Recruitment of PCM1 to the centrosome by the cooperative action of DISC1 and BBS4: a candidate for psychiatric illnesses - PubMed (original) (raw)

. 2008 Sep;65(9):996-1006.

doi: 10.1001/archpsyc.65.9.996.

Perciliz L Tan, Ken-ichiro Kubo, Caitlin Engelhard, Koko Ishizuka, Akiharu Kubo, Sachiko Tsukita, Ann E Pulver, Kazunori Nakajima, Nicola G Cascella, Nicholas Katsanis, Akira Sawa

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Recruitment of PCM1 to the centrosome by the cooperative action of DISC1 and BBS4: a candidate for psychiatric illnesses

Atsushi Kamiya et al. Arch Gen Psychiatry. 2008 Sep.

Abstract

Context: A role for the centrosome has been suggested in the pathology of major mental illnesses, especially schizophrenia (SZ).

Objectives: To show that pericentriolar material 1 protein (PCM1) forms a complex at the centrosome with disrupted-in-schizophrenia 1 (DISC1) and Bardet-Biedl syndrome 4 protein (BBS4), which provides a crucial pathway for cortical development associated with the pathology of SZ. To identify mutations in the PCM1 gene in an SZ population.

Design: Interaction of DISC1, PCM1, and BBS proteins was assessed by immunofluorescent staining and coimmunoprecipitation. Effects of PCM1, DISC1, and BBS on centrosomal functions and corticogenesis in vivo were tested by RNA interference. The PCM1 gene was examined by sequencing 39 exons and flanking splice sites.

Setting: Probands and controls were from the collection of one of us (A.E.P.).

Patients: Thirty-two probands with SZ from families that had excess allele sharing among affected individuals at 8p22 and 219 white controls.

Main outcome measures: Protein interaction and recruitment at the centrosome in cells; neuronal migration in the cerebral cortex; and variant discovery in PCM1 in patients with SZ.

Results: PCM1 forms a complex with DISC1 and BBS4 through discrete binding domains in each protein. DISC1 and BBS4 are required for targeting PCM1 and other cargo proteins, such as ninein, to the centrosome in a synergistic manner. In the developing cerebral cortex, suppression of PCM1 leads to neuronal migration defects, which are phenocopied by the suppression of either DISC1 or BBS4 and are exacerbated by the concomitant suppression of both. Furthermore, a nonsense mutation that segregates with SZ spectrum psychosis was found in 1 family.

Conclusions: Our data further support for the role of centrosomal proteins in cortical development and suggest that perturbation of centrosomal function contributes to the development of mental diseases, including SZ.

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Figures

Figure 1

Figure 1. PCM1, DISC1, and BBS proteins interact, localizing with γ-tubulin at the centrosome

(A) PCM1 interacts with DISC1. HA-tagged PCM1 was co-expressed with myc-tagged DISC1 in HEK293 cells. Cell extracts were immunoprecipitated with an anti-HA antibody. Immunoprecipitates were analyzed by Western blotting with an anti-myc antibody (upper panel). The input of each protein is also shown (middle and bottom panels). (B) BBS proteins interact with DISC1 in co-immunopecipitation in HEK293 cells. Myc-tagged BBS1, 2, 4, 5, 6, 7, and 8 all bind to HA-tagged DISC1. Consistent results were observed in both under low stringency (150mM NaCl) washing condition (upper panels) as well as high stringency (500mM NaCl) washing condition (lower panels). The input of each protein is shown in the middle and bottom panels. (C) Localization of BBS1, BBS4, DISC1, and PCM1 in immature cortical neurons at 3 days in vitro (DIV3). Endogenous BBS1, BBS4, and DISC1 (red) are co-localized with γ-tubulin at the centrosome (arrowheads). Endogenous PCM1 (red) mainly occurs just adjacent to γ-tubulin with slight overlap with each other. Blue, nucleus; green, γ-tubulin. Scale bar, 10 µm.

Figure 2

Figure 2. PCM1, DISC1, and BBS4 interact with each other through distinct binding domains

