Abstract

Schizophrenia is a complex mental disorder with a fairly high degree of heritability. Although the causes of schizophrenia remain unclear, it is now widely accepted that it is a neurodevelopmental and neurodegenerative disorder involving disconnectivity and disorder of the synapses. Disrupted-in-schizophrenia 1 (DISC1) is a promising candidate susceptibility gene involved in neurodevelopment, including maturation of the cerebral cortex. To identify other susceptibility genes for schizophrenia, we screened for DISC1-interacting molecules [NudE-like (NUDEL), Lissencephaly-1 (LIS1), 14-3-3epsilon (YWHAE), growth factor receptor bound protein 2 (GRB2) and Kinesin family 5A of Kinesen1 (KIF5A)], assessing a total of 25 tagging single-nucleotide polymorphisms (SNPs) in a Japanese population. We identified a YWHAE SNP (rs28365859) that showed a highly significant difference between case and control samples, with higher minor allele frequencies in controls (Pallele = 1.01 × 10−5 and Pgenotype = 4.08 × 10−5 in 1429 cases and 1728 controls). Both messenger RNA transcription and protein expression of 14-3-3epsilon were also increased in the lymphocytes of healthy control subjects harboring heterozygous and homozygous minor alleles compared with homozygous major allele subjects. To further investigate a potential role for YWHAE in schizophrenia, we studied Ywhae+/− mice in which the level of 14-3-3epsilon protein is reduced to 50% of that in wild-type littermates. These mice displayed weak defects in working memory in the eight-arm radial maze and moderately enhanced anxiety-like behavior in the elevated plus-maze. Our results suggest that YWHAE is a possible susceptibility gene that functions protectively in schizophrenia.

INTRODUCTION

Recent neuroimaging studies show that structural brain abnormalities are an established feature of schizophrenia and are characterized by decreased total gray matter volume (1,2). These morphological correlates of schizophrenia range from a reduction in brain size to localized alterations in the morphology and molecular composition of specific neuronal, synaptic and glial populations in specific brain areas such as the hippocampus, dorsolateral prefrontal cortex and dorsal thalamus. These findings have fostered the current view of schizophrenia as a disorder of connectivity (3,4) and of the synapse (5). Although the mechanism underlying the neurodevelopmental/neurodegenerative process is still unclear, a way forward is provided by the recent identification of several putative susceptibility genes, such as Neuregulin 1 (6), Dysbindin (7), G72 (8), Catechol-O-methyltransferase (COMT) (9–11) and others (12,13). For none of these genes, however, has a causative allele or the mechanism by which it predisposes to schizophrenia been identified.

Disrupted-in-schizophrenia 1 (DISC1) was first described as a strong candidate gene in a large Scottish family in which a balanced chromosomal translocation segregates with schizophrenia and other psychiatric disorders (12,14,15). The translocation mutation may result in loss of DISC1 function via haploinsufficiency or dominant-negative effects of a predicted mutant DISC1 truncated protein product. DISC1 has been implicated in neurodevelopment, including maturation of the cerebral cortex (16).

DISC1 interacts with several proteins, including NudE-like (NUDEL) (17–19), lissencephaly-1 (LIS1, also called PAFAH1B1) (20), fasciculation and elongation protein zeta 1 (FEZ1) (21) and phosphodiesterase 4B (PDE4B) (16). Recently, we identified several novel DISC1-interacting molecules, including 14-3-3epsilon, Kinesin family 5A of Kinesen1 (KIF5A) and Growth factor receptor bound protein 2 (Grb2) by affinity column chromatography (22,23). Furthermore, we confirmed that DISC1 regulates the localization of the NUDEL/LIS1/14-3-3epsilon complex or Grb2 into axons as a cargo receptor (22,23) and it also regulates Neurotrophin-induced axon elongation by Grb2 (23).

In this study, we screened for the genetic association of DISC1-interacting molecules—NUDEL (17p13.1, OMIM: *607538), LIS1 (17p13.3, OMIM: #607432), 14-3-3epsilon (17p13.3, OMIM: *605066), Grb2 (17p24-q23, OMIM: *108355) and KIF5A (12q13, OMIM: *602821)—with schizophrenia, and identified the gene encoding 14-3-3epsilon (YWHAE) as a possible susceptibility gene. Our results show that a SNP of YWHAE, which influence the expression of 14-3-3epsilon RNA and protein, is associated with schizophrenia and seems to work protectively. We also investigated the behavioral phenotype of mice with ∼50% reduction in 14-3-3epsilon protein expression and found that these mice displayed weak phenotypes consistent with some aspects of human schizophrenia.

RESULTS

Screening analysis of DISC1-related genes and identification of YWHAE as a possible susceptibility gene for schizophrenia

To investigate whether novel DISC1-interacting molecules such as NUDEL, LIS1, YWAHE, GRB2 and KIF5A are associated with schizophrenia, we performed genetic association analyses using a Japanese population.

We failed to develop the genotyping of three SNPs in LIS1 (rs8082331, rs12938775 and rs4790348) and one SNP in GRB2 (rs16967795), therefore a total of 25 SNPs were assessed in this analysis.

Though genotype distributions of two SNPs significantly deviated from Hardy–Weinberg Equilibrium (HWE, PHWE = .0143: rs4789172 in case sample, and P HWE = .0171: rs11172247 in control sample), those of the other markers were in HWE. Six tagging SNPs in YWHAE were significantly associated with schizophrenia and also YWHAE showed gene-wide significance (permutation P = 0.0021), whereas we found no association of tagging SNPs in NUDEL, LIS1, GRB2 or KIF5A (Table 1).

Table 1.

