Mamoru Fukuchi

Valproic acid induces up- or down-regulation of gene expression responsible for the neuronal excitation and inhibition in rat cortical neurons through its epigenetic actions

Valproic acid (VPA), a drug used to treat epilepsy and bipolar mood disorder, inhibits histone deacetylase (HDAC), which is associated with the epigenetic regulation of gene expression. Using a microarray, we comprehensively examined which genes are affected by stimulating cultured rat cortical neurons with VPA, and found that the VPA-treatment markedly altered gene expression (up-regulated; 726 genes, down-regulated; 577 genes). The mRNA expression for brain-derived neurotrophic factor (BDNF) and the alpha4 subunit of the GABA(A) receptor (GABA(A)Ralpha4), known to be involved in epileptogenesis, was up-regulated, with the increase in BDNF exon I-IX mRNA expression being remarkable, whereas that for GABA(A)Rgamma2, GAD65 and 67, and the K(+)/Cl(-) co-transporter KCC2, which are responsible for the development of GABAergic inhibitory neurons, was down-regulated. The number of GAD67-positive neurons decreased upon VPA-treatment. Similar changes of up- and down-regulation were obtained by trichostatin A. VPA did not affect the intracellular Ca(2+) concentration and the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), suggesting its direct action on HDAC. The acetylation of histones H3 and H4 was increased in the promoters of up-regulated but not down-regulated genes. Thus, VPA may disrupt a balance between excitatory and inhibitory neuronal activities through its epigenetic effect.

Robust stimulation of TrkB induces delayed increases in BDNF and Arc mRNA expressions in cultured rat cortical neurons via distinct mechanisms.

In cultures of rat cortical neurons, we found that stimulation of tyrosine receptor kinase B (TrkB) with brain‐derived neurotrophic factor (BDNF) induced a biphasic expression of BDNF exon IV–IX mRNA, which became obvious 1–3 h (primary induction) and 24–72 h (delayed induction) after the stimulation, and characterized the delayed induction in relation to the mRNA expression of activity‐regulated cytoskeleton‐associated protein (Arc). Withdrawal of BDNF from the medium after stimulation for 3 h allowed the delayed induction, which was caused at the transcriptional level and dependent upon the initial contact between exogenously added BDNF and TrkB, the effect of which was time‐ and dose‐dependent. The primary induction was controlled by the extracellular signal‐regulated kinase/mitogen‐activated protein kinase (ERK/MAPK) whereas the secondary induction by the calcium (Ca2+) signaling pathway. The enhanced Arc or Zif268 mRNA expression was controlled by activation of the ERK/MAPK pathway, both of which were repressed by blocking the binding of endogenously synthesized BDNF to TrkB. Thus, robust stimulation of TrkB autonomously induces delayed BDNF mRNA expression in an activity‐dependent manner in rat cortical neurons, resulting in the stimulation of Arc mRNA expression through endogenously synthesized BDNF, the process being orchestrated by the Ca2+ and ERK/MAPK signaling pathways.

Inactivation of aconitase during the apoptosis of mouse cerebellar granule neurons induced by a deprivation of membrane depolarization

During the excitotoxic neuronal cell death which accompanies an overflow of extracellular Ca2+ into neurons, aconitase, an oxidative stress‐sensitive enzyme of the tricarboxylic acid (TCA)‐cycle in mitochondria, is inactivated due to the generation of oxidative stress (Patel et al. [1996] Neuron 16:345–355). In this study, we investigated whether aconitase could be inactivated during the apoptosis of mouse cerebellar granule cells (CGCs), which was caused by a deprivation of membrane depolarization followed by a stoppage of Ca2+ influx into CGCs. Upon lowering the potassium (K+) concentration in medium from 25 to 5 mM (low K+), aconitase was inactivated in accordance with the decrease in methylthiazoletetrazolium (MTT)‐reducing activity although its mRNA expression did not change. The blockade of Ca2+ influx into CGCs mediated by nicardipine at 25 mM KCl also caused the inactivation of aconitase, accompanying induction of the apoptosis of CGCs. Suppression of the apoptosis of CGCs mediated by the Ca2+ influx or neurotrophic factors such as brain‐derived neurotrophic factor (BDNF) and adenylate cyclase activating polypeptide‐38 (PACAP‐38) attenuated the aconitase inactivation as well as the lactate dehydrogenase (LDH)‐release and the decrease in MTT reduction. On the other hand, the levels of intracellular glutathione and manganese superoxide dismutase‐2 mRNA decreased under the low K+ condition, supporting a cause for oxidative stress at low K+ due to a loss of anti‐oxidant activity. Thus, the inactivation of aconitase is also caused by a deprivation of Ca2+ influx into neurons, suggesting that aconitase is a key mitochondrial enzyme influencing the viability of neurons in response to oxidative stress.

