Genetic and pharmacological evidence for schizophrenia-related Disc1 interaction with GSK-3

Abstract

Recent studies have identified disrupted-in-schizophrenia-1 (DISC1) as a strong genetic risk factor associated with schizophrenia. Previously, we have reported that a mutation in the second exon of the DISC1 gene [leucine to proline at amino acid position 100, L100P] leads to the development of schizophrenia-related behaviors in mice. Glycogen synthase kinase-3 (GSK-3) is a serine/threonine protein kinase that interacts with the N-terminal region of DISC1 (aa 1-220) and has been implicated as an important downstream component in the etiology of schizophrenia. Here, for the first time, we show that pharmacological and genetic inactivation of GSK-3 reverse prepulse inhibition and latent inhibition deficits as well as normalizing the hyperactivity of Disc1-L100P mutants. In parallel to these observations, interaction between DISC1 and GSK-3α and β is reduced in Disc1-L100P mutants. Our data provide genetic, biochemical, and behavioral evidence for a molecular link between DISC1 and GSK-3 in relation to psychopathology and highlights the value of missense mutations in dissecting the underlying and complex molecular mechanisms of neurological disorders.

Fig. 1

A–C. Effects of TDZD-8 (0mg/kg, 2.5 mg/kg, 7.5 mg/kg and 15 mg/kg) on locomotor activity in WT and Disc1-L100P mutants. Locomotor activity curve in (A) - WT (n = 6–8 per group) and (B) - Disc1-L100P mice (n = 6–9 per group) depicted with 5 min intervals over 30 min, expressed as traveled distance in centimeters (cm); (C) total locomotor activity. # - p < 0.001 in comparison with vehicle-treated WT mice; ** - p < 0.01; *** - p < 0.001 – in comparison with vehicle-treated mice within Disc1-L100P group.

Fig. 2

A–D. Effects of TDZD-8 on (A) sensorimotor gating, (B) Acoustic Startle Response (n = 7–12 per group), (C) Latent Inhibition (n = 8–9 per group) in WT and Disc1-L100P mice and on LI in C57BL/6 mice with 4 conditioning trials (n = 8–10 per group) (D). (A) Figure depicts percent PPI at three prepulses (69, 73 and 81 dB) in WT and Disc1-L100P mice. Background noise was set of 65 dB. (B) Figure depicts the magnitude of the acoustic startle response in WT and Disc1-L100P mice. Startle intensity was 120 dB. # - p < 0.001 in comparison with vehicle-treated WT mice; * - p < 0.001 in comparison with vehicle-treated Disc1-L100P mutants. (C). Mean suppression ratios of PE and NPE WT and Disc1-L100P mice. TDZD-8 at 7.5 mg/kg was administrated before preexposure and conditioning sessions and reversed the LI deficit in Disc1-L100P mice due to the improved capacity to ignore irrelevant tone of PE group (p < 0.001). * - p < 0.001 in comparison with PE group; # - p < 0.001 in comparison with PE vehicle-treated Disc1-L100P mice. (D). Mean suppression ratios of C57BL/6 mice in LI procedure with 40 PE and 4 conditioning trials. TDZD-8 at 7.5 mg/kg facilitated the disrupted LI (p < 0.001). * - p < 0.001 – in comparison with PE group, # - p < 0.001 in comparison with PE vehicle-treated C57BL/6 mice.

Fig. 3

A–D. Genetic inactivation of GSK-3α rescued schizophrenia-related phenotypes of Disc1-L100P mutant mice. (A–B) Ambulation in the open field of Disc1-L100P mice (WT, HET or MUT) carrying one or both GSK-3α allele (n = 8–17 per group). * - p < 0.01 – in comparison with WT mice; # - p < 0.001 - in comparison with Disc1-L100P MUT. (C) percent of PPI at three prepulses of ASR in genetic crosses between Disc1-L100P and GSK-3α-HET mice (n = 9–17 per group). ** - p 0.01, *** - p < 0.001 - in comparison with WT mice; # - p < 0.01, ## - p < 0.001 - in comparison with Disc1-L100P HET and Disc1-L100P MUT, respectively. (D) Mean suppression ratios of PE and NPE mice (n = 7–14 per group). PE groups of Disc1-L100P HET and MUT animals without one GSK-3α allele expressed LI. * - p < 0.001 – in comparison with PE group within each genotype, # - p < 0.001 – in comparison with PE group of Disc1-L100P HET or Disc1-L100P MUT. Balanced groups of male and female mice were used and gender effect was not detected in any of the behavioral tests, hence data from different genders were analyzed together. Numbers of animals (n) for each condition are indicated.

