BACKGROUND:Treatment-Resistant Depression (TRD) features persistent socio-affective deficits poorly responsive to monoaminergic antidepressants. We investigated Kamikihito, mesenchymal stem cells, or their combination in a two-hit rat model recapitulating TRD-like social withdrawal and empathic impairment (prenatal ethanol exposure + adolescent corticosterone) Methods: Pregnant Wistar rats were exposed to ethanol from D10 to D14 twice a day. Offspring received corticosterone from weaning to PND 42. In late adolescence, they were treated with the traditional Japanese herbal formula Kamikihito (KMK), intravenous Mesenchymal Stem Cells (MSCs), or a combination of both, as compared to a vehicle-treated control. At nine weeks of age, we assessed social interaction and rescue‑task performance, followed by rt-qPCR to measure the gene expression of VGLUT1 and VGAT in the Anterior Cingulate Cortex (ACC) and OXTR and Cd38 in the paraventricular nucleus (PVN). RESULTS:The stressed saline-treated rats showed significant reductions in social and empathic behaviours. MSCs alone produced modest improvements, whereas KMK (alone or combined with MSCs) significantly restored rescue‑task performance, with the highest composite behavioural scores in the combination group. All active treatments tended to normalize the ACC excitatory/ inhibitory balance (as reflected in the VGLUT1/VGAT ratio) and significantly upregulated OXTR and Cd38 expression in the PVN compared with stressed controls. DISCUSSION:Developmental stress induces persistent socio-affective dysfunction linked to alterations in cortical excitatory/inhibitory balance and oxytocinergic signaling. The greater efficacy of KMK, particularly in combination with MSCs, supports complementary neuromodulatory and neuroimmune mechanisms relevant to TRD. CONCLUSION:KMK, especially combined with MSCs, partially reverses stress-induced socio-affective deficits and modulates key molecular pathways, supporting a promising strategy for targeting socio-affective dysfunction in TRD.

Long interspersed nuclear element-1 (LINE-1 or L1) is the only autonomously active retrotransposon in the human genome and produces both sense and antisense transcripts from its 5' untranslated region (5'UTR) of L1Hs, a human-specific L1 subfamily. Among these, ORF0 is an antisense transcript-derived protein implicated in retrotransposition activity, yet its mRNA expression has been difficult to quantify because strand discrimination is required for accurate detection. Here, we developed a strand-specific quantitative polymerase chain reaction (qPCR) method incorporating multiplex gene-specific tagged primers in the reverse transcription (RT) step, which enables simultaneous quantification of antisense ORF0 and sense 5'UTR and ORF2 transcripts. Validation in 5-aza-2'-deoxycytidine-treated neuroblastoma cells confirmed dose-dependent increases in expression of ORF0 and sense transcripts, demonstrating the strand specificity and functionality of this method. Application to human postmortem prefrontal cortex and cerebellum samples revealed stable expression of ORF0 and sense transcripts. This assay provides a robust and scalable tool for the precise quantification of strand-specific L1 transcription, complementing locus-specific analyses, and offering a platform for future studies on the role of ORF0 and L1 antisense transcription.

Schizophrenia is a severe neurodevelopmental disorder whose etiology remains incompletely understood. Epidemiological studies of the Dutch Hunger Winter demonstrated that maternal famine during early gestation increased the risk of schizophrenia in offspring, implicating the Developmental Origins of Health and Disease (DOHaD) framework. However, the molecular mechanisms underlying this association remain unclear. Here, we developed a novel DOHaD-based schizophrenia model by subjecting pregnant mice to transient fasting restricted to the peri-implantation period, a critical window of global epigenomic reprogramming. Male offspring of fasted dams exhibited schizophrenia-related phenotypes, including impaired sensorimotor gating, abnormal behavioral patterns, and reduced dendritic spine density in the medial prefrontal cortex. Multi-omics profiling, integrating bulk RNA sequencing, Visium HD spatial transcriptomics, and DNA methylation arrays, revealed convergent alterations in synaptic organization, protein homeostasis, and oxidative stress pathways. These findings highlight how brief maternal fasting reprograms the epigenome and reshapes neural circuitry. Our work establishes the first animal model that directly mirrors early gestational famine exposure linked to schizophrenia risk, providing a unique platform for uncovering epigenetic mechanisms underlying the developmental origins of psychiatric disorders.

