased numbers of mature mushroom spines both in vitro and in vivo (Kim et al. 2013; Nakazawa et al. 2016; Rosario et al. 2012; Schuster et al. 2015; Shen et al. 2011). This spine phenotype of TCGAP may also be largely rescued by Cdc42, which implies a role for TCGAP in the inhibition of Cdc42 to mediate spine formation (Kim et al. 2013; Rosario et al. 2012). In addition, according to a new report, TCGAP may interact with SORT1 and they together mediate TrkB trafficking, which is essential for synapse development (Nakazawa et al. 2016). 2.1.6. Oligophrenin-1 OPHN1, which encodes the RhoGAP Oligophrenin-1, was initially identified to be involved in X-linked intellectual disability (Billuart et al. 1998). Oligophrenin-1 contains an N-terminal BAR domain, a PH domain, a Rho GAP domain and three proline-rich regions. Oligophrenin-1 is capable of inactivating RhoA, Rac1 and Cdc42, but acts predominantly on RhoA in vivo (Fig. 2) (Fauchereau et al. 2003; Govek et al. 2004) (Table. 1). To date, its function remains incompletely understood. Enriched in the brain, Oligophrenin-1 is widely expressed in both pre- and post-synapses (Fauchereau et al. 2003; Govek et al. 2004). In both hippocampal slices and cultured neurons, the knockdown of Oligophrenin-1 leads to dendritic spine shortening and reduces the dendritic spine density (Govek et al. 2004; Khelfaoui et al. 2007; Nakano-Kobayashi et al. 2009). The phenotype can be mimicked by activated RhoA and reversed by a Rho-kinase (ROCK) inhibitor, which suggests decreased Oligophrenin-1 regulates spine morphology by

enhancing RhoA/ROCK (Govek et al. 2004; Khelfaoui et al. 2007; Nadif et al. 2009). Oligophrenin-1 has been determined to associate with Homer (Govek et al. 2004), a postsynaptic adaptor protein, which can guide glutamate receptors to multiple intracellular targets (Xiao et al. 2000), suggesting that Oligophrenin-1 may serve as a key transducer between postsynaptic receptors and downstream cytoskeletons. Together, these findings are consistent with the established view that Oligophrenin-1 alters dendritic spine morphology. It has also been reported that Oligophrenin-1 is involved in the activity dependent synaptic plasticity and maturation in a GAP dependent manner. Defective Oligophrenin-1 causes the impairments of synaptic plasticity and NMDA receptors due to the destabilization of AMPA receptors; these findings suggest that Oligophrenin-1 is indispensable for activity-triggered glutamatergic synapse development (Nadif et al. 2009). In addition, Oligophrenin-1 is involved in the regulation of AMPA receptor trafficking (Khelfaoui et al. 2009). Through interactions with SH3-containing endocytosis proteins, Oligophrenin-1 is enriched in endocytic sites to downregulate the RhoA/ROCK signals on endocytosis (Khelfaoui et al. 2009; Nakano-Kobayashi et al. 2009). The loss of Oligophrein-1 function affected AMPA receptor endocytosis and synaptic plasticity, which may be rescued by a ROCK inhibitor (Khelfaoui et al. 2009). In addition, Oligophrenin-1 has been implicated in metabotropic glutamate receptor (mGluR) induced LTD (Nadif et al. 2011). It should be noted that this role of Oligophrenin-1 is independent from its functions on the basal synaptic strength, which needs the RhoGAP activity of Oligophrenin-1 and interaction with Homer1b/c (Govek et al. 2004; Nadif et al. 2009; Nadif et al. 2011). Recently, Nakano-Kobayashi et al. have demonstrated that an Oligophrenin-1-Homer1b/c interaction is important for the guiding the endocytic zones adjacent to the PSD (Nakano-Kobayashi et al. 2014). They showed that when the binding between Oligophrein-1 and Homer1b/c was interrupted, endocytic zones were displaced from the PSD, which thereby led to the impaired AMPAR recycling as well as less AMPAR at synapses (Nakano-Kobayashi et al. 2014). Thus, Oligophrenin-1 plays a pivotal role in neuronal development via the mediation of synapse formation and plasticity, AMPAR trafficking and endocytosis of synaptic vesicles (Table. 1). 