Chronic Corticosterone Treatment Decreases Extracellular pH and Increases Lactate Release via PDK4 Upregulation in Cultured Astrocytes.

The pathogenesis of stress-related disorders involves aberrant glucocorticoid secretion, and decreased pH and increased lactate in the brain are common phenotypes in several psychiatric disorders. Mice treated with glucocorticoids develop these phenotypes, but it is unclear how glucocorticoids affect brain pH. Therefore, we investigated the effect of corticosterone (CORT), the main glucocorticoid in rodents, on extracellular pH and lactate release in cultured astrocytes, which are the main glial cells that produce lactate in the brain. CORT treatment for one week decreased the extracellular pH and increased the extracellular lactate level via glucocorticoid receptors. CORT also increased the intracellular pyruvate level and upregulated pyruvate dehydrogenase kinase 4 (PDK4), while PDK4 overexpression increased extracellular lactate and decreased the extracellular pH. Furthermore, PDK4 inhibition suppressed the increase in extracellular lactate and the decrease in extracellular pH induced by CORT. These results suggest that increased lactate release via accumulation of intracellular pyruvate in astrocytes by chronic glucocorticoid exposure contributes to decreased brain pH.

Chronic social defeat stress induces anxiety-like behaviors via downregulation of serotonin transporter in the prefrontal serotonergic system in mice.

The serotonergic (5-HTergic) system is closely involved in the pathophysiology of mood and anxiety disorders and the responsibility of this system may differ for each symptom. In this study, we examined the relationship between the dysfunction of the 5-HTergic system and abnormal behaviors in the social defeat stress model, an animal model of mood and anxiety disorders and in mice with knockdown of Slc6a4, the gene encoding SERT. Monoamine content, serotonin (5-HT) release, 5-HT uptake, 5-HT transporter (SERT) protein levels, and behaviors were investigated in mice subjected to chronic social defeat stress and in mice with knockdown of Slc6a4, in 5-HTergic neurons projecting to the prefrontal cortex (PFC). Furthermore, DNA methylation of Slc6a4 was examined in mice subjected to chronic social defeat stress. Increased turnover, increased extracellular basal levels, decreased release and decreased uptake of 5-HT, and decreased SERT protein levels were observed in the PFC of the stressed mice. The decreased 5-HT uptake correlated with anxiety-like behavior characterized by decreased time spent in the open arms of the elevated plus maze. DNA methylation was increased in the CpG island of Slc6a4 in 5-HTergic neurons projecting to the PFC of the stressed mice. Similar to the stressed mice, mice with Slc6a4 knockdown in 5-HTergic neurons projecting to the PFC also showed decreased release and uptake of 5-HT in the PFC and increased anxiety-like behavior. Chronic stress may induce anxiety due to dysfunction in the prefrontal 5-HTergic system via decreased SERT expression in the PFC.

2'-Fucosyllactose Alleviates Early Weaning-Induced Anxiety-Like Behavior, Amygdala Hyperactivity, and Gut Microbiota Changes.

Early life stress has a significant impact on development of the central nervous system (CNS), with lasting rather than transient consequences; therefore, it is important to alleviate these effects. In recent years, functional communication between the CNS and gut microbiota through the so-called brain-gut-microbiota axis has been examined, and it is likely that prebiotics contribute to development of the CNS through the gut microbiota. In this study, we performed behavioral, neurohistological, and fecal microbiota analyses in early-weaned mice to examine the effects of 2'-fucosyllactose (2'-FL), a human milk oligosaccharide, on anxiety induced by early life stress. Mice weaned at 17 d old (17-d mice) showed anxiety-like behaviors, such as decreased time in the open arms in the elevated plus maze test, compared to mice weaned at 24 d old (24-d mice). The number of cells that were positive for the neuronal activity marker c-Fos in the amygdala was also higher in 17-d mice. The behavioral and neural abnormalities caused by early weaning were alleviated by post-weaning ingestion of 2'-FL. The composition of the fecal microbiota differed among control diet-fed 24-d and 17-d mice, and 2'-FL diet-fed 17-d mice. These findings indicate that human milk oligosaccharides 2'-FL alleviate early stress-induced anxiety, amygdala hyperactivity, and gut microbiota changes.