Fig. 4

(A–G). Characterization of the DISC1-GSK-3a/b complex formation in Disc1-L100P mutant mice. A. Mice striatal brain samples were incubated with DISC1 antibody for coimmunoprecipitation. Precipitated proteins were subject to SDS-PAGE and then immunoblotted with GSK-3α or GSK-3β antibody. Each coimmunoprecipitation was in parallel with the directly immunoprecipitated DISC1 proteins. B–C. The densitometry evaluation of coimmunoprecipitation of GSK-3α (left) and GSK-3β (right). Data are means ±SEM, and were normalized to direct IP of DISC1 (D). Mice striatal brain samples were incubated with GSK-3β antibody for coimmunoprecipitation. Precipitated proteins were subject to SDS-PAGE and then immunoblotted with DISC1 antibody. Each coimmunoprecipitation was in parallel with the directly immunoprecipitated GSK-3β proteins. (E). The densitometry evaluation of coimmunoprecipitation of DISC1. Data are means ±SEM., and were normalized to direct IP of GSK3β. (F). Mice striatal brain samples were incubated with GSK-3α antibody for coimmunoprecipitation. Precipitated proteins were subject to SDS-PAGE and then immunoblotted with DISC1 antibody. Each coimmunoprecipitation was in parallel with the directly immunoprecipitated GSK-3α proteins. (G). The densitometry evaluation of coimmunoprecipitation of DISC1. Data are means ±SEM, and were normalized to direct IP of GSK3α. All data were analyzed with t-test (n = 5 per group; * - p < 0.05 – in comparison with WT). WT: Wild type; L100P – Disc1-L100P mutants.

Fig. 5

A–D. Biochemical characterization of GSK-3 functional state in the Striatum of Disc1-L100P mice. (A) Representative immunoblots are shown, probed with an antibody against total GSK-3α and GSK-3β and with antibodies to p-GSK-3α at Ser21, p-GSK-3β at Ser9 as well as GAPDH as a loading control. (B) Optical densities are shown, reflecting normalized phosphorylation of GSK-3α and GSK-3β over the total GSK-3α and GSK-3β, respectively (n = 7–15 per group). ** - p < 0.01 – in comparison with WT (C). Representative immunoblots are shown, probed with an antibody against β-catenin, total GSK-3α and GSK-3β and with antibodies to p-GSK-3α at Tyr 276, p-GSK-3β at Tyr 216, as well as GAPDH as a loading control. Equal loading of the lanes was assessed using an anti-GAPDH antibody and the blots are representative of 2 independent experiments. (D). GSK-3 kinase activities in WT and L100P Disc1 mutant mice. Striatum tissues were extracted from WT and L100P Disc 1 mice, lysed with Buffer H and immunoprecipitated for GSK-3α or GSK-3β with their activities assayed as described in materials and methods. GSK-3 kinase activity is expressed relative to that for WT control (which is set at 100% for each GSK-3 isoform assayed) and corrected for potential differences in immunoprecipitated GSK-3 amount (E). Values are the means +/− SEM of results for four different tissue samples.

Fig. 6

A–D. Biochemical characterization of GSK-3 functional state in the Hippocampus of Disc1-L100P mice. (A) Representative immunoblots are shown, probed with an antibody against total GSK-3α and GSK-3β and with antibodies to p-GSK-3α at Ser21, p-GSK-3β at Ser9 as well as GAPDH as a loading control. (B) Optical densities are shown, reflecting normalized phosphorylation of GSK-3α and GSK-3β over the total GSK-3α and GSK-3β, respectively (n = 7 per group). (C). Representative immunoblots are shown, probed with an antibody against β-catenin, total GSK-3α and GSK-3β and with antibodies to p-GSK-3α at Tyr 276, p-GSK-3β at Tyr 216, as well as GAPDH as a loading control. Equal loading of the lanes was assessed using an anti-GAPDH antibody and the blots are representative of 2 independent experiments. (D). GSK-3 kinase activities in WT and L100P Disc1 mutant mice. Hippocampal tissues were extracted from WT and L100P Disc 1 mice, lysed with Buffer H and immunoprecipitated for GSK-3α or GSK-3β with their activities assayed as described in materials and methods. GSK-3 kinase activity is expressed relative to that for WT control (which is set at 100% for each GSK-3 isoform assayed) and corrected for potential differences in immunoprecipitated GSK-3 amount (E). Values are the means +/− SEM of results for four different tissue samples.