＜文献概要＞女性の更年期障害は,卵巣機能低下によるエストロゲン分泌減少などの生物学的要因に加え,社会心理的ストレスなどの環境要因が複雑に関与し,身体的症状や精神的症状を呈する。これらの症状に対し,女性三大漢方薬の1つである加味逍遙散(KSS)が汎用され,特にうつ・不安,イライラなどの精神的症状改善において臨床的評価が高い。本研究は,更年期うつ症状を改善するKSSの作用機序解明を目的とし,更年期うつ病モデルマウスを用いた解析を行った結果,KSSが海馬におけるセロトニン1A(5-HT1A)受容体発現回復およびPKA-CREB-BDNF経路の活性化を介してうつ症状を改善している可能性が示唆された。

＜文献概要＞女性の更年期障害は,卵巣機能低下によるエストロゲン分泌減少などの生物学的要因に加え,社会心理的ストレスなどの環境要因が複雑に関与し,身体的症状や精神的症状を呈する。これらの症状に対し,女性三大漢方薬の1つである加味逍遙散(KSS)が汎用され,特にうつ・不安,イライラなどの精神的症状改善において臨床的評価が高い。本研究は,更年期うつ症状を改善するKSSの作用機序解明を目的とし,更年期うつ病モデルマウスを用いた解析を行った結果,KSSが海馬におけるセロトニン1A(5-HT1A)受容体発現回復およびPKA-CREB-BDNF経路の活性化を介してうつ症状を改善している可能性が示唆された。

Major depressive disorder (MDD) is a multifactorial disease affected by several environmental factors. Although several potential onset hypotheses have been identified, the molecular mechanisms underlying the pathogenesis of this disorder remain unclear. Several recent studies have suggested that among many environmental factors, inflammation and immune abnormalities in the brain or the peripheral tissues are associated with the onset of MDDs. Furthermore, several stress-related hypotheses have been proposed to explain the onset of MDDs. Thus, inflammation or immune abnormalities can be considered stress responses that occur within the brain or other tissues and are regarded as one of the mechanisms underlying the stress hypothesis of MDDs. Therefore, we introduce several current advances in inflammation studies in the brain that might be related to the pathophysiology of MDD due to stress exposure in this review.

Protein arginine methylation has been recognized as one of key posttranslational modifications for refined protein functions, mediated by protein arginine methyltransferases (Prmts). Coactivator-associated arginine methyltransferase (Carm1, also known as Prmt4) participates in various cellular events, such as cell survival, proliferation, and differentiation through its protein arginine methylation activities. Carm1 regulates cell proliferation of a neuronal cell line and is reportedly expressed in the mammalian brain. However, its detailed function in the central nervous system, particularly in glial cells, remains largely unexplored. In this study, Carm1 exhibited relatively high expression in oligodendrocyte (OL) lineage cells present in the corpus callosum of the developing brain, followed by a remarkable downregulation after active myelination. The suppression of Carm1 activity by inhibitors in isolated oligodendrocyte precursor cells (OPCs) reduced the number of Ki67-expressing and BrdU-incorporated proliferating cells. Furthermore, Carm1 inactivation attenuated OL differentiation, as determined by the expression of Plp, a reliable myelin-related marker. It also impaired the extension of OL processes, accompanied by a significant reduction in gene expression related to OL differentiation and myelination, such as Sox10, Cnp, Myrf, and Mbp. In addition, OLs co-cultured with embryonic dorsal root ganglia neurons demonstrated that Carm1 activity is required for the appropriate formation of myelin processes and myelin sheaths around neuronal axons, and the induction of the clustering of Caspr, a node of Ranvier structural molecule. Thus, we propose that Carm1 is an essential molecule for the development of OPCs and OLs during brain development.

Females are well known to suffer disproportionately more than males from stress-related neuropsychiatric disorders, especially during perimenopausal and postmenopausal periods. In addition to a decline in serum estradiol levels, environmental stress and social stress likely contribute to the development of neuropsychiatric symptoms in perimenopausal and postmenopausal women. Kamishoyosan (KSS) is a traditional Japanese Kampo medicine, composed of a specified mixture of 10 crude compounds derived from plant sources, widely used for various neuropsychiatric symptoms in perimenopausal and postmenopausal women. However, the molecular mechanisms underlying KSS-mediated attenuation of neuropsychological symptoms and stress-response behaviors in perimenopausal and postmenopausal women remain unknown. In the present study, we first established a mouse model for postmenopausal depression-like signs using chronic water-immersion and restraint-stressed ovariectomized (OVX) mice to investigate the underlying molecular mechanism of KSS. We found that continuous administration of KSS to these mice normalized the activation of the hypothalamic-pituitary-adrenal (HPA) axis, ameliorated stress-induced depressive behavior, and prevented a decrease of neurogenesis in the hippocampus. As previous studies have implicated dysfunction of the hippocampal 5-HT1A receptor (5-HT1AR) in depressive disorders, we also evaluated the effect of KSS on 5-HT1AR expression and the protein kinase A- (PKA-) cAMP response element-binding- (CREB-) brain-derived neurotrophic factor (BDNF) signaling pathway in the hippocampus in this model. The level of 5-HT1AR in the hippocampus decreased in chronic stress-exposed OVX mice, while KSS treatment normalized the stress-induced decrease in 5-HT1AR expression in the hippocampus of chronic stress-exposed OVX mice. Furthermore, we found that KSS treatment upregulated the expression levels of phosphorylated PKA (p-PKA), phosphorylated CREB (p-CREB), and BDNF in the hippocampus in chronic stress-exposed OVX mice. These results suggest that KSS improves neuropsychiatric symptoms through 5-HT1AR and PKA-CREB-BDNF signaling in the hippocampus in postmenopausal women.

