A loss-of-function variant in SUV39H2 identified in autism-spectrum disorder causes altered H3K9 trimethylation and dysregulation of protocadherin ß-cluster genes in the developing brain.

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 ß (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.

Glyoxalase I disruption and external carbonyl stress impair mitochondrial function in human induced pluripotent stem cells and derived neurons.

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.

Role of an Atypical Cadherin Gene, Cdh23 in Prepulse Inhibition, and Implication of CDH23 in Schizophrenia.

We previously identified quantitative trait loci (QTL) for prepulse inhibition (PPI), an endophenotype of schizophrenia, on mouse chromosome 10 and reported Fabp7 as a candidate gene from an analysis of F2 mice from inbred strains with high (C57BL/6N; B6) and low (C3H/HeN; C3H) PPI levels. Here, we reanalyzed the previously reported QTLs with increased marker density. The highest logarithm of odds score (26.66) peaked at a synonymous coding and splice-site variant, c.753G>A (rs257098870), in the Cdh23 gene on chromosome 10; the c.753G (C3H) allele showed a PPI-lowering effect. Bayesian multiple QTL mapping also supported the same variant with a posterior probability of 1. Thus, we engineered the c.753G (C3H) allele into the B6 genetic background, which led to dampened PPI. We also revealed an e-QTL (expression QTL) effect imparted by the c.753G>A variant for the Cdh23 expression in the brain. In a human study, a homologous variant (c.753G>A; rs769896655) in CDH23 showed a nominally significant enrichment in individuals with schizophrenia. We also identified multiple potentially deleterious CDH23 variants in individuals with schizophrenia. Collectively, the present study reveals a PPI-regulating Cdh23 variant and a possible contribution of CDH23 to schizophrenia susceptibility.

Peroxisome proliferator-activated receptor α as a novel therapeutic target for schizophrenia.

BACKGROUND: The pathophysiology of schizophrenia, a major psychiatric disorder, remains elusive. In this study, the role of peroxisome proliferator-activated receptor (PPAR)/retinoid X receptor (RXR) families, belonging to the ligand-activated nuclear receptor superfamily, in schizophrenia, was analyzed. METHODS: The PPAR/RXR family genes were screened by exploiting molecular inversion probe (MIP)-based targeted next-generation sequencing (NGS) using the samples of 1,200 Japanese patients with schizophrenia. The results were compared with the whole-genome sequencing databases of the Japanese cohort (ToMMo) and the gnomAD. To reveal the relationship between PPAR/RXR dysfunction and schizophrenia, Ppara KO mice and fenofibrate (a clinically used PPARα agonist)-administered mice were assessed by performing behavioral, histological, and RNA-seq analyses. FINDINGS: Our findings indicate that c.209-2delA, His117Gln, Arg141Cys, and Arg226Trp of the PPARA gene are risk variants for schizophrenia. The c.209-2delA variant generated a premature termination codon. The three missense variants significantly decreased the activity of PPARα as a transcription factor in vitro. The Ppara KO mice exhibited schizophrenia-relevant phenotypes, including behavioral deficits and impaired synaptogenesis in the cerebral cortex. Oral administration of fenofibrate alleviated spine pathology induced by phencyclidine, an N-methyl-d-aspartate (NMDA) receptor antagonist. Furthermore, pre-treatment with fenofibrate suppressed the sensitivity of mice to another NMDA receptor antagonist, MK-801. RNA-seq analysis revealed that PPARα regulates the expression of synaptogenesis signaling pathway-related genes. INTERPRETATION: The findings of this study indicate that the mechanisms underlying schizophrenia pathogenesis involve PPARα-regulated transcriptional machinery and modulation of synapse physiology. Hence, PPARα can serve as a novel therapeutic target for schizophrenia.

A potential role of fatty acid binding protein 4 in the pathophysiology of autism spectrum disorder.

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.

Evaluation of the role of fatty acid-binding protein 7 in controlling schizophrenia-relevant phenotypes using newly established knockout mice.

