Molecular Psychiatry (2011) 16, 1105–1116 & 2011 Macmillan Publishers Limited All rights reserved 1359-4184/11 www.nature.com/mp ORIGINAL ARTICLE Dysbindin-1, a schizophrenia-related protein, facilitates neurite outgrowth by promoting the transcriptional activity of p53 X Ma, E Fei, C Fu, H Ren and G Wang Laboratory of Molecular Neuropathology, Key Laboratory of Brain Function and Diseases, School of Life Sciences, University of Science and Technology of China, Chinese Academy of Sciences, Anhui, People’s Republic of China

Genetic variations in the DTNBP1 gene (encoding the protein dysbindin-1) have been implicated as risk factors in the pathogenesis of schizophrenia. Previous studies have indicated that dysbindin-1 functions in the regulation of synaptic activity. Recently, dysbindin-1 has also been documented to be involved in neuronal development. In this study, we identified necdin as a binding partner of dysbindin-1 using a yeast two-hybrid screen. Dysbindin-1 recruits necdin to the cytoplasm, thereby attenuating the repressive effects of necdin on p53 transcriptional activity. Knockdown of dysbindin-1, like knockdown of p53, greatly decreases the expressions of the p53 target genes coronin 1b and rab13, which are required for neurite outgrowth. Moreover, overexpression of p53 restores the neurite outgrowth blocked by dysbindin-1 knockdown. In brains of dysbindin-1 null mice (the sandy strain), p21, Coronin 1b and Rab13 levels are reduced. Furthermore, primary cultured cortical neurons from sandy mice display neurite outgrowth defects when compared with those from wild-type mice. Thus, our data provide evidence that dysbindin-1 has an important role in neurite outgrowth through its regulation of p53’s transcriptional activity. Molecular Psychiatry (2011) 16, 1105–1116; doi:10.1038/mp.2011.43; published online 19 April 2011 Keywords: dysbindin-1; schizophrenia; p53; necdin; neurite outgrowth

Introduction Previous studies have shown that dysbindin-1 functions both pre- and postsynaptically through Schizophrenia is a severe mental disorder that affects interaction with partners11,12 to regulate neurotrans- 0.5–1% of the population and has a strong hereditary mitter release and signal transduction. Decreased component.1 To date, many susceptibility genes have dysbindin-1 expression is related to intrinsic gluta- been found to be associated with schizophrenia by matergic alterations in the hippocampi of schizophre- genetic studies.2 Genetic variations in the DTNBP1 nia patients.8 Furthermore, knockdown of endogenous gene encoding dysbindin-1 have been linked to dysbindin-1 in cultured cells leads to a decrease of schizophrenia in multiple populations.3–6 Some of glutamine release.13 It has also been reported that these genetic variations are associated with a de- knockdown of dysbindin-1 increases cell surface creased level of dysbindin-1 mRNA in the prefrontal dopamine receptor (D2DR) levels,14 and that a cortex of schizophrenia patients.7 In the hippocam- decrease of dysbindin-1 expression increases both pus of schizophrenia patients, the protein levels of the D2DR recycling rate and the rate of its insertion dysbindin-1 are significantly reduced.8 Functional into cell surface.15 Thus, loss of dysbindin-1 function studies have revealed that genetic variations of may contribute to the pathogenesis of schizophrenia DTNBP1 modulate prefrontal brain function9 and due to altered neurotransmitter signaling. contribute to impairments in spatial working memory In addition to the role of neurotransmitters in the and executive function.10 These studies have shown a etiology schizophrenia, evidence for a defect in strong connection between dysbindin-1 function and neurite formation during development caused by the pathogenesis of schizophrenia. susceptibility gene products has been obtained.16 Of these gene products, DISC1 (disrupted-in-schizophre- Correspondence: Dr G Wang, Professor of Neurobiology, Labora- nia-1) was reported to regulate neurite outgrowth 17,18 tory of Molecular Neuropathology, Key Laboratory of Brain during neuron differentiation and NRG1 (neu- Function and Diseases, School of Life Sciences, University of regulin-1) was reported to induce neurite formation.19 Science and Technology of China, Hefei, Anhui 230027, People’s The expression level of dysbindin-1 in the mouse Republic of China. brain is developmentally regulated, suggesting that E-mail: [email protected] Received 31 October 2010; revised 4 March 2011; accepted 7 dysbindin-1 may be involved in neuronal develop- March 2011; published online 19 April 2011 ment.11 Downregulation of dysbindin-1 by small Regulation of neurite outgrowth by dysbindin-1 XMaet al 1106 interfering RNA in SH-SY-5Y cells causes shorter Culture and transfection of primary cultured neurons neurite length and aberrant organization of the actin Dissociated rat or mouse cortex cultures were pre- cytoskeleton at the tips of neurites.20 Primary hippo- pared from postnatal 0- to 1-day-old Sprague–Dawley campal neurons derived from sandy mice, which lack rats or C57BL/6J mice. Cortical cells were gently dysbindin-1 protein due to the deletion of two exons dissociated with a plastic pipette after digestion with in the DTNBP1 gene,21,22 exhibit abnormal cytoskele- 0.5% trypsin (GIBCO, Los Angeles, CA, USA) at 37 1C tal organization in the growth cone.20 It was recently for 20 min. The dissociated cells were plated at a final reported that dysbindin-1 regulates dendrite spine density of 5 Â 105 cmÀ2 on polyethyleneimine-coated formation through its interaction with WAVE2 and 24-well plates (Corning, Inc., Corning, NY, USA) and Abi-1.23 Moreover, dysbindin-1 is a subunit of the cultured in Neurobasal medium (GIBCO) containing biogenesis of lysosome-related organelles complex-1, 1 Â B27 supplement (GIBCO) and 3 mgmlÀ1 glutamine which is required for the trafficking of lysosome- (Sigma, Saint Louis, MO, USA). After 3 days of related organelles.24 The dysbindin-1-containing bio- culturing, 5-fluoro-20-deoxy-uridine (FUDR) was genesis of lysosome-related organelles complex-1 is added to a final concentration of 10 mM (Sigma) to also involved in neurite outgrowth.11 These studies repress the growth of glial cells. highlight functions of dysbindin-1 in neurite out- For transfection, cultured cells were washed with growth; however, the underlying mechanisms are still Opti-MEM (GIBCO, Los Angeles, CA, USA) medium largely unknown. and then transfected with small interfering RNA As an established tumor suppressor protein, p53 using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, binds to specific DNA sequences to transcriptionally USA) reagent in Opti-MEM medium without serum. regulate the expression of apoptosis-related proteins Primary cell culture medium was added to the culture (such as bax and puma) when cells are exposed to medium at 6 h after transfection. At 4 days after apoptotic stressors.25 Besides from its role in cell transfection, the cells were subjected to reverse death, non-apoptotic functions of p53 are also transcription-PCR analysis. required during neuronal development.26 Recently, increasing evidence has shown that p53 transcrip- Animal experiments tional activity is critical for neurite outgrowth. In Sandy mutant (sdy/sdy) and wild-type (WT; þ / þ ) PC12 cells, p53 transcriptional activity is necessary mice were derived from heterozygotes obtained from for nerve growth factor (NGF)-mediated differentia- the Jackson Laboratory (Bar Harbor, ME, USA). To tion.27 Moreover, two proteins that are transcription- determine the genotypes of þ / þ , þ /sdy and sdy/ ally activated by p53, the small GTPase Rab13 and the sdy, we performed PCR assays to genotype the mice F-actin-binding protein Coronin 1b, are required for based on the nature of the deletion mutation in the neurite outgrowth and nervous system regenera- DTNBP1 gene.22 The cortex and hippocampus of tion.28,29 The effects of these two proteins on neurite mouse brains were homogenized in cell lysis buffer outgrowth are dependent on p53 transcriptional (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP-40 activity.28,29 The p53 target protein cyclic guanosine and 0.5% sodium deoxycholate) in the presence of a monophosphate (cGMP)-dependent protein kinase protein inhibitor cocktail (Roche, Mannheim, Ger- type I, which influences growth cone navigation, many). The lysates were subjected to immunoblot was also reported to be regulated by p53 during analysis. For total RNA extraction, the hippocampus neuronal maturation.30 Thus, the regulation of multi- of mice was homogenized in lysis buffer using a ple neuronal development-related proteins by p53 protein and RNA extraction kit (Takata, Shiga, Japan), ensures neurite outgrowth until axon formation or and total RNA was isolated according to the manu- maturation.27,29,30 In addition, factors that influence facturer’s recommendations. All mice were main- p53 transcriptional activity can manipulate the fate of tained in a specific pathogen-free room, and all neurons. For example, Kru¨ ppel-like factor-4, which animal experiments were approved by the Animal was identified as a transcriptional repressor of p53, Welfare Advisory Committee of the University of inhibits neurite outgrowth during central nervous Science and Technology of China. system neuron development.31 Here, we investigated the role dysbindin-1- Statistical analysis mediated regulation of p53 activity in neurite out- Relative density analysis of immunoblots and reverse growth. transcription-PCR products from three independent experiments was performed using Adobe Photoshop Materials and methods CS 8.0 (Adobe, San Jose, CA, USA). Data were analyzed using Origin 7.5 (OriginLab, Northampton, Information about plasmid construction, yeast two- MA, USA). hybrid screening, glutathione S-transferase pull-down assays, immunoprecipitation, antibodies, immuno- Neurite outgrowth assay cytochemistry, RNA interference, reverse transcrip- N2a cells were seeded onto 12-well plates (5 Â 105 tion-PCR assays, cell culture and transfection, and cells per well) and cultured for 24 h before dual-luciferase reporter gene assays are provided in transfection in Dulbecco’s modied Eagle’s medium the Supplementary Materials and Methods. containing 10% fetal bovine serum. Plasmids or small

