Brain-derived Neurotrophic Factor Promoter Region Research Articles

The Polyunsaturated Fatty Acid EPA, but Not DHA, Enhances Neurotrophic Factor Expression through Epigenetic Mechanisms and Protects against Parkinsonian Neuronal Cell Death.

ω-3 Polyunsaturated fatty acids (PUFAs) have been found to exert many actions, including neuroprotective effects. In this regard, the exact molecular mechanisms are not well understood. Parkinson's disease (PD) is the second most common age-related neurodegenerative disease. Emerging evidence supports the hypothesis that PD is the result of complex interactions between genetic abnormalities, environmental toxins, mitochondrial dysfunction, and other cellular processes, such as DNA methylation. In this context, BDNF (brain-derived neurotrophic factor) and GDNF (glial cell line-derived neurotrophic factor) have a pivotal role because they are both involved in neuron differentiation, survival, and synaptogenesis. In this study, we aimed to elucidate the potential role of two PUFAs, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), and their effects on BDNF and GDNF expression in the SH-SY5Y cell line. Cell viability was determined using the MTT assay, and flow cytometry analysis was used to verify the level of apoptosis. Transmission electron microscopy was performed to observe the cell ultrastructure and mitochondria morphology. BDNF and GDNF protein levels and mRNA were assayed by Western blotting and RT-PCR, respectively. Finally, methylated and hydroxymethylated DNA immunoprecipitation were performed in the BDNF and GDNF promoter regions. EPA, but not DHA, is able (i) to reduce the neurotoxic effect of neurotoxin 6-hydroxydopamine (6-OHDA) in vitro, (ii) to re-establish mitochondrial function, and (iii) to increase BNDF and GDNF expression via epigenetic mechanisms.

LncRNA ANRIL mediates endothelial dysfunction through BDNF downregulation in chronic kidney disease

Endothelial dysfunction is common in patients with chronic kidney disease (CKD), but the mechanism is unknown. In this study, we found that the circulating ANRIL level was increased and correlated with vascular endothelial dysfunction in patients with CKD, also negatively correlated with plasma brain-derived neurotrophic factor (BDNF) concentration. We constructed the ANRIL knockout mice model, and found that ANRIL deficiency reversed the abnormal expression of BDNF, along with endothelial nitric oxide synthase (eNOS), vascular adhesion molecule 1 (VCAM-1) and Von Willebrand factor (vWF). Meanwhile, mitochondrial dynamics-related proteins, Dynamin-related protein 1 (Drp1) and mitofusins (Mfn2) level were also recovered. In addition, in vitro, serum derived from CKD patients and uremia toxins induced abnormal expression of ANRIL. By making use of the gain- and loss-of-function approaches, we observed that ANRIL mediated endothelial dysfunction through BDNF downregulation. To explore the specific mechanism, RNA pull-down and RNA-binding protein immunoprecipitation (RIP) were used to explore the binding of ANRIL to histone methyltransferase Enhancer of zeste homolog 2 (EZH2). Further experiments found increased EZH2 and histone H3 lysine 27 trimethylation (H3K27me3) levels at the BDNF promoter region. Collectively, we demonstrated that ANRIL mediate BDNF transcriptional suppression through recruitment of EZH2 to the BDNF promoter region, then regulated the proteins expression related to endothelial function and mitochondrial dynamics. This study provides new insights for the study of endothelial dysfunction in CKD.

Effects of Thermal Conditioning and Folic Acid on Methylation of the BDNF Promoter Region in Chicks.

