From the Neurobiology Program, Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario K1A 0R6, Canada
Received for publication, November 12, 2002 , and in revised form, May 8, 2003.
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ABSTRACT |
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INTRODUCTION |
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There is a growing interest in BDNF as a potential therapeutic agent for neurodegenerative diseases, because its deficiency was found in brains of both Alzheimer's and Parkinson's patients (610). Indeed, BDNF treatment has been shown not only to potentiate synaptic transmission in vivo (11, 12) but also to increase neuronal survival and augment some behavioral changes in animal models (13, 14). However, more recent data indicate that BDNF can also induce behavioral sensitization by causing an overexpression of dopamine D3 receptors and could, actually, contribute to the amplification of pathophysiologies associated with conditions such as epilepsy, drug addiction, schizophrenia, and Parkinson's disease (15, 16). Clearly, further work is required to resolve some of these potential side-effects.
In contrast to a large body of work on the temporal and spatial patterns of BDNF expression in neurodevelopment and neurodegeneration, relatively little is known about the transcriptional regulation of the human BDNF gene. This is partially due to the fact that the genomic structure of the human gene has not yet been fully elucidated. The gene was first localized to chromosome11p13 and predicted to consist of multiple exons (17), but the existence of multiple transcripts, derived from different exons, was demonstrated only recently by Aoyama et al. (18) in human neuroblastoma cells. A more detailed transcript mapping of an 810-kb region of chromosome 11p13-14 further defined its genomic localization, although no additional information on the actual structure of the gene itself was presented (19).
The existence of multiple human BDNF transcripts (18) is consistent with a genomic structure similar to that of the rat gene, which consists of four short 5' exons, each controlled by a distinct promoter, and one 3' exon encoding the mature BDNF protein (20, 21). In the rat, the four promoters direct expression of the BDNF gene in a tissue-specific manner, i.e. promoters I and II are active preferentially in neurons, whereas promoters III and IV are active both in neurons and in a limited number of non-neuronal tissues such as lung and heart (20, 21). Thus far, only a limited characterization of a 3.2-kb genomic fragment of the human gene containing some structural elements of a promoter was reported (22).
Recently, two transcription factors, CREB and a calcium-responsive factor, were identified as positive regulators of the rat promoter III (2325), and the neuron-restrictive silencer factor as a negative regulator of promoters I and II (20, 26). It is still not known whether the regulatory machinery controlling the rat BDNF gene is conserved in humans.
Several studies indicate a direct involvement of neurotransmitters such as glutamate, GABA, acetylcholine, serotonin, and dopamine in the transcriptional regulation of the BDNF gene (2731) but the molecular components involved in this regulation have not yet been revealed. However, because DA exerts its effects through G protein-coupled transmembrane receptors, of which the D1-like class can stimulate adenylyl cyclase and increase production of cyclic AMP (cAMP) which, in turn, activates the downstream protein kinase A (PKA) signaling, it is possible that these events are involved in the transcriptional response of BDNF gene to DA via the transcription factor CREB.
In this study, we have derived the genomic structure of human BDNF gene from the Human Genome by a direct sequence and structure comparison with the rat gene and proceeded to characterize the molecular mechanisms of its transcriptional activation in Ntera2/D1 teratocarcimona (NT2) cells. The NT2 cells can be differentiated into post-mitotic NT2/N neurons by a treatment with retinoic acid (32, 33). We have established the presence of CRE-like element in promoter III of the gene and examined the role of DA signaling in its regulation. Recently, it has been established that NT2/N neurons express functional DA receptors of both D1-like and D2-like classes and are responsive to dopamine (3436).
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MATERIALS AND METHODS |
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Mouse Neuro-2a (N2a) neuroblastoma cells (ATCC CCL-131) were cultured in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% FCS (Wisent Inc.) and used for transfections at density of 50% confluence.
