The solute carrier 27A (SLC27A) gene family encodes fatty acid transport proteins (FATPs) and includes 6 members. During fetal and postnatal periods of development, the growing brain requires a reliable supply of fatty acids. Because autism spectrum disorders (ASD) are now recognized as disorders caused by impaired early brain development, it is possible that functional abnormalities of SLC27A genes may contribute to the pathogenesis of ASD. Here, we confirmed the expression of SLC27A3 and SLC27A4 in human neural stem cells derived from human induced pluripotent stem cells, which suggested their involvement in the developmental stage of the central nervous system. Additionally, we resequenced the SLC27A3 and SLC27A4 genes using 267 ASD patient and 1140 control samples and detected 47 (44 novel and 29 nonsynonymous) and 30 (17 novel and 14 nonsynonymous) variants for the SLC27A3 and SLC27A4, respectively, revealing that they are highly polymorphic with multiple rare variants. The SLC27A4 Ser209 allele was more frequently represented in ASD samples. Furthermore, we showed that a SLC27A4 Ser209 mutant resulted in significantly higher fluorescently-labeled fatty acid uptake into bEnd3 cells, a mouse brain capillary-derived endothelial cell line, compared with SLC27A4 Gly209, suggesting that the functional change may contribute to ASD pathophysiology.

Histone H3 methylation at lysine 9 (H3K9) is a conserved epigenetic signal, mediating heterochromatin formation by trimethylation, and transcriptional silencing by dimethylation. Defective GLP (Ehmt1) and G9a (Ehmt2) histone lysine methyltransferases, involved in mono and dimethylation of H3K9, confer autistic phenotypes and behavioral abnormalities in animal models. Moreover, EHMT1 loss of function results in Kleefstra syndrome, characterized by severe intellectual disability, developmental delays and psychiatric disorders. We examined the possible role of histone methyltransferases in the etiology of autism spectrum disorders (ASD) and suggest that rare functional variants in these genes that regulate H3K9 methylation may be associated with ASD.

Disturbances of lipid metabolism have been implicated in psychiatric illnesses. We previously reported an association between the gene for fatty acid binding protein 7 (FABP7) and schizophrenia. Furthermore, we identified and reported several rare non-synonymous polymorphisms of the brain-expressed genes FABP3, FABP5 and FABP7 from schizophrenia and autism spectrum disorder (ASD), diseases known to part share genetic architecture. Here, we conducted further studies to better understand the contribution these genes make to the pathogenesis of schizophrenia and ASD. In postmortem brains, we detected altered mRNA expression levels of FABP5 in schizophrenia, and of FABP7 in ASD and altered FABP5 in peripheral lymphocytes. Using a patient cohort, comprehensive mutation screening identified six missense and two frameshift variants from the three FABP genes. The two frameshift proteins, FABP3 E132fs and FABP7 N80fs, formed cellular aggregates and were unstable when expressed in cultured cells. The four missense mutants with predicted possible damaging outcomes showed no changes in intracellular localization. Examining ligand binding properties, FABP7 S86G and FABP7 V126L lost their preference for docosahexaenoic acid to linoleic acid. Finally, mice deficient in Fabp3, Fabp5 and Fabp7 were evaluated in a systematic behavioral test battery. The Fabp3 knockout (KO) mice showed decreased social memory and novelty seeking, and Fabp7 KO mice displayed hyperactive and anxiety-related phenotypes, while Fabp5 KO mice showed no apparent phenotypes. In conclusion, disturbances in brain-expressed FABPs could represent an underlying disease mechanism in a proportion of schizophrenia and ASD sufferers.

