Inhibition of protein translation by the DISC1-Boymaw fusion gene from a Scottish family with major psychiatric disorders

Abstract

The t(1; 11) translocation appears to be the causal genetic lesion with 70% penetrance for schizophrenia, major depression and other psychiatric disorders in a Scottish family. Molecular studies identified the disruption of the disrupted-in-schizophrenia 1 (DISC1) gene by chromosome translocation at chromosome 1q42. Our previous studies, however, revealed that the translocation also disrupted another gene, Boymaw (also termed DISC1FP1), on chromosome 11. After translocation, two fusion genes [the DISC1-Boymaw (DB7) and the Boymaw-DISC1 (BD13)] are generated between the DISC1 and Boymaw genes. In the present study, we report that expression of the DB7 fusion gene inhibits both intracellular NADH oxidoreductase activities and protein translation. We generated humanized DISC1-Boymaw mice with gene targeting to examine the in vivo functions of the fusion genes. Consistent with the in vitro studies on the DB7 fusion gene, protein translation activity is decreased in the hippocampus and in cultured primary neurons from the brains of the humanized mice. Expression of Gad67, Nmdar1 and Psd95 proteins are also reduced. The humanized mice display prolonged and increased responses to the NMDA receptor antagonist, ketamine, on various mouse genetic backgrounds. Abnormal information processing of acoustic startle and depressive-like behaviors are also observed. In addition, the humanized mice display abnormal erythropoiesis, which was reported to associate with depression in humans. Expression of the DB7 fusion gene may reduce protein translation to impair brain functions and thereby contribute to the pathogenesis of major psychiatric disorders.

Figure 1.

MTT reduction and rapid degradation of DB7 fusion protein. (A) pAAV plasmid constructs. Each expression construct was tagged with HA epitope. Western blot analyses revealed abundant expression of full-length DISC1 (FL), truncated DISC1 (Trunc) and BD13 (Boymaw-DISC1). The expression of the DB7 (DISC1-Boymaw) was very low (black arrowhead), and restricted to the pellet fraction. Equal amounts of proteins were loaded for the western blot (see β-actin control). (B) MTT reduction assays at multiple time points. There were 4–6 replica wells per construct per time point. The mean value of the MTT reduction in cells transfected with GFP was used as the control (100%) to calculate relative MTT reduction for all other constructs at each time point. ANOVA analysis revealed a significant gene effect [F(4,126) = 28.09, P < 0.0001]. Post hoc analyses (Tukey studentized range test) revealed that cells transfected with the DISC1-Boymaw (DB7) construct displayed a significant decrease in MTT reduction at all time points except 45 h post-transfection. Error bar: SEM. (*P < 0.05, **P < 0.01, ***P < 0.001). (C) Bi-cistronic constructs in pTimer1 plasmid vector. Each gene was linked with a fluorescence marker (timer) gene via an IRES site. Green fluorescence was examined 48 h after transfection of HEK293T cells. There was no difference between any of the four gene constructs. Scale bar: 25 µm. (D) The FT E5 was in-frame fused with DISC1-FL, truncated DISC1, DB7 and BD13 in pTimer plasmid vector. Expression of the fusion proteins was examined 48 h after transfection of HEK293T cells. Both green and red fluorescence were readily detected and co-localized in DISC1-FL-FT, DISC1-trunc-FT and BD13-FT. Weak green, and no red, fluorescence was detected in comparable number of cells expressing DB7-FT only after overexposure. Scale bar: 25 µm. (E) In iTRAQ experiments, proteins from cells expressing the full-length DISC1 gene were labeled with 114 and 115 Da mass tags; proteins from cells expressing the DB7 gene were labeled with 116 and 117 Da mass tags. Abundant human DISC1 peptides were also detected in cells expressing the DB7 gene.

Figure 2.

