Yoshihiro Tsuruo

Catecholamines and estrogen are involved in the pathogenesis of emotional stress-induced acute heart attack.

Emotional stress triggers takotsubo cardiomyopathy in postmenopausal women. Clinical analysis of autonomic nervous function has revealed a transient increase of sympathetic nervous activity and decrease of vagal nervous activity. Immobilization (IMO) stress of rats can reproduce the electrocardiographic and left ventriculographic changes that occur in takotsubo cardiomyopathy, both of which are prevented by combined blockade of α‐ and β‐adrenoceptors. Estrogen supplementation partially attenuated these cardiac changes. It also attenuated the IMO‐induced increase of c‐Fos immunoreactivity, or c‐fos mRNA expression in the lateral septum, medial amygdaloid nucleus, paraventricular hypothalamic nucleus, dorsomedial hypothalamic nucleus, laterodorsal tegmental nucleus, and locus ceruleus; these regions contain central sympathetic neurons and neurons with immunoreactive estrogen receptors. It also downregulated c‐fos mRNA expression in the adrenal gland and the heart, suggesting an increase of estrogen attenuated the stress‐induced hypothalamo‐sympathoadrenal outflow from the central nervous system to the target organs. Estrogen treatment also upregulated the levels of cardioprotective substances, such as atrial natriuretic peptide and heat shock protein 70, in the heart. These data suggest that reduction of estrogen levels following menopause might be involved in the primary cause of takotsubo cardiomyopathy both by indirect action on the nervous system and by direct action on the heart.

Adaptive HNE-Nrf2-HO-1 pathway against oxidative stress is associated with acute gastric mucosal lesions

Disturbance of the microcirculation and generation of reactive oxygen species are crucial in producing acute gastric mucosal lesions (AGML). To understand the protective mechanism against mucosal injury and oxidative stress in the stomach, we investigated sequential expression and localization of a product of lipid peroxidation and a chemical mediator of the oxidative response array, 4-hydroxynonenal (HNE), transcriptional factor, NF-E2-related factor (Nrf2), and the inducible heme oxygenase (HO-1) in the injured stomach. AGML was produced by intragastric administration of 0.6 N HCl in male rats. Expression and localization of HNE, Nrf2, and HO-1 were investigated by Western blotting, immunohistochemistry, real-time RT-PCR, and in situ hybridization histochemistry. Mucosal lesions and expression of HNE and HO-1 were assessed by prior treatment with the PGI2 analog beraprast or after sensory denervation by pretreatment with capsaicin. Mucosal lesions were assessed by prior treatment with a HO-1 inhibitor, zinc protoporphyrin (ZnPP). After AGML, increased generation of HNE was observed in the injured mucosa and the surrounding submucosa, followed by nuclear translocation of Nrf2 and upregulation of HO-1 in the macrophages located in the margin of the injured mucosa and in the submucosa. Pretreatment with beraprost attenuated AGML and downregulated the expression of HNE and HO-1, while sensory denervation aggravated AGML and upregulated the expression of HNE and HO-1. Pretreatment with ZnPP also aggravated AGML. The sequential HNE-Nrf2-HO-1 pathway in the gastric mucosal cells and the macrophages is involved in an adaptive mechanism against oxidative stress after AGML.

Estrogen alters c-Fos response to immobilization stress in the brain of ovariectomized rats

Estrogen receptors are widely expressed in the brain, where estrogen modulates central nervous function. In this study, we investigated the effect of estrogen on the emotional stress response in the brain by comparing the CNS patterns of c-Fos expression in response to immobilization stress (IMO) in ovariectomized rats with placebo treatment (OVX + Pla) vs. ovariectomized rats supplemented with 17beta-estradiol (OVX + E2). Increased c-Fos immunoreactive neurons in response to IMO were observed in cerebral cortex, septum, thalamus, hypothalamus, midbrain, pons and medulla oblongata in accordance with previous findings. When OVX + E2/Stress were compared with OVX + Pla/Stress, the numbers of c-Fos immunoreactive cells were significantly lower in the lateral septum, paraventricular hypothalamic nucleus, dorsomedial hypothalamic nucleus, medial amygdaloid nucleus, lateral periaqueductal gray, laterodorsal tegmental nucleus and locus coeruleus, while they were significantly higher in paraventricular thalamic nucleus and nucleus of the solitary tract. These data suggest that neuronal activities in these areas are influenced bidirectionally by systemic estrogen level.

Immunocytochemical localization of lung-type prostaglandin F synthase in the rat spinal cord.

Prostaglandin F synthase, producing prostaglandin F(2 alpha) and 9 alpha,11 beta-prostaglandin F(2), has at least two isozymes, lung-type and liver-type ones. The present study including double immunolabelling with microtubule-associated protein 2 indicated that the lung-type isozyme was present in neuronal dendrites and somata of gray matter (relatively intense in lamina I and II in dorsal horn, and IX in ventral horn) and vascular endothelial cells in the rat spinal cord at all segmental levels.

