Long-Term Epigenetic Regulation of Foxo3 Expression in Neonatal Valproate-Exposed Rat Hippocampus with Sex-Related Differences.

Perinatal exposure to valproic acid is commonly used for autism spectrum disorder (ASD) animal model development. The inhibition of histone deacetylases by VPA has been proposed to induce epigenetic changes during neurodevelopment, but the specific alterations in genetic expression underlying ASD-like behavioral changes remain unclear. We used qPCR-based gene expression and epigenetics tools and Western blotting in the hippocampi of neonatal valproic acid-exposed animals at 4 weeks of age and conducted the social interaction test to detect behavioral changes. Significant alterations in gene expression were observed in males, particularly concerning mRNA expression of Foxo3, which was significantly associated with behavioral changes. Moreover, notable differences were observed in H3K27ac chromatin immunoprecipitation, quantitative PCR (ChIP-qPCR), and methylation-sensitive restriction enzyme-based qPCR targeting the Foxo3 gene promoter region. These findings provide evidence that epigenetically regulated hippocampal Foxo3 expression may influence social interaction-related behavioral changes. Furthermore, identifying sex-specific gene expression and epigenetic changes in this model may elucidate the sex disparity observed in autism spectrum disorder prevalence.

Acute valproate exposure affects proneural factor expression by increasing FOXO3 in the hippocampus of juvenile mice with a sex-based difference.

Valproic acid (VPA), an anticonvulsant and mood stabilizer, may affect Notch signaling and mitochondrial function. In a previous study, acute VPA exposure induced increased expression of FOXO3, a transcription factor that shares common targets with pro-neuronal ASCL1. In this study, intraperitoneal acute VPA (400 mg/kg) administration in 4-week-old mice increased and decreased FOXO3 and ASCL1 expression, respectively, in the hippocampus, associated with sex-based differences. Treatment of Foxo3 siRNA increased the mRNA expression levels of Ascl1, Ngn2, Hes6, and Notch1 in PC12 cells. Furthermore, VPA exposure induced significant expression changes of mitochondria-related genes, including COX4 and SIRT1, in hippocampal tissues, associated with sex-based differences. This study suggests that acute VPA exposure differently affects proneural gene expression via FOXO3 induction in the hippocampus based on sex.

Neonatal Exposure to Valproate Induces Long-Term Alterations in Steroid Hormone Levels in the Brain Cortex of Prepubertal Rats.

Valproic acid (VPA) is a known drug for treating epilepsy and mood disorders; however, it is not recommended for pregnant women because of its possible teratogenicity. VPA affects neurotransmission and gene expression through epigenetic mechanisms by acting as a histone deacetylase inhibitor and has been used to establish animal models of autism spectrum disorder (ASD). However, studies on the long-term effects of early exposure to VPA on glucocorticoid and neurosteroid synthesis in the brain are lacking. Therefore, this study aimed to investigate the long-term changes in metabolic alterations and gene expression regulation according to sex, using metabolic steroid profiling data from cerebral cortex samples of rats four weeks after VPA exposure (400 mg/kg). In neonatal VPA-exposed models, estradiol levels decreased, and cytochrome P450 19A1 gene (Cyp19a1) expression was reduced in the prepubertal male cortex. Progesterone and allopregnanolone levels decreased, and 3ß-hydroxysteroid dehydrogenase 1 gene (Hsd3b1) expression was also downregulated in the prepubertal female cortex. Furthermore, cortisol levels increased, and mRNA expression of the nuclear receptor subfamily 3 group C member 1 gene (Nr3c1) was downregulated in the cortices of both sexes. Unlike the neonatal VPA-exposed models, although a decrease in progestin and estradiol levels was observed in females and males, respectively, no differences were observed in cortisol levels in the cortex tissues of 8-week-old adult rats administered VPA for four weeks. These results indicate that early environmental chemical exposure induces long-term neurosteroid metabolic effects in the brain, with differences according to sex.

Brain Region and Sex-specific Changes in Mitochondrial Biogenesis Induced by Acute Trimethyltin Exposure.