Alzheimer's disease (AD) is a major cause of dementia, with polygenic risk scores (PRSs) widely used to capture cumulative genetic risk. While PRSs have been associated with cognitive decline, their relevance to clinically accessible measures in general populations is not yet fully established, particularly in non-European cohorts. In this study, we investigated the association between AD PRSs and cognitive function assessed by the Mini-Mental State Examination (MMSE) in a community-dwelling Japanese older population ( N = 1,301). Three PRSs were constructed using genome-wide association study (GWAS) summary statistics derived from European and Japanese populations. Among the PRSs, the score based on Japanese GWAS showed the strongest and most consistent association with MMSE score, whereas those based on European GWAS showed weaker or no associations. Stratification analyses further demonstrated that individuals with higher PRS exhibited lower MMSE scores and a higher prevalence of cognitive impairment. Notably, these associations were attenuated after excluding participants with dementia, suggesting that PRS primarily reflects clinically relevant cognitive decline. No significant associations were observed between PRSs and hippocampal volume in our cohort. These findings highlight the importance of population-specific PRS and suggest its potential utility for stratifying cognitive impairment using simple clinical measures in community-based settings. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This work was funded by the Japan Society for the Promotion of Science (JSPS) Grants-in-Aid for Scientific Research (KAKENHI) grant numbers JP23H02840, JP23H03838, JP23K16578, JP22K07583, JP25K10839, JP25H01309, and JP25H01314, and by Japan Science and Technology Agency (JST) Moonshot R&D grant number JPMJMS2021, and by Japan Agency for Medical Research and Development (AMED) grant numbers JP24wm0625302 and JP24wm0625001. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Ethics committee of Kumamoto University gave ethical approval for this work. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present work are contained in the manuscript.

Age-related cognitive decline and depressive symptoms are prevalent in later life, yet the genetic determinants of vulnerability remain unclear. Here, we investigated how genetic and epigenetic regulation of the serotonin transporter gene SLC6A4 contributes to susceptibility to these age-related conditions in later life. In community-dwelling older adults in Japan ( N = 1,317), functional stratification of the serotonin transporter-linked polymorphic region (5-HTTLPR) revealed that participants with low-activity genotypes showed a robust co-occurrence of cognitive decline and depressive symptoms, whereas this comorbid pattern was not observed in those with the high-activity genotype. The genotype-dependent co-occurrence was consistently replicated across seven independent population-based cohorts (total N = 7,889). DNA methylation at a functional promoter CpG site increased with age and partially mediated age-related cognitive decline specifically among low-activity genotypes. In contrast, the high-activity genotype was associated with relative resistance to these functional declines, partly mediated by a protective effect on hippocampal volume during aging. Notably, genotype-dependent effects on hippocampal volume were absent in adolescence, indicating that the influence of SLC6A4 emerges in an aging-specific manner. Together, these findings identify SLC6A4 promoter activity as a key genetic factor modulating vulnerability and resilience in later life. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This study was partly supported by Grants-in-Aid for Scientific Research (KAKENHI) from the Japan Society for the Promotion of Science (JSPS) and the Japan Science and Technology Agency (JST) (JP22K07583, JP23K27531, JP25H01314, JP25H01309, JP23H03838, JP24K22098, JP25K10839, JP23K16578, JP24K02378, JPMJMS2021, and JPMJFR231Q). This study was also partly supported by a grant from the Center for Metabolic Regulation of Healthy Aging (CMHA) in Kumamoto University. This study was also supported by the Japan Agency for Medical Research and Development (AMED) (JP24dk0207053, JP24wm0625302, and JP24wm0625001), and Suntory Holdings Limited (Osaka, Japan). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Ethics committee of Kyushu University and Kumamoto University gave ethical approval for this work. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present work are contained in the manuscript.