Screening analysis of DISC1-related genes

Gene SNPs  Positiona Missing rate (%) MAF
 
P-value
 
     Cases Controls Allele Genotype 
NUDEL rs3744652 C>T 8280008 0.3 33.0 35.9 0.250 0.274 
 rs8064655 C>T 8301185 33.2 36.3 0.228 0.246 
LIS1 rs1266474 A>G 2481460 0.4 9.72 12.4 0.110 0.0876 
 rs4790356 G>A 2532979 10.6 11.7 0.528 0.730 
 rs7212450 C>G 2538690 42.3 41.7 0.821 0.907 
YWHAE rs34041110 C>T 1193642 48.9 42.5 0.0166 0.00563 
 rs9393 A>G 1195142 27.3 27.9 0.805 0.868 
 rs8064578 C>T 1201625 48.5 43.4 0.0562 0.117 
 rs7224258 G>C 1202252 2.1 15.0 20.3 0.0102 0.0342 
 rs3752826 G>T 1211814 48.6 42.1 0.0139 0.0175 
 rs7214541 T>C 1220072 44.6 49.4 0.0725 0.107 
 rs11655548 A>G 1230748 2.3 29.3 38.3 0.000418 0.00162 
 rs2131431 A>C 1241645 0.3 13.2 18.5 0.00598 0.0176 
 rs1873827 A>G 1247690 42.4 49.6 0.00732 0.0136 
 rs12452627 C>T 1249222 17.7 19.6 0.367 0.662 
GRB2 rs7219 T>C 70826963 9.07 6.85 0.125 0.239 
 rs8079197 C>G 70828274 0.6 8.45 6.60 0.190 0.308 
 rs4789172 C>T 70853307 0.6 24.9c 26.1 0.617 0.659 
 rs2053156 T>G 70890035 6.04 4.53 0.206 0.344 
 rs930296 G>A 70915763 5.91 4.66 0.298 0.432 
KIF5A rs11172247 C>G 56232777 39.4 38.3b 0.676 0.609 
 rs11172254 G>A 56255005 0.3 19.5 21.2 0.422 0.679 
 rs775250 C>A 56263307 20.8 21.7 0.672 0.690 
 rs775251 C>T 56265007 0.4 27.7 32.2 0.0713 0.129 
 rs1678536 C>G 56265457 0.1 47.9 47.4 0.833 0.644 
Gene SNPs  Positiona Missing rate (%) MAF
 
P-value
 
     Cases Controls Allele Genotype 
NUDEL rs3744652 C>T 8280008 0.3 33.0 35.9 0.250 0.274 
 rs8064655 C>T 8301185 33.2 36.3 0.228 0.246 
LIS1 rs1266474 A>G 2481460 0.4 9.72 12.4 0.110 0.0876 
 rs4790356 G>A 2532979 10.6 11.7 0.528 0.730 
 rs7212450 C>G 2538690 42.3 41.7 0.821 0.907 
YWHAE rs34041110 C>T 1193642 48.9 42.5 0.0166 0.00563 
 rs9393 A>G 1195142 27.3 27.9 0.805 0.868 
 rs8064578 C>T 1201625 48.5 43.4 0.0562 0.117 
 rs7224258 G>C 1202252 2.1 15.0 20.3 0.0102 0.0342 
 rs3752826 G>T 1211814 48.6 42.1 0.0139 0.0175 
 rs7214541 T>C 1220072 44.6 49.4 0.0725 0.107 
 rs11655548 A>G 1230748 2.3 29.3 38.3 0.000418 0.00162 
 rs2131431 A>C 1241645 0.3 13.2 18.5 0.00598 0.0176 
 rs1873827 A>G 1247690 42.4 49.6 0.00732 0.0136 
 rs12452627 C>T 1249222 17.7 19.6 0.367 0.662 
GRB2 rs7219 T>C 70826963 9.07 6.85 0.125 0.239 
 rs8079197 C>G 70828274 0.6 8.45 6.60 0.190 0.308 
 rs4789172 C>T 70853307 0.6 24.9c 26.1 0.617 0.659 
 rs2053156 T>G 70890035 6.04 4.53 0.206 0.344 
 rs930296 G>A 70915763 5.91 4.66 0.298 0.432 
KIF5A rs11172247 C>G 56232777 39.4 38.3b 0.676 0.609 
 rs11172254 G>A 56255005 0.3 19.5 21.2 0.422 0.679 
 rs775250 C>A 56263307 20.8 21.7 0.672 0.690 
 rs775251 C>T 56265007 0.4 27.7 32.2 0.0713 0.129 
 rs1678536 C>G 56265457 0.1 47.9 47.4 0.833 0.644 

YWHAE showed gene-wide significance (permutation P = 0.0021).

Bold numbers represent significant P-values (<0.05).

aBased on HapMap database release#21.

bdeviated from Hardy–Weinberg equilibrium.

MAF, minor allele frequency.

Since six tagging SNPs in YWHAE located in the intron region, we performed denaturing high-performance liquid chromatography (dHPLC) analysis in 5′ flanking regions and entire exon regions of YWHAE to identify the possible causal polymorphism, and detected two SNPs: one in the 5′ flanking region (−261 bp from the initial exon: rs28365859) and the other one in the 3′-UTR (rs9393). Since the 5′ flanking region SNP might have a functional effect due to its position, we focused on this SNP in the following analysis [linkage disequilibrium (LD) structure of first-set samples in YWHAE can be seen in Fig. 1).

Figure 1.

Tagging SNPs and LD evaluation of YWHAE for first-set screening samples. rs28365859 was included. Vertical bars represent exons. Numbers in boxes represent r2 values, which should be expressed as decimals. r2 values of 1.0 are not shown. Color scheme was based on GOLD format. Additional information is provided at the Haploview website.

Figure 1.

Tagging SNPs and LD evaluation of YWHAE for first-set screening samples. rs28365859 was included. Vertical bars represent exons. Numbers in boxes represent r2 values, which should be expressed as decimals. r2 values of 1.0 are not shown. Color scheme was based on GOLD format. Additional information is provided at the Haploview website.

First, to examine the association of this SNP, we expanded the sample size (1065 cases and 1386 controls in a second set of confirmation samples, for a total of 1429 cases and 1728 controls including the first set of screening samples, call rates were 100%), and significant association was obtained (Pallele = 1.01 × 10−5 and Pgenotype = 4.08 × 10−5). Furthermore, the significance could be detected in either set independently (Table 2). The commonly observed feature of these analyses was that the minor allele frequencies (MAFs) of this SNP were higher in controls than in schizophrenia patients. There was no discrepancy out of 380 randomly selected samples (190 cases and 190 controls) genotyped in duplicate and by another method (TaqMan Assay: C12125119) for this marker, suggesting it is unlikely that genotyping error had occurred.

