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Genes to Cells (2006) 11, 607-614. doi:10.1111/j.1365-2443.2006.00966.x
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Role of the Rho GTPase-activating protein RICS in neurite outgrowth

Yukiko Nasu-Nishimura1, Tomoatsu Hayashi1, Tomohiro Ohishi1, Toshio Okabe2, Susumu Ohwada2, Yoshimi Hasegawa3, Takao Senda3, Chikashi Toyoshima4, Tsutomu Nakamura1 and Tetsu Akiyama1,*

1 Laboratory of Molecular and Genetic Information, Institute for Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
2 Second Department of Surgery, Gunma University School of Medicine, 3-39-22 Showa-machi, Maebashi, Gunma 371, Japan
3 Department of Anatomy I, Fujita Health University School of Medicine, 1-98 Dengakugakubo, kutsukake-cho, Toyoake, Aichi 470-1192, Japan
4 Laboratory of Supramolecular Structure, Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
The Rho family of small GTPases, including RhoA, Rac1 and Cdc42, are critical regulators of the actin cytoskeleton. In neuronal systems, Rho GTPase-activating proteins (RhoGAPs) and their substrates, Rho GTPases, have been implicated in regulating multiple processes in the morphological development of neurons, including axonal growth and guidance, dendritic elaboration and formation of synapses. RICS is mainly expressed in the brain and functions as a RhoGAP protein for Cdc42 and Rac1 in vitro. To examine the biological function of RICS, we disrupted the RICS gene in mice. RICS knockout mice developed normally and were fertile. However, when cultured in vitro, Cdc42 activity in RICS–/– neurons was higher than that in wild-type neurons. Consistent with this finding, hippocampal and cerebellar granule neurons derived from RICS–/– mice bore longer neurites than those from wild-type mice. These findings suggest that RICS plays an important role in neurite extension by regulating Cdc42 in vivo.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
In the developing nervous system, formation of appropriate connections among neurons is a prerequisite for the establishment and maturation of neuronal circuits. The development of functional nerve networks involves several discrete morphological steps. Newborn neurons must migrate to their characteristic locations, extend axons and dendrites into their proper target regions and then form synapses with appropriate partners. These seemingly different processes all depend on the regulation of the cytoskeleton in response to extra- or intra-cellular cues that orchestrate the morphological development of neurons.

The Rho family of small GTPases such as Rho GTPases, RhoA, Rac1 and Cdc42 are critical regulators of the actin cytoskeleton in eukaryotic cells from yeast to humans, and have been implicated in morphological changes in various cellular functions (Hall 1998; Symons & Settleman 2000). The Rho GTPases function as binary molecular switches shuttling between a GDP-bound inactive state and GTP-bound active state in response to a variety of extracellular stimuli. Three classes of regulatory proteins are involved in balancing between the active and inactive states of the Rho GTPases: the guanine nucleotide exchange factors (GEFs) of the Dbl family, which promote the release of bound GDP and facilitate GTP binding; the Rho GTPase-activating proteins (GAPs), which increase the intrinsic GTPase activity of the Rho GTPases, thus accelerating the return of the proteins to the inactive state; and the guanine nucleotide dissociation inhibitors (GDIs), which sequester the GDP-bound form of Rho GTPases and also retain the Rho GTPases in the cytoplasm (Sasaki & Takai 1998).

In neuronal systems, RhoGAPs and their substrate Rho GTPases have been implicated in regulating multiple processes in the morphological development of neurons, including axonal growth and guidance, dendritic elaboration and formation of synapses (Threadgill et al. 1997; Albertinazzi et al. 1998; Lehmann et al. 1999; Ruchhoeft et al. 1999; Luo 2000). Recent studies of p190RhoGAP-deficient mice showed that RhoGAP plays an important role in axon growth, guidance and fasciculation and neural development (Brouns et al. 2000, 2001). The discovery of the RhoGAP oligopherenin-1, which is associated with X-linked mental retardation, further highlights the importance of RhoGAP in the nervous system (Billuart et al. 1998).