2.1.7. α- and β-chimaerin Chimaerins are RhoGAPs with specific activity for Rac1 that contain an N-terminal C1 phorbol ester- and diacyglyerol (DAG)-binding domain besides a C-terminal RhoGAP domain (Fig. 2). There are four members of this family, including α1-, α2-, β1- and β2-chimaerin, which are produced from α-chimaerin and β-chimaerin genes (Dong et al. 1995; Hall et al. 1990; Hall et al. 1993). Furthermore, α2-chimaerin and β2-chimaerin variants have an additional SH2 domain in the N-terminus (Fig. 2). All α1-, α2- and β2-chimaerins are high expressed in the CNS, with the exception of β1-chimaerin, which is predominantly expressed in the testis (Table 1). α1-chimaerin has been shown to regulate spine and dendritic morphogenesis (Van de Ven et al. 2005). In vitro, the knockdown of α1chimaerin increases the spine density and leads to increased growth (Buttery et al. 2006; Van de Ven et al. 2005). Similarly, α1-chimaerin overexpression in Purkinje cells resulted in substantial alterations of the dendritic morphology, which exhibited a simplified dendritic arbors, a loss of normal spines and a reduction in dendritic length and branching (Buttery et al. 2006; Van de Ven et al. 2005). However, α2chimaerin facilitates neurite outgrowth, contrary to that of pruning of α1-chimaerin (Buttery et al. 2006). The opposite role of these two isoforms may be due to the difference in SH2 domain, which may regulate the GAP activity via an auto-inhibitory mechanism. Distinct from other RhoGAPs, chimaerins may be regulated by lipid binding, which therefore indicates they represent a linker between lipid signaling and Rac1 inactivation (Brose et al. 2004; Caloca et al. 2001). Treatment with phorbol esters or DAG may promote the recruitment and translocation of chimaerins to the plasma membrane through its C1 domain

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(Buttery et al. 2006; Colon-Gonzalez and Kazanietz 2006). The role of α1-chimaerin in dendritic pruning relies on both the GAP domain and the DAG-binding C1 domain (Buttery et al. 2006). α1-chimaerin may also be involved in NMDAR-dependent developmental plasticity because it has been shown to interact with NMDAR to regulate spine formation in vitro (Van de Ven et al. 2005). There is strong evidence that α1-chimaerin is involved in spine development in vitro (Buttery et al. 2006; Van de Ven et al. 2005); however, whether α1-chimaerin plays the same role in vivo has remained unknown until recent years. Using a series of mice with global and conditional KOs of α-chimaerin isoforms (α1-chimaerin and α2chimaerin), Iwata et al. have determined that α1-chimaerin KO does not affect the development of spine formation and cognitive ability, whereas α2-chimaerin disruption results in an increased size and density of spines in the hippocampus (Iwata et al. 2015; Iwata et al. 2014). The reason may be a result of their expression patterns because α1chimaerin is mainly expressed in adults, whereas α2-chimaerin is mainly expressed in developmental stages (Hall et al. 2001; Lim et al. 1992). In addition to its previously described role in spine formation, α2chimaerin also regulates Sema 3A-induced growth cone collapse during axon outgrowth. In early research, Brown et al. demonstrated that phorbol ester induced α2-chimaerin activation led to growth cone collapse (Brown et al. 2004). α2-chimaerin with GAP mutation or α2chimaerin interrupted the binding site with phosphotyrosine (SH2 domain) blocked the Sema 3A-induced collapse, which suggests α2chimaerin took part in Sema 3A signals via the SH2–phosphotyrosine binding (Brown et al. 2004). In 2007, several different follow-up groups simultaneously demonstrated that α2-chimaerin interacts with EphA4 via its SH2 domain, transduces EphA4 forward signals to the actin cytoskeleton through Rac1 activity modulation, and regulates EphA4-dependent growth cone collapse (Beg et al. 2007; Iwasato et al. 2007; Shi et al. 2007; Wegmeyer et al. 2007). Importantly, α2-chimaerin mutant mice exhibit abnormal midline