Age-Dependent and Aß-Induced Dynamic Changes in the Subcellular Localization of HMGB1 in Neurons and Microglia in the Brains of an Animal Model of Alzheimer's Disease.

HMGB1 is a prototypical danger-associated molecular pattern (DAMP) molecule that co-localizes with amyloid beta (Aß) in the brains of patients with Alzheimer's disease. HMGB1 levels are significantly higher in the cerebrospinal fluid of patients. However, the cellular and subcellular distribution of HMGB1 in relation to the pathology of Alzheimer's disease has not yet been studied in detail. Here, we investigated whether HMGB1 protein levels in brain tissue homogenates (frontal cortex and striatum) and sera from Tg-APP/PS1 mice, along with its cellular and subcellular localization in those regions, differed. Total HMGB1 levels were increased in the frontal cortices of aged wildtype (7.5 M) mice compared to young (3.5 M) mice, whereas total HMGB1 levels in the frontal cortices of Tg-APP/PS1 mice (7.5 M) were significantly lower than those in age-matched wildtype mice. In contrast, total serum HMGB1 levels were enhanced in aged wildtype (7.5 M) mice and Tg-APP/PS1 mice (7.5 M). Further analysis indicated that nuclear HMGB1 levels in the frontal cortices of Tg-APP/PS1 mice were significantly reduced compared to those in age-matched wildtype controls, and cytosolic HMGB1 levels were also significantly decreased. Triple-fluorescence immunohistochemical analysis indicated that HMGB1 appeared as a ring shape in the cytoplasm of most neurons and microglia in the frontal cortices of 9.5 M Tg-APP/PS1 mice, indicating that nuclear HMGB1 is reduced by aging and in Tg-APP/PS1 mice. Consistent with these observations, Aß treatment of both primary cortical neuron and primary microglial cultures increased HMGB1 secretion in the media, in an Aß-dose-dependent manner. Our results indicate that nuclear HMGB1 might be translocated from the nucleus to the cytoplasm in both neurons and microglia in the brains of Tg-APP/PS1 mice, and that it may subsequently be secreted extracellularly.

Extracellular Vesicles Released by Lactobacillus paracasei Mitigate Stress-induced Transcriptional Changes and Depression-like Behavior in Mice.

Various probiotic strains have been reported to affect emotional behavior. However, the underlying mechanisms by which specific probiotic strains change brain function are not clearly understood. Here, we report that extracellular vesicles derived from Lactobacillus paracasei (Lpc-EV) have an ability to produce genome-wide changes against glucocorticoid (GC)-induced transcriptional responses in HT22 hippocampal neuronal cells. Genome-wide analysis using microarray assay followed by Rank-Rank Hypergeometric Overlap (RRHO) method leads to identify the top 20%-ranked 1,754 genes up- or down-regulated following GC treatment and their altered expressions are reversed by Lpc-EV in HT22 cells. Serial k-means clustering combined with Gene Ontology enrichment analyses indicate that the identified genes can be grouped into multiple functional clusters that contain functional modules of "responses to stress or steroid hormones", "histone modification", and "regulating MAPK signaling pathways". While all the selected genes respond to GC and Lpc-EV at certain levels, the present study focuses on the clusters that contain Mkp-1, Fkbp5, and Mecp2, the genes characterized to respond to GC and Lpc-EV in opposite directions in HT22 cells. A translational study indicates that the expression levels of Mkp-1, Fkbp5, and Mecp2 are changed in the hippocampus of mice exposed to chronic stress in the same directions as those following GC treatment in HT22 cells, whereas Lpc-EV treatment restored stress-induced changes of those factors, and alleviated stress-induced depressive-like behavior. These results suggest that Lpc-EV cargo contains bioactive components that directly induce genome-wide transcriptional responses against GC-induced transcriptional and behavioral changes.

Lactobacillus-derived extracellular vesicles counteract Aß42-induced abnormal transcriptional changes through the upregulation of MeCP2 and Sirt1 and improve Aß pathology in Tg-APP/PS1 mice.