Dampened prepulse inhibition (PPI) is a consistent observation in psychiatric disorders, including schizophrenia and qualifies as a robust endophenotype for genetic evaluation. Using high PPI C57BL/6NCrlCrlj (B6Nj) and low PPI C3H/HeNCrlCrlj (C3HNj) inbred mouse strains, we have previously reported a quantitative trait locus (QTL) for PPI at chromosome 10 and identified Fabp7 as a candidate gene for regulating PPI and schizophrenia pathogenesis using Fabp7-deficient mice (B6.Cg-Fabp7 KO). Here, considering a possibility of carryover of residual genetic materials from embryonic stem (ES) cells used in generating knockout (KO) mice, we set out to re-address the genotype-phenotype correlation in a uniform genetic background. By generating a new Fabp7 KO mouse model in C57BL/6NCrl (B6N) background using the CRISPR-Cas9 nickase system, we evaluated the impact of Fabp7 ablation on schizophrenia-related behavioral phenotypes. To our surprise, we found no significant differences in PPI or any of the schizophrenia-related behavioral scores, as observed in our previous B6.Cg-Fabp7 KO mice. We identified several C3H/He mouse strain-specific alleles within the interval of chromosome 10-QTL, which are shared with 129/Sv mouse strains. These alleles, derived from 129/Sv ES cells, were retained in the B6.Cg-Fabp7 KO, despite multiple backcrossing and are thought to be responsible for the dampened PPI. In summary, our study demonstrates a precise genotype-phenotype relation for Fabp7 loss-of-function in a uniform B6N background, and raises the necessity of further analysis of the effects of genomic variants flanking the Fabp7 interval on phenotypes.

Excess hydrogen sulfide and polysulfides production underlies a schizophrenia pathophysiology.

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.

Investigation of betaine as a novel psychotherapeutic for schizophrenia.

BACKGROUND: 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. METHODS: Using Chdh (a gene for betaine synthesis)-deficient mice and betaine-supplemented inbred mice, we assessed the role of betaine in psychiatric pathophysiology, and its potential as a novel psychotherapeutic, by leveraging metabolomics, behavioral-, transcriptomics and DNA methylation analyses. FINDINGS: The Chdh-deficient mice revealed remnants of psychiatric behaviors along with schizophrenia-related molecular perturbations in the brain. Betaine supplementation elicited genetic background-dependent improvement in cognitive performance, and suppressed methamphetamine (MAP)-induced behavioral sensitization. Furthermore, betaine rectified the altered antioxidative and proinflammatory responses induced by MAP and in vitro phencyclidine (PCP) treatments. Betaine also showed a prophylactic effect on behavioral abnormality induced by PCP. Notably, betaine levels were decreased in the postmortem brains from schizophrenia, and a coexisting elevated carbonyl stress, a form of oxidative stress, demarcated a subset of schizophrenia with "betaine deficit-oxidative stress pathology". We revealed the decrease of betaine levels in glyoxylase 1 (GLO1)-deficient hiPSCs, which shows elevated carbonyl stress, and the efficacy of betaine in alleviating it, thus supporting a causal link between betaine and oxidative stress conditions. Furthermore, a CHDH variant, rs35518479, was identified as a cis-expression quantitative trait locus (QTL) for CHDH expression in postmortem brains from schizophrenia, allowing genotype-based stratification of schizophrenia patients for betaine efficacy. INTERPRETATION: The present study revealed the role of betaine in psychiatric pathophysiology and underscores the potential benefit of betaine in a subset of schizophrenia. FUND: This study was supported by the Strategic Research Program for Brain Sciences from AMED (Japan Agency for Medical Research and Development) under Grant Numbers JP18dm0107083 and JP19dm0107083 (TY), JP18dm0107129 (MM), JP18dm0107086 (YK), JP18dm0107107 (HY), JP18dm0107104 (AK) and JP19dm0107119 (KH), by the Grant-in-Aid for Scientific Research on Innovative Areas from the MEXT under Grant Numbers JP18H05435 (TY), JP18H05433 (AH.-T), JP18H05428 (AH.-T and TY), and JP16H06277 (HY), and by JSPS KAKENHI under Grant Number JP17H01574 (TY). In addition, this study was supported by the Collaborative Research Project of Brain Research Institute, Niigata University under Grant Numbers 2018-2809 (YK) and RIKEN Epigenetics Presidential Fund (100214-201801063606-340120) (TY).

