Brain-derived Neurotrophic Factor Gene Promoter Research Articles

A set of differentially expressed miRNAs, including miR-30a-5p, act as post-transcriptional inhibitors of BDNF in prefrontal cortex

Expression of brain-derived neurotrophic factor (BDNF) is developmentally regulated in prefrontal cortex (PFC). The underlying molecular mechanisms, however, remain unclear. Here, we explore the role of microRNAs (miRNAs) as post-transcriptional inhibitors of BDNF. A sequential approach involving in silico, miRNA microarray, in situ hybridization and qRT-PCR studies identified a group of 10 candidate miRNAs, segregating into five miRNA families (miR-30a-5p/b/c/d, miR-103/107, miR-191, miR-16/195, miR-495), which exhibited distinct developmental and lamina-specific expression in human PFC. Luciferase assays confirmed that at least two of these miRNAs, miR-30a-5p and miR-195, target specific sequences surrounding the proximal polyadenylation site within BDNF 3'-untranslated region. Furthermore, neuronal overexpression of miR-30a-5p, a miRNA enriched in layer III pyramidal neurons, resulted in down-regulation of BDNF protein. Notably, a subset of seven miRNAs, including miR-30a-5p, exhibited an inverse correlation with BDNF protein levels in PFC of subjects age 15-84 years. In contrast, the role of transcriptional mechanisms was more apparent during the transition from fetal to childhood and/or young adult stages, when BDNF mRNA up-regulation was accompanied by similar changes in (open chromatin-associated) histone H3-lysine 4 methylation at BDNF gene promoters I and IV. Collectively, our data highlight the multiple layers of regulation governing the developmental expression of BDNF in human PFC and suggest that miRNAs are involved in the fine-tuning of this neurotrophin particularly in adulthood.

Synaptic Plasticity-Regulated Gene Expression: a Key Event in the Long-Lasting Changes of Neuronal Function

"Neuronal activity"-dependent transcriptional activation is required for the long-lasting, functional changes that are involved in memory consolidation or drug addiction. Elucidation of the molecular mechanisms underlying the neuronal activity-dependent transcription of synaptic plasticity-related genes has helped towards understanding neuronal function and disorders as well in identifying new target molecules for drug design. In this study, we focused on neurotrophin and neuropeptide, which both have the ability to modulate neuronal survival and function. We also examined the molecular mechanisms by which underlying neurotrophin genes are regulated by neuronal activity. Brain-derived neurotrophic factor (BDNF) is a neurotrophin family member that has important roles in neuronal survival and plasticity as well as in psychiatric disorders. Transcriptional activation of the BDNF gene is commonly regulated by a key transcription factor, cAMP response element-binding protein (CREB), and this at least in part contributes to neuronal activity-dependent neuronal survival. Among at least four distinct promoters of the BDNF gene, promoters I and III are differentially activated by Ca2+ signals via NMDA receptors and L-type voltage-dependent Ca2+ channels. Especially, BDNF gene promoter I activation requires the cooperative binding of and upstream stimulatory factor (USF) and CREB to a CRE/USF binding site. By contrast, NT-3 gene transcription is regulated by Sp3/4. An important future direction will be to elucidate how long-lasting changes in neuronal plasticity are "epigenetically" and "structurally" controlled. Our studies on the relationships between long-lasting neuronal responses and gene expressions should help guide research into novel drugs for neuronal or psychiatric disorders.

Histone modifications around individual BDNF gene promoters in prefrontal cortex are associated with extinction of conditioned fear

Extinction of conditioned fear is an important model both of inhibitory learning and of behavior therapy for human anxiety disorders. Like other forms of learning, extinction learning is long-lasting and depends on regulated gene expression. Epigenetic mechanisms make an important contribution to persistent changes in gene expression; therefore, in these studies, we have investigated whether epigenetic regulation of gene expression contributes to fear extinction. Since brain-derived neurotrophic factor (BDNF) is crucial for synaptic plasticity and for the maintenance of long-term memory, we examined histone modifications around two BDNF gene promoters after extinction of cued fear, as potential targets of learning-induced epigenetic regulation of gene expression. Valproic acid (VPA), used for some time as an anticonvulsant and a mood stabilizer, modulates the expression of BDNF, and is a histone deacetylase (HDAC) inhibitor. Here, we report that extinction of conditioned fear is accompanied by a significant increase in histone H4 acetylation around the BDNF P4 gene promoter and increases in BDNF exon I and IV mRNA expression in prefrontal cortex, that VPA enhances long-term memory for extinction because of its HDAC inhibitor effects, and that VPA potentiates the effect of weak extinction training on histone H4 acetylation around both the BDNF P1 and P4 gene promoters and on BDNF exon IV mRNA expression. These results suggest a relationship between histone H4 modification, epigenetic regulation of BDNF gene expression, and long-term memory for extinction of conditioned fear. In addition, they suggest that HDAC inhibitors may become a useful pharmacological adjunct to psychotherapy for human anxiety disorders.