This study aimed to investigate the effects of thermal conditioning and folic acid on the methylation levels of the avian brain-derived neurotrophic factor (BDNF) promoter region at the M3 and M9 positions in the early life of broiler chicks. In Experiment 1, male broiler chicks (day 3 of life) were orally injected with methyl cellulose solution with or without folic acid (25 mg). The chicks in the heat-treatment groups were immediately exposed to a high ambient temperature (40±0.5°C) for 12 h, while chicks in the non-heat treatment groups were left in the thermoneutral zone (30±0.5°C). The groups were as follows: 1) no thermal conditioning group without folic acid (control), 2) thermal conditioning group without folic acid, 3) no thermal conditioning group with folic acid, and 4) thermal conditioning group with folic acid. In Experiment 2, treatments were similar to those in Experiment 1, except for the usage of female chicks. After the treatments, the methylation levels of the BDNF promoter in chicks were determined using semiquantitative PCR. There were no significant differences between groups in the levels of methylation at the M3 position in both males and females as a result of thermal conditioning and folic acid treatment. Interestingly, significant effects of thermal conditioning and folic acid treatment on methylation at the M9 position were found. BDNF methylation levels at M9 significantly decreased following thermal conditioning, while folic acid suppressed demethylation in both male and female chicks. These data suggest that folic acid and thermal conditioning affects DNA methylation patterns in the central nervous system of chicks, regardless of sex.

Electroacupuncture ameliorates post-traumatic stress disorder in rats via a mechanism involving the BDNF-TrkB signaling pathway

Post-traumatic stress disorder(PTSD) is a mental health condition triggered by a terrifying event, causing flashbacks, nightmares and severe anxiety. It develops in individuals who have experienced a shocking, scary, or dangerous event. Electroacupuncture is reported to be effective for the treatment of PTSD. The present study was carried out to investigate the protective effect of electroacupuncture in a rat model of PTSD, and the mechanism involved. Specific-pathogen-free male Sprague Dawley rats (n = 30) weighing 180 - 220 g (mean weight = 200 ± 20 g) were randomly assigned to three groups of ten rats each: control group, single-prolonged stress (SPS) group, and treatment group. The treatment group rats received electroacupuncture. Changes in PTSD-like behavior were assessed using locomotor activity, elevated plus-maze (EPM) and fear conditioning tests. The mRNA and protein expressions of brain-derivedneurotrophicfactor (BDNF) and tropomyosin receptor kinase B (TrkB) were determined using real-time quantitative polymerase chain reaction (qRT-PCR) and Western blotting, respectively. Co-immunoprecipitation (Co-IP) was used to measure BDNF and TrkB binding interaction, while chromatin immunoprecipitation (ChIP) was used to evaluate the binding between cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB) and its target genes. Electroacupuncture significantly increased locomotor activity and exploratory behavior, but significantly reduced general fear and anxiety in SPS rats (p < 0.05). It also significantly upregulated the mRNA and protein expressions of BDNF and TrkB, and increased the binding of BDNF to its receptor TrkB (p < 0.05). Electroacupuncture significantly increased the binding of CREB to BDNF promoter region (p < 0.05). Electroacupuncture ameliorates PTSD in rats via a mechanism involving the BDNF-TrkB signaling pathway.

Chronic tinnitus and BDNF/GDNF CpG promoter methylations: a case-control study.

Brain-derived neurotrophic factor (BDNF) and Glial-derived neurotrophic factor (GDNF) are neurotrophic factors that play key roles in the auditory pathway. While the relationship between serum levels and polymorphisms of BDNF/GDNF and chronic tinnitus is emphasized in the literature, there is no study showing the link between the promoter methylations of these genes and tinnitus. For this purpose, the relationship between chronic tinnitus and peripheral blood derived BDNF/GDNF promoter methylations was investigated to identify their role in the pathophysiology of tinnitus. In this case-control study, we examined the possible effects of BDNF/GDNF methylations in the blood samples of patients with tinnitus complaints for more than 3months. Sixty tinnitus subjects between the ages of 18-55 and 50 healthy control subjects in the same age group who were free of any otorhinolaryngology and systemic disease were selected for examination. Methylation of total 12 CpG sites in BDNF and GDNF promoter regions were determined by the bisulfite-pyrosequencing method. Statistically significant differences were detected between BDNF CpG6 and GDNF CpG3-5-6 methylation ratios in the comparison of control group and the chronic tinnitus patients (P = 0.002, 0.0005, 0.00003, and 0.0029, respectively). To our knowledge, this is the first study in the literature investigating the relationship between chronic tinnitus and peripheral blood derived BDNF/GDNF promoter methylations. It is believed that the current results might be supported by investigating the relationships between BDNF/GDNF methylations and genotypes in future research using higher sample sizes.