Mounting evidence suggests that probiotics are beneficial for treating Alzheimer's disease (AD). However, the mechanisms by which specific probiotics modify AD pathophysiology are not clearly understood. In this study, we investigated whether Lactobacillus paracasei-derived extracellular vesicles (Lpc-EV) can directly act on neuronal cells to modify amyloid-beta (Aß)-induced transcriptional changes and Aß pathology in the brains of Tg-APP/PS1 mice. Lpc-EV treatment in HT22 neuronal cells counteracts Aß-induced downregulation of Brain-derived neurotrophic factor (Bdnf), Neurotrophin 3 (Nt3), Nt4/5, and TrkB receptor, and reverses Aß-induced altered expression of diverse nuclear factors, including the downregulation of Methyl-CpG binding protein 2 (Mecp2) and Sirtuin 1 (Sirt1). Systematic siRNA-mediated knockdown experiments indicate that the upregulation of Bdnf, Nt3, Nt4/5, and TrkB by Lpc-EV is mediated via multiple epigenetic factors whose activation converges on Mecp2 and Sirt1. In addition, Lpc-EV reverses Aß-induced downregulation of the Aß-degrading proteases Matrix metalloproteinase 2 (Mmp-2), Mmp-9, and Neprilysin (Nep), whose upregulation is also controlled by MeCP2 and Sirt1. Lpc-EV treatment restores the downregulated expression of Bdnf, Nt4/5, TrkB, Mmp-2, Mmp-9, and Nep; induces the upregulation of MeCP2 and Sirt1 in the hippocampus; alleviates Aß accumulation and neuroinflammatory responses in the brain; and mitigates cognitive decline in Tg-APP/PS1 mice. These results suggest that Lpc-EV cargo contains a neuroactive component that upregulates the expression of neurotrophic factors and Aß-degrading proteases (Mmp-2, Mmp-9, and Nep) through the upregulation of MeCP2 and Sirt1, and ameliorates Aß pathology and cognitive deficits in Tg-APP/PS1 mice.

Extracellular Vesicles from Gram-positive and Gram-negative Probiotics Remediate Stress-Induced Depressive Behavior in Mice.

Chronic stress causes maladaptive changes in the brain that lead to depressive behavior. In the present study, we investigate whether chronic stress alters gut microbiota compositions that are related to stress-induced maladaptive changes in the brain. Mice treated with daily 2-h restraint for 14 days (CRST) exhibit depressive-like behavior. Sequence readings of 16S rRNA genes prepared from fecal samples taken from CRST-treated mice suggest that chronic stress induces gut microbiota changes that are pronounced in the post-stress period, relative to those that occur in the 14-day stress phase. The genus Lactobacillus is one such microbiota substantially changed following chronic stress. In contrast, intraperitoneal injection of extracellular vesicles (EVs) isolated from culture media of the Gram-positive probiotic Lactobacillus plantarum is sufficient to ameliorate stress-induced depressive-like behavior. Interestingly, EVs from the Gram-positive probiotic Bacillus subtilis and EVs from the Gram-negative probiotic Akkermansia muciniphila also produce anti-depressive-like effects. While chronic stress decreases the expression of MeCP2, Sirt1, and/or neurotrophic factors in the hippocampus, EVs from the three selected probiotics differentially restore stress-induced changes of these factors. These results suggest that chronic stress produces persistent changes in gut microbiota composition, whereas purified EVs of certain probiotics can be used for treatment of stress-induced depressive-like behavior.

Behavioral Engagement With Playable Objects Resolves Stress-Induced Adaptive Changes by Reshaping the Reward System.