Repeated stress is a risk factor for mental disorders and can also lead to sleep disturbances. Although the effects of stress on sleep architecture have been investigated in rodents, the length of the stress exposure period in most studies has been limited to about 10 days, and few studies have analyzed the effects of chronic stress over a longer period. Here we investigated how sleep is affected in a mouse model of depression induced by 3 weeks of daily water immersion and restraint stress (WIRS). Sleep was recorded after 1, 2, and 3 weeks of stress exposure. Some stress-induced changes in several sleep measures were maintained across the 3 weeks, whereas other changes were most prominent during the 1st week. The total amount of non-rapid eye movement sleep (NREMS) was increased and the total amount of time spent awake was decreased across all 3 weeks. On the other hand, the amount of REMS during the dark phase was significantly increased in the 1st week compared with that at baseline or the 2nd and 3rd weeks. Electroencephalogram (EEG) power in the delta range was decreased during NREMS, although the total amount of NREMS was increased. These findings indicate that repeated WIRS, which eventually leads to a depression-like phenotype, differentially affects sleep between the early and subsequent periods. The increase in the amount of REMS during the dark phase in the 1st week significantly correlated with changes in body weight. Our results show how sleep changes throughout a long period of chronic stress in a mouse model of depression.

Oligodendrocytes, the myelin-forming cells in the central nervous system (CNS), undergo morphological differentiation characterized by elaborated branched processes to enwrap neuronal axons. However, the basic molecular mechanisms underlying oligodendrocyte morphogenesis remain unknown. Herein, we describe the essential roles of Nuclear Distribution E Homolog 1 (NDE1), a dynein cofactor, in oligodendrocyte morphological differentiation. In the mouse corpus callosum, Nde1 mRNA expression was detected in oligodendrocyte lineage cells at the postnatal stage. In vitro analysis revealed that downregulation of NDE1 by siRNA impaired the outgrowth and extensive branching of oligodendrocyte processes and led to a decrease in the expression of myelin-related markers, namely, CNPase and MBP. In myelinating co-cultures with dorsal root ganglion (DRG) neurons, NDE1-knockdown oligodendrocyte precursor cells (OPCs) failed to develop into MBP-positive oligodendrocytes with multiple processes contacting DRG axons. Immunoprecipitation studies showed that NDE1 interacts with the dynein intermediate chain (DIC) in oligodendrocytes, and an overexpressed DIC-binding region of NDE1 exerted effects on oligodendrocyte morphogenesis that were similar to those following NDE1 knockdown. Furthermore, NDE1-knockdown-impaired oligodendrocyte process formation was rescued by siRNA-resistant wild-type NDE1 but not by DIC-binding region-deficient NDE1 overexpression. These results suggest that NDE1 plays a crucial role in oligodendrocyte morphological differentiation via interaction with dynein.

© 2018 The Authors Abnormalities in limbic neural circuits have been implicated in the onset of anxiety disorders. However, the molecular pathogenesis underlying anxiety disorders remains poorly elucidated. Here, we demonstrate that myristoylated alanine-rich C-kinase substrate like 1 (MARCKSL1) regulates amygdala circuitry to control the activity of the hypothalamic-pituitary-adrenal (HPA) axis, as well as induces anxiety-like behaviors in mice. MARCKSL1 expression was predominantly localized in the prefrontal cortex (PFC), hypothalamus, hippocampus, and amygdala of the adult mouse brain. MARCKSL1 transgenic (Tg) mice exhibited anxiety-like behaviors dependent on corticotropin-releasing hormone. MARCKSL1 increased spine formation in the central amygdala, and downregulation of MARCKSL1 in the amygdala normalized both increased HPA axis activity and elevated anxiety-like behaviors in Tg mice. Furthermore, MARCKSL1 expression was increased in the PFC and amygdala in a brain injury model associated with anxiety-like behaviors. Our findings suggest that MARCKSL1 expression in the amygdala plays an important role in anxiety-like behaviors.

Repeated stressful events are associated with the onset of major depressive disorder (MDD). We previously showed oligodendrocyte (OL)-specific activation of the serum/glucocorticoid-regulated kinase (SGK)1 cascade, increased expression of axon-myelin adhesion molecules, and elaboration of the oligodendrocytic arbor in the corpus callosum of chronically stressed mice. In the current study, we demonstrate that the nodes and paranodes of Ranvier in the corpus callosum were narrower in these mice. Chronic stress also led to diffuse redistribution of Caspr and Kv 1.1 and decreased the activity in white matter, suggesting a link between morphological changes in OLs and inhibition of axonal activity. OL primary cultures subjected to chronic stress resulted in SGK1 activation and translocation to the nucleus, where it inhibited the transcription of metabotropic glutamate receptors (mGluRs). Furthermore, the cAMP level and membrane potential of OLs were reduced by chronic stress exposure. We showed by diffusion tensor imaging that the corpus callosum of patients with MDD exhibited reduced fractional anisotropy, reflecting compromised white matter integrity possibly caused by axonal damage. Our findings suggest that chronic stress disrupts the organization of the nodes of Ranvier by suppressing mGluR activation in OLs, and that specific white matter abnormalities are closely associated with MDD onset.

Major depression, one of the most prevalent mental illnesses, is thought to be a multifactorial disease related to both genetic and environmental factors. However, the genes responsible for and the pathogenesis of major depression at the molecular level remain unclear. Recently, we reported that stressed mice with elevated plasma corticosterone levels show upregulation and activation of serum glucocorticoid-regulated kinase (Sgk1) in oligodendrocytes. Active Sgk1 causes phosphorylation of N-myc downstream-regulated gene 1 (Ndrg1), and phospho-Ndrg1 increases the expression of N-cadherin, alpha-catenin, and beta-catenin in oligodendrocytes. This activation of the Sgk1 cascade results in morphological changes in the oligodendrocytes of nerve fiber bundles, such as those present in the corpus callosum. However, little is known about the molecular functions of the traditional and/or desmosomal cadherin superfamily in oligodendrocytes. Therefore, in this study, we aimed to elucidate the functions of the desmosomal cadherin superfamily in oligodendrocytes. Desmoglein (Dsg) 1, Dsg2, and desmocollin 1 (Dsc1) were found to be expressed in the corpus callosum of mouse brain, and the expression of a subtype of Dsg1, Dsg1c, was upregulated in oligodendrocytes after chronic stress exposure. Furthermore, Dsg1 proteins were localized around the plasma membrane regions of oligodendrocytes. A study in primary oligodendrocyte cultures also revealed that chronic upregulation of Sgk1 by dexamethasone administration is involved in upregulation of Dsg1c mRNA. These results may indicate that chronic stress induced Sgk1 activation in oligodendrocytes, which increases Dsg1 expression near the plasma membrane. Thus, Dsg1 upregulation may be implicated in the molecular mechanisms underlying the morphological changes in oligodendrocytes in response to chronic stress exposure. (C) 2015 Elsevier Inc. All rights reserved.