Background Heart failure with preserved ejection fraction (HfpEF) is increasingly recognized as a multisystem disorder linked to the cardiovascular-kidney-metabolic (CKM) syndrome. While the falling heart undergoes metabolic reprogramming, the interorgan crosstalk regulating myocardial substrate preference in HFpEF remains elusive. We aimed to clarify the role of systemic and local ketogenesis in the pathogenesis of cardiac hypertrophy and HFpEF. Methods A mouse model of HFpEF was employed using a high-fat diet combined with NG-Nitro-L-arginine methyl ester hydrochloride (L-NAME). Cardiac hypertrophy and systemic metabolic profiling including ketogenesis were evaluated. To dissect the role of site-specific ketogenesis, we generated inducible cardiomyocyte-specific (Hmgcs2ΔiCM) and hepatocyte-specific (Hmgcs2ΔHep) knockout mice of HMG-CoA synthase 2 (Hmgcs2), deficient in the rate-limiting enzyme for ketogenesis. Cardiomyocyte -specific nuclei were isolated for transcriptomic (RNA-seq) and in vitro assays in H9C2 cells were used to elucidate molecular mechanisms. Results The HFpEF model successfully exhibited diastolic dysfunction, impaired exercise capacity and cardiac hypertrophy with elevated circulating ketone body concentration. Myocardial metabolomics and snRNA-seq identified a profound metabolic shift characterized by the accumulation of long-chain fatty acids and Krebs cycle intermediates, coupled with the transcriptional downregulation of insulin signaling and fatty acid degradation pathways. Although circulating ketone body level was upregulated, Hmgcs2ΔiCM mice showed no exacerbation of the HFpEF phenotype. In contrast, Hmgcs2ΔHep mice exhibited significantly aggravated cardiac hypertrophy (HW/TL; Hmgcs2flox: 7.41 ± 0.87: Hmgcs2ΔHep: 8.29 ± 0.73; p = 0.0154). Mechanistically, hepatic ketogenesis was required to maintain circulating beta-hydroxybutyrate (BHB) levels, which directly modulated cardiomyocyte metabolism. BHB acted as a metabolic signal to dampen fatty acid overload and facilitate glucose utilization. Conclusions Our study identifies a critical "liver-heart axis" where hepatic ketogenesis serves as an essential regulator of myocardial metabolic resilience. Impaired hepatic ketogenesis creates a metabolic mismatch that drives pathological cardiac remodeling. These findings highlight the liver as a therapeutic target within the CKM syndrome framework, suggesting that restoring the hepato-cardiac metabolic bridge may ameliorate HFpEF progression. ### Competing Interest Statement The authors have declared no competing interest. Japan Society for the Promotion of Science, 24K18380, 23KK0150, 25K02647 Japan Agency for Medical Research and Development, https://ror.org/004rtk039, JP24fk0210119, JP256f0137011 Takeda Science Foundation, https://ror.org/02y123g31 Suzuken Memorial Foundation Astellas Foundation

Retrotransposon long interspersed nuclear element-1 (LINE-1, L1) constitutes a large proportion of the mammalian genome. A fraction of L1s, which have no deleterious mutations in the structure, can amplify their copies via a process called retrotransposition (RT). RT affects genome stability and gene expression and is involved in the pathogenesis of many hereditary diseases. Measuring expression of RT-capable L1s (rc-L1s) among the hundreds of thousands of non rc-L1s is an essential step to understand the impact of RT. We developed mobile element-originated read enrichment from RNA-seq data (MORE-RNAseq), a pipeline for calculating expression of rc-L1s using manually curated L1 references in humans and mice. MORE-RNAseq allows for quantification of expression levels of overall (sum of the expression of all rc-L1s) and individual rc-L1s with consideration of the genomic context. We applied MORE-RNAseq to publicly available RNA-seq data of human and mouse cancer cell lines from the studies that reported increased L1 expression. We found the significant increase of rc-L1 expressions at the overall level in both inter- and intragenic contexts. We also identified differentially expressed rc-L1s at the locus level, which will be the important candidates for downstream analysis. We also applied our method to young and aged human muscle RNA-seq data with no prior information about L1 expression, and found a significant increase of rc-L1 expression in the aged samples. Our method will contribute to understand the role of rc-L1s in various physiological and pathophysiological conditions using standard RNA-seq data. All scripts are available at https://github.com/molbrain/MORE-RNAseq.