crossing and projections of axons in the CNS, a phenotype similar to EphA4 mutant mice (Beg et al. 2007; Iwasato et al. 2007; Wegmeyer et al. 2007). Recent studies have further confirmed that α2-chimaerin serves as a signal convergent point of the Sema 3A and EphA-mediated axon guidance signaling pathway (Ferrario et al. 2012; Kao et al. 2015). Compared with α-chimaerin, there are fewer reports on β2chimaerin in neuronal development. Riccomagno el al. have reported the inhibition of Rac1 by β2-chimaerin mediates Sema3F-dependent axon pruning in vitro and in vivo (Riccomagno et al. 2012). Interestingly, semaphorin signaling is well-established in the mediation of axon pruning, axon repulsion and spine remodeling; however, β2chimaerin is only necessary for axon pruning, but dispensable for axon guidance and spine formation (Riccomagno et al. 2012), which suggests a unique role of β2-chimaerin in modulating specific neuronal development. It should be noted that the previously described functions of chimaerins depend on the GAP activity, which may be regulated by phospholipids or protein-protein binding. The mediation is based on an auto-inhibitory mechanism in which the N terminus of β2-chimaerin protrudes into the GAP domain, which sterically blocks Rac1 binding, and the C1 domain is covered due to the intramolecular interactions with SH2 domain, GAP domains and intermediator regions (Canagarajah et al. 2004). When the C1 domain binds to the phospholipids, it induces the division of the interactions and leads to the exposure of the GAP domain to mediate Rac1 (Canagarajah et al. 2004; Hall et al. 2005). Furthermore, the GAP activity is mediated by protein binding and Src/Cdk5-mediated phosphorylation events, which may interfere with the conformational changes of chimaerins to stimulate GAP activity (Beg et al. 2007; Canagarajah et al. 2004; Iwasato et al. 2007; Kai et al. 2007; Qi et al. 2004). Taken together, with a specific DAG binding C1 domain and an auto-inhibitory mechanism mediated by intramolecular interactions,

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chimaerins function in diverse aspects of neuronal development and exhibit different roles. 2.1.8. p190RhoGAP The p190RhoGAP family consists of p190-A (originally referred to as p190) and p190-B (Bernards and Settleman 2005; Ellis et al. 1990; Tcherkezian and Lamarche-Vane 2007). p190GAP is a conserved protein specific for RhoA and is expressed in humans, rats, flies, and mice (Billuart et al. 2001; Settleman et al. 1992). p190RhoGAP is composed of a GTP-binding domain followed by four consecutive FF protein-interaction domains, proline-rich regions, and a GAP domain (Haskell et al. 2001; Ridley et al. 1993) (Fig. 2 and Table. 1). p190RhoGAP is specifically expressed at a high level throughout the developing nervous system (Brouns et al. 2000; Brouns et al. 2001). p190RhoGAP overexpression promotes extensive neurite outgrowth (Brouns et al. 2001), whereas dominant negative p190RhoGAP significantly reduced neurite outgrowth by increasing GTP-RhoA levels (Jeon et al. 2012); these findings indicate that it may be a vital regulator of RhoA-mediated cytoskeleton rearrangement. p190RhoGAP mutant mice exhibited several developmental deficits in the CNS, including abnormal axon guidance, outgrowth and fasciculation (Brouns et al. 2000; Matheson et al. 2006), in a GAP-activity-dependent manner (Brouns et al. 2000; Brouns et al. 2001; Matheson et al. 2006). Similarly, impaired axon guidance and target recognition were also detected in the p190RhoGAP-deficient Drosophila (Jeong et al. 2012). Studies indicate that phosphorylation of p190RhoGAP can regulate its GAP activity. Protein kinases, such as Src, can phosphorylate p190RhoGAP in response to stimulations (Haskell et al. 2001), which in turn leads to a conformational change of p190RhoGAP and activates the GAP to downregulate Rho GTPases. The deletion of Src kinases exhibits analogous defects to p190RhoGAP mutant mice (Brouns et al. 2001). Studies have indicated