Experimental TreatmentsBoth undifferentiated and post-mitotic NT2/N neurons were treated with 0.2 mM Bt2cAMP for up to 3 h and harvested for RNA and protein extractions as described below. Stimulation of D1 class DA receptors with SKF-81297 agonist was performed as follows. The NT2/N neurons were placed in fresh medium 1012 h prior to the treatment, and 20 µM SKF-81297 (freshly prepared) was then added to the cultures for periods of time up to 3 h. We have shown previously that this concentration of SKF-81297 was the most effective in increasing the intracellular cAMP level (36). In some experiments 10 µM of the PKA inhibitor H-89 (from a 10 mM stock in Me2SO) was added together with 20 µM SKF-81297 for up to 3 h. The cells were subsequently harvested for the RNA extraction.
The cells were also subjected to oxygen-glucose deprivation (OGD) according to the following protocol. Post-mitotic NT2/N neurons, cultured in 12-well plates, were pretreated for 30 min with either 0.2 mM Bt2cAMP, 10 µM DA, or 20 µM SKF-81297. Subsequently, the cells were washed once with glucose-free DME medium, placed in glucose-free DME supplemented with 10% FCS and containing the same concentrations of Bt2cAMP, DA, or SKF-81297, and were exposed to 2 h of OGD as described below. In some experiments 2 µg/ml anti-TrkB antibody was added to the cultures for 1.5 h prior to the addition of Bt2cAMP and the OGD treatment. The cells were incubated for 2 h at 37 °C in a Gas Pak 100 chamber containing Gas Pak Plus gas generator envelopes (BD Biosciences). The generator envelopes catalytically reduce the oxygen concentration while providing a humidified atmosphere with 5% CO2. Control cells were subjected to the same 2-h OGD period but without any pretreatments. Cell viability was assessed at the end of the OGD treatment (0 time) and after a 24-h recovery period under normoxic conditions. At the same time points the cells were also harvested for RNA and protein extractions.
Cell Viability AssaysThe 5-carboxyfluorescein diacetate (CFDA) assay was used as an indicator of live cells and Hoechst 33342 dye staining to identify the dead cells. For the CFDA assay the cells were washed with PBS and incubated at 37 °C for 30 min with 5 µg/ml 5-CFDA, AM (Sigma) in Earle's balanced salt solution (Sigma). Fluorescence was quantified using a CytoFluorTM 2300/2350 fluorescence measurement system (Millipore, Bedford, MA) with an excitation filter at 480/20 nm and an emission filter at 530/25 nm. For the Hoechst staining assay the cells were rinsed off the flasks with medium and pelleted by centrifugation. The collected cells were fixed in suspension with 3% paraformaldehyde in PBS buffer (0.13 M NaCl, 5 mM Na2HPO4, 1.6 mM KH2PO4). The cell suspensions were drawn through 26-gauge needles to disperse them and were stained with 0.2 µg/ml Hoechst 33342 dye in PBS. An aliquot of each sample was mounted on glass slides, and these were viewed on an Olympus BX50 fluorescence microscope with an Olympus x40, numerical aperture 1.0, Planapo oil immersion objective.
RNA Extraction, RT-PCR, and Real-time Quantitative PCRRNA
was extracted with TriReagent according to the manufacturer's protocol
(Molecular Research Center, Inc., Cincinnati, OH) and was reverse-transcribed
into single-stranded cDNA with 400 units of Superscript II reverse
transcriptase (Invitrogen, Bethesda, MD) using 0.3 µg of random primers
(Invitrogen) in a 40-µl reaction containing 2 µg of total RNA. The cDNA
reaction mixtures were diluted 5-fold, and 3 µl of diluted cDNA was
used for each PCR amplification. Each 25-µl PCR reactions contained
1x reaction buffer (Invitrogen), 2 µM of primers, 0.2
mM dNTP, 1.5 mM MgCl2, and 2.5 units of
Taq polymerase (Invitrogen). PCR amplification for both CREB and BDNF
was carried out for 25 cycles and for
-actin for 19 cycles, which were
in the linear range of amplification. Each cycle consisted of the following
steps: 94 °C for 30 s, 60 °C for 1 min, and 72 °C for 1 min. Total
RNA in the samples was normalized to the amount of
-actin. The PCR
products were separated by electrophoresis on agarose gels. Ethidium
bromide-stained gels were photographed using the Ultra.Lum gel documentation
system (Claremont, CA) and quantified, when applied, using Scion image
software (Frederick, MD).
