Yutaka Yamamuro

Choline availability modulates human neuroblastoma cell proliferation and alters the methylation of the promoter region of the cyclin-dependent kinase inhibitor 3 gene

Choline is an important methyl donor and a component of membrane phospholipids. In this study, we tested the hypothesis that choline availability can modulate cell proliferation and the methylation of genes that regulate cell cycling. In several other model systems, hypomethylation of cytosine bases that are followed by a guanosine (CpG) sites in the promoter region of a gene is associated with increased gene expression. We found that in choline‐deficient IMR‐32 neuroblastoma cells, the promoter of the cyclin‐dependent kinase inhibitor 3 gene (CDKN3) was hypomethylated. This change was associated with increased expression of CDKN3 and increased levels of its gene product, kinase‐associated phosphatase (KAP), which inhibits the G1/S transition of the cell cycle by dephosphorylating cyclin‐dependent kinases. Choline deficiency also reduced global DNA methylation. The percentage of cells that accumulated bromodeoxyuridine (proportional to cell proliferation) was 1.8 times lower in the choline‐deficient cells than in the control cells. Phosphorylated retinoblastoma (p110) levels were 3 times lower in the choline‐deficient cells than in control cells. These findings suggest that the mechanism whereby choline deficiency inhibits cell proliferation involves hypomethylation of key genes regulating cell cycling. This may be a mechanism for our previously reported observation that stem cell proliferation in hippocampus neuroepithelium is decreased in choline‐deficient rat and mouse fetuses.

Rabies Virus-Induced Activation of Mitogen-Activated Protein Kinase and NF-κB Signaling Pathways Regulates Expression of CXC and CC Chemokine Ligands in Microglia

ABSTRACT Following virus infection of the central nervous system, microglia, the ontogenetic and functional equivalents of macrophages in somatic tissues, act as sources of chemokines, thereby recruiting peripheral leukocytes into the brain parenchyma. In the present study, we have systemically examined the growth characteristics of rabies virus (RV) in microglia and the activation of cellular signaling pathways leading to chemokine expression upon RV infection. In RV-inoculated microglia, the synthesis of the viral genome and the production of virus progenies were significantly impaired, while the expression of viral proteins was observed. Transcriptional analyses of the expression profiles of chemokine genes revealed that RV infection, but not exposure to inactivated virions, strongly induces the expression of CXC chemokine ligand 10 (CXCL10) and CC chemokine ligand 5 (CCL5) in microglia. RV infection triggered the activation of signaling pathways mediated by mitogen-activated protein kinases, including p38, extracellular signal-regulated kinases 1 and 2 (ERK1/2), and c-Jun N-terminal kinase, and nuclear factor κB (NF-κB). RV-induced expression of CXCL10 and CCL5 was achieved by the activation of p38 and NF-κB pathways. In contrast, the activation of ERK1/2 was found to down-regulate CCL5 expression in RV-infected microglia, despite the fact that it was involved in partial induction of CXCL10 expression. Furthermore, NF-κB signaling upon RV infection was augmented via a p38-mediated mechanism. Taken together, these results indicate that the strong induction of CXCL10 and CCL5 expression in microglia is precisely regulated by the activation of multiple signaling pathways through the recognition of RV infection.

Downregulation of estrogen receptor gene expression by exogenous 17β-estradiol in the mammary glands of lactating mice

The biological actions of estrogen are mostly conveyed through interaction with the nuclear estrogen receptor (ER). Previous evidence indicated that estrogen participates in self-regulation through the modulation of the expression of its own receptors. However, the self-regulation of estrogen against ER in the mammary gland during established lactation has not yet been investigated. The present study evaluated ER gene expression in the lactating gland activated by large doses of 17β-estradiol (E2). Repeated E2 treatments dose-dependently decreased the gene, expression of ER, especially its subtype ER-α mRNA, Which was decreased to 10% of the vehicle-injected control by 1 μg E2 injection, whereas it was decreased by 73% for another subtype, ER-β. A single injection of 5 μg of E2 drastically downregulated both ER genes within 12 hrs of injection, and they did not recover to pretreatment level within 48 hrs. Western blot analysis verified that E2 treatment inhibited the phosphorylation of Stat5, which is a potent transcriptional regulator for ER mRNA. The present findings demonstrate that E2 treatment decreases the gene expression of its own receptor in the mammary gland during galactopoesis and induces an apparent transition of the ER profile in the mammary gland during lactation into postlactation.