Objective: In this study, we investigated sex- and region-specific effects of acute trimethyltin (TMT) exposure on mitochondrial biogenesis. Methods: We treated TMT to primary neuronal cultures and 4-week-old male and female mice. We measured the mitochondrial DNA copy numbers using the quantitative polymerase chain reaction method. We also measured mitochondrial biogenesis related genes (sirtuin-1, estrogen-related receptor alpha, cytochrome C oxidase subunit IV) by western blotting. Results: The mitochondrial DNA copy number increased in the primary hippocampal neuron; however, it decreased in the primary cortical neuron. The mitochondrial copy number increased in the hippocampus and decreased in the cortex in the TMT treated female mice, though the mitochondrial copy number increased in both cortex and hippocampus in the TMT treated male mice. TMT treatment increased sirtuin-1 expression in the male hippocampus but did not in the female brain. In the female brain, estrogen-related receptor alpha expression decreased in the cortex though there is no significant change in the male brain. The protein level of mitochondrial protein, cytochrome C oxidase subunit IV, increased in both cortex and hippocampus after TMT injection in male mice brain, but not in female mice brain. Conclusion: Our data suggest that acute TMT exposure induces distinct sex-specific metabolic characteristics in the brain before significant sexual maturation.

Comparison of Peripheral Biomarkers and Reduction of Stress Response in Patients With Major Depressive Disorders vs. Panic Disorder.

Alteration in stress response seems to affect the development of psychiatric disorders. In this study, we aimed to investigate whether baseline peripheral biomarkers could predict the reduction of stress response among patients with major depressive disorder (MDD) and panic disorder (PD). Patients with MDD (n = 41) and PD (n = 52) and healthy controls (HC, n = 59) were selected and regularly followed up with five visits for 12 weeks. The severity of stress at every visit was assessed using the Stress Response Inventory (SRI), and peripheral biomarkers were measured by blood tests at baseline and 2, 4, 8, and 12 weeks. Interleukin (IL)-6, IL-10, tumor necrosis factor (TNF)-α, interferon (IFN)-γ, C-reactive protein (CRP), adiponectin, and leptin levels were analyzed using enzyme-linked immunosorbent assays. Reduction of stress response was defined as the difference in SRI score between baseline and 12 weeks divided by the baseline score. SRI scores were significantly (p < 0.0001) higher in patients with MDD and PD than in HC at every visit after adjusting for variables. In multivariable linear regression, adiponectin levels at baseline were significantly associated with reduction of stress response in patients with PD. When adiponectin increased 1 mg/l, stress response decreased 0.781 points (ß = -0.781, S.E. = 0.220, p = 0.001). Among the subscales of SRI, somatization had a moderate negative correlation with adiponectin levels (r = -0.469). There was no significant association between baseline peripheral biomarkers and reduction of stress response in patients with MDD. Our study showed an inverse association between baseline adiponectin levels and stress response changes in patients with PD, but not in patients with MDD. Thus, differentiated approaches for assessing and treating stress responses of patients with PD and MDD might be helpful. Larger and longitudinal studies are necessary to establish the role and mechanism of action of adiponectin in regulating stress responses in PD.

Acute Valproate Exposure Induces Mitochondrial Biogenesis and Autophagy with FOXO3a Modulation in SH-SY5Y Cells.

Valproic acid (VPA) is an antiepileptic drug found to induce mitochondrial dysfunction and autophagy in cancer cell lines. We treated the SH-SY5Y cell line with various concentrations of VPA (1, 5, and 10 mM). The treatment decreased cell viability, ATP production, and mitochondrial membrane potential and increased reactive oxygen species production. In addition, the mitochondrial DNA copy number increased after VPA treatment in a dose-dependent manner. Western blotting showed that the levels of mitochondrial biogenesis-related proteins (PGC-1α, TFAM, and COX4) increased, though estrogen-related receptor expression decreased after VPA treatment. Further, VPA treatment increased the total and acetylated FOXO3a protein levels. Although SIRT1 expression was decreased, SIRT3 expression was increased, which regulated FOXO3 acetylation in the mitochondria. Furthermore, VPA treatment induced autophagy via increased LC3-II levels and decreased p62 expression and mTOR phosphorylation. We suggest that VPA treatment induces mitochondrial biogenesis and autophagy via changes in FOXO3a expression and posttranslational modification in the SH-SY5Y cell line.

Prenatal Trimethyltin Exposure Induces Long-Term DNA Methylation Changes in the Male Mouse Hippocampus.