Real-time Q-PCR was performed with 10 ng of cDNA, purified on a QIAquick
Spin column (Qiagen, Mississauga, Ontario, Canada) and quantified with an
Oligreen® single-stranded DNA quantitation reagent kit (Molecular Probes,
Eugene, OR), using the ABI GeneAmp 5700 sequence detection system (ABI, Foster
City, CA) and Quantitect SYBR Green PCR kit (Qiagen). PCR primers were the
same as those used for RT-PCR and are listed in
Table I. Thermal cycling was
initiated with a 2-min incubation at 50 °C, followed by a first
denaturation step of 15 min at 95 °C and then 40 cycles of 95 °C for
15 s (denaturing) and 60 °C for 1 min (annealing and extension). The
real-time PCR amplification data were collected continuously and analyzed with
the Sequence Detection System (ABI, Foster City, CA). The copy number of
target sequences in the tested samples was inversely proportional to and,
hence, can be deduced from the CT (the thresh-hold cycle)
level at which a significant increase in fluorescence signal was first
detected. The amount of amplified products of the treated samples was
normalized to the untreated controls and was calculated using the term,
2CT.
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Preparation of Protein ExtractsCells were trypsinized, collected by centrifugation, washed once with PBS, and lysed with radioimmune precipitation assay buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% deoxycholate, 1% Triton X-100, 1 mM NaF, 1 mM Na3OVO4, and 1x Protease Inhibitor Cocktail (Roche Diagnostics, Indianapolis, IN). The lysates were vortexed, incubated for 15 min on ice, centrifuged at 13,000 rpm (IEC/MicroMax) for 20 min, and the supernatants (total cell lysates) were used for further analysis.
To prepare nuclear protein extracts, cells were lysed in 2 mM KH2PO4/KOH buffer, pH 6.55, containing 0.15 M NaCl, 1 mM EGTA 5 mM MgCl2, 0.1 mM DTT, 0.3% Triton X-100, and 1x Protease Inhibitor Cocktail (Roche Diagnostics). Nuclei were collected by centrifugation and washed once with the cell lysis buffer, and nuclear proteins were extracted with 20 mM HEPES buffer, pH 7.9, containing 25% v/v glycerol, 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, and 1x Protease Inhibitor Cocktail.
Western BlottingProtein extracts (50 µg/lane) were boiled in Laemmli/SDS sample buffer (2x buffer: 62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 0.01% bromphenol blue) for 5 min, separated by electrophoresis on 10% SDS-PAGE, and electrotransferred onto Hybond-C extra membranes (Amersham Biosciences, Piscataway, NJ). The membranes were first incubated overnight at 4 °C with primary antibodies diluted 1:1000 v/v in TBST (50 mM Tris, pH 7.5, 150 mM NaCl, 0.05% Tween 20, and 5% instant skim milk powder) and for an additional hour at room temperature with horseradish peroxidase-conjugated secondary antibodies (dilution 1:5000 v/v in TBST). The antigen-antibody complexes were visualized using the ECL detection system (Amersham Biosciences). The following antibodies were used: affinity-purified rabbit polyclonal antibody raised against a C terminus peptide of human CREB-1 (Santa Cruz Biotechnology, Santa Cruz, CA); anti-phospho-CREB (anti-p-CREB), an affinity-purified rabbit polyclonal antibody raised against a synthetic phosphopeptide corresponding to residues 123136 of rat CREB coupled to keyhole limpet hemocyanin (Upstate Biotechnology, Lake Placid, NY); affinity-purified rabbit polyclonal anti-BDNF antibody (Santa Cruz Biotechnology).