Remote control of activity-dependent BDNF gene promoter-I transcription mediated by REST/NRSF

To know the role of repressor element-1 (RE-1)-silencing transcription factor (REST) in activity-dependent gene transcription in neurons, we investigated whether the Ca2+ signal-induced transcription of brain-derived neurotrophic factor promoter-I (BDNF-PI) is repressed by RE-1 located in exon II from far downstream of BDNF promoter-II (BDNF-PII). By constructing plasmids in which the location between BDNF-PI, -PII, and -RE-1 is maintained, we found, by conducting promoter assays with cortical neurons, that the promoter activity was constitutively repressed through the actions of BDNF-RE-1 but activated by Ca2+ signals evoked via membrane depolarization, which was due to BDNF-PI but not to BDNF-PII. The over-expression of REST reduced the level of transcriptional activation through the N- and C-terminals, suggesting the recruitment of a histone deacetylase. On over-expression of REST, an increased depolarization did not allow the activation. Thus, REST remotely represses activity-dependent gene transcription, the level of which controls the magnitude of the repression.

Involvement of the 3′‐untranslated region of the brain‐derived neurotrophic factor gene in activity‐dependent mRNA stabilization

J. Neurochem. (2010) 115, 1222–1233.

Involvement of the Serum Response Factor Coactivator Megakaryoblastic Leukemia (MKL) in the Activin-regulated Dendritic Complexity of Rat Cortical Neurons

Dynamic changes in neuronal morphology and transcriptional regulation play crucial roles in the neuronal network and function. Accumulating evidence suggests that the megakaryoblastic leukemia (MKL) family members, which function not only as actin-binding proteins but also as serum response factor (SRF) transcriptional coactivators, regulate neuronal morphology. However, the extracellular ligands and signaling pathways, which activate MKL-mediated morphological changes in neurons, remain unresolved. Here, we demonstrate that in addition to MKL1, MKL2, highly enriched in the forebrain, strongly contributes to the dendritic complexity, and this process is triggered by stimulation with activin, a member of the transforming growth factor β (TGF-β) superfamily. Activin promoted dendritic complexity in a SRF- and MKL-dependent manner without drastically affecting MKL localization and protein levels. In contrast, activin promoted the nuclear export of suppressor of cancer cell invasion (SCAI), which is a corepressor for SRF and MKL. Furthermore, overexpression of SCAI blocked activin-induced SRF transcriptional responses and dendritic complexity. Collectively, these results strongly suggest that activin-SCAI-MKL signaling is a novel pathway that regulates the dendritic morphology of rat cortical neurons by excluding SCAI from the nucleus and activating MKL/SRF-mediated gene expression.

Extracellular adenosine 5'-triphosphate elicits the expression of brain-derived neurotrophic factor exon IV mRNA in rat astrocytes.

A growing body of recent evidence indicates that ATP plays an important role in neuronal–glial communications. In this study, the authors demonstrated that extracellular ATP elicits the gene expression of brain‐derived neurotrophic factor (BDNF), especially BDNF exon IV mRNA, in primary cultured rat cortical astrocytes but not in neurons. To investigate the mechanism by which ATP induces BDNF exon IV mRNA expression, the authors used immortalized astrocyte cell line RCG‐12. ATP dose‐dependently increased the expression of BDNF exon IV mRNA and activated BDNF promoter IV. P2Y receptor agonists (ADP and 2MeS‐ADP) but not a P2X receptor agonist (αβMeATP) induced the expression of BDNF exon IV mRNA. Moreover, ATP‐induced BDNF exon IV mRNA upregulation was inhibited by a P2Y antagonist (MRS2179) but not by P2X antagonists (TNP‐ATP and PPADS). These findings suggest the involvement of P2Y receptors in the ATP‐induced transcription of the BDNF gene. Among the signal transduction inhibiters examined in this study, intracellular Ca2+ chelator (BAPTA‐AM) and Ca2+/calmodulin‐dependent kinase (CaM kinase) inhibitors (KN‐93 and W‐7) attenuated ATP‐induced BDNF exon IV mRNA upregulation. ATP transiently induced the phosphorylation of cAMP‐responsive element‐binding protein (CREB). ATP‐induced CREB phosphorylation was repressed by P2Y antagonists, BAPTA‐AM, and CaM kinase inhibitors. Overexpression of dominant negative CREB mutants reduced the activation of BDNF promoter IV and attenuated the upregulation of BDNF exon IV mRNA expression. These results suggest that ATP induces BDNF expression through P2Y receptor followed by the activation of CaM kinase and CREB in astrocytes. These mechanisms are likely to contribute to the enhancement of neuronal–glial networks.