Excess maternal fructose consumption impairs hippocampal function in offspring via epigenetic modification of BDNF promoter.

Recent increases in fructose consumption have raised concerns about the potential adverse intergenerational effects of excess fructose intake. In the present study, we investigated whether excess maternal fructose intake affects hippocampal function in offspring. Female Sprague-Dawley rats were divided into 3 experimental groups: one group received distilled water, one group received 20% fructose water, and one group received 20% glucose water in addition to standard chow during gestation and lactation. Hippocampal function of offspring was evaluated by using novel object recognition and fear conditioning tests. Impaired cognitive performance was observed in the offspring of fructose-fed dams at postnatal d 60, potentially a result of decreased hippocampal neurogenesis. Real-time PCR analysis demonstrated that offspring from fructose-fed dams exhibited decreased brain-derived neurotrophic factor ( BDNF) gene expression, whereas pyrosequencing assays revealed increased DNA methylation at the BDNF promoter. The potential association between BDNF transcription and levels of DNA methylation was confirmed on the basis of luciferase activity. Furthermore, longitudinal analysis revealed that increased methylation of the BDNF promoter region was maintained at least until rats reached maturity. These results indicate that epigenetic changes associated with BDNF may underlie hippocampal dysfunction that is induced by early-life exposure to excess maternal fructose consumption.-Yamazaki, M., Yamada, H., Munetsuna, E., Ishikawa, H., Mizuno, G., Mukuda, T., Mouri, A., Nabeshima, T., Saito, K., Suzuki, K., Hashimoto, S., Ohashi, K. Excess maternal fructose consumption impairs hippocampal function in offspring via epigenetic modification of BDNF promoter.

Resveratrol Preconditioning Induces a Novel Extended Window of Ischemic Tolerance in the Mouse Brain.

Prophylactic treatments that afford neuroprotection against stroke may emerge from the field of preconditioning. Resveratrol mimics ischemic preconditioning, reducing ischemic brain injury when administered 2 days before global ischemia in rats. This protection is linked to silent information regulator 2 homologue 1 (Sirt1) and enhanced mitochondrial function possibly through its repression of uncoupling protein 2. Brain-derived neurotrophic factor (BDNF) is another neuroprotective protein associated with Sirt1. In this study, we sought to identify the conditions of resveratrol preconditioning (RPC) that most robustly induce neuroprotection against focal ischemia in mice. We tested 4 different RPC paradigms against a middle cerebral artery occlusion model of stroke. Infarct volume and neurological score were calculated 24 hours after middle cerebral artery occlusion. Sirt1-chromatin binding was evaluated by ChIP-qPCR. Percoll gradients were used to isolate synaptic fractions, and changes in protein expression were determined via Western blot analysis. BDNF concentration was measured using a BDNF-specific ELISA assay. Although repetitive RPC induced neuroprotection from middle cerebral artery occlusion, strikingly one application of RPC 14 days before middle cerebral artery occlusion showed the most robust protection, reducing infarct volume by 33% and improving neurological score by 28%. Fourteen days after RPC, Sirt1 protein was increased 1.5-fold and differentially bound to the uncoupling protein 2 and BDNF promoter regions. Accordingly, synaptic uncoupling protein 2 level decreased by 23% and cortical BDNF concentration increased 26%. RPC induces a novel extended window of ischemic tolerance in the brain that lasts for at least 14 days. Our data suggest that this tolerance may be mediated by Sirt1 through upregulation of BDNF and downregulation of uncoupling protein 2.

Inhibition of histone methyltransferases SUV39H1 and G9a leads to neuroprotection in an in vitro model of cerebral ischemia.

Cerebral ischemia induces a complex transcriptional response with global changes in gene expression. It is essentially regulated by transcription factors as well as epigenetic players. While it is well known that the inhibition of transcriptionally repressive histone deacetylases leads to neuroprotection, the role of histone methyltransferases in the postischemic transcriptional response remains elusive. We investigated the effects of inhibition of the repressive H3K9 histone methyltransferases SUV39H1 and G9a on neuronal survival, H3K9 promoter signatures and gene expression. Their inhibition either with the specific blocker chaetocin or by use of RNA interference promoted neuronal survival in oxygen glucose deprivation (OGD). Brain-derived neurotrophic factor (BDNF) was upregulated and BDNF promoter regions showed an increase in histone marks characteristic for active transcription. The BDNF blockade with K252a abrogated the protective effect of chaetocin treatment. In conclusion, inhibition of histone methyltransferases SUV39H1 and G9a confers neuroprotection in a model of hypoxic metabolic stress, which is at least in part mediated by BDNF.