In our previous study, RICS was identified as a novel RhoGAP interacting with ß-catenin via its armadillo repeats 10–12, and was demonstrated to possess GAP activity for Cdc42 and Rac1 (Okabe et al. 2003). RICS was also identified in other groups as Grit/p200RhoGAP/p250RhoGAP/GCGAP (Nakamura et al. 2002; Moon et al. 2003; Nakazawa et al. 2003; Zhao et al. 2003). The RICS-ß-catenin complex was found to be associated with N-cadherin, N-methyl-D-aspartate (NMDA) receptors and PSD-95, and localized to the postsynaptic density (PSD), suggesting that RICS is involved in synaptic adhesion- and NMDA-mediated organization of cytoskeletal networks and signal transduction. In this study, we generated RICS knockout (RICS–/–) mice by a targeted gene disruption strategy, and examined the biological functions of RICS in neurite outgrowth.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
We generated knockout mice, in which the mouse RICS gene was replaced with a bacterial ß-galactosidase (LacZ) reporter gene, using a standard gene targeting strategy (Fig. 1A). Genotyping was verified by competitive PCR and Southern blot analysis (Fig. 1B,C). RICS–/– mice were found to be fertile and to produce litters of normal size (data not shown). Northern blot analysis revealed a doublet of 9.8- and 10-kbp mRNA. RICS mRNA was found in almost all parts of the brain except the spinal cord, medulla and corpus callosum (Fig. 2A). Consistent with these results, ß-Gal staining of RICS–/– adult brain revealed that RICS is expressed in the cerebral cortex, thalamus, amygdale, caudate putamen and hippocampus (Fig. 2B). Immunoblotting analysis using anti-RICS antibody revealed that RICS protein is present in the adult brain of wild-type mice but not RICS–/– mice (Fig. 1D). Truncated RICS proteins were not detected in RICS–/– mice. Histological analysis of the brain, kidney, heart, liver, and colon of RICS–/– mice showed no differences between RICS–/– mice and wild-type mice (data not shown).


Figure 1
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  		Figure 1  Targeted disruption of the mouse RICS gene. (A) Structure of the wild-type allele, targeting construct, and recombinant locus are shown. The probe P1 used for detection of homologous recombinants is represented by the blank bar, and the probe P2 used for detection of a single insertion of the neomycine gene is represented by the black bar. The primer sets used for detection of recombinants are also represented by blank and black arrowheads, respectively. (B) PCR genotyping of RICS–/– mice. Competitive PCR was done on genomic tail DNA with three primers: one common 3' primer and 5' RICS- and LacZ-specific primers. A 1.1 kbp band was generated from the WT allele, and a 670-bp band was generated from the KO allele. (C) Southern blot analysis of RICS+/+, RICS+/–, RICS–/– mice. Southern blot was carried out on EcoRI-digested genomic DNA. Blots were probed with probe P1 (top) or probe P2 (bottom). For P1, the WT allele results in a 2.1 kbp band and the KO allele results in a 4.6 kbp band. P2 is a 187-bp sequence within the Neo gene. A 1.5 kbp band was detected with the homologous recombinant allele. (D) Western blot analysis of brain extracts from adult wild-type and RICS–/– mice. RICS was not detected in the brains of knockout mice.

 

Figure 2
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  		Figure 2  Expression of RICS in mouse adult tissues. (A) MTN blots of mouse adult tissues (Clontech) were probed with a cDNA encoding RICS. The positions of the RICS mRNAs are indicated by arrowheads. (B) ß-Gal staining on frozen sections of RICS–/– adult brain. RICS was expressed in the cerebral cortex, thalamus, amygdale, caudate putamen and hippocampus. Sections of wild-type adult brain were used as negative controls. Cx, cortex; Am, amygdala; Th, thalamus; Cp, caudate putamen; Hp, hippocampus.

