- Email: [email protected]
Identifying inducers of BDNF gene expression from pharmacologically validated compounds; antipyretic drug dipyrone increases BDNF mRNA in neurons
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Identifying inducers of BDNF gene expression from pharmacologically validated compounds; antipyretic drug dipyrone increases BDNF mRNA in neurons Mamoru Fukuchi Laboratory of Molecular Neuroscience, Faculty of Pharmacy, Takasaki University of Health and Welfare, 60 Nakaorui-machi, Takasaki-shi, Gunma, 3700033, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 January 2020 Accepted 5 February 2020 Available online xxx
Low levels of brain-derived neurotrophic factor (BDNF), a key regulator of synaptic plasticity, are associated with neurological diseases, including depression and Alzheimer’s disease. Therefore, BDNF is a drug target for these diseases. Here we screened for inducers of neuronal Bdnf expression from a pharmacologically validated compound library using our recently developed screening assay based on luciferase activity in cultured cortical neurons. We identified 18 pharmacologically validated compounds, most of which were inferred to induce Bdnf expression by their validated pharmacological actions, such as Gs-coupled receptor activation or neuronal excitation. Unexpectedly, the screening assay identified the antipyretic drug, dipyrone, to increase Bdnf expression. Dipyrone induced endogenous Bdnf expression by Ca2þ influx evoked via L-type voltage-dependent Ca2þ channels and the N-methyl-D-aspartate receptor, indicating that dipyrone induced activity-regulated Bdnf expression in neurons. However, dipyrone-induced Bdnf expression is independent of validated pharmacological effects. Although our screening assay is difficult to reveal how active compounds induce Bdnf expression, this method is convenient to identify inducers of Bdnf expression in primary neurons. Our screening assay evaluated neuronal BDNF induction and can be used to screen for drug re-positioning, as well as novel candidate drugs, for neurological diseases that have low levels of BDNF in the brain. © 2020 Elsevier Inc. All rights reserved.
Keywords: BDNF Dipyrone Drug re-positioning Luciferase Screening
1. Introduction Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin family and plays fundamental roles in central nervous system processes, such as learning and memory [1]. In addition, brain BDNF is associated with neurological diseases. For example, BDNF levels are reduced in postmortem Alzheimer’s disease brains [2]. In addition, higher expression levels of BDNF in postmortem brains are associated with slower aging-related cognitive decline [3]. BDNF levels are also elevated in postmortem brains of major depressive disorder patients who were treated with antidepressant medication [4]. Thus, high levels of BDNF in the brain appear to be
Abbreviations: BDNF, brain-derived neurotrophic factor; Bdnf-Luc, Bdnf-Luciferase; CB1, cannabiloid receptor type 1; COX, cyclooxygenase; CREB, cAMP-response element-binding protein; CRTC1, CREB-regulated transcriptional coactivator 1; GPCR, G protein-coupled receptor; L-VDCC, L-type voltage-dependent Ca2þ channel; NMDAR, N-methyl-D-aspartate receptor. E-mail address: [email protected].
beneficial for preserving cognitive functions but are also associated with the progression of neurological diseases. We previously generated a novel transgenic mouse strain, termed Bdnf-Luciferase (Bdnf-Luc), to measure changes in Bdnf expression in vitro [5] and in vivo [6]. In addition, we recently developed a screening assay to identify inducers of neuronal Bdnf expression using primary cultures of cortical neurons prepared from Bdnf-Luc mouse embryos [7]. The aim of this study was to identify inducers of Bdnf expression in neurons from a pharmacologically validated compound library, as part of a drug repositioning strategy to re-develop drugs for use in a different disease. We evaluated the ability of 1280 validated compounds to induce Bdnf expression in neurons. We found that an antipyretic drug, dipyrone, increased activity-regulated Bdnf expression. Although dipyrone is known as an analgesic non-opioid drug, it may have beneficial effects on symptoms of neurological diseases that have decreased levels of BDNF in the brain. Thus, our screening assay to identify inducers of BDNF expression is useful for drug re-positioning.
https://doi.org/10.1016/j.bbrc.2020.02.019 0006-291X/© 2020 Elsevier Inc. All rights reserved.