Activation of tyrosine hydroxylase (TH) gene transcription induced by brain-derived neurotrophic factor (BDNF) and its selective inhibition through Ca2+ signals evoked via the N-methyl-D-aspartate (NMDA) receptor

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the biosynthesis of catecholamine but its transcriptional regulation is not fully understood. Using a reporter assay with cultured rat cortical neurons, we demonstrated that the TH gene promoter was activated by brain-derived neurotrophic factor (BDNF), through its specific receptor TrkB and the ERK/MAP kinase pathway. Using a series of mutant TH gene promoters, we found that the cAMP-response element (CRE) plays a crucial role in the TH promoter activity and the Egr-1-responsive element (ERE), at least in part, is responsible for the BDNF-induced activation. Notably, the influx of Ca(2+) evoked via the N-methyl-D-aspartate receptor (NMDA-R) but not via the L-type voltage-dependent Ca(2+) channel (L-VDCC) selectively antagonized the activation of the gene promoter, suggesting a new link between the catecholaminergic and glutamatergic systems. The Ca(2+) signals evoked via NMDA-R did not affect the phosphorylation of ERK1/2 induced by BDNF. These results suggest that the TH genes transcription is positively regulated by BDNF, through the CRE and ERE of the promoter, but selectively antagonized by the Ca(2+) signals evoked via NMDA-R without disturbing the ERK/MAP kinase pathway, the regulation by which may underlie the development of the catecholaminergic system in the brain.

Neuromodulatory Effect of Gαs- or Gαq-Coupled G-Protein-Coupled Receptor on NMDA Receptor Selectively Activates the NMDA Receptor/Ca2+/Calcineurin/cAMP Response Element-Binding Protein-Regulated Transcriptional Coactivator 1 Pathway to Effectively Induce Brain-Derived Neurotrophic Factor Expression in Neurons

Although coordinated molecular signaling through excitatory and modulatory neurotransmissions is critical for the induction of immediate early genes (IEGs), which lead to effective changes in synaptic plasticity, the intracellular mechanisms responsible remain obscure. Here we measured the expression of IEGs and used bioluminescence imaging to visualize the expression of Bdnf when GPCRs, major neuromodulator receptors, were stimulated. Stimulation of pituitary adenylate cyclase-activating polypeptide (PACAP)-specific receptor (PAC1), a Gαs/q-protein-coupled GPCR, with PACAP selectively activated the calcineurin (CN) pathway that is controlled by calcium signals evoked via NMDAR. This signaling pathway then induced the expression of Bdnf and CN-dependent IEGs through the nuclear translocation of CREB-regulated transcriptional coactivator 1 (CRTC1). Intracerebroventricular injection of PACAP and intraperitoneal administration of MK801 in mice demonstrated that functional interactions between PAC1 and NMDAR induced the expression of Bdnf in the brain. Coactivation of NMDAR and PAC1 synergistically induced the expression of Bdnf attributable to selective activation of the CN pathway. This CN pathway-controlled expression of Bdnf was also induced by stimulating other Gαs- or Gαq-coupled GPCRs, such as dopamine D1, adrenaline β, CRF, and neurotensin receptors, either with their cognate agonists or by direct stimulation of the protein kinase A (PKA)/PKC pathway with chemical activators. Thus, the GPCR-induced expression of IEGs in coordination with NMDAR might occur via the selective activation of the CN/CRTC1/CREB pathway under simultaneous excitatory and modulatory synaptic transmissions in neurons if either the Gαs/adenylate cyclase/PKA or Gαq/PLC/PKC-mediated pathway is activated.

Identification of genetic networks involved in the cell growth arrest and differentiation of a rat astrocyte cell line RCG‐12

The purpose of the present study is to establish and characterize a conditionally immortalized astrocyte cell line and to clarify the genetic networks responsible for the cell growth arrest and differentiation. A conditionally immortalized astrocyte cell line, RCG‐12, was established by infecting primary cultured rat cortical glia cells with a temperature‐sensitive simian virus 40 large T‐antigen. At a permissive temperature of 33°C, the large T‐antigen was expressed and cells grew continuously. On the other hand, the down‐regulation of T‐antigen at a non‐permissive temperature of 39°C led to growth arrest and differentiation. The cells expressed astrocyte‐expressed genes such as glial fibrillary acidic protein. Interestingly, the differentiated condition induced by the non‐permissive temperature significantly elevated the expression levels of several astrocyte‐expressed genes. To identify the detailed mechanisms by which non‐permissive temperature‐induced cell growth arrest and differentiation, we performed high‐density oligonucleotide microarray analysis and found that 556 out of 15,923 probe sets were differentially expressed 2.0‐fold. A computational gene network analysis revealed that a genetic network containing up‐regulated genes such as RB, NOTCH1, and CDKN1A was associated with the cellular growth and proliferation, and that a genetic network containing down‐regulated genes such as MYC, CCNB1, and IGF1 was associated with the cell cycle. The established cell line RCG‐12 retains some characteristics of astrocytes and should provide an excellent model for studies of astrocyte biology. The present results will also provide a basis for understanding the detailed molecular mechanisms of the growth arrest and differentiation of astrocytes. J. Cell. Biochem. 102: 1472–1485, 2007.