Recent evidence has documented the potential roles of histone-modifying enzymes in autism-spectrum disorder (ASD). Aberrant histone H3 lysine 9 (H3K9) dimethylation resulting from genetic variants in histone methyltransferases is known for neurodevelopmental and behavioral anomalies. However, a systematic examination of H3K9 methylation dynamics in ASD is lacking. Here we resequenced nine genes for histone methyltransferases and demethylases involved in H3K9 methylation in individuals with ASD and healthy controls using targeted next-generation sequencing. We identified a novel rare variant (A211S) in the SUV39H2, which was predicted to be deleterious. The variant showed strongly reduced histone methyltransferase activity in vitro. In silico analysis showed that the variant destabilizes the hydrophobic core and allosterically affects the enzyme activity. The Suv39h2-KO mice displayed hyperactivity and reduced behavioral flexibility in learning the tasks that required complex behavioral adaptation, which is relevant for ASD. The Suv39h2 deficit evoked an elevated expression of a subset of protocadherin β (Pcdhb) cluster genes in the embryonic brain, which is attributable to the loss of H3K9 trimethylation (me3) at the gene promoters. Reduced H3K9me3 persisted in the cerebellum of Suv39h2-deficient mice to an adult stage. Congruently, reduced expression of SUV39H1 and SUV39H2 in the postmortem brain samples of ASD individuals was observed, underscoring the role of H3K9me3 deficiency in ASD etiology. The present study provides direct evidence for the role of SUV39H2 in ASD and suggests a molecular cascade of SUV39H2 dysfunction leading to H3K9me3 deficiency followed by an untimely, elevated expression of Pcdhb cluster genes during early neurodevelopment.

In schizophrenia (SCZ), neurons in the brain tend to undergo gross morphological changes, but the related molecular mechanism remains largely elusive. Using Kif3b+/- mice as a model with SCZ-like behaviors, we found that a high-betaine diet can significantly alleviate schizophrenic traits related to neuronal morphogenesis and behaviors. According to a deficiency in the transport of collapsin response mediator protein 2 (CRMP2) by the KIF3 motor, we identified a significant reduction in lamellipodial dynamics in developing Kif3b+/- neurons as a cause of neurite hyperbranching. Betaine administration significantly decreases CRMP2 carbonylation, which enhances the F-actin bundling needed for proper lamellipodial dynamics and microtubule exclusion and may thus functionally compensate for KIF3 deficiency. Because the KIF3 expression levels tend to be downregulated in the human prefrontal cortex of the postmortem brains of SCZ patients, this mechanism may partly participate in human SCZ pathogenesis, which we hypothesize could be alleviated by betaine administration.

The early growth response gene 2 (EGR2) has been recently reported to be associated with bipolar disorder in the Korean population. However replication studies in independent cohorts of same and different ethnicities are essential for establishing the credibility of a genotype-phenotype association. With this notion, in the present study we have performed a replication study of the reported association of SNPs in EGR2 in a case-control study comprising of 867 unrelated Japanese bipolar disorder patients and 895 age-, sex- and ethnicity-matched controls. Results showed no significant differences in allele and genotype frequencies of EGR2 SNPs between bipolar disorder patients and controls and also in a sex-stratified genetic analysis. The haplotype and meta-analyses also showed no significant association with bipolar disorder. In conclusion, this is the first replication study of the previously reported association of EGR2 with bipolar disorder in a larger sample set and the results showed that the EGR2 gene is unlikely to contribute to the susceptibility of bipolar disorder in a Japanese cohort.

Neural recognition molecule NB-2/contactin 5 is expressed transiently during the first postnatal week in glutamatergic neurons of the central auditory system. Here, we investigated the effect of NB-2 deficiency on the auditory brainstem in mouse. While almost all principal neurons are wrapped with the calyces of Held in the medial nucleus of the trapezoid body (MNTB) in wild type, 8% of principal neurons in NB-2 knockout (KO) mice lack the calyces of Held at postnatal day (P) 6. At P10 and P15, apoptotic principal neurons were detected in NB-2 KO mice, but not in wild type. Apoptotic cells were also increased in the ventral cochlear nucleus (VCN) of NB-2 KO mice, which contains bushy neurons projecting to the MNTB and the lateral superior olive (LSO). At the age of 1 month, the number of principal neurons in the MNTB and of glutamatergic synapses in the LSO was reduced in NB-2 KO mice. Finally, interpeak latencies for auditory brainstem response waves II-III and III-IV were significantly increased in NB-2 KO mice. Together, these findings suggest that NB-2 deficiency causes a deficit in synapse formation and then induces apoptosis in MNTB and VCN neurons, affecting auditory brainstem function.