Association between DNA Methylation of the BDNF Promoter Region and Clinical Presentation in Alzheimer's Disease

Background/Aims: In the present study, we examined whether DNA methylation of the brain-derived neurotrophic factor (BDNF) promoter is associated with the manifestation and clinical presentation of Alzheimer's disease (AD). Methods: Of 20 patients with AD and 20 age-matched normal controls (NCs), the DNA methylation of the BDNF promoter (measured using peripheral blood samples) was completely analyzed in 12 patients with AD and 6 NCs. The resulting methylation levels were compared statistically. Next, we investigated the correlation between the DNA methylation levels and the clinical presentation of AD. Results: The total methylation ratio (in %) of the 20 CpG sites was significantly higher in the AD patients (5.08 ± 5.52%) than in the NCs (2.09 ± 0.81%; p < 0.05). Of the 20 CpG sites, the methylation level at the CpG4 site was significantly higher in the AD subjects than in the NCs (p < 0.05). Moreover, the methylation level was significantly and negatively correlated with some neuropsychological test subscores (registration, recall, and prehension behavior scores; p < 0.05). Conclusion: These results suggest that the DNA methylation of the BDNF promoter may significantly influence the manifestation of AD and might be associated with its neurocognitive presentation.

BDNF promoter I methylation correlates between post-mortem human peripheral and brain tissues

Several psychiatric disorders have been associated with CpG methylation changes in CG rich promoters of the brain-derived neurotrophic factor (BDNF) mainly by extracting DNA from peripheral blood cells. Whether changes in peripheral DNA methylation can be used as a proxy for brain-specific alterations remains an open question. In this study we aimed to compare DNA methylation levels in BDNF promoter regions in human blood cells, muscle and brain regions using bisulfite-pyrosequencing. We found a significant correlation between the levels of BDNF promoter I methylation measured in quadriceps and vPFC tissues extracted from the same individuals (n=98, Pearson, r=0.48, p=4.5×10−7). In the hippocampus, BDNF promoter I and IV methylation levels were strongly correlated (Pearson, n=37, r=0.74, p=1.4×10−7). We found evidence for sex-dependent effect on BDNF promoter methylation levels in the various tissues and blood samples. Taken together, these data indicate a strong intra-individual correlation between peripheral and brain tissue. They also suggest that sex determines methylation patterns in BDNF promoter region across different types of tissue, including muscle, brain, and blood.

Association of brain-derived neurotrophic factor DNA methylation and reduced white matter integrity in the anterior corona radiata in major depression

Considerable evidence suggests a crucial role for the epigenetic regulation of brain-derived neurotrophic factor (BDNF) in the pathophysiology of major depressive disorder (MDD). However, the relationship between BDNF DNA methylation and white matter (WM) integrity in MDD has not yet been investigated. In the current study, we examined the association between the DNA methylation status of the BDNF promoter region and WM integrity in MDD. Sixty patients with MDD and 53 healthy controls underwent T1-weighted structural magnetic resonance imaging (MRI), including diffusion tensor imaging (DTI), to assess their WM integrity. BDNF DNA methylation at 4 CpG sites of the promoter region was also measured.As compared to healthy controls, the MDD group demonstrated reduced fractional anisotropy (FA) in the bilateral anterior and posterior corona radiata (ACR and PCR), genu of the corpus callosum, and the bilateral posterior thalamic radiations. We observed a significant inverse correlation between the DNA methylation of the BDNF promoter region and the FA of the right ACR in MDD patients.Our findings demonstrate a relationship between methylation of the BDNF promoter region and the integrity of the ACR, a key structural component of the emotional and cognitive control network involved in the pathophysiology of MDD. This correlation suggests that BDNF DNA methylation may contribute to structural WM changes in MDD patients.