Electrophoretic Mobility Shift AssayA 19-bp probe corresponding to the CRE-like element (boldface letters) of the human BDNF promoter III was synthesized as complementary oligonucleotide strands (5'-GACAGCGCACGTCAAGGCA-3', 5'-GGTGCCTTGACGTGCGCTGT-3'). Complementary oligonucleotides containing mutated CRE were also applied (5'-GACAGCCAGCTGCAAGGCA-3', 5'-GGTGCCTTGCAGCTGGCTGT-3'). The strands were annealed and labeled with [32P]dCTP using a nucleotide fill-in method catalyzed by the Klenow enzyme (Amersham Biosciences). The protein binding assay was carried out in a 30-µl reaction mixture containing 20 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 40 mM KCl, 5 mM DTT, 10% glycerol, 30 or 60 ng of poly(dI-dC), 3 µg of bovine serum albumin, 6 µg of nuclear protein extract, and about 2 ng of 32P-labeled probe (5060,000 cpm/assay). The mixture was incubated for 20 min at room temperature, and the reaction was stopped by an addition of 1/10 volume of 10x sample loading buffer (250 mM Tris-HCl, pH 7.5, 40% glycerol, and 0.2% bromphenol blue). In the competition assays, an excess (50x) of unlabeled probe was added to the reaction mixture 30 min prior to the addition of the labeled oligonucleotides. In the antibody supershift assays, reaction mixtures were preincubated for 30 min at room temperature with 1 µl of rabbit polyclonal anti-CREB antibody (Rockland Inc., Gilbertsville, PA) or 1 µl of anti-ATF1 antibody (F11, Santa Cruz Biotechnology, Santa Cruz, CA) prior to the addition of the labeled probe. DNA-protein complexes were resolved by electrophoresis on a non-denaturing 6% polyacrylamide gel run in 50 mM Tris, 1 mM EDTA, and 300 mM glycine running buffer (pH 8.68.7) and were visualized by autoradiography on Kodak BioMax MS film.
Plasmid ConstructsGenomic DNA prepared from NT2 cells was used as PCR template to amplify the 548-bp fragment of human BDNF promoter III with a forward primer, 5'-TCGAGCTCTATACGTGTGTTTGCTG-3', and a reverse primer, 5'-ATCTCGAGCCTTTTCAGTCACTACT-3'. The fragment was cloned into pGL3-Basic luciferase reporter vector (Promega, Madison, WI) between SacI and XhoI sites to generate plasmid pGL3-PIII. The CRE mutation (pGL3-PIII_CREm) was introduced into PIII using the in vitro site-directed mutagenesis system GeneEditorTM (Promega, Madison, WI) with a primer, 5'-TATCATATGACAGCGACCTGCAAGGCACCGTGGAGC-3', in which the CRE site was mutated (underlining indicates CRE-site changes). Both pGL3-PIII and pGL3-PIII_CREm plasmids were sequenced to assure the correctness.
Cell Transfection and Luciferase Activity AssayTransient
transfections were carried out on neuroblastoma N2a cells using
Lipofect-AMINETM 2000 (Invitrogen Canada, Inc., Burlington). The
transfection efficiency of NT2 cells was too low to obtain any meaningful
data. Briefly, cells growing in 6-well plates were co-transfected with 5 µg
of pGL3 reporter plasmid and 2 µg of pSV--Gal (Promega) per well
following the manufacturer's protocols. Two hours after transfection, cells
were placed in serum-free DME, and 24 h later they were treated with 0.2
mM Bt2cAMP for 2 h. The cells were then washed twice
with cold Ca2+/Mg2+-free PBS and
lysed with 500 µl/well of Glo lysis buffer (1x) (Promega). One
hundred microliters of cell lysate was used for Bright-GloTM luciferase
assay system (Promega). The luciferase activity was measured using a
luminescence counter (1450 MicroBeta Trilux, Wallac). Thirty microliters of
cell lysate was used for analysis with the
-Galactosidase Enzyme Assay
System (Promega). The activity of
-galactosidase was detected by
absorption at 420 nm using a spectrometer (Spectra Max 340, Molecular Devices,
Sunnyvale, CA).
Data AnalysisData were analyzed and plotted using Jandel Sigmaplot 2000 software.