Recently several potential susceptibility genes for major psychiatric disorders (schizophrenia and major depression) such as disrupted-in-schizophrenia 1(DISC1), dysbindin and pituitary adenylate cyclase-activating polypeptide (PACAP) have been reported. DISC1 is involved in neural development directly via adhesion molecules or via its binding partners of DISC1 such as elongation protein zeta-1 (FEZ1), DISC1-binding zinc-finger protein (DBZ) and kendrin. PACAP also regulates neural development via stathmin 1 or via regulation of the DISC1-DBZ binding. Dysbindin is also involved in neural development by regulating centrosomal microtubule network formation. All such molecules examined to date are involved in neural development. Thus, these findings provide new molecular insights into the mechanisms of neural development and neuropsychiatric disorders. On the other hand, in addition to neurons, both DISC and DBZ have been detected in oligodendrocytes and implicated in regulating oligodendrocyte differentiation. DISC1 inhibits the differentiation of oligodendrocyte precursor cells into oligodendrocytes, while DBZ has a positive regulatory role in oligodendrocyte differentiation. Evidence suggesting that disturbance of oligodendrocyte development causes major depression is also described.

Stressful events are known to down-regulate expression levels of glucocorticoid receptors (GRs) in the brain. Recently, we reported that stressed mice with elevated plasma levels of corticosterone exhibit morphological changes in the oligodendrocytes of nerve fiber bundles, such as those in the corpus callosum. However, little is known about the molecular mechanism of GR expression regulation in oligodendrocytes after stress exposure. A previous report has suggested that GR protein levels might be regulated by microRNA (miR)-18 and/or -124a in the brain. In this study, we aimed to elucidate the GR regulation mechanism in oligodendrocytes and evaluate the effects of yokukansan (YKS), a Kampo medicine, on GR protein regulation. Acute exposure to stress increased plasma corticosterone levels, decreased GR protein expression, and increased miR-124a expression in the corpus callosum of adult male mice, though the GR mRNA and miR-18 expression levels were not significant changes. YKS normalized the stress-induced changes in the plasma corticosterone, GR protein, and miR124a expression levels. An oligodendrocyte primary culture study also showed that YKS down-regulated miR-124a, but not miR-18, expression levels in dexamethasone-treated cells. These results suggest that the down-regulation of miR124a expression might be involved in the normalization of stress-induced decreases in GR protein in oligodendrocytes by YKS. This effect may imply the molecular mechanisms underlying the ameliorative effects of YKS on psychological symptoms and stress-related behaviors. (C) 2015 Elsevier Inc. All rights reserved.

The major psychiatric disorders such as schizophrenia (SZ) and major depressive disorder (MDD) are thought to be multifactorial diseases related to both genetic and environmental factors. However, the genes responsible and the molecular mechanisms underlying the pathogenesis of SZ and MDD remain unclear. We previously reported that abnormalities of disrupted-in-Schizophrenia-1 (DISC1) and DISC1 binding zinc finger (DBZ) might cause major psychiatric disorders such as SZ. Interestingly, both DISC and DBZ have been further detected in oligodendrocytes and implicated in regulating oligodendrocyte differentiation. DISC1 negatively regulates the differentiation of oligodendrocytes, whereas DBZ plays a positive regulatory role in oligodendrocyte differentiation. We have reported that repeated stressful events, one of the major risk factors of MDD, can induce sustained upregulation of plasma corticosterone levels and serum/glucocorticoid regulated kinase 1 (Sgk1) mRNA expression in oligodendrocytes. Repeated stressful events can also activate the SGK1 cascade and cause excess arborization of oligodendrocyte processes, which is thought to be related to depressive-like symptoms. In this review, we discuss the expression of DISC1, DBZ, and SGK1 in oligodendrocytes, their roles in the regulation of oligodendrocyte function, possible interactions of DISC1 and DBZ in relation to SZ, and the activation of the SGK1 signaling cascade in relation to MDD.

It is well known that glucocorticoid receptor (GR) signaling regulates the hypothalamic-pituitary-adrenal (HPA) axis, and GR expression level is associated with HPA axis activity. Recent studies revealed that microRNA-(miR-) 18 and/or 124a are candidate negative regulators of GR in the brain. The Kampo medicine Yokukansan (YKS) can affect psychological symptoms such as depression and anxiety that are associated with stress responses. In this study, we evaluated the effect of YKS on miR-18 and 124a and GR levels in mice exposed to stress. We found that YKS pretreatment normalized elevated plasma corticosterone levels in stress-exposed mice. In addition, GR mRNA levels were downregulated in the brain following stress exposure. While miR-124a expression levels were not altered in the hypothalamus of stress-exposed mice, miR-18 levels decreased in the hypothalamus of YKS-pretreated mice after stress exposure. Finally, GR protein levels in the paraventricular nucleus (PVN) of the hypothalamus after stress exposure recovered in YKS-pretreated mice. Collectively, these data suggest that YKS normalizes GR protein levels by regulating miR-18 expression in the hypothalamus, thus normalizing HPA axis activity following stress exposure.

Recent studies have shown changes in myelin genes and alterations in white matter structure in a wide range of psychiatric disorders. Here we report that DBZ, a central nervous system (CNS)-specific member of the DISC1 interactome, positively regulates the oligodendrocyte (OL) differentiation in vivo and in vitro. In mouse corpus callosum (CC), DBZ mRNA is expressed in OL lineage cells and expression of DBZ protein peaked before MBP expression. In the CC of DBZ-KO mice, we observed delayed myelination during the early postnatal period. Although the myelination delay was mostly recovered by adulthood, OLs with immature structural features were more abundant in adult DBZ-KO mice than in control mice. DBZ was also transiently upregulated during rat OL differentiation in vitro before myelin marker expression. DBZ knockdown by RNA interference resulted in a decreased expression of myelin-related markers and a low number of cells with mature characteristics, but with no effect on the proliferation of oligodendrocyte precursor cells. We also show that the expression levels of transcription factors having a negative-regulatory role in OL differentiation were upregulated when endogenous DBZ was knocked down. These results strongly indicate that OL differentiation in rodents is regulated by DBZ.