Table 2.

Association analysis of promoter SNP in YWHAE (rs28365859)

Samplesa Phenotype n Genotype
 
 P-values
 
   G/G G/C C/C MAF (%) HWEb Allele Genotype 
Combined Cases 1429 921 457 51 19.6 0.537 1.01 × 10−5 4.08 × 10−5 
 Controls 1728 1000 620 108 24.2 0.366   
First-set Cases 364 245 106 13 18.1 0.715 0.00108 0.00545 
 Controls 342 192 127 23 25.3 0.748   
Second-set Cases 1065 676 351 38 20.0 0.359 0.00123 0.00280 
 Controls 1386 808 493 85 23.9 0.399   
Samplesa Phenotype n Genotype
 
 P-values
 
   G/G G/C C/C MAF (%) HWEb Allele Genotype 
Combined Cases 1429 921 457 51 19.6 0.537 1.01 × 10−5 4.08 × 10−5 
 Controls 1728 1000 620 108 24.2 0.366   
First-set Cases 364 245 106 13 18.1 0.715 0.00108 0.00545 
 Controls 342 192 127 23 25.3 0.748   
Second-set Cases 1065 676 351 38 20.0 0.359 0.00123 0.00280 
 Controls 1386 808 493 85 23.9 0.399   

First-set samples were identical to those used in screening analysis.

Second-set samples were independent set of samples to increase the sample size.

aCombined samples = first-set+second-set samples.

HWE, Hardy–Weinberg equilibrium.

Functional analysis of the promoter SNP in YWHAE: in vitro and in vivo expression assays

We first investigated the influence of rs28365859 on YWHAE expression by dual-luciferase assay, although there is no evidence that the region where this SNP is located on is evolutionally conserved and that any regions in YWHAE are match as a core promoter by in silico promoter detection software. As shown in Fig. 2, a trend for significance in a promoterless vector and significance in a promoter vector were obtained in the different cell lines. The constructs containing a minor allele (C allele) showed higher expression in the promoter vector, suggesting that the C allele plays a possible enhancer role in these cell lines.

Figure 2.

In vitro and in vivo expression assays. (A) Promoterless vector (basic vector) and (B) promoter vector in CHO and COS7 cells. Firefly luciferase activities were normalized with Renilla luciferase activities. Relative expression was calculated as 100 for the major allele (G allele) of rs28365839. *P < 0.05, **P < 0.01, ***P < 0.001 (in Student's t-test). Results of (C) real-time RT–PCR and (D) western blot analysis. Expression levels of 14-3-3epsilon RNA and protein were normalized with GAPDH expression. Relative expression was calculated as 100 for the major allele homozygous genotype (G/G genotype) or major allele (G allele) of rs28365839. Number of individuals with the distinct genotypes of rs28365839 were 16 for G/G, 8 for G/C and 3 for C/C. *P < 0.05, **P < 0.01 (in post hoc comparison with the Dunnett test for genotype-wise analysis, and t-test for allele-wise analysis).

Figure 2.

In vitro and in vivo expression assays. (A) Promoterless vector (basic vector) and (B) promoter vector in CHO and COS7 cells. Firefly luciferase activities were normalized with Renilla luciferase activities. Relative expression was calculated as 100 for the major allele (G allele) of rs28365839. *P < 0.05, **P < 0.01, ***P < 0.001 (in Student's t-test). Results of (C) real-time RT–PCR and (D) western blot analysis. Expression levels of 14-3-3epsilon RNA and protein were normalized with GAPDH expression. Relative expression was calculated as 100 for the major allele homozygous genotype (G/G genotype) or major allele (G allele) of rs28365839. Number of individuals with the distinct genotypes of rs28365839 were 16 for G/G, 8 for G/C and 3 for C/C. *P < 0.05, **P < 0.01 (in post hoc comparison with the Dunnett test for genotype-wise analysis, and t-test for allele-wise analysis).

Next, to examine the role of this SNP in peripheral blood of healthy control subjects, real-time RT–PCR and western blot analysis were performed. Similar to the luciferase assays, heterozygous and homozygous minor allele (G/C and C/C genotype) subjects showed higher expression levels of 14-3-3epsilon than did homozygous major allele (G/G genotype) subjects (one-way analysis of variance, ANOVA, P = 0.0251 and 0.0014 in real-time RT–PCR and western blot analysis, respectively). Experimental analysis were performed to examine the differences under an additive model (G/G versus G/C+C/C), again significant associations were obtained.

Furthermore, haplotype trend regression test was applied to check the effects of haplotypes of rs28365859 and other four SNPs in intron 1 (rs11655548, rs2131431, rs1873827 and rs12452627), which might also be in an enhancer region. This showed significant association in either analysis (P = 0.0282 and 0.0186 in real-time RT–PCR and western blot analysis, respectively), however, each SNP in intron 1 was not correlated with the expression level (data not shown).

Effect of reduction of 14-3-3epsilon protein on the cognitive functions of mice

14-3-3 proteins are highly conserved across species, from bacteria to humans, and bind to phosphoserine/phosphothreonine motifs in a sequence-specific manner (24–28). Previously we reported that 14-3-3epsilon binds to CDK5/p35-phosphorylated NUDEL and maintains NUDEL phosphorylation. To examine the protective effect of 14-3-3epsilon on schizophrenia using mice, we should investigate whether overexpression of 14-3-3epsilon results in resistance for the onset of schizophrenic symptoms. However, an assay system to evaluate the effect of a gene on the onset of schizophrenia in mice has not yet been developed. Thus, in support of a role for YWHAE in schizophrenia, we investigated Ywhae knockout mice. Null mice of Ywhae gene (Ywhae−/−) show a severe cell migration defect in both the cortex and the hippocampus, whereas Ywhae+/− mice, in which the expression level of 14-3-3epsilon protein is reduced to ∼50% compared with their wild-type littermates, show a milder migration defect (29). Because most Ywhae−/− mice die at birth as previously reported (29), Ywhae+/− mice and their wild-type littermates were analyzed by a comprehensive behavioral test battery to investigate whether the reduction in 14-3-3epsilon protein affects behavior (30,31). Ywhae+/− mice appeared normal, healthy and fertile (Table 3).