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RESULTS |
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Identification of a CRE-like Sequence in Promoter III of the Human GeneIt is well known that the expression of the rat BDNF gene is directed by four distinct promoters of which the activation of promoter III involves CRE and CaRE1 elements activated by the transcription factors CREB and calcium-responsive factor (20, 21, 2325). Subsequently, we used the rat promoter III and exon III sequence (GI 557911) (21) to search against the human genome (Human Genome browser, April 1, 2001 freeze) and identified the corresponding human promoter III sequence. The two sequences were further aligned using the ClustalW program (www2.ebi.ac.uk/clustalw). As shown in Fig. 2, the alignment revealed that the human promoter III contained a CRE-like sequence, 5'-GCACGTCA-3', located upstream from the exon III transcription initiation site. This element differed from the rat CRE by one nucleotide, 5'-TCACGTCA-3' (boldface letter), and from the palindromic CRE consensus sequence of by two nucleotides, 5'-TGACGTCA-3' (boldface letters) (37). The promoter also contained a CaRE1 element, 5'-CTATTTCGAG-3', identical to that of the rat, although its functionality will not be addressed in the present study. We did not find a TATA box consensus sequence. Instead, the promoter contained a pyrimidine-rich sequence, 5'-CTCCCACCACTTTCCCATTCACC-3'(Inr), suggested previously to function as an initiator of the rat exon III transcription (20).
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Expression Pattern of BDNF Gene during Differentiation of NT2 CellsWe used two separate sets of primers to distinguish transcripts generated by activation of different promoters. Accordingly, the F1/R1 primers were located in exon V and would amplify all BDNF transcripts, whereas the F2/R2 primers would amplify messages containing exons III and V, generated by promoter III (Table I and Fig. 1B). The expression of the gene, as measured by the level of exon V transcripts, was relatively low in both the undifferentiated and the 3- and 4-week RA-treated cells (Fig. 3, lanes 13; top panel), but it significantly increased and remained high in the post-mitotic NT2/N neurons treated with DNA synthesis inhibitors for 13 weeks (lanes 46, top panel). The expression pattern of exons IIIV mRNA was very similar. The level of these transcripts was also much lower in the undifferentiated and RA-treated cells (Fig. 3, lanes 13, middle panel) than in neurons maintained in culture in the presence of mitotic inhibitors (lanes 46, middle panel), suggesting that the activity of promoter III was significantly up-regulated in the post-mitotic NT2/N neurons.
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Interaction of CRE-like Element with CREBAlthough other regulatory elements were found in the promoter III, this study was designed to establish the functionality of the CRE. Initially, we performed the EMSA using the 32P-labeled 19-bp probe containing the CRE sequence, to determine its protein binding properties. In this in vitro assay two complexes were formed with nuclear proteins extracted from NT2/N cells (Fig. 4, lane 2, complexes I and II). An excess of unlabeled oligonucleotides representing both unaltered and crumbled CRE was used to test the specificity of these complexes. As shown in Fig. 4, a 50-fold excess of wild-type CRE eliminated both complexes (lane 3), but the same excess of mutated CRE affected only complex I (lane 4), suggesting this complex might not be CRE-specific. Subsequently, we performed the EMSA in the presence of anti-CREB and anti-ATF1 antibodies to probe the complexes for these transcription factors. The supershift of complex II, but not complex I, was observed with the anti-CREB antibody (compare lanes 2 and 5), the anti-ATF1 antibody had no effect under these conditions (compare lanes 2 and 6). This further confirmed the specificity of complex II and indicated that CREB, but not the closely related CREB family member ATF1, interacted with this CRE sequence in vitro. The electrophoretic mobility of complex I remained unchanged under these conditions suggesting that this binding was due to nuclear proteins other than these two CREB family members.
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Expression Pattern of CREB during NT2 Cell
DifferentiationThe interaction of CREB with the CRE-containing
fragment of BDNF promoter III in vitro
(Fig. 4), indicated its likely
involvement in the regulation of BDNF transcription. Therefore, it was
important to establish whether the expression level and/or transcriptional
competence of this transcription factor was responsible for the temporal
pattern of BDNF expression during the differentiation of NT2 cells. It is
known that the alternative splicing of the CREB gene generates several
isoforms, of which CREB and CREB
are the most abundant and
functionally most significant in mammalian cells. We used a pair of primers
that amplified both CREB
and CREB
. The amplified products could
be distinguished by their size on agarose gels
(Fig. 5A). The
CREB
mRNA remained unchanged during the entire differentiation paradigm
(Fig. 5A, upper
band), but the expression CREB
increased significantly following
exposure of the cells to RA (compare lanes 1 with lanes 2
and 3) and remained elevated in the post-mitotic neurons (lanes
4 and 5).