a role for p190RhoGAP in neuronal development and neuritogenesis via the mediation of Src-dependent manner in regulating Rho GTPase activity (Brouns et al. 2000; Brouns et al. 2001). Interestingly, a recent study reported that p190RhoGAP binds to the intracellular part of semaphorin and mediates its reverse signaling to control axon guidance and defasciculation (Jeong et al. 2012). This characteristic is different from other RhoGAPs, such as α2-chimaerin and srGAP3, which mediates a transduction pathway downstream of Sema-Plexin and SlitRobo signaling to inactivate Rho GTPase. This finding uncovered a novel potential molecular mechanism of p190RhoGAP. In addition to its role in axons, p190RhoGAP is expressed in the dendrite spine and contributes to spine maturation via the inactivation of RhoA (Duman et al. 2015; Sfakianos et al. 2007; Zhang and Macara 2008). The knockdown of p190RhoGAP by shRNA or a GAP-deficient mutant of p190RhoGAP reduced the spine density compared with the control (Zhang and Macara 2008), which indicates its necessity for spine formation via the GAP domain. As previously discussed, the GAP activity of p190RhoGAP can be modulated by phosphorylation. Furthermore, with respect to synapse formation, instead of being phosphorylated by kinases, such as Src (Haskell et al. 2001), p190RhoGAP may be regulated by the tyrosine kinase Arg (Bradley et al. 2006; Kerrisk and Koleske 2013). Arg KO mice exhibited reduced p190RhoGAP phosphorylation, which resulted in increased RhoA activity and the loss of synapses (Sfakianos et al. 2007). Moreover, p190RhoGAP and Arg mutations exhibited a synergistic effect on dendritic regression, whereas the down-regulation of the Rho effector ROCKII reversed the dendritic regression of Arg KO mice (Sfakianos et al. 2007); these findings indicate the coordination of Arg and p190RhoGAP acts in the regulation of spine maturation and stability through RhoA pathways (Kerrisk and Koleske 2013; Sfakianos et al. 2007). p190RhoGAP is also involved in the polarity protein Par6 mediated downstream pathways, and targeting RhoA is able to restore the changes of spine morphology in Par6 deficient neurons (Tolias et al. 2011; Zhang and Macara 2008). These findings provide insights into the functions of p190RhoGAP in molecular interactions, signaling pathways and the regulation of spine formation.

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2.1.9. Vilse Vilse, also referred to as CrossGAP, was identified as a GAP that mediates Slit-Robo signaling in axon repulsion in Drosophila (Hu et al. 2005; Lundstrom et al. 2004). A protein sequence analysis indicated that Vilse contains two WW motifs at its N-terminus, MyTH4 (myosin tail homology 4) and a RhoGAP domain at the C-terminal (Fig. 2). Vilse is active both in vitro and in vivo towards Rac1 and Cdc42 but not RhoA as reported by Lundstrom et al. (Lundstrom et al. 2004); Hu et al. reported Vilse strongly and specifically down regulates the activity of Rac1 but not Cdc42 and RhoA (Hu et al. 2005) (Table. 1). Vilse was first identified as a mediator of Robo repulsion through a genetic screen for embryonic tracheal phenotypes in Drosophila (Lundstrom et al. 2004). Subsequent research confirmed that Vilse participated in axon guidance, serving as a downstream protein in Robomediated repulsion in Drosophila (Hu et al. 2005). Via its WW domain, Vilse may directly interact with the CC2 proline-rich region of Robo and act as a GAP specifically for Rac1 to regulate midline crossing (Hu et al. 2005). Intriguingly, the biological role of Vilse in Robo signaling appears to be different in midline neurons compared with the trachea (Hu et al. 2005; Lundstrom et al. 2004). Furthermore, in mammals, Vilse overexpression inhibited spine formation and exhibited few dendrites (Lim et al. 2014). Regarding the mechanism, Vilse may interact with the scaffold protein CNK2 through its WW domain, thereby modulating Rac1 activity to mediate spine formation (Lim et al. 2014). Recently, we determined that Vilse inhibited neural stem cell proliferation through the Wnt/β-catenin pathway via its GAP domain, which extended the functions of Vilse in the CNS (Huang et al. 2016). As a conserved protein with WW, MyTH4 and RhoGAP domains in different species, Vilse is involved in Robo-mediated axon guidance, scaffold protein-mediated spine formation and Wnt pathway-mediated stem cell proliferation (Hu et al. 2005; Huang et al. 2016; Lim et al. 2014; Lundstrom et al. 2004). Nevertheless, additional studies are required to fully explore its role in neuronal development and the potential implications in CNS diseases. 