Key role of soluble epoxide hydrolase in the neurodevelopmental disorders of offspring after maternal immune activation.

Maternal infection during pregnancy increases risk of neurodevelopmental disorders such as schizophrenia and autism spectrum disorder (ASD) in offspring. In rodents, maternal immune activation (MIA) yields offspring with schizophrenia- and ASD-like behavioral abnormalities. Soluble epoxide hydrolase (sEH) plays a key role in inflammation associated with neurodevelopmental disorders. Here we found higher levels of sEH in the prefrontal cortex (PFC) of juvenile offspring after MIA. Oxylipin analysis showed decreased levels of epoxy fatty acids in the PFC of juvenile offspring after MIA, supporting increased activity of sEH in the PFC of juvenile offspring. Furthermore, expression of sEH (or EPHX2) mRNA in induced pluripotent stem cell-derived neurospheres from schizophrenia patients with the 22q11.2 deletion was higher than that of healthy controls. Moreover, the expression of EPHX2 mRNA in postmortem brain samples (Brodmann area 9 and 40) from ASD patients was higher than that of controls. Treatment with 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl)urea (TPPU), a potent sEH inhibitor, in juvenile offspring from prenatal day (P) 28 to P56 could prevent cognitive deficits and loss of parvalbumin (PV) immunoreactivity in the medial PFC of adult offspring after MIA. In addition, dosing of TPPU to pregnant mothers from E5 to P21 could prevent cognitive deficits, and social interaction deficits and PV immunoreactivity in the medial prefrontal cortex of juvenile offspring after MIA. These findings suggest that increased activity of sEH in the PFC plays a key role in the etiology of neurodevelopmental disorders in offspring after MIA. Therefore, sEH represents a promising prophylactic or therapeutic target for neurodevelopmental disorders in offspring after MIA.

Thiosulfate promotes hair growth in mouse model.

The present study describes the hair growth-promoting effects of sodium thiosulfate (STS), a widely used compound, in mice. STS accelerated hair growth in the "telogen model", suggesting that it stimulates telogen hair follicles to reenter the anagen phase of hair growth. In the same model, STS potentiated hair growth in an additive manner with minoxidil (MXD), a drug used for the treatment of androgenic alopecia. Furthermore, in the "anagen model", STS promoted hair growth, probably by promoting hair follicle proliferation. Since STS elevated the skin surface temperature, its hair growth-promoting activity may be partly due to vasorelaxation, similar to MXD. In addition, STS is known to generate a gaseous mediator, H2S, which has vasorelaxation and anti-inflammatory/anti-oxidative stress activities. Therefore, STS and/or provisionally its metabolite, H2S, may aid the hair growth process. Collectively, these results suggest that salts of thiosulfate may represent a novel and beneficial remedy for hair loss.

Investigation of the fatty acid transporter-encoding genes SLC27A3 and SLC27A4 in autism.

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.

Narrow-band imaging observation of colorectal lesions using NICE classification to avoid discarding significant lesions.

AIM: To assess the risk of failing to detect diminutive and small colorectal cancers with the "resect and discard" policy. METHODS: Patients who received colonoscopy and polypectomy were recruited in the retrospective study. Probable histology of the polyps was predicted by six colonoscopists by the use of NICE classification. The incidence of diminutive and small colorectal cancers and their endoscopic features were assessed. RESULTS: In total, we found 681 cases of diminutive (1-5 mm) lesions in 402 patients and 197 cases of small (6-9 mm) lesions in 151 patients. Based on pathology of the diminutive and small polyps, 105 and 18 were non-neoplastic polyps, 557 and 154 were low-grade adenomas, 18 and 24 were high-grade adenomas or intramucosal/submucosal (SM) scanty invasive carcinomas, 1 and 1 were SM-d carcinoma, respectively. The endoscopic features of invasive cancer were classified as NICE type 3 endoscopically. CONCLUSION: The risk of failing to detect diminutive and small colorectal invasive cancer with the "resect and discard" strategy might be avoided through the use of narrow-band imaging observation with the NICE classification scheme and magnifying endoscopy.