Characterization of the Boymaw gene. (A) Boymaw was fused with a FT tagged with HA in pTimer plasmid vector. The pTimer-HA plasmids (FT) were used as controls. (B) Expression of the FT-Boymaw fusion proteins was examined 48 h after transfection of HEK293T cells. While green and red fluorescence of the control FT proteins were very strong, little green and no red fluorescence of FT-Boymaw was detected. Scale bar: 15 µm. (C) Western blot analysis barely detected expression of the FT-Boymaw fusion proteins (black arrowhead) in comparison with abundant control FT proteins. (D) MTT assays revealed decreased MTT reduction by expression of the FT-Boymaw fusion gene in comparison with the controls and DISC1-FL [F(2,66) = 145.12, P < 0.0001]. Post hoc analyses (Tukey studentized range test) revealed that cells transfected with the FT-Boymaw construct displayed a significant decrease in MTT reduction at all time points except 45 h post-transfection. There were 4–6 replica wells per construct per time point. The mean value of the MTT reduction in cells transfected with FT was used as the control (100%) to calculate relative MTT reduction for the other two constructs at each time point. (E) A series of C-terminal deletion of FT-Boymaw fusion gene. Western blot analysis revealed expression of each deletion construct. (F) MTT assays were conducted 48 h after transfection of each deletion construct. There was a significant group effect on MTT reduction [F(8, 27) = 92.14, P < 0.000001]. Post hoc analyses (Tukey studentized range test) revealed significant MTT reduction in cells transfected with FT-Boymaw, C3-del and C4-del. There were four replica wells per construct. The mean value of the MTT reduction in cells transfected with FT was used as the control (100%) to calculate relative MTT reduction for all other constructs. Error bar: SEM. (*P < 0.05, **P < 0.01, ***P < 0.001).

Figure 3.

Reduced NADH oxidoreductase activities. (A) HEK293T cells were transfected with DISC1-FL and DB7, respectively. Two days after transfection, MTT reduction was performed in both living cells and cell lysate, respectively. Student's t-tests (unpaired, two-tailed) were used to compare MTT reduction between DISC1-FL and DB7 in living cells and cell lysate, respectively. The mean value of the MTT reduction in cells transfected with DISC1-FL was used as the control (100%) to calculate relative MTT reduction for DB7 in each group. Significantly decreased MTT reduction in DB7 transfection was observed in living cells (n = 3) [t(4) = 10.74, P < 0.001].and cell lysate (n = 6) [t(10) = 4.92, ^###P < 0.001]. (B) Reversal of MTT reduction deficit in cell lysate. There were 12 replica wells for each gene and treatment. Unpaired student's t-test (two-tailed) was used for statistical analysis. Significantly decreased MTT reduction was observed in HEK293T cell lysate from DB7 transfection in comparison with DISC1-FL [t(22) = 11.50, P < 0.001]. E. coli ADH can catalyze MTT reduction in the presence of NADH. Addition of the ADH did not completely reverse the deficit of MTT reduction in DB7 lysate in comparison with DISC1-FL lysate [t(22) = 4.26, ^###P < 0.001]. However, addition of recombinant E. coli ADH significantly improved MTT reduction in DB7 cell lysate [t(22) = 8.66, ^$$$P < 0.001 (comparison between DB7 lysate with and without ADH)]. No difference was found in MTT reduction of the DISC1-FL cell lysate with or without ADH. The mean value of the MTT reduction of DISC1-FL samples without ADH was used as the control (100%) to calculate relative MTT reduction for DB7, DISC1-FL+ADH and DB7+ADH. (C) Purification of mitochondria and endoplasmic reticulum (ER) from transfected HEK293T cells using density-gradient ultracentrifugation. Large amount of cells was transfected for each purification experiment. Cytochrome c and cytochrome p450 reductase were used as marker proteins for mitochondria and ER respectively. MTT reduction assays were conducted in purified mitochondria and ER from HEK293T cells transfected with DISC1-FL and DB7 constructs. The value of MTT reduction was further normalized with total amount of proteins in mitochondria and ER, respectively. Significantly reduced MTT reduction was observed in both mitochondria (n = 6)[t(10) = 5.53, two-tailed, P < 0.001) and ER (n = 6)[t(10) = 6.59, two-tailed, ^###P < 0.001] isolated from cells transfected with DB7 expression construct. (D) The level of CYB5R3 protein expression was examined with western blot analysis, and normalized with the expression of ER marker cytochrome p450 reductase (CYPOR). CYB5R3 expression was reduced in the ER of the cells expressing the DB7 fusion gene. No significant difference in CYB5R3 expression was observed between DISC1-FL and DB7 in either cell homogenate or mitochondria after normalization with mitochondrial marker cytochrome c. (E) CYB5R3 expression was quantified in ER preparations of each construct (n = 4). Unpaired student's t-test (two-tailed) analysis revealed a significant reduction of CYB5R3 in DB7 [t(6) = 18.84, P < 0.001] Error bar: SEM. (*P < 0.05, **P < 0.01, ***P < 0.001).

Figure 4.

