Deficiency of schnurri-2, an MHC enhancer binding protein, induces mild chronic inflammation in the brain and confers molecular, neuronal, and behavioral phenotypes related to schizophrenia

Abstract

Schnurri-2 (Shn-2), an nuclear factor-κB site-binding protein, tightly binds to the enhancers of major histocompatibility complex class I genes and inflammatory cytokines, which have been shown to harbor common variant single-nucleotide polymorphisms associated with schizophrenia. Although genes related to immunity are implicated in schizophrenia, there has been no study showing that their mutation or knockout (KO) results in schizophrenia. Here, we show that Shn-2 KO mice have behavioral abnormalities that resemble those of schizophrenics. The mutant brain demonstrated multiple schizophrenia-related phenotypes, including transcriptome/proteome changes similar to those of postmortem schizophrenia patients, decreased parvalbumin and GAD67 levels, increased theta power on electroencephalograms, and a thinner cortex. Dentate gyrus granule cells failed to mature in mutants, a previously proposed endophenotype of schizophrenia. Shn-2 KO mice also exhibited mild chronic inflammation of the brain, as evidenced by increased inflammation markers (including GFAP and NADH/NADPH oxidase p22 phox), and genome-wide gene expression patterns similar to various inflammatory conditions. Chronic administration of anti-inflammatory drugs reduced hippocampal GFAP expression, and reversed deficits in working memory and nest-building behaviors in Shn-2 KO mice. These results suggest that genetically induced changes in immune system can be a predisposing factor in schizophrenia.

Figure 1

Schizophrenia-related behavioral abnormalities in Shn-2 KO mice. (a, b) In the spatial working memory version of the eight-arm radial maze, Shn-2 KO mice performed significantly worse with respect to the number of different arm choices in the first eight entries (genotype effect: F_1,24=62.104, P<0.0001) and made significantly more revisiting errors than controls (genotype effect: F_1,24=45.597, P<0.0001; genotype × trial block interaction: F_12,228=1.470, P=0.1345). (c) Mutant mice also showed poor working memory performance in the T-maze forced-alternation task (genotype effect: F_1,21=20.497, P=0.0002; genotype × session interaction: F_7,147=3.273, P=0.0029). (d) With increased delay, Shn-2 KO mice exhibited a lower correct percentage than controls (delay=3, 10, 30, and 60 s; P=0.0010, P=0.0047, P=0.0083, and P=0.0026, respectively). (e) Shn-2 KO and wild-type mice were comparable in the left–right discrimination task (genotype effect: F_1,19=0.209, P=0.6529) and reversal learning (genotype effect: F_1,19=5.917, P=0.0251). (f) The amplitude of the acoustic startle response was not significantly different between genotypes (Shn-2^+/+, +Veh vs Shn-2^−/−, Veh, F_1,73=1.371, P=0.2454). (g) PPI of the acoustic startle response was impaired in Shn-2 KO mice (Shn-2^+/+, Veh vs Shn-2^−/−, Veh, 110 dB startle, P=0.0027; 120 dB startle, P=0.0003). Administration of haloperidol improved the PPI of Shn-2^−/− mice (Shn-2^−/−, Veh vs Shn-2^−/−,1 mg/kg Hal, 110 dB, P=0.0145; 120 dB, P=0.0059; Shn-2^−/−, Veh vs Shn-2^−/−, 3 mg/kg Hal, 120 dB, P=0.0044). Post hoc Bonferroni's test after two-way repeated-measures ANOVA (level of significance was set at P<0.0167). (h) Shn-2 KO mice display a lower level of social approach in the sociability test. (i) Shn-2 KO mice did not show social novelty preference. (j) Shn-2 KO mice displayed decreased social interaction in a novel environment (total contact duration: F_1,14=11.569, P=0.0043). (k) Administration of clozapine (1 mg/kg, i.p.) reversed hyperactivity in mutant mice (genotype effect: P=0.0012, drug effect: P=0.0003, genotype × drug interaction: P=0.0574, Shn-2^−/−, Clz. vs Shn-2^+/+, Veh., P=0.4221). (l) Administration of haloperidol (0.3 mg/kg, i.p.) also reduced hyperactivity in Shn-2 KO mice (genotype effect: P<0.0001, drug effect: P<0.0001, genotype × drug interaction: P=0.0275, Shn-2^−/−, Hal. vs Shn-2^+/+, Veh., P=0.8957). (m, n) Nest building was impaired in Shn-2 KO mice (P<0.0001). Veh, Vehicle; Clz, Clozapine; Hal, Haloperidol.