Background Long interspersed nuclear element-1 (LINE1 or L1) is a major type of retrotransposons constituting about 20% of mammalian genomes, some of which still have retrotransposition ability. L1 is the only transposon that can retrotranspose itself using its own proteins. Although retrotransposition is strictly repressed by the host-defense mechanism, it is known that somatic L1 retrotransposition is transiently activated in early neural development. We previously reported the increased L1 copy number in the brains of patients as well as in animal models of psychiatric disorders (Bundo et al., Neuron 2014), suggesting the possibility that aberrant expression of L1 increases the risk for psychiatric disorders.L1 has its own internal promoter at the 5’ UTR. Several transcription factors that bind to the L1 5’ UTR play a critical role in the regulation of L1 expression. A YY1 binding site exists near the start of L1 at about +10 to +20. This site is shown to be important for the precise transcription start. Its effect on L1 expression, however, is controversial in studies using a teratocarcinoma cell line. One study showed that the promoter missing the site is 5 times less active, whereas another study found only a minor effect in a retrotransposition assay. Importantly, there are L1 copies missing the YY1 binding site in the human genome, and they are reported less methylated, evading the repression in the adult human brain. Thus, it is important to elucidate the effect of YY1 binding site on the L1 retrotransposition activity in the brain. Aims & Objectives In this study, we developed transgenic mice to investigate the effect of truncation of YY1 binding site on the L1 retrotransposition in the mouse brain. Method We utilized the L1EGFP construct for monitoring L1 retrotransposition. L1EGFP was made from human L1 and EGFP. In the system, the cells are labeled by EGFP expression only after the retrotransposition of the construct. We created 2 lines of L1EGFP mice by inserting intact or YY1 binding site-truncated L1EGFP (trL1EGFP) construct into the Rosa26 locus. We selected 5 mice from each line with the same sex and similar average age for this experiment. We perfused the mice and fixed the isolated brains overnight with 10% formalin neutral buffer solution. Then, the brain samples were placed in 30% sucrose solution and kept at -4°. The samples were sectioned with microtome and immunostained for EGFP. Results There was a considerable number of EGFP-labeled neurons in L1EGFP mice, mostly gathered in the neocortex and dentate gyrus. We also found EGFP-labeled neuroglia in the brain such as astrocytes. We also detected EGFP-labeled neurons in trL1EGFP mice. We compare the frequency and patterns of EGFP- positive cells between two strains and will present them on the poster. Discussion & Conclusion By analyzing the results, functions of the YY1 binding site on the L1 retrotransposition will be revealed. The trL1EGFP mice will be a useful tool to investigate the role of L1 retrotransposition in neurological disorders, psychiatric disorders, and aging in human. References Bundo, M. et al (2014) ‘Increased L1 Retrotransposition in the Neuronal Genome in Schizophrenia,’ Neuron, 81(2), pp. 306–313.

BACKGROUND:Major depression (MD) is caused by both genetic and environmental factors. Epigenetic mechanisms, particularly DNA methylation (5mC) and hydroxymethylation (5hmC), are thought to mediate gene - environment interactions. However, findings in mouse models remain dispersed. OBJECTIVE:This review evaluates the studies on 5mC and 5hmC in mouse models of depression. METHODS:We systematically searched PubMed, Scopus, and Web of Science using terms related to 5mC/5hmC, depression, and mouse, until December 2024. We grouped the articles as candidate, global, cellular, and comprehensive studies and summarized the findings accordingly. RESULTS:Sixty-eight studies met inclusion criteria. The main findings were environmental models, especially chronic stress paradigms, which were most frequently used to induce depression models. Candidate gene studies focused on Bdnf and Nr3c1, while global and cellular assays revealed both regional and widespread 5mC/5hmC changes. Genome-wide approaches revealed that epigenetic changes are not limited to isolated loci rather affect broad genomic regions involved in neural development and plasticity. CONCLUSION:This review provides a comprehensive summary of existing research on epigenetic changes in terms of DNA methylation in mouse models of depression. Broader application of standardized, integrative, and cell-type-specific approaches is needed to fully elucidate the role of epigenetic regulation in the pathology of MD.