Background: Oxidative stress has been suggested as a mechanism underlying skin aging, as it triggers apoptosis in various cell types, including fibroblasts, which play important roles in the preservation of healthy, youthful skin. Catechins, which are antioxidants contained in green tea, exert various actions such as anti-inflammatory, anti-bacterial, and anti-cancer actions. In this study, we investigated the effect of (+)-catechin on apoptosis induced by oxidative stress in fibroblasts. Methods: Fibroblasts (NIH3T3) under oxidative stress induced by hydrogen peroxide (0.1 mM) were treated with either vehicle or (+)-catechin (0-100 mu M). The effect of (+)-catechin on cell viability, apoptosis, phosphorylation of c-Jun terminal kinases (JNK) and p38, and activation of caspase-3 in fibroblasts under oxidative stress were evaluated. Results: Hydrogen peroxide induced apoptotic cell death in fibroblasts, accompanied by induction of phosphorylation of JNK and p38 and activation of caspase-3. Pretreatment of the fibroblasts with (+)-catechin inhibited hydrogen peroxide-induced apoptosis and reduced phosphorylation of JNK and p38 and activation of caspase-3. Conclusion: (+)-Catechin protects against oxidative stress-induced cell death in fibroblasts, possibly by inhibiting phosphorylation of p38 and JNK. These results suggest that (+)-catechin has potential as a therapeutic agent for the prevention of skin aging.

L-Arginine is considered a conditionally essential amino acid and has been shown to enhance wound healing. However, the molecular mechanisms through which arginine stimulates cutaneous wound repair remain unknown. Here, we evaluated the effects of arginine supplementation on fibroblast proliferation, which is a key process required for new tissue formation. We also sought to elucidate the signaling pathways involved in mediating the effects of arginine on fibroblasts by evaluation of extracellular signal-related kinase (ERK) 1/2 activation, which is important for cell growth, survival, and differentiation. Our data demonstrated that addition of 6 mM arginine significantly enhanced fibroblast proliferation, while arginine deprivation increased apoptosis, as observed by enhanced DNA fragmentation. In vitro kinase assays demonstrated that arginine supplementation activated ERK1/2, Akt, PKA and its downstream target, cAMP response element binding protein (CREB). Moreover, knockdown of GPRC6A using siRNA blocked fibroblast proliferation and decreased phosphorylation of ERK1/2, Akt and CREB. The present experiments demonstrated a critical role for the GPRC6A-ERK1/2 and PI3K/Akt signaling pathway in arginine-mediated fibroblast survival. Our findings provide novel mechanistic insights into the positive effects of arginine on wound healing.

X-linked mental retardation (XLMR) is a common cause of moderate to severe intellectual disability in males. XLMR protein related to neurite extension (Xpn, also known as KIAA2022) has been implicated as a gene responsible for XLMR in humans. Although Xpn is highly expressed in the developing brain and is involved in neurite outgrowth in PC12 cells and neurons, little is known about the functional role of Xpn. Here, we show that Xpn regulates cell-cell and cell-matrix adhesion and migration in PC12 cells. Xpn knockdown enhanced cell-cell and cell-matrix adhesion mediated by N-cadherin and beta 1-integrin, respectively. N-Cadherin and beta 1-integrin expression at the mRNA and protein levels was significantly increased in Xpn knockdown PC12 cells. Furthermore, overexpressed Xpn protein was strongly expressed in the nuclei of PC12 and 293T cells. Finally, depletion of Xpn perturbed cellular migration by enhancing N-cadherin and beta 1-integrin expression in a PC12 cell wound healing assay. We conclude that Xpn regulates cell-cell and cell-matrix adhesion and cellular migration by regulating the expression of adhesion molecules. (C) 2013 Elsevier Ltd. All rights reserved.

Increased expression of thioredoxin-interacting protein (TXNIP) has recently been proved to be a crucial event for irremediable endoplasmic reticulum (ER) stress resulting in the programmed cell death (apoptosis) of pancreatic beta-cells. The present study demonstrated that treatment with 1-10 mu g/ml tunicamycin, a potent revulsant of ER stress, drastically induced TXNIP expression accompanied by the generation of cleaved caspase-3 as an indicator of apoptosis in SK-N-SH human neuroblastoma cells. This result substantiated that TXNIP is also involved in neurodegeneration triggered by ER stress. Moreover, we evaluated the effects of nobiletin, a citrus polymethoxyflavonoid, on tunicamycin-induced apoptosis and TXNIP expression in SK-N-SH cells, because we reported previously that this flavonoid might be able to reduce TXNIP expression. Co-treatment of SK-N-SH cells with 100 mu M nobiletin and 1 mu g/ml tunicamycin for 24 h strongly suppressed apoptosis and increased TXNIP expression induced by 1 mu g/ml tunicamycin treatment alone. In addition, we proved that the ability of 100 mu M nobiletin treatment to reduce TXNIP expression is exerted from 3 h after the onset of treatment. Therefore, the protective and ameliorative effects of nobiletin on neuronal degeneration and impaired memory, which several studies using animal models have demonstrated, might arise in part from nobiletin's ability to repress TXNIP expression (C) 2013 Elsevier Ireland Ltd. All rights reserved.

The Golgi apparatus is important for the transport of secretory cargo. Glycosylation is a major post-translational event. Recognition of O-glycans on proteins is necessary for glycoprotein trafficking. In this study, specific inhibition of O-glycosylation (Golgi stress) induced the expression of endoplasmic reticulum (ER)-resident heat shock protein (HSP) 47 in NIH3T3 cells, although cell death was not induced by Golgi stress alone. When HSP47 expression was downregulated by siRNA, inhibition of O-glycosylation caused cell death. Three days after the induction of Golgi stress, the Golgi apparatus was disassembled, many vacuoles appeared near the Golgi apparatus and extended into the cytoplasm, the nuclei had split, and cell death assay-positive cells appeared. Six hours after the induction of Golgi stress, HSP47-knockdown cells exhibited increased cleavage of Golgi-resident caspase-2. Furthermore, activation of mitochondrial caspase-9 and ER-resident unfolded protein response (UPR)-related molecules and efflux of cytochrome c from the mitochondria to the cytoplasm was observed in HSP47-knockdown cells 24 h after the induction of Golgi stress. These findings indicate that (i) the ER-resident chaperon HSP47 protected cells from Golgi stress, and (ii) Golgi stress-induced cell death caused by the inhibition of HSP47 expression resulted from caspase-2 activation in the Golgi apparatus, extending to the ER and mitochondria.