Figure 2

Comparison of gene expression profiles between Shn-2 KO mice and individuals with schizophrenia. (a) Venn diagram of genes differentially expressed in the medial prefrontal cortex (mPFC) of Shn-2 KO mice and Brodmann area (BA) 10 of postmortem schizophrenia brain (Schizo.). (b) P-values of overlap between Shn-2 KO mouse and schizophrenia data sets. (c) Scatter plot of gene expression fold change values in Shn-2 KO mice and schizophrenia. (d) Genes differentially expressed in both Shn-2 KO mice and schizophrenia. Red indicates gene upregulation and blue indicates downregulation in both Shn-2 KO mice and schizophrenia. The top 40 genes are included.

Figure 3

Schizophrenia-related alterations in the Shn-2 KO mouse brain. (a–d) The number of parvalbumin-positive cells is decreased in mPFC (P=0.0088) (a, b) and CA1 (P=0.0029) (c, d) of Shn-2 KO mice. (e, f) The expression of GAD67 in MFs of the hippocampus decreased in mutants (P=0.011). (g, h) Reduced CNPase expression in the DG of Shn-2 KO mice compared with controls (molecular layer, P<0.0001; hilus, P<0.0001). (i, j) DG cell number was evaluated by staining cell nuclei with Hoechst dye. Cell-packing density was higher in Shn-2 KO mice (P=0.0061) (j). GAD67, glutamic acid decarboxylase 67; CNPase, 2′,3-cyclic nucleotide 3′-phosphodiesterase; ML, molecular layer. Scale bars indicate 200 μm (a, c, g), 500 μm (e), 20 μm (i).

Figure 4

Reduced Arc induction in Shn-2 KO mice was observed after foot shocks in a novel environment. (a–c) Shn-2 KO mice were mated with transgenic mice expressing dVenus under the Arc promoter. Representative images of both genotypes are shown. (d) In Shn-2 KO mice, Arc-dVenus expression was greatly reduced in the DG and other regions of cortex and amygdala. Arc, activity-regulated cytoskeleton-associated protein. Scale bars indicate 1 mm (a), 250 μm (b), and 100 μm (c). MO, medial orbital cortex; FA, frontal association cortex; PL, prelimbic cortex; M1, primary motor cortex; CG, cingulate cortex; S1, somatosensory cortex; CA, central amygdaloid nucleus; LA, lateral amygdaloid nucleus.

Figure 5

Abnormalities in the cortex of Shn-2 KO mice. (a, b) The cortex of Shn-2 KO mice was thinner than that of wild-type mice. Cortical cell density was also reduced in the prelimbic cortex (PrL) and primary visual cortex (V1) in Shn-2 KO mice (c). (d) Theta band power increased and gamma power decreased in Shn-2 KO mice. (e) A mouse with the Neurologger, a head-mounted EEG data logger device. M1, primary motor cortex; S1, primary somatosensory cortex. Scale bar indicates 1 mm (a).

Figure 6

Dentate granule cells fail to mature in Shn-2 KO mice. (a) The hippocampal transcriptome pattern of Shn-2 KO mice was similar to that of α-CaMKII^+/− mice, which also demonstrated maturation failure in the DG. Genes showing differential expression between genotypes at P<0.005 in both experiments were plotted. (b) Normalized gene expression of differentially expressed genes in Shn-2 KO and α-CaMKII^+/− mice. The top 10 genes are indicated in the graphs. (c) The number of cells expressing the mature neuronal marker calbindin was decreased in Shn-2 KO mice. (d) The expression of the immature-neuronal marker calretinin was markedly increased. (e–k) Physiological properties of granule cells in the DG of Shn-2 KO mice and controls. Physiological features of DG neurons in the mutants were strikingly similar to those of immature DG neurons in normal rodents. Cell capacitance was small in the granule cells of Shn-2 KO mice (e, P<0.0001), whereas input resistance was high (f, P=0.0007), and the threshold current to induce spikes was low (g, P<0.0001). In the current injection (320 pA) experiments, the latency-to-burst spike was shorter (h, P<0.0001) and the number of spikes was lower (i, P=0.0004) compared with that in wild-type mice. (j) The efficacy of basal transmission at the MF synapse was increased in mutant mice (P<0.0001). The ratio of the peak EPSP amplitude to fiber volley amplitude is shown. (k) Shn-2 KO mice display greatly reduced frequency facilitation at 1 Hz (k, genotype effect: P<0.0001 at steady level). Scale bars indicate 500 μm (a), 250 μm (d).