Please cite this article as: M. Fukuchi, Identifying inducers of BDNF gene expression from pharmacologically validated compounds; antipyretic drug dipyrone increases BDNF mRNA in neurons, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2020.02.019
2
M. Fukuchi / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
2. Materials and methods 2.1. Animals All animal care and experimental protocols were approved by the Animal Experiment Committee of Takasaki University of Health and Welfare (Authorization No. 1733, 1809, 1913), and were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of Takasaki University of Health and Welfare. Mice were housed under standard laboratory conditions (12 he12 h/light-dark cycle, 22 ± 2 C) and had free access to food and water. The generation of Bdnf-Luc mice has been described previously [5]. Sprague-Dawley rats at embryonic day 17 were purchased from Japan SLC (Shizuoka, Japan). 2.2. Reagents and library Dipyrone, aspirin, ibuprofen, and phenacetin were purchased from Santa Cruz Biotechnology. (Dallas, TX, USA). Nicardipine, D-APV, 4aminoantipyrine, and 4-methylaminoantipyrine were purchased from Sigma-Aldrich (St. Louis, MO, USA). (þ)-WIN 55,212-2 mesylate was purchased from Calbiochem (San Diego, CA, USA). Pharmacologically validated compound libraries were kindly donated by Drug Discovery Initiative (The University of Tokyo, Tokyo, Japan). 2.3. Screening assay A screening assay using primary cultures of cortical neurons prepared from Bdnf-Luc embryos was performed as described previously [7]. Briefly, dissociated cortical neurons were seeded and cultured in poly-L-lysine-coated 96-well culture plates. At 13 days in culture, neurons were treated with each compound (final concentration: 10 mM) for 6 h, and the luciferase activity of each well was then measured using the Steady-Glo Luciferase Assay System (Promega, Madison, WI, USA).
luciferase activity of vehicle (DMSO)-treated neurons to that of test compound-treated neurons. Compounds that increased luciferase activity by more than 2-fold were considered active compounds, as previously reported [7]. As shown in Fig. 1, we identified that 18 compounds that increased luciferase activity. Among these active compounds, apomorphine acts as a dopamine agonist, and fenoterol, dobutamine, metaproterenol, isoproterenol, ractopamine, and formoterol are b adrenergic agonists. We previously showed that activation of Gs- or Gq-coupled G protein-coupled receptors (GPCRs) increases Bdnf expression through an N-methyl-D-aspartate receptor (NMDAR)/calcineurin/cAMP-response elementbinding protein (CREB)-regulated transcriptional coactivator 1(CRTC1)/CREB-dependent pathway [5], indicating that these compounds identified here induce Bdnf expression via this pathway. Furthermore, in our recent study, we identified apomorphine, dobutamine, methylergometrine, gabazine, metaproterenol, picrotoxinin, aconitine, and isoproterenol as active agents using commercially available neurotransmitter libraries and herbal medicine-derived compound library [7]. Bdnf expression is controlled, in part, by the cholinergic system [8], supporting our finding that cholinergic agonists, carbachol and methacholine (Fig. 1B), induced Bdnf expression. We also found that the GABA antagonists, gabazine, picrotoxin, and thiocolchicoside, were active compounds (Fig. 1B). Suppression of the inhibitory action of GABA in mature neurons results in induction of activity-regulated Bdnf expression [9]; therefore, it is plausible that gabazine, picrotoxin, and thiocolchicoside induce activity-regulated Bdnf expression. Resibufogenin is an inhibitor of Naþ/Kþ-ATPase (Fig. 1B) that induces membrane depolarization [10], suggesting that resibufogenin-induced depolarization caused activity-regulated Bdnf expression in neurons. Although several compounds decreased luciferase activity (Fig. 1A), this decrease would be due to the reduction of cell viability, as previously reported [7]. 3.2. Dipyrone increases endogenous Bdnf expression in cultured cortical neurons
2.4. RT-PCR Primary cultures of rat cortical neurons were prepared from embryonic day 17 Sprague-Dawley rats, as described previously [5]. Briefly, dissociated cortical neurons were seeded and cultured in poly-L-lysine-coated 6-well plates. At 13 days in culture, neurons were treated with dipyrone or other reagents, and RT-PCR was carried out, as described previously [7]. Detailed experimental schedules are described in the figure legends. 2.5. Statistics All data are presented as the mean ± the standard error of the mean (S.E.M.). Statistical analyses were performed using Prism 7 software (GraphPad, San Diego, CA, USA). Detailed information is shown in the figure legends. 3. Results 3.1. Screening for Bdnf inducers from a validated compound library We recently constructed a screening assay for identifying inducers of Bdnf expression using primary cultures of cortical neurons prepared from Bdnf-Luc mice embryos [7]. Here, we used this screening assay to identify pharmacologically validated compounds that increase Bdnf expression in neurons. We used a library consisting of 1280 validated compounds. Cortical neurons prepared from Bdnf-Luc mice were cultured for 13 days, and then the neurons were treated with each compound for 6 h. We compared the