Carbonyl stress, a specific form of oxidative stress, is reported to be involved in the pathophysiology of schizophrenia; however, little is known regarding the underlying mechanism. Here, we found that disruption of GLO1, the gene encoding a major catabolic enzyme scavenging the carbonyl group, increases vulnerability to external carbonyl stress, leading to abnormal phenotypes in human induced pluripotent stem cells (hiPSCs). The viability of GLO1 knockout (KO)-hiPSCs decreased and activity of caspase-3 was increased upon addition of methylglyoxal (MGO), a reactive carbonyl compound. In the GLO1 KO-hiPSC-derived neurons, MGO administration impaired neurite extension and cell migration. Further, accumulation of methylglyoxal-derived hydroimidazolone (MG-H1; a derivative of MGO)-modified proteins was detected in isolated mitochondria. Mitochondrial dysfunction, including diminished membrane potential and dampened respiratory function, was observed in the GLO1 KO-hiPSCs and derived neurons after addition of MGO and hence might be the mechanism underlying the effects of carbonyl stress. The susceptibility to MGO was partially rescued by the administration of pyridoxamine, a carbonyl scavenger. Our observations can be used for designing an intervention strategy for diseases, particularly those induced by enhanced carbonyl stress or oxidative stress.

Genome-wide association studies have successfully identified several common variants showing robust association with schizophrenia. However, individually, these variants only produce a weak effect. To identify genetic variants with larger effect sizes, increasing attention is now being paid to uncommon and rare variants.

Maternal immune activation (MIA) contributes to behavioral abnormalities relevant to schizophrenia in adult offspring, although the molecular mechanisms underlying MIA-induced behavioral changes remain unclear. Here we demonstrated that dietary intake of glucoraphanin (GF), the precursor of a natural antioxidant sulforaphane, during juvenile and adolescent stages prevented cognitive deficits and loss of parvalbumin (PV) immunoreactivity in the medial prefrontal cortex (mPFC) of adult offspring after MIA. Gene set enrichment analysis by RNA sequencing showed that MIA caused abnormal expression of centrosome-related genes in the PFC and hippocampus of adult offspring, and that dietary intake of GF improved these abnormal gene expressions. Particularly, MIA increased the expression of suppressor of fermentation-induced loss of stress resistance protein 1 (Sfi1) mRNA in the PFC and hippocampus of adult offspring, and dietary intake of GF prevented the expression of Sfi1 mRNA in these regions. Interestingly, we found altered expression of SFI1 in the postmortem brains and SFI1 mRNA in hair follicle cells from patients with schizophrenia compared with controls. Overall, these data suggest that centrosome-related genes may play a role in the onset of psychosis in offspring after MIA. Therefore, dietary intake of GF-rich vegetables in high-risk psychosis subjects may prevent the transition to psychosis in young adulthood.

NB-2 is a neuronal cell recognition molecule that is preferentially expressed in auditory pathways. Mice deficient in the NB-2 gene exhibit aberrant responses to acoustic stimuli. Here we examined the expression and localization of NB-2 in the auditory brainstem during development in the rat. NB-2 was strongly expressed in the ventral cochlear nucleus (VCN), ventral acoustic stria, lateral and medial superior olivary complex (SOC), superior paraolivary nucleus, medial nucleus of the trapezoid body (MNTB), ventrolateral lemniscus, and central nucleus of the inferior colliculus (CIC). In the VCN and CIC, NB-2 was expressed in the regions that are known to respond to high frequencies. In situ hybridization combined with immunohistochemistry suggested that NB-2 is expressed only in neurons. NB-2 was colocalized with glutamatergic elements in the neuropil and the calyces of Held but not with glycinergic or GABAergic elements. NB-2 expression in the SOC was first detected at embryonic day (E)19, reached a maximum level at postnatal day (P)7, and declined thereafter. Immunolabeling with VGLUT1 and VGLUT2, markers for mature and premature glutamatergic synapses, respectively, in combination with NB-2 immunolabeling revealed that NB-2 is expressed at glutamatergic synapses. Collectively, our findings suggest that NB-2 plays a key role in maturation of glutamatergic synapses in the brainstem during the final stages of auditory development.

Betaine is known to act against various biological stresses and its levels were reported to be decreased in schizophrenia patients. We aimed to test the role of betaine in schizophrenia pathophysiology, and to evaluate its potential as a novel psychotherapeutic.

