Disrupted-in- 1–mediated guidance involves TRIO-RAC-PAK small GTPase pathway signaling

Shih-Yu Chena, Pei-Hsin Huangb,c,1, and Hwai-Jong Chenga,1

aCenter for Neuroscience, University of California, Davis, CA 95618; bGraduate Institute of Pathology, College of Medicine, National Taiwan University, Taipei 100, Taiwan; and cDepartment of Pathology, National Taiwan University Hospital, Taipei 100, Taiwan

Edited by Mu-ming Poo, University of California, Berkeley, California, and approved March 3, 2011 (received for review December 7, 2010) Defects in neuronal connectivity of the brain are well documented that DISC1 regulates axon guidance through activation of RAC- among schizophrenia patients. Although the schizophrenia sus- PAK signaling pathways. Importantly, we confirmed this finding in ceptibility Disrupted-in-Schizophrenia 1 (DISC1) has been im- mammalian cultured cells and demonstrated that DISC1 can in- plicated in various neurodevelopmental processes, its role in teract with mammalian TRIO to activate the RAC-PAK signal regulating axonal connections remains elusive. Here, a heterolo- pathway. gous DISC1 transgenic system in the relatively simple and well- characterized motor neurons has been es- Results tablished to investigate whether DISC1 regulates axon guidance Heterologous Mouse DISC1 (mDISC1) Induces Axon Guidance Defects during development. Transgenic DISC1 in C. elegans motor neurons in C. elegans. A GFP and mDISC1 fusion (GFP:: is enriched in the migrating growth cones and causes guidance mDISC1) was expressed in C. elegans D-type dorsal (DD) and defects of their growing . The abnormal axonal phenotypes ventral (VD) motor neurons under a GABAergic-specific unc-25 induced by DISC1 are similar to those by gain-of-function rac promoter. These neurons are located along the ventral midline . In vivo genetic interaction studies revealed that the UNC- of the worm body and project their commissural axons dorsally 73/TRIO-RAC-PAK signaling pathway is activated by ectopic DISC1 (Fig. 1 A and B). In these mDISC1 transgenic worms, some of in C. elegans motor axons. Using in vitro GST pull-down and coim- the commissures failed to reach their dorsal destinations. We munoprecipitation assays, we found that DISC1 binds specifically quantified the severity of axon guidance defect by calculating the to the amino half of spectrin repeats of TRIO, thereby preventing percentage of commissures that failed to reach the dorsal cord at NEUROSCIENCE TRIO’s amino half of spectrin repeats from interacting with its first young adult stages. In mDISC1 transgenic animals, approxi- guanine nucleotide exchange factor (GEF) domain, GEF1, and fa- mately 40% of commissures showed guidance defects, compared cilitating the recruitment of RAC1 to TRIO. In cultured mammalian with less than 5% in control animals expressing GFP alone. (Fig. cells, RAC1 is activated by increased TRIO’s GEF activity when 1 C–E). DISC1 is present. These results together indicate that the TRIO- RAC-PAK signaling pathway can be exploited and modulated by Transgenic mDISC1 in C. elegans Motor Neurons Functions in a Similar DISC1 to regulate axonal connectivity in the developing brain. Fashion as in Vertebrate Neurons. To validate the use of heterol- ogous mDISC1 in C. elegans,wefirst characterized its subcellular genetic model | rolipram localization in motor neurons. In vertebrate neurons, DISC1 is known to be present in the growth cone (11). Similarly, in chizophrenia is a neurodevelopmental disorder with genetic mDISC1 transgenic worms, GFP::mDISC1 was observed in mi- Spredispositions (1, 2). Although the etiology and neuropa- grating VD growth cones at larval stage 2 (L2) (75%, n = 20) thology of schizophrenia are still elusive, functional and neuroan- (Fig. S1A) and in the tips of mature axons at L4 or young adult fi atomical studies from patients have documented various abnor- stage after the motor axons had already reached their nal tar- malities in the diseased brain. In particular, defects in neuronal gets (Fig. S1B). The local