NRG treatment affects DISC1 expression in an isoform-specific manner. (A) Western blotting for DISC1 in NRG-treated neurons. Western blotting with D27 shows increase in isoform of DISC1 at 130 kDa (arrow) following treatment with NRG1 (mean ± SD, 1.77 ± 0.23-fold increase, P = 0.007, n = 5) and NRG2 (1.53 ± 0.13-fold increase, P = 0.008, n = 4), whereas bands at 100 and 105 kDa were unaffected (arrowheads). GAPDH was used as a loading control. (B) Immunofluorescent cell staining and quantification of DISC1 in NRG1-treated neurons. Cell staining for DISC1 with antibody mExon3 was quantified and normalized to neurite length indicated by β-tubulin staining in primary neurons treated with NRG1. DISC1 in neurites increased after NRG1 treatment (1.72 ± 0.33-fold increase, P = 0.01, n = 3). Magnification, 20×. (Scale bar, 50 μm.)

NRG1 signaling knockdown affects DISC1 expression in vivo. (A) Western blotting for DISC1 in NRG1-KO mice. Immunoblotting with DISC1 antibody D27 shows reduced DISC1 expression (arrow) in cortex of heterozygous P0 NRG1-KO mice compared with wild-type littermates (23.6% decrease). A C-terminal–directed NRG1 antibody was used to verify a reduction in cleaved NRG1 in these mice. GAPDH was used as loading control. (B) Immunohistochemistry for DISC1 in NRG1 knockout mice. Neurons (Tuj1) and radial glia (RC2) in E10.5 telencephalon of WT and homozygous NRG1-KO mice were colabeled with DISC1. Both neurons and radial glia express DISC1, as seen in outlined cells; however, DISC1 is down-regulated in NRG1-deficient cells. Quantification of fluorescent intensity (arbitrary units) verifies decreased DISC1 in neurons (NRG1^−/−: mean ± SD, 9.62 ± 1.24, WT: 34.91 ± 1.17), and radial glia (NRG1^−/−: 8.04 ± 0.9, WT: 15.87 ± 1.2). Magnification, 630×. (Scale bar, 10 μm.) (C) Western blotting for DISC1 in BACE1 knockout mice. Immunoblotting with DISC1 antibody D27 shows reduced DISC1 expression (arrow) in cortex of homozygous P0 BACE1-KO mice compared with wild-type littermates (44.1% decrease, n = 3). A C-terminal–directed NRG1 antibody was used to verify a reduction in cleaved NRG1 in these mice. GAPDH used as loading control. (D) Immunohistochemistry for DISC1 in BACE1 knockout mice. Staining with DISC1 antibody mExon3 shows reduced DISC1 in the cortex of homozygous P0 BACE1-KO mice compared with wild-type littermates, with a reduction in the percent of neurons with visibly stained neurites (76.4% reduction, n = 3). Magnification 630×. (Scale bar, 50 μm and 10 μm.)

NRG1 signaling affects DISC1 expression via ErbB3/ErbB2 and Akt activation. (A) Western blotting for DISC1 after NRG1 treatment with receptor knockdown by RNAi. Primary cortical neurons infected with lentiviral shRNA against ErbB4, ErbB3, and ErbB2 (Fig. S3A) and treated with NRG1. Western blotting was performed for 130 kDa DISC1 with DISC1 antibodies D27 and mExon3 and quantified (*P < 0.01, n = 3). (B) Western blotting for DISC1 after NRG1 treatment with pharmacological blockade of secondary signaling pathways. Primary cortical neurons were treated with LY294002 and AG490, then treated with NRG1. Western blotting was done for 130 kDa DISC1 with DISC1 antibodies D27 and mExon3 and quantified (*P < 0.01, n = 3). Magnification 200× and 400×. (Scale bar, 50 μm and 5 μm.)

DISC1 expression in different cell types. (A) Immunohistochemistry for DISC1 and cell type–specific markers in human cortical sections. Costaining for human DISC1 (brown) with purified monoclonal antibody 3D4 and the following markers (green) was performed: MAP2 (neurons), GFAP (astrocytes), nogoA (oligodendrocytes), and KP-1 (microglia). Magnification 100×. (Scale bar, 50 μm.) (B) Immunofluorescent cell staining for DISC1 and cell type–specific markers in rat primary cortical cultures. Costaining for DISC1 with antibody mExon3 (red) and the following markers (green) was performed: MAP2 (neurons), GFAP (astrocytes), A2B5 (oligodendrocytes), and CD11b (microglia). Magnification 200×. (Scale bar, 10 μm.)