Most active compounds identified by our screening assay were inferred to activate Bdnf expression by their validated pharmacological actions, such as activation of Gs-coupled receptors or enhancement of neuronal excitation. However, several active compounds identified by the screening assay (dipyrone, digoxigenin, and vinorelbine) could not be inferred to activate Bdnf expression by their actions (Fig. 1B). Among them, we here focused on the effects of dipyrone, a cyclooxygenase (COX) inhibitor, on endogenous Bdnf expression in primary cultures of rat cortical neurons. As shown in Fig. 2A, the levels of Bdnf mRNA increased in a dipyrone dose-dependent manner. Levels of Bdnf mRNA increased in response to 50 mM dipyrone and peaked after 3 h (Fig. 2B). The increased mRNA levels were observed after dipyrone treatment for 6 h, but not for 1 h or 12 h (Fig. 2B). Rodent Bdnf gene consists of nine exons, and promoters are located upstream of each exon [11]. Because of these multiple promoters and alternative splicing, a number of Bdnf transcripts are synthesized [11]. We examined which 5’ exon-specific Bdnf mRNAs were increased by dipyrone treatment in cultured rat cortical neurons. As shown in Fig. 2C, expression levels of exon I-, exon II-, exon IV-, exon VI-, and exon IXA-containing Bdnf mRNAs were significantly increased by dipyrone. Expression levels of exon III-, exon V-, and exon VIII-containing mRNAs were low and those of exon VII-containing mRNA were barely detectable (Fig. S1). Among these transcripts, the expression levels of exon IV-containing Bdnf mRNA were highly induced by dipyrone (Fig. 2C). Bdnf promoter IV, which controls transcription of exon IVcontaining Bdnf mRNA, is a main promoter regulating neuronal
Please cite this article as: M. Fukuchi, Identifying inducers of BDNF gene expression from pharmacologically validated compounds; antipyretic drug dipyrone increases BDNF mRNA in neurons, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2020.02.019
M. Fukuchi / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
3
Fig. 1. Screening for inducers of neuronal Bdnf expression from a pharmacologically validated compound library. (A) After culture for 13 days, primary cultures of cortical neurons prepared from Bdnf-Luc mice were treated with each compound at a final concentration of 10 mM for 6 h. Luciferase activity in each well was then measured. Compared to luciferase activity of vehicle (DMSO)-treated neurons, compounds that increased luciferase activity by more than 2-fold (shown as black circle) were considered active compounds. Indicated numbers correspond to those in Fig. 1B. (B) Active compounds screened from a validated compound library that induce Bdnf expression.
activity-dependent Bdnf expression [12,13]; therefore, we predicted that dipyrone would activate Bdnf expression mediated by Ca2þ signals. To address this, we examined effects of nicardipine, an Ltype voltage-dependent Ca2þ channel (L-VDCC) blocker and APV, an NMDAR antagonist, on the dipyrone-induced Bdnf expression. As shown in Fig. 2D, dipyrone-induced Bdnf expression was significantly suppressed by nicardipine and APV. In particular, APV completely blocked the induction. These results indicated that dipyrone activates Ca2þ signaling pathways evoked via L-VDCC and NMDAR, resulting in the induction of Bdnf expression.
3.3. Neither COX inhibition nor active dipyrone metabolites contribute to the induction of Bdnf expression We next investigated how dipyrone affects Bdnf induction. Dipyrone mainly inhibits COX-3 and COX-1, but not COX-2 [14]. To examine whether the effect of dipyrone on Bdnf expression results from inhibition of COXs, we used several COX inhibitors: aspirin (a COX-1 and COX-2 inhibitor), ibuprofen (a COX-1, COX-2, and COX-3 inhibitor), and phenacetin (a COX-3 inhibitor) [14]. Although dipyrone increased levels of Bdnf mRNA in a dipyrone dose-dependent
Please cite this article as: M. Fukuchi, Identifying inducers of BDNF gene expression from pharmacologically validated compounds; antipyretic drug dipyrone increases BDNF mRNA in neurons, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2020.02.019
4
M. Fukuchi / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
Fig. 2. Upregulation of Bdnf expression by dipyrone in cultured cortical neurons. (A) Dipyrone increased Bdnf mRNA levels in a dose-dependent manner. Neurons were treated with dipyrone at the indicated concentrations for 3 h, and changes in Bdnf mRNA levels were examined by RT-PCR. Means ± SEM (n ¼ 3), ***p < 0.001 and ****p < 0.0001 vs. 0 mM (one-way ANOVA with Dunnett’s multiple comparisons test). (B) Time-course of changes in Bdnf mRNA levels after dipyrone treatment. Neurons were treated with 50 mM dipyrone, and total RNA was prepared at the indicated time points. Means ± SEM (n ¼ 3), *p < 0.05 and ****p < 0.0001 vs. DMSO at the same time point (two-way ANOVA with Tukey’s multiple comparisons test). (C) Effect of dipyrone on expression of 50 exon-specific Bdnf mRNAs. Neurons were treated with 50 mM dipyrone, and total RNA was prepared 3 h after the treatment. Means ± SEM (n ¼ 3), *p < 0.05, **p < 0.01, and ***p < 0.001 vs. DMSO (unpaired t-test). (D) Effects of nicardipine and APV on dipyrone-induced Bdnf expression. Nicardipine (Nica, 5 mM) or APV (200 mM) was added 10 min before dipyrone treatment. Total RNA was prepared 3 h after dipyrone treatment for RT-PCR. Means ± SEM (n ¼ 6), ****p < 0.0001 vs. DMSO/Vehicle, yyyyp < 0.0001 vs. Dipyrone/Vehicle (two-way ANOVA with Tukey’s multiple comparisons test).