Table 3.

Comprehensive behavioral test battery

Test  Ywhae+/+ Ywhae+/− P-value F value 
General health      
 Weight (g)  28.6 29.682 0.0262* 1.335 
 Rectal temperature (°C)  37.033 36.688 0.0435* 4.406 
Pain test      
 Hot plate (latency, s)  6.206 5.053 0.1142 2.633 
Motor tests      
 Grip strength (n 1.044 1.085 0.2825 1.194 
 Wire hang (latency to fall, s)  60 50 0.0234* 5.65 
 Rotarod (latency to fall, s; average of six trials)  161.759 182.618 0.3391 0.941 
Anxiety-like behavior      
 Light/dark transition      
  Distance travelled (cm) Light side 484.983 617.782 0.0728 3.434 
Dark side 1095.389 1099.288 0.97 0.001 
  Stay time in light side (s)  214.972 231.176 0.6043 0.274 
  Transitions (times)  35.111 33.588 0.6827 0.17 
  Latency to light side (s)  31.444 34.941 0.6683 0.187 
 Elevated plus-maze      
  Number of entries (times)  32.556 25.118 0.0075** 8.126 
  Entries onto open arms (%)  31.824 26.648 0.2044 1.677 
  Distance travelled (cm)  1323.722 1194.329 0.1071 2.744 
  Time on open arms (%)  15.269 12.971 0.3798 0.793 
 Time on closed arms (%)  50.87 63.196 0.0195* 6.034 
  Time on center area (%)  35.034 23.283 0.0012* 12.495 
Depression model      
 Porsolt forced swim (immobility, %) Day 1 59.369 65.648 0.0661 3.614 
 Day 2 77.026 78.564 0.6256 0.243 
 Tail suspension (immobility, %)  26.194 22.774 0.6267 0.241 
Locomotor activity      
 Open field      
  Total distance travelled (cm)  8745.222 9258.941 0.5822 0.309 
  Vertical activity (times)  208.722 393.824 0.047* 4.259 
  Center time (s/min)  1.432 1.107 0.6505 0.209 
  Stereotypic counts (times)  7260.944 6124.118 0.2251 1.528 
Sensory motor gating      
 Acoustic startle response  3.021 2.704 0.32 1.02 
 Prepulse inhibition (startle stimulus, %)      
  110-dB startle  48.887 47.697 0.8496 0.037 
  120-dB startle  13.566 16.6 0.5617 0.344 
Working memory      
 8-arm radial maze      
  Training      
  Different arm choice in first 8 entries (times)  6.209 6.016 0.3325 0.967 
  Revisiting errors (times)  6.12 7.613 0.1557 2.11 
 Delay 30 s      
  Different arm choice in first 8 entries (times)  6.5 6.471 0.8972 0.017 
  Revisiting errors (times)  3.417 3.676 0.7599 0.095 
 Delay 120 s      
  Different arm choice in first 8 entries (times)  5.882 0.6476 0.213 
  Revisiting errors (times)  4.944 6.735 0.1715 1.954 
 Delay 300 s      
  Different arm choice in first 8 entries (times)  6.167 5.971 0.5077 0.448 
  Revisiting errors (times)  3.778 6.294 0.0229* 5.698 
Reference memory      
 T-maze (correct, %) Training 80.648 77.157 0.0696 3.519 
 Reverse 61.759 59.314 0.4567 0.567 
Cued and contextual fear conditioning      
 Conditioning (freezing, %)  28.324 29.29 0.7581 0.096 
 Context testing (freezing, %)  50.998 46.611 0.5754 0.32 
 Cued testing with altered context freezing, %)  53.641 52.926 0.8342 0.045 
Social interaction      
 Total duration of contact (s)  118.386 153.383 0.1239 2.776 
 Number of contacts (times)  49.429 53.333 0.4968 0.494 
 Total duration of active contacts (s)  14.257 18.733 0.0809 3.693 
 Mean duration/contact  2.443 3.017 0.2889 1.241 
 Distance travelled (cm)  2789.357 2882.167 0.7206 0.135 
Test  Ywhae+/+ Ywhae+/− P-value F value 
General health      
 Weight (g)  28.6 29.682 0.0262* 1.335 
 Rectal temperature (°C)  37.033 36.688 0.0435* 4.406 
Pain test      
 Hot plate (latency, s)  6.206 5.053 0.1142 2.633 
Motor tests      
 Grip strength (n 1.044 1.085 0.2825 1.194 
 Wire hang (latency to fall, s)  60 50 0.0234* 5.65 
 Rotarod (latency to fall, s; average of six trials)  161.759 182.618 0.3391 0.941 
Anxiety-like behavior      
 Light/dark transition      
  Distance travelled (cm) Light side 484.983 617.782 0.0728 3.434 
Dark side 1095.389 1099.288 0.97 0.001 
  Stay time in light side (s)  214.972 231.176 0.6043 0.274 
  Transitions (times)  35.111 33.588 0.6827 0.17 
  Latency to light side (s)  31.444 34.941 0.6683 0.187 
 Elevated plus-maze      
  Number of entries (times)  32.556 25.118 0.0075** 8.126 
  Entries onto open arms (%)  31.824 26.648 0.2044 1.677 
  Distance travelled (cm)  1323.722 1194.329 0.1071 2.744 
  Time on open arms (%)  15.269 12.971 0.3798 0.793 
 Time on closed arms (%)  50.87 63.196 0.0195* 6.034 
  Time on center area (%)  35.034 23.283 0.0012* 12.495 
Depression model      
 Porsolt forced swim (immobility, %) Day 1 59.369 65.648 0.0661 3.614 
 Day 2 77.026 78.564 0.6256 0.243 
 Tail suspension (immobility, %)  26.194 22.774 0.6267 0.241 
Locomotor activity      
 Open field      
  Total distance travelled (cm)  8745.222 9258.941 0.5822 0.309 
  Vertical activity (times)  208.722 393.824 0.047* 4.259 
  Center time (s/min)  1.432 1.107 0.6505 0.209 
  Stereotypic counts (times)  7260.944 6124.118 0.2251 1.528 
Sensory motor gating      
 Acoustic startle response  3.021 2.704 0.32 1.02 
 Prepulse inhibition (startle stimulus, %)      
  110-dB startle  48.887 47.697 0.8496 0.037 
  120-dB startle  13.566 16.6 0.5617 0.344 
Working memory      
 8-arm radial maze      
  Training      
  Different arm choice in first 8 entries (times)  6.209 6.016 0.3325 0.967 
  Revisiting errors (times)  6.12 7.613 0.1557 2.11 
 Delay 30 s      
  Different arm choice in first 8 entries (times)  6.5 6.471 0.8972 0.017 
  Revisiting errors (times)  3.417 3.676 0.7599 0.095 
 Delay 120 s      
  Different arm choice in first 8 entries (times)  5.882 0.6476 0.213 
  Revisiting errors (times)  4.944 6.735 0.1715 1.954 
 Delay 300 s      
  Different arm choice in first 8 entries (times)  6.167 5.971 0.5077 0.448 
  Revisiting errors (times)  3.778 6.294 0.0229* 5.698 
Reference memory      
 T-maze (correct, %) Training 80.648 77.157 0.0696 3.519 
 Reverse 61.759 59.314 0.4567 0.567 
Cued and contextual fear conditioning      
 Conditioning (freezing, %)  28.324 29.29 0.7581 0.096 
 Context testing (freezing, %)  50.998 46.611 0.5754 0.32 
 Cued testing with altered context freezing, %)  53.641 52.926 0.8342 0.045 
Social interaction      
 Total duration of contact (s)  118.386 153.383 0.1239 2.776 
 Number of contacts (times)  49.429 53.333 0.4968 0.494 
 Total duration of active contacts (s)  14.257 18.733 0.0809 3.693 
 Mean duration/contact  2.443 3.017 0.2889 1.241 
 Distance travelled (cm)  2789.357 2882.167 0.7206 0.135 