ABSTRACT Aims This study aimed to investigate whether multi‐timepoint DNA methylation levels at the SLC6A4 gene during early adolescence are associated with psychopathological and behavioral clusters, SLC6A4 encodes the serotonin transporter, which regulates the concentration of serotonin in the synaptic cleft. The clusters were previously identified by deep learning analysis of self‐ and parental‐report questionnaires from participants in the Tokyo Teen Cohort (TTC) study in Japan. Methods We extracted genomic DNA from saliva samples of a subset of TTC participants (N = 122) at ages 11, 13, and 15. DNA methylation levels at the functional CpG sites within the SLC6A4 promoter were measured using bisulfite pyrosequencing. Five psychopathological and behavioral clusters were applied from the previous study: minimal problems, persistent or worsening internalizing problems, subjective problems overlooked by caregivers, persistent externalizing problems, and chronic severe problems across symptoms. Linear mixed‐effects models were applied to assess the associations between DNA methylation levels and psycho‐behavioral clusters. Results Males exhibited significantly lower mean methylation levels compared to females across all time points. Males classified as persistent externalizing problems showed notably lower methylation levels than those classified as minimal problems. Conclusions DNA methylation levels in the SLC6A4 could potentially serve as epigenetic signatures for male adolescents exhibiting externalizing behavioral problems. To our knowledge, this is the first study to track SLC6A4 methylation at three developmental time points across early to mid‐adolescence. Further epigenetic research is warranted to understand the role of environmental and genetic factors in the manifestation of adolescent behavioral problems.

Sex differences shape human physiology and disease risk, yet their autosomal epigenetic basis remains incompletely understood. Using whole genome bisulfite sequencing data from iMETHYL database (∼100 adults) across three purified blood cell types (CD4⁺ T cells, monocytes, neutrophils), we performed comprehensive mapping of cell-type specific sex-associated DNA methylation. Using genome-wide Z -test and confidence-interval method, we identified thousands of autosomal differentially methylated sites (DMSs), the majority of which were cell type-specific. We found that neutrophils exhibited pronounced female-biased hypermethylation, whereas CD4⁺ T cells and monocytes showed more balanced patterns. DMSs were enriched within gene bodies and annotated to neuronal and adhesion functions, with T-cell activation uniquely associated in CD4⁺ T cells. Transcription factor binding site enrichment indicated hematopoietic regulators, suggesting that sex-associated methylation is tightly linked to early developmental processes within blood lineages. Differentially methylated regions overlapped genome-wide association study loci for lifetime smoking, multiple sclerosis, and psoriasis, indicating convergence between sex-associated epigenetic states and genetic susceptibility. Evolutionary analysis revealed limited conservation but identified a conserved intronic CpG within FIGN . This study provides the first genome-wide, cell type-resolved map of autosomal sex-associated DNA methylation in human blood and establishes a foundation for mechanistic and translational studies in sex-informed biology and medicine. ![Figure][1] ### Competing Interest Statement The authors have declared no competing interest. Japan Society for the Promotion of Science, JP22K07583, JP23K27531, JP25H01314, JP25H01309, JP23H03838, JP24K22098, and JP25K10839 Japan Agency for Medical Research and Development, JP19dm0207074 and JP24wm0625302 [1]: pending:yes

Antipsychotic drugs are increasingly recognized to exert therapeutic effects not only through neurotransmitter modulation but also, in part, through epigenetic mechanisms. Risperidone is an atypical antipsychotic drug widely used to treat schizophrenia, yet its in vivo epigenomic effects remain poorly understood. We investigated genome-wide DNA methylation changes induced by chronic risperidone administration in the common marmoset, a non-human primate with high translational relevance. Adult males were treated orally with risperidone for 28 days. DNA methylation was analyzed in four brain regions (frontal cortex, hippocampus, cerebellum, and caudate nucleus) and two peripheral tissues (blood and liver) using a HumanMethylation450K BeadChip adapted to the marmoset genome. Risperidone induced region- and tissue-specific methylation alterations. The brain showed predominant hypermethylation, and the hippocampus had the largest number of differentially methylated probes. Rank-rank hypergeometric overlap analysis revealed partial hypermethylation-hypermethylation concordance between the hippocampus or caudate and peripheral tissues, indicating partially coordinated changes. We also found that risperidone-treated neuroblastoma cells showed methylation patterns closely resembling those of the hippocampus, suggesting that the epigenetic changes are partly conserved in the cell models. These findings offer a framework for understanding the molecular basis of antipsychotic actions and for identifying potential epigenetic markers relevant to clinical effects.