 
We previously showed that RICS has RhoGAP activity toward Cdc42 and Rac1 in vitro (Okabe et al. 2003). To examine whether RICS shows the same specificity in vivo, we measured the amounts of activated Cdc42 and Rac1 in cerebellar granule neurons that had been cultured for 24 h. Activated Cdc42 was more abundant in RICS–/– neurons than in wild-type neurons (Fig. 3A), whereas no difference was detected in the abundance of activated Rac1 between RICS–/– and wild-type neurons (Fig. 3B). These results suggest that RICS functions as a GAP for Cdc42 in vivo. In addition, we examined whether RICS interacts with Cdc42, since it has been reported that some GAP proteins interact with Rho GTPases (Brill et al. 1996; Chiang et al. 2003). We generated the following mutants of Rho GTPases fused to GST and examined their ability to interact with RICS: T17N, inactive mutants of Cdc42 and Rac1, in which Thr-17 was replaced with Asn; Q61L, constitutively active mutants of Cdc42 and Rac1, in which Gln-61 was replaced with Leu; T19N, an inactive mutant of RhoA, in which Thr-19 was replaced with Asn; and G14V, a constitutively active mutant of RhoA, in which Gly-14 was replaced with Val. When mouse brain lysates were incubated with GST-Rho GTPases immobilized to Sepharose beads, RICS was found to co-precipitate with GST-Cdc42-Q61L (Fig. 3C). By contrast, RICS did not co-precipitate with GST-Rac1-Q61L, -T17N, GST-RhoA-G14V, -T19N or GST-Cdc42-T17N.


Figure 3
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  		Figure 3  Hyperactivity of Cdc42 in primary-cultured RICS–/– neurons. (A, B) The levels of active GTP-bound (A) Cdc42 and (B) Rac1 were measured in primary-cultured cerebellar granule neurons from wild-type or RICS–/– mice. GTPase activities of Cdc42 and Rac1 were assessed using a GST fusion protein derived from PAK3 (PBD), which selectively binds GTP-bound Rac1 and Cdc42. GST-PBD precipitates were immunoblotted with antibodies to Cdc42 (A, upper) or Rac1 (B, upper). (A, B, bottom) Western blots were quantified with the NIH image software. The bars represent the mean ± SEM of triplicate assays. Symbols indicate the results of t-test analysis; *P < 0.02. (C) Association of RICS with Cdc42. Mouse brain lysates were incubated with GST-Rho GTPases immobilized to Sepharose beads, and precipitated proteins were subjected to immunoblotting analysis with anti-RICS antibody (upper panel). The amount of loaded GST fusion protein was visualized by Coomassie Brilliant Blue staining (bottom panel).

 
It has been reported that Cdc42 is localized at growth cones and modulates their morphological changes during neurite elongation (Eaton et al. 1995). We therefore performed immunofluorescent staining of mouse hippocampal neuronal cultures to examine the intracellular localizaiton of RICS. When hippocampal neuronal cells were cultured for 1.5 days and double-labeled with anti-RICS antibody and Rhodamine-Phalloidin, the immunostaining pattern of RICS was found to overlap extensively with that of actin, concentrating at growth cones in addition to cell bodies (Fig. 4A). This finding is consistent with the fact that RICS contains two tyrosine-based sorting motifs (YXXLE), which have been implicated in axonal growth cone sorting (Kamiguchi & Lemmon 1998).


Figure 4
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  		Figure 4  RICS is involved in neurite outgrowth. (A) Hippocampal neurons cultured for 1.5 days were double-labeled with anti-RICS antibody and Rhodamine-Phalloidine. Growth cones are indicated with arrowheads. Bar, 20 µm. (B, C) Neurite bearing assay of (B) hippocampal and (C) cerebellar granule neurons from RICS–/– brain. Cells were cultured for 36 h and labeled with Rhodamine-Phalloidine and ToPro3. Bar, 100 µm. More than three separate experiments were performed for each neuron and at least 150 cells were counted for each experiment. Neurons having neurites whose lengths were longer than their cell body lengths were scored as neurite bearing. Results are expressed as the mean percentage of neurite-bearing cells with the standard error. Symbols indicate the results of t-test analysis; *P < 0.001 (cerebellar neurons), P < 0.05 (hippocampal neurons), compared with a control. (D) Effects of exogenous expression of RICS on the neurite outgrowth of RICS–/– cerebellar granule neurons. Cerebellar granule neurons were transfected with wild-type or mutant RICS as indicated and cultured for 24 h. Cells were stained with Rhodamine-Phalloidine and FITC-anti-FLAG antibody. The length of the longest neurite in a transfected cell was measured. The panel shows the percentages of cells with indicated neurite length.