Transcriptional Regulation of Neuronal Genes and Its Effect on Neural Functions: Cumulative mRNA Expression of PACAP and BDNF Genes Controlled by Calcium and cAMP Signals in Neurons

Although it is widely accepted that an activity-dependent gene transcription is induced by the calcium (Ca2+) signals in neurons, it is still unclear how the particular mRNA moieties are transiently accumulated in response to synaptic transmission that evokes multiple intracellular signals including Ca2+ and cAMP ones. Promoters of the brain-derived neurotrophic factor (BDNF) and the pituitary adenylate cyclase-ativating polypeptide (PACAP) can commonly be activated through the cAMP-responsive element (CRE), to which the CRE-binding protein (CREB) predominantly bound. The activation of BDNF gene promoter I and III (BDNF-PI and -PIII, respectively) was mediated not only by the CREB but also by the upstream stimulatory factor, whereas that of PACAP gene promoter (PACAP-P) was mediated by only one CRE located at around −200. The PACAP-P was synergistically enhanced by Ca2+ and cAMP signals through the CRE, whereas the BDNF-PI did not show such a synergistic activation upon the stimulation with both signals. In addition, we found that the half-lives of PACAP and BDNF mRNA were prolonged by the Ca2+ influx into neurons but not that of Arc mRNA, indicating an activity-dependent stabilization of particular mRNA species in neurons. Thus, the activity-dependent gene expression is co-ordinately controlled by Ca2+ and cAMP signals not only at the transcriptional level but also at the post-transcriptional level for the cumulative mRNA expression in neurons.

Transcriptional Activation of the Human Brain-derived Neurotrophic Factor Gene Promoter III by Dopamine Signaling in NT2/N Neurons

We have identified a functional cAMP-response element (CRE) in the human brain-derived neurotrophic factor (BDNF) gene promoter III and established that it participated in the modulation of BDNF expression in NT2/N neurons via downstream signaling from the D1 class of dopamine (DA) receptors. The up-regulation of BDNF expression, in turn, produced neuroprotective signals through receptor tyrosine kinase B (TrkB) and promoted cell survival under the conditions of oxygen and glucose deprivation. To our knowledge this is the first evidence showing the presence of a functional CRE in the human BDNF gene and the role of DA signaling in establishing transcriptional competence of CRE in post-mitotic NT2/N neurons. This ability of DA to regulate the expression of the BDNF survival factor has a profound significance for the nigrostriatal pathway, because it indicates the existence of a feedback loop between the neutrophin, which promotes both the maturation and survival of dopaminergic neurons, and the neurotransmitter, which the mature neurons ultimately produce and release.

Pathways to care in early psychosis

Recent studies have suggested that first and second generation antipsychotics (FGAs and SGAs) have different neuroprotective effects. However, the molecular mechanisms of SGAs are not fully understood, and investigations into changes in intracellular signaling related to their neuroprotective effects remain scarce. In the present study, we compared the SGA aripiprazole with the FGA haloperidol in SH-SY5Y human neuroblastoma cells via brain-derived neurotrophic factor (BDNF)-mediated signaling, notably BDNF, glycogen synthase kinase-3β (GSK-3β), and B cell lymphoma protein-2 (Bcl-2). We examined the effects of aripiprazole (five and 10 μM) and haloperidol (one and 10 μM) on BDNF gene promoter activity in SH-SY5Y cells transfected with a rat BDNF promoter fragment (− 108 to + 340) linked to the luciferase reporter gene. The changes in BDNF, p-GSK-3β, and Bcl-2 levels were measured by Western blot analysis. The haloperidol was not associated with a significant difference in BDNF promoter activity. In contrast, aripiprazole was associated with increased BDNF promoter activity only with a dose of 10 μM (93%, p < 0.01). Treatment with aripiprazole at 10 μM increased the levels of BDNF by 85%, compared with control levels (p < 0.01), whereas haloperidol had no effect. Moreover, cells treated with aripirazole effectively increased the levels of GSK-3β phosphorylation and Bcl-2 at doses of five and 10 μM (30% and 58% and 31% and 80%, respectively, p < 0.05 or p < 0.01). However, haloperidol had no effects on p-GSK-3 β and Bcl-2 expression. This study showed that aripiprazole, but not haloperidol, appeared to offer neuroprotective effects on human neuronal cells. The actions of signaling systems associated with BDNF may represent key targets for both aripiprazole and haloperidol, but the latter may be associated with distinct effects. These differences might be related to the different therapeutic effects of FGAs and SGAs in patients with schizophrenia.