Methyl Donor Supplementation Prevents a Folate Deficiency-induced Depression-like State and Neuronal Immaturity of the Dentate Gyrus in Mice.

We have recently shown that folate deficiency induces depression-like behavior and neuronal immaturity in the dentate gyrus (DG) in mice. We also revealed that folate deficiency inhibits neuronal maturation, hypomethylates the promoter of certain neuronal genes and decreases intracellular levels of S-adenosylmethionine (SAM), a methyl donor, in cultured neural stem and progenitor cells. Based on these findings, we hypothesized that SAM reduction may be involved in a folate deficiency-induced depressive state and neural immaturity. In this study, we examined whether SAM supplementation prevents depression-like behavior and neural immaturity in low folate diet-fed mice. Intraperitoneal administration of SAM (50 mg/kg/day) for 14 days from 7 weeks old prevented increased immobility in low folate diet-fed mice. SAM supplementation also prevented an increase in the number of doublecortin (an immature neuron marker)-positive cells and a decrease in the number of NeuN (a mature neuron marker)/5-bromo-2'-deoxyuridine (a proliferation marker)-double positive cells in the DG of these mice. Furthermore, neurofunctional and neuromorphological abnormalities in the DG of low folate diet-fed mice, such as decreases in stress-induced expression of c-Fos (a neuronal activity marker), dendritic complexity and the number of mature spines, were improved by SAM supplementation. The disrupted expression of transcription factors involved in neuronal differentiation and maturation was also normalized by SAM supplementation. These results suggest that SAM reduction may be involved in a folate deficiency-induced depressive state.

Kihito prevents corticosterone-induced brain dysfunctions in mice.

Kihito (KIT; Gui Pi Tang) is a traditional herbal medicine that is used for treatment of neuropsychiatric disorders such as depression, anxiety, neurosis and insomnia in China and Japan. Recently, it has also been shown that KIT improves cognitive dysfunction in patients with Alzheimer's disease. In this study, to investigate the mechanisms underlying the effects of KIT on stress-induced brain dysfunctions such as a depressed state and memory impairment, we examined whether KIT prevents behavioral and neurophysiological abnormalities in mice treated chronically with corticosterone (CORT). CORT (40 mg/kg/day, s.c.) and KIT (1000 mg/kg/day, p.o.) were given to 7-week-old male ddY mice for 14 days. Twenty-four hours after the last treatment, depression-like behavior in the forced swim test, spatial memory in the Barnes maze test, cell survival and the number of new-born immature neurons, dendritic spine density and expression levels of mRNA for neurotrophic factors were analyzed. Depression-like behavior and spatial memory impairment were observed in CORT-treated mice without KIT treatment. Hippocampal cell survival, the number of hippocampal new-born immature neurons, hippocampal and accumbal dendritic spine density and mRNA levels for neurotrophic factors such as glial cell line-derived neurotrophic factor (GDNF) were decreased in CORT-treated mice without KIT treatment. KIT prevented CORT-induced depression-like behavior, spatial memory impairment, and decreases in hippocampal cell survival, the number of hippocampal new-born immature neurons, accumbal dendritic spine density and GDNF mRNA. KIT may ameliorate stress-induced brain dysfunctions via prevention of adverse effects of CORT on cell survival, new-born immature neurons, spine density and neurotrophic factors.

Low folate induces abnormal neuronal maturation and DNA hypomethylation of neuronal differentiation-related genes in cultured mouse neural stem and progenitor cells.

Folate deficiency in a fetus is well known to cause neurodevelopment defects and development disorders. A low level of folate is also thought to be a risk for depression in adults. We have previously shown that post-weaning low folate induces neuronal immaturity in the dentate gyrus in mice, which suggests that low folate causes neuropsychological disorders via inhibition of neuronal maturation. In this study, we examined the effects of low folate on expression and epigenetic modification of genes involved in neuronal differentiation and maturation in primary mouse neural stem/progenitor cells (NSPCs) in vitro. An increase in Nestin (NSPC marker)-positive cells was observed in cells differentiated in a low folate medium for 3 days. An increase in ßIII-tubulin (Tuj1: immature neuron marker)-positive cells and a decrease in microtubule-associated protein 2 (MAP2: mature neuron marker)-positive cells were observed in cells differentiated in a low folate medium for 7 days. In these cells, mRNA levels for genes involved in neuronal differentiation and maturation were altered. Hypomethylation of DNA, but not of histone proteins, was also observed at some promoters of these neuronal genes. The level of S-adenosylmethionine (SAM), a methyl donor, was decreased in these cells. The abnormalities in neural maturation and changes in gene expression in culture under low folate conditions were partially normalized by addition of SAM (5 µM). Based on these results, decreased SAM may induce DNA hypomethylation at genes involved in neuronal differentiation and maturation under low folate conditions, and this hypomethylation may be associated with low folate-induced neuronal immaturity.

