ORIGINAL ARTICLE Recruitment of PCM1 to the Centrosome by the Cooperative Action of DISC1 and BBS4 A Candidate for Psychiatric Illnesses Atsushi Kamiya, MD, PhD; Perciliz L. Tan, BSc; Ken-ichiro Kubo, MD, PhD; Caitlin Engelhard, BSc; Koko Ishizuka, MD, PhD; Akiharu Kubo, MD, PhD; Sachiko Tsukita, PhD; Ann E. Pulver, ScD; Kazunori Nakajima, MD, PhD; Nicola G. Cascella, MD; Nicholas Katsanis, PhD; Akira Sawa, MD, PhD Context: A role for the centrosome has been suggested Main Outcome Measures: Protein interaction and re- in the pathology of major mental illnesses, especially cruitment at the centrosome in cells; neuronal migra- schizophrenia (SZ). tion in the cerebral cortex; and variant discovery in PCM1 in patients with SZ. Objectives: To show that pericentriolar material 1 pro- tein (PCM1) forms a complex at the centrosome with dis- Results: PCM1 forms a complex with DISC1 and BBS4 rupted-in-schizophrenia 1 (DISC1) and Bardet-Biedl syn- through discrete binding domains in each protein. DISC1 drome 4 protein (BBS4), which provides a crucial pathway and BBS4 are required for targeting PCM1 and other cargo for cortical development associated with the pathology proteins, such as ninein, to the centrosome in a syner- of SZ. To identify mutations in the PCM1 gene in an SZ gistic manner. In the developing cerebral cortex, sup- population. pression of PCM1 leads to neuronal migration defects, which are phenocopied by the suppression of either DISC1 Design: Interaction of DISC1, PCM1, and BBS proteins was assessed by immunofluorescent staining and coim- or BBS4 and are exacerbated by the concomitant sup- munoprecipitation. Effects of PCM1, DISC1, and BBS on pression of both. Furthermore, a nonsense mutation centrosomal functions and corticogenesis in vivo were that segregates with SZ spectrum psychosis was found tested by RNA interference. The PCM1 gene was exam- in 1 family. ined by sequencing 39 exons and flanking splice sites. Conclusions: Our data further support for the role of Setting: Probands and controls were from the collec- centrosomal proteins in cortical development and sug- tion of one of us (A.E.P.). gest that perturbation of centrosomal function contrib- utes to the development of mental diseases, including SZ. Patients: Thirty-two probands with SZ from families that had excess allele sharing among affected individuals at 8p22 and 219 white controls. Arch Gen Psychiatry. 2008;65(9):996-1006 ECENT GENETIC STUDIES PCM1 is a component of centriolar sat- have suggested that centro- ellites and acts as a scaffold to target sev- somal dysfunction under- eral proteins to the centrosome in a dynein lies risks for various neu- motor-dependent manner and regulate mi- ropsychiatric disorders, crotubular dynamics.18-20 PCM1 also inter- Rbecause variants in some genes that en- acts with Bardet-Biedl syndrome 4 protein code centrosomal proteins have been as- (BBS4), which is encoded by one of the sociated with schizophrenia (SZ) and causative genes for Bardet-Biedl syn- bipolar disorder (BP).1-4 These genes in- drome (BBS), an inherited disorder char- clude pericentriolar material 1 (PCM1) on acterized by renal dysfunction, obesity, chromosome 8p22,2 one of the reproduc- polydactyly, and diverse neuropsychiatric ible linkage loci for SZ and BP,5-8 and dis- symptoms.21-24 Bardet-Biedl syndrome is ge- rupted-in-schizophrenia 1 (DISC1).3,4 The netically heterogeneous, with 12 genes centrosome plays a role in organizing mi- identified to date, but mutations in each of crotubules, contributing to cell cycle pro- these genes lead to similar pathology in hu- gression, cell polarization, and ciliogen- mans, suggesting that BBS proteins func- esis.9-12 Consequently, the centrosome is tion through a common molecular path- Author Affiliations are listed at required for proper neurodevelopment, es- way. Consistent with this notion, all BBS the end of this article. pecially in the cerebral cortex.13-17 proteins investigated to date localize pri- (REPRINTED) ARCH GEN PSYCHIATRY/ VOL 65 (NO. 9), SEP 2008 WWW.ARCHGENPSYCHIATRY.COM 996 ©2008 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/25/2021 marily at the centrosome and the basal body of ciliated cells, CELL CULTURE AND TRANSFECTION where they contribute to the maintenance of microtubu- lar dynamics, as well as intracellular transport and ciliary HEK293 cells were maintained in Dulbecco’s modified Eagle function.25-30 medium with 10% fetal bovine serum and 1% penicillin- We have reported previously that DISC1, a major sus- streptomycin. PC12 cells were maintained in Dulbecco’s modi- ceptibility factor for SZ and BP, plays a crucial role at the fied Eagle medium with 10% fetal bovine serum, 5% horse se- 31,32 rum, and 1% penicillin-streptomycin. Transfection