Identifying beneficial surrogate genetic markers in psychiatric disorders is crucial but challenging.

The post-synaptic density (PSD) of glutamatergic synapses harbors a multitude of proteins critical for maintaining synaptic dynamics. Alteration of protein expression levels in this matrix is a marked phenomenon of neuropsychiatric disorders including schizophrenia, where cognitive functions are impaired. To investigate the genetic relationship of genes expressed in the PSD with schizophrenia, a family-based association analysis of genetic variants in PSD genes such as DLG4, DLG1, PICK1 and MDM2, was performed, using Japanese samples (124 pedigrees, n = 376 subjects). Results showed a significant association of the rs17203281 variant from the DLG4 gene, with preferential transmission of the C allele (p = 0.02), although significance disappeared after correction for multiple testing. Replication analysis of this variant, found no association in a Chinese schizophrenia cohort (293 pedigrees, n = 1163 subjects) or in a Japanese case-control sample (n = 4182 subjects). The DLG4 expression levels between postmortem brain samples from schizophrenia patients showed no significant changes from controls. Interestingly, a five marker haplotype in DLG4, involving rs2242449, rs17203281, rs390200, rs222853 and rs222837, was enriched in a population specific manner, where the sequences A-C-C-C-A and G-C-C-C-A accumulated in Japanese (p = 0.0009) and Chinese (p = 0.0007) schizophrenia pedigree samples, respectively. However, this could not be replicated in case-control samples. None of the variants in other examined candidate genes showed any significant association in these samples. The current study highlights a putative role for DLG4 in schizophrenia pathogenesis, evidenced by haplotype association, and warrants further dense screening for variants within these haplotypes.

Enhanced carbonyl stress underlies a subset of schizophrenia, but its causal effects remain elusive. Here, we elucidated the molecular mechanism underlying the effects of carbonyl stress in iPS cells in which the gene encoding zinc metalloenzyme glyoxalase I (GLO1), a crucial enzyme for the clearance of carbonyl stress, was disrupted. The iPS cells exhibited significant cellular and developmental deficits, and hyper-carbonylation of collapsing response mediator protein 2 (CRMP2). Structural and biochemical analyses revealed an array of multiple carbonylation sites in the functional motifs of CRMP2, particularly D-hook (for dimerization) and T-site (for tetramerization), which are critical for the activity of the CRMP2 tetramer. Interestingly, carbonylated CRMP2 was stacked in the multimer conformation by irreversible cross-linking, resulting in loss of its unique function to bundle microtubules. Thus, the present study revealed that the enhanced carbonyl stress stemmed from the genetic aberrations results in neurodevelopmental deficits through the formation of irreversible dysfunctional multimer of carbonylated CRMP2.

Abnormalities of lipid metabolism contributing to the autism spectrum disorder (ASD) pathogenesis have been suggested, but the mechanisms are not fully understood. We aimed to characterize the lipid metabolism in ASD and to explore a biomarker for clinical evaluation.

Mice with the C3H background show greater behavioral propensity for schizophrenia, including lower prepulse inhibition (PPI), than C57BL/6 (B6) mice. To characterize as-yet-unknown pathophysiologies of schizophrenia, we undertook proteomics analysis of the brain in these strains, and detected elevated levels of Mpst, a hydrogen sulfide (H2 S)/polysulfide-producing enzyme, and greater sulfide deposition in C3H than B6 mice. Mpst-deficient mice exhibited improved PPI with reduced storage sulfide levels, while Mpst-transgenic (Tg) mice showed deteriorated PPI, suggesting that "sulfide stress" may be linked to PPI impairment. Analysis of human samples demonstrated that the H2 S/polysulfides production system is upregulated in schizophrenia. Mechanistically, the Mpst-Tg brain revealed dampened energy metabolism, while maternal immune activation model mice showed upregulation of genes for H2 S/polysulfides production along with typical antioxidative genes, partly via epigenetic modifications. These results suggest that inflammatory/oxidative insults in early brain development result in upregulated H2 S/polysulfides production as an antioxidative response, which in turn cause deficits in bioenergetic processes. Collectively, this study presents a novel aspect of the neurodevelopmental theory for schizophrenia, unraveling a role of excess H2 S/polysulfides production.