Ferulic acid alleviates abnormal behaviors in isolation-reared mice via 5-HT1A receptor partial agonist activity.

RATIONALE: Preclinical and clinical reports suggest that ferulic acid (FA), a plant-derived phenylpropanoid, is effective against mental health problems such as agitation, anxiety, and irritability in humans, without causing adverse side effects. However, the mechanism of action is unknown. OBJECTIVE: The aim of the study is to investigate the mechanism underlying the ameliorative effects of FA on mental health problems such as agitation, anxiety, and irritability, using in vivo behavioral analysis, in vitro pharmacological analysis, and in silico binding analysis. METHODS: The effects of FA (10 mg/kg, 50 mg/kg, and 250 mg/kg) on hyperactivity and aggressive behaviors of isolation-reared mice were examined. The effects of FA (50 mg/kg and 250 mg/kg) on extracellular levels of monoamines such as serotonin (5-HT), dopamine, and noradrenaline were analyzed by in vivo microdialysis. The effects of FA (10-13-10-6 M) on 5-HT1A and 5-HT2A receptors were analyzed using a luciferase reporter gene assay. Binding of FA to the mouse 5-HT1A receptor was evaluated by in silico analysis. RESULTS: The behavioral analysis showed that administration of FA (50 mg/kg) 1 h before experiments significantly alleviated hyperactivity and aggressive behaviors in isolation-reared mice. These alleviative effects were abolished by pretreatment with the 5-HT1A receptor antagonist WAY-100635 (1 mg/kg). In vivo microdialysis analysis showed that FA (50 mg/kg) did not change extracellular monoamine levels in the prefrontal cortex of mice. The luciferase reporter gene assay indicated that FA activated 5-HT1A receptors, but not 5-HT2A receptors, in a dose-dependent manner. The maximal response of 5-HT1A receptors to FA was weaker than that to 8-hydroxy-2-dipropylaminotetralin (8-OH-DPAT), a 5-HT1A receptor full agonist. In silico binding analysis showed that FA binds to the orthosteric site of 5-HT1A receptors. CONCLUSION: Taken together, these results suggest that FA ameliorates agitation-, anxiety-, and irritability-like behaviors such as hyperactivity and aggressive behaviors in isolation-reared mice via 5-HT1A receptor partial agonist activity. These findings support the efficacy of FA on mental health problems that have been suggested in preclinical and clinical practice.

Adenovirus vector-based incorporation of a photo-cross-linkable amino acid into proteins in human primary cells and cancerous cell lines.

The site-specific incorporation of cross-linkable designer amino acids into proteins is useful for covalently bonding protein complexes upon exposure to light. This technology can be used to study networks of protein-protein interactions in living cells; however, to date it has only been applicable for use with a narrow range of cell types, due to the limited availability of plasmid-based transfection protocols. In the present study, we achieved adenovirus-based expression of a variant of an archaeal pyrrolysyl-tRNA synthetase and UAG-recognising tRNA pair, which was used to incorporate unnatural amino acids into proteins at sites defined by in-frame UAG codons within genes. As such, the site-specific photo-cross-linking method is now applicable to a wide variety of mammalian cells. In addition, we repositioned the reactive substituent of a useful photo-cross-linker, Nε-(para-trifluoromethyl-diazirinyl-benzyloxycarbonyl)-l-lysine (pTmdZLys), to the meta position, which improved its availability at low concentration. Finally, we successfully applied this system to analyse the formation of a protein complex in response to a growth signal in human cancerous cells and human umbilical vein endothelial cells. This adenovirus-based system, together with the newly designed cross-linkable amino acid, will facilitate studies on molecular interactions in various cell lines of medical interest.