LD structure of NDEL1 haplotype block. LD (D′) structure using Haploview 3.32 (46). One SNP, rs 2012190, demonstrated extremely rare (<05%) minor allele frequency and was excluded from further analyses. Of the remaining SNPs, four formed a tight haplotype block spanning ∼36 kb, encompassing the NDEL1 gene.

LD structure of NDE1 haplotype block. LD (D′) structure using Haploview 3.32 (24). Four of the six SNPs formed a tight haplotype block spanning ∼30 kb, within the NDE1 gene.

Protein interaction of DISC1, NDEL1 and NDE1. (A) Self-association of NDEL1 and hetero-oligomerization of NDEL1 and NDE1. HA-tagged DISC1, NDEL1 or GAPDH were transfected with myc-tagged NDEL1 or NDE1 in HEK293 cells. Immunoprecipitates with an anti-HA antibody were analyzed by western blotting with an anti-myc antibody. Myc-tagged NDEL1 is co-precipitated with HA-tagged DISC1 and NDEL1, but not with GAPDH (top panel). NDE1 is also co-precipitated with HA-tagged NDEL1, but not with GAPDH (top panel), suggesting that NDEL1 self-associates and forms a hetero-oligomer with NDE1. For a negative control in co-immunoprecipitation, we used a rabbit IgG and observed no precipitation of target proteins (second panel). The inputs of each protein in co-immunoprecipitation are also shown (bottom panels). (B) The specificities of monoclonal antibodies against NDE1 (see Materials and Methods section) and NDEL1 (24) were evaluated by western blotting. Anti-NDE1 antibody recognizes exogenous NDE1 protein but not exogenous NDEL1 protein. On the other hand, anti-NDEL1 antibody detects exogenous NDEL1 protein but not exogenous NDE1 protein. (C) Endogenous protein interaction of NDE1–DISC1 was confirmed by co-immunoprecipitation with the tissue extract of rat adult cerebral cortex. A monoclonal antibody against NDE1 was used for immunoprecipitation. (D) Endogenous protein interaction of NDE1–NDEL1 was confirmed by co-immunoprecipitation with the cell extract of undifferentiated PC12 cells. A monoclonal antibody against NDEL1 was used for immunoprecipitation. (E) Amino acids 802–835 of DISC1 are critical for DISC1–NDEL1 interaction. HEK293 cells were transfected with myc-tagged NDEL1 and HA-tagged DISC1 or DISC1 lacking amino acids 802–835 [DISC1Δ(802–835)]. Lysates were then immunoprecipitated with an anti-myc antibody and analyzed by western blotting with an anti-HA antibody. Deletion of amino acids 802–835 of DISC1, which is a critical domain for DISC1–NDEL1 interaction we previously reported, completely abolished the interaction of exogenous DISC1 [DISC1Δ(802–835)] with NDEL1 (top panel). The inputs of each protein are shown (bottom panels). (F) Amino acids 802–835 of DISC1 are critical for DISC1–NDE1 interaction. Myc-tagged NDE1 was transfected with HA-tagged DISC1 or HA-tagged DISC1Δ(802–835) in HEK293 cells. Protein extracts were immunoprecipitated with an anti-myc antibody. Deletion of amino acids 802–835 of DISC1 dramatically weakened the interaction with NDEL1 in co-immunoprecipitation (top panel). The inputs are shown (bottom panels). (G) Protein expression of NDE1 and NDEL1 in the cerebral cortex. NDE1 protein was expressed constantly at a low level from E14 to adulthood. Expression of NDEL1 is increased according to brain development, with the highest expression at P7.

Effect of NDEL1 rs1391768 genotype in a DISC1 Ser704Cys background. Subjects are grouped by genotype at both NDEL1 rs1391768 and DISC1 Ser704Cys. The X-axis represents NDEL1 genotype group. The Y-axis represents OR. These data depict the DISC1–NDEL1 genotype interaction graphically. Sample sizes for each group are as follows: NDEL1 G carrier × DISC1 Ser704Ser (SZ = 74, HC = 43); NDEL1 AA × DISC1 Ser704Ser (SZ = 31, HC = 44); NDEL1 G carrier × DISC1 Cys carrier (SZ = 52, HC = 42) and NDEL1 AA × DISC1 Cys carrier (SZ = 83, HC = 65).

Effect of NDE1 rs3784859 genotype in a DISC1 Ser704Cys background. Subjects are grouped by genotype at both NDE1 rs3784859 and DISC1 Ser704Cys. The X-axis represents NDE1 genotype group. The Y-axis represents OR. These data depict the DISC1–NDE1 genotype interaction graphically. Sample sizes for each group are as follows: NDE1 G carrier × DISC1 Ser704Ser (SZ = 45, HC = 35); NDE1 AA × DISC1 Ser704Ser (SZ = 28, HC = 25); NDE1 G carrier × DISC1 Cys carrier (SZ = 75, HC = 41) and NDE1 AA × DISC1 Cys carrier (SZ = 22, HC = 24).

Possible competition of NDE1 and NDEL1 for DISC1. (A) Change in subcellular distribution of NDE1 by overexpression of DISC1. Exogenous expression of DISC1 (DISC1-Ser704) (red) leads to punctuated distribution of GFP-tagged NDE1 (green) in COS7 cells. Scale bar, 10 µm. (B) NDE1 affects the accumulation of NDEL1 to the centrosome in PC12 cells. Endogenous NDEL1 (red) is localized in the perinuclear region, including the centrosome, in undifferentiated PC12 cells. Overexpression of NDE1 (green, upper panels), but not GAPDH (green, lower panels), redistributes endogenous NDEL1 (red) from the perinuclear regions (lower panels). To semi-quantify the localization change, immunointensity of NDEL1 in the centrosome area (white circle) relative to that in the whole cell region surrounded by green line was examined. Bars represent averages of each group of cells in blinded experiments (*P < 0.005). Error bars represent SEM. Representative images are shown. Blue, nucleus; red, NDEL1; green, NDE1 or GAPDH; white, γ-tubulin (indicated by arrowheads). Scale bar, 10 µm. (C) Protein interactions of DISC1–NDE1 and DISC1–NDEL1 were not affected in the presence of U0126, which is known to block NGF-induced activation of ERK1/2 in PC12 cells. Error bars represent SEM. (D) HEK 293 cells were transfected with HA-tagged DISC1^Ser or DISC1^Cys together with myc-tagged NDE1. Lysates were then immunoprecipitated with an anti-myc antibody and western-blotted with an antibody to HA to analyze the DISC1–NDE1 interaction. Interaction of DISC1^Ser with NDE1 is slightly stronger than that with DISC1^Cys as shown in co-immunoprecipitation with an anti-myc antibody (top panel). The inputs are also shown (bottom panels). (E) Influence of the Ser704Cys variation of DISC1 on the DISC1–NDEL1 and DISC1–NDE1 protein interactions. NDE1 binds to DISC1^Ser more tightly than to DISC1^Cys (left graph and D), whereas NDEL1 interacts with DISC1^Ser less tightly than with DISC1^Cys as published previously (middle graph). The opposite binding pattern for NDE1 and NDEL1 with regard to DISC1 Ser704Cys results in about 1.6-fold difference between DISC1^Ser and DISC1^Cys (right graph) (P = 0.054). Error bars represent SEM.