of expression centrosome, while another group has reported con- constructs or RNAi constructs was carried out with Lipofect- sistently that DISC1 interacts with kendrin, a compo- amine 2000 (Invitrogen, Carlsbad, California) for PC12 cells 33 nent of pericentriolar material. Consequently, DISC1 and with PolyFect Transfection Reagent (Qiagen, Valencia, Cali- is required for neurite outgrowth and proper develop- fornia) for HEK293 cells. The molar ratio of pEGFP-F to RNAi ment of the cerebral cortex, such as neuronal migration plasmid(s) was 1:3 for the transfection. Rodent primary corti- and dendritic arborization.31 Therefore, we hypoth- cal neurons were prepared as described previously.37 esized that PCM1, DISC1, and the BBS proteins may in- teract and play a role in the centrosome and that such COIMMUNOPRECIPITATION AND interactions might be relevant both to the DISC- CELL EXTRACTION associated neurodevelopmental functions and to the etio- pathology of SZ. Immunoprecipitation Herein, we provide biological and genetic evidence that PCM1-DISC1-BBS proteins form a centrosomal path- Cells were lysed in a RIPA buffer (50mM TRIS–hydrogen chlo- way, potentially associating with major mental ill- ride, pH 7.4, 150mM sodium chloride, 5mM magnesium nesses, such as SZ. These proteins form a complex at the chloride, 5mM dithiothreitol, 1mM phenylmethylsulfonyl fluo- centrosome through discrete binding domains. DISC1 and ride, 1mM ethylene diamine tetraacetic acid, 1% Triton X-100, BBS4 act synergistically to recruit PCM1 and associated and protease inhibitor mixture [Roche, Basel, Switzerland]). proteins to the centrosome. Disruption of the PCM1- Precleared supernatants (500 µg) from crude cell lysates cen- DISC1-BBS4 pathway leads to profound defects in neu- trifuged at 14 000ϫg for 10 minutes were incubated with pri- ronal migration during cortical development. Finally, we mary antibodies (1 µg/mL of rabbit polyclonal antibody against report a pedigree in which a nonsense mutation in the HA-tag or against myc-tag) overnight, which was followed by the addition of TrueBlot anti-Rabbit Ig IP Beads (eBioscience, PCM1 gene segregates with SZ spectrum psychosis. San Diego, California) (30 µL) or Protein G Plus/Protein A Aga- rose (Calbiochem, Darmstadt, Germany) (30 µL) for 1 hour. The immunoprecipitates were washed 3 times by a TRIS- METHODS buffered saline–based buffer with 0.05% Tween 20 and ana- lyzed with sodium dodecyl sulfate polyacrylamide gel electro- phoresis (SDS-PAGE)/Western blotting. In the stringent wash PLASMIDS AND ANTIBODIES conditions, we added sodium chloride up to the final concen- tration at 500mM. ProFound Mammalian HA Tag IP/Co-IP Kit All the deletion DISC1 and PCM1 expression constructs were (Pierce, Rockford, Illinois) was also used. made by polymerase chain reaction–based mutagenesis proto- col.34 The deletion BBS4 expression constructs were made as Cell Extraction described previously.21 pEGFP-F was purchased from BD Bio- science Clontech (Mountain View, California). Rabbit poly- Cells were sonicated in ice-cold lysis buffer (50mM TRIS– clonal antibodies against PCM1, ninein, BBS1, BBS4, and BBS8 hydrogen chloride, pH 7.4, 150mM sodium chloride, 1% NP- 20,21,25,35 antibody were prepared as described previously. The 40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, following antibodies were also used: mouse monoclonal anti- and a protease inhibitor mixture). Extracted cells were mixed ␤ ␥ bodies against -tubulin and -tubulin (Sigma-Aldrich, with SDS-PAGE loading buffer after protein concentrations were St Louis, Missouri); mouse monoclonal antibodies against measured. Each protein sample (10 µg) was analyzed with HA-tag and myc-tag (BAbCO, Berkeley, California); rabbit SDS-PAGE followed by Western blotting. polyclonal antibody against HA-tag (Clontech); rabbit poly- clonal antibody against myc-tag (Santa Cruz Biotechnology, Santa Cruz, California); affinity-purified rabbit antiserum IMMUNOFLUORESCENT STAINING against green fluorescent protein (GFP) (Molecular Probes, Eugene, Oregon); and mouse monoclonal antibody against Cells were fixed with ice-cold methanol at −20°C 3 days after GFP (Nacalai Tesque, Kyoto, Japan). The rabbit polyclonal transfection. After blocking with 1.5% bovine serum albumin anti-DISC1 antibody (D27) was a gift from Nicholas J. Bran- and 0.5% normal goat serum in phosphate-buffered saline, cells don, PhD (Wyeth Discovery Neuroscience). Plasmids express- were treated with primary antibodies (dilution: ␥-tubulin, 1:100; ing interfering short hairpin RNA (shRNA)36 were generated to DISC1, 1:200; PCM1, 1:500; ninein, 1:500; BBS1, 1:300; BBS4, suppress endogenous DISC1, PCM1, and BBS4 protein expres- 1:500; BBS8, 1:500) for 1 hour followed by