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The CREB protein was also present in the undifferentiated, RA-only-treated, and mature NT2/N cells (Fig. 5B, upper panel). Furthermore, the level of CREB phosphorylated at Ser-133 (p-CREB), which is considered to be transcriptionally competent, was very similar in the RA-treated cells (Fig. 5B, lane 2, lower panel), in which the promoter III was inactive (Fig. 3, lanes 2 and 3), and in the post-mitotic neurons (Fig. 5B, lane 3, lower panel), which expressed high levels of BDNF message (Fig. 3, lanes 4 and 5). Thus, the data showed no direct correlation between the levels of either CREB or p-CREB and the pattern of promoter III-driven gene transcription, suggesting that the one or more additional molecular factors were involved in the regulation of this process.
Effects of Bt2cAMP on BDNF ExpressionThe data presented above raised a question of when promoter III became accessible to cAMP-generated stimuli during the process of NT2 cells differentiation. To establish this, both undifferentiated and differentiated cells were treated with Bt2cAMP, and its effects on CREB phosphorylation and promoter III-driven transcription were analyzed 2 h later (Fig. 6). Clearly, Bt2cAMP had no effect of on BDNF transcription in undifferentiated cells (Fig. 6A). There was no induction of the message (Fig. 6A, left panel) and no change in CREB phosphorylation either (Fig. 6A, right panel) over the time period studied. A very different picture was seen in the post-mitotic NT2/N neurons (Fig. 6B). Although these cells already expressed BDNF mRNA at levels higher than they did in undifferentiated cells (Fig. 3), they responded to Bt2cAMP treatment further by both the up-regulation of CREB phosphorylation (Fig. 6B, right panel) and the expression of BDNF (Fig. 6B, left panel). The up-regulation of BDNF expression in response to Bt2cAMP was also verified by real-time PCR (Q-PCR) (Fig. 6B, left panel) and confirmed by Western blot (Fig. 6B, right panel). These results indicated that the undifferentiated NT2 cells possibly lacked one or more regulatory molecules essential for coupling the stimulus to the transcriptional apparatus.
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To determine the functionality of the promoter III CRE element
(5'-GCACGTCA-3'), a 548-bp genomic DNA sequence containing about
210 bp of 5' flanking region and exon III
(Fig. 2) was inserted into
plasmid pGL3-basic, which contained a firefly luciferase reporter gene. In
parallel a substitution mutant, in which the CRE sequence was changed to
5'-GACCTGCA-3' to eliminate the CREB binding, was also generated.
Each reporter plasmid was transiently transfected into mouse N2a neuroblastoma
cells. 24 h after transfections, the cells were treated with 0.2 mM
Bt2cAMP for 2 h. As shown in
Fig. 6C, the
expression of luciferase gene was driven by the CRE-containing promoter III
fragment, even in the absence of the stimulation, and the reporter gene
expression was significantly higher following the exposure of the cells to
cAMP. Furthermore, the substitution mutations within the CRE not only severely
affected the basal activity of promoter III (by 5-fold) but also
eliminated its responsiveness to Bt2cAMP. These findings strongly
suggested that the CRE element was an important mediator of BDNF exon III
transcription.
Activation of BDNF Expression by Dopamine SignalingIn neuronal cells the neurotransmitter DA triggers an elevation of intracellular cAMP and activation of PKA pathway through the D1 class of receptors. We have shown recently that the postmitotic NT2/N cells express functional DA receptors of both D1 and D2 classes capable of modulating the intracellular level of cAMP NT2/N neurons (36). We treated the NT2/N cells with 20 µM of the D1 class receptor agonist SKF-81297 (at this concentration SKF-81297 was effective in elevating the intracellular cAMP levels in the NT2/N cells, Sodja et al. (36)) and examined its effect on BDNF transcription (Fig. 7). Consistently, the treatment resulted both in the increased levels of exon III-containing BDNF transcripts in RNA samples collected at 2 and 3 h after the treatment (Fig. 7A) and p-CREB in nuclear protein samples collected at the same time points (Fig. 7B). The SKF-81297 treatment was completely ineffective in inducing the BDNF message in the presence of the PKA inhibitor H89 (Fig. 7C). Changes in BDNF expression in response to these treatments were also verified by Q-PCR. This data clearly demonstrated that the transcriptional activation of promoter III through the D1 class of receptors required downstream PKA signaling.