Molecular Psychiatry Regulation of neurite outgrowth by dysbindin-1 XMaet al 1107 interfering RNA were transfected into N2a cells using BD-dysbindin-1-(1–189) and AD vector did not Lipofectamine 2000 (for plasmids) or Oligo Lipofec- (Figure 1a). These results suggest that dysbindin-1 tamine (for small interfering RNA). At 6 h after interacts with full-length necdin in yeast. transfection, the cells were then cultured with To determine whether there is a direct interaction Dulbecco’s modied Eagle’s medium containing 2% between necdin and dysbindin-1, we performed a fetal bovine serum for 24 h to induce neurite out- glutathione S-transferase pull-down assay. Glu- growth. The formation of neurites was observed using tathione S-transferase–dysbindin-1, but not glu- a microscope. The longest neurite length of each cell tathione S-transferase alone, pulled down His– was measured by ImageJ software as described else- necdin (Supplementary Figure S1A). To determine where.17,29,32 Each well was evaluated using three whether dysbindin-1 interacts with necdin in mam- different fields under a microscope. The average malian cells, we performed a coimmunoprecipitation neurite length of approximately 200–250 cells is assay in human embryonic kidney cells (HEK293A) presented as mean±s.e. For cultured primary neu- expressing a combination of FLAG–dysbindin-1 and rons, the cortical neurons from newborn C57BL/6J enhanced green fluorescence protein (EGFP) or WT mice or sandy mice were used. These cells were necdin–EGFP. FLAG–dysbindin-1 coprecipitated cultured in NB/B27 (neurobasal medium containing with necdin–EGFP when necdin–EGFP was immu- 1  B27 and glutamine) medium for 5 days. Neurite noprecipitated with anti-GFP antibodies; however, it analysis was performed using Image J software after did not precipitate with EGFP alone (Supplementary cells were immunostained with anti-Tuj1 antibody. Figure S1B). Similar results were obtained using anti- The length of the longest neurite of each neuron was dysbindin-1 antibodies to immunoprecipitate endo- measured from the edge of each soma to neurite tips. genous dysbindin-1 in a neuroblastoma cell line (N2a For each group, three independent experiments were cells) transfected with HA–necdin. When endogenous performed. In each replicate, 100–110 cells were dysbindin-1 was immunoprecipitated, HA–necdin counted from several randomly selected microscope coprecipitated (Figure 1b). fields using an  40 objective. Relative average neurite lengths of B330 cells from three independent Influence of dysbindin-1 on the intracellular experiments were analyzed using OriginLab software distribution of necdin and p53 transcriptional activity and are presented as mean±s.e. For soma size Necdin was originally reported to be a nuclear analysis, the soma area of each neuron was measured protein,35 but it was subsequently reported to localize by Image J according to its user manual. The relative to both the cytoplasm and the nucleus.36,37 We average soma areas of B300 cells from three inde- hypothesized that the interaction between dysbin- pendent experiments were analyzed using OriginLab din-1 and necdin might alter the cellular distribution software and are presented as mean±s.e. of one or both proteins. To assess this possibility, we cotransfected HA–necdin together with EGFP or dysbindin-1–EGFP into HEK293A cells. HA–necdin Results appeared to be cytoplasmic in cells expressing Interactions between dysbindin-1 and necdin dysbindin-1–EGFP, but it was diffusely distributed To investigate dysbindin-1 function, a yeast two- in both the cytoplasm and the nucleus in cells hybrid screen was performed to identify novel expressing EGFP (Figure 1c) and in cells transfected partners of dysbindin-1. Because full-length dysbin- with HA–necdin alone (data not shown). din-1 caused autoactivation in yeast strain AH109,33 Necdin was previously reported to interact with we used the N terminus of dysbindin-1 (amino acids transcription factors, such as p53 and E2F1, in the 1–189) as the bait for a screen (Figure 1a). A total of 20 nucleus and to have an important role during the ter- positive transformants were obtained, including sna- minal differentiation of neurons.34,38,39 It suppresses the pin, homer1, 14-3-3 zeta, clusterin and a fragment of transcriptional activity of p53 by interacting with it.38 necdin containing amino acids 43–284. Snapin is a We found that the cellular distribution of necdin is reported partner of dysbindin-112 and necdin is a altered by dysbindin-1. We thus employed a dual- protein associated with neuronal differentiation.34 We luciferase assay to investigate whether dysbindin-1 therefore chose necdin for further study. To further influences p53 transcriptional activity. In human confirm the necdin interaction with dysbindin-1 in colon tumor cells that lack p53 (HCT116 p53À/À), yeast, we constructed a full-length version of activa- overexpression of p53 greatly increased p53-respon- tion domain (AD)-fused necdin and retransformed it sive element-containing reporter activity to B18 into yeast with binding domain (BD)-dysbindin-1-(1– times that of the basal level, and coexpression of 189) or BD vector. Yeast cotransformed with BD- necdin repressed p53 transcriptional activity. How- dysbindin-1-(1–189) and AD-necdin, as well as ever, overexpression of dysbindin-1 significantly positive control yeast (cotransformed with BD-p53 blocked the repressive effects of necdin on p53 and pTD1-1 that were supplied with the kit), grew transcriptional activity (Figure 1d). To further well on culture medium lacking both histidine and confirm the effects of dysbindin-1 on p53 transcrip- adenine, indicating activation of each of the reporter tional activity, we tested whether dysbindin-1 in- genes, but negative control yeast (cotransformed with creases endogenous p53 transcriptional activity. In a BD and AD vectors) or the yeast cotransformed with human colon tumor cell line with WT p53 (HCT116