Figure 7

Inflammatory-like phenomena in the hippocampus of Shn-2 KO mice. (a–d) The hippocampal transcriptome pattern of Shn-2 KO mice was similar to the transcriptome data from LPS treatment (a), injury (b), prion infection (c), and aging (d). The Gene Expression Omnibus (GEO) accession numbers for the transcriptome data used for the graphs are GSE23182 (a, c), GSE5296 (b), and GSE13799 (d). Genes that showed differential expression between conditions at P<0.005 in Shn-2 KO mice and P<0.025 in LPS treatment were plotted in (a). Genes that showed differential expression between conditions at P<0.005 in both transcriptome data sets are plotted in (b–d). (e, f) The expression of p22 phox, a component of NADH/NADPH oxidase, was increased in the DG (ML, P=0.027; GCL, P=0.048; HI, P=0.039) and CA1 (Or, P=0.011; Py, P=0.019) of Shn-2 KO mice. (g, h) The expression of vimentin in the DG of mutants was higher than that of the controls. (i–k) The expression of GFAP was increased in the DG of Shn-2 KO mice. Although the area of GFAP-positive cells increased (j), the number of GFAP-positive cells remained unchanged (k, l), which was confirmed by Hoechest nuclear counterstaining. Or, oriens layer; Py, pyramidal cell layer; Rad, stratum radiatum; ML, molecular layer; GCL, granule cell layer; HI, hilus. Scale bars indicate 500 μm (e), 100 μm (g), and 200 μm (i).

Figure 8

Anti-inflammatory treatment rescued neuronal and behavioral phenotypes of Shn-2 KO mice. Mice were chronically treated with rolipram (Rpm, 4 mg/kg) and ibuprofen (Ib, 400 p.p.m.) for 3 weeks. The treatment significantly decreased GFAP immunoreactivity in the DG of Shn-2 KO mice (a, genotype effect: P<0.0001; genotype × drug interaction: P=0.014; Shn-2^−/−, Veh vs Shn-2^−/−, Rpm+Ib, P=0.0323). There was a tendency of decrease in GFAP expression in the mutant CA1 (b, genotype effect: P<0.0001; genotype × drug interaction: P=0.053; Shn-2^−/−, Veh vs Shn-2^−/−, Rpm+Ib, P=0.051). The treatment did not reverse GFAP expression in the PFC of Shn-2 KO mice (c, genotype effect: P=0.0717; genotype × drug interaction: P=0.3662). The reductions of parvalbumin in CA1 (d, genotype effect: P<0.0001) or PFC (e, genotype effect: P=0.0002) were not rescued by the treatment. Increased expression of doublecortin (f, genotype effect: P=0.0014; genotype × drug interaction: P=0.0014; Shn-2^−/−, Veh vs Shn-2^−/−, Rpm+Ib, P=0.0109) and calretinin (g, genotype effect: P<0.0001; genotype × drug interaction: P=0.0019; Shn-2^−/−, Veh vs Shn-2^−/−, Rpm+Ib, P=0.0028) in the DG of Shn-2 KO mice were attenuated by the treatment, while decreased expressions of calbindin (h, genotype effect: P<0.0001) or GAD67 (i, genotype effect: P<0.0001) in the mutant DG were not rescued by the treatment. Working memory (j, genotype effect: P<0.0001; genotype × drug interaction: P=0.0504; Shn-2^−/−, Veh vs Shn-2^−/−, Rpm+Ib, P=0.0042) and nest-building behavior (k, genotype effect: P<0.0001; genotype × drug interaction: P=0.1542; Shn-2^−/−, Veh vs Shn-2^−/−, Rpm+Ib, P=0.0407) were significantly improved by the anti-inflammatory treatment in Shn-2 KO mice. On the other hand, hyperlocomotor activity (l), impaired PPI (m), or anhedonia in the sucrose preference test (n, genotype effect: P=0.006) were not improved. n=5–10 per group. Rpm, rolipram; Ib, ibuprofen; ns, not significant; *P<0.05, **P<0.01.