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Neuroprotective Effects BDNF against OGDTo establish
whether BDNF expression in the post-mitotic NT2/N neurons could promote their
survival, the cells were pre-treated for 30 min with 0.2 mM
Bt2cAMP, 10 µM DA, or 20 µM SKF-81297
to up-regulate the BDNF expression (Fig.
7), and, subsequently, they were subjected to the OGD treatment to
activate the cell death process. According to the data obtained from two
independent tests, the CFDA viability assay
(Fig. 8A) and counting
of Hoechst dye-stained condensed nuclei
(Fig. 8B), the 2-h OGD
treatment reproducibly resulted in 3540% of cell loss within 24 h.
Based on the morphological assessment of Hoechst-stained nuclei the OGD
treatment triggered neuronal apoptosis
(Fig. 8B). The cAMP
pretreatment was evidently neuroprotective, because only 10% of cells
died under the same OGD regimen and within the same time frame. Similar
results were obtained for cells exposed to either DA or SKF-81297 treatments,
no significant viability loss was detected at 24 h after the OGD
(Fig. 8A). Cell
analysis at the end of the OGD treatment showed much higher p-CREB and BDNF
message levels in Bt2cAMP treated than in control cells
(Fig. 8C), indicating
that this neuroprotective pathway might involve BDNF itself. To further
confirm this, we blocked the interaction of BDNF molecule with its receptor
TrkB by preincubating cells with anti-TrkB antibody prior to the
Bt2cAMP/OGD treatment. As shown in
Fig. 8A, the anti-TrkB
antibody completely reversed the neuroprotective effects of
Bt2cAMP, presumably by eliminating the downstream signaling of
BDNF.
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DISCUSSION |
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We have established that the overall structure of the human gene is very similar to that of the rat, with at least five exons and four introns (Fig. 1) clearly pointing out the complexity of structure and transcriptional regulation. However, to date there has been only one study on a genomic fragment containing some structural elements of the human gene promoter (22). According to the sequence alignment performed in this study (Fig. 1A), this fragment represents promoter IV of the human gene.
Multiple promoters have been shown to regulate rat BDNF gene expression,
but the activity-dependent transcription appears to be controlled mainly by
the promoter III
(2325).
However, it has been reported recently that a CRE located in the rat BDNF gene
promoter I also plays a role in its activation, although, the activity of
promoter I is 8-fold lower than that of the promoter III
(44). Our unpublished
data2 indicated that
neither exon I nor exon II of human BDNF gene was expressed in NT2 cells,
hence these promoters did not contribute to the BDNF message level in our
system (data not shown). The present study was undertaken to establish whether
the same structural and molecular components that regulate rat BDNF gene
promoter III were also involved in the control of the human gene. The human
promoter III contained the same pyrimidine-rich sequence identified by Timmusk
et al. (20) as the
initiator of the rat exon III transcription and no identifiable TATA box,
although it has been reported by Nakayama et al.
(21) that the rat promoter III
contains a potential TATA element, TATAAT, located between the CaRE1 and CRE
elements. This finding, however, is somewhat controversial, because this
element is not found in the sequence published by Timmusk et al.
(20). The latter authors
report a TATCAT sequence at this location, which is also present in the human
promoter (Fig. 2). It is
possible, therefore, that the conclusion of Nakayama et al.
(21) results from a sequencing
error.
The human promoter III contained both the CaRE and CRE elements (Fig. 2), confirming that the structural elements critical for activity-dependent regulation of transcription in rat (2325) were also conserved in the human gene promoter. The CRE sequence, 5'-GCACGTCA-3', located upstream from the exon III transcription initiation site, differed slightly from the palindromic 5'-TGACGTCA-3' consensus sequence (37). However, the two bases C and G at the center of the element (boldface letters) that discriminate between CREB binding and members of the AP-1 family (45, 46) were conserved. Indeed, in the in vitro EMSA the binding of CREB but not the closely related CREB family member ATF1 to this CRE sequence was established (Fig. 4).