 
It is well known that Cdc42, Rac1 and RhoG activate neurite extension, while RhoA mediates neurite retraction (Kozma et al. 1997; Katoh et al. 2000; Yamaguchi et al. 2001). We examined the role of RICS in neurite extension using primary-cultured neurons from the littermates obtained by intercrossing RICS heterozygous mice. We found that hippocampal neurons from RICS–/– mice are more abundant in neurite-bearing cells than those from wild-type mice (Fig. 4B). Also, the RICS–/– neurons had much longer neurites than those of wild-type mice. To further confirm these results, we also examined neurite extension in primary cerebellar granule neurons. In accordance with the results from hippocampal neurons, cerebellar granule neurons from RICS–/– mice were found to bear longer neurites than those from wild-type mice (Fig. 4C). These data suggest that RICS regulates neurite outgrowth.

To further confirm these findings, we examined the effect of exogenous expression of RICS on the phenotype of RICS–/– cerebellar granule neurons. We found that exogenous expression of RICS rescued the phenotype of RICS–/– cerebellar granule neurons; RICS–/– neurons transfected with RICS had shorter neurites than those of untransfected neurons (Fig. 4D). In contrast, an inactive mutant RICS, RICS-R58I, in which Arg-58 was replaced with Ile (Okabe et al. 2003), did not affect the length of neurites. On the other hand, expression of another mutant form of RICS, RICS-{Delta}ß, which lacks the ß-catenin-binding domain, resulted in an increase in the length of neurites. Taken together, these results suggest that RICS regulates neurite outgrowth as a RhoGAP and that the ß-catenin-binding domain may be important for this activity.


   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
In an attempt to elucidate the function of RICS, we generated mice lacking RICS expression by gene targeting. Mice heterozygous and homozygous for the targeted allele developed normally and were grossly indistinguishable from their normal littermates. This finding was unexpected, especially because regulation of the activity of Rho GTPases is considered crucial for neuronal proliferation and migration (Luo 2000). For example, it has been reported that mice lacking p190 RhoGAP exhibit aberrant tissue morphogenesis (Brouns et al. 2000). However, when we examined the activation of Cdc42 and Rac1 in cultured primary cerebellar granule neurons, Cdc42 activity in RICS–/– neurons was found to be higher than that in wild-type neurons (Fig. 3A). Consistent with this finding, RICS interacted directly with Cdc42. These results suggest that RICS functions as a GAP for Cdc42 in at least hippocampal and cerebellar granule neurons. This is the first report describing abnormal activation of Rho GTPases in RhoGAP-null cells. Although we also examined Rac1 activation, the differences between wild-type and RICS–/– neurons were not detected (Fig. 3B).

RICS was found to be localized at the developing growth cones of cultured hippocampal neuronal cells. Consistent with this finding, RICS contains two tyrosine-based sorting motifs (YXXLE), which have been implicated in axonal growth cone sorting (Kamiguchi & Lemmon 1998). Since Cdc42 activation is believed to promote growth cone advance (Luo 2000; Dickson 2001), it is possible that RICS functions as a regulator of Rho GTPases during neurite outgrowth. Indeed, we found that cerebellar granule and hippocampal neurons from RICS–/– mice had longer neurites than wild-type neurons. Furthermore, exogenous expression of RICS, but not RICS-R58I, rescued the phenotype of RICS–/– cerebellar granule neurons. These findings suggest that RICS plays an important role in neurite extension. In this regard, it is interesting to note that RICS was also identified as a protein (Grit) that interacts with TrkA, a high-affinity receptor for NGF (Nakamura et al. 2002). It has been reported that over-expression of the TrkA-binding region of RICS/GRIT inhibits NGF-induced neurite elongation in PC12 cells. We also found that over-expression of dominant-negative mutants of RICS in PC12 cells enhances NGF-induced neurite extension (data not shown). Thus, endogenous RICS may be involved in the regulation of NGF-induced neurite extension.