manner (Fig. 3A), other COX inhibitors did not significantly affect Bdnf expression at the same concentrations (Fig. 3B), indicating that COX inhibition did not increase Bdnf expression. 4-aminoantipyrine and 4-methylaminoantipyrine are active metabolites of dipyrone [15]. Therefore, we also examined whether these active metabolites of dipyrone affect Bdnf expression. However, these metabolites did not significantly affect levels of Bdnf mRNA (Fig. 3C). Taken together, dipyrone-induced Bdnf expression is independent of COX inhibition and active dipyrone metabolites. Previously, it has been reported that endocannabinoid systems, CB1 receptors in particular, are involved in the effects of dipyrone
[16]. Therefore, we next investigated whether activation of CB1 affects Bdnf expression in neurons. As shown in Fig. 4, CB1 agonist WIN 55,212-2 significantly decreased Bdnf mRNA expression in a dose-dependent manner. Because activation of CB1 negatively regulates Bdnf expression in neurons, it is suggested that CB1 did not contribute to the dipyrone-induced Bdnf expression. 4. Discussion In this study, we identified several pharmacologically validated compounds that upregulate Bdnf expression using a recently
Please cite this article as: M. Fukuchi, Identifying inducers of BDNF gene expression from pharmacologically validated compounds; antipyretic drug dipyrone increases BDNF mRNA in neurons, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2020.02.019
M. Fukuchi / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
5
Fig. 3. Effects of cyclooxygenase inhibitors or active dipyrone metabolites on expression levels of Bdnf in cultured cortical neurons. After 13 days in culture, dipyrone (A), COX inhibitors [B, COXi (aspirin, ibuprofen, and phenacetin)], or active dipyrone metabolites (C, 4-aminoantipyrine and 4methylaminoantipyrine), were added at the indicated concentrations. Total RNA was prepared 3 h after the addition, and changes in Bdnf mRNA were examined by RT-PCR. Means ± SEM (n ¼ 3), **p < 0.01 and ****p < 0.0001 vs. 0 mM (one-way ANOVA with Dunnett’s multiple comparisons test).
Fig. 4. Effects of canncabinoid CB1 agonist on expression levels of Bdnf in cultured cortical neurons. After 13 days in culture, WIN 55,212-2, cannabinoid CB1 agonist, was added at the indicated concentrations. Total RNA was prepared 3 h after the addition, and changes in Bdnf mRNA were examined by RT-PCR. Means ± SEM (n ¼ 3), *p < 0.05 and **p < 0.01 vs. 0 mM (one-way ANOVA with Dunnett’s multiple comparisons test).
developed screening assay comprising primary cultures of cortical neurons prepared from Bdnf-Luc mouse embryos [7]. Among these compounds, we focused on dipyrone, a COX inhibitor, and confirmed that dipyrone increased endogenous Bdnf expression in cultured cortical neurons. Among the active compounds identified using the screening assay, most were b adrenergic agonists. Considering previous
findings regarding Gs- or Gq-coupled GPCR signaling-induced Bdnf expression through an NMDAR/calcineurin/CRTC1/CREBdependent pathway [5], it is plausible that b adrenergic agonists increase Bdnf expression via Gs-coupled b adrenergic receptors. b adrenergic agonists reduced expression of a-synuclein, excess expression of which is thought to be a risk factor in Parkinson’s disease [17,18], and a clinically used b adrenergic agonist, salbutamol, is associated with lower risk of Parkinson’s disease [19]. In addition, reduced BDNF expression has been reported in the substantia nigra in Parkinson’s disease [20]. These findings and those of the present study indicate that b adrenergic agonists may exert protective effects in the Parkinson’s disease brain by reducing asynuclein and inducing BDNF expression. We also found that the analgesic non-opioid drug, dipyrone, increased endogenous Bdnf expression in cultured cortical neurons. We examined effects of COX inhibition and active dipyrone metabolites on Bdnf expression but detected no increase in Bdnf expression. The analgesic effects of dipyrone are mediated via cannabinoid CB1 receptors [16]. CB1 is Gi/o-copuled GPCR and widely expressed in axon terminals of the nervous systems, and activation of CB1 causes inhibition of glutamatergic and cholinergic neurotransmitter release [21]. In support of the inhibitory effects of CB1 on neurotransmissions, we found that CB1 agonist significantly decreased the levels of Bdnf mRNA in neurons. In addition, our present results strongly indicate that dipyrone increased Bdnf expression by Ca2þ signals evoked via L-VDCC and NMDAR. This supports the idea that CB1 is not involved in dipyrone-induced Bdnf expression. On the other hand, a series of previous reports suggested that activity-dependent Bdnf expression would be observed within 1 h [22,23]. Although the dipyrone-induced Bdnf expression is likely activity-dependent manner, the induction was observed after dipyrone treatment for 3 h, but not 1 h. Therefore, it is likely that dipyrone indirectly induces neuronal activity, resulting in the delayed induction of Bdnf expression via L-VDCC and NMDAR. Further investigations are necessary to uncover the molecular mechanisms underlying the dipyrone-induced Bdnf expression in neurons. Depressive behaviors in mice induced by unpredictable chronic mild stress are suppressed by chronic administration of dipyrone
Please cite this article as: M. Fukuchi, Identifying inducers of BDNF gene expression from pharmacologically validated compounds; antipyretic drug dipyrone increases BDNF mRNA in neurons, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2020.02.019
6
M. Fukuchi / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
[24], indicating that dipyrone has an anti-depressant-like effect. Levels of BDNF are reduced in the brains of depression patients, and BDNF levels increase in patients treated with antidepressant medications [25]. Furthermore, brain BDNF levels are reduced in some neurological disease cases [26]. Therefore, dipyrone may have beneficial and protective effects on neurological diseases that have reduced BDNF levels in the brain, although it should be demonstrated whether Bdnf expression in the brain is increased by administration of dipyrone in vivo. Although our screening assay is difficult to demonstrate how active compound induces Bdnf expression, this method is convenient to identify inducers of Bdnf expression in primary neuronal cells. Numerous studies support the idea that enhancing Bdnf expression could be beneficial for neurodegenerative and neuropsychiatric diseases. Our screening assay using Bdnf-Luc cortical neurons is applicable for mining inducers of Bdnf expression from pharmacologically validated compound libraries, as the first screen of drug re-positioning, as well as for finding novel candidates for ameliorating neurological disorders that have low levels of BDNF in the brain, such as dementia, depression, and so on.