NB-2/contactin-5 plays an important role in synapse formation in the developing auditory system of rodents. In this study, to further elucidate the molecular role of NB-2 in synapse formation, we examined the interaction between NB-2 and amyloid precursor-like protein 1 (APLP1), as well as their possible co-localization at the synapse. Pull-down assays and cell surface binding assays demonstrated that NB-2 interacts with APLP1. Furthermore, the protein expression profile of APLP1 in western blots was similar to that of NB-2, and localization of APLP1 mRNA partially overlapped that of NB-2 mRNA. In cultured hippocampal neurons, immunofluorescence signals for both NB-2 and APLP1 overlapped with synapsin, a presynaptic marker. Biochemical analysis showed that both NB-2 and APLP1 were enriched in the presynaptic fraction. These results indicate that NB-2 forms a cis-complex with APLP1 on the presynaptic membrane.

The neural cell recognition molecule NB-3, also referred to as contactin-6, is expressed prominently in the developing nervous system after birth and its deficiency has been shown to cause impairment in motor coordination. Here, we investigated the contribution of NB-3 to cerebellar development, focusing on lobule 3 where NB-3 was expressed in granule cells but not in Purkinje cells. In the developing molecular layer, the neural cell recognition molecules TAG-1, L1, and NB-3 formed distinct expression zones from the external granule cell layer to the internal granule cell layer (IGL), respectively. The NB-3-immunoreactive zone did not overlap with TAG-1-immunoreactive zone. By contrast, the L1-immunoreactive zone overlapped with both the TAG-1- and NB-3-immunoreactive zones. NB-3-positive puncta overlapped with vesicular glutamate transporter 1, a presynaptic marker and were apposed close to metabotropic glutamate receptor 1A, a postsynaptic marker, indicating that NB-3 is localized presynaptically at glutamatergic synapses between parallel fibers and Purkinje cells. In NB-3 knockout mice, L1 immunoreactive signals were increased in the IGL at postnatal day (P) 5, suggesting the increase in the number of immature granule cells of the IGL. In addition, the density of parallel fiber synaptic terminals was reduced in NB-3 knockout mice relative to wild-type mice at P5 to P10. In parallel with these findings, caspase-dependent cell death was significantly increased in the NB- 3-deficient cerebellum at P15. Collectively, our results indicate that NB-3 deficiency affects synapse formation during postnatal cerebellar development.

Autism spectrum disorder is a neurodevelopmental disorder characterized by difficulties in social communication and interaction, as well as repetitive and characteristic patterns of behaviour. Although the pathogenesis of autism spectrum disorder is unknown, being overweight or obesity during infancy and low weight at birth are known as risks, suggesting a metabolic aspect. In this study, we investigated adipose tissue development as a pathophysiological factor of autism spectrum disorder by examining the serum levels of adipokines and other metabolic markers in autism spectrum disorder children (n = 123) and typically developing children (n = 92) at 4-12 years of age. Among multiple measures exhibiting age-dependent trajectories, the leptin levels displayed different trajectory patterns between autism spectrum disorder and typically developing children, supporting an adipose tissue-dependent mechanism of autism spectrum disorder. Of particular interest, the levels of fatty acid binding protein 4 (FABP4) were significantly lower in autism spectrum disorder children than in typically developing subjects, at preschool age (4-6 years old: n = 21 for autism spectrum disorder and n = 26 for typically developing). The receiver operating characteristic curve analysis discriminated autism spectrum disorder children from typically developing children with a sensitivity of 94.4% and a specificity of 75.0%. We re-sequenced the exons of the FABP4 gene in a Japanese cohort comprising 659 autism spectrum disorder and 1000 control samples, and identified two rare functional variants in the autism spectrum disorder group. The Trp98Stop, one of the two variants, was transmitted to the proband from his mother with a history of depression. The disruption of the Fabp4 gene in mice evoked autism spectrum disorder-like behavioural phenotypes and increased spine density on apical dendrites of pyramidal neurons, which has been observed in the postmortem brains of autism spectrum disorder subjects. The Fabp4 knockout mice had an altered fatty acid composition in the cortex. Collectively, these results suggest that an 'adipo-brain axis' may underlie the pathophysiology of autism spectrum disorder, with FABP4 as a potential molecule for use as a biomarker.