Although RICS was identified as a ß-catenin-binding protein, the significance of this interaction has not been elucidated. However, it is interesting that RICS-{Delta}ß, which lacks the ß-catenin-binding domain, was unable to rescue the phenotype of RICS–/– cerebellar granule neurons. This finding raises the possibility that the interaction of RICS with ß-catenin is important for its ability to regulate neuritegenesis. However, one cannot exclude the possibility that deletion of this domain resulted in the loss of other important functions. Also, the reason why RICS-{Delta}ß stimulated neurite outgrowth of RICS–/– neurons remains to be elucidated.

RICS is highly expressed in the cortex and amygdala and is localized at spines. Furthermore, we have previously found that RICS is a component of the NMDA receptor complex (Okabe et al. 2003). All these results raise the possibility that RICS plays an important role in higher functions such as learning and emotional memory. Further studies on RICS–/– animals may reveal not only the molecular mechanism of neuritegenesis but also those of some higher functions.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Construction of the targeting vector and generation of RICS–/– mice

A genomic clone containing the first methionine of the RICS gene was isolated from a C57BL/6 mouse genomic library (Incyte Genomics, Inc). The targeting vector was constructed by replacing a 12 kb fragment containing the ATG start codon with a LacZ-Neo cassette. The targeting construct was electroporated into TT2 ES cells and the G418-resistant clones were tested for a targeted event by Southern blot analysis. Two probes were used for analyzing ES cell and mouse tail DNA as shown in Fig. 1A. P1 is a 419-bp fragment corresponding to a region 434-bp upstream of the homologous region. P2 is a 187-bp fragment within the neomycine resistance gene. Six independent RICS heterozygous (RICS+/–) ES clones were identified and expanded. The RICS+/– ES cells were aggregated with or injected into ICR morulas or blastocysts, respectively. Two independent germ line-transmitting chimeras were obtained and bred to C57BL/6 N to generate an inbred colony. We performed experiments with two independently derived knockout lines and obtained similar results.

Antibodies

Antibodies to RICS were prepared as previously described (Okabe et al. 2003). Antibodies to Cdc42 and Rac1 were purchased from SantaCruz Biotechnology and BD Transduction Laboratories, respectively. Anti-{alpha} tubelin antibody was obtained from Oncogene. Anti-FLAG M2 antibody was from Sigma.

Cell culture

Mouse hippocampal and cerebellar granule neurons were grown in Neurobasal medium (Invitrogen) supplemented with B-27 supplement (Invitrogen). In cerebellar granule neuron culture, 25 mM KCl was included.

PBD assay

A GST fusion protein of the p21-binding domain (amino acids 65–136) of mouse PAK 3 (PBD) was prepared. RICS–/– and wild-type cerebellar granule cells were seeded on 6 cm dish/mouse. After 24 h incubation, cells were lyzed with lysis buffer A (1% TritonX-100, 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10 mM MgCl2, 1 mM PMSF, 10 µg/mL aprotinin, 10 µg/mL leupeptin, 0.5% sodium deoxycholate, 0.1% SDS). GTP-bound forms of endogenous Cdc42 and Rac1 were affinity purified from the clarified lysates containing 200 µg (for Cdc42GAP) or 100 µg (for RacGAP) of protein with 20 µg GST-PBD. Bound Cdc42 and Rac1 were detected by immunoblotting with antibodies against Cdc42 or Rac1, respectively (Ren et al. 1999; del Pozo et al. 2000). In lanes labeled total Cdc42 or Rac1, 20 µg of lysates used for immunoprecipitation were loaded. Each experiment was independently repeated 3 times.