[5]
[6]
[7]
[8]
[9]
[10]
Declaration of competing interest [11]
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This work was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Nos. JP25870256 [Grant-in-Aid for Young Scientists (B) to M.F.], 16K12894 (Grant-in-Aid for Challenging Exploratory Research to M.F.), and 16H05275 [Grant-in- Aid for Scientific Research (B) to M.F.], the Takeda Science Foundation (to M.F.), the Mochida Memorial Foundation for Medical and Pharmaceutical Research (to M.F.), a Hokugin Research Grant (to M.F.), and a research grant from the Toyama First Bank Foundation (to M.F.). We appreciate the contribution of members of Professor Mori’s laboratory (Department of Molecular Neuroscience, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama) in the generation of Bdnf-Luc mice. This work was supported by the Platform Project for Supporting in Drug Discovery and Life Science Research from the Japan Agency for Medical Research and Development (AMED). We thank Jeremy Allen, PhD, from Edanz Group (www.edanzediting.com/ac) for editing a draft of this manuscript.
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
Appendix A. Supplementary data [21]
Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2020.02.019. References [1] M. Miranda, J.F. Morici, M.B. Zanoni, et al., Brain-derived neurotrophic factor: a key molecule for memory in the healthy and the pathological brain, Front. Cell. Neurosci. 13 (2019) 363, https://doi.org/10.3389/fncel.2019.00363. [2] Y. Du, H.T. Wu, X.Y. Qin, et al., Postmortem brain, cerebrospinal fluid, and blood neurotrophic factor levels in Alzheimer’s disease: a systematic review and meta-analysis, J. Mol. Neurosci. (2018) 289e300, https://doi.org/10.1007/ s12031-018-1100-8. [3] A.S. Buchman, L. Yu, P.A. Boyle, et al., Higher brain BDNF gene expression is associated with slower cognitive decline in older adults, Neurology 86 (2016) 735e741, https://doi.org/10.1212/WNL.0000000000002387. [4] A. Sheldrick, S. Camara, M. Ilieva, et al., Brain-derived neurotrophic factor (BDNF) and neurotrophin 3 (NT3) levels in post-mortem brain tissue from
[22]
[23]
[24]
[25]
[26]
patients with depression compared to healthy individuals - a proof of concept study, Eur. Psychiatr. 46 (2017) 65e71, https://doi.org/10.1016/ j.eurpsy.2017.06.009. M. Fukuchi, A. Tabuchi, Y. Kuwana, et al., Neuromodulatory effect of Gas- or Gaq-coupled G-protein-coupled receptor on NMDA receptor selectively activates the NMDA receptor/Ca2þ/calcineurin/cAMP response element-binding protein-regulated transcriptional coactivator 1 pathway to effectively induce brain-derived neurotrophic factor expression in neurons, J. Neurosci. 35 (2015) 5606e5624, https://doi.org/10.1523/JNEUROSCI.3650-14.2015. M. Fukuchi, H. Izumi, H. Mori, et al., Visualizing changes in brain-derived neurotrophic factor (BDNF) expression using bioluminescence imaging in living mice, Sci. Rep. 7 (2017) 4949, https://doi.org/10.1038/s41598-01705297-x. M. Fukuchi, Y. Okuno, H. Nakayama, et al., Screening inducers of neuronal BDNF gene transcription using primary cortical cell cultures from BDNFluciferase transgenic mice, Sci. Rep. 9 (2019) 11833, https://doi.org/10.1038/ s41598-019-48361-4. n, et al., Cholinergic regulation of M. da Penha Berzaghi, J. Cooper, E. Castre brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) but not neurotrophin-3 (NT-3) mRNA levels in the developing rat hippocampus, J. Neurosci. 13 (1993) 3818e3826, https://doi.org/10.1523/JNEUROSCI.13-0903818.1993. M. Fukuchi, Y. Kirikoshi, A. Mori, et al., Excitatory GABA induces BDNF transcription via CRTC1 and phosphorylated CREB-related pathways in immature cortical cells, J. Neurochem. 