Inhibition of rRNA expression and protein translation. (A) Reduction of total RNA in cells expressing the DB7 gene [unpaired, two-tailed student's t-test, t(10) = 9.84, P < 0.001]. There were three to six replica wells for each gene construct. Significant reduction of rRNA expression was observed between DB7 and DISC1-FL. The same amount of rRNA reduction was further confirmed in later transfection experiments. Expression of total RNA and rRNA was normalized against the number of cells. The level of total RNA and rRNA expression of the DISC1-FL was used as the reference (100%) to calculate relative expression of the other three groups. (B) Reduction of total RNA and rRNA in cells expressing FT-Boymaw fusion gene compared with the control FT gene [unpaired, two-tailed student's t-test, t(8) = 7.6, P < 0.001). There were five replica wells for each construct. Significant reduction of rRNA was observed in cells expressing the FT-Boymaw fusion gene. (C) Pulse-labeling of new protein synthesis via incorporation of puromycin. Anti-puromycin antibody has no cross-reactions with HEK293T cellular proteins. A 10 min pulse-labeling generated massive incorporation of puromycin in western blot analysis. However, the presence of cycloheximide, an inhibitor of protein translation, dramatically reduced the puromycin labeling. (D) Inhibition of protein translation. Protein translation activities were measured with SUnSET in cells at 48 h post-transfection. Remarkable reduction of puromycin labeling was observed in cells expressing the DB7 construct in two representative sets of transfection experiments. (E) Quantification of reduction of puromycin incorporation. There were four replica dishes for each construct. There was a significant reduction of protein translation in cells expressing the DB7 construct in comparison with the DISC1-FL construct [unpaired, two-tailed student's t-test, t(6) = 5.03, P < 0.01]. The mean value of the incorporation of puromycin in DISC1-FL samples was used as the control (100%) to calculate relative intensity of puromycin incorporation in the other three genes. No difference was found in protein translation activity between the other three constructs. (F) There was a reduction of protein translation in cells expressing the FT-Boymaw fusion gene compared with the control FT gene in SUnSET. (G) There were six replica dishes for each construct. Significant reduction of puromycin incorporation was observed in cells expressing the FT-Boymaw fusion gene [unpaired, two-tailed student's t-test, t(10) = 4.48, P < 0.01]. Error bar: SEM. (*P < 0.05, **P < 0.01, ***P < 0.001).

Figure 5.

Generation of humanized DISC1-Boymaw Mice. (A) Both the DISC1-Boymaw and the Boymaw-DISC1 fusion genes were knocked-in to replace mouse endogenous disc1 gene. E1 and E2 are exon 1 and 2 of mouse disc1 gene. (B) RT-PCR analysis of the expression of the DB7 fusion genes in humanized DISC1-Boymaw mice (DISC1^H). The two PCR primers were localized in exon 1 and DB7, respectively (arrows). The expression of the bi-cistronic gene generated a 150 bp cDNA fragment after RT-PCR. Correct splicing was confirmed after sequencing. (C) Western blot analysis of the DB7 fusion protein in cultured primary neurons. Expression of the DISC1-Boymaw (DB7) fusion proteins was detected from the primary neurons isolated from the humanized DISC1-Boymaw mice. The 75 kDa protein band is very close to predicted molecular size (∼73 kDa). (D) Immunocytochemical staining of the DB7 fusion proteins in the primary neurons. Primary neurons were isolated from postnatal Day 1 mice, and cultured 4 days in vitro before immunostaining. Co-localization between MAP2 and DB7 proteins was observed. Scale bar: 20 µm. (E) Decreased MTT reduction in brain homogenate of neonatal heterozygous mice (DISC1^H) (n = 14) in comparison with wild-type control littermates (n = 12) after normalization with total amount of proteins (t(24) = 2.984, P < 0.01, unpaired, two-tailed student's t-test). (F) Reduced expression of Cyb5r3 in the ER isolated from the brains of neonatal DISC1-Boymaw mice (n = 16) and wild-type littermates (n = 18) after normalization with ER marker Cypor (unpaired, two-tailed student's t-test, t(32) = 2.58, P < 0.05). Error bar: SEM. (*P < 0.05, **P < 0.01).

Figure 6.