2.1.10. Nadrin Nadrin (ARHGAP17, RICH-1) was first identified and named neuronassociated developmentally regulated protein because its expression is neuron-specific and developmentally regulated (Harada et al. 2000). A subsequent sequence analysis suggested that Nadrin contains a GAP domain, a serine/threonine domain, a proline-rich region and an N-terminal BAR domain involved in membrane deformation events (Furuta et al. 2002; Harada et al. 2000) (Fig. 2). Nadrin acts on RhoA, Rac1, and Cdc42 in vitro. In previous studies, Nadrin has been shown to play a role in the regulation of Ca2 +-dependent exocytosis to inhibit neurite outgrowth (Furuta et al. 2002; Harada et al. 2000). It has also been reported that Nadrin has variants; five murine Nadrin variants have been identified to date (Furuta et al. 2002). Nadrin isoforms selectively act on specific Rho GTPases. For example, both Nadrin2 and Nadrin5 regulate RhoA and Rac1, whereas only Nadrin5 can inactivate Cdc42 (Beck et al. 2013). The BAR domain was considered to regulate membrane curvature (Peter et al. 2004). However, emerging studies indicate that the BAR domain has other roles (Eberth et al. 2009). Recently, the BAR domain of Nadrin is reported to mediate its GAP activity and target the GAP domain to its membrane substrate, indicating a potential auto-inhibitory mechanism of Nadrin (Beck et al. 2013). Moreover, Nadrin can be phosphorylated by Src, which also affects the GAP activity (Beck et al. 2014). Interestingly, Src-mediated tyrosine phosphorylation affects the GAP activity in an isoform- and target-specific manner. For example, phosphorylated Nadrin5 by Src results in the inactivation of Cdc42, whereas phosphorylated Nadrin2 by Src leads to the activation of RhoA and Rac1 (Beck et al. 2014). The reasons for these differences remain elusive. Taken together, at least two mechanisms are involved in the mediation of Nadrin function, including the BAR-mediated auto-inhibitory mechanism and the Src-mediated phosphorylation mechanism, in an

isoform- and target-specific manner. In addition, we cannot exclude other potential regulating mechanism involved in mediating Nadrin GAP activity, for instance protein–protein interactions. 2.2. RhoGAP in CNS diseases The essential functions of RhoGAPs in the mediation of nervous system development suggest that abnormal regulation of RhoGAPs may be the basis of specific neurodegenerative diseases. The dysregulation of RhoGAPs has been reported in various neurodegenerative diseases, including intellectual disability (ID), autism, Alzheimer's disease (AD), schizophrenia and (Table 1). 2.2.1. Intellectual disability In general, ID is one of the neurodevelopmental disorders featured by impaired cognitive abilities (Chelly et al. 2006). The causes of ID are multifactorial, ranging from environmental factors to gene mutations (Chelly et al. 2006). Numerous ID-associated genes have been identified in recent years (Gilissen et al. 2014; Grozeva et al. 2015; Kochinke et al. 2016; Najmabadi et al. 2011; Piton et al. 2013), many of which are Rho GTPases related downstream effectors or mediators, including RhoGAPs (Chelly et al. 2006; Inlow and Restifo 2004). As previously discussed, OPHN1 was firstly characterized in a mild ID patient with a balanced translocation t(X; 12) (Bienvenu et al. 1997; Billuart et al. 1998). Its involvement in X-linked intellectual disability (XLID) was simultaneously