131 (2014) 134e146, https://doi.org/10.1111/ jnc.12801. Z.J. Wang, L. Sun, T. Heinbockel, Resibufogenin and cinobufagin activate central neurons through an ouabain-like action, PloS One 9 (2014), e113272, https://doi.org/10.1371/journal.pone.0113272. T. Aid, A. Kazantseva, M. Piirsoo, et al., Mouse and rat BDNF gene structure and expression revisited, J. Neurosci. Res. 85 (2007) 525e535, https://doi.org/ 10.1002/jnr.21139. E.J. Hong, A.E. McCord, M.E. Greenberg, A biological function for the neuronal activity-dependent component of Bdnf transcription in the development of cortical inhibition, Neuron 60 (2008) 610e624, https://doi.org/10.1016/ j.neuron.2008.09.024. K. Sakata, N.H. Woo, K. Martinowich, et al., Critical role of promoter IV-driven BDNF transcription in GABAergic transmission and synaptic plasticity in the prefrontal cortex, Proc. Natl. Acad. Sci. U. S. A 106 (2009) 5942e5947, https:// doi.org/10.1073/pnas.0811431106. N.V. Chandrasekharan, H. Dai, K.L. Roos, et al., COX-3, a cyclooxygenase-1 variant inhibited by acetaminophen and other analgesic/antipyretic drugs: cloning, structure, and expression, Proc. Natl. Acad. Sci. U. S. A 99 (2002) 13926e13931, https://doi.org/10.1073/pnas.162468699. A. Jasiecka, T. Maslanka, J.J. Jaroszewski, Pharmacological characteristics of metamizole, Pol. J. Vet. Sci. 17 (2014) 207e214. https://10.2478/pjvs-20140030. F. Crunfli, F.C. Vilela, A. Giusti-Paiva, Cannabinoid CB1 receptors mediate the effects of dipyrone, Clin. Exp. Pharmacol. Physiol. 42 (2015) 246e255, https:// doi.org/10.1111/1440-1681.12347. A.B. Singleton, M. Farrer, J. Johnson, et al., alpha-Synuclein locus triplication causes Parkinson’s disease, Science 302 (2003) 841, https://doi.org/10.1126/ science.1090278. M. Farrer, J. Kachergus, L. Forno, et al., Comparison of kindreds with parkinsonism and alpha-synuclein genomic multiplications, Ann. Neurol. 55 (2004) 174e179, https://doi.org/10.1002/ana.10846. S. Mittal, K. Bjørnevik, D.S. Im, et al., b2-Adrenoreceptor is a regulator of the asynuclein gene driving risk of Parkinson’s disease, Science 357 (2017) 891e898, https://doi.org/10.1126/science.aaf3934. M. Mogi, A. Togari, T. Kondo, et al., Brain-derived growth factor and nerve growth factor concentrations are decreased in the substantia nigra in Parkinson’s disease, Neurosci. Lett. 270 (1999) 45e48, https://doi.org/10.1016/ S0304-3940(99)00463-2. A. Busquets-Garcia, J. Bains, G. Marsicano, CB1 receptor signaling in the brain: extracting specificity from ubiquity, Neuropsychopharmacology 43 (2018) 4e20, https://doi.org/10.1038/npp.2017.206. X. Tao, S. Finkbeiner, D.B. Arnold, et al., Ca2þ influx regulates BDNF transcription by a CREB family transcription factor-dependent mechanism, Neuron 20 (1998) 709e726, https://doi.org/10.1016/S0896-6273(00)81010-7. X. Tao, A.E. West, W.G. Chen, et al., A calcium-responsive transcription factor, CaRF, that regulates neuronal activity-dependent expression of BDNF, Neuron 33 (2002) 383e395, https://doi.org/10.1016/S0896-6273(01)00561-X. lu, K.E. Demirkol, F. Berktas¸, et al., Dipyrone ameliorates behavioural O.E. Kirog changes induced by unpredictable chronic mild stress: gender differences, Clin. Invest. Med. 39 (2016) 27494, https://doi.org/10.25011/cim.v39i6.27494. B. Chen, D. Dowlatshahi, G.M. MacQueen, et al., Increased hippocampal BDNF immunoreactivity in subjects treated with antidepressant medication. https:// doi.org/10.1016/S0006-3223(01)01083-6, 2001. T. Numakawa, H. Odaka, N. Adachi, Actions of brain-derived neurotrophin factor in the neurogenesis and neuronal function, and its involvement in the pathophysiology of brain diseases, Int. J. Mol. Sci. 19 (2018), https://doi.org/ 10.3390/ijms19113650 pii: E3650.