Behavioral test battery was performed in the following order: general health/neurological screen, wire hang, grip strength test, light/dark transition, open field, elevated plus-maze, hot plate, social interaction (novel environment), rotarod, prepulse inhibition, Porsolt forced swim, eight arm radial maze, T-maze, cued and contextual fear condition test, latent inhibition, tail suspension test.

*P < 0.05, **P < 0.01.

To examine whether reduction in 14-3-3epsilon was associated with cognitive deficits, we analyzed Ywhae+/− mice and their wild-type littermates in working memory and reference memory tasks (Table 3). To assess working memory of Ywhae+/− mice, we used a spatial working memory version of the 8-arm radial maze task (32,33). The mice were trained for 26 trials. During training, both control and mutant mice improved their performance and no significant difference was observed (P = 0.3325) (Fig. 3A). The number of revisiting errors of Ywhae+/− mice was significantly more than their wild-type littermates during trials with a delay of 300 s (P = 0.0229) (Fig. 3C). The number of different arms chosen during the first eight choices, which is considered a measure of working memory that is relatively independent of locomotor activity levels and the total number of choices, was not significantly affected by the deficit of 14-3-3epsilon protein during training and trials with 30, 120 and 300 s of delay (P = 0.3325, 0.8972, 0.6476 and 0.5077, respectively) (Fig. 3B and D). These results suggest that Ywhae+/− mice show weak defects in working memory.

Figure 3.

Behavioral abnormality of Ywhae+/− mice. (AD) Working memory test of Ywhae+/− mice in the 8-arm radial maze (A and B). Total number of revisiting errors (A) and the number of different arms chosen in the first 8-arm visits (B) across training were counted. Data are presented as 2-day/trial averages. (C and D) Total number of revisiting errors (C) and the number of different arms chosen in the first 8-arm visits (D) of mice after training; exposure to delays of 30, 120 or 300 s after four pellets had been taken were counted (see Materials and Methods). (E and F) Anxiety-like behavior test of Ywhae+/− mice in the elevated plus-maze. (E) Number of total entries. (F) Time spent on closed arms. Number of total entries was lower and time spent on closed arms were greater in Ywhae+/− mice than in controls. Asterisks indicate a difference from the values of control mice. *P < 0.05, **P < 0.01.

Figure 3.

Behavioral abnormality of Ywhae+/− mice. (AD) Working memory test of Ywhae+/− mice in the 8-arm radial maze (A and B). Total number of revisiting errors (A) and the number of different arms chosen in the first 8-arm visits (B) across training were counted. Data are presented as 2-day/trial averages. (C and D) Total number of revisiting errors (C) and the number of different arms chosen in the first 8-arm visits (D) of mice after training; exposure to delays of 30, 120 or 300 s after four pellets had been taken were counted (see Materials and Methods). (E and F) Anxiety-like behavior test of Ywhae+/− mice in the elevated plus-maze. (E) Number of total entries. (F) Time spent on closed arms. Number of total entries was lower and time spent on closed arms were greater in Ywhae+/− mice than in controls. Asterisks indicate a difference from the values of control mice. *P < 0.05, **P < 0.01.

Next, we analyzed reference memory of Ywhae+/− mice, using the left–right discrimination test version of the T-maze. Ywhae+/− mice and their wild-type littermates were trained for 6 trials; then the correct side was reversed. The next 6 trials were performed under the reversal-learning condition. No significant difference was observed in the percentage of correct choices at the sixth trial (Ywhae+/+, 80.647%; Ywhae+/−, 77.157%; P = 0.7516), and no significant difference was observed under the reversal-learning condition (P = 0.4567) (Table 3). These results suggest that a decrease in the 14-3-3epsilon protein results in weak defects, specifically in spatial working memory.