accumulation of mDISC1 inside the connectivity during development have been proposed as an im- growth cone was also observed in dissociated neurons from portant precipitating factor for schizophrenia, which is thought to mDISC1 transgenic animals (Fig. S1C), suggesting that extrinsic be unmasked by other developmental events or environmental factors are not involved in the mDISC1 localization. By testing stressors later in life (3). It is therefore likely that some of the serial deletion constructs of mDISC1, we found that such accu- major schizophrenia susceptibility genes are important for regu- mulation at axon tips was regulated by its C terminus (Fig. S1D), lating axonal connections during development. as has been suggested in vertebrate cells (4). In recent years, the effort to search for genes susceptible to DISC1 is known to interact with more than a dozen vertebrate schizophrenia has led to the identification of the Disrupted-in- genes (4). Among these genes, homologs of two well-known Schizophrenia 1 (DISC1) gene, whose mutation is highly asso- mDISC1-interacting genes, pde-4 and lis-1, are present in the ciated with schizophrenia and other major human mental dis- C. elegans motor neurons (12) (Fig. S2). To validate studying eases (4). DISC1 has roles in neuron proliferation, neuron mDISC1-mediated signaling pathways in the C. elegans heterol- migration, axon outgrowth, and formation/maturation ogous system, we tested whether the phenotype caused by ectopic (5–8). In our previous studies, we identified an unexpected role mDISC1 was suppressed by knockdown of these two C. elegans of DISC1 in regulating the guidance of developing axons in the genes. We found the axon guidance defects in mDISC1 trans- adult-born hippocampal dentate granule cells (5, 9). These effects expand the known functions of DISC1 but sheds little Author contributions: S.-Y.C., P.-H.H., and H.-J.C. designed research; S.-Y.C. performed light on how DISC1 effects axon guidance. research; S.-Y.C., P.-H.H., and H.-J.C. analyzed data; and S.-Y.C., P.-H.H., and H.-J.C. wrote To systemically study the role of DISC1 in axon guidance and the paper. to identify the signaling pathways that might be involved, we set The authors declare no conflict of interest. out to establish a heterologous genetic system in the nematode This article is a PNAS Direct Submission. Caenorhabditis elegans, in which no endogenous DISC1 homol- 1To whom correspondence may be addressed. E-mail: [email protected] or hjcheng@ ogous gene is identified (10). In this genetic model, DISC1 is ac- ucdavis.edu. cumulated in the growth cone, and transgenic animals exhibited This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. axon guidance defects. Further genetic interaction studies showed 1073/pnas.1018128108/-/DCSupplemental.

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Fig. 1. Expression of mDISC1 in C. elegans motor neurons causes axon guidance defects. (A–D) Confocal images of the commissural axons from DD and VD motor neurons at L4. In wild-type animals (A and B), all of the commissures reach the dorsal nerve cord. In mDISC1 transgenic animals (C and D), the commissures exhibit guidance defects (arrowheads). Boxed areas in A and C are magnified in B and D, respectively. (Scale bars: A and C,20μm; B and D,10 μm.) (E) The adult mDISC1 animals have an average of 40% axon guidance defect. The L1 transgenic animals exhibit similar percentage of defects, indicating that the phenotype is caused by a defect in the guidance of developing axons rather than to abnormal regrowth from degenerated axons. (F) The axon guidance defects caused by expressing mDISC1 are suppressed by RNAi knockdown of C. elegans homologs of known mammalian DISC1-interacting molecules (n =25–60). col-86, negative control gene. (G) Incubation of the mDISC1 animals with a PDE4-specific inhibitor, rolipram, suppresses the guidance defects (n = 60). Bars represent the SE. *P < 0.05, **P < 0.001 (Student’s t test). In all pictures, anterior is to the left and dorsal is up.

