Disrupted-in-Schizophrenia-1 (DISC-1): mutant truncation prevents binding to NudE-like (NUDEL) and inhibits neurite outgrowth

Abstract

Disrupted-in-Schizophrenia-1 (DISC-1) is a gene whose mutant truncation is associated with major psychiatric illness with a predominance of schizophrenic symptomatology. We have cloned and characterized rodent DISC-1. DISC-1 expression displays pronounced developmental regulation with the highest levels in late embryonic life when the cerebral cortex develops. In yeast two-hybrid analyses, DISC-1 interacts with a variety of cytoskeletal proteins. One of these, NudE-like (NUDEL), is associated with cortical development and is linked to LIS-1, the disease gene for a form of lissencephaly, a disorder of cortical development. The disease mutant form of DISC-1 fails to bind NUDEL. Expression of mutant, but not wild-type, DISC-1 in PC12 cells reduces neurite extension. As schizophrenia is thought to reflect defects in cortical development that are determined by cytoskeletal protein activities, the cellular disturbances we observe with mutant DISC-1 may be relevant to psychopathologic mechanisms.

Figure 1

Cloning of rodent DISC-1. (A) Schematic diagram of the structure of DISC-1 proteins from the three species and the homology among them. GD denotes the putative globular domain, and CCD denotes the coiled-coil motif-containing domain. (B) Genomic organization of mouse DISC-1, mapped on mouse chromosome 8. The drawing was made by comparing the full-length coding sequence of mouse DISC-1 with a mouse genomic sequence, GA_x6k02T2NUPS, derived from the Celera mouse genome database (www.celera.com). Filled boxes correspond to the coding region of each exon, and open boxes are untranslated regions. Nucleotide numbers in each exon, exon numbers, and physical interval between neighboring exons are indicated. Numbers in parentheses are those that correspond only to coding nucleotides within exons 1 and 13. The entire gene spans >200 kb.

Figure 2

DISC-1 protein expression in tissues and cells. Major forms of DISC-1 (100 and 75 kDa) were detected by antibodies against both the N-terminal side (amino acids 347–600 and 212–537) and C-terminal side (amino acids 601–854) of the breakpoint. The specificities were also confirmed by preabsorption experiments. (A) High expression of DISC-1 protein in brain, heart, liver, and kidney, and weak expression in thymus. DISC-1 of 100 kDa is predominant in brain and heart. In adult brains, DISC-1 is enriched in the forebrain. DISC-1 expression is developmentally regulated with an enhanced expression of the 100-kDa band at E20.5. (B) DISC-1 protein expression in cell lines. DISC-1 is expressed in both neuronal and glial cells, as well as some peripheral cells. In PC12 cells, differentiation is associated with augmented expression. (C) DISC-1 distribution is analyzed by subcellular fractionation and immunofluorescent staining. Endogenous DISC-1 protein is enriched in P1 and P2 fractions of differentiated PC12 cells and in P2 of HeLa cells. Exogenously expressed full-length (FL) DISC-1 tagged with HA also occurs in P1 and P2 fractions, whereas mutant DISC-1 with the C-terminal deletion (Mut-HA, ΔC) distributes in P1, P2, and P3 fractions. Another mutant of DISC-1 lacking 290 amino acids of the N-terminal region (ΔN) distributes in all fractions, including the supernatant. Immunofluorescent staining of DISC-1 was analyzed under a confocal microscope. Endogenous and exogenous DISC-1 was stained in red; the nucleus was stained in blue with Hoechst 33258. Dotted DISC-1 staining is observed in the cytoplasm, especially in the perinuclear regions of undifferentiated PC12 cells and HeLa cells. In differentiated PC12 cells, DISC-1 occurs not only in cytoplasm, but also in neurites with higher density at the terminal regions (white arrow indicates DISC-1 in the terminus). Exogenously expressed DISC-1 tagged with HA in COS cells was stained with an anti-HA Ab (red). The dotted staining in perinuclear regions similar to centrosomes reflects full-length DISC-1 (DISC-1 FL). Mutant DISC-1 with the C-terminal deletion occurs in the cytoplasm with smaller dotted structures than DISC-1 FL. The N-terminal truncated form of DISC-1 (DISC-1 ΔN) distributes diffusely in the cytoplasm of COS cells.

Figure 3

Possible DISC-1 interactors identified by yeast two-hybrid screening. Interactors include cytoskeletal proteins, such as NUDEL and Citron, that are associated with brain development.

Figure 4

Confirmation and characterization of DISC-1–NUDEL interaction. (A) DISC-1–NUDEL interaction by in vitro binding. The interaction is specific, as no interaction of DISC-1 and GAPDH is observed. Representative data from one of three assays are presented. (B) Coimmunoprecipitation of DISC-1 and NUDEL in HEK293 cells. Mutant DISC-1 with the C-terminal deletion fails to bind to NUDEL. Representative data from one of three assays are presented. (C) Mutational analyses of DISC-1–NUDEL interaction. Binding critically depends on the C-terminal end of DISC-1 (amino acids 796–854) and the middle portion of NUDEL (amino acids 201–280) outside of its coiled-coil region.

Figure 5

Truncated DISC-1 inhibits neurite outgrowth of PC12 cells. (A) Representative morphology of a PC12 cell transfected with constructs for GAPDH together with GFP (Left). Representative morphology of PC12 cells transfected with constructs of the full-length DISC-1 and GFP (Center). Representative morphology of PC12 cells with the C-terminal truncated mutant DISC-1 with GFP (Right). (B) Mean percentages ± SE of PC12 cells harboring neurites (longer than three cell body diameters) 5 days after transfection of full-length DISC-1, mutant DISC-1, or GAPDH. Mutant, but not full-length, DISC-1 alters neurite outgrowth (P < 0.01). (C) No obvious signs of apoptotic cell death (nuclear condensation and fragmentation) in cells transfected with the mutant or the full-length DISC-1.