Molecular Psychiatry Regulation of neurite outgrowth by dysbindin-1 XMaet al 1108 ab Leucine - - - Tryptophan - -- Histidine ++- - ndin 3-AT - + - Adenine+ + - trol IgG -dysbi  BD+AD con HA-Ndn BD-dysbindin-(1-189)+AD IP dysbindin BD-dysbindin-(1-189)+AD-Ndn Input HA-Ndn BD-p53+pTD1-1

c HA-Ndn Hochest Merge ** 100 ABCD 80

60 EGFP 40

EFGH 20 HA-Ndn in nucleus 0 % double positive cells shown Dys-EGFP

EGFP+HA-Ndn

Dys-EGFP+HA-Ndn

d e f * * ** 1.4 24 22 1.2 NS 20 1.2 1.0 18 * * 16 1.0 0.8 14 0.8 12 0.6 10 0.6 8 0.4 6 0.4 4 0.2 0.2 Relative Luciferase Activity

Relative Luciferase Activity 2 0 0 0 Relative Luciferase Activity FLAG-p53 - + + + FLAG-Ndn - - + + HA + - FLAG-Ndn - - + + HA-dysbindin - + - + HA-dysbindin - + HA-dysbindin - - - + Figure 1 Dysbindin-1 interacts with necdin, recruits it to the cytoplasm and thereby prevents it from repressing the transcriptional activity of p53. (a) Binding domain (BD)-dysbindin-1-(1–189) was cotransformed with activation domain (AD) or AD-necdin (AD-Ndn) into yeast strain AH109. The double transformants were grown on ÀLeu/ÀTrp/ þ His (left row), ÀLeu/ÀTrp/ÀHis/ þ 3-AT (middle row) or ÀLeu/ÀTrp/ÀHis/ÀAde (right row) Sprague–Dawley plates. Double transformants containing BD and AD or BD-p53 and pTD1-1 served as negative controls and positive controls, respectively. (b) Immunoprecipitation assays were performed showing that HA–Ndn interacts with endogenous dysbindin-1 in N2a cells. (c) Immunofluorescence analyses indicating that dysbindin-1–enhanced green fluorescence protein (EGFP) recruits HA–Ndn to the cytoplasm of HEK293A cells coexpressing HA–Ndn with EGFP (A–D) or dysbindin-1–EGFP (E–H). Bar: 10 mm; green: EGFP or dysbindin-1–EGFP; red: HA–Ndn; blue: nuclei. The ratio of cells with nuclear HA–Ndn in EGFP- or dysbindin-1– EGFP-positive cells was quantified for three independent experiments by one-way analysis of variance (ANOVA), **P < 0.005. (d) Dual-luciferase p53 reporter assays using HCT116 p53-null cells transfected with expression plasmids (as indicated) were performed and they show that HA–dysbindin-1 blocks the repressive effects of necdin on p53 activity. Quantitative data from three independent experiments were quantified by one-way ANOVA, *P < 0.05. (e) Reporter gene assays similar to those in d using HCT116 p53-wild-type cells were performed to show the effects of dysbindin-1 or necdin on endogenous p53 activity. Quantitative data from three independent experiments were quantified by one-way ANOVA, *P < 0.05. (f) Reporter gene assays similar to those in d using HCT116 p53-null cells were performed to show that HA– dysbindin-1 has no effect on p53 report genes without p53. Quantitative data from three independent experiments were quantified by one-way ANOVA, NS. NS, not significant.