genic animals were suppressed by RNAi knockdown of pde-4 the axon guidance defects, but expression of wild-type rac genes (Fig. 1F). RNAi of lis-1 caused embryonic lethality as previously enhanced the phenotype (Fig. 3B). reported (12). However, some escapers that had milder RNAi As summarized in Fig. 3A, the two rac genes, ced-10 and mig-2, effects showed significant suppression of the guidance defect function redundantly in motor axons. Although single mutants of (Fig. 1F). Together, these studies demonstrate mDISC1 rac genes cause no axon guidance defects, double rac mutants in the C. elegans motor neurons behave similarly to those in exhibit severe axon guidance defects. On the other hand, the vertebrate neurons. roles of the two pak genes, pak-1 and max-2, are slightly differ- ent: MAX-2 has RAC-dependent and RAC-independent roles, mDISC1 Causes Axon Guidance Defects and Ectopic Branching in C. but PAK-1 functions completely in the RAC signaling pathway, elegans Motor Neurons by Activating the UNC-73-RAC-PAK Signaling which is redundant to MAX-2 (Fig. 3A). Consistent with the rac Pathway. UNC-73-RAC-PAK signaling has previously been (dn) experiments, single mutants of mig-2 or ced-10 partially but characterized in the guidance of C. elegans motor commissural significantly suppressed the axon guidance defects (Fig. 3C). It is axons (13). Interestingly, the C. elegans motor axons expressing important to note that mutants with severe axon guidance the mDISC1 gene exhibited a unique branching defect very sim- defects would preclude us from performing genetic suppression ilar to those in the transgenic worms expressing gain-of-function experiments. We therefore were unable to test double mutants (gf) rac genes (14) (Fig. 2). In wild-type worms, each DD or VD axon has two branch points: the first point is located at the start such as ced-10;mig-2 that, by themselves, showed severe axon of the commissural branch that extends from the ventral axon guidance defects (13, 14). However, double mutants of pak-1;ced- process, and The second branch point is at the end of the 10, which did not exhibit axon guidance defects by themselves commissure that branches into anteriorly and posteriorly ex- (13), suppressed the defects of mDISC1 transgenic animals more fi tended processes (Fig. 2 A and E). The average branch points in signi cantly than either pak-1 or ced-10 single mutants (Fig. 3E). many axon guidance mutants such as max-1 remain unchanged Our previous genetic studies have shown that, in RAC-PAK (Fig. 2 B and E). However, in both mDISC1 and rac(gf) trans- signaling, PAK-1 is the preferential effector for MIG-2, whereas genic animals, affected axons not only exhibited a similar ab- MAX-2 functions preferentially downstream of CED-10 (Fig. normal branching pattern but also had significantly more branch 3A) (13). Consistent with this finding, pak-1;ced-10 double mu- points (Fig. 2 C–E). In addition to the branching phenotype, tant was a stronger suppressor than pak-1;mig-2 double mutant both mDISC1 and rac(gf) transgenic animals also displayed axon in transgenic mDISC1 background (Fig. 3E). guidance defects with misguided commissures failing to reach We reasoned that, if activation of RAC signaling by ectopic the dorsal cord. Involvement of RAC signaling by mDISC1 was mDISC1 is a mechanism by which axon guidance is affected, further supported by the observation that expression of dominant- mutants of genes that transduce signals by RAC-independent negative (dn) rac genes in mDISC1 transgenic animals suppressed pathways would be predicted to enhance the axon guidance

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1018128108 Chen et al. Downloaded by guest on September 23, 2021 A B mDISC1 Activates RAC Signaling by Direct Binding with TRIO/UNC-73 in Mammalian Cells. To validate and further investigate the interactions between mDISC1 and UNC-73-RAC-PAK signaling in mammalian cells, we used mammalian homologous proteins wildtype max-1 to study the molecular interactions. We found that DISC1 coimmunoprecipitated with TRIO, an UNC-73 mammalian ho- molog (Fig. 4D), as independently reported in a previous yeast two-hybrid study (17). TRIO is a large Dbl family protein, whose structure consists of two GEF domains: the RAC1-specific first GEF (GEF1) and the RHO-specific second GEF (GEF2) (Fig. C D 4B). Domain mapping indicated that N-terminal globular do- main of mDISC1 bound to the amino half of spectrin repeats (SPn) and the GEF2 of human TRIO (hTRIO) (Fig. S3). Be- cause the GEF2 domain is dispensable for its function in guiding mDISC1 ced-10(gf) commissural motor axons in