Consumption of coffee is associated with a reduced risk of colorectal cancer (CRC); however, the underlying mechanisms are not fully understood. Gut serotonin (5-HT) plays a complex role in CRC development. Intestinal 5-HT levels are regulated by the serotonin transporter (SERT), in intestinal epithelial cells. Recent evidence suggests a correlation between SERT expression and DNA methylation of a functional CpG site (CpG3) in the promoter region of SLC6A4 , the SERT gene. This study investigated the effects of coffee on SERT expression in Caco-2 cells, an intestinal epithelial cell model. Exposure of Caco-2 cells to instant coffee solution 1-10% (v/v) for 48 h was found to result in a concentration-dependent decrease in SERT-mediated 5-HT uptake and SERT mRNA expression. This effect was observed for different instant coffee brands and coffee bean species, and were not reproduced by exposure to major coffee components such as caffeine and chlorogenic acid, or by extracts from unroasted green coffee beans. Pyrosequencing revealed that coffee exposure altered the DNA methylation of CpG3. This preliminary study suggests a novel mechanism by which coffee protects against CRC: suppression of SERT expression in intestinal epithelial cells, possibly via epigenetic modification of SLC6A4 .

Serotonin-transporter-linked polymorphic region (5-HTTLPR), a variable number of tandem repeats in the promoter region of serotonin transporter gene, is classified into short (S) and long (L) alleles. Initial case-control association studies claiming the risks of the S allele in depression and anxiety were not completely supported by recent studies. However, most studies, especially those on East Asian populations, have overlooked the complexity of 5-HTTLPR, which involves multiple different alleles with distinct functional properties. To address this issue, distinguishing multiple 5-HTTLPR alleles is essential. Here, using the 5-HTTLPR genotypes previously determined by exhaustive Sanger sequencing of approximately 1,500 Japanese subjects and their comprehensive SNP data, we constructed a method for 5-HTTLPR genotype imputation. We identified 28 tag SNPs for the imputation of four major 5-HTTLPR alleles, which collectively account for 97.6% of 5-HTTLPR alleles in the Japanese population. Our imputation method, achieved an accuracy of 0.872 in cross-validation, will contribute to association analysis of 5-HTTLPR in the Japanese subjects.

Background: Ketone body metabolism, known for its multifaceted effects, is gaining attention as a potential therapeutic target for cardiovascular diseases. However, the impact of ketone bodies on cardiac hypertrophy and Heart Failure with Preserved Ejection Fraction (HFpEF) remains unclear. Research Questions: To elucidate the effects of ketone bodies on cardiac hypertrophy. The aim of the present research is to investigate the impact of ketone bodies on cardiac hypertrophy induced by metabolic abnormalities using organ-specific ketone body synthesis-deficient mice. Methods: Obesity and hypertension were induced by a high-fat diet combined with NG-Nitro-L-arginine methyl ester hydrochloride (L-NAME) (Combined Stress), and the resultant cardiac hypertrophy and ketone body synthesis were evaluated. Subsequently, organ-specific knockout mice of HMG-CoA synthase 2 ( Hmgcs2 ), a rate-limiting enzyme in ketone body synthesis, were subjected to Combined Stress to assess the impact on cardiac hypertrophy. To conduct a metabolism-focused analysis, cell type-specific nuclei were isolated and subjected to RNA sequencing (RNA-seq) and comprehensive metabolomics analysis. To evaluate the direct effects of ketone bodies, H9C2 cells, rat cardiac cells ,were treated with β-hydroxybutyrate, followed by analysis of oxygen consumption and metabolomics. Results: Combined Stress resulted in increased myocardial cross-sectional area and enhanced ketone body synthesis in the liver and heart. Hepatocyte-specific Hmgcs2 knockout mice ( Hmgcs2 ΔHep ) subjected to Combined Stress exhibited exacerbated myocardial hypertrophy (cardiomyocyte cell size; Hmgcs2 flox : 322.8 ± 88.3 μm 2 : Hmgcs2 ΔHep : 444.0 ± 118.5 μm 2 ; p < 0.0001). RNA-seq analysis revealed that the upregulation of glycolytic genes induced by Combined Stress did not occur in Hmgcs2 ΔHep mice, and a metabolic phenotype favoring fatty acid oxidation persisted. In the liver, hepatocyte destruction and increased serum fatty acid levels were observed. Additionally, H9C2 cells treated with β-hydroxybutyrate showed decreased fatty acid utilization and increased glucose utilization. Conclusion: In cardiac hypertrophy induced by obesity and hypertension, ketone body synthesis mitigates fatty acid overload and promotes glucose utilization, thereby exerting an anti-hypertrophic effect.