BACKGROUND: The reward system regulates motivated behavior, and repeated practice of specific motivated behavior might conversely modify the reward system. However, the detailed mechanisms by which they reciprocally regulate each other are not clearly understood. METHODS: Mice subjected to chronic restraint stress show long-lasting depressive-like behavior, which is rescued by continual engagement with playable objects. A series of molecular, pharmacological, genetic, and behavioral analyses, combined with microarray, liquid chromatography, and chemogenetic tools, are used to investigate the neural mechanisms of antidepressive effects of playable objects. RESULTS: Here, we show that repeated restraint induces dopamine surges into the nucleus accumbens-lateral shell (NAc-lSh), which cause upregulation of the neuropeptide PACAP in the NAc-lSh. As repeated stress is continued, the dopamine surge by stressors is adaptively suppressed without restoring PACAP upregulation, and the resulting enhanced PACAP inputs from NAc-lSh neurons to the ventral pallidum facilitate depressive-like behaviors. Continual engagement with playable objects in mice subjected to chronic stress remediates reduced dopamine response to new stressors, enhanced PACAP upregulation, and depressive-like behaviors. Overactivation of dopamine D1 receptors over the action of D2 receptors in the NAc-lSh promotes depressive-like behaviors. Conversely, inhibition of D1 receptors or PACAP upregulation in the NAc-lSh confers resilience to chronic stress-induced depressive-like behaviors. Histochemical and chemogenetic analyses reveal that engagement with playable objects produces antidepressive effects by reshaping the ventral tegmental area-to-NAc-lSh and NAc-lSh-to-ventral pallidum circuits. CONCLUSIONS: These results suggest that behavioral engagement with playable objects remediates depressive-like behaviors by resolving stress-induced maladaptive changes in the reward system.

Repeated exposure with short-term behavioral stress resolves pre-existing stress-induced depressive-like behavior in mice.

Chronic stress induces adaptive changes in the brain via the cumulative action of glucocorticoids, which is associated with mood disorders. Here we show that repeated daily five-minute restraint resolves pre-existing stress-induced depressive-like behavior in mice. Repeated injection of glucocorticoids in low doses mimics the anti-depressive effects of short-term stress. Repeated exposure to short-term stress and injection of glucocorticoids activate neurons in largely overlapping regions of the brain, as shown by c-Fos staining, and reverse distinct stress-induced gene expression profiles. Chemogenetic inhibition of neurons in the prelimbic cortex projecting to the nucleus accumbens, basolateral amygdala, or bed nucleus of the stria terminalis results in anti-depressive effects similarly to short-term stress exposure, while only inhibition of neurons in the prelimbic cortex projecting to the bed nucleus of the stria terminalis rescues defective glucocorticoid release. In summary, we show that short-term stress can reverse adaptively altered stress gains and resolve stress-induced depressive-like behavior.

Hyperoxygenation Treatment Reduces Beta-amyloid Deposition via MeCP2-dependent Upregulation of MMP-2 and MMP-9 in the Hippocampus of Tg-APP/PS1 Mice.

Recently we reported that hyperoxygenation treatment reduces amyloid-beta accumulation and rescues cognitive impairment in the Tg-APP/PS1 mouse model of Alzheimer's disease. In the present study, we continue to investigate the mechanism by which hyperoxygenation reduces amyloid-beta deposition in the brain. Hyperoxygenation treatment induces upregulation of matrix metalloproteinase-2 (MMP-2), MMP-9, and tissue plasminogen activator (tPA), the endopeptidases that can degrade amyloid-beta, in the hippocampus of Tg-APP/PS1 mice. The promoter regions of the three proteinase genes all contain potential binding sites for MeCP2 and Pea3, which are upregulated in the hippocampus after hyperoxygenation. Hyperoxygenation treatment in HT22 neuronal cells increases MeCP2 but not Pea3 expression. In HT22 cells, siRNA-mediated knockdown of Mecp2 decreases Mmp-9 expression and to a lesser extent, Mmp-2 and tPA expression. In mice, siRNA-mediated Mecp2 knockdown in the hippocampus reduces Mmp-9 expression, but not significantly Mmp-2 and tPA expression. The ChIP assay indicates that hyperoxygenation treatment in Tg-APP/PS1 mice increases MeCP2 binding to the promoter regions of Mmp-2 , Mmp-9 and tPA genes in the hippocampus. Together, these results suggest that hyperoxygenation increases the expression of MMP-2, MMP-9, and tPA, of which MMP-9 is upregulated via MeCP2 in neuronal cells, and MMP-2 and tPA are upregulated through MeCP2 and other nuclear factors.