C. elegans (15), we focused on the interaction of DISC1 with TRIO-SPn and asked how such an interaction affected RAC-PAK signaling. First, in a series of in vitro pull-down assays, we found that the RAC1-specific TRIO-GEF1 specifically bound to TRIO-SPn but not to the carboxyl half of spectrin repeats (SPc) or TRIO-GEF2 E ** stn ** (Fig. 4E). Second, the binding between TRIO-GEF1 and its ef- ioP hcnarB fo rebmuN fo hcnarB ioP 5 ** fector RAC1 was significantly compromised in the presence of ** 4 exogenous TRIO-SPn (Fig. 4F). These results indicate that the 3 recruitment of RAC1 to TRIO’s GEF1 domain is inhibited by 2 the binding of its SPn. Next, we investigated the effect of 1 mDISC1 on the interaction between RAC1 and hTRIO-B, 0 ) e 1CSID )fg(01 a mammalian equivalent to the C. elegans unc-73b isoform that is py 9 3uj t sufficient to rescue the axon guidance defect caused by unc-73 NEUROSCIENCE li d (1 w -xa -d m mutants (15). In vitro pull-down experiments showed that ec m mDISC1 itself did not bind RAC1 (Fig. S4), but hTRIO-B recruited more RAC1 in the presence of mDISC1 (Fig. 4G). Fig. 2. mDISC1 transgenic animals exhibit a similar ectopic branching phe- fi notype seen in the transgenic animals expressing rac(gf) genes. (A) A con- To con rm the in vitro biochemical results, a RAC1 activity focal image of the representative wild-type DD motor neuron at L1 stage. assay was performed in COS cells. As shown in Fig. 4H, active The DD motor neuron first extends its short axon process along the ventral GTP-bound RAC1 was dramatically increased in the presence of cord, from which the commissure branches out and migrates circum- both DISC1 and TRIO. In addition, when unc-73b was expressed in ferentially to the dorsal side of the body. When the commissure hits the wild-type C. elegans motor neurons, only very mild axon guidance dorsal cord, it branches again and extends both anteriorly and posteriorly defect was observed. However, expression of unc-73b in the motor along the dorsal cord. The two branch points are indicated by arrows. (B)In neurons of mDISC1 transgenic animals strongly enhanced the axon max-1 mutant animals, although the commissural axons often fail to reach guidance defects (Fig. 4I). Together, we conclude that DISC1 can the dorsal cord, these axons exhibit two branch points as seen in the wild interact with TRIO’s SPn, which results in activation of UNC-73/ type (arrows). (C and D) The misguided DD axons in either mDISC1 (C)orrac (gf)(D) transgenic worms exhibit significantly more branch points (arrows). TRIO-RAC-PAK signaling to induce axon guidance defects. All images are taken from a single DD neuron at L1 stage. (Scale bar: 5 μm.) (E) Quantification of the branch points for each genetic background. The Discussion branch point is defined as a point with an additional neurite extending more A heterologous DISC1 genetic model has been established in C. than 1 μm. Bars represent the SE (n = 21). **P < 0.001 (Student’s t test). In all elegans motor neurons. Using this system, we studied how DISC1 pictures, anterior is to the left and dorsal is up. is involved in the signaling pathways of axon guidance. We showed that DISC1 can activate the RAC-PAK signaling pathway via interacting with UNC-73/TRIO in C. elegans motor axons. defects in mDISC1 transgenic animals. Indeed, consistent with Importantly, this DISC1-mediated signaling pathway is phyloge- its additional RAC-independent role, max-2 single mutant en- netically conserved. In mammalian cultured cells, DISC1 directly hanced the defects (Fig. 3D). In addition, the axon guidance interacts with the SPn domain of TRIO. This interaction facili- defects of mDISC1 transgenic animals were enhanced by the tates the recruitment of RAC1 to the GEF1 domain of TRIO and mutation of a RAC-independent gene, max-1 (Fig. 3D). Collec- results in activation of RAC1 activity. tively, these data not only support mDISC1 activation of RAC- C. elegans is a relatively simple genetic system for investigating PAK signaling but also suggest mDISC1 acts as an upstream molecular signaling pathways. By establishing the mDISC1 regulator of the RAC-PAK signaling. transgenic animal, we were able to dissect the molecular inter- Given that UNC-73 is the main guanine nucleotide exchange actions in detail. We found that, in C. elegans motor neurons, factor (GEF) activating the RAC-PAK signaling in C. elegans DISC1 interacts with a GEF, UNC-73/TRIO, and activates the ’ motor axons (15, 16), we investigated whether DISC1 activates RAC signaling to regulate axon guidance. TRIO s role in axon guidance has been well studied, and its functions are