Untranslated regions of brain-derived neurotrophic factor mRNA control its translatability and subcellular localization.

Brain-derived neurotrophic factor (BDNF) promotes neuronal survival and growth during development. In the adult nervous system, BDNF is important for synaptic function in several biological processes such as memory formation and food intake. In addition, BDNF has been implicated in development and maintenance of the cardiovascular system. The Bdnf gene comprises several alternative untranslated 5' exons and two variants of 3' UTRs. The effects of these entire alternative UTRs on translatability have not been established. Using reporter and translating ribosome affinity purification analyses, we show that prevalent Bdnf 5' UTRs, but not 3' UTRs, exert a repressive effect on translation. However, contrary to previous reports, we do not detect a significant effect of neuronal activity on BDNF translation. In vivo analysis via knock-in conditional replacement of Bdnf 3' UTR by bovine growth hormone 3' UTR reveals that Bdnf 3' UTR is required for efficient Bdnf mRNA and BDNF protein production in the brain, but acts in an inhibitory manner in lung and heart. Finally, we show that Bdnf mRNA is enriched in rat brain synaptoneurosomes, with higher enrichment detected for exon I-containing transcripts. In conclusion, these results uncover two novel aspects in understanding the function of Bdnf UTRs. First, the long Bdnf 3' UTR does not repress BDNF expression in the brain. Second, exon I-derived 5' UTR has a distinct role in subcellular targeting of Bdnf mRNA.

An 840 kb distant upstream enhancer is a crucial regulator of catecholamine-dependent expression of the Bdnf gene in astrocytes.

Brain-derived neurotrophic factor (BDNF) plays a fundamental role in the developing and adult nervous system, contributing to neuronal survival, differentiation, and synaptic plasticity. Dysregulation of BDNF synthesis, secretion or signaling has been associated with many neurodevelopmental, neuropsychiatric, and neurodegenerative disorders. Although the transcriptional regulation of the Bdnf gene has been extensively studied in neurons, less is known about the regulation and function of BDNF in non-neuronal cells. The most abundant type of non-neuronal cells in the brain, astrocytes, express BDNF in response to catecholamines. However, genetic elements responsible for this regulation have not been identified. Here, we investigated four potential Bdnf enhancer regions and based on reporter gene assays, CRISPR/Cas9 engineering and CAPTURE-3C-sequencing we conclude that a region 840 kb upstream of the Bdnf gene regulates catecholamine-dependent expression of Bdnf in rodent astrocytes. We also provide evidence that this regulation is mediated by CREB and AP1 family transcription factors. This is the first report of an enhancer coordinating the transcription of Bdnf gene in non-neuronal cells.

Differential Regulation of the BDNF Gene in Cortical and Hippocampal Neurons.

Brain-derived neurotrophic factor (BDNF) is a widely expressed neurotrophin that supports the survival, differentiation, and signaling of various neuronal populations. Although it has been well described that expression of BDNF is strongly regulated by neuronal activity, little is known whether regulation of BDNF expression is similar in different brain regions. Here, we focused on this fundamental question using neuronal populations obtained from rat cerebral cortices and hippocampi of both sexes. First, we thoroughly characterized the role of the best-described regulators of BDNF gene - cAMP response element binding protein (CREB) family transcription factors, and show that activity-dependent BDNF expression depends more on CREB and the coactivators CREB binding protein (CBP) and CREB-regulated transcriptional coactivator 1 (CRTC1) in cortical than in hippocampal neurons. Our data also reveal an important role of CREB in the early induction of BDNF mRNA expression after neuronal activity and only modest contribution after prolonged neuronal activity. We further corroborated our findings at BDNF protein level. To determine the transcription factors regulating BDNF expression in these rat brain regions in addition to CREB family, we used in vitro DNA pulldown assay coupled with mass spectrometry, chromatin immunoprecipitation (ChIP), and bioinformatics, and propose a number of neurodevelopmentally important transcription factors, such as FOXP1, SATB2, RAI1, BCL11A, and TCF4 as brain region-specific regulators of BDNF expression. Together, our data reveal complicated brain region-specific fine-tuning of BDNF expression.SIGNIFICANCE STATEMENT To date, majority of the research has focused on the regulation of brain-derived neurotrophic factor (BDNF) in the brain but much less is known whether the regulation of BDNF expression is universal in different brain regions and neuronal populations. Here, we report that the best described regulators of BDNF gene from the cAMP-response element binding protein (CREB) transcription factor family have a more profound role in the activity-dependent regulation of BDNF in cortex than in hippocampus. Our results indicate a brain region-specific fine tuning of BDNF expression. Moreover, we have used unbiased determination of novel regulators of the BDNF gene and report a number of neurodevelopmentally important transcription factors as novel potential regulators of the BDNF expression.

CREB Family Transcription Factors Are Major Mediators of BDNF Transcriptional Autoregulation in Cortical Neurons.