Mitochondria are dynamic organelles that change in response to extracellular stimuli. These changes are essential for normal mitochondrial/cellular function and are controlled by a tight balance between two antagonistic pathways that promote fusion and fission. Although some molecules have been identified to mediate the mitochondrial fusion and fission process, the underlying mechanisms remain unclear. Tumor necrosis factor receptor-associated protein 1 (TRAP1) is a mitochondrial molecule that regulates a variety of mitochondrial functions. Here, we examined the role of TRAP1 in the regulation of morphology. Stable TRAP1 knockdown cells showed abnormal mitochondrial morphology, and we observed significant decreases in dynamin-related protein 1 (Drp1) and mitochondrial fission factor (Mff), mitochondrial fission proteins. Similar results were obtained by transient knockdown of TRAP1 in two different cell lines, SH-SY5Y neuroblastoma cells and KNS-42 glioma cells. However, TRAP1 knockdown did not affect expression levels of fusion proteins. The reduction in Drp1 and Mff protein levels was rescued following treatment with the proteasome inhibitor MG132. These results suggest that TRAP1 regulates the expression of fission proteins and controls mitochondrial fusion/fission, which affects mitochondrial/cellular function.

KIAA2022 has been implicated as a gene responsible for expressing X-linked mental retardation (XLMR) proteins in humans. However, the functional role of KIAA2022 in the human brain remains unclear. Here, we revealed that depletion of Kiaa2022 inhibits neurite outgrowth of PC12 cells, indicating that the gene participates in neurite extension. Thus, we termed Kiaa2022 as an XLMR protein related to neurite extension (Xpn). Using the mouse brain as a model and ontogenetic analysis of Xpn by real-time PCR, we clearly demonstrated that Xpn is expressed transiently during the late embryonic and perinatal stages. In situ hybridization histochemistry further revealed that Xpn-expressing neurons could be categorized ontogenetically into three types. The first type showed transient expression of Xpn during development. The second type maximally expressed Xpn during the late embryonic or perinatal stage. Thereafter, Xpn expression in this type of neuron decreased gradually throughout development. Nevertheless, a significant level of Xpn expression was detected even into adulthood. The third type of neurons initiated expression of Xpn during the embryonic stage, and continued to express the gene throughout the remaining developmental stages. Subsequent immunohistochemical analysis revealed that Xpn was localized to the nucleus and cytoplasm throughout brain development. Our findings indicate that Xpn may participate in neural circuit formation during developmental stages via nuclear and cytoplasmic Xpn. Moreover, disturbances of this neuronal circuit formation may play a role in the pathogenesis of mental retardation. (c) 2012 IBRO. Published by Elsevier Ltd. All rights reserved.

Stress in mitochondria or the endoplasmic reticulum (ER) independently causes cell death. Recently, it was reported that ER stress causes mitochondrial dysfunction via p53-upregulated modulator of apoptosis (PUMA). However, little is known regarding the mitochondria molecules that mediate ER dysfunction. The present study revealed that tumor necrosis factor receptor-associated protein 1 (TRAP1), which localizes in the mitochondria, is associated with the unfolded protein response (UPR) in the ER. TRAP1 knockdown activated the ER-resident caspase-4, which is activated by ER stress, to induce cell death in humans. However, TRAP1 knockdown cells did not show a significant increase in the level of cell death at least within 24 h after early phase of ER stress in comparison with that of the control cells. This finding could be attributed to a number of reasons. TRAP1 knockdown failed to activate caspase-9, which is activated by activated caspase-4. In addition, TRAP1 knockdown increased the basal level of GRP78/BiP expression, which protects cells, and decreased the basal level of C/EBP homologous protein (CHOP) expression, which induces cell death, even under ER stress. Thus, the present study revealed that mitochondria could be a potential regulator of the UPR in the ER through mitochondrial TRAP1. (C) 2011 Elsevier Ltd. All rights reserved.

Repeated stressful events are known to be associated with onset of depression. Further, stress activates the hypothalamic-pituitary-adrenocortical (HPA) system by elevating plasma cortisol levels. However, little is known about the related downstream molecular pathway. In this study, by using repeated water-immersion and restraint stress (WIRS) as a stressor for mice, we attempted to elucidate the molecular pathway induced by elevated plasma corticosterone levels. We observed the following effects both, in vivo and in vitro: (1) repeated exposure to WIRS activates the 3-phosphoinositide-dependent protein kinase (PDK1)-serum glucocorticoid regulated kinase (SGK1)-N-myc downstream-regulated gene 1 (NDRG1)-adhesion molecule (i.e., N-cadherin, alpha-catenin, and beta-catenin) stabilization pathway via an increase in plasma corticosterone levels; (2) the activation of this signaling pathway induces morphological changes in oligodendrocytes; and (3) after recovery from chronic stress, the abnormal arborization of oligodendrocytes and depression-like symptoms return to the control levels. Our data strongly suggest that these abnomalities of oligodendrocytes are possibly related to depression-like symptoms.

Background: Recently, several studies have reported Yokukansan (Tsumura TJ-54), a traditional Japanese medicine, as a potential new drug for the treatment of Alzheimer's disease (AD). Endoplasmic reticulum (ER) stress is known to play an important role in the pathogenesis of AD, particularly in neuronal death. Therefore, we examined the effect of Yokukansan on ER stress-induced neurotoxicity and on familial AD-linked presenilin-1 mutation-associated cell death. Methods: We employed the WST-1 assay and monitored morphological changes to evaluate cell viability following Yokukansan treatment or treatment with its components. Western blotting and PCR were used to observe the expression levels of GRP78/BiP, caspase-4 and C/EBP homologous protein. Results: Yokukansan inhibited neuronal death during ER stress, with Cnidii Rhizoma (Senkyu), a component of Yokukansan, being particularly effective. We also showed that Yokukansan and Senkyu affect the unfolded protein response following ER stress and that these drugs inhibit the activation of caspase-4, resulting in the inhibition of ER stress-induced neuronal death. Furthermore, we found that the protective effect of Yokukansan and Senkyu against ER stress could be attributed to the ferulic acid content of these two drugs. Conclusions: Our results indicate that Yokukansan, Senkyu and ferulic acid are protective against ER stress-induced neuronal cell death and may provide a possible new treatment for AD.