Trimethyltin (TMT) is an irreversible neurotoxicant. Because prenatal TMT exposure has been reported to induce behavioral changes, this study was conducted to observe gender differences and epigenetic changes using a mouse model. In behavioral testing of offspring at 5 weeks of age, the total times spent in the center, corner, or border zones in the male prenatal TMT-exposed mice were less than those of control unexposed mice in the open-field test. Female TMT-exposed mice scored lower on total numbers of arm entries and percentages of alternations than controls in the Y-maze test with lower body weight. We found that only TMT-exposed males had fewer copies of mtDNA in the hippocampus and prefrontal cortex region than controls. Additional epigenetic changes, including increased 5-methyl cytosine/5-hydroxymethyl cytosine levels in the male TMT hippocampus, were observed. After methylation binding domain (MBD) sequencing, multiple signaling pathways related to metabolism and neurodevelopment, including FoxO signaling, were identified by pathway analysis for differentially methylated regions (DMRs). Increased FOXO3 and decreased ASCL1 expression were also observed in male TMT hippocampi. This study suggests that sex differences and epigenetics should be more carefully considered in prenatal toxicology studies.

Sex-specific Changes in Brain Estrogen Metabolism Induced by Acute Trimethyltin Exposure.

BACKGROUND/AIM: In this study, we investigated sex-specific effects of acute exposure to trimethyltin, a known neurotoxicant on metabolic steroids. MATERIALS AND METHODS: We administered intraperitoneally 2.3 mg/kg trimethyltin to 4-week-old male mice and measured the levels of metabolic steroids 24 h after treatment. We also measured mRNA and protein levels of cytochrome P450 1B1 using real-time polymerase chain reaction and western blotting. RESULTS: Cortisol levels in the cortex increased in both sexes following acute trimethyltin exposure. The estradiol levels decreased, and the 4-hydroxyestradiol levels increased only in females. We also observed increased cytochrome P450 1B1 mRNA and protein levels only in the female cortex. CONCLUSION: Acute trimethyltin exposure induces distinct sex-specific metabolic changes in the brain before significant sexual maturation.

Predictive inflammatory biomarkers for change in suicidal ideation in major depressive disorder and panic disorder: A 12-week follow-up study.

Previous studies have investigated the role of inflammatory markers in suicidality of patients with major depressive disorder (MDD) or panic disorder (PD). However, few studies have investigated associations between serum inflammatory cytokine levels and suicidality. We hypothesized that MDD and PD status might be significantly associated with serum inflammatory cytokines and that we could predict levels of improvement in suicide ideation intensity using serum inflammatory biomarkers in patients with MDD and PD. For this study, 41 patients with MDD, 52 patients with PD, and 59 healthy control (HC) subjects were enrolled. Psychological measurements and serum inflammatory markers such as interleukin (IL) -6, -10, interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and C reactive protein (CRP) were examined. A total of five visits were completed during 12 weeks. After controlling for confounding factors, log-transformed IL-6 (ln_IL-6) at baseline (MDD: 0.297 ± 0.626; PD: 0.342 ± 0.723; HC: -0.121 ± 0.858; p = 0.007, >0.0017, 0.05/30) and mean ln_IL-6 (MDD: 0.395 ± 0.550, PD: 0.249 ± 0.544, HC: -0.139 ± 0.622, p = 0.002, >0.0017, 0.05/30) levels were trends towards significantly higher in patients with MDD and PD than in HC. In MDD patients, a higher level of basal ln_TNF-α was a significant predictor of ΔSSI (changes in SSI scores between baseline and week 12) even after controlling for changes of depression symptoms and baseline SSI scores (standardized ß = 0.541, p = 0.002 < 0.0028, 0.05/18). In conclusion, we could predict ΔSSI using baseline inflammatory biomarkers for patients with MDD.

Pre-treatment peripheral biomarkers associated with treatment response in panic symptoms in patients with major depressive disorder and panic disorder: A 12-week follow-up study.

OBJECTIVE: Peripheral biomarkers have been studied to predict treatment response of panic symptoms. We hypothesized that depressive disorder (MDD) vs. panic disorder (PD) would exhibit different peripheral biomarkers, and their correlation with severity of panic attacks (PA) would also differ. METHODS: Forty-one MDD patients, 52 PD patients, and 59 healthy controls were followed for 12 weeks. We measured peripheral biomarkers along with the Panic Disorder Severity Scale (PDSS) at each visit-pre-treatment, 2, 4, 8, and 12 weeks on a regular schedule. Peripheral biomarkers including serum cytokines, plasma and serum brain-derived neurotrophic factor (BDNF), leptin, adiponectin, and C-reactive protein (CRP) were quantified using enzyme-linked immunosorbent assay (ELISA). RESULTS: Patients with MDD and PD demonstrated significantly higher levels of pre-treatment IL-6 compared to controls, but no differences were seen in plasma and serum BDNF, leptin, adiponectin, and CRP. Pre-treatment leptin showed a significant clinical correlation with reduction of panic symptoms in MDD patients at visit 5 (p=0.011), whereas pre-treatment IL-6 showed a negative correlation with panic symptom reduction in PD patients (p=0.022). An improvement in three panic-related items was observed to be positively correlated with pre-treatment leptin in MDD patients: distress during PA, anticipatory anxiety, and occupational interference. CONCLUSION: Higher pre-treatment leptin was associated with better response to treatment regarding panic symptoms in patients with MDD, while higher IL-6 was associated with worse response regarding panic symptoms in PD patients. Different predictive peripheral biomarkers observed in MDD and PD suggest the need for establishing individualized predictive biomarkers, even in cases of similar symptoms observed in different disorders.