Aging-Dependent Downregulation of SUV39H1 Histone Methyltransferase Increases Susceptibility to Stress-Induced Depressive Behavior.

Aging induces cellular and molecular changes including gene expression alteration in the brain, which might be associated with aging-induced decrease in stress coping ability. In the present study, we investigate how aging changes the ability to cope with stress and increases sensitivity to stress. Aged mice show decreased expression of SUV39H1 histone methyltransferase and increased expression of Mkp-1 in the hippocampus. The siRNA-mediated knockdown of SUV39H1 increases Mkp-1 expression and suppresses p-CREB and Bdnf expression in HT22 cells and in the hippocampus of mice. Chromatin immunoprecipitation assays indicate that the levels of SUV39H1 and methylated histone-H3 bound to the promoter of the Mkp-1 in the hippocampus are reduced in aged mice. Aged mice exhibit depression-like behavior following weak stress that does not induce depressive behavior in young mice. Rosmarinic acid, a phenolic compound that increases SUV39H1 expression, reverses stress-induced changes of SUV39H1, Mkp-1, and Bdnf expression in the hippocampus via an overlapping but distinct mechanism from those of fluoxetine and imipramine and produces anti-depressive effects. These results suggest that aging increases susceptibility to stress via downregulation of SUV39H1 and resulting changes in SUV39H1-regulated signaling pathways in the hippocampus.

Humulus japonicus rescues autistic­like behaviours in the BTBR T+ Itpr3tf/J mouse model of autism.

Humulus japonicus (HJ) is a traditional herbal medicine that exhibits anti­inflammatory, antimicrobial and anti­tumor effects that is used for the treatment of hypertension, pulmonary disease and leprosy. Recently, it has also been reported that HJ demonstrates neuroprotective properties in animal models of neurodegenerative diseases. The current study hypothesised that the administration of HJ would exhibit therapeutic effects in autism spectrum disorder (ASD), a neurodevelopmental disorder with lifelong consequences. The BTBR T+ Itpr3tf/J mouse model of ASD was used to investigate the anti­autistic like behavioural effects of HJ. Chronic oral administration of the ethanolic extract of HJ significantly increased social interaction, attenuated repetitive grooming behaviour and improved novel­object recognition in BTBR mice. Anti­inflammatory effects of HJ in the brain were analysed using immunohistochemistry and reverse­transcription quantitative PCR analysis. Microglia activation was markedly decreased in the striatum and hippocampus, and pro­inflammatory cytokines, including C­C Motif Chemokine Ligand 2, interleukin (IL)­1ß and IL­6, were significantly reduced in the hippocampus following HJ treatment. Moreover, HJ treatment normalised the phosphorylation levels of: N­methyl­D­aspartate receptor subtype 2B and calcium/calmodulin­dependent protein kinase type II subunit α in the hippocampus of BTBR mice. The results of the present study demonstrated that the administration of HJ may have beneficial potential for ameliorating behavioural deficits and neuroinflammation in ASD.

Hyperoxygenation Ameliorates Stress-induced Neuronal and Behavioral Deficits.

Hyperoxygenation therapy remediates neuronal injury and improves cognitive function in various animal models. In the present study, the optimal conditions for hyperoxygenation treatment of stress-induced maladaptive changes were investigated. Mice exposed to chronic restraint stress (CRST) produce persistent adaptive changes in genomic responses and exhibit depressive-like behaviors. Hyperoxygenation treatment with 100% O2 (HO2) at 2.0 atmospheres absolute (ATA) for 1 h daily for 14 days in CRST mice produces an antidepressive effect similar to that of the antidepressant imipramine. In contrast, HO2 treatment at 2.0 ATA for 1 h daily for shorter duration (3, 5, or 7 days), HO2 treatment at 1.5 ATA for 1 h daily for 14 days, or hyperbaric air treatment at 2.0 ATA (42% O2) for 1 h daily for 14 days is ineffective or less effective, indicating that repeated sufficient hyperoxygenation conditions are required to reverse stress-induced maladaptive changes. HO2 treatment at 2.0 ATA for 14 days restores stress-induced reductions in levels of mitochondrial copy number, stress-induced attenuation of synaptophysin-stained density of axon terminals and MAP-2-staining dendritic processes of pyramidal neurons in the hippocampus, and stress-induced reduced hippocampal neurogenesis. These results suggest that HO2 treatment at 2.0 ATA for 14 days is effective to ameliorate stress-induced neuronal and behavioral deficits.