Involvement of an Upstream Stimulatory Factor as Well as cAMP-responsive Element-binding Protein in the Activation of Brain-derived Neurotrophic Factor Gene Promoter I

The use of different brain-derived neurotrophic factor (BDNF) gene promoters results in the differential production of 5'-alternative transcripts, suggesting versatile functions of BDNF in neurons. Among four BDNF promoters I, II, III, and IV (BDNF-PI, -PII, -PIII, and -PIV), BDNF-PI was markedly activated, as well as BDNF-PIII, by Ca(2+) signals evoked via neuronal activity. However, little is known about the mechanisms for the transcriptional activation of BDNF-PI. Using rat cortical neurons in culture, we assigned the promoter sequences responsible for the Ca(2+) signal-mediated activation of BDNF-PI and found that the Ca(2+)-responsive elements were located in two separate (distal and proximal) regions and that the DNA sequences in the proximal region containing cAMP-responsive element (CRE), which is overlapped by the upstream stimulatory factor (USF)-binding element, were largely responsible for the activation of BDNF-PI. CRE-binding protein (CREB) family transcription factors and USF1/USF2 bind to this overlapping site, depending upon their preferred sequences which also control the magnitude of the activation. Overexpression of dominant negative CREB or USF reduced the BDNF-PI activation. These findings support that not only CREB but also USF1/USF2 contributes to Ca(2+) signal-mediated activation of BDNF-PI through the recognition of an overlapping CRE and USF-binding element.

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 Ca(2+) signals evoked via Ca(2+) influx into neurons, little is known about how the activation of alternative BDNF gene promoters is controlled by the Ca(2+) signals evoked via N-methyl-d-aspartate receptors (NMDA-R) and L-type voltage-dependent Ca(2+) 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 Ca(2+) influx. Although the promoter activations at 25 and 50 mm KCl were different, BDNF-PIII was activated by either the Ca(2+) influx through NMDA-R or L-VDCC, whereas BDNF-PI was predominantly by the Ca(2+) 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 Ca(2+) signals evoked via NMDA-R and L-VDCC.

Trophic regulation of synaptic plasticity

Trophins are now known to acutely regulate synaptic plasticity, in addition to exerting long-recognized actions on neuronal survival and development. To begin elucidating underlying mechanisms, cultured dissociated hippocampal neurons were examined by whole-cell patch-clamp recording. BDNF (brain derived neurotrophic factor) specifically and selectively increased the integrated synaptic current twofold within five minutes. The effect was mediated postsynaptically by the trkB receptor and intracellular phosphorylation-dependent mechanisms. Iontophoresis combined with patch-clamp recording indicated that BDNF increased activity of NMDA (N-methyl-D-aspartate) receptors. Single channel-channel recording indicated that BDNF increased channel open probability by increasing opening frequency. In parallel, the trophin specifically increased phosphorylation of NR1 and NR2B subunits of NMDA receptors in the postsynaptic density, providing potential mechanisms eliciting receptor activation. A model is proposed in which activity-driven experience activates specific BDNF gene promoters, leading to enhanced transcription, elevated trophin levels, postsynaptic NMDA receptor activation and increased synaptic transmission. These sequential mechanisms may contribute to enhanced long-term potentiation, a correlate of learning and memory. © 1999 John Wiley & Sons, Inc. J Neurobiol 41: 108–118, 1999

Trophic regulation of synaptic plasticity

Trophins are now known to acutely regulate synaptic plasticity, in addition to exerting long-recognized actions on neuronal survival and development. To begin elucidating underlying mechanisms, cultured dissociated hippocampal neurons were examined by whole-cell patch-clamp recording. BDNF (brain derived neurotrophic factor) specifically and selectively increased the integrated synaptic current twofold within five minutes. The effect was mediated postsynaptically by the trkB receptor and intracellular phosphorylation-dependent mechanisms. Iontophoresis combined with patch-clamp recording indicated that BDNF increased activity of NMDA (N-methyl-D-aspartate) receptors. Single channel-channel recording indicated that BDNF increased channel open probability by increasing opening frequency. In parallel, the trophin specifically increased phosphorylation of NR1 and NR2B subunits of NMDA receptors in the postsynaptic density, providing potential mechanisms eliciting receptor activation. A model is proposed in which activity-driven experience activates specific BDNF gene promoters, leading to enhanced transcription, elevated trophin levels, postsynaptic NMDA receptor activation and increased synaptic transmission. These sequential mechanisms may contribute to enhanced long-term potentiation, a correlate of learning and memory.

Inducibility of BDNF Gene Promoter I Detected by Calcium-Phosphate-Mediated DNA Transfection Is Confined to Neuronal but Not to Glial Cells

Although DNA transfection with calcium-phosphate/DNA precipitates for promoter analysis of genes has been previously applied to primary cultures of neuronal cells, it remains uncertain whether the expression of the introduced genes in glial cells, which are also in primary culture, affects the transcriptional signals obtained. Using plasmid DNA containing the brain-derived neurotrophic factor (BDNF) gene promoter I or c-fospromoter, we investigated the optimum conditions for calcium-phosphate-mediated DNA transfection in primary culture of rat cortical neuronal cells. Normalizing the firefly luciferase activity of the experimental reporter gene to theRenillaluciferase activity of the internal control increased experimental reliability. Maximum expression of firefly luciferase activity in the cells stimulated by membrane depolarization required 6–12 hrs of incubation after stimulation. Differences in the ratio of the experimental reporter gene to the internal control did not affect experimental gene expression. Under our optimal conditions, the activation of the BDNF gene promoter I was detected in neuronal but not in glial cells. Calcium-phosphate-mediated DNA transfection should be widely applicable for promoter analysis of inducible genes in neurons.