BDNF signaling via its transmembrane receptor TrkB has an important role in neuronal survival, differentiation, and synaptic plasticity. Remarkably, BDNF is capable of modulating its own expression levels in neurons, forming a transcriptional positive feedback loop. In the current study, we have investigated this phenomenon in primary cultures of rat cortical neurons using overexpression of dominant-negative forms of several transcription factors, including CREB, ATF2, C/EBP, USF, and NFAT. We show that CREB family transcription factors, together with the coactivator CBP/p300, but not the CRTC family, are the main regulators of rat BDNF gene expression after TrkB signaling. CREB family transcription factors are required for the early induction of all the major BDNF transcripts, whereas CREB itself directly binds only to BDNF promoter IV, is phosphorylated in response to BDNF-TrkB signaling, and activates transcription from BDNF promoter IV by recruiting CBP. Our complementary reporter assays with BDNF promoter constructs indicate that the regulation of BDNF by CREB family after BDNF-TrkB signaling is generally conserved between rat and human. However, we demonstrate that a nonconserved functional cAMP-responsive element in BDNF promoter IXa in humans renders the human promoter responsive to BDNF-TrkB-CREB signaling, whereas the rat ortholog is unresponsive. Finally, we show that extensive BDNF transcriptional autoregulation, encompassing all major BDNF transcripts, occurs also in vivo in the adult rat hippocampus during BDNF-induced LTP. Collectively, these results improve the understanding of the intricate mechanism of BDNF transcriptional autoregulation.SIGNIFICANCE STATEMENT Deeper understanding of stimulus-specific regulation of BDNF gene expression is essential to precisely adjust BDNF levels that are dysregulated in various neurological disorders. Here, we have elucidated the molecular mechanisms behind TrkB signaling-dependent BDNF mRNA induction and show that CREB family transcription factors are the main regulators of BDNF gene expression after TrkB signaling. Our results suggest that BDNF-TrkB signaling may induce BDNF gene expression in a distinct manner compared with neuronal activity. Moreover, our data suggest the existence of a stimulus-specific distal enhancer modulating BDNF gene expression.

AP-1 Transcription Factors Mediate BDNF-Positive Feedback Loop in Cortical Neurons.

Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, regulates both survival and differentiation of several neuronal populations in the nervous system during development, as well as synaptic plasticity in the adult brain. BDNF exerts its biological functions through its receptor TrkB. Although the regulation of BDNF transcription by neuronal activity has been widely studied, little is known about TrkB signaling-dependent expression of BDNF. Using rat primary cortical neuron cultures, we show that the BDNF gene is a subject to an extensive autoregulatory loop, where TrkB signaling upregulates the expression of all major BDNF transcripts, mainly through activating MAPK pathways. Investigating the mechanisms behind this autoregulation, we found that AP-1 transcription factors, comprising Jun and Fos family members, participate in the induction of BDNF exon I, III, and VI transcripts. AP-1 transcription factors directly upregulate the expression of exon I transcripts by binding two novel AP-1 cis-elements in promoter I. Moreover, our results show that the effect of AP-1 proteins on the activity of rat BDNF promoters III and VI is indirect, because AP-1 proteins were not detected to bind the respective promoter regions by chromatin immunoprecipitation (ChIP). Collectively, we describe an extensive positive feedback system in BDNF regulation, adding a new layer to the elaborate control of BDNF gene expression. SIGNIFICANCE STATEMENT: Here, we show for the first time that in rat primary cortical neurons the expression of all major BDNF transcripts (exon I, II, III, IV, VI, and IXa transcripts) is upregulated in response to TrkB signaling, and that AP-1 transcription factors participate in the induction of exon I, III, and VI transcripts. Moreover, we have described two novel functional AP-1 cis-elements in BDNF promoter I, responsible for the activation of the promoter in response to TrkB signaling. Our results indicate the existence of a positive feedback loop for obtaining sufficient BDNF levels necessary for various TrkB signaling-dependent physiological outcomes in neurons.

Revisiting the expression of BDNF and its receptors in mammalian development.

Brain-derived neurotrophic factor (BDNF) promotes the survival and functioning of neurons in the central nervous system and contributes to proper functioning of many non-neural tissues. Although the regulation and role of BDNF have been extensively studied, a rigorous analysis of the expression dynamics of BDNF and its receptors TrkB and p75NTR is lacking. Here, we have analyzed more than 3,600 samples from 18 published RNA sequencing datasets, and used over 17,000 samples from GTEx, and ~ 180 samples from BrainSpan database, to describe the expression of BDNF in the developing mammalian neural and non-neural tissues. We show evolutionarily conserved dynamics and expression patterns of BDNF mRNA and non-conserved alternative 5' exon usage. Finally, we also show increasing BDNF protein levels during murine brain development and BDNF protein expression in several non-neural tissues. In parallel, we describe the spatiotemporal expression pattern of BDNF receptors TrkB and p75NTR in both murines and humans. Collectively, our in-depth analysis of the expression of BDNF and its receptors gives insight into the regulation and signaling of BDNF in the whole organism throughout life.

Intronic enhancer region governs transcript-specific Bdnf expression in rodent neurons.

Brain-derived neurotrophic factor (BDNF) controls the survival, growth, and function of neurons both during the development and in the adult nervous system. Bdnf is transcribed from several distinct promoters generating transcripts with alternative 5' exons. Bdnf transcripts initiated at the first cluster of exons have been associated with the regulation of body weight and various aspects of social behavior, but the mechanisms driving the expression of these transcripts have remained poorly understood. Here, we identify an evolutionarily conserved intronic enhancer region inside the Bdnf gene that regulates both basal and stimulus-dependent expression of the Bdnf transcripts starting from the first cluster of 5' exons in mouse and rat neurons. We further uncover a functional E-box element in the enhancer region, linking the expression of Bdnf and various pro-neural basic helix-loop-helix transcription factors. Collectively, our results shed new light on the cell-type- and stimulus-specific regulation of the important neurotrophic factor BDNF.