Decreased rRNA expression and protein translation. (A) Hippocampal DNA and RNA were isolated from 3-week old heterozygous DISC1-Boymaw (n = 8) and wild-type control mice (n = 12) respectively. No difference in total amount of DNA was observed between the two genotypes. However, significant reduction of total RNA was observed [unpaired, two-tailed student's t-test, t(18) = 3.33, P < 0.01]. After gel electrophoresis, 28S and 18S rRNA displayed significant reduction in the hippocampus of the heterozygous DISC1-Boymaw mice. (B) Chromogenic RNA in situ hybridization of 18S rRNA was conducted in the mouse brains at postnatal Day 7 (scale bar: 400 µm). Reduction of 18S rRNA was observed in both hippocampal neuronal cells (scale bar: 100 µm) as well as cortical neuronal cells (scale bar: 10 µm). Nuclear rRNA was readily detected in the nucleolus of the cortical neurons. (C) Protein translation activities in hippocampus. Intracerebroventricular injection of puromycin generated massive labeling of new protein synthesis in mouse hippocampus. Decreased puromycin labeling can be readily observed in the heterozygous mice. (D) Significant reduction of protein synthesis activities was found in the hippocampus of the heterozygous DISC1-Boymaw mice in comparison with wild-type sibling mice [unpaired, two-tailed student's t-test, t(4) = 9.28, P < 0.001]. (E) Primary neurons were isolated from postnatal Day 1 mice and cultured at 4 days in vitro before SUnSET experiments. After pulse-labeling, western blot revealed less puromycin labeling in the primary neurons from the heterozygous DISC1-Boymaw mice than their wild-type siblings. (F) Significant reduction of protein synthesis was detected [unpaired, two-tailed student's t-test, t(4) = 4.74, P < 0.01]. Error bar: SEM. (*P < 0.05, **P < 0.01, ***P < 0.001).

Figure 7.

Reduction of Gad67, Nmdar1 and Psd95 Proteins. (A) Q-PCR analysis of mRNA transcripts of Gad67, Nmdar1 and Psd95 genes in adult mouse brain. There was no significant difference between wild-type and the heterozygous DISC1-Boymaw mice in Gad67 [unpaired, two-tailed student's t-test, t(19) = 0.73, n.s.], Nmdar1 [unpaired, two-tailed student's t-test, t(19) = −1.44, n.s.] and Psd95 [unpaired, two-tailed student's t-test, t(19) = −1.17, n.s.] expression. (B) Western blot analysis and quantification of Gad67, synaptophysin (Syp), Nmdar1 and Psd95 in the brains of the adult heterozygous DISC1-Boymaw (n = 9) and wild-type (n = 9) mice. Unpaired two-tailed student's t-test was used for statistical analysis. No significant reduction of synaptophysin was observed between the two genotypes. Significant reduction of Gad67 [t(16) = 2.37, P < 0.05], Nmdar1 [t(16) = 2.55, P < 0.05] and Psd95 [t(16) = 2.58, P < 0.05] proteins was observed after normalization with either total amount of loading protein or β-actin expression. (C) In primary neurons isolated from cortex and striatum of postnatal Day 1 mice, significant reduction of both Gad67 (t(4) = 11.92, P < 0.001) and Psd95 [t(4) = 3.30, P < 0.05) proteins was observed in cultured primary neurons at 4 days in vitro from the DISC1-Boymaw mice (n = 3) in comparison with wild-type controls (n = 3) (unpaired, two-tailed student's t-test). Reduction of both proteins was further confirmed in the primary neuron culture (isolated from cortex and striatum) at 4 days in vitro from E18.5 embryos. (D) Immunohistochemical analysis of parvalbumin, Gad67, and Nmdar1 proteins in the heterozygous DISC1-Boymaw mice (anti-Psd95 antibodies did not work on paraffin sections). Reduction of Gad67 and Nmdar1 was observed in both hippocampus (scale bar: 100 µm) and cortex (scale bar: 20 µm). No difference was detected in parvalbumin expression. White ‘spots’ were nuclei not stained by antibodies. Error bar: SEM. (*P < 0.05, **P < 0.01, ***P < 0.001).

Figure 8.

Behavioral analyses of the humanized DISC1-Boymaw mice on F1 129S/C57 genetic background. (A) The breeding scheme of mice on F1 129S/C57 background. In the F1 generation, there is no genetic segregation between individual mice. Both wild-type and the heterozygous DISC1-Boymaw mice carry one wild-type copy of mouse Disc1 gene. (B) The cohort of F1 mice consists of 28 wild-type and 26 heterozygous DISC1-Boymaw mice. There was no sex effect or gene effect on PPI [F(1,52) = 0.00, n.s.]. (C) There was no gene effect on startle habituation [F(1,50) = 0.21, n.s.]. (D) A large startle variation was observed between individual mice despite the fact that they are genetically identical. Dots represent individual mice (black = females, pale = males). (E) Males were tested for their responses to ketamine in the BPM. There were 15 wild-type and 14 heterozygous age-matched males. A significant gene by ketamine interaction was observed [F(1,27) = 4.30, P < 0.05]. Post hoc analyses (Tukey studentized range test) revealed that the heterozygous mice displayed significantly increased responses to ketamine in comparison with wild-type male controls in the first three blocks. Error bar: SEM. #P < 0.10; *P < 0.05; **P < 0.01.