Molecular Psychiatry Regulation of neurite outgrowth by dysbindin-1 XMaet al 1109 p53 þ / þ ), overexpression of dysbindin-1 increased dysbindin-1 mutants and performed coimmunopreci- endogenous p53 transcriptional activity over the basal pitation assays. In contrast to full-length dysbindin-1, level (Figure 1e). However, simultaneous overexpres- which interacted with necdin, neither dysbindin- sion of necdin repressed endogenous p53 transcrip- 1DCCD–EGFP nor dysbindin-1-DLZM–EGFP inter- tional activity, and these effects were partially acted with necdin (Figure 2c), suggesting that amino blocked by overexpressing dysbindin-1 (Figure 1e). acids 97–118, the LZM of dysbindin-1, are critical for Moreover, overexpressing HA–dysbindin-1 in its interaction with necdin. To assess whether the HCT116 p53-null cells failed to increase p53 reporter LZM of dysbindin-1 (amino acids 97–118) are activity (Figure 1f), suggesting that dysbindin-1 involved in the effects of dysbindin-1 on necdin- cannot activate p53 reporter activity without p53. mediated p53 transcriptional regulation, we per- To further explore the influence of dysbindin-1 on formed dual-luciferase assays. In HCT116 p53-null the transcriptional activation of p53, we next tested cells, dysbindin-1–EGFP blocked the repressive whether dysbindin-1 influences the expression of effects of necdin on p53 transcriptional activity. downstream target gene transactivated by p53. We However, dysbindin-1DLZM–EGFP did not examined p21, a well-known transcriptional target of (Figure 2d). As dysbindin-1DLZM–EGFP failed to p53.40,41 In HCT116 WT cells transfected with dys- regulate necdin-mediated repression of p53 activity, bindin-1, both the protein and the mRNA levels of we next asked whether it had effects on the cellular p21 increased (Supplementary Figure S2A and B). In distribution of necdin. We transfected HEK293A cells contrast to dysbindin-1 overexpression, knockdown with HA–necdin along with EGFP-tagged full-length of endogenous dysbindin-1 decreased p21 levels in dysbindin-1 or dysbindin-1DLZM and examined the cells treated or not treated with doxorubicin, an distribution of necdin using immunocytochemical inducer of p53 (Supplementary Figure S2C). More- staining. In contrast to full-length dysbindin-1, which over, in HCT116 p53-null cells cotransfected with caused a translocation of necdin from the nucleus to p53–EGFP and FLAG or FLAG–necdin, along with or the cytoplasm, dysbindin-1DLZM–EGFP, similarly to without HA–dysbindin-1 as indicated, necdin over- EGFP, did not alter the subcellular distribution of expression decreased both the protein and mRNA necdin (Figure 2e), suggesting that amino acids 97– levels of p21 that had been induced by p53 over- 118 are critical for the ability of dysbindin-1 to alter expression. However, in the presence of dysbindin-1, necdin’s subcellular distribution. Taken together with necdin failed to repress p21 levels (Supplementary the coimmunoprecipitation and reporter gene assay Figure S2D), suggesting that dysbindin-1 influences results (Figures 1 and 2), these data suggest that the effects of necdin on p53 target gene expression. dysbindin-1 regulates transcriptional activity of p53 by altering the subcellular distribution of necdin Amino acids 97–118 of dysbindin-1 are required for its through its LZM domain. interaction with and effects on necdin To further identify which domain of dysbindin-1 is Regulation of downstream targets of p53 and neurite critical for its interaction with necdin, we generated outgrowth by dysbindin-1 several deletion mutants of dysbindin-1 (Figure 2a). p53 is a well-known tumor suppressor; however, it We already showed that amino acids 1–189 of was recently reported to be associated with neuronal dysbindin-1 interact with necdin in a yeast two- differentiation.27,43 Multiple neural development-re- hybrid screen (Figure 1a), suggesting that dysbindin-1 lated genes have been found to be transcriptionally may interact with necdin though its N terminus. To regulated by p53. For instance, the F-actin-binding confirm this, we constructed an N terminus of proteins Coronin 1b and GTPase Rab13, two proteins dysbindin-1 containing amino acids 1–217 (dysbin- critical for neurite outgrowth, are transcriptionally din-1-N–EGFP) and a C terminus of dysbindin-1 regulated by p53.29 Importantly, dysbindin-1 was also containing amino acids 216–351 (dysbindin-1-C– reported to be involved in neurite outgrowth in EGFP). We then transfected HEK293A cells with cultured cells and primary neurons.20 We thus FLAG–necdin together with either EGFP, EGFP- wondered whether dysbindin-1 facilitates neurite tagged full-length dysbindin-1, dysbindin-1-N or outgrowth in a pathway similar to that of p53. We dysbindin-1-C. Necdin coimmunoprecipitated with employed a mouse neuroblastoma cell line (N2a both EGFP-tagged full-length dysbindin-1 and dys- cells), a commonly used cell line whose differentia- bindin-1-N, but not with EGFP alone or EGFP-tagged tion and neurite outgrowth can be induced by retinoic dysbindin-1-C (Figure 2b). acid or serum deprivation,44,45 as a model for neurite Amino acids 1–217 of dysbindin-1 contain a coiled- outgrowth assays. After induction with a low con- coil domain (CCD) located in amino acids 89–18842 centration of serum, the average neurite length was and a putative leucine zipper motif (LZM) in amino much shorter in p53 knockdown cells (Figure 3a). acids 97–118 that is predicted by PROSITE (http:// Similar results were obtained in dysbindin-1 knock- us.expasy.org/prosite/). We therefore generated two down cells (Figure 3b). Consistent with previous deletion mutants in which amino acids 89–188 or 97– findings,29 the mRNA levels of Coronin 1b and Rab13, 118 were deleted (Figure 2a). We transfected HEK293 two factors associated with neurite outgrowth, were cells with FLAG–necdin along with either EGFP, significantly decreased in p53 knockdown cells EGFP-tagged full-length dysbindin-1 or EGFP-tagged treated with retinoic acid or low-concentration serum

Molecular Psychiatry Regulation of neurite outgrowth by dysbindin-1 XMaet al 1110 ab EGFP+ - - - dysbindin-EGFP - + - - 1 89 97 118 188 351 dysbindin-N-EGFP - - + - dysbindin dysbindin-C-EGFP - - - + 1 217 FLAG-Ndn +++ + kDa dysbindin-N IB:-FLAG 216 351 dysbindin-C 85 1 89 188 351 dysbindinCCD IP: 49 -EGFP  189 97 118188 351 IB: -EGFP 34 dysbindinLZM

LZM: Leucin zipper motif 26 CCD: Coiled coil domain IB:-FLAG Input

c EGFP + --- d dysbindin-EGFP - + - - dysbindinCCD-EGFP - -+-  ** ** dysbindin LZM-EGFP - - - + 18 FLAG-Ndn + +++ + + ** ** 16 IB:-FLAG 14 12 10 85 8 IP: 6  IB:-EGFP 49 -EGFP 4 2 Relative Luciferase Activity 34 0 FLAG-p53 - +++++ + + + FLAG-Ndn - - + + + 26 dysbindin-EGFP - - - + - dysbindinLZM-EGFP - - - - + IB:-FLAG Input

e HA-Ndn Hochest Merge ABCD EGFP

EFGH Dys-EGFP

IJKL LZM-  EGFP Dys

Figure 2 Amino acids 97–118 of dysbindin-1 are essential for its ability to interact with and influence necdin. (a) Schematic representation of dysbindin-1–enhanced green fluorescence protein (EGFP) and its mutants. (b) Immunoprecipitation assays were performed to show that dysbindin-1–EGFP or its N terminus, but not its C terminus, interacts with FLAG–necdin (Ndn) in HEK293A cells. (c) Immunoprecipitation assays were performed showing that the LZM of dysbindin-1 (amino acids 97–118) is required for its interaction with necdin. (d) Dual-luciferase p53 reporter assays using HCT116 p53-null cells transfected with expression plasmids (as indicated) were performed to show that dysbindin-1DLZM fails to antagonize necdin’s repressive effects on a p53 reporter gene. Quantitative data from three independent experiments were quantified by one-way analysis of variance, **P < 0.005. (e) Immunofluorescence analyses were performed to show that dysbindin-1DLZM–EGFP fails to recruit HA–Ndn to the cytoplasm of HEK293A cells coexpressing HA–Ndn and dysbindin-1DLZM–EGFP (I–L), whereas dysbindin-1–EGFP recruits HA–Ndn to the cytoplasm (E–H); bar: 10 mm. LZM, leucine zipper motif.