Protein arginine N-methyltransferase 3 (PRMT3) is a cytoplasmic enzyme that utilizes S-adenosyl-L-methionine (AdoMet) to methylate specific proteins, most of which contain GAR (glycine-arginine rich) motifs. PRMT3 has been shown to play a role in the proper maturation of the 80S ribosome by binding to and catalyzing the methylation of rpS2, a component of the 40S ribosomal subunit. However, the other roles of PRMT3 are fairly unclear, particularly in the brain, which is abundant in methylated proteins. In this study, we perturbed PRMT3 expression in cultured rat hippocampal neurons by transiently introducing siRNA oligonucleotides that were designed to hybridize with PRMT3 mRNA and then we examined the morphological and functional effects of neuronal PRMT3 depletion. PRMT3-defective neurons showed deformed spines without any change in spine number; less BDNF-induced protein translation of alpha CaMKII; and diminished rpS2 protein stability. Furthermore, overexpression of a methylation-resistant rpS2, whose methylated arginine residues were deleted, produced phenotypes that were similar to those associated with PRMT3 downregulation. These findings demonstrated that PRMT3 possibly plays a pivotal role in neuronal translation by interaction with rpS2 and that it contributes to activity-dependent changes in the dendritic spines. (C) 2010 Published by Elsevier B.V.

Fibroblast proliferation and migration play important roles in wound healing. bFGF is known to promote both fibroblast proliferation and migration during the process of wound healing. However, the signal transduction of bFGF-induced fibroblast migration is still unclear, because bFGF can affect both proliferation and migration. Herein, we investigated the effect of bFGF on fibroblast migration regardless of its effect on fibroblast proliferation. We noticed involvement of the small GTPases of the Rho family, PI3-kinase, and JNK. bFGF activated RhoA, Rac1, PI3-kinase, and JNK in cultured fibroblasts. Inhibition of RhoA did not block bFGF-induced fibroblast migration, whereas inhibition of Rac1, PI3-kinase, or JNK blocked the fibroblastmigration significantly. PI3-kinase- inhibited cells down-regulated the activities of Rac1 and JNK, and Rac1-inhibited cells down-regulated JNK activity, suggesting that PI3-kinase is upstream of Rac1 and that JNK is downstream of Rac1. Thus, we concluded that PI3-kinase, Rac1, and JNK were essential for bFGF-induced fibroblast migration, which is a novel pathway of bFGF-induced cell migration.

Background: An accumulating body of evidence suggests that Dtnbp1 (Dysbindin) is a key susceptibility gene for schizophrenia. Using the yeast-two-hybrid screening system, we examined the candidate proteins interacting with Dysbindin and revealed one of these candidates to be the transcription factor NF-YB. Methods: We employed an immunoprecipitation (IP) assay to demonstrate the Dysbindin-NF-YB interaction. DNA chips were used to screen for altered expression of genes in cells in which Dysbindin or NF-YB was down regulated, while Chromatin IP and Reporter assays were used to confirm the involvement of these genes in transcription of Myristoylated alanine-rich protein kinase C substrate (MARCKS). The sdy mutant mice with a deletion in Dysbindin, which exhibit behavioral abnormalities, and wild-type DBA2J mice were used to investigate MARCKS expression. Results: We revealed an interaction between Dysbindin and NF-YB. DNA chips showed that MARCKS expression was increased in both Dysbindin knockdown cells and NF-YB knockdown cells, and Chromatin IP revealed interaction of these proteins at the MARCKS promoter region. Reporter assay results suggested functional involvement of the interaction between Dysbindin and NF-YB in MARCKS transcription levels, via the CCAAT motif which is a NF-YB binding sequence. MARCKS expression was increased in sdy mutant mice when compared to wild-type mice. Conclusions: These findings suggest that abnormal expression of MARCKS via dysfunction of Dysbindin might cause impairment of neural transmission and abnormal synaptogenesis. Our results should provide new insights into the mechanisms of neuronal development and the pathogenesis of schizophrenia.

Protein arginine N-methyltransferase (PRMT) 8 was first discovered from a database search for genes harboring four conserved methyltransferase motifs, which shares more than 80% homology to PRMT1 in amino acid [Lee J, Sayegh J, Daniel J, Clarke S, Bedford MT (2005) PRMT8, a new membrane-bound tissue-specific member of the protein arginine methyltransferase family. J Biol Chem 280:32890-32896]. Interestingly, its tissue distribution is strikingly restricted to mouse CNS. To characterize the function in the CNS neurons, we raised an antiserum against PRMT8 to perform immunohistochemistry (IHC) and Western blot analysis. By IHC, the immunoreactivity of endogenous PRMT8 was broadly distributed in the CNS neurons with markedly intense signals in the cerebellum, hippocampal formation, and cortex, but was not detected in the cerebellar granular layer. In some subset of the neurons, the immunoreactivity was observed in the dendrites and axon bundles. The subcellular localization of the immunoreactivity was dominantly nuclear, arguing against the original report that exogenously expressed PRMT8 localizes to the plasma membrane via the N-terminal myristoylation. A series of the exogenously expressed proteins with different in-frame translation initiation codons was tested for comparison with the endogenous protein in molecular size. The third initiator codon produced the protein that was equivalent in size to the endogenous and showed a similar localizing pattern in PC12 cells. In conclusion, PRMT8 is a neuron-specific nuclear enzyme and the N-terminus does not contain the glycine end for myristoylation target. (C) 2009 IBRO. Published by Elsevier Ltd. All rights reserved.

Neurite outgrowth is one of the Crucial events in the formation of neural circuits. The majority of studies on neurite outgrowth have focused on signal transduction processes based on phosphorylation and acetylation: a few studies have suggested the involvement of other molecular mechanisms. Recent progress in understanding the nature of protein arginine N-methyltransferases (PRMTs) raises the possibility of the involvement of protein methylation accompanied by cell shape changes during neuronal differentiation. Here, we show that PRMT1 play a pivotal role in the neurite outgrowth of Neuro2a cells. Our results revealed that PRMT1 depletion specifically affected neurite outgrowth but not the physiological processes involved in cell growth and differentiation. Furthermore, we demonstrated that Btg2, one of the PRMT1 binding partner, depletion clown-regulated arginine methylation in the nucleus and inhibited neurite outgrowth. These results indicate that protein arginine methylation by PRMT1 in the nucleus is an important step in neuritogenesis. (c) 2008 Elsevier Ireland Ltd. All rights reserved.

