Akiko Tabuchi

Differential Activation of Brain-derived Neurotrophic Factor Gene Promoters I and III by Ca2+ Signals Evoked vial-type Voltage-dependent andN-Methyl-d-aspartate Receptor Ca2+Channels

Although the brain-derived neurotrophic factor (BDNF) gene is activated by the intracellular Ca2+signals evoked via Ca2+ influx into neurons, little is known about how the activation of alternative BDNF gene promoters is controlled by the Ca2+ signals evoked viaN-methyl-d-aspartate receptors (NMDA-R) and L-type voltage-dependent Ca2+ channels (L-VDCC). There is a critical range in the membrane depolarization caused by high K+ concentrations (25–50 mm KCl) for effective BDNF mRNA expression and transcriptional activation of BDNF gene promoters I and III (BDNF-PI and -PIII, respectively) in rat cortical culture. The increase in BDNF mRNA expression induced at high K+ was repressed not only by nicardipine, an antagonist for L-VDCC, but also by dl-amino-5-phosphonovalerate, an antagonist for NMDA-R, which was supported by the effects of antagonists on the Ca2+ influx. Although the promoter activations at 25 and 50 mm KCl were different, BDNF-PIII was activated by either the Ca2+ influx through NMDA-R or L-VDCC, whereas BDNF-PI was predominantly by the Ca2+influx through L-VDCC. Direct stimulation of NMDA-R supported the activation of BDNF-PIII but not that of BDNF-PI. Thus, the alternative BDNF gene promoters responded differently to the intracellular Ca2+ signals evoked via NMDA-R and L-VDCC.

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.

Involvement of endogenous PACAP expression in the activity-dependent survival of mouse cerebellar granule cells

Membrane depolarization causes Ca2+ influx through L-type voltage-dependent calcium channels (L-VDCC), which promotes the activity-dependent survival of mouse cerebellar granule cells (CGCs). Although exogenously added pituitary adenylate cyclase activating polypeptide (PACAP) is effective in promoting the survival of CGCs, it is unknown whether PACAP is synthesized in CGCs and involved in the activity-dependent survival of CGCs. In this study, we found that the PACAP gene was activated in depolarized CGCs cultured at 25 mM KCl (high K+), independently of de novo protein synthesis. In addition, the PACAP immunoreactivity increased through the activation of L-VDCC in depolarized CGCs, indicating that PACAP is concomitantly produced with PACAP mRNA in an activity-dependent manner. Exogenously added PACAP attenuated the apoptosis of CGCs through a specific interaction with PACAP receptors. Furthermore, a PACAP receptor antagonist, PACAP(6-38), reduced the survival of CGCs at high K+. These findings indicate that endogenous PACAP production induced by Ca2+ signals exerts a survival effect on CGCs via PACAP receptors, which, at least in part, accounts for the activity-dependent survival of CGCs.

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.

Regulation of neurotrophin‐3 gene transcription by Sp3 and Sp4 in neurons

Neurotrophin‐3 (NT‐3), a neurotrophin member, plays crucial roles in neuronal development, function and plasticity. Previous studies have demonstrated that NT‐3 gene transcription is driven by alternative promoters A and B, located upstream of exons 1A (EIA) and 1B (EIB), respectively. However, the transcription factors and DNA elements that drive NT‐3 gene transcription remain to be identified. Here, we analysed the promoter region of the NT‐3 gene and found that an NT‐3 transcript containing EIB is predominantly expressed in cortical neurons which preferentially utilize promoter B, and two tandemly repeated GC‐boxes, located between −100 and −60 base pairs within promoter B, are required for the transcription. Electrophoretic mobility shift and chromatin immunoprecipitation assays revealed that both specificity protein (Sp)3 and Sp4 were able to bind to the Sp1 binding sequences within the GC boxes. Expression of dominant‐negative Sp3 and Sp4 small interfering RNA in cortical neurons reduced the activity of the NT‐3 gene promoter. Over‐expression of Sp1 family members, especially Sp4, resulted in an increase of the NT‐3 gene promoter. These findings indicate that the NT‐3 gene is a target gene for Sp4 that is abundantly expressed in the brain.

Developmental expression of the SRF co-activator MAL in brain: role in regulating dendritic morphology.

The dynamic changes in dendritic morphology displayed by developing and mature neurons have stimulated interest in deciphering the signaling pathways involved. Recent studies have identified megakaryocytic acute leukemia (MAL), a serum response factor (SRF) co‐activator, as a key component of a signaling pathway linking changes in the actin cytoskeleton to SRF‐mediated transcription. To help define the role of this pathway in regulating dendritic morphology, we have characterized the pattern of MAL expression in the developing and adult brain, and have examined its role in regulating dendritic morphology in cultured cortical neurons. In histological studies of mouse brain, we found prominent expression of MAL in neurons in adult hippocampus and cerebral cortex. MAL immunostaining revealed localization of this protein in neuronal cell bodies and apical dendrites. During development, an increase in MAL expression occurs during the second post‐natal week. Expression of dominant negative MAL constructs or MAL siRNA in cortical neurons grown in primary culture reduces the number of dendritic processes and decreases the basal level of SRF‐mediated transcription. Taken together, these findings indicate that the MAL‐SRF signaling pathway plays a key role in regulating dendritic morphology.

Coactivation of secretogranin-II and BDNF genes mediated by calcium signals in mouse cerebellar granule cells

In primary culture of mouse cerebellar granule cells, the brain-derived neurotrophic factor (BDNF) gene is activated in an activity-dependent manner, accompanying Ca2+ influx into neurons through voltage-dependent calcium channels (VDCCs). In this study, we investigated the inducibility of secretogranin-II (Sg-II) gene in terms of Ca2+ signals evoked via VDCCs, by a comparison with BDNF and c-fos genes. Deprivation and subsequent induction of membrane depolarization by lowering and reelevating the extracellular concentration of potassium chloride (KCl), respectively, led to an decrease and then an increase in the Sg-II, BDNF and c-fos mRNA expression. The increase in Sg-II mRNA expression was detected as early as but was slower than that of BDNF one. The increase in Sg-II mRNA expression was induced depending upon the extracellular Ca2+ and inhibited by nicardipine, indicating a requirement of Ca2+ influx through VDCCs for the Sg-II as well as BDNF gene induction. Inhibition of de novo protein synthesis by cycloheximide did not affect the Sg-II induction. The response of Sg-II gene to the changes in extracellular KCl concentration was the same as that of BDNF but different from that of c-fos gene. Thus, Sg-II gene is coactivated with BDNF gene in response to the intracellular Ca2+ signals evoked via Ca2+ influx through VDCCs.

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.