Steroid 5α-reductase type 1 immunolocalized in the adrenal gland of normal, gonadectomized, and sex hormone-supplemented rats

Abstract Steroid 5α-reductase in the rat adrenal gland is supposed to play a role in the catabolism of adrenal steroids. We showed immunohistochemically the cellular and subcellular localization of 5α-reductase in the rat adrenal gland, using a polyclonal antibody against 5α-reductase rat type 1. In the adrenal cortex, positive immunoreaction was found in the cells of the zonae fasciculata and reticularis but was absent in those of the zona glomerulosa. The positive staining was restricted to the cytoplasm but not to the nucleus, Golgi complexes or mitochondria. The staining intensity showed a marked change depending on the steroidal milieu. Gonadectomy for 6 weeks increased the immunoreaction, regardless of the sex. Testosterone replacement for the last 2 weeks considerably reduced the immunoreaction in 6-week-castrated males, and estradiol supplement for the last 2 weeks also resulted in the marked reduction of immunostaining in 6-week-gonadectomized females. In the adrenal medulla, the immunoreaction was localized in the supporting cells and the Schwann cells but not in the chromaffin cells. In these cells, the immunoreaction was not affected by steroidal treatments. These findings suggest that the expression of 5α-reductase in the rat adrenal cortex is regulated by sex hormones from the gonads, and the enzyme may participate in the conversion of adrenal steroids depending on the steroidal environment, although the functional significance of the enzyme in the adrenal medulla remains unclarified.

Nrf2-inducing anti-oxidation stress response in the rat liver--new beneficial effect of lansoprazole.

Lansoprazole is a potent anti-gastric ulcer drug that inhibits gastric proton pump activity. We identified a novel function for lansoprazole, as an inducer of anti-oxidative stress responses in the liver. Gastric administration of lansoprazole (10–100 mg/kg) to male Wistar rats produced a dose-dependent increase in hepatic mRNA levels of nuclear factor, erythroid-derived 2, -like 2 (Nrf2), a redox-sensitive transcription factor, at 3 h and Nrf2 immunoreactivity (IR) in whole hepatic lysates at 6 h. Conversely, the levels of Kelch-like ECH-associated protein (Keap1), which sequesters Nrf2 in the cytoplasm under un-stimulated conditions, were unchanged. Translocation of Nrf2 into the nuclei of hepatocytes was observed using western blotting and immunohistochemistry. Expression of mRNAs for Nrf2-dependent antioxidant and phase II enzymes, such as heme oxygenase 1 (HO-1), NAD (P) H dehydrogenase, quinone 1 (Nqo1), glutathione S-transferase A2 (Gsta2), UDP glucuronosyltransferase 1 family polypeptide A6 (Ugt1a6), were dose-dependently up-regulated at 3 h. Furthermore, the levels of HO-1 IR were dose-dependently increased in hepatocytes at 6 h. Subcutaneous administration of lansoprazole (30 mg/kg/day) for 7 successive days resulted in up-regulation and nuclear translocation of Nrf2 IR in hepatocytes and up-regulation of HO-1 IR in the liver. Pretreatment with lansoprazole attenuated thioacetamide (500 mg/kg)-induced acute hepatic damage via both HO-1-dependent and -independent pathways. Up-stream networks related to Nrf2 expression were investigated using microarray analysis, followed by data mining with Ingenuity Pathway Analysis. Up-regulation of the aryl hydrocarbon receptor (AhR)-cytochrome P450, family 1, subfamily a, polypeptide 1 (Cyp1a1) pathway was associated with up-regulation of Nrf2 mRNA. In conclusion, lansoprazole might have an alternative indication in the prevention and treatment of oxidative hepatic damage through the induction of both phase I and phase II drug-metabolizing systems, i.e. the AhR/Cyp1a1/Nrf2 pathway in hepatocytes.

Adrenergic activation of steroid 5α-reductase gene expression in rat C6 glioma cells

Steroid 5α-reductase (5α-R) is well known as the enzyme converting progesterone and other steroid hormones to their 5α-reduced metabolites and has been reported to be localized in both neuronal and glial cells in the brain. Previously, the enzyme activity in glial cells has been shown to be enhanced either by coculturing with neuronal cells or by adding the conditioned medium of neuronal cells, suggesting a possible implication of neuro-glial interactions in the regulation of neurosteroid metabolism in the brain. In the present studies, the effects of adrenergic agonists on 5α-R mRNA and protein levels in rat C6 glioma cells were examined as one of the model experiments for investigating the influence of neuronal activity on the expression of 5α-R gene in the glial cell. The direct challenge of β-adrenergic agonists to glioma cells resulted in the rapid and transient elevation of 5α-R mRNA levels through the activation of the cyclic AMP (cAMP)/protein kinase A-mediated signaling pathway. Further studies showed that cAMP-induced 5α-R mRNA expression was completely abolished by pretreatment of cells with actinomycin D and also indicated that the elevation of 5α-R mRNA levels was accompanied by an increase in enzyme protein in the cells. These findings provide strong evidence that the stimulation of β-adrenergic receptors might induce the transcriptional activation of 5α-R gene expression in glial cells, proposing the possibility that neuronal activity might be involved in the production of neuroactive 5α-reduced steroids in the brain.