reported when an XLID family was identified with a frameshift of the GAP domain caused by a 1 bp deletion in the coding sequence of OPHN1 gene (Billuart et al. 1998). The impaired Oligophrenin-1 activity leads to cognitive damage in humans (Billuart et al. 1998). Subsequent clinical reports and animal model studies indicated OPHN1 mutations with ID also associated with other clinical symptoms, such as cerebellar hypoplasia and lateral ventricle enlargement and/or epilepsy (Khelfaoui et al. 2007; Philip et al. 2003; Pirozzi et al. 2011; Zanni et al. 2005). To date, all OPHN1 mutations, including three nonsense mutations, one splice mutation, three deletions and one insertion mutation, have been reported or predicted to be the loss of function (Al-Owain et al. 2011; Bergmann et al. 2003; Pirozzi et al. 2011; Zanni et al. 2005), and mice with oligophrenin-1 inactivation have been demonstrated to exhibit similar phenotypes with human patients in aspects of behavior, social connection, and cognitive function (Khelfaoui et al. 2007). The cognitive impairment caused by OPHN1 mutation is associated with both pre- and postsynaptic changes, including spine immaturity and altered short-term synaptic plasticity (Khelfaoui et al. 2007). Another RhoGAP linked to ID is the MEGAP gene, also referred to as srGAP3, which resides on chromosome 3p25. MEGAP gene was identified in a patient with severe ID and 3p-syndrome (Endris et al. 2002). Subsequent studies have also indicated that 3p-syndrome patients with MEGAP deletion exhibit several clinical manifestations, such as hypotonia and microcephaly, in addition to severe ID (Mowrey et al. 1993). Carlson et al. reported that a srGAP3 conditional KO mouse exhibits cognitive dysfunctions in learning and memory, including impaired abilities in the novel object recognition, the delayed probe test, the water maze test, and the passive avoidance test (Carlson et al. 2011). These cognitive impairments are a result of abnormal dendritic spine development and formation. Together, srGAP3-mediated dendritic spine formation is required for normal intellectual abilities. Functional disruption of the srGAP3 protein is associated with severe ID in 3p-syndrome (Bacon et al. 2009; Endris et al. 2002). 2.2.2. Autism spectrum disorder Autism spectrum disorder (ASD), a highly heritable brain disorder, is characterized by difficulty with social interaction, repetitive behaviors as well as disability in verbal and non-verbal communication.

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Recently, Akshoomoff et al. predicted p250GAP as an important candidate gene in ASD due to its expression pattern and its pivotal role in regulating neuronal morphology (Akshoomoff et al. 2015). Nakamura et al. determined that p250GAP-deficient mice exhibit ASD phenotypes, including poor or inappropriate social interactions, as well as ASD-related comorbidities, including epilepsy and impaired motor coordination (Nakamura et al. 2016) (Table 1). Furthermore, treatment with a GABAAR agonist improved several autistic-like phenotypes (Nakamura et al. 2016), which indicates that malfunction of p250GAPmediated GABAAR trafficking is a potential cause of the behavioral deficits. Similar to p250GAP, NOMA-GAP KO mice also exhibited autismlike social behavior, which may be caused by the marked loss of surface AMPARs and defective synaptic transmission (Schuster et al. 2015). Moreover, there are many other genes associated with ASD, such as SYNGAP1 (Synaptic Ras GTPase-activating protein 1). In recent years, different mutations of the SYNGAP1 gene, including a de novo splicing mutation, missense mutation or gene deletion, have been identified in ASD patients (Berryer et al. 2013; Clement et al. 2012; Cook 2011; Hamdan et al. 2011; Pinto et al. 2010), which indicates it is a risk gene for ASD. Its pivotal role in the regulation of fundamental molecular changes in dendritic spine synaptic morphology and function, including NMDA receptor-mediated synaptic plasticity and AMPAR membrane insertion, underlies its implication in ASD (Jeyabalan and Clement 2016; Kim et al. 2005; Krapivinsky et al. 2004; Rumbaugh et al. 2006). 