[Bacillus cereus septicemia and necrotizing fasciitis in a patient with liver cirrhosis: a case report].

A 54-year-old woman with hematemesis was referred to our hospital. She had a history of liver cirrhosis and diabetes mellitus. After inserting a Sengstaken-Blakemore tube, we performed endoscopic variceal ligation for ruptured esophageal varices. On the third day of admission, she developed septicemia and necrotizing fasciitis caused by Bacillus cereus. She was successfully treated with early debridement of both lower extremities and intravenous treatment with vancomycin, ciprofloxacin, and clindamycin. Although B. cereus is an attenuate bacterium, it can occasionally cause fatal infection in immuno-compromised individuals, such as those with liver cirrhosis.

Functional characterization of FABP3, 5 and 7 gene variants identified in schizophrenia and autism spectrum disorder and mouse behavioral studies.

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.

Epha4 controls the midline crossing and contralateral axonal projections of inferior olive neurons.

The guidance of axonal projections to ipsilateral and contralateral regions is essential for integration of bilateral sensory information and coordination of movement. In the development of olivocerebellar projections, newborn neurons of inferior olivary (IO) nuclei ventrally migrate from the hindbrain rhombic lip to the floor plate (FP). The cell bodies of IO neurons cannot cross the FP but their axons can, and thus IO neurons project their axons only to the contralateral cerebellar cortex. The molecular mechanisms determining the contralateral axonal projections of IO neurons, however, are obscure. The IO neurons and their axons express EphA4, whereas the FP expresses an EphA4 ligand, EphrinB3, from embryonic day 12.5. Therefore, we tested whether EphA4-deficient mice (EphA4(-/-) ) would show impairment in the development of olivocerebellar projections. We found that, in EphA4(-/-) embryos, some of the IO neurons projected their axons to the ipsilateral cerebellar cortex because the cell bodies of the IO neurons abnormally crossed the FP. Furthermore, even in adults, EphA4(-/-) cerebella were bilaterally innervated by unilateral IO subnuclei. These observations indicate that EphA4 is involved in the contralateral axonal projections of IO neurons by preventing their cell bodies from crossing the midline FP.

Directional gene-transfer into the brain by an adenoviral vector tagged with magnetic nanoparticles.

Adenoviral (Ad) vectors are useful for in vivo gene transfer into the brain. If Ad vectors are injected into the ventricle of mouse embryonic brain, Ad vectors introduce a foreign gene into neural progenitor cells on the surface of ventricle. However, Ad vectors were unable to deliver a foreign gene to a targeted region of the embryonic brain because Ad vectors evenly infected the neural progenitor cells on the surface of ventricle. Therefore, the Ad infection to the neural progenitor cells was uncontrollable. To develop a directional gene-transfer with Ad vector, we generated Ad vector tagged with magnetic nanoparticles (Ad-mag) by linking a biotinylated adenovirus vector with a streptavidin-conjugated magnetic nanoparticle. Ad-mags were attracted by magnetic force in vitro and in vivo. When Ad-mags were injected into the ventricle of mouse embryo and a strong magnet was attached to the head of the embryo, Ad-mags were attracted to the restricted direction or region where the magnet was placed. As a result, Ad-mags efficiently introduced a foreign gene into the restricted region of the brain.

[Myoclonus following spinal and epidural anesthesia--a case report].

We report a case of spinal myoclonus following cesarean section. The patient was a 34-year-old woman without history of neurologic disorders. In the operating room, after placement of an epidural catheter at T12-L1, bupivacaine 2.4 ml was administered intrathecally via a 25 G needle at L2-3. Epidural administration of ropivacaine (0.13%, 4 ml x hr(-1)) was started 72 min after spinal anesthesia. The intra- and postoperative courses were otherwise uneventful. The patient complained of involuntary jerky movements of her lower legs 195 min after the start of the spinal anesthesia. The sensory level was T12 and she could move her legs on command but could not stop her involuntary movements. The myoclonic movements ceased 150 min later without medication and did not reappear, despite restarting the epidural anesthesia with ropivacaine.