Propionic acid induces mitochondrial dysfunction and affects gene expression for mitochondria biogenesis and neuronal differentiation in SH-SY5Y cell line.

Studies in animal models have shown that the short-chain fatty acid, propionic acid (PPA), interferes with mitochondrial metabolism leading to mitochondrial dysfunction and behavioral abnormalities. The aim of this study was to investigate the effects of PPA on mitochondrial function and gene expression in neuronal cells. SH-SY5Y cells and normal human neural progenitor (NHNP) cells were exposed to 1, 5 mM PPA for 4 or 24 h and we found that the mitochondrial potential measured in SH-SY5Y cells decreased in a dose-dependent manner after PPA treatment. Electron microscopy analysis revealed that the size of the mitochondria was significantly reduced following PPA treatment. A dose-dependent increase in the mitochondrial DNA copy number was observed in the PPA-treated cells. The expression of the mitochondrial biogenesis-related proteins PGC-1α, TFAM, SIRT3, and COX4 was significantly increased after PPA treatment. Transcriptome analysis revealed that mRNA expression in the notch signaling-related genes ASCL1 and LFNG changed after PPA treatment and the positive correlated protein expression changes were also observed. These results revealed that PPA treatment may affect neurodevelopment by altering mitochondrial function and notch signaling-related gene expression.

Impaired learning and memory in CD38 null mutant mice.

CD38 is an enzyme that catalyzes the formation of cyclic ADP ribose and nicotinic acid adenine dinucleotide phosphate, both of which are involved in the mobilization of Ca(2+) from intracellular stores. Recently, CD38 has been shown to regulate oxytocin release from hypothalamic neurons. Importantly, CD38 mutations are associated with autism spectrum disorders (ASD) and CD38 knockout (CD38(-/-)) mice display ASD-like behavioral phenotypes including deficient parental behavior and poor social recognition memory. Although ASD and learning deficits commonly co-occur, the role of CD38 in learning and memory has not been investigated. We report that CD38(-/-) mice show deficits in various learning and memory tasks such as the Morris water maze, contextual fear conditioning, and the object recognition test. However, either long-term potentiation or long-term depression is not impaired in the hippocampus of CD38(-/-) mice. Our results provide convincing evidence that CD38(-/-) mice show deficits in various learning and memory tasks including spatial and non-spatial memory tasks. Our data demonstrate that CD38 is critical for regulating hippocampus-dependent learning and memory without modulating synaptic plasticity.

Effect of midazolam on memory during fiberoptic gastroscopy under conscious sedation.

OBJECTIVE: As the fiberoptic gastroscopy using midazolam is being in widespread use, the exact nature of midazolam on memory should be clarified. We intended to examine whether midazolam causes selective anterograde amnesia and what impact it has on other aspects of memory and general cognitive function. METHODS: We recruited healthy subjects undergoing fiberoptic gastroscopy under conscious sedation. At baseline, history taking for retrograde amnesia and the Korean version of the Montreal Cognitive Assessment were performed. A man's name and address were given immediately after intravenous midazolam administration. After gastroscopy, the subjects were asked to recall those items. By the time they had fully recovered consciousness, the same test was repeated along with the Korean version of the Montreal Cognitive Assessment and a test for retrograde amnesia. RESULTS: A total of 30 subjects were enrolled in this study. Subjects with high-dose midazolam showed lower scores in the immediate and delayed recall of "a man's name and address" compared with those with low-dose midazolam. The midazolam dose was inversely correlated with the delayed recall scores of "a man's name and address." On full recovery of consciousness, the subjects did not exhibit any of anterograde or retrograde amnesia. CONCLUSIONS: These findings suggest that midazolam causes transient selective anterograde amnesia in a dose-dependent manner.