Moderately enhanced anxiety-like behavior in Ywhae+/− mice in the elevated plus-maze test

To examine the effect of 14-3-3epsilon deficit on anxiety-like behavior, Ywhae+/− mice and their wild-type littermates were analyzed in light/dark transition and elevated plus-maze tests. In light/dark transition, no significant difference was observed between Ywhae+/− mice and their wild-type littermates (Table 3). In the elevated plus-maze test, Ywhae+/− mice showed a smaller number of total entries (P = 0.0075) (Fig. 3E), increased time spent on closed arms (P = 0.0195) (Fig. 3F) and decreased time spent on center area (P = 0.0012) (Table 3). A significant difference was not observed in the number of entries onto open arms, total distance travelled or time spent on open arms (P = 0.2044, 0.1071, 0.3798, respectively) (Table 3). Thus, it is conceivable that Ywhae+/− mice have moderately enhanced anxiety-like behavior that could be detected only by the elevated plus-maze test but not by the light/dark transition or by the open-field tests.

DISCUSSION

Association between YWHAE and schizophrenia

In this study, we have identified YWHAE, the gene encoding 14-3-3epsilon, which forms a complex with DISC1 in vivo, as a possible susceptibility gene for schizophrenia. Genetic and expression evidence indicates that the SNP in 5′ flanking region (rs28365859) is associated with schizophrenia through influencing the expression level of YWHAE. Subjects with the C allele of rs28365859 were thought to have a reduced risk of schizophrenia [odds ratio of combined subjects = 0.76 (95% confidence interval: 0.68-0.86)]. Our sample size was relatively large (3157samples consisting of 706 first-set and 2451 second-set samples: 1429 schizophrenics and 1728 controls), making our results reliable. In addition, another research assessing the genetic association of YWHAE with suicide victims [two of SNPs (rs3752826 and rs9393) are identical SNPs in our study and another SNP (rs1532976) can be captured by rs3752826 using HapMap information] can support our results, since it showed the same trends in the distributions of MAFs (MAFs of these SNPs were higher in controls than in cases) (34). However, a couple of limitations should be outlined. First, our results that show statistical significances may be derived from unknown population stratification, since Genomic Control was not included in this analysis. Secondly, there could be a possible effect of differential age distribution between cases and controls in the association analysis.

The in vitro luciferase assay suggests that the C allele might act as an enhancer, since significant luciferase induction could not be seen with the use of a promoterless vector, but luciferase activity (LA) could be assayed from the vector containing a promoter. Further, in vivo expression assays of RNA and protein in peripheral blood samples clarified the functional relevance of this SNP: Subjects who were heterozygous and homozygous with the C allele had higher expression of 14-3-3epsilon. Of note, our samples were control subjects not on medication; therefore, we could avoid the bias related to drug effects, which may be seen when studying schizophrenia subjects.

Also, haplotype trend regression analysis showed that the haplotypes consisted of five SNPs located in 5′ flanking region (rs28365859) and intron1 (rs11655548, rs2131431, rs1873827 and rs12452627) were correlated with the expression level of YWHAE, whereas each SNP in intron 1 was not correlated with the expression. Therefore, we speculate that this significant result in haplotype-wise analysis may be derived mainly from the effects of rs28365859.

We analyzed for the homology of genome sequence between human and mice Ywhae gene using 500 bp upstream region from start ATG. About 200 bp upstream region from start ATG shows high identity, however, a region containing rs2836589 SNP does not show homology. This result suggests that this SNP is not evolutionally conserved. We searched for functional motif on the sequence in the 5′ upstream region of YWHAE including rs28365859 using TESS: Transcription Element Search System (http://www.cbil.upenn.edu/cgi-bin/tess/tess). In minor allele (C allele), ubiquitously expressed cellular upstream stimulatory factor (USF) -interacting motif ‘CCACGT’ was detected in this in silico analysis. This result may explain a possible functional effect of this SNP, an upregulation of 14-3-3epsilon in C allele-harboring people, however, further analysis would be needed to provide definitive conclusion.

Role of 14-3-3epsilon in neuronal development

Several observations of the postmortem brain suggest that alterations in neuronal cell migration, and synaptic, dendritic and axonal organizations occur in schizophrenia patients (35,36). Ywhae+/− mice show milder migration defects in both the cortex and the hippocampus, whereas Ywhae−/− mice display severe neuronal migration defects (29). Primary hippocampal neurons from Ywhae−/− mice display shorter axons and a defect in accumulation of the NUDEL/LIS1 complex in the distal part of axons (29). We confirmed that knockdown of 14-3-3epsilon by RNAi impairs not only the NUDEL/LIS1 complex transport into axons but also axon elongation (data not shown). Previously, we identified 14-3-3epsilon as an interacting molecule of DISC1 (22). DISC1 is required to transport the NUDEL/LIS1/14-3- 3epsilon complex into axons (22). Of note, depletion of endogenous DISC1 by RNAi results in a severe neuronal migration defect in the developing neocortex via regulation of the dynein complex (37). These results and reports suggest that both DISC1 and 14-3-3epsilon are required for neuronal development via transport of the NUDEL/LIS1 complex. To clarify the functional relationship between 14-3-3 epsilon and DISC1 on neuronal development via transport of the NUDEL/LIS1 complex, further genetic analysis using knockout mice will be required.