Aging increases vulnerability to stress-induced depression via upregulation of NADPH oxidase in mice.

Brain aging proceeds with cellular and molecular changes in the limbic system. Aging-dependent changes might affect emotion and stress coping, yet the underlying mechanisms remain unclear. Here, we show aged (18-month-old) mice exhibit upregulation of NADPH oxidase and oxidative stress in the hippocampus, which mirrors the changes in young (2-month-old) mice subjected to chronic stress. Aged mice that lack p47phox, a key subunit of NADPH oxidase, do not show increased oxidative stress. Aged mice exhibit depression-like behavior following weak stress that does not produce depressive behavior in young mice. Aged mice have reduced expression of the epigenetic factor SUV39H1 and its upstream regulator p-AMPK, and increased expression of Ppp2ca in the hippocampus-changes that occur in young mice exposed to chronic stress. SUV39H1 mediates stress- and aging-induced sustained upregulation of p47phox and oxidative stress. These results suggest that aging increases susceptibility to stress by upregulating NADPH oxidase in the hippocampus.

Early-Life Stress in D2 Heterozygous Mice Promotes Autistic-like Behaviors through the Downregulation of the BDNF-TrkB Pathway in the Dorsal Striatum.

A number of specific genetic variants including gene mutations and single nucleotide variations have been identified in genomewide association studies of autism spectrum disorder (ASD). ASD phenotypes in individuals carrying specific genetic variations are manifest mostly in a heterozygous state. Furthermore, individuals with most genetic variants show incomplete penetrance and phenotypic variability, suggesting that non-genetic factors are also involved in developing ASD. However, the mechanisms of how genetic and environmental factors interactively promote ASD are not clearly understood. In the present study, we investigated whether early-life stress (ELS) in D2 dopamine receptor heterozygous knockout (D2+/-) mice induces ASD-like symptoms. To address that, we exposed D2 heterozygous pups to maternal separation stress for 3 h daily for 13 days beginning on postnatal day 2. D2+/- adult mice that had experienced ELS exhibited impaired sociability in the three-chamber test and home-cage social interaction test and increased grooming behavior, whereas wildtype littermates exposed to ELS did not show those phenotypes. ELS-exposed D2+/- mice had decreased levels of BDNF, TrkB, phospho-ERK1/2 and phospho-CREB in the dorsal striatum. Administration of the TrkB agonist 7,8-dihydroxyflavone (7,8-DHF) to ELS-exposed D2+/- mice rescued the sociability deficits and repetitive behavior. In contrast, behavioral rescue by 7,8-DHF in ELS-exposed D2+/- mice was blocked when TrkB expression in the dorsal striatum was locally inhibited by the injection of TrkB-siRNA. Together, our results suggest that the interaction between ELS and defective D2 gene function promotes autistic-like behaviors by downregulating the BDNF-TrkB pathway in the dorsal striatum.

Stress-Induced Epigenetic Changes in Hippocampal Mkp-1 Promote Persistent Depressive Behaviors.