Please cite this article as: M. Fukuchi, Identifying inducers of BDNF gene expression from pharmacologically validated compounds; antipyretic drug dipyrone increases BDNF mRNA in neurons, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2020.02.019
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Identifying inducers of BDNF gene expression from pharmacologically validated compounds; antipyretic drug dipyrone increases BDNF mRNA in neurons Mamoru Fukuchi Laboratory of Molecular Neuroscience, Faculty of Pharmacy, Takasaki University of Health and Welfare, 60 Nakaorui-machi, Takasaki-shi, Gunma, 3700033, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 January 2020 Accepted 5 February 2020 Available online xxx
Low levels of brain-derived neurotrophic factor (BDNF), a key regulator of synaptic plasticity, are associated with neurological diseases, including depression and Alzheimer’s disease. Therefore, BDNF is a drug target for these diseases. Here we screened for inducers of neuronal Bdnf expression from a pharmacologically validated compound library using our recently developed screening assay based on luciferase activity in cultured cortical neurons. We identified 18 pharmacologically validated compounds, most of which were inferred to induce Bdnf expression by their validated pharmacological actions, such as Gs-coupled receptor activation or neuronal excitation. Unexpectedly, the screening assay identified the antipyretic drug, dipyrone, to increase Bdnf expression. Dipyrone induced endogenous Bdnf expression by Ca2þ influx evoked via L-type voltage-dependent Ca2þ channels and the N-methyl-D-aspartate receptor, indicating that dipyrone induced activity-regulated Bdnf expression in neurons. However, dipyrone-induced Bdnf expression is independent of validated pharmacological effects. Although our screening assay is difficult to reveal how active compounds induce Bdnf expression, this method is convenient to identify inducers of Bdnf expression in primary neurons. Our screening assay evaluated neuronal BDNF induction and can be used to screen for drug re-positioning, as well as novel candidate drugs, for neurological diseases that have low levels of BDNF in the brain. © 2020 Elsevier Inc. All rights reserved.
Keywords: BDNF Dipyrone Drug re-positioning Luciferase Screening
1. Introduction Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin family and plays fundamental roles in central nervous system processes, such as learning and memory [1]. In addition, brain BDNF is associated with neurological diseases. For example, BDNF levels are reduced in postmortem Alzheimer’s disease brains [2]. In addition, higher expression levels of BDNF in postmortem brains are associated with slower aging-related cognitive decline [3]. BDNF levels are also elevated in postmortem brains of major depressive disorder patients who were treated with antidepressant medication [4]. Thus, high levels of BDNF in the brain appear to be
Abbreviations: BDNF, brain-derived neurotrophic factor; Bdnf-Luc, Bdnf-Luciferase; CB1, cannabiloid receptor type 1; COX, cyclooxygenase; CREB, cAMP-response element-binding protein; CRTC1, CREB-regulated transcriptional coactivator 1; GPCR, G protein-coupled receptor; L-VDCC, L-type voltage-dependent Ca2þ channel; NMDAR, N-methyl-D-aspartate receptor. E-mail address: [email protected].
beneficial for preserving cognitive functions but are also associated with the progression of neurological diseases. We previously generated a novel transgenic mouse strain, termed Bdnf-Luciferase (Bdnf-Luc), to measure changes in Bdnf expression in vitro [5] and in vivo [6]. In addition, we recently developed a screening assay to identify inducers of neuronal Bdnf expression using primary cultures of cortical neurons prepared from Bdnf-Luc mouse embryos [7]. The aim of this study was to identify inducers of Bdnf expression in neurons from a pharmacologically validated compound library, as part of a drug repositioning strategy to re-develop drugs for use in a different disease. We evaluated the ability of 1280 validated compounds to induce Bdnf expression in neurons. We found that an antipyretic drug, dipyrone, increased activity-regulated Bdnf expression. Although dipyrone is known as an analgesic non-opioid drug, it may have beneficial effects on symptoms of neurological diseases that have decreased levels of BDNF in the brain. Thus, our screening assay to identify inducers of BDNF expression is useful for drug re-positioning.
https://doi.org/10.1016/j.bbrc.2020.02.019 0006-291X/© 2020 Elsevier Inc. All rights reserved.