Cognitive dysfunction of Ywhae+/− mice

Ywhae+/− mice, in which the expression of 14-3-3epsilon protein was reduced to ∼50% compared with their wild-type littermates, showed weak cognitive dysfunction specifically in working memory (Table 3). Interestingly, missense mutant mice of the DISC1 gene show defects in working memory (38). Reduction of DISC1 or 14-3-3epsilon results in developmental defects of hippocampal neurons. These results and reports suggest that impairment of DISC1 or 14-3-3epsilon cause neuronal developmental defects, that result in cognitive dysfunction. Interestingly, impairment of working memory is one of the prominent features of schizophrenia symptomatology (39–41). Non-synonymous polymorphism of DISC1 that consists of a serine to cysteine substitution at codon 704 (DISC1Ser704Cys) is reported to correlate with variations in hippocampal size and cognitive function including working memory, and is associated with schizophrenia (42). Although relation between 14-3-3epsilon and cognitive function in human is not known, 14-3- 3epsilon could be implicated in cognitive function that is associated with DISC1. Another prominent feature of schizophrenia symptomatology, prepulse inhibition (43), did not differ in Ywhae+/− mice compared with their wild-type littermates (Table 3). Schizophrenia is a complex disorder with a variety of pathology and risk factor genes. It is a reasonable assumption that modification of a single gene does not mimic all features of schizophrenia symptomatology. We think that our results using Ywhae+/− mice partly support our genetic data. However, further analysis would be required to clarify a role of 14-3-3epsilon on cognitive functions and functional relationship between YWHAE and DISC1.

YWHAE as a possible susceptibility gene for schizophrenia

In this study, we found that a SNP of YWHAE that correlates the expression of 14-3-3epsilon is associated with schizophrenia, and that this SNP would reduce the risk of schizophrenia. Perhaps, increased 14-3-3epsilon expression in humans affected by the identified SNP is protective, whereas decreased 14-3-3epsilon expression due to 50% reduction by heterozygous knockout in mice results in behavioral deficits. At this point, we do not know why higher expression levels of 14-3-3epsilon reduce the risk of schizophrenia, or why lower expression levels of this gene result in increase of the risk in human and behavioral changes in mice. By its susceptibility genes, schizophrenia seems to be a complex disorder with multiple symptoms and genetic risk factors. We predict that schizophrenia would be divided into several classes by its susceptibility genes. Each class would have its own molecular/signaling pathway that plays important roles in the pathogenesis. DISC1 and its interacting molecules are required in neuronal developments and adult neurongenesis (44), and would play critical roles in pathogenesis of specific classes of schizophrenia. In other classes of schizophrenia, the DISC1-pathway would not be implicated in the pathogenesis. Some genes could have redundant functions. 14-3-3epsilon is a member of adaptor proteins that interact with phosphorylated serine or threonine residue of target proteins. More than 100 of 14-3-3-binding partners involved in signal transduction, cell cycle regulation, apoptosis, stress responses and malignant transformation have been identified (45). Proteomic analysis of synapse revealed that 14-3-3epsilon forms a complex with NMDA receptor (46). Placing these results and reports in the context of the pathogenesis of schizophrenia, 14-3-3epsilon could be a susceptibility gene of not only DISC1-implicated, but also wide range of schizophrenia because of its wide variety of interacting partners. 14-3-3epsilon would be a key molecule to understand molecular mechanisms of susceptibility genes for schizophrenia.

MATERIALS AND METHODS

Subjects in genetic association analyses

In the genetic association analyses, two independent sets of subjects were examined. The first screening analysis included 364 patients with schizophrenia (188 male and 176 female; mean age ± SD 42.5 ± 14.8 years) and 342 healthy controls (191 male and 151 female; 35.0 ± 13.6 years). Patients for the second confirmation analysis included 1065 patients with schizophrenia (562 male and 503 female; 48.9 ± 14.7 years) and 1386 controls (714 male and 672 female; 42.6 ± 14.6 years). All subjects were unrelated to each other and reported to be of Japanese ethnicity. Forty patients with schizophrenia were used as subjects for a mutation search; these subjects were also included in the first-set screening scan. The schizophrenia patients were diagnosed according to criteria in the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition after at least two experienced psychiatrists reached consensus on the diagnosis on the basis of unstructured interviews and review of medical records. All healthy controls were also psychiatrically screened on the basis of unstructured interviews; to exclude subjects with any brain disorder, or psychotic disorder, or who had first-degree relatives with psychotic disorders, trained psychiatrist interviewed them to assess current and/or past mental states (psychotic, mood, anxiety, obsessive-compulsive symptoms) and family history. After description of the study, written informed consent was obtained from each subject. This study was approved by the ethics committees at Fujita Health University, Teikyo University, Okayama University, and Nagoya University Graduate School of Medicine.

SNP selection and genotyping

For LD-based association analysis using the first set of screening samples, we first consulted the HapMap and dbSNP databases to pick-up ‘tagging SNPs’. From the HapMap database (Data Release #21: population JPT: MAF of >0.05: regions 8275000.8320000 for NUDEL, 2440000.2537000 for LIS1, 1193000.1256000 for YWHAE, 70823000.70917000 for GRB2, 56227000.56266000 for KIF5A), we selected a total of 27 tagging SNPs (one SNP for NUDEL, six SNPs for LIS1, nine SNPs for YWHAE, six SNPs for GRB2 and five SNPs for KIF5A) with a threshold criterion of r2 > 0.8 in pairwise tagging mode using Tagger software (47). Two SNPs (one for NUDEL, rs3744652 and one for YWHAE, rs34041110) were added for denser mapping.

All SNPs were genotyped by TaqMan assays, primer extension using dHPLC and polymerase chain reaction–restriction fragment length polymorphism assays as described previously (48). More detailed assay information can be found in Supplementary Material, Table S1.

Mutation search

After we detected significant association of YWHAE in screening samples, we used dHPLC analysis for a mutation search, the details of which are described in a previous paper (48). Primer pairs (Supplementary Material, Table S2) were designed with the use of information from the GenBank sequence (accession number: NT 010718.15) into 10 amplified regions, which covered all the coding regions, the branch sites and the 5′ flanking region 1026 bp upstream from the initial exon of YWHAE.