Chronic stress induces persistent depressive behaviors. Stress-induced transcriptional alteration over the homeostatic range in stress hormone-sensitive brain regions is believed to underlie long-lasting depressive behaviors. However, the detailed mechanisms by which chronic stress causes those adaptive changes are not clearly understood. In the present study, we investigated whether epigenetic changes regulate stress-induced depressive behaviors. We found that chronic stress in mice downregulates the epigenetic factors HDAC2 and SUV39H1 in the hippocampus. A series of follow-up analyses including ChIP assay and siRNA-mediated functional analyses reveal that glucocorticoids released by stress cumulatively increase Mkp-1 expression in the hippocampus, and increased Mkp-1 then debilitates p-CREB and PPARγ, which in turn suppress the epigenetic factors HDAC2 and SUV39H1. Furthermore, HDAC2 and SUV39H1 normally suppress the transcription of the Mkp-1, and therefore the reduced expression of HDAC2 and SUV39H1 increases Mkp-1 expression. Accordingly, repeated stress progressively strengthens a vicious cycle of the Mkp-1 signaling cascade that facilitates depressive behaviors. These results suggest that the hippocampal stress adaptation system comprising HDAC2/SUV39H1-regulated Mkp-1 signaling network determines the vulnerability to chronic stress and the maintenance of depressive behaviors.

Extracellular Vesicles Derived from Lactobacillus plantarum Increase BDNF Expression in Cultured Hippocampal Neurons and Produce Antidepressant-like Effects in Mice.

Gut microbiota play a role in regulating mental disorders, but the mechanism by which gut microbiota regulate brain function remains unclear. Gram negative and positive gut bacteria release membrane-derived extracellular vesicles (EVs), which function in microbiota-host intercellular communication. In the present study, we investigated whether Lactobacillus plantarum derived EVs (L-EVs) could have a role in regulating neuronal function and stress-induced depressive-like behaviors. HT22 cells treated with the stress hormone glucocorticoid (GC; corticosterone) had reduced expression of Bdnf and Sirt1, whereas L-EV treatment reversed GC-induced decreased expression of Bdnf and Sirt1. The siRNA-mediated knockdown of Sirt1 in HT22 cells decreased Bdnf4, a splicing variant of Bdnf, and Creb expression, suggesting that Sirt1 plays a role in L-EV-induced increase of BDNF and CREB expression. Mice exposed to restraint for 2-h daily for 14 days (CRST) exhibited depressive-like behaviors, and these CRST-treated mice had reduced expression of Bdnf and Nt4/5 in the hippocampus. In contrast, L-EV injection prior to each restraint treatment blocked the reduced expression of Bdnf and Nt4/5, and stress-induced depressive-like behaviors. Furthermore, L-EV treatment in CRST-treated mice also rescued the reduced expression of Bdnf, and blocked stress-induced depressive-like behaviors. These results suggest that Lactobacillus derived EVs can change the expression of neurotropic factors in the hippocampus and afford antidepressant-like effects in mice with stress-induced depression.

Reciprocal interactions across and within multiple levels of monoamine and cortico-limbic systems in stress-induced depression: A systematic review.

The monoamine hypothesis of depression, namely that the reduction in synaptic serotonin and dopamine levels causes depression, has prevailed in past decades. However, clinical and preclinical studies have identified various cortical and subcortical regions whose altered neural activities also regulate depressive-like behaviors, independently from the monoamine system. Our systematic review indicates that neural activities of specific brain regions and associated neural circuitries are adaptively altered after chronic stress in a specific direction, such that the neural activity in the infralimbic cortex, lateral habenula and amygdala is upregulated, whereas the neural activity in the prelimbic cortex, hippocampus and monoamine systems is downregulated. The altered neural activity dynamics between monoamine systems and cortico-limbic systems are reciprocally interwoven at multiple levels. Furthermore, depressive-like behaviors can be experimentally reversed by counteracting the altered neural activity of a specific neural circuitry at multiple brain regions, suggesting the importance of the reciprocally interwoven neural networks in regulating depressive-like behaviors. These results promise for reshaping altered neural activity dynamics as a therapeutic strategy for treating depression.

Hyperoxygenation revitalizes Alzheimer's disease pathology through the upregulation of neurotrophic factors.