Dopamine signaling is mediated in neuronal cells by the activation or
inhibition of adenylyl cyclase. DA exerts its action through a subset of
specific membrane receptors classified as seven-transmembrane domain
G-protein-coupled receptors. Thus far, five distinct DA receptors,
D1 to D5, have been identified and characterized, and
based on their biochemical and pharmacological properties the receptors have
been subdivided into two families, D1-like (the D1 and
D5) and D2-like (D2, D3, and D4).
The D1-like receptor subtypes are positive and D2-like are negative regulators
of the cAMP pathway (for reviews, see Refs.
47 and
48). We have shown previously
that the NT2/N cells express functional D1R and D5R
receptors, and their activation results in an increase of 2-fold in the
cAMP level (36). Thus, the
activation of the D1 class of receptors by SKF-81297 agonist led to the
up-regulation of promoter III-driven BDNF transcription in the post-mitotic
NT2/N neurons, signifying the requirements for functional D1R signaling and
the CRE element of the promoter in this process
(Fig. 7A). This
activation did not occur in the presence of PKA inhibitor H-89
(Fig. 7C), suggesting
activation of PKA was involved. Consistently, the stimulation of promoter III
by cAMP was evident in the post-mitotic NT2/N neurons but not in
undifferentiated or RA-only-treated cells
(Fig. 3), indicating clearly
that this process required additional intracellular components, notably D1
class receptor signaling, capable of coupling the stimulus to
transcription.
It has been documented in previous studies (4951) that BDNF modulates the survival and differentiation of mesencephalic dopaminergic neurons, including those that degenerate in Parkinson's disease, and promotes the development of dopaminergic networks in rodent retina (52). In our study, coupling of the downstream D1R signaling with CRE-modulated gene expression in the post-mitotic NT2/N neurons produced a BDNF-dependent neuroprotection against oxidative stress (the OGD treatment, Fig. 8). Similar conclusions can be discerned from the studies on the role of DA in the development and maturation of mouse striatal GABAergic neurons (5355). In this cell system DA was also shown to up-regulate BDNF expression through the D1-like receptors (31).
In summary, we have put together the full structure of the human BDNF gene and have identified the cis-regulatory elements of the promoter III. Subsequently, we focused our attention on the CRE element and established that it participated in the modulation of BDNF expression in NT2/N neurons via downstream signaling from the D1 class of DA receptors via cAMP, PKA, and CREB. The up-regulation of BDNF expression, in turn, produced neuroprotective signals promoting cell survival under the oxidative stress generated by the OGD conditions. Significantly, our data pointed to the existence of a feedback loop between the neutrophin that promotes the maturation and survival of dopaminergic neurons and the neurotransmitter, which the mature neurons ultimately produce and release. Clearly, this ability of DA to regulate the expression of the survival factor has a profound significance for the nigrostriatal network. For, as documented in the Parkinsonian brain (9, 10, 56), the loss of dopaminergic neurons and the impairments of DA input would contribute to decreased BDNF expression and, consequently, to the uncoupling of cell death/survival controlling mechanisms.
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FOOTNOTES |
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To whom correspondence should be addressed: Neurobiology Program, Institute
for Biological Sciences, National Research Council of Canada, M-54, 1500
Montreal Rd., Ottawa, Ontario K1A 0R6, Canada. Tel.: 613-990-0891; Fax:
613-941-4475; E-mail:
hung.fang{at}nrc.ca.
1 The abbreviations used are: BDNF, brain-derived neurotrophic factor;
Bt2cAMP, dibutyryl cyclic AMP; TrkB, tyrosine kinase B; GABA,
-aminobutyric acid; PKA, protein kinase A; FCS, fetal calf serum; RA,
all-trans-retinoic acid; N2a, Neuro-2a neuroblastoma cells; DA,
dopamine; OGD, oxygen-glucose deprivation; DME, Dulbecco's modified Eagle's
medium; CFDA, 5-carboxyfluorescein diacetate; PBS, phosphate-buffered saline;
RT, reverse transcription; EMSA, electrophoretic mobility shift assay; Q-PCR,
real-time quantitative PCR.
2 H. Fang, J. Chartier, C. Sodja, A. Desbois, M. Ribecco-Lutkiewicz, P. Roy
Walker, and M. Sikorska, unpublished data.
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