phyloge- RAC-PAK signaling through unc-73. The first GEF domain, netically conserved (15, 18, 19). Thus, our results were readily GEF1, of UNC-73 is specific for activating RAC, which is both fi applicable to the mammalian system. Indeed, we demonstrated necessary and suf cientforthefunctionofUNC-73inaxonguid- similar interactions of DISC1 with the TRIO-RAC signaling in ance (15). The axon guidance defects of the mDISC1 transgenic mammalian cultured cells. TRIO is a Dbl family protein, which worms were suppressed by a hypomorphic unc-73 allele, rh40, has two distinct GEF domains (20). Our analysis suggests an which has a missense mutation in its GEF1 domain (Fig. 4C). inhibitory control of GEF1 activity by the SPn domain of TRIO. Thus, in heterologous C. elegans motor axons, mDISC1 is likely Consistent with these findings, the N terminus of TRIO has been to activate RAC-PAK signaling through UNC-73. shown to act as a dominant-negative inhibitor of TRIO’s activity

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Fig. 3. mDISC1 in C. elegans motor neurons interacts genetically with RAC-PAK signaling pathways to regulate axon guidance. (A) Schematic diagram summarizing the known RAC-dependent and RAC-independent signaling pathways in developing C. elegans motor neurons (13). The RAC-dependent pathway involves unc-73 (a GEF gene), two functional redundant rac genes, ced-10 and mig-2, and two downstream pak genes, pak-1 and max-2. The RAC- independent pathway is simplified by showing a completely RAC-independent gene, max-1. Note that the max-2 gene also has a RAC-independent function. Solid arrows indicate direct activation, and dashed arrows indicate weaker activation. (B) The severity of axon guidance defects in mDISC1 transgenic animals is significantly suppressed by ced-10(dn)ormig-2(dn), whereas expression of wild-type ced-10 or mig-2 enhances the axon guidance defects of mDISC1 transgenic animals. (C) Single mutants of ced-10 or mig-2 suppress the axon guidance defects of mDISC1 transgenic animals. (D) Axon guidance defects in mDISC1 transgenic animals are greatly enhanced by loss of genes that can transduce signals independent of the RAC signaling pathway. (E) Double mutants of pak-1;ced-10 suppress the defects more significantly than either pak-1 or ced-10 single mutants. They also suppress the defects more significantly than pak- 1;mig-2 double mutants, consistent with the preferential uses of the rac-pak genes in the motor axons. In all diagrams, bars represent the SE (n =32–62). *P < 0.05, **P < 0.001 (Student’s t test).

(21), and similar inhibitory mechanisms have been reported in of genes involved in the RAC-PAK signaling pathway might cause other Dbl family proteins (22, 23). Thus, despite its heterologous schizophrenia or other related mental illnesses. Indeed, abnormal nature, the established mDISC1 genetic system has proven use- expressions or mutations of genes in this RAC-PAK pathway have ful for studying DISC1’s functions. been reported in human genomic studies of patients with psychotic Neuronal dysconnection has been demonstrated in the brains symptoms (32–34). of schizophrenic patients (24, 25). However, the causes of such Several transgenic C. elegans lines have been successfully abnormalities are still unknown. Recent evidence has begun to established to study human neurodegenerative disorders (35– show that schizophrenia susceptibility genes are essential for ax- 40). Such heterologous genetic models have proven to be very onal connectivity during development. For example, neuregulin, useful for understanding disease pathogenesis and developing a well known schizophrenia susceptibility gene, is required for therapeutic strategy. For instance, the heterologous model for thalamocortical projections in mice (26, 27). Our studies in mouse polyglutamine aggregation in C. elegans has demonstrated that hippocampi and in C. elegans motor neurons together also show chronic expression of aggregation-prone proteins can disrupt the that DISC1 plays a role in regulating axonal connections (9). homeostasis of protein folding and cause pleiotropic cytotoxicity Because DISC1 is involved in essentially all aspects of neuro- (35). The establishment of this transgenic DISC1 model expands development (4–7, 11), and because more than a dozen cytosolic the repertoire of C. elegans disease models to study human proteins are reported to interact with DISC1 (6, 8, 28–30), it is neurodevelopmental disorders. One important application of likely that DISC1 acts as a scaffold protein inside neurons, re- disease models in C. elegans is to identify drug targets or screen