Molecular Psychiatry Regulation of neurite outgrowth by dysbindin-1 XMaet al 1111 (Figure 3c). Interestingly, similar changes were ob- down (Supplementary Figure S3A). Furthermore, served in dysbindin-1 knockdown cells (Figure 3d). overexpression of p53 restored the neurite outgrowth Moreover, no alterations in p53 or necdin expression that was repressed by dysbindin-1 knockdown levels were observed when dysbindin-1 was knocked (Figure 3e).

ac ** si-NC + - + - si-p53 - + - + 6 m) Coronin 1b si-NC si-p53  5 Rab13 4

3 p53

2 -actin 1 lower serum RA Average neuirte length ( Average neuirte 0 d si-NC si-p53 si-NC +- + - si-dys - + - + b ** 8 Coronin 1b

si-NC si-dys m) 7  Rab13 6 dysbindin 5 -actin 4 3 lower serum RA

2 si-NC si-dys 1 * * * * Average neuirte length ( 1.0 0 si-NC si-dys 0.8

0.6 e 04

Phase EGFP/p53- EGFP Merge Relative density 0.2 ABC 0

13 13 Rab Rab

Coronin 1b Coronin 1b lower serum RA si-NC+EGFP

* 4.0 DEF * EGFP + + - m)

 3.5 p53-EGFP - - + si-NC + -- 3.0 si-dys - + + 2.5

si-dys+EGFP 2.0 dysbindin 15 p53-EGFP GHI 1.0 GAPDH

Average neurite length ( Average neurite 0.5 0 P si-dys+p53-EGFP si-NC+EGFsi-dys+EGFP

si-dys+p53-EGFP Figure 3 For caption see next page.

Molecular Psychiatry Regulation of neurite outgrowth by dysbindin-1 XMaet al 1112 Influence of dysbindin-1 on p53 target gene expression dysbindin-1 interacts directly with necdin (Supple- and neurite outgrowth mentary Figure S1A). Dysbindin-1 overexpression To further explore the physiological roles of dysbin- results in necdin translocation to the cytoplasm din-1 in p53 target gene expression, we examined (Figure 1c). Because necdin functions in the nucleus Coronin 1b and Rab13 levels in primary cultured to repress p53 transcriptional activity, recruitment of neurons and in sandy mice. In primary cultured necdin to the cytoplasm by dysbindin-1 may cause neurons from rat cortex, knockdown of dysbindin-1 necdin to fail to repress p53 transcriptional activity. decreased both Coronin 1b and Rab13 mRNA levels Furthermore, evidence for the involvement of these (Figure 4a). In sandy mice in which dysbindin-1 is interactions in the regulation of p53 transcriptional not expressed, the mRNA levels of p21, Rab13 and activity comes from observations that the repressive Coronin 1b were significantly decreased (Figure 4b). effects of necdin on p53 cannot be blocked by The protein levels of p21 in the hippocampi of sandy dysbindin-1DLZM–EGFP, a deletion mutant that mice were also lower than in control mice (Figure 4c). neither interacts with necdin nor changes necdin’s Moreover, we observed no alteration in the mRNA cellular distribution (Figures 2d and e). These data levels of p53 or necdin in dysbindin-1 knockdown demonstrate that dysbindin-1 upregulates p53 tran- primary cortical neurons or in sandy mice (Supple- scriptional activity by interacting with necdin and mentary Figure S3B and C). Finally, to test the recruiting it to the cytoplasm (Figure 5). possibility that dysbindin-1 might influence neurite Although p53 promotes the expression of many outgrowth in cultured primary neurons, we cultured apoptosis-related proteins when cells are exposed to cortical neurons derived from newborn WT mice or apoptotic stressors, a non-apoptotic role of p53 in sandy mice (postnatal days 0 and 1) for neurite neuronal development has also been proposed. It has outgrowth. Cortical neurons from sandy mice showed been well documented that p53 can act as either an shorter neurite lengths than WT mice after being apoptotic or non-apoptotic factor based on its post- cultured for 5 days (B24% decrease, P = 0.0088). transcriptional modification pattern.26 In our experi- Moreover, no alteration was observed in neuronal mental system, overexpression of p53 in N2a cells soma size between WT mice and sandy mice failed to induce cell death in either normal or (Figure 4d and Supplementary Figure S4). These data differentiation conditions (Supplementary Figure reveal that dysbindin-1-deficient neurons have a S5), because the cells were not exposed to apoptotic defect in neurite outgrowth. stressors. Moreover, it has been well documented that there is a tissue-specific restriction of the p53 response.52,53 Furthermore, high levels of p53 mRNA Discussion are present in some areas in the developing brain, but In this study, we demonstrate a novel function of the cells do not undergo p53-dependent apopto- dysbindin-1, whereby it regulates p53 activity and sis.54,55 Interestingly, in p53-deficient mice, defects subsequently influences neurite outgrowth. We de- of the neural tube have been observed,56 indicating monstrate that dysbindin-1 upregulates p53 transcrip- that p53 has non-apoptotic functions during neural tional activity to promote neurite outgrowth by development. interacting with necdin and recruiting it to the We observed that dysbindin-1 knockdown, simi- cytoplasm. Necdin is a member of the melanoma larly to p53 knockdown, represses neurite outgrowth. antigen-encoding gene family.46,47 It is predominantly p53 overexpression restores neurite outgrowth in expressed in postmitotic neurons and is associated dysbindin-1 knockdown cells (Figure 3). These data with Prader–Willi syndrome, a neurodevelopmental suggest that dysbindin-1 functions in the p53 path- disorder.48,49 Necdin has been identified as a way to regulate neurite outgrowth. In vivo studies multifunctional protein that interacts with and further show that the mRNA levels of Coronin 1b and represses transcription factors such as E2F1 and p53 Rab13, two p53 target genes, are both downregulated in the nucleus.38,50,51 In our study, we found that in sandy mice (Figure 4b). Furthermore, cortical

Figure 3 Effect of dysbindin-1 on p53-mediated neurite outgrowth. (a) Neurite outgrowth assays were performed showing that knockdown of p53 by small interfering RNA (siRNA) inhibits neurite outgrowth in N2a cells. The bar graphs show the average neurite length of each group measured by Image J software from three independent experiments (bar: 10 mm, number of cells: B200, **P < 0.005, one-way analysis of variance (ANOVA)). (b) Neurite outgrowth assays were performed showing that knockdown of dysbindin-1 by siRNA inhibits neurite outgrowth in N2a cells. The bar graphs show the average neurite length of each group from three independent fields (bar: 10 mm, number of cells: B200, **P < 0.005, one-way ANOVA). (c) Reverse transcription-PCR (RT-PCR) assays were performed showing that knockdown of p53 decreases Coronin 1b and Rab13 mRNA levels in N2a cells. (d) RT-PCR assays were performed showing that knockdown of dysbindin-1 decreases Coronin 1b and Rab13 mRNA levels in SH-SY-5Y cells. Densitometric analyses from three independent experiments were quantified by one-way ANOVA, *P < 0.05. (e) Neurite outgrowth assays were performed showing that p53–enhanced green fluorescence protein (EGFP; G–I), but not EGFP alone (D–F), restores the neurite outgrowth that is blocked by dysbindin-1 knockdown in N2a cells. The bar graphs show the average neurite length of each group from three independent experiments (bar: 10 mm, number of EGFP-positive cells: B200, *P < 0.05, one-way ANOVA). The protein levels of p53–EGFP, dysbindin-1 and glyceraldehyde-3-phosphate dehydrogenase were also detected using immunoblot analyses.