(A) The middle portion of DISC1 (amino acids 349–600) is crucial for DISC1-BBS4 protein interaction. The N-terminal portion (amino acids 1–348) and the C-terminal portion (amino acids 601–854) of DISC1 are important for the DISC1-PCM1 binding. HA-tagged three DISC1 protein fragments [DISC1 (N-348), DISC1 (349–600), and DISC1 (601-C)] were expressed in HEK293 cells for co-immunoprecipitation with an anti-HA antibody. The middle portion of DISC1, but not the N- nor C-terminal DISC1, binds to each of BBS1, 4, and 8, whereas the N- and C-terminal DISC1 bind to PCM1 (upper panels). The inputs of each protein are shown at the right and bottom panels. IB indicates antibodies used for Western blotting. (B) The C-terminus domain of DISC1 for interaction with PCM1 is distinct from the NDEL1 binding domain of DISC1. Deletion of DISC1-NDEL1 binding region [DISC1Δ(802–835)] had no effect on the interaction of DISC1 with PCM1. The inputs are shown in the middle and bottom panels. (C) The second TPR motif of BBS4 is crucial for the BBS4-DISC1 interaction. A series of myc-tagged BBS4 truncation mutants were co-expressed with HA-tagged DISC1 in HEK293 cells for co-immunoprecipitation with an anti-HA antibody. Deletion of the N-terminal region in [BBS4 (1-13-C)] and further deletion of the first TRP motif [BBS4 (2-13-C)] does not affect the BBS4-DISC1 interaction. By contrast, BBS4 mutants with further deletion of the second TRP motif [BBS4 (3-13-C) and BBS4 (4-13-C)] did not bind with DISC1. BBS4 lacking the C-terminal region [BBS4 (N-1-9)] bind to DISC1. The inputs are also shown (middle and bottom panels). (D) The third TPR motif of BBS4 is important for the BBS4-PCM1 interaction. Deletion of the third TPR motif [BBS4 (4-13-C)] dramatically weakened the interaction of myc-tagged BBS4 with HA-tagged PCM1. An anti-HA antibody was used for co-immunoprecipitation. The inputs are shown in the middle and bottom panels. (E) The middle portion of PCM1 is important for DISC1-PCM1 interaction. Myc-tagged DISC1 was co-expressed with HA-tagged PCM1 protein fragments in HEK293 cells for co-immunoprecipitation with an anti-myc antibody. PCM1 (741–1420) has stronger binding affinity to DISC1 than PCM1 (N-740) and PCM1 (1421-C). The inputs of each protein are also shown in middle and bottom panels. (F) Schematic of DISC1, BBS4, and PCM1 interaction shows that these proteins may interact with each other through distinct binding domains.

Figure 3

Figure 3. Synergistic effect of DISC1 and BBS4 on recruitment of PCM1 and ninein to the centrosome

(A) Efficient suppression of DISC1, BBS4, and PCM1 by RNAi. RNAi to DISC1, BBS4, and PCM1 suppress 78, 65, and 78% of endogenous DISC1, BBS4, and PCM1 expression, respectively, in PC12 cells (top panels). RNAi to DISC1 or BBS4 does not affect the levels of endogenous PCM1 (middle panels). IB: antibodies used for Western blotting. (B) Suppression of DISC1 and BBS4 reduces accumulation of PCM1 to the centrosome in PC12 cells in a synergistic manner. To quantify the accumulation, immunointensity of PCM1 in the centrosome area (white circle) relative to that in the whole cell region surrounded by green line was quantified. Bars represent averages of each group of cells in three independent and blinded experiments (***P<0.001, ** P<0.01, * P<0.05). Error bars represent SEM. Representative images are shown. Blue, nucleus; red, PCM1; green, pEGFP-F; white, γ-tubulin (also indicated by arrowheads). Scale bar, 10 µm. (C) Accumulation of ninein at the centrosome is disturbed by synergistic application of DISC1 and BBS4 RNAi, or PCM1 RNAi. Although neither application of DISC1 RNAi nor BBS4 RNAi leads to a significant effect on ninein, the synergistic application of both RNAi reduces accumulation of ninein to the centrosome, resembling the phenotype in the presence of RNAi to PCM1. To quantify the accumulation, immunointensity of ninein in the centrosome area (white circle) relative to that in the whole cell region surrounded by the green line was quantified. Bars represent averages of each group of cells in three independent and blinded experiments (* P<0.005, ** P<0.0001). Error bars represent SEM. Representative images of PC12 cells are shown. Blue, nucleus; red, ninein; green, pEGFP-F; white, γ-tubulin (also indicated by arrowheads). Scale bar, 10 µm.

Figure 4

Figure 4. Knockdown of DISC1, BBS4, and PCM1 leads to neuronal migration defects in the developing cerebral cortex

(A) RNAi constructs and GFP expression vectors were electroporated into the ventricular zone (VZ) at E15 and analyzed at P0. In brains with control RNAi (Con RNAi), 40% of GFP-labeled cells exited the VZ, and 25% of GFP-labeled cells completed migration and formed the superficial layers of the cortex that correspond to bins 9 and 10. By contrast, only less than 15% of GFP-positive cells reached the superficial layers in brain slices with DISC1 RNAi, BBS4 RNAi, or PCM1 RNAi, with the majority of GFP-positive cells remaining in the intermediate zone (IZ), subventricular zone (SVZ), and VZ. Green, cells co-transfected with GFP and RNAi constructs; purple, propidium iodide (PI). Scale bar: 100 µm. (B) A migration distance is shown. Silencing of DISC1, BBS4, or PCM1 induces delayed radial migration (** P<0.0001). Silencing of both DISC1 and BBS4 expression leads to a more severe defect compared with that with either DISC1 RNAi or BBS4 RNAi. * P<0.05. Values are mean ± SEM.

Figure 5

Figure 5. A nonsense mutation in PCM1 in a family with SZ and schizoaffective disorder

Mutation analysis of a Caucasian family JHU37007 shows a heterozygous 4057G→T mutation in exon 24 of PCM1, introducing a premature termination codon (E1353X); genotypes are shown below each individual, as are sequence traces. The psychiatric phenotype (if any) of each family member is also shown.

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