cruiting various signaling components to exert its specific function for new drugs (41–43). Recently, a large-scale small-molecule at different developmental stages. It is therefore not surprising screen in C. elegans has successfully isolated a calcium channel that, in contrast to our results, DISC1 has recently been reported blocker. Subsequent suppressor genetic screens also identify the to interact with another RAC1-GEF, -7, and to inhibit the α1-subunit of the L-type calcium channel as a potential drug target RAC1 signaling during dendritic spine morphogenesis (31). De- (44). Interestingly, when the mDISC1 transgenic animals were in- spite the differences, these studies together suggest that mutations cubated with rolipram, a well-known phosphodiesterase 4 (PDE4)–

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Fig. 4. In mammalian systems, DISC1 binds to TRIO and activates RAC1 signaling. (A) Schematic diagram summarizing mDISC1 constructs used to generate His-tagged recombinants. mDISC1 contains an N-terminal globular head domain and multiple coiled-coil domains in the C terminus. The numbers indicate the beginning and ending amino acids for each construct. (B) Schematic diagram summarizing hTRIO constructs used to generate GST-tagged recombinants. hTRIO consists of a Sec14 homolog domain, a spectrin repeats (SP) domain, two GEF domains (GEF1 and GEF2), and a kinase domain. (C) Axon guidance defects in mDISC1 transgenic worms are significantly suppressed by rh40, a hypomorphic mutant allele of unc-73/trio (n =50–69). (D) mDISC1 and hTRIO are coimmunoprecipitated as a protein complex when expressed in COS cells. (E) The SPn of hTRIO, but not the SPc or the GEF2 domain, binds to its GEF1 as shown in the GST pull-down assays. The loading inputs are 5% of the total purified His-tagged proteins. (F) hTRIO-SPn specifically inhibits the binding of human RAC1 (hRAC1) to hTRIO-GEF1 domain in the GST pull-down assays. The ratios of hRAC1 pulled down by hTRIO-GEF1 in the presence of hTRIO-SPn or hTRIO-SPc were quantified and are shown in the histogram (n = 3). The ratio is indicated as fold increase relative to that without adding hTRIO-SP proteins. (G) hTRIO pulls down more hRAC1 in the presence of mDISC1 full-length (mDISC1-FL) but not a control mDISC1 fragment (mDISC1-MD) that does not bind to hTRIO. The ratios of hRAC1 pull-down were quantified as in F (n = 3). (H) The RAC1 activities in COS cells are significantly increased when hTRIO is coexpressed with mDISC1. The activity of RAC1 was assayed by the amount of GTP-RAC1 pulled-down by GST-PAK1 70–117 (45). (I) Expression of unc-73b significantly enhances the motor axon guidance defects in mDISC1 transgenic animals (n =54–60). In all diagrams, bars represent the SE. *P < 0.05, **P < 0.001 (Student’s t test).

specific inhibitor with anti-psychotic effects, the axon guidance Descriptions of C. elegans strains, constructs, in vitro binding assay, RAC1 defects were apparently suppressed (Fig. 1G). Thus, this estab- activity assay, and other general techniques are provided in SI Materials lished mDISC1 genetic system could be used in the future to screen and Methods. for small molecules or chemicals with potential therapeutic effects ACKNOWLEDGMENTS. We thank the International C. elegans Gene Knock- on DISC1-related abnormalities. out Consortium for strains. We are grateful to Yuji Kohara, Erik Lundquist, Ken-Ichi Ogura, Yoshio Goshima, Hongjun Song, Gary Ruvkun, Andy Fire, Materials and Methods and Anne Debant for materials. We thank Mark Lucanic, Noelle L’Etoile, Phenotypic Analysis. The axon guidance defects were quantified by calculating Ting-Wen Cheng, Damien O’Halloran, Scott Hamilton, Bi-Tzeng Juang, Karl ’ the percentage of commissures from DD and VD motor neurons that failed to Murray, Hai-Gwo Hwu, Chih-Min Liu, members of the L Etoile and Cheng laboratories, and members of the “Super Worm Group” at the University of reach the dorsal cord within a single animal. For branching phenotype, the fi California, Davis, for comments and advice. We also thank Wei-Wen Liu, branch point was de ned as a point with an additional neurite extending more Kimberly Zhou, and Abraham Noorbakhsh for technical help. This work μ than 1 m in length. Multiple comparisons were performed with the two- was supported in part by a startup fund and a health system grant from tailed Student’s t test and a Benjamini and Hochberg correction. In all figures, the University of California, Davis (to H.-J.C.), and a grant from the National statistically significant differences are indicated by asterisks. Taiwan University Hospital (to P.-H.H.).