Alzheimer's disease (AD) is a neurodegenerative disease characterized by Aß-induced pathology and progressive cognitive decline. The incidence of AD is growing globally, yet a prompt and effective remedy is not available. Aging is the greatest risk factor for AD. Brain aging proceeds with reduced vascularization, which can cause low oxygen (O2 ) availability. Accordingly, the question may be raised whether O2 availability in the brain affects AD pathology. We found that Tg-APP/PS1 mice treated with 100% O2 at increased atmospheric pressure in a chamber exhibited markedly reduced Aß accumulation and hippocampal neuritic atrophy, increased hippocampal neurogenesis, and profoundly improved the cognitive deficits on the multiple behavioral test paradigms. Hyperoxygenation treatment increased the expression of BDNF, NT3, and NT4/5 through the upregulation of MeCP2/p-CREB activity in HT22 cells in vitro and in the hippocampus of mice. In contrast, siRNA-mediated inhibition of MeCP2 or TrkB neurotrophin receptors in the hippocampal subregion, which suppresses neurotrophin expression and neurotrophin action, respectively, blocked the therapeutic effects of hyperoxygenation on the cognitive impairments of Tg-APP/PS1 mice. Our results highlight the importance of the O2 -related mechanisms in AD pathology, which can be revitalized by hyperoxygenation treatment, and the therapeutic potential of hyperoxygenation for AD.

A Group of Descending Glutamatergic Neurons Activated by Stress in Corticolimbic Regions Project to the Nucleus Accumbens.

The nucleus accumbens (NAc) is the major component of the ventral striatum that regulates stress-induced depression. The NAc receives dopaminergic inputs from the ventral tegmental area (VTA), and the role of VTA-NAc neurons in stress response has been recently characterized. The NAc also receives glutamatergic inputs from various forebrain structures including the prelimbic cortex (PL), basolateral amygdala (BLA), and ventral hippocampus (vHIP), whereas the role of those glutamatergic afferents in stress response remains underscored. In the present study, we investigated the extent to which descending glutamatergic neurons activated by stress in the PL, BLA, and vHIP project to the NAc. To specifically label the input neurons into the NAc, fluorescent-tagged cholera toxin subunit B (CTB), which can be used as a retrograde neuronal tracer, was injected into the NAc. After two weeks, the mice were placed under restraint for 1 h. Subsequent histological analyses indicated that CTB-positive cells were detected in 170~680 cells/mm2 in the PL, BLA, and vHIP, and those CTB-positive cells were mostly glutamatergic. In the PL, BLA, and vHIP regions analyzed, stress-induced c-Fos expression was found in 20~100 cells/mm2. Among the CTB-positive cells, 2.6% in the PL, 4.2% in the BLA, and 1.1% in the vHIP were co-labeled by c-Fos, whereas among c-Fos-positive cells, 7.7% in the PL, 19.8% in the BLA, and 8.5% in the vHIP were co-labeled with CTB. These results suggest that the NAc receives a significant but differing proportion of glutamatergic inputs from the PL, BLA, and vHIP in stress response.

Proteomic Analysis of Hippocampus in a Mouse Model of Depression Reveals Neuroprotective Function of Ubiquitin C-terminal Hydrolase L1 (UCH-L1) via Stress-induced Cysteine Oxidative Modifications.

Chronic physical restraint stress increases oxidative stress in the brain, and dysregulation of oxidative stress can be one of the causes of major depressive disorder. To understand the underlying mechanisms, we undertook a systematic proteomic analysis of hippocampus in a chronic restraint stress mouse model of depression. Combining two-dimensional gel electrophoresis (2D-PAGE) for protein separation with nanoUPLC-ESI-q-TOF tandem mass spectrometry, we identified sixty-three protein spots that changed in the hippocampus of mice subjected to chronic restraint stress. We identified and classified the proteins that changed after chronic stress, into three groups respectively functioning in neural plasticity, metabolic processes and protein aggregation. Of these, 5 proteins including ubiquitin C-terminal hydrolase L1 (UCH-L1), dihydropyrimidinase-related protein 2 (DPYL2), haloacid dehalogenase-like hydrolase domain-containing protein 2 (HDHD2), actin-related protein 2/3 complex subunit 5 (ARPC5) and peroxiredoxin-2 (PRDX2), showed pI shifts attributable to post-translational modifications. Further analysis indicated that UCH-L1 underwent differential oxidations of 2 cysteine residues following chronic stress. We investigated whether the oxidized form of UCH-L1 plays a role in stressed hippocampus, by comparing the effects of UCH-L1 and its Cys mutants on hippocampal cell line HT-22 in response to oxidative stress. This study demonstrated that UCH-L1 wild-type and cysteine to aspartic acid mutants, but not its cysteine to serine mutants, afforded neuroprotective effects against oxidative stress; there were no discernible differences between wild-type UCH-L1 and its mutants in the absence of oxidative stress. These findings suggest that cysteine oxidative modifications of UCH-L1 in the hippocampus play key roles in neuroprotection against oxidative stress caused in major depressive disorder.