Progressive myoclonus epilepsy (PME) is a syndrome characterized by myoclonic seizures (lightning-like jerks), generalized convulsive seizures, and varying degrees of neurological decline, especially ataxia and dementia. Previously, we characterized three pedigrees of individuals with PME and ataxia, where either clinical features or linkage mapping excluded known PME loci. This report identifies a mutation in PRICKLE1 (also known as RILP for REST/NRSF interacting LIM domain protein) in all three of these pedigrees. The identified PRICKLE1 mutation blocks the PRICKLE1 and REST interaction in vitro and disrupts the normal function of PRICKLE1 in an in vivo zebrafish overexpression system. PRICKLE1 is expressed in brain regions implicated in epilepsy and ataxia in mice and humans, and, to our knowledge, is the first molecule in the noncanonical WNT signaling pathway to be directly implicated in human epilepsy.

The Drosophila planar cell polarity (PCP) gene prickle has been previously indicated as one of the regulators of gastrulation in the early embryonic stage. However, the functional role of prickle in the brain in particular is not known. We first indicated that mouse Prickle1 and Prickle2 are continually expressed in the brain throughout the embryonic stages and are observed to be specifically expressed in the postmitotic neurons. Furthermore, Prickle1 or Prickle2 depletion effectively decreases the neurite outgrowth levels of mouse neuroblastoma Neuro2a cells. These results indicate that mouse Prickle1 and Prickle2 possibly regulate positive neurite formation during brain development. (c) 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

The regional distribution of PRMT8 transcript was examined in mouse brain using in situ hybridization (ISH) histochemistry. The PRMT8 cRNA probe was specifically hybridized with CNS and the signals were observed only in the neurons. The distribution of the neurons expressing PRMT8 mRNA was not even throughout the brain. All of the regions related to general somatosensory system expressed PRMT8 mRNA strongly. Most of the relay nuclei intervening the special somatosensory system, such as the auditory, visual, and vestibular systems, were packed with PRMT8 mRNA expressing neurons. Forebrain limbic areas and thalamic nuclei relevant to limbic areas were also strongly labeled with the probe. Some areas related to the motor system, such as the caudate putamen, Purkinje cells, inferior olivary nucleus and cerebellar nuclei expressed PRMT8 mRNA strongly. These findings suggest that PRMT8 is chiefly involved in the somatosensory and limbic systems, and a part of motor system.

HuD is an RNA-binding protein that has been shown to induce neuronal differentiation by stabilizing labile mRNAs carrying AU-rich instability elements. Here, we show a novel mechanism of arginine methylation of HuD by coactivator-associated arginine methyltransferase 1 (CARM1) that affected mRNA turnover of p21(cip1/waf1) mRNA in PC12 cells. CARM1 specifically methylated HuD in vitro and in vivo and colocalized with HuD in the cytoplasm. Inhibition of HuD methylation by CARM1 knockdown elongated the p21(cip1/waf1) mRNA half-life and resulted in a slow growth rate and robust neuritogenesis in response to nerve growth factor (NGF). Methylation-resistant HuD bound more p21(cip1/waf1) mRNA than did the wild type, and its overexpression upregulated p21(cip1/waf1) protein expression. These results suggested that CARM1-methylated HuD maintains PC12 cells in the proliferative state by committing p21(cip1/waf1) mRNA to its decay system. Since the methylated population of HuD was reduced in NGF-treated PC12 cells, downregulation of HuD methylation is a possible pathway through which NGF induces differentiation of PC12 cells.

The distribution of protein arginine N-methyltransferase 3 (PRMT3) was investigated in the mouse brain using indirect immunofluorescence. PRMT3 was observed to be localized in the cell bodies and dendrites of neurons but not in the axons and glial cells, indicating that PRMT3 is involved in neuronal function. The distribution of the immunoreactive neurons in the brain was uneven, indicating that PRMT3 plays a role in specific neuronal systems such as the motor and limbic systems, as well as functions related to the cerebellum. The present ontogenetic analysis of PRMT1 and PRMT3 using Western blot methodology clearly revealed that PRMT3 develops during the perinatal stage and its expression is maintained even in adulthood. PRMT1, on the other hand, is expressed transiently during the early embryonic stage. These findings indicate that PRMT3 is related with neuronal function in both young and adult brains, while PRMT1 has roles in the immature brain, such as the formation of neural circuits. (c) 2006 IBRO. Published by Elsevier Ltd. All rights reserved.

Localprotein synthesis in neuronal dendrites is one of the mechanisms that may mediate a rapid and synapse-specific mobilization of proteins from the resident mRNAs. A great deal of effort has been made in analyzing the dynamic state of protein synthesis in the living cells chiefly by quantifying protein level. However, the protein level cannot mirror the spatiotemporal alteration of translation because it can be affected, not only by protein synthesis, but also by other factors, like degradation. Therefore, it is problematic to visualize the dynamic state of translation by the present methods'. To solve the problem, we applied fluorescence resonance energy transfer (FRET) technique to in situ detection of the assembly and disassembly cycle among a pair of translation initiation factors [eukaryotic initiation factors (elFs)], thereby showing that BDNF and ephrin could potentiate local protein synthesis in the dendrites of hippocampal neurons. (c) 2004 Elsevier B.V All rights reserved.

Recent papers have reported that neuronal death in patients with Alzheimer's disease, Parkinson's disease, and cerebral ischemia has its origin in the endoplasmic reticulum (ER). IRE lot is one of the ER stress transducers that detect the accumulation of unfolded proteins in the ER. IRE1alpha mediates two major cellular responses, which are the unfolded protein response (UPR), a defensive response, and apoptosis that leads to cell death. However, little is known about the regulatory mechanisms that select between the UPR and apoptosis. We identified Jun activation domain-binding protein-1 (JAB1) as a molecule that interacts with IRE1alpha using a yeast two-hybrid system. We demonstrated that JAB1 binds to IRE1alpha in the absence of stress, but that binding is decreased by ER stress inducers. Moreover, mutant JAB1 down-regulates the UPR signaling pathway through tight binding with IRE1alpha. These results suggested that JAB1 may act as a key molecule in selecting the UPR or cell death by association and dissociation with IRE I (X. (C) 2004 Elsevier Ltd. All rights reserved.