2.2.3. Alzheimer's disease Alzheimer’s disease (AD) is a cognitive impairment related neurodegeneration disease (Bloom 2014). Impaired synapses have been considered as one of the major cause of cognitive changes in AD (Bloom 2014). Lots of evidence have demonstrated that dysregulated Rho GTPase activity are associated with Aβ production and synapse plasticity in AD (Stankiewicz and Linseman 2014). Previous studies have reported that the activity of Rho GTPases (including Rac1 and RhoA) is altered in the hippocampus of AD patients or mice (Huesa et al. 2010; Stankiewicz and Linseman 2014). Abnormal activation of Rho GTPase may lead to neurite retracting and increased toxic Aβ (Boo et al. 2008; Stankiewicz and Linseman 2014). As a mediator of Rho GTPase activity, the altered expressions of RhoGAPs, such as α1-chimaerin and TAGAP (T-cell activation Rho GTPase activating protein), have been identified in the brains of AD patients or mice (Chang and Hsiao 2005; Kato et al. 2015). These findings suggested a potential role for RhoGAPs contributing to the progression of AD. To date, limited information is available regarding the function of RhoGAPs in AD, which remains to be more clearly defined in further studies. 2.2.4. Schizophrenia Schizophrenia is a mental disorder with abnormal emotion, cognition and social behaviors. With a high rate of heritability, schizophrenia is related to dysfunctional neuronal development caused by specific genes (Tsuang 2000). RhoGAPs, acting as key regulators of developmental processes, may represent strong candidate genes for association with schizophrenia (Table 1). srGAP3 KO mice have been shown to exhibit schizophrenia-related phenotypes, such as impaired spontaneous alternation and thicker white matter tracts (Waltereit et al. 2012). In addition to srGAP3, based on a genetic association analysis, Ohi et al. reported that a p250GAP genetic variant was related to the risk for schizophrenia (Ohi et al. 2012) (Table 1). Furthermore, in healthy person, p250GAP gene was also relevant to schizotypal personality traits (Ohi et al. 2012). In addition to p250GAP, a missense polymorphism of the β-chimaerin gene has been demonstrated to be potentially associated with schizophrenia in a population of Japanese men (Hashimoto et al. 2005). The relation between the p250GAP or β-chimaerin and schizophrenia may be a result of the effect of dysfunction of p250GAP or β-chimaerin on axon and spine formation.

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Similar to p250GAP, TCGAP KO mice exhibited neuropsychiatric disorder-related behaviors, as well as impaired spine morphogenesis (Nakazawa et al. 2016). Based on a genetic analysis of schizophrenic patients, Nakazawa et al. determined that TCGAP is genetically associated with schizophrenia (Nakazawa et al. 2016). Mechanistically, TCGAP acts with SORT1 to mediate TrkB trafficking, which may be responsible for synapse formation and schizophrenia (Nakazawa et al. 2016).

3. Conclusions The understanding of RhoGAPs has evolved notably from when they were considered to be only Rho GTPase signal terminators in neuronal development and CNS diseases. Clearly, in addition to the inactivation of Rho GTPases, RhoGAPs interact with other proteins to mediate multiple processes of neuronal development through diverse signaling pathways. Moreover, accumulating studies have indicated that RhoGAPs play pivotal roles in several CNS diseases, which provide novel insights into the treatment of these diseases. The substantial number and structural complexity of RhoGAPs indicate we remain far from a complete understanding of how they operate in neuronal development and CNS diseases spatially and temporally. Thus, detection of variants within GAP genes in patients might facilitate the diagnosis and treatment of CNS diseases in the future.

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