Abstract
Brain-derived neurotrophic factor (BDNF) is an active neurotrophin abundantly expressed throughout the nervous system. It plays an important role in synaptic transmission, plasticity, neuronal proliferation, differentiation, survival, and death. The Bdnf gene in rodents has eight non-coding exons and only a single coding exon (IX). Despite its recognized regulation by neuronal activity, relatively little is known about its transcriptional regulation, and even less about the transcription factor candidates that may play such a role. The goal of the present study was to probe for such a candidate that may regulate exon IX in the rat Bdnf gene. Our in silico analysis revealed tandem binding sites for nuclear respiratory factor 2 (NRF-2) on the promoter of exon IX. NRF-2 is of special significance because it co-regulates the expressions of mediators of energy metabolism (cytochrome c oxidase) and mediators of neuronal activity (glutamatergic receptors). To test our hypothesis that NRF-2 also regulates the Bdnf gene, we performed electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation (ChIP), promoter cloning, and site-directed mutagenesis, real-time quantitative PCR (RT-qPCR), and Western blotting analysis. Results indicate that NRF-2 functionally regulates exon IX of the rat Bdnf gene. The binding sites of NRF-2 are conserved between rats and mice. Overexpressing NRF-2 up-regulated the expression of Bdnf exon IX, whereas knocking down NRF-2 down-regulated such expression. These findings are consistent with our hypothesis that NRF-2, in addition to regulating the coupling between neuronal activity and energy metabolism, also regulates the expression of BDNF, which is intimately associated with energy-demanding neuronal activity.
Highlights
Brain-derived neurotrophic factor (BDNF)2 is regarded as the most widely expressed, active, and well characterized neurotrophin in developing and adult mammalian central nervous system [1,2,3]
Overexpressing nuclear respiratory factor 2 (NRF-2) up-regulated the expression of Bdnf exon IX, whereas knocking down NRF-2 down-regulated such expression. These findings are consistent with our hypothesis that NRF-2, in addition to regulating the coupling between neuronal activity and energy metabolism, regulates the expression of BDNF, which is intimately associated with energy-demanding neuronal activity
The regulation of BDNF expression by neuronal activity has received a great deal of attention, relatively little is known about the transcriptional regulation of the coding exon of the Bdnf gene, nor about transcription factor candidates that may play a significant role
Summary
Brain-derived neurotrophic factor (BDNF) is regarded as the most widely expressed, active, and well characterized neurotrophin in developing and adult mammalian central nervous system [1,2,3]. The Bdnf gene consists of nine exons, including eight non-coding exons and a single coding one (exon IX) These yield a single, identical, mature BDNF protein (reviewed in Ref. 18). The regulation of BDNF expression by neuronal activity has received a great deal of attention, relatively little is known about the transcriptional regulation of the coding exon of the Bdnf gene, nor about transcription factor candidates that may play a significant role. By means of a combination of bioinformatics, biochemical, and molecular biological approaches, including in silico analysis, electrophoretic mobility shift and supershift assays, realtime quantitative PCR (RT-qPCR), chromatin immunoprecipitation, promoter mutational analysis, overexpression, shRNA, functional assays, and Western blot analysis, we have documented that NRF-2 functionally regulates the transcription of exon IX of the Bdnf gene in rat cortical neurons
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