1. Lewis DA, Levitt P (2002) Schizophrenia as a disorder of neurodevelopment. Annu Rev 4. Chubb JE, Bradshaw NJ, Soares DC, Porteous DJ, Millar JK (2008) The DISC in Neurosci 25:409–432. psychiatric illness. Mol Psychiatry 13:36–64. 2. Ross CA, Margolis RL, Reading SAJ, Pletnikov M, Coyle JT (2006) Neurobiology of 5. Duan X, et al. (2007) Disrupted-In-Schizophrenia 1 regulates integration of newly schizophrenia. Neuron 52:139–153. generated neurons in the adult brain. Cell 130:1146–1158. 3. Lewis DA, Lieberman JA (2000) Catching up on schizophrenia: Natural history and 6. Mao Y, et al. (2009) Disrupted in schizophrenia 1 regulates neuronal progenitor neurobiology. Neuron 28:325–334. proliferation via modulation of GSK3β/β-catenin signaling. Cell 136:1017–1031.

Chen et al. PNAS Early Edition | 5of6 Downloaded by guest on September 23, 2021 7. Kamiya A, et al. (2005) A schizophrenia-associated mutation of DISC1 perturbs 27. López-Bendito G, et al. (2006) Tangential neuronal migration controls axon guidance: cerebral cortex development. Nat Cell Biol 7:1167–1178. A role for neuregulin-1 in thalamocortical axon navigation. Cell 125:127–142. 8. Ozeki Y, et al. (2003) Disrupted-in-Schizophrenia-1 (DISC-1): Mutant truncation 28. Millar JK, et al. (2005) DISC1 and PDE4B are interacting genetic factors in schizophrenia prevents binding to NudE-like (NUDEL) and inhibits neurite outgrowth. Proc Natl that regulate cAMP signaling. Science 310:1187–1191. Acad Sci USA 100:289–294. 29. Brandon NJ, et al. (2004) Disrupted in Schizophrenia 1 and Nudel form a neurodevelop- 9. Faulkner RL, et al. (2008) Development of hippocampal mossy fiber synaptic outputs mentally regulated protein complex: Implications for schizophrenia and other major by new neurons in the adult brain. Proc Natl Acad Sci USA 105:14157–14162. neurological disorders. MolCellNeurosci25:42–55. 10. Bord L, et al. (2006) Primate disrupted-in-schizophrenia-1 (DISC1): High divergence of 30. Singh KK, et al. (2010) Dixdc1 is a critical regulator of DISC1 and embryonic cortical a gene for major mental illnesses in recent evolutionary history. Neurosci Res 56: development. Neuron 67:33–48. 286–293. 31. Hayashi-Takagi A, et al. (2010) Disrupted-in-Schizophrenia 1 (DISC1) regulates spines 11. Miyoshi K, et al. (2003) Disrupted-In-Schizophrenia 1, a candidate gene for of the glutamate synapse via Rac1. Nat Neurosci 13:327–332. schizophrenia, participates in neurite outgrowth. Mol Psychiatry 8:685–694. 32. Rejeb I, et al. (2008) A novel splice mutation in PAK3 gene underlying mental 12. Dawe AL, Caldwell KA, Harris PM, Morris NR, Caldwell GA (2001) Evolutionarily retardation with neuropsychiatric features. Eur J Hum Genet 16:1358–1363. conserved nuclear migration genes required for early embryonic development in 33. Allen KM, et al. (1998) PAK3 mutation in nonsyndromic X-linked mental retardation. Caenorhabditis elegans. Dev Genes Evol 211:434–441. Nat Genet 20:25–30. 13. Lucanic M, Kiley M, Ashcroft N, L’Etoile N, Cheng H-J (2006) The Caenorhabditis 34. Aston C, Jiang L, Sokolov BP (2005) Transcriptional profiling reveals evidence for elegans P21-activated kinases are differentially required for UNC-6/netrin-mediated signaling and oligodendroglial abnormalities in the temporal cortex from patients commissural motor axon guidance. Development 133:4549–4559. with major depressive disorder. Mol Psychiatry 10:309–322. 