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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.

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.

Takashi Tanaka, Shoko Shimizu, Masaki Ueno, Yoshitaka Fujihara, Masahito Ikawa, Shingo Miyata, MARCKSL1 Regulates Spine Formation in the Amygdala and Controls the Hypothalamic-Pituitary-Adrenal Axis and Anxiety-Like Behaviors, EBioMedicine, Volume 30, 2018, Pages 62-73, ISSN 2352-3964, https://doi.org/10.1016/j.ebiom.2018.03.018., 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.

Sterol regulatory element-binding proteins (SREBPs) are key transcription factors that stimulate the expression of genes involved in fatty acid and cholesterol biosynthesis. Here, we demonstrate that a prenylated flavonoid in hops, xanthohumol (XN), is a novel SREBP inactivator that reduces the de novo synthesis of fatty acid and cholesterol. XN independently suppressed the maturation of SREBPs of insulin-induced genes in a manner different from sterols. Our results suggest that XN impairs the endoplasmic reticulum-to-Golgi translocation of the SREBP cleavage-activating protein (SCAP)-SREBP complex by binding to Sec23/24 and blocking SCAP/SREBP incorporation into common coated protein II vesicles. Furthermore, in diet-induced obese mice, dietary XN suppressed SREBP-1 target gene expression in the liver accompanied by a reduction of the mature form of hepatic SREBP-1, and it inhibited the development of obesity and hepatic steatosis. Altogether, our data suggest that XN attenuates the function of SREBP-1 by repressing its maturation and that it has the potential of becoming a nutraceutical food or pharmacological agent for improving metabolic syndrome.

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.

Neurotrophin receptor p75 (p75NTR) knockout mice (p75(−/−) mice) provide a good animal model of progressive-onset hearing loss.1 Cell loss of the spiral ganglion neurons (SGNs) and hair cell degeneration at the basal turn of the cochlea are seen in p75(−/−) mice from 3 months of age. Furthermore, from 3 to 6 months of age, the hearing thresholds of p75(−/−) mice are gradually raised; and after 6 months of age, the mice mostly exhibit hearing loss.1 Several previous reports have indicated that electroacupuncture (EA) stimulation may improve subjective symptoms of tinnitus or hearing loss.2 ,3 However, the molecular mechanisms underlying the therapeutic effect of EA stimulation for hearing loss remain unclear. Thus, to determine whether EA stimulation is useful for the prevention of hearing loss, we used a p75(−/−) mouse model of progressive-onset hearing loss. The Ting Gong (SI19; Small Intestine 19) and the Yifeng (TE17; Triple Energiser meridian 17) are …

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 αCaMKII; 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.

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.

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.

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. TheKampomedicine 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.