Effects of histone acetyltransferase inhibitors on L-DOPA-induced dyskinesia in a murine model of Parkinson's disease.

Histone acetylation is a key regulatory factor for gene expression in cells. Modulation of histone acetylation by targeting of histone acetyltransferases (HATs) effectively alters many gene expression profiles and synaptic plasticity in the brain. However, the role of HATs on L-DOPA-induced dyskinesia of Parkinson's disease (PD) has not been reported. Our aim was to determine whether HAT inhibitors such as anacardic acid, garcinol, and curcumin from natural plants reduce severity of L-DOPA-induced dyskinesia using a unilaterally 6-hydroxydopamine (6-OHDA)-lesioned PD mouse model. Anacardic acid 2 mg/kg, garcinol 5 mg/kg, or curcumin 100 mg/kg co-treatment with L-DOPA significantly reduced the axial, limb, and orofacial (ALO) score indicating less dyskinesia with administration of HAT inhibitors in 6-OHDA-lesioned mice. Additionally, L-DOPA's efficacy was not altered by the compounds in the early stage of treatment. The expression levels of c-Fos, Fra-2, and Arc were effectively decreased by administration of HAT inhibitors in the ipsilateral striatum. Our findings indicate that HAT inhibitor co-treatment with L-DOPA may have therapeutic potential for management of L-DOPA-induced dyskinesia in patients with PD.

Alarmin HMGB1 induces systemic and brain inflammatory exacerbation in post-stroke infection rat model.

Post-stroke infection (PSI) is known to worsen functional outcomes of stroke patients and accounts to one-third of stroke-related deaths in hospital. In our previous reports, we demonstrated that massive release of high-mobility group box protein 1 (HMGB1), an endogenous danger signal molecule, is promoted by N-methyl-D-aspartic acid-induced acute damage in the postischemic brain, exacerbating neuronal damage by triggering delayed inflammatory processes. Moreover, augmentation of proinflammatory function of lipopolysaccharides (LPS) by HMGB1 via direct interaction has been reported. The aim of this study was to investigate the role of HMGB1 in aggravating inflammation in the PSI by exacerbating the function of LPS. PSI animal model was produced by administrating a low-dose LPS at 24 h post-middle cerebral artery occlusion (MCAO). Profound aggravations of inflammation, deterioration of behavioral outcomes, and infarct expansion were observed in LPS-injected MCAO animals, in which serum HMGB1 surge, especially disulfide type, occurred immediately after LPS administration and aggravated brain and systemic inflammations probably by acting in synergy with LPS. Importantly, blockage of HMGB1 function by delayed administrations of therapeutic peptides known to inhibit HMGB1 (HMGB1 A box, HPep1) or by treatment with LPS after preincubation with HMGB1 A box significantly ameliorated damages observed in the rat PSI model, demonstrating that HMGB1 plays a crucial role. Furthermore, administration of Rhodobacter sphaeroides LPS, a selective toll-like receptor 4 antagonist not only failed to exert these effects but blocked the effects of LPS, indicating its TLR4 dependence. Together, these results indicated that alarmin HMGB1 mediates potentiation of LPS function, exacerbating TLR4-dependent systemic and brain inflammation in a rat PSI model and there is a positive-feedback loop between augmentation of LPS function by HMGB1 and subsequent HMGB1 release/serum. Therefore, HMGB1 might be a valuable therapeutic target for preventing post-stroke infection.