Methylation of BDNF in women with bulimic eating syndromes: associations with childhood abuse and borderline personality disorder.

DNA methylation allows for the environmental regulation of gene expression and is believed to link environmental stressors to such mental-illness phenotypes as eating disorders. Numerous studies have shown an association between bulimia nervosa (BN) and variations in brain-derived neurotrophic factor (BDNF). BDNF has also been linked to borderline personality disorder (BPD) and to such traits as reward dependence. We examined the extent to which BDNF methylation corresponded to bulimic or normal-eater status, and also to the presence of comorbid borderline personality disorder (BPD) and childhood abuse. Our sample consisted of 64 women with BN and 32 normal-eater (NE) control women. Participants were assessed for eating-disorder symptoms, comorbid psychopathology, and childhood trauma, and then they were required to provide blood samples for methylation analyses. We observed a significant site×group (BN vs. NE) interaction indicating that women with BN showed increases in methylation at specific regions of the BDNF promoter. Furthermore, examining effects of childhood abuse and BPD, we observed significant site×group interactions such that groups composed of individuals with childhood abuse or BPD had particularly high levels of methylation at selected CpG sites. Our findings suggest that BN, especially when co-occurring with childhood abuse or BPD, is associated with a propensity towards elevated methylation at specific BDNF promoter region sites. These findings imply that hypermethylation of the BDNF gene may be related to eating disorder status, developmental stress exposure, and comorbid psychopathology.

Genomic Organization and Identification of Promoter Regions for the BDNF Gene in the Pond Turtle Trachemys scripta elegans

Brain-derived neurotrophic factor (BDNF) is an important regulator of neuronal development and synaptic function. The BDNF gene undergoes significant activity-dependent regulation during learning. Here, we identified the BDNF promoter regions, transcription start sites, and potential regulatory sequences for BDNF exons I-III that may contribute to activity-dependent gene and protein expression in the pond turtle Trachemys scripta elegans (tBDNF). By using transfection of BDNF promoter/luciferase plasmid constructs into human neuroblastoma SHSY5Y cells and mouse embryonic fibroblast NIH3T3 cells, we identified the basal regulatory activity of promoter sequences located upstream of each tBDNF exon, designated as pBDNFI-III. Further, through chromatin immunoprecipitation (ChIP) assays, we detected CREB binding directly to exon I and exon III promoters, while BHLHB2, but not CREB, binds within the exon II promoter. Elucidation of the promoter regions and regulatory protein binding sites in the tBDNF gene is essential for understanding the regulatory mechanisms that control tBDNF gene expression.

Epigenetic modifications in frontal cortex from Alzheimer's disease and bipolar disorder patients

Alzheimer's disease (AD) and bipolar disorder (BD) are progressive brain disorders. Upregulated mRNA and protein levels of neuroinflammatory and arachidonic acid (AA) markers with loss of synaptic markers (synaptophysin and drebrin) have been reported in brain tissue from AD and BD patients. We hypothesized that some of these changes are associated with epigenetic modifications of relevant genes. To test this, we measured gene-specific CpG methylation, global DNA methylation and histone modifications in postmortem frontal cortex from BD (n=10) and AD (n=10) patients and respective age-matched controls (10 per group). AD and BD brains showed several epigenetic similarities, including global DNA hypermethylation, and histone H3 phosphorylation. These changes were associated with hypo- and hypermethylation of CpG islands in cyclooxygenase-2 and brain-derived neurotrophic factor promoter regions, respectively. Only the AD brain showed hyper- and hypomethylated CpG islands in promoter regions for cAMP response element-binding protein and nuclear transcription factor kappa B genes, respectively. Only the BD brain demonstrated increased global histone H3 acetylation and hypermethylation of the promotor region for the drebrin-like protein gene. There was no significant epigenetic modification for 12-lipooxygenase or p450 epoxygenase in either illness. Many observed epigenetic changes were inversely related to respective changes in mRNA and protein levels. These epigenetic modifications involving neuroinflammatory, AA cascade and synaptic markers may contribute to progression in AD and BD and identify new targets for drug development.

Selective DNA methylation of BDNF promoter in bipolar disorder: differences among patients with BDI and BDII.