In vivo binding assay

Mouse brain was lyzed in lysis buffer 10 mM Tris-HCl (pH 8.0), 140 mM NaCl, 1 mM EDTA, 1% TritonX-100, 10 µg/mL aprotinin, 10 µg/mL leupeptin, 1 mM NaF and 1 mM Na3VO4 and 200 µg of total protein was incubated with GST-fusion proteins immobilized to GSH-Sepharose beads. The beads were then washed 4 times with lysis buffer, and once with TEN buffer (10 mM Tris-HCl (pH 8.0), 140 mM NaCl, 1 mM EDTA). Samples were then boiled in 15 µL of 1 x SDS sample buffer and analyzed by immunoblotting. Each experiment was independently repeated 3 times.

Immunostaining

Mouse hippocampal neurons were cultured for 36 h and then fixed with 2% paraformaldehyde in PBS for 10 min at RT, and stained with anti-RICS antibody. The staining patterns obtained with anti-RICS antibody were visualized with FITC-labeled anti-rabbit antibody (ICN Biomedicals). The cells were also costained with Rhodamine-Phalloidine (Molecular Probe). The cells were then photographed with a Carl Zeiss LSM510 laser scanning microscope.

ß-Gal staining

Ten-week-old male mice were anesthetized and perfused with PBS (pH 7.4). Brains were dissected, fixed in 4% paraformaldehyde/PBS for 1 h, and then re-fixed in 4% paraformaldehyde/PBS containing 0.2% glutaraldehyde, 0.02% NP-40 for 1 h. After fixation, brains were embedded in Tissue-TEK® O.C.T. compound. Frozen brain sections were stained overnight in the staining solution (44 mM HEPES (pH 7.0), 3 mM K3[Fe(CN)6], 15 mM NaCl, 1.3 mM MgCl2, 0.5 mg/mL X-gal/DMFO, in PBS).

Neurite bearing assay of primary neurons

Hippocampal and cerebellar granule neurons were prepared from postnatal day 0–1 or day 7–9 mice, respectively. Cells were plated on glass coverslips coated with poly-L-lysine (50 µg/mL, Sigma) in 24-well plates at 4 wells/mouse. After 36 h incubation, cells were fixed and labeled with Rhodamine-Phalloidine and ToPro3. The cells were photographed with a Carl Zeiss LSM510 laser scanning microscope and the length of neurites was measured using the LSM510 software. Each experiment was independently repeated 3 times.

Neurite bearing assay of primary neurons transfected with RICS mutants

Cerebellar granule neurons were prepared from postnatal day 8–9 mice. Cells were plated on glass coverslips coated with poly L-lysine (50 µg/mL, Sigma) in 24-well plates at 4 wells/mouse. Cells were transfected with the FLAG-tagged constructs indicated in Fig. 4D using mouse neuron nucleofector kit (Amaxa) and incubated for 24 h. Cells were fixed in 4% paraformaldehyde/PBS for 10 min at RT, then stained with anti-FLAG antibody. The cells were photographed with a Carl Zeiss LSM510 laser scanning microscope and the length of neurites was measured using the LSM510 software. Each experiment was independently repeated 3 times.


   Acknowledgements
 
Supported by Grants-in-Aid for Scientific Research on Priority Areas and the Organization for Pharmaceutical Safety and Research.


   Footnotes
 
Communicated by: Kohei Miyazono

* Correspondence: E-mail: akiyama{at}iam.u-tokyo.ac.jp


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 References
 
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T. Nakamura, T. Hayashi, Y. Nasu-Nishimura, F. Sakaue, Y. Morishita, T. Okabe, S. Ohwada, K. Matsuura, and T. Akiyama
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T. Hayashi, T. Okabe, Y. Nasu-Nishimura, F. Sakaue, S. Ohwada, K. Matsuura, T. Akiyama, and T. Nakamura
PX-RICS, a novel splicing variant of RICS, is a main isoform expressed during neural development
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M. Rosario, R. Franke, C. Bednarski, and W. Birchmeier
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X. Shang, S. Y. Moon, and Y. Zheng
p200 RhoGAP Promotes Cell Proliferation by Mediating Cross-talk between Ras and Rho Signaling Pathways
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