Please cite this article as: M. Fukuchi, Identifying inducers of BDNF gene expression from pharmacologically validated compounds; antipyretic drug dipyrone increases BDNF mRNA in neurons, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2020.02.019
2
M. Fukuchi / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
2. Materials and methods 2.1. Animals All animal care and experimental protocols were approved by the Animal Experiment Committee of Takasaki University of Health and Welfare (Authorization No. 1733, 1809, 1913), and were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of Takasaki University of Health and Welfare. Mice were housed under standard laboratory conditions (12 he12 h/light-dark cycle, 22 ± 2 C) and had free access to food and water. The generation of Bdnf-Luc mice has been described previously [5]. Sprague-Dawley rats at embryonic day 17 were purchased from Japan SLC (Shizuoka, Japan). 2.2. Reagents and library Dipyrone, aspirin, ibuprofen, and phenacetin were purchased from Santa Cruz Biotechnology. (Dallas, TX, USA). Nicardipine, D-APV, 4aminoantipyrine, and 4-methylaminoantipyrine were purchased from Sigma-Aldrich (St. Louis, MO, USA). (þ)-WIN 55,212-2 mesylate was purchased from Calbiochem (San Diego, CA, USA). Pharmacologically validated compound libraries were kindly donated by Drug Discovery Initiative (The University of Tokyo, Tokyo, Japan). 2.3. Screening assay A screening assay using primary cultures of cortical neurons prepared from Bdnf-Luc embryos was performed as described previously [7]. Briefly, dissociated cortical neurons were seeded and cultured in poly-L-lysine-coated 96-well culture plates. At 13 days in culture, neurons were treated with each compound (final concentration: 10 mM) for 6 h, and the luciferase activity of each well was then measured using the Steady-Glo Luciferase Assay System (Promega, Madison, WI, USA).
luciferase activity of vehicle (DMSO)-treated neurons to that of test compound-treated neurons. Compounds that increased luciferase activity by more than 2-fold were considered active compounds, as previously reported [7]. As shown in Fig. 1, we identified that 18 compounds that increased luciferase activity. Among these active compounds, apomorphine acts as a dopamine agonist, and fenoterol, dobutamine, metaproterenol, isoproterenol, ractopamine, and formoterol are b adrenergic agonists. We previously showed that activation of Gs- or Gq-coupled G protein-coupled receptors (GPCRs) increases Bdnf expression through an N-methyl-D-aspartate receptor (NMDAR)/calcineurin/cAMP-response elementbinding protein (CREB)-regulated transcriptional coactivator 1(CRTC1)/CREB-dependent pathway [5], indicating that these compounds identified here induce Bdnf expression via this pathway. Furthermore, in our recent study, we identified apomorphine, dobutamine, methylergometrine, gabazine, metaproterenol, picrotoxinin, aconitine, and isoproterenol as active agents using commercially available neurotransmitter libraries and herbal medicine-derived compound library [7]. Bdnf expression is controlled, in part, by the cholinergic system [8], supporting our finding that cholinergic agonists, carbachol and methacholine (Fig. 1B), induced Bdnf expression. We also found that the GABA antagonists, gabazine, picrotoxin, and thiocolchicoside, were active compounds (Fig. 1B). Suppression of the inhibitory action of GABA in mature neurons results in induction of activity-regulated Bdnf expression [9]; therefore, it is plausible that gabazine, picrotoxin, and thiocolchicoside induce activity-regulated Bdnf expression. Resibufogenin is an inhibitor of Naþ/Kþ-ATPase (Fig. 1B) that induces membrane depolarization [10], suggesting that resibufogenin-induced depolarization caused activity-regulated Bdnf expression in neurons. Although several compounds decreased luciferase activity (Fig. 1A), this decrease would be due to the reduction of cell viability, as previously reported [7]. 3.2. Dipyrone increases endogenous Bdnf expression in cultured cortical neurons
2.4. RT-PCR Primary cultures of rat cortical neurons were prepared from embryonic day 17 Sprague-Dawley rats, as described previously [5]. Briefly, dissociated cortical neurons were seeded and cultured in poly-L-lysine-coated 6-well plates. At 13 days in culture, neurons were treated with dipyrone or other reagents, and RT-PCR was carried out, as described previously [7]. Detailed experimental schedules are described in the figure legends. 2.5. Statistics All data are presented as the mean ± the standard error of the mean (S.E.M.). Statistical analyses were performed using Prism 7 software (GraphPad, San Diego, CA, USA). Detailed information is shown in the figure legends. 3. Results 3.1. Screening for Bdnf inducers from a validated compound library We recently constructed a screening assay for identifying inducers of Bdnf expression using primary cultures of cortical neurons prepared from Bdnf-Luc mice embryos [7]. Here, we used this screening assay to identify pharmacologically validated compounds that increase Bdnf expression in neurons. We used a library consisting of 1280 validated compounds. Cortical neurons prepared from Bdnf-Luc mice were cultured for 13 days, and then the neurons were treated with each compound for 6 h. We compared the
Most active compounds identified by our screening assay were inferred to activate Bdnf expression by their validated pharmacological actions, such as activation of Gs-coupled receptors or enhancement of neuronal excitation. However, several active compounds identified by the screening assay (dipyrone, digoxigenin, and vinorelbine) could not be inferred to activate Bdnf expression by their actions (Fig. 1B). Among them, we here focused on the effects of dipyrone, a cyclooxygenase (COX) inhibitor, on endogenous Bdnf expression in primary cultures of rat cortical neurons. As shown in Fig. 2A, the levels of Bdnf mRNA increased in a dipyrone dose-dependent manner. Levels of Bdnf mRNA increased in response to 50 mM dipyrone and peaked after 3 h (Fig. 2B). The increased mRNA levels were observed after dipyrone treatment for 6 h, but not for 1 h or 12 h (Fig. 2B). Rodent