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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.

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.

Sterol regulatory element-binding proteins (SREBPs) are transcription factors that regulate a wide variety of genes involved in fatty acid and cholesterol synthesis. In the present study, we identified that isoxanthohumol (IXN) suppressed SREBP activity. Low concentrations of IXN (10 and 30 μM) reduced the amount of mature forms of SREBPs, while high concentration of IXN (100 μM) reduced both precursor and mature forms of SREBPs in Huh-7 cells. The IXN-mediated decrease in the precursor forms of SREBPs in Huh-7 cells was completely abolished by culturing cells under sterol-supplemented conditions and was partly abolished by treatment with a proteasome inhibitor, MG132, but not a lysosome inhibitor, NH4Cl. Moreover, IXN accelerated the ubiquitination of the precursor forms of SREBP-1a. These results suggest that IXN suppresses SREBP activity, at least in part, via ubiquitin-proteasome-dependent degradation of the precursor forms of SREBPs. Abbreviations: ACC1: acetyl-CoA carboxylase 1; DMEM: Dulbecco’s modified Eagle’s medium; ER: endoplasmic reticulum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; 25-HC: 25-hydroxycholesterol; HMGCR: HMG-CoA reductase; HMGCS: HMG-CoA synthase; Insig: insulin-induced gene; IXN: isoxanthohumol; LPDS: lipoprotein-deficient serum; SCAP: SREBP cleavage-activating protein; SCD1: stearoyl-CoA desaturase; SREBPs: sterol regulatory element-binding proteins; XN: xanthohumol

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.

[Purpose] The purpose of this study was to examine the effects of task-specific plantar flexor training on walking ability indices in a patient with a paretic ankle. [Subject and Methods] The subject was a 65-year-old male patient with right hemiplegia due to a left medullary ventral infarction. An ABA' single-subject design was adopted. The independent variable was a task-specific plantar flexor training exercise, similar to that during walking, targeting the paretic ankle. The dependent variables were the isometric ankle plantar flexor strength, maximum walking speed, step length, and trailing limb angle in the paretic terminal stance phase. The B study phase was divided into B1 and B2 phases. A two standard-deviation-band method was used to evaluate improvement. [Results] Improvements in the paretic plantar flexor strength, maximum walking speed, step length, and trailing limb angle in the B2 phase were observed. The improvements in the maximum walking speed, step length, and trailing limb angle were sustained in the A' study phase. [Conclusion] These results suggest that task-specific plantar flexor training exercise is efficacious in improving the walking ability index of a paretic ankle.

Myelination and remyelination in the central nervous system (CNS) are essential for rapid conduction of action potentials and for appropriate neuronal communications supporting higher brain functions. Myelination is dependent on developmental stage and is controlled by neuronal axons-oligodendrocyte (OL) signaling. Numerous studies of the initial myelination and remyelination stages in the CNS have demonstrated several key cytoskeletal signals in axons and OLs. In this review, we focus on cytoskeletal signal-regulated OL myelination and remyelination, with particular attention to neuronal Notch proteins, bidirectional Eph/ephrin signaling, OL integrin and cadherin superfamily proteins, OL actin rearrangement, and OL tyrosine kinase Fyn substrate proteins during the initial myelination and remyelination stages in the CNS.

Sterol regulatory element-binding proteins (SREBPs) are a family of transcription factors that regulate lipid homeostasis by controlling the expression of genes involved in fatty acid and cholesterol synthesis. In this study, we used a stable cell line that expresses a luciferase reporter gene driven by an SRE-containing fatty acid synthase promoter to identify allyl isothiocyanate (AITC), one of the major isothiocyanates in cruciferous vegetables, as a novel SREBP inactivator. We found that AITC downregulated the proteolytic processing of SREBPs and the expression of their target genes in human hepatoma Huh-7 cells. Furthermore, AITC reduced the de novo synthesis of both fatty acids and cholesterol. Our results indicate a novel physiological function of AITC in lipid metabolism regulation.

Myelination and remyelination in the central nervous system (CNS) are essential for rapid conduction of action potentials and for appropriate neuronal communications supporting higher brain functions. Myelination is dependent on developmental stage and is controlled by neuronal axons–oligodendrocyte (OL) signaling. Numerous studies of the initial myelination and remyelination stages in the CNS have demonstrated several key cytoskeletal signals in axons and OLs. In this review, we focus on cytoskeletal signal-regulated OL myelination and remyelination, with particular attention to neuronal Notch proteins, bidirectional Eph/ephrin signaling, OL integrin and cadherin superfamily proteins, OL actin rearrangement, and OL tyrosine kinase Fyn substrate proteins during the initial myelination and remyelination stages in the CNS.

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.

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 ζ-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.

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.

A high level of plasma low-density lipoprotein (LDL) cholesterol is considered a risk factor for atherosclerosis. Because the hepatic LDL receptor (LDLR) is essential for clearing plasma LDL cholesterol, activation of LDLR is a promising therapeutic target for patients with atherosclerotic disease. Here we demonstrated how the flavonoid kaempferol stimulated the gene expression and activity of LDLR in HepG2 cells. The kaempferol-mediated stimulation of LDLR gene expression was completely inhibited by knockdown of Sp1 gene expression. Treatment of HepG2 cells with kaempferol stimulated the recruitment of Sp1 to the promoter region of the LDLR gene, as well as the phosphorylation of Sp1 on Thr-453 and Thr-739. Moreover, these kaempferol-mediated processes were inhibited in the presence of U0126, an ERK pathway inhibitor. These results suggest that kaempferol may increase the activity of Sp1 through stimulation of Sp1 phosphorylation by ERK1/2 and subsequent induction of LDLR expression and activity.

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.

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.

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.

〔目的〕求心性収縮（COC）と遠心性収縮（ECC）時の収縮特性を筋酸素動態の変化から検討した。〔対象〕健常男性16名を対象とした。〔方法〕等速性運動機器を使用しCOC，ECCでの膝関節屈伸運動を行い，同時に近赤外線分光装置にて外側広筋の筋酸素動態の変化を記録した。得られたデータより，運動中の酸素飽和度（SdO2）最下点までの低下量，SdO2最下点までの到達時間，SdO2の回復時間を算出し，COC，ECC間で検討した。〔結果〕筋力はECCの方が高値であった。筋酸素動態では低下量は有意差を認めず，最下点到達時間はECCの方が遅く，回復時間は短かった。〔結語〕ECCではCOCよりも総仕事量が多いにもかかわらず酸素飽和度の低下は遅く，回復が早いという結果となった。これはECCでは機械的効率が高く，また筋血流量は比較的維持されていたためであると思われた。

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 down-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.

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.

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.

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.

μ + spin relaxation measurements on antiferromangetic CeB 2 C 2 , with T N1 = 7.1 K and T N2 = 6.4 K, were performed under zero-field and longitudinal fields (LF). The values of the nuclear dipolar width and the relaxation rate associated with Ce 4f localized electronic moments are estimated. The muon spin polarization at t = 0 decreases sharply at T N1 , indicating magnetic long-range order. The LF dependence of the time spectrum at T = 3.1 K ( T N2 ) is different. At T = 3.1 K, the observed spectra depend strongly on the LF and a decoupling process is observed, while that at T = 6.8 K they do not change their shape up to the maximum field applied in this work. It is suggested that the local fields in the intermediate phase fluctuate strongly.

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.

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.

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.