Interleukin-18 deficiency reduces neuropeptide gene expressions in the mouse amygdala related with behavioral change.

In this study, we examined the effects of IL-18 deficiency on behaviors and gene expression profiles in 6 brain regions. IL-18(-/-) mice reduced depressive-like behavior and changed gene expressions predominantly in the amygdala compared with wild-type mice. Pathway analysis of the differentially expressed genes ranked behavior as the top-scored biological function. Of note, the absence of IL-18 decreased Avp, Hcrt, Oxt, and Pmch mRNA levels and the number of arginine vasopressin- and oxytocin-positive cells in the amygdala, but not in the hypothalamus. Our results suggest that IL-18-dependent vasopressinergic and oxytocinergic circuitry in the amygdala may regulate depressive-like behaviors in mice.

A DEAD-box RNA helicase Ddx54 protein in oligodendrocytes is indispensable for myelination in the central nervous system.

We recently reported that a new monoclonal antibody, 4F2, which labels oligodendroglial lineage cells, recognizes a DEAD‐box RNA helicase Ddx54 and that Ddx54 binds to myelin basic protein (MBP) in brain and cultured oligodendrocytes. To elucidate the biological function of Ddx54, we generated a recombinant adenovirus, Ad‐shRNA:Ddx54, expressing a short hairpin RNA to silence endogenous Ddx54 protein. The virus was intraventricularly injected into the brains of mice on postnatal day (PD) 2. The brains at PD 9 were then analyzed by immunohistochemistry. In untreated normal brain sections, as well as control brains that had been injected with Ad‐β‐Gal, myelination of axons occurred in the corpus callosum with filamentous patterns of immunosignals of myelin‐associated glycoprotein (MAG) and MBP. In Ad‐shRNA:Ddx54‐injected brain, substantial amounts of MAG and MBP immunosignals were present, but MBP immunosignals accumulated in the subplate layer and did not intrude into the emerging white matter. Immunoblot analysis revealed that Ddx54 knockdown caused a significant decrease in the level of 21.5 kDa MBP isoform and Ddx54, but the amount of Olig2; 2′,3′‐cyclic nucleotide 3′ phosphodiesterase; MAG; three MBP isoforms (14, 17.5, and 18 kDa); and QKI‐5, QKI‐6, and QKI‐7 proteins remained unchanged. Transfection of the Ddx54 expression vector into luciferase reporter‐introduced neuroepithelial cells resulted in upregulated MBP promoter activity. Immunoprecipitation of Ddx54 protein in MBP‐transfected HEK293 cells indicated that Ddx54 may directly interact with MBP mRNA. These results suggest that Ddx54 protein play an important role in central nervous system myelination, presumably in myelin sheath formation after the differentiation of oligodendrocytes.

A new monoclonal antibody, 4F2, specific for the oligodendroglial cell lineage, recognizes ATP‐dependent RNA helicase Ddx54: Possible association with myelin basic protein

Recent research in neural development has highlighted the importance of markers to discriminate phenotypic alterations of neural cells at various developmental stages. We isolated a new monoclonal antibody, 4F2, which was shown to be specific for an oligodendrocyte lineage. In primary cultures of oligodendroglial and mixed neural cells, the 4F2 antibody labeled a large proportion of Sox2+, Sox10+, A2B5+, NG2+, Olig2+, O4+, and myelin basic protein (MBP)+ cells but did not label any GFAP+ or NeuN+ cells. In immunohistochemisty of rat embryos, the 4F2 antibody labeled a portion of neuroepithelial cells of the neural tube at embryonic day 9. The 4F2‐positive cells were located initially in the ventricular zone as Musashi1+ Tuj1− populations and distributed throughout the striatum; thereafter, they populated the whole brain and spinal cord. These cells showed ramified processes during embryonal development. The 4F2 antigen was associated with all four isoforms of MBP in coimmunoprecipitation experiments using brain homogenates or cell lysates of cultured oligodendrocytes. Immunoscreening of a brain cDNA library identified the antigen as DEAD (Asp‐Glu‐Ala‐Asp) box polypeptide 54 (Ddx54), a member of the DEAD box family of RNA helicases involved in RNA metabolism, transcription, and translation. Cotransfection of the Ddx54 gene with MBP isoform genes increased the nuclear localization of the 21.5‐kDa MBP isoform, which has been reported to function as a nuclear signal transduction molecule. These data indicate that Ddx54 might be not only a useful marker for investigating the ontogeny of oligodendrocytes but also an important factor in oligodendrocyte differentiation and myelination. Journal of Neuroscience Research (2011)