Figure 9.

Behavioral characterization of the humanized DISC1-Boymaw mice on 129S background. (A) Breeding of wild-type and the heterozygous DISC1-Boymaw mice on 129S pure genetic background. 129S mice carry a frame-shift mutation in exon 6 of the disc1 gene. (B) The cohort consisted of 14 wild-type females, 14 heterozygous females, 13 wild-type males and 15 heterozygous males. The age range of the birth date between individual mice was less than a week. Body weight was compared between the wild-type and the heterozygous mice. Heterozygous males displayed slightly, but significantly, reduced body weight at postnatal Day 103 [unpaired, two-tailed student's t-test, t(26) = 2.132, P < 0.05]. No difference was found in body weight between female wild-type and heterozygous mice. (C) There was a significant gene effect [F(1,54) = 4.41, P < 0.05] in PPI at 69 dB prepulse. A significant gene X ISI interaction was also observed [F(4,216) = 2.90, P < 0.05]. Post hoc analysis revealed that the heterozygous DISC1-Boymaw mice displayed increased PPI (or impaired prepulse facilitation) with ISI at 25 ms. No sex difference was observed. (D) The male heterozygous DISC1-Boymaw mice display a significant gene effect [F(1,26) = 6.26, P < 0.05] as well as a gene X ketamine interaction [F(1,26) = 8.61, P < 0.01]. Post hoc analyses (Tukey studentized range test) revealed that the male heterozygous mice displayed significantly increased and prolonged responses to ketamine in comparison with wild-type male controls in all time blocks except the first and last one. (E) Total immobility time (seconds) was recorded during a 6 min suspension test. Five wild-type male mice were lost because of fighting in cages after previous tests. The heterozygous DISC1-Boymaw mice (n = 29) displayed significantly more immobility time than the wild-type control mice (n = 22) [unpaired, two-tailed student's t-test, t(49) = 2.22, P < 0.05). (F) Two or three mice were grouped in a single cage according to genotype. Saccharin preference test was conducted for four consecutive days. Consumption of saccharin was measured each day, and compared between the wild-type and the heterozygous mice in each sex. Significant gene effect was detected [F(1,11) = 17.85, P < 0.01] between female wild-type (n = 7) and the DISC1-Boymaw heterozygous mouse cages (n = 6). Post hoc analysis revealed that the female heterozygous mice displayed significantly less saccharin consumption than female wild-type controls mice at Day 2 and Day 4. Error bar: SEM. #P < 0.10; *P < 0.05; **P < 0.01.

Figure 10.

Increased RDW in the humanized DISC1-Boymaw mice. (A) Schematic pathway of erythropoiesis. (B) Concentration of hemoglobin (HB) was measured in the blood of individual male mice. Severe, moderate and mild anemia was diagnosed according to hemoglobin concentrations (49). Two heterozygous DISC1-Boymaw mice suffered from severe and moderate anemia. All wild-type mice were normal. Each dot represents an individual mouse. (C) Homogeneity of variances of RDW data was first assessed with Levene's test (P = 0.057). Significantly higher RDW was found in the heterozygous DISC1-Boymaw mice [F(1,19) = 8.451, **P < 0.01]. Most of these mice have RDW above the upper limit of the normal RDW range of their wild-type sibling mice (dashed line). (D) Six wild-type and six heterozygous DISC1-Boymaw male mice were re-examined 1 week later (t2) for their RDW stability, and compared with their initial (t1) RDW measurement. There was little change in RDW of the wild-type mice during this period. However, high magnitude of RDW fluctuation was observed in the heterozygous DISC1-Boymaw mice, indicating unstable erythropoiesis.

Figure 11.

Hypothetical pathways of the DB7 fusion gene. Cell metabolism is tightly coupled with protein synthesis. Reduction of NADH oxidoreductase activity may alter production of ATP and NAD⁺ which can trigger at least two known pathways (AMPK and Sirtuin) to down-regulate rRNA transcription and protein translation. AMPK, AMP-activated protein kinase; TSC1 and TSC2, tuberous sclerosis complex 1 and 2; mTOR, mammalian target of rapamycin.