Oxidative stress is a major risk factor for Alzheimer's disease (AD) and other neurodegenerative disorders. Metals are known to be one of the factors that contribute to oxidative stress. Recently, we reported that the aberrant splicing isoform (PS2V) generated by skipping exon5 of the presenilin-2 (PS2) gene is a diagnostic feature of sporadic AD (SAD). PS2V is inducible by exposure of human neuroblastoma to hypoxia. We examined whether this aberrant splicing was caused by metal-induced oxidative stress, such as exposure to aluminum. As a result, we demonstrated that exposure to aluminum accelerated PS2V production induced by hypoxia. This acceleration of the production of PS2V to hypoxia was caused by chronic aluminum exposure, but was not related to the intracellular content of aluminum. HMGA1a is a mediator of PS2V production, and it was induced by aluminum as well as by hypoxia. Induction of HMGA1a was increased by chronic exposure to aluminum, and a nuclear extract containing HMGA1a bound to a specific sequence on exon5 of PS2 pre-mRNA, as reported previously. Finally, the acceleration of PS2V production induced by aluminum under hypoxic conditions reflected, but has not yet been directly shown to cause, vulnerability to endoplasmic reticulum stress. These results suggest that exposure to some metals can accelerate and enhance PS2V generation, and that hypoxia plus chronic exposure to metals may promote the development of AD.

The human ADP-ribosylation factor-like protein, ARF4L is a member of the ARF family, which are small GTP-binding proteins that play significant roles in vesicle transport and protein secretion. However, little is known about the physiological roles of ARF4L. In this study, to understand the biological functions of ARF4L, we carried out immunocytochemical analysis of ARF4L molecules with mutations in the functional domains. ARF4L was shown to be distributed to the plasma membrane following binding to GTP (Q80L), and into endosomes following binding to GDP (T35N). Moreover, the inactive-form of ARF4L ( T35N) causes localization of transferrin receptors to the endosomal compartment, while the active form ( Q80L) causes transport to the plasma membrane. These findings indicate that ARF4L drive the transport of cargo protein and subsequent fusion of recycling vesicles with the plasma membrane for maintenance of the cell surface.

Neurotrophins including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) are known to play important roles in the survival, proliferation, differentiation, and/or maintenance of function in several tissues including neuronal tissues. The role of neurotrophins in liver tissue, however, has not yet been clarified. In the present study, we assessed the temporal change in gene expression of neurotrophins, NGF, BDNF, and NT-3, and their receptors, low affinity neurotrophin receptor (p75NTR) and Trks A, B, and C, by RT-PCR technique in the liver of rats treated with lead nitrate (LN; 0.1 mmol/kg body weight), an inducer of liver hyperplasia. The mRNAs for NGF, BDNF with exon 4, NT-3, p75NTR, and all Trk members were detected in the LN-untreated liver. LNtreatment resulted in increases in the levels of NGF, BDNF with exon 4, NT-3, p75NTR, and TrkA mRNAs and further led to expression of BDNF mRNA with exon 3. The increase in gene expression of neurotrophins and their receptors was closely correlated with those in liver weight. In this report, we propose for the first time that neurotrophins may play crucial roles in LN-induced liver hyperplasia. (C) 2000 Academic Press.

We have previously reported that in cultured rat vascular smooth muscle cells (VSMCs), neurotrophin-3 (NT-3) gene expression was suppressed by TPA (12-O-tetradecanoyl phorbol-13-acetate), which induces an AP-1 transcription factor. In the present study, to clarify the mechanism for TPA-mediated downregulation of NT-3 gene expression, effects of cycloheximide and dexamethasone (Dex) on the TPA-mediated downregulation were examined in VSMCs. Pretreatment with cycloheximide, an inhibitor of protein synthesis, or simultaneous treatment with Dex, an inhibitor of AP-1, suppressed the TPA-mediated downregulation of NT-3 gene expression. Furthermore, co-transfection of c-fos and c-jun expression vectors into VSMCs resulted in decrease in the NT-3 gene expression. The present findings suggest that TPA-induced AP-1 de novo synthesis causes the downregulation of NT-3 gene expression in VSMCs. (C) 1999 Elsevier Science B.V. All rights reserved.

THE neurotrophin-3 (NT-3) gene has previously been reported to consist of three exons including two 5' short untranslated exons and a 3' long exon encoding the entire protein, and to give rise to two classes of transcripts by alternative splicing of the 5' exons to the 3' coding exon. In the present study, we demonstrated the presence of at least four new classes of transcripts of the NT-3 gene, in addition to the two known transcripts. The present finding proposes the further complexity of the regulational mechanism for NT-3 expression. NeuroReport 9: 3675-3679 (C) 1998 Lippincott Williams & Wilkins.

Most previous researches on neurotrophins including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) have focused on the nervous system, because their receptors are widely distributed in neuronal tissues. Recently, however, the participation of neurotrophins in inflammation and atherosclerosis has been proposed. Therefore, the gene expression of neurotrophins is now an urgent issue is to be investigated in nonneuronal tissues. Here, we evaluated the gene expression of neurotrophins and their receptors in rat cultured vascular smooth muscle cells (VSMCs) by the reverse transcriptase-polymerase chain reaction method, The transcripts of NGF, NT-3, and TrkC thigh-affinity receptor for NT-3), and two BDNF alternative spliced transcript variants with exons 3 and 4 were clearly detected in VSMCs cultured under conventional culture conditions. The upregulation of mRNA levels for NGF, two BDNF variants with exons 1 and 2, low-affinity neurotrophin receptor, and high-affinity receptors, TrkA (for NGF) and TrkB (for BDNF), was observed in response to the treatment with serum and phorbol-ester following the serum-starvation. In contrast, the expression of NT-3 and TrkC genes was downregulated under these conditions. Go-expression of these factors and their receptors and the characteristic regulation of their gene transcriptions suggest that these factors play crucial roles in the function of VSMCs through an autocrine mechanism. (C) 1998 Academic Press.