Differences in Electrophysiological Properties of Angiotensinergic Pathways from the Subfornical Organ to the Median Preoptic Nucleus between Normotensive Wistar-Kyoto and Spontaneously Hypertensive Rats

Electrophysiological properties of angiotensinergic pathways from the subfornical organ (SFO) to the median preoptic nucleus (MnPO) were investigated in normotensive Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) under urethane anesthesia. The activity of SFO neurons that were antidromically activated by electrical stimulation of the MnPO was compared between WKY (n = 28) and SHR (n = 27). No significant differences were observed between WKY and SHR in the latency, conduction velocity, or threshold of antidromic activation. The firing rate was significantly shorter in SHR. The activity of MnPO neurons was tested for a response to microiontophoretic application of angiotensin II (ANG II) and electrical stimulation of the SFO. Sixteen of 46 MnPO neurons tested in WKY and 15 of 47 MnPO neurons tested in SHR were excited by both ANG II applied iontophoretically and SFO stimulation, and the excitatory responses were prevented by iontophoretically applied saralasin, a specific ANG II antagonist. In these MnPO neurons that demonstrated the excitation to both SFO stimulation and ANG II, the firing rate was significantly higher and the threshold current required to evoke the SFO stimulus-induced excitation was significantly lower in SHR. The sensitivity to SFO stimulation was much greater in SHR than in WKY. These results provide evidence that there are marked alterations in the physiological properties of the angiotensinergic circuit from the SFO to the MnPO between WKY and SHR and imply that a disorder in the neural circuit may contribute, in part, to hypertension.

Double-stranded RNA stimulates chemokine expression in microglia through vacuolar pH-dependent activation of intracellular signaling pathways

During neurotropic virus infection, microglia act as a source of chemokines, thereby regulating the recruitment of peripheral leukocytes and the multicellular immune response within the CNS. Herein, we present a comprehensive study on the chemokine production by microglia in response to double‐stranded RNA (dsRNA), a conserved molecular pattern of virus infection. Transcriptional analyses of chemokine genes revealed that dsRNA strongly induces the expression of CXC chemokine ligand 10 (CXCL10) and CC chemokine ligand 5 (CCL5) in microglia. We also observed that the dsRNA stimulation triggered the activation of signaling pathways mediated by nuclear factor κB (NF‐κB) and mitogen‐activated protein kinases (MAPK), including extracellular signal‐regulated kinases 1 and 2 (ERK1/2), p38, and c‐Jun N‐terminal kinase (JNK). The microglial CXCL10 response to dsRNA was induced via NF‐κB, p38, and JNK pathways, whereas the dsRNA‐induced CCL5 production was dependent on JNK, but not on the other signal‐transducing molecules tested. In addition, the acidic environment of intracellular vesicles was required for the activation of cellular signaling in response to dsRNA. Taken together, these results suggest that the recognition of dsRNA structure selectively induces the CXCL10 and CCL5 responses in microglia through vacuolar pH‐dependent activation of NF‐κB and MAPK signaling pathways.

Responses of subfornical organ neurons projecting to the hyphotalamic paraventricular nucleus to hemorrhage

The activity of subfornical organ (SFO) neurons that were antidromically identified by electrical stimulation of the rat hypothalamic paraventricular nucleus (PVN) was tested for a response to microiontophoretic application of angiotensin II (ANG II) or hemorrhage (10 ml/kg b.w.t.). Microiontophoretically (MIPh) applied ANG II caused an increased excitability in 24 out of 28 neurons tested and the excitation was blocked by MIPh-applied saralasin (Sar), a specific ANG II antagonist. Of these neurons that responded to ANG II, 14 displayed an increase in neuronal firing in response to hemorrhage, while 10 were unresponsive. The excitatory response to hemorrhage in 5 out of 14 neurons tested was prevented by MIPh-applied Sar, whereas the response of the remaining neurons was not affected. These results show that part of SFO neurons projecting to the PVN may receive neural inputs from the peripheral baroreceptors, and suggest that the inputs may be partially attributable to the involvement of central angiotensinergic circuits.