The etiology of bipolar disorder (BD) is still poorly understood, involving genetic and epigenetic mechanisms as well as environmental contributions. This study aimed to investigate the degree of DNA methylation at the promoter region of the brain-derived neurotrophic factor (BDNF) gene, as one of the candidate genes associated with major psychoses, in peripheral blood mononuclear cells isolated from 94 patients with BD (BD I=49, BD II=45) and 52 healthy controls. A significant BDNF gene expression downregulation was observed in BD II 0.53±0.11%; P<0.05), but not in BD I (1.13±0.19%) patients compared with controls (CONT: 1±0.2%). Consistently, an hypermethylation of the BDNF promoter region was specifically found in BD II patients (CONT: 24.0±2.1%; BDI: 20.4±1.7%; BDII: 33.3±3.5%, P<0.05). Of note, higher levels of DNA methylation were observed in BD subjects on pharmacological treatment with mood stabilizers plus antidepressants (34.6±4.2%, predominantly BD II) compared with those exclusively on mood-stabilizing agents (21.7±1.8%; P<0.01, predominantly BD I). Moreover, among the different pharmacological therapies, lithium (20.1±3.8%, P<0.05) and valproate (23.6±2.9%, P<0.05) were associated with a significant reduction of DNA methylation compared with other drugs (35.6±4.6%). Present findings suggest selective changes in DNA methylation of BDNF promoter in subjects with BD type II and highlight the importance of epigenetic factors in mediating the onset and/or susceptibility to BD, providing new insight into the mechanisms of gene expression. Moreover, they shed light on possible mechanisms of action of mood-stabilizing compounds vs antidepressants in the treatment of BD, pointing out that BDNF regulation might be a key target for their effects.

P01-199 - Selective DNA methylation of BDNF promoter and nociceptin gene in bipolar disorder

IntroductionThe etiology of bipolar disorder (BD) is still poorly understood and it has been proposed that altered expression of multiple mRNAs, affecting neurotransmission, in psychotic subjects may be due to epigenetic mechanisms (e.g., DNA methylation or histone modification).AimsThe present collaborative study was aimed to investigate, in subjects with BD, dysregulation of DNA methylation, in the brain-derived neurotrophic factor (BDNF), previously associated with psychosis and linked to epigenetic changes at promoter regions, and in the nociceptin gene (N/OFQ), a new proposed target in psychiatric disorders.MethodsDNA was isolated from blood of patients diagnosed with BD (either type I [n = 19] or II [n = 20]) according to DSM-IV criteria, and from healthy controls (n = 20) and, thereafter, bisulfite conversion was performed. Real-Time Methylation Specific PCR (MSP) was used for the quantification of the methylated promoters in all samples.ResultsA hypermethylation of BDNF promoter region was observed in BD II patients (but not in BD I) compared to controls (CONT: 19.0 ± 3.2%; BD I: 27.2 ± 4.0%; BD II: 36.3 ± 6.2% * P = 0.0167; ANOVA F = 3.464; P = 0.0384; Bonferroni’s post hoc test). No significant differences were found for DNA methylation of N/OFQ promoter.ConclusionsPresent preliminary findings suggest selective changes in DNA methylation of BDNF promoter in type II bipolar patients and highlight the importance of epigenetic factors in mediating the onset and/or susceptibility to BD, providing new insight into the mechanisms of gene expression.

Long‐lasting depression‐like behavior and epigenetic changes of BDNF gene expression induced by perinatal exposure to methylmercury

Substantial evidence indicates that predisposition to diseases can be acquired during early stages of development and interactions between environmental and genetic factors may be implicated in the onset of many pathological conditions. Data collected over several decades have shown that chemicals are among the relevant factors that can endanger CNS. We previously showed that perinatal exposure to methylmercury (MeHg) causes persistent changes in learning and motivational behavior in mice. In this study, we report that the depression-like behavior in MeHg-exposed male mice is reversed by chronic treatment with the antidepressant fluoxetine. Behavioral alterations are associated with a decrease in brain-derived neurotrophic factor (BDNF) mRNA in the hippocampal dentate gyrus and fluoxetine treatment restores BDNF mRNA expression. We also show that MeHg-exposure induces long-lasting repressive state of the chromatin structure at the BDNF promoter region, in particular DNA hypermethylation, an increase in histone H3-K27 tri-methylation and a decrease in H3 acetylation at the promoter IV. While fluoxetine treatment does not alter hypermethylation of H3-K27, it significantly up-regulates H3 acetylation at the BDNF promoter IV in MeHg-exposed mice. Our study shows that developmental exposure to low levels of MeHg predisposes mice to depression and induces epigenetic suppression of BDNF gene expression in the hippocampus.