Molecular Psychiatry Regulation of neurite outgrowth by dysbindin-1 XMaet al 1113 absi-NC WT si-dys Sdy

si-NC + - * * * * * si-dys - + 1.0 WT Sandy 1.0 Coronin 1b 0.8 p21 0.8

Rab13 0.6 Rab13 0.6 -actin 0.4 Coronin 1b 0.4 Relative density dysbindin Relative density 0.2 -actin 0.2 0 0

p21 nin 1b Rab13 Rab13

Coro Coronin 1b

c * 1.0

WT Sandy 0.8 p21 0.6

GAPDH 0.4

dysbindin Relative density 0.2 0 y WT Sand

d Tuj1 Hochest Merge

A B C

*

100 WT mice 80

60

D EF 40 cortical nerons 20 Neurite length, % of WT

0 WT Sandy Sandy mice

Figure 4 Influence of dysbindin-1 on p53 target gene expression in vivo and neurite outgrowth defects in cortical neurons of sandy mice. (a) Reverse transcription-PCR (RT-PCR) assays were performed to show that knockdown of dysbindin-1 in rat primary cortex neurons decreases the mRNA levels of Coronin 1b and Rab13. Densitometric data from three independent experiments were quantified by one-way analysis of variance (ANOVA), *P < 0.05. (b) RT-PCR assays were performed to show that the mRNA levels of p21, Coronin 1b and Rab13 are all lower in the hippocampi of sandy mice than in those of wild-type (WT) mice of the same age. Densitometric data from three independent experiments were quantified by one-way ANOVA, *P < 0.05. (c) Immunoblot analyses were performed to show that p21 protein levels are lower in the hippocampi of sandy mice than in those of WT mice of the same age. Densitometric data from three independent experiments were quantified by one-way ANOVA, *P < 0.05. (d) Representative image of cortical neurons from WT mice (C57BL/6J) and dysbindin-1- deficient sandy mice in NB/B27 (neurobasal medium containing 1 Â B27 and glutamine) medium for 5 days. Neurons were immunostained with anti-Tuj1 antibody (an anti neuron-specific class III b-tubulin antibody) to show neurons (red) and Hoechst to show nuclei (blue). The bar graphs show the relative average neurite length of B330 neurons from WT or sandy mice from three independent experiments (WT is 100%, bar: 10 mm, *P < 0.05, one-way ANOVA).

neurons of sandy mice display neurite outgrowth It has been reported that Rab13 promotes neurite defects compared with those of WT mice (Figure 4d). outgrowth by interacting with and counteracting Taken together, we propose that dysbindin-1 neurite outgrowth inhibitors.57 Depletion of Coronin may activate p53 by attenuating necdin repression, 1b from cells alters lamellipodial dynamics due to allowing the maintenance of p53 transcriptional increased actin filament nucleation in the leading activity and the gene expression necessary for neurite edge.58 Interestingly, in cultured hippocampal neu- outgrowth. rons from sandy mice, there is a derangement of actin

Molecular Psychiatry Regulation of neurite outgrowth by dysbindin-1 XMaet al 1114 Dys decrease in dysbindin-1 protein in schizophrenia nucleus patients may lead to susceptibility to illness through abnormal development of the neural system. Ndn p53 coronin 1b, rab13 In conclusion, this study reveals a novel function of dysbindin-1 in association with neurite outgrowth. cytoplasm DysDs Ndn Conflict of interest Dysbindin-1 recruits necdin in cytoplasm Ndn The authors declare no conflict of interest. Dys nucleus Acknowledgments Ndn p53 coronin 1b, rab13 This work was supported, in part, by the National Natural Sciences Foundation of China (no. 30770664 cytoplasm and 30970921) and the CAS Knowledge Innovation Dys Ndn Ndn Project (KSCX2-YW-R138).