Involvement of histone acetylation in the regulation of choline acetyltransferase gene in NG108-15 neuronal cells.

Post-translational modification of histone such as acetylation of N-terminal of lysine residues influences gene expression by modulating the accessibility of specific transcription factors to the promoter region, and is essential for a wide variety of cellular processes in the development of individual tissues, including the brain. However, few details concerning the acquisition of specific neurotransmitter phenotype have been obtained. In the present study, we investigated the possible involvement of histone acetylation in the gene expression of choline acetyltransferase (ChAT), a specific marker for cholinergic neuron and its function, in NG108-15 neuronal cells as an in vitro model of cholinergic neuron. Treatment with the histone deacetylase (HDAC) inhibitor trichostatin A (TSA), which induces global histone hyper-acetylation of the cells, resulted in marked increase in the expression of ChAT gene in proliferating NG108-15 cells. Furthermore, RT-PCR analysis using primer pairs for individual variants of ChAT mRNA (R1-4, N1, and M type) revealed that M type, not R1-4 and N1 type, ChAT mRNA were mainly transcribed, and chromatin immunoprecipitation assay indicated that the promoter region of M type ChAT gene was highly acetylated, in the dibutyryl cyclic AMP-induced neuronal differentiation of NG108-15 cells. The present findings demonstrate that the acquisition of neurotransmitter phenotype is epigenetically, at least the hyper-acetylation on the core promoter region of ChAT gene, regulated in NG108-15 neuronal cells.

Acetylcholine in the hippocampus during the discrimination learning performance in a rat model of chronic cerebral ischaemia

Acetylcholine (ACh) release in the hippocampus of Wistar strain rats with permanent bilateral occlusion of the common carotid arteries was examined during a discrimination learning task using a microdialysis method. Such occlusion resulted in obvious impairment of the discrimination performance. The state, the basal value and released patterns, of ACh in the hippocampus differed in the sham-operated control and the experimental group, while ACh release was elevated during the dialysis experiment in both groups. These findings suggested that the bilateral occlusion produced persistent learning deficits from an early stage after the operation and that the impaired discrimination learning performance might be related to the diffusion of ACh in the hippocampus.

Effects of discrimination learning on the rat striatal dopaminergic activity: a microdialysis study.

THE prefrontal cortex (PFC) has anatomical and functional relationships with the striatum. In a previous study we showed that dopamine (DA) turnover in the PFC of rats is enhanced during the performance of a discrimination task. In the present study, we used an in vivo microdialysis method to examine whether DAergic activity in the striatum could also be altered by the discrimination task. The results showed a substantial and sustained suppression of DAergic activity during and after the discriminative behaviour. The fact that thediscriminative performance induced opposite changes in DAergic activity in the striatum and the PFC is consistent with the results of biochemical studies, suggesting that the suppressed DA turnover in the striatum may be induced by the enhanced DAergic activity in the PFC during the discrimination task.

Valproate administration to mice increases hippocampal p21 expression by altering genomic DNA methylation.

Although valproate (VPA) is used widely in the treatment of bipolar mood disorder and epilepsy, the precise mechanism of action in the brain remains elusive. In this study, we investigated the effects of subchronic VPA administrations on the expression of the cyclin-dependent kinase inhibitor (Cdkn) family in the hippocampus of adult mice. The administration of VPA specifically increased hippocampal p21 expression involving both mRNA and protein levels, but other members of the Cdkn family were not affected. We identified two CpG islands in the p21 gene regulatory region, located distal and proximal to the transcription start site. VPA altered genomic DNA methylation patterns in the distal region, but not in the proximal promoter region. However, no change was found in DNA methyltransferase (Dnmt) 1 or Dnmt3a protein levels, suggesting an involvement in active demethylation mechanisms. These findings suggest that VPA alters the gene expression of cell cycle regulators by modulating promoter DNA methylation, and this resulted in altered hippocampal cell proliferation. These findings promote understanding of the actions of VPA in the brain.