Rodent BDNF genes, novel promoters, novel splice variants, and regulation by cocaine

Results from studies using molecular and genetic methods in humans and rodents suggest that brain-derived neurotrophic factor (BDNF) is involved in the behavioral effects of abused drugs, making understanding of its genomic structure and regulation of substantial interest. Recently, we have reported that the human BDNF gene contains seven upstream exons that can each be spliced independently to the major BDNF coding exon to form diverse bipartite BDNF transcripts. We also identified a novel “BDNFOS” gene that is transcribed to produce alternatively spliced natural antisense transcripts (NATs); its fifth exon overlaps with the protein coding exon VIII of human BDNF. To better understand BDNF's genomic structure and differential regulation, we now describe the rodent BDNF gene and transcripts. This gene includes six bipartite transcripts that are generated by six independently transcribed exons, each of which is spliced to a major coding exon and a tripartite transcript that is composed of two upstream exons and one coding exon. In addition, we found no evidence for antisense, opposite strand BDNFOS gene transcripts in mice or rats. The BDNF rodent splice variants display specific patterns of differential expression in different brain regions and peripheral tissues. Acute cocaine administration increased striatal expression of a specific BDNF4 splice variant by up to 5-fold. Interestingly, however, neither experimenter- nor self-administered chronic cocaine administration enhanced striatal BDNF expression. These data suggest a role of specific BDNF promoter regions and regulatory sequences in stimulant-induced alterations in BDNF expression, and in the alterations that changed BDNF expression is likely to confer in the brain.

Identification of brain-derived neurotrophic factor promoter regions mediating tissue-specific, axotomy-, and neuronal activity-induced expression in transgenic mice.

The structure of rat brain-derived neurotrophic factor (BDNF) gene is complex; four 5' exons are linked to separate promoters and one 3' exon is encoding the BDNF protein. To analyze the relative importance of the regulatory regions in vivo, we have generated transgenic mice with six different promoter constructs of the BDNF gene fused to the chloramphenicol acetyl transferase reporter gene. High level and neuronal expression of the reporter gene, that in many respects recapitulated BDNF gene expression, was achieved by using 9 kb of genomic sequences covering the promoter regions that lie adjacent to each other in the genome (promoters I and II and promoters III and IV, respectively) and by including sequences of BDNF intron-exon splice junctions and 3' untranslated region in the constructs. The genomic regions responsible for the in vivo upregulation of BDNF expression in the axotomized sciatic nerve and in the brain after kainic acid-induced seizures and KCl-induced spreading depression were mapped. These data show that regulation of the different aspects of BDNF expression is controlled by different regions in vivo, and they suggest that these promoter constructs may be useful for targeted expression of heterologous genes to specific regions of the central and peripheral nervous systems in an inducible manner.

Characterization of the 5′-flanking region of the human brain-derived neurotrophic factor gene

The 5'-flanking region of the human brain-derived neurotrophic factor (BDNF) gene was isolated from a human placental genomic library using the cDNA fragment for the 5'-noncoding region of human BDNF as a probe. A 3.2 Kbp genomic fragment containing the 5'-flanking region, the first exon and a portion of the first intron was isolated and sequenced. The transcriptional initiation site, identified by S1 nuclease mapping, was located 26 bp downstream from the TATA-like sequence. Several expression plasmids, in which the BDNF promoter regions were fused to the chloramphenicol acetyltransferase (CAT) gene, were constructed. Transient expression in human glioma Hs683 cells demonstrated that a fragment of about 0.5 Kbp from the transcriptional initiation site was sufficient for promoter activity.