14. Struckhoff EC, Lundquist EA (2003) The -binding protein UNC-115 is an effector 35. Gidalevitz T, Ben-Zvi A, Ho KH, Brignull HR, Morimoto RI (2006) Progressive disruption of Rac signaling during axon pathfinding in C. elegans. Development 130:693–704. of cellular protein folding in models of polyglutamine diseases. Science 311: 15. Steven R, et al. (1998) UNC-73 activates the Rac GTPase and is required for cell and 1471–1474. growth cone migrations in C. elegans. Cell 92:785–795. 36. Faber PW, Alter JR, MacDonald ME, Hart AC (1999) Polyglutamine-mediated 16. Wu Y-C, Cheng T-W, Lee M-C, Weng N-Y (2002) Distinct rac activation pathways dysfunction and apoptotic death of a Caenorhabditis elegans sensory neuron. Proc control Caenorhabditis elegans and axon outgrowth. Dev Biol 250: Natl Acad Sci USA 96:179–184. 145–155. 37. Faber PW, Voisine C, King DC, Bates EA, Hart AC (2002) Glutamine/proline-rich PQE-1 17. Camargo LM, et al. (2007) Disrupted in Schizophrenia 1 : Evidence for the proteins protect Caenorhabditis elegans neurons from huntingtin polyglutamine close connectivity of risk genes and a potential synaptic basis for schizophrenia. Mol neurotoxicity. Proc Natl Acad Sci USA 99:17131–17136. Psychiatry 12:74–86. 38. Hamamichi S, et al. (2008) Hypothesis-based RNAi screening identifies neuroprotective 18. Newsome TP, et al. (2000) Trio combines with dock to regulate Pak activity during genes in a Parkinson’s disease model. Proc Natl Acad Sci USA 105:728–733. photoreceptor axon pathfinding in Drosophila. Cell 101:283–294. 39. Kraemer BC, et al. (2003) Neurodegeneration and defective neurotransmission in 19. Briançon-Marjollet A, et al. (2008) Trio mediates netrin-1-induced Rac1 activation in a Caenorhabditis elegans model of tauopathy. Proc Natl Acad Sci USA 100:9980–9985. axon outgrowth and guidance. Mol Cell Biol 28:2314–2323. 40. Link CD (1995) Expression of human β-amyloid peptide in transgenic Caenorhabditis 20. Bateman J, Van Vactor D (2001) The Trio family of guanine-nucleotide-exchange elegans. Proc Natl Acad Sci USA 92:9368–9372. factors: Regulators of axon guidance. J Cell Sci 114:1973–1980. 41. Giacomotto J, et al. (2009) Evaluation of the therapeutic potential of carbonic anhydrase 21. Estrach S, et al. (2002) The human Rho-GEF trio and its target GTPase RhoG are inhibitors in two animal models of dystrophin deficient muscular dystrophy. Hum Mol involved in the NGF pathway, leading to neurite outgrowth. Curr Biol 12:307–312. Genet 18:4089–4101. 22. Rossman KL, Der CJ, Sondek J (2005) GEF means go: Turning on RHO GTPases with 42. Kaminsky R, et al. (2008) A new class of anthelmintics effective against drug-resistant guanine nucleotide-exchange factors. Nat Rev Mol Cell Biol 6:167–180. nematodes. Nature 452:176–180. 23. Yu B, et al. (2010) Structural and energetic mechanisms of cooperative autoinhibition 43. Kwok TCY, et al. (2008) A for dihydropyridine (DHP)-resistant worms and activation of Vav1. Cell 140:246–256. reveals new residues required for DHP-blockage of mammalian calcium channels. 24. Stephan KE, Baldeweg T, Friston KJ (2006) Synaptic plasticity and dysconnection in PLoS Genet 4:e1000067. schizophrenia. Biol Psychiatry 59:929–939. 44. Kwok TCY, et al. (2006) A small-molecule screen in C. elegans yields a new calcium 25. Stephan KE, Friston KJ, Frith CD (2009) Dysconnection in schizophrenia: From channel antagonist. Nature 441:91–95. abnormal synaptic plasticity to failures of self-monitoring. Schizophr Bull 35:509–527. 45. Zahir N, et al. (2003) Autocrine laminin-5 ligates α6β4 integrin and activates RAC and 26. Li B, Woo R-S, Mei L, Malinow R (2007) The neuregulin-1 receptor erbB4 controls NFκB to mediate anchorage-independent survival of mammary tumors. J Cell Biol 163: glutamatergic synapse maturation and plasticity. Neuron 54:583–597. 1397–1407.

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