Glucocorticoids decrease astrocyte numbers by reducing glucocorticoid receptor expression in vitro and in vivo.

Glucocorticoids are stress hormones released from the adrenal cortex and their concentration is controlled by the hypothalamic-pituitary-adrenal axis. In this study, we investigated the effect of glucocorticoids on the number of astrocytes and glucocorticoid receptor (GR) expression in vitro and in vivo. Proliferation of cultured astrocytes was reduced following treatment with corticosterone and dexamethasone for 72 h. Corticosterone and dexamethasone also reduced GR expression in astrocytes. RU486, a GR antagonist, inhibited the reduction in both astrocyte proliferation and GR expression. Furthermore, GR knockdown by siRNA inhibited astrocyte proliferation. We also examined the effect of excessive glucocorticoid release on GR expression and the number of astrocytes in vivo by administering adrenocorticotropic hormone to rats for 14 days. GR expression was reduced in the prefrontal cortex and hippocampus and the number of astrocytes was reduced in the frontal cortex. Overall, our results suggest that glucocorticoids decrease the number of astrocytes by reducing GR expression.

A neuronal cell-based reporter system for monitoring the activity of HDAC2.

Given that histone acetylation via histone acetyltransferases (HATs) and histone deacetylases (HDACs) is significant in memory formation, HDAC2 has been thoroughly investigated as a potential therapeutic target for the treatment of cognitive dysfunction. Although HDAC inhibitors have been discovered through in vitro enzyme assay, off-target effects on other HDACs are common due to their conserved catalytic domains. Each HDAC could be regulated by specific intracellular molecular mechanisms, raising the possibility that a cell-based assay could identify selective inhibitors targeting specific HDACs through their regulatory mechanisms. Here, we propose a versatile, cell-based reporter system for screening HDAC2 inhibitors. Through RNA-sequencing from human cultured neuronal cells, we determined that expression of a transcriptional repressor, inhibitor of DNA binding 1 (ID1), is increased by knockdown of HDAC2. We also established the knock-in neuronal cell lines of a bioluminescence reporter gene to ID1. The knock-in cell lines showed significant reporter activity by known HDAC inhibitors and by HDAC2-knockdown but not by HDAC1-knockdown. Thus, our neuronal cell-based reporter system is a promising method for screening the specific inhibitors of HDAC2 but not HDAC1, by potentially targeting not only HDAC2, but also the regulatory mechanisms of HDAC2 in neurons.

Identification of a novel planarian G-protein-coupled receptor that responds to serotonin in Xenopus laevis oocytes.

Planarians are useful animals for regenerative and neuroscience research at the molecular level. Previously, we have reported the distribution and function of neurotransmitter-synthesizing neurons in the planarian central nervous system. In order to understand the neural projections and connections, it is important to understand the distribution of neurotransmitter receptors. In this study, we isolated a serotonin receptor gene and named it DjSER-7 (Dugesia japonica serotonin receptor type 7). DjSER-7-expressing cells were distributed in the planarian brain. According to electrophysiological analysis using Xenopus oocytes, current response was detected upon exposure to serotonin, but not other neurotransmitters in oocytes that were co-injected with mRNAs of both DjSER-7 and Galpha chimera B-2, which can interact with either Gq-, Gs- or Gi-coupled receptor. In contrast, current response was not detected after exposure to neurotransmitters in oocytes injected with only DjSER-7 mRNA. Our results indicated that DjSER-7 responds to serotonin, as indicated by electrophysiological analysis using Xenopus oocytes.

Acceleration of serotonin transporter transport-associated current by 3,4-methylenedioxymethamphetamine (MDMA) under acidic conditions.

3,4-Methylenedioxymethamphetamine (MDMA) selectively releases serotonin (5-HT) from neurons after uptake by the serotonin transporter (SERT) and causes psychostimulant effects accompanied by hyperthermia. Since rapid increases in CNS lactate levels are suggested in response to MDMA, we investigated the effects of the physiologically-relevant increase in H(+) concentration on the two conducting states of SERT expressed in Xenopus oocytes. Perfusion with SERT substrates at pH 7.4 dose-dependently evoked a transport-associated inward current response with the following rank order of potency: 5-HT>MDMA>dopamine>>norepinephrine. In the absence of transport substrates, a step hyperpolarization pulse activated a transient Na(+) leak current, which was inhibited by SERT substrates with the same rank, as well as by a SERT inhibitor, citalopram. At pH 6.4, the K(m) values of 5-HT and dopamine for the transport-associated current were not altered, while that of MDMA was three-fold lower. In contrast, the K(i) values of all these substrates for transient leak current were unchanged at pH 6.4, suggesting that the affinities to SERT binding sites are not influenced. These results suggest that the effect of MDMA on SERT is enhanced by acidic conditions.

Heme oxygenase-1 contributes to pathology associated with thrombin-induced striatal and cortical injury in organotypic slice culture.

The blood coagulation factor thrombin that leaks from ruptured vessels initiates brain tissue damage after intracerebral hemorrhage. We have recently shown that mitogen-activated protein kinases (MAPKs) activated by thrombin exacerbate hemorrhagic brain injury via supporting survival of neuropathic microglia. Here, we investigated whether induction of heme oxygenase (HO)-1 is involved in these events. Zinc protoporphyrin IX (ZnPP IX), a HO-1 inhibitor, attenuated thrombin-induced injury of cortical cells in a concentration-dependent manner (0.3-3 microM) and tended to inhibit shrinkage of the striatal tissue at 0.3 microM. HO-1 expression was induced by thrombin in microglia and astrocytes in both the cortex and the striatum. The increase of HO-1 protein was suppressed by a p38 MAPK inhibitor SB203580, and early activation of p38 MAPK after thrombin treatment was observed in neurons and microglia in the striatum. Notably, concomitant application of a low concentration (0.3 microM) of ZnPP IX with thrombin induced apoptotic cell death in striatal microglia and significantly decreased the number of activated microglia in the striatal region. On the other hand, a carbon monoxide releaser reversed the protective effect of ZnPP IX on thrombin-induced injury of cortical cells. Overall, these results suggest that p38 MAPK-dependent induction of HO-1 supports survival of striatal microglia during thrombin insults. Thrombin-induced cortical injury may be also regulated by the expression of HO-1 and the resultant production of heme degradation products such as carbon monoxide.