AIM:Schizophrenia (SZ) is a severe psychiatric disorder caused by the interaction of genetic and environmental factors. Although somatic mutations that occur in the brain after fertilization may play an important role in the cause of SZ, their frequencies and patterns in the brains of patients and related animal models have not been well studied. This study aimed to find somatic mutations related to the pathophysiology of SZ. METHODS:We performed whole-exome sequencing (WES) of neuronal and nonneuronal nuclei isolated from the postmortem prefrontal cortex of patients with SZ (n = 10) and controls (n = 10). After detecting somatic mutations, we explored the similarities and differences in shared common mutations between two cell types and cell type-specific mutations. We also performed WES of prefrontal cortex samples from an animal model of SZ based on maternal immune activation (MIA) and explored the possible impact of MIA on the patterns of somatic mutations. RESULTS:We did not find quantitative differences in somatic mutations but found higher variant allele fractions of neuron-specific mutations in patients with SZ. In the mouse model, we found a larger variation in the number of somatic mutations in the offspring of MIA mice, with the occurrence of somatic mutations in neurodevelopment-related genes. CONCLUSION:Somatic mutations occurring at an earlier stage of brain cell differentiation toward neurons may be important for the cause of SZ. MIA may affect somatic mutation profiles in the brain.

The majority of peripheral serotonin (5-HT) is produced in the digestive tract and plays a crucial role in controlling intestinal peristalsis and energy metabolism. The serotonin transporter (SERT), expressed in intestinal epithelial cells, regulates the available amount of 5-HT. Recently, a functional CpG site (CpG3) was identified in the SERT gene promoter region, and its expression may be subject to epigenetic regulation. Coffee is the most widely consumed beverage worldwide and exhibits a U- or J-shaped relationship with the risk of a variety of diseases. While caffeine has traditionally been recognized as a key component of coffee, the physiological activity of dietary polyphenols, which can alter DNA methylation patterns, has recently gained attention.

Maternal immune activation is one of the environmental risk factors for offspring to develop psychiatric disorders. A synthetic viral mimetic immunogen, polyinosinic-polycytidylic acid (poly(I:C)), is used to induce maternal immune activation in animal models of psychiatric disorders. In the mouse poly(I:C) model, the existence of segment filamentous bacteria (SFB) in the maternal intestine has been reported to be important for the induction of ASD-related behavioral alterations as well as atypical cortical development called cortical patches. This study aimed to elucidate the effect of a single poly(I:C) injection during embryonic day (E) 9 to E16 on offspring's behavior in the ensured absence of maternal SFB by vancomycin drinking in C57BL/6N mice. The cortical patches were not found at either injection timings with poly(I:C) or PBS vehicle, tested in male or female offspring at postnatal day 0 or 1. Prepulse inhibition was decreased in male adult offspring most strongly at poly(I:C) injection timings later than E11, whereas a modest but significant decrease was observed in female offspring with an injection during E12 to E15. The decrease in social interaction was observed in female offspring most conspicuously at injection timings later than E11, whereas a significant decrease was observed in male offspring with an injection during E12 to E15. In conclusion, this study indicated that behavioral alterations could be induced without maternal SFB. The effect on behavior was substantially different between males and females.

Long interspersed nuclear element-1 (LINE-1, L1) affects the transcriptome landscape in multiple ways. Promoter activity within its 5'UTR plays a critical role in regulating diverse L1 activities. However, the epigenetic status of L1 promoters in adult brain cells and their relationship with psychiatric disorders remain poorly understood. Here, we examined DNA methylation and hydroxymethylation of the full-length L1s in neurons and nonneurons and identified "epigenetically active" L1s. Notably, some of epigenetically active L1s were retrotransposition competent, which even had chimeric transcripts from the antisense promoters at their 5'UTRs. We also identified differentially methylated L1s in the prefrontal cortices of patients with psychiatric disorders. In nonneurons of bipolar disorder patients, one L1 was significantly hypomethylated and showed an inverse correlation with the expression level of the overlapping gene NREP. Finally, we observed that altered DNA methylation levels of L1 in patients with psychiatric disorders were not affected by the surrounding genomic regions but originated from the L1 sequences. These results suggested that altered epigenetic regulation of the L1 5'UTR in the brain was involved in the pathophysiology of psychiatric disorders.