Hu proteins have been identified as target antigens in the sera of patients with paraneoplastic encephalomyelitis, an autoimmune disease associated with small-cell lung cancer and neuroblastoma (20, 64). The four members of the Hu protein family have been identified as RNA-binding proteins (RBPs) that show homology to the Drosophila melanogaster ELAV protein (27, 28, 42, 52, 56). These mammalian Hu/ELAV proteins, with the exception of HuR, are expressed exclusively in neurons (20, 45, 52). Hu family proteins share the characteristic of three RNA recognition motif domains (RRMs), with a hinge region intervening between the second and the third RRMs (27, 28, 56). In previous reports, Hu proteins had been reported to bind to long poly(A)+ tails (1, 46), but recent studies demonstrated that they recognize AU-rich elements (AREs) which reside in the 3′-untranslated regions (3′-UTRs) of some labile mRNA species (33, 40, 42, 53, 54) and determine their stability or translational efficiency (6, 7, 23, 29, 37). HuD, one of the Hu family proteins, has been shown to bind to AREs found in the 3′-UTRs of several mRNAs, including c-fos (14), tau (9), GAP43 (15), and p21cip1/waf1 (30), and to the U-rich element found in the p27 mRNA 5′-UTR (36). Previously, it was reported that overexpression of HuD induces neuronal differentiation in PC12 cells, cortical primary culture neurons, and retinoic acid-induced teratocarcinoma cell lines (5). On the other hand, antisense-mediated knockdown of HuD resulted in the inhibition of neurite extension in PC12 cells (48) and HuD-deficient mice exhibited a larger population of dividing stem cells in the adult subventricular zone (3). These findings indicated that HuD is required for neuronal differentiation processes, including growth arrest and cell fate acquisition of neural stem/progenitor cells, and possibly for sprouting and regeneration of mature neurons. Given that HuD-bound gene products are involved in cell cycle arrest (p21cip1/waf1 and p27), neurite outgrowth (GAP43 and tau), and functional differentiation (choline acetyltransferase) (21), HuD is presumed to induce the neuronal cell shape by exerting a protective effect on these ARE-containing labile mRNAs by antagonizing ARE-mediated mRNA decay. In the case of nerve growth factor (NGF)-induced differentiation of PC12 cells, NGF alters the RNA binding property of HuD towards AREs in the course of differentiation. However, there is no evidence revealing how HuD-ARE interactions are regulated under the NGF signal transduction pathway. We note that HuR, a ubiquitously distributed Hu protein, was arginine methylated by coactivator-associated arginine methyltransferase 1 (CARM1) in the myeloid cell line when the cells were stimulated by lipopolysaccharide (41). However, functional differences between methylated and unmethylated HuR have not yet been elucidated. Since the four mammalian Hu proteins (HuR, HuB, HuC, and HuD) are quite akin to each other in amino acid sequence (27, 52), we explored the possibility that HuD is also methylated at the corresponding arginine residue to HuR. RBPs are the major substrate group for protein arginine methyltransferases (PRMTs) (8, 26, 34, 37, 43, 47, 49, 50, 57, 60, 61, 67). Most RBPs contain GAR domains, which consist of a repetition of RGG or RXR (X is an aliphatic residue) (11, 58) and are the canonical targets for type I PRMTs that catalyze the formation of asymmetric NG,NG-dimethylarginine residues (37, 49). Type I enzymes PRMT1 and PRMT3 favor GAR domains as their substrates (18, 25, 66), and especially PRMT1 has a promiscuity to methylate arginine residues encompassed by GAR domains (65, 66). On the other hand, another type I enzyme, CARM1, methylates a narrow spectrum of proteins, histone H3 (12, 44), p300/CBP (69), PABP1, and TARPP (39), all of which lack GAR-like domains around the arginine residues. HuR also lacks the canonical GAR domain but instead has an alanine residue 2 residues N-terminal before the methylated arginine, which is common to most of the CARM1 substrates (41). In this report, we first demonstrated that HuD is also an in vivo and in vitro substrate for CARM1 by 3H labeling and immunodetection of the methylarginine residue of which Arg236 is mapped as the methylated residue by CARM1. Though CARM1 was so far reported to reside predominantly in the cell nuclei, in PC12 cells CARM1 distribution ranges from the nuclei to the cytoplasm, including the cell peripheries, and CARM1 is colocalized with HuD in the cytoplasm. To examine the biological significance of HuD methylation by CARM1, we established CARM1-depleted PC12 cell lines and investigated the effect of CARM1 loss on HuD-regulated gene expression. In a series of CARM1-depleted cell lines, methylated HuD was completely lost, with the total HuD level being unchanged, and the p21cip1/waf1 protein levels was remarkably increased compared with levels in parental PC12 cells. Further, we demonstrated that unmethylated HuD bound more p21cip1/waf1 mRNA than did methylated HuD and led to prolongation of p21cip1/waf1 mRNA half-life. This phenomenon was reproduced in the PC12 cells overexpressing R236K methylation-resistant HuD. p21cip1/waf1 cyclin-dependent kinase inhibitor has been shown to inhibit the proliferation of PC12 cells and accelerate neurite outgrowth in response to NGF (22, 71, 72). As anticipated, these cells exhibited a slower growth rate in the growth medium and accelerated neuritogenesis in response to NGF than did the parental and mock-transfected PC12 cells. These findings indicated that CARM1 negatively regulates neuronal differentiation of PC12 cells by methylating HuD to prevent p21cip1/waf1 mRNA from entering into the decay pathway. The overlapped distribution of CARM1 with BrdU-positive cells in the subventricular zone of the adult mouse generalizes the inhibitory role of CARM1 for the differentiation of neural progenitor/precursor cells as well as 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.

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.

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.

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.

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 β1-integrin, respectively. N-Cadherin and β1-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 β1-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.

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.

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.

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, α-catenin, and β-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 abnornalities of oligodendrocytes are possibly related to depression-like symptoms.

BackgroundRecently, 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.MethodsWe 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.ResultsYokukansan 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.ConclusionsOur 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.

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 fibroblast migration 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.

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.

Local protein 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 (eIFs)], thereby showing that BDNF and ephrin could potentiate local protein synthesis in the dendrites of hippocampal neurons.

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.

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 (high-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. Co-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.

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 μM). 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.