In vivo and in vitro expression assays

We used a dual-luciferase assay, real-time RT–PCR and western blot analysis to examine the influence of SNP rs28365859 in the 5′ flanking region on expression levels of YWHAE. For the dual-luciferase assay, 497-bp fragments that included rs28365859 were PCR amplified (Supplementary Material, Table S1). Genomic DNAs with identified genotypes were used as templates, and PCR products of either genotypes were cloned into a pGL3-basic vector and a pGL3-promoter vector (Promega, WI). These vectors with both alleles, the Renilla luciferase vector and the phRL-TK vector, were transiently transfected into Chinese hamster ovary (CHO) cells and COS-7 cells with the use of Lipofectamine 2000™ (Invitrogen, CA). All inserts were sequenced to confirm the containing alleles. After 48 h, cell extracts were prepared and assayed for firefly LA (LAF) and Renilla LA (LAR) as described by the manufacturer (PikkaGene Dual SeaPansy™ Luminescence Kit, Tokyo Ink, Japan) on a Fluoroskan Ascent FL (Thermo Labsystems, Finland).

For in vitro assays (real-time RT–PCR and western blot analysis), we processed and analyzed a total of 27 peripheral blood samples from normal control subjects to determine the amount of YWHAE transcript or protein: 16 subjects with homozygous major alleles (G/G genotype: 7 male and 9 female; 32.6 ± 6.4 years) in rs28365859; 8 subjects with heterozygous major alleles (G/C genotype: 4 male and 4 female; 33.5 ± 7.7 years) and 3 subjects with homozygous minor alleles (C/C genotype: 1 male and 2 female; 51.3 ± 17.0 years). These subjects were healthy controls who had not received any medication within at least 1 month before the collection of RNA and protein.

In the real-time RT–PCR assay, total RNA was isolated with the use of a QIAamp RNA Blood Mini Kit (QIAGEN, Inc., CA). Complementary DNA was generated with the use of a High-Capacity cDNA Archive Kit (Applied Biosystems). Real-time PCR constituents were 50 ng DNA, 2× TaqMan Universal Master Mix and 20× primer/probe mix (Hs00356749_g1, Applied Biosystems) in a 50-μl final volume. The amplification was done according to the manufacturer's instructions, and signals were recorded during PCR with the use of an ABI PRISM 7900 instrument. All gene expression results were normalized to the expression of glyceryl aldehyde-3-phosphate dehydrogenase (GAPDH).

In the western blot analysis, lymphocytes were purified (Axis-Shield, Oslo, Norway) and protein concentrations were determined with bovine serum albumin as the reference protein. The antibody against 14-3-3epsilon and alpha-tubulin were purchased (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Proteins were subjected to SDS–PAGE, followed by immunoblot analyses with anti-14-3-3epsilon or anti-alpha-tubulin antibody. The amount of 14-3-3epsilon was detected by chemiluminescence in a linear range using serial dilutions of standards and was estimated with Densitograph (ATTO, Tokyo, Japan). Alpha-tubulin was used as the standard for quantification. The results of these in vivo and in vitro expression assays were representative of three independent experiments.

Animals and experimental design

Ywhae+/− mice and their wild-type littermates were obtained as previously reported (29). Genetic background of mice is mixed 129/S6 × NIH Black Swiss. All behavioral tests (8-arm radial maze test, elevated plus-maze test, T-maze test, light/dark transition test and startle response/prepulse inhibition tests) were carried out with male mice that were 9–10 weeks old at the start of the testing. Heterozygous knockout mice and wild-type littermates were compared in experiments. Mice were housed in a room with a 12-h light/dark cycle (lights on at 7:00 a.m.) with access to food and water ad libitum. Behavioral testing was performed between 9:00 a.m. and 6:00 p.m. After the tests, all apparatus was cleaned with super hypochlorous water to prevent a bias on the basis of olfactory cues with the apparatus. Detailed description of each behavioral test (neurological screen, neuromascular strength, rotarod test, open-field test, light/dark transition test, elevated plus-maze test, hot plate test, startle response/prepulse inhibition tests, social interaction test in a novel environment, sociability and social novelty preference test, social interaction test in home cage, T-maze test and contextual and cued fear conditioning) can be seen in Supplementary methods.

Statistical analysis

Tests for HWE and marker-trait association were evaluated by χ2 test (SAS/Genetics, release 8.2, SAS Institute Japan Inc., Tokyo, Japan). Gene-wide significance of single-SNP test was estimated by permuting phenotype status to generate 10 000 data set of SNPs in each gene under null hypothesis of no association (49). Differences in relative expression between alleles (for luciferase assay) and genotypes (for real-time PCR and western blot) were evaluated by a two-tailed Student's t-test and one-way ANOVA, respectively (JMP5.1J, SAS Institute Japan Inc.). When a significant difference was obtained in ANOVA, post hoc comparison with the Dunnett test [with homozygous major alleles (G/G genotype) set as controls] was used to identify specific group differences. Also to check the effects of haplotypes on gene expression, haplotype trend regression test with permutation (10 000 times) was applied (Power Marker V3.25 by Jack Liu, www://powermarker.net/). In behavior analysis, statistical analysis was conducted by using STATVIEW (SAS Institute, Cary, NC). Data were analyzed by ANOVA or repeated-measures ANOVA. Values in graphs were expressed as mean ± SEM. The level of significance was set at 0.05.

FUNDING

This research was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT), a Grant-in-Aid for Creative Scientific Research from Japan Society for the Promotion of Science, the MEXT 21st Century Center of Excellence Program, a research grant for Nervous and Mental Disorders from the Ministry of Health, Labour and Welfare, a Grant-in-Aid for Scientific Research on Pathomechanisms of Brain Disorders from the MEXT (17025021), the Japan Health Sciences Foundation (Research on Health Sciences Focusing on Drug Innovation), the Core Research for Evolutional Science and Technology, Grant-in-Aid for Scientific Research on Priority Areas—Integrative Brain Research (Shien)—from MEXT in Japan, Grant-in-Aid from Neuroinformatics Japan Center (NIJC), RIKEN and Grant-in-Aid from Institute for Bioinformatics Research and Development (BIRD) of Japan Science and Technology Agency (JST).

SUPPLEMENTARY MATERIAL

Supplementary Material is available at HMG Online.

Conflict of Interest statement: None declared.

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Author notes

The authors wish it to be known that, in their opinion, the first three authors should be regarded as joint First authors.

Supplementary data