References Expression level of Coronin 1b and Rab13 increased 1 Freedman R. Schizophrenia. N Engl J Med 2003; 349: 1738–1749. 2 Ross CA, Margolis RL, Reading SA, Pletnikov M, Coyle JT. Neurite Outgrowth Neurobiology of schizophrenia. Neuron 2006; 52: 139–153. 3 Riley B, Kendler KS. Molecular genetic studies of schizophrenia. Figure 5 A diagram summarizing our findings: recruit- Eur J Hum Genet 2006; 14: 669–680. ment of necdin to the cytoplasm by dysbindin-1 promotes 4 Schwab SG, Knapp M, Mondabon S, Hallmayer J, Borrmann- p53 transcriptional activity when cells undergo differentia- Hassenbach M, Albus M et al. Support for association of tion. This increased p53 transcriptional activity promotes schizophrenia with genetic variation in the 6p22.3 gene, dysbin- the expression of neurite outgrowth-related genes (coronin din, in sib-pair families with linkage and in an additional sample 1b and rab13), which in turn induce neurite outgrowth. of triad families. Am J Hum Genet 2003; 72: 185–190. 5 Tang JX, Zhou J, Fan JB, Li XW, Shi YY, Gu NF et al. Family-based association study of DTNBP1 in 6p22.3 and schizophrenia. Mol Psychiatry 2003; 8: 717–718. filaments in the growth cone and an abnormality of 6 Funke B, Finn CT, Plocik AM, Lake S, DeRosse P, Kane JM et al. 20 Association of the DTNBP1 locus with schizophrenia in a U.S. neurite morphogenesis. In this study, we show that population. Am J Hum Genet 2004; 75: 891–898. dysbindin-1 knockdown reduces the expression of 7 Weickert CS, Rothmond DA, Hyde TM, Kleinman JE, Straub RE. both Rab13 and Coronin 1b (Figure 3d). It is notable Reduced DTNBP1 (dysbindin-1) mRNA in the hippocampal that the expression of dysbindin-1 is developmentally formation of schizophrenia patients. Schizophr Res 2008; 98: 105–110. regulated in association with biogenesis of lysosome- 8 Talbot K, Eidem WL, Tinsley CL, Benson MA, Thompson EW, related organelles complex-1 during neuronal devel- Smith RJ et al. Dysbindin-1 is reduced in intrinsic, glutamatergic opment.11 It is therefore possible that the regulation of terminals of the hippocampal formation in schizophrenia. J Clin these two gene products by dysbindin-1 has an Invest 2004; 113: 1353–1363. 9 Fallgatter AJ, Herrmann MJ, Hohoff C, Ehlis AC, Jarczok TA, important role in neurite outgrowth during neuronal Freitag CM et al. DTNBP1 (dysbindin) gene variants modulate development. Further studies on the correlation prefrontal brain function in healthy individuals. Neuropsycho- between dysbindin-1 and p53 target gene products pharmacology 2006; 31: 2002–2010. necessary for neurite outgrowth during brain devel- 10 Donohoe G, Morris DW, Clarke S, McGhee KA, Schwaiger S, opment would contribute to our understanding of the Nangle JM et al. Variance in neurocognitive performance is associated with dysbindin-1 in schizophrenia: a preliminary detailed functions of dysbindin-1. study. Neuropsychologia 2007; 45: 454–458. Recently, multiple lines of evidence have suggested 11 Ghiani CA, Starcevic M, Rodriguez-Fernandez IA, Nazarian R, that neuronal developmental events may be involved Cheli VT, Chan LN et al. The dysbindin-containing complex in the pathogenesis of schizophrenia.59 Several (BLOC-1) in brain: developmental regulation, interaction with susceptibility genes for schizophrenia,16 including SNARE proteins and role in neurite outgrowth. Mol Psychiatry 17,18 19 60 2010; 15: 115, 204–115. DISC1, NRG1 and Akt, are tightly associated 12 Talbot K, Cho DS, Ong WY, Benson MA, Han LY, Kazi HA et al. with neuronal development. In postmortem brains of Dysbindin-1 is a synaptic and microtubular protein that binds schizophrenia patients, spine density is lower than in brain snapin. Hum Mol Genet 2006; 15: 3041–3054. normal controls.59 In primary cultured neurons, 13 Numakawa T, Yagasaki Y, Ishimoto T, Okada T, Suzuki T, Iwata N et al. Evidence of novel neuronal functions of dysbindin, a knockdown of dysbindin-1 results in abnormal susceptibility gene for schizophrenia. Hum Mol Genet 2004; 13: 23 dendritic spine formation, suggesting an involve- 2699–2708. ment of dysbindin-1 in neuronal development. Our 14 Iizuka Y, Sei Y, Weinberger DR, Straub RE. Evidence that the study shows the involvement of dysbindin-1 in BLOC-1 protein dysbindin modulates dopamine D2 receptor internalization and signaling but not D1 internalization. J Neurosci neurite outgrowth through the p53 pathway, provid- 2007; 27: 12390–12395. ing a mechanistic explanation for the association of 15 Ji Y, Yang F, Papaleo F, Wang HX, Gao WJ, Weinberger DR et al. dysbindin-1 with neuronal development. Thus, a Role of dysbindin in dopamine receptor trafficking and

Molecular Psychiatry Regulation of neurite outgrowth by dysbindin-1 XMaet al 1115 cortical GABA function. Proc Natl Acad Sci USA 2009; 106: suppressor necdin modulates proliferation and collagen expres- 19593–19598. sion. Proc Natl Acad Sci USA 2003; 100: 10008–10013. 16 Bellon A. New genes associated with schizophrenia in neurite 36 Kuwajima T, Nishimura I, Yoshikawa K. Necdin promotes formation: a review of cell culture experiments. Mol Psychiatry GABAergic neuron differentiation in cooperation with Dlx home- 2007; 12: 620–629. odomain proteins. J Neurosci 2006; 26: 5383–5392. 17 Kamiya A, Tomoda T, Chang J, Takaki M, Zhan C, Morita M et al. 37 Liu X, Wang Y, Zhang Y, Zhu W, Xu X, Niinobe M et al. DISC1-NDEL1/NUDEL protein interaction, an essential compo- Nogo-A inhibits necdin-accelerated neurite outgrowth by nent for neurite outgrowth, is modulated by genetic variations of retaining necdin in the cytoplasm. Mol Cell Neurosci 2009; 41: DISC1. Hum Mol Genet 2006; 15: 3313–3323. 51–61. 18 Ozeki Y, Tomoda T, Kleiderlein J, Kamiya A, Bord L, Fujii K et al. 38 Taniura H, Matsumoto K, Yoshikawa K. Physical and functional Disrupted-in-schizophrenia-1 (DISC-1): mutant truncation pre- interactions of neuronal growth suppressor necdin with p53. J Biol vents binding to NudE-like (NUDEL) and inhibits neurite out- Chem 1999; 274: 16242–16248. growth. Proc Natl Acad Sci USA 2003; 100: 289–294. 39 Taniura H, Taniguchi N, Hara M, Yoshikawa K. Necdin, a 19 Gerecke KM, Wyss JM, Carroll SL. Neuregulin-1beta induces postmitotic neuron-specific growth suppressor, interacts with neurite extension and arborization in cultured hippocampal viral transforming proteins and cellular transcription factor neurons. Mol Cell Neurosci 2004; 27: 379–393. E2F1. J Biol Chem 1998; 273: 720–728. 20 Kubota K, Kumamoto N, Matsuzaki S, Hashimoto R, Hattori T, 40 Sherr CJ, Roberts JM. CDK inhibitors: positive and negative Okuda H et al. Dysbindin engages in c-Jun N-terminal kinase regulators of G1-phase progression. Genes Dev 1999; 13: activity and cytoskeletal organization. Biochem Biophys Res 1501–1512. Commun 2009; 379: 191–195. 41 Pechnick RN, Zonis S, Wawrowsky K, Pourmorady J, 21 Feng YQ, Zhou ZY, He X, Wang H, Guo XL, Hao CJ et al. Chesnokova V. p21Cip1 restricts neuronal proliferation in the Dysbindin deficiency in sandy mice causes reduction of snapin subgranular zone of the dentate gyrus of the hippocampus. Proc and displays behaviors related to schizophrenia. Schizophr Res Natl Acad Sci USA 2008; 105: 1358–1363. 2008; 106: 218–228. 42 Benson MA, Newey SE, Martin-Rendon E, Hawkes R, Blake DJ. 22 Li W, Zhang Q, Oiso N, Novak EK, Gautam R, O’Brien EP et al. Dysbindin, a novel coiled-coil-containing protein that interacts Hermansky-Pudlak syndrome type 7 (HPS-7) results from mutant with the dystrobrevins in muscle and brain. J Biol Chem 2001; 276: dysbindin, a member of the biogenesis of lysosome-related 24232–24241. organelles complex 1 (BLOC-1). Nat Genet 2003; 35: 84–89. 43 Brynczka C, Labhart P, Merrick BA. NGF-mediated transcriptional 23 Ito H, Morishita R, Shinoda T, Iwamoto I, Sudo K, Okamoto K targets of p53 in PC12 neuronal differentiation. BMC Genomics et al. Dysbindin-1, WAVE2 and Abi-1 form a complex that 2007; 8: 139. regulates dendritic spine formation. Mol Psychiatry 2010; 15: 44 Bani-Yaghoub M, Amundsen DE. Study and simulation of 976–986. reaction-diffusion systems affected by interacting signaling path- 24 Starcevic M, Dell’Angelica EC. Identification of snapin ways. Acta Biotheor 2008; 56: 315–328. and three novel proteins (BLOS1, BLOS2, and BLOS3/reduced 45 Wu PY, Lin YC, Chang CL, Lu HT, Chin CH, Hsu TT et al. pigmentation) as subunits of biogenesis of lysosome- Functional decreases in P2X7 receptors are associated with related organelles complex-1 (BLOC-1). J Biol Chem 2004; 279: retinoic acid-induced neuronal differentiation of neuro-2a neuro- 28393–28401. blastoma cells. Cell Signal 2009; 21: 881–891. 25 Harms K, Nozell S, Chen X. The common and distinct target genes 46 Maruyama K, Usami M, Aizawa T, Yoshikawa K. A novel brain- of the p53 family transcription factors. Cell Mol Life Sci 2004; 61: specific mRNA encoding nuclear protein (necdin) expressed in 822–842. neurally differentiated embryonal carcinoma cells. Biochem 26 Tedeschi A, Di Giovanni S. The non-apoptotic role of p53 in Biophys Res Commun 1991; 178: 291–296. neuronal biology: enlightening the dark side of the moon. EMBO 47 Uetsuki T, Takagi K, Sugiura H, Yoshikawa K. Structure and Rep 2009; 10: 576–583. expression of the mouse necdin gene. Identification of a 27 Zhang J, Yan W, Chen X. p53 is required for nerve growth factor- postmitotic neuron-restrictive core promoter. J Biol Chem 1996; mediated differentiation of PC12 cells via regulation of TrkA 271: 918–924. levels. Cell Death Differ 2006; 13: 2118–2128. 48 Jay P, Rougeulle C, Massacrier A, Moncla A, Mattei MG, Malzac P 28 Di Giovanni S, De Biase A, Yakovlev A, Finn T, Beers J, Hoffman et al. The human necdin gene, NDN, is maternally imprinted and EP et al. In vivo and in vitro characterization of novel neuronal located in the Prader-Willi syndrome chromosomal region. Nat plasticity factors identified following spinal cord injury. J Biol Genet 1997; 17: 357–361. Chem 2005; 280: 2084–2091. 49 MacDonald HR, Wevrick R. The necdin gene is deleted in Prader- 29 Di Giovanni S, Knights CD, Rao M, Yakovlev A, Beers J, Catania J Willi syndrome and is imprinted in human and mouse. Hum Mol et al. The tumor suppressor protein p53 is required for neurite Genet 1997; 6: 1873–1878. outgrowth and axon regeneration. EMBO J 2006; 25: 4084–4096. 50 Taniura H, Kobayashi M, Yoshikawa K. Functional domains 30 Tedeschi A, Nguyen T, Steele SU, Feil S, Naumann U, Feil R et al. of necdin for protein-protein interaction, nuclear matrix The tumor suppressor p53 transcriptionally regulates cGKI targeting, and cell growth suppression. J Cell Biochem 2005; 94: expression during neuronal maturation and is required for 804–815. cGMP-dependent growth cone collapse. J Neurosci 2009; 29: 51 Kurita M, Kuwajima T, Nishimura I, Yoshikawa K. Necdin 15155–15160. downregulates CDC2 expression to attenuate neuronal apoptosis. 31 Moore DL, Blackmore MG, Hu Y, Kaestner KH, Bixby JL, Lemmon J Neurosci 2006; 26: 12003–12013. VP et al. KLF family members regulate intrinsic axon regeneration 52 Midgley CA, Owens B, Briscoe CV, Thomas DB, Lane DP, Hall PA. ability. Science 2009; 326: 298–301. Coupling between gamma irradiation, p53 induction and the 32 MacGillavry HD, Stam FJ, Sassen MM, Kegel L, Hendriks WT, apoptotic response depends upon cell type in vivo. J Cell Sci 1995; Verhaagen J et al. NFIL3 and cAMP response element-binding 108(Pt 5): 1843–1848. protein form a transcriptional feedforward loop that controls 53 Clarke AR, Gledhill S, Hooper ML, Bird CC, Wyllie AH. p53 neuronal regeneration-associated gene expression. J Neurosci dependence of early apoptotic and proliferative responses within 2009; 29: 15542–15550. the mouse intestinal epithelium following gamma-irradiation. 33 Benson MA, Tinsley CL, Blake DJ. Myospryn is a novel Oncogene 1994; 9: 1767–1773. binding partner for dysbindin in muscle. J Biol Chem 2004; 279: 54 Komarova EA, Chernov MV, Franks R, Wang K, Armin G, 10450–10458. Zelnick CR et al. Transgenic mice with p53-responsive lacZ: p53 34 Takazaki R, Nishimura I, Yoshikawa K. Necdin is required for activity varies dramatically during normal development and terminal differentiation and survival of primary dorsal root determines radiation and drug sensitivity in vivo. EMBO J 1997; ganglion neurons. Exp Cell Res 2002; 277: 220–232. 16: 1391–1400. 35 Hu B, Wang S, Zhang Y, Feghali CA, Dingman JR, Wright TM. A 55 Gottlieb E, Haffner R, King A, Asher G, Gruss P, Lonai P et al. nuclear target for interleukin-1alpha: interaction with the growth Transgenic mouse model for studying the transcriptional activity

Molecular Psychiatry Regulation of neurite outgrowth by dysbindin-1 XMaet al 1116 of the p53 protein: age- and tissue-dependent changes in 58 Cai L, Marshall TW, Uetrecht AC, Schafer DA, Bear JE. Coronin 1B radiation-induced activation during embryogenesis. EMBO J coordinates Arp2/3 complex and cofilin activities at the leading 1997; 16: 1381–1390. edge. Cell 2007; 128: 915–929. 56 Armstrong JF, Kaufman MH, Harrison DJ, Clarke AR. High- 59 Beneyto M, Lewis DA. Insights into the neurodevelopmental frequency developmental abnormalities in p53-deficient mice. origin of schizophrenia from postmortem studies of prefrontal Curr Biol 1995; 5: 931–936. cortical circuitry. Int J Dev Neurosci 2010; doi:10.1016/j.ijdevneu. 57 Sakane A, Honda K, Sasaki T. Rab13 regulates neurite outgrowth 2010.08.003. in PC12 cells through its effector protein, JRAB/MICAL-L2. Mol 60 Read DE, Gorman AM. Involvement of Akt in neurite outgrowth. Cell Biol 2010; 30: 1077–1087. Cell Mol Life Sci 2009; 66: 2975–2984.

Supplementary Information accompanies the paper on the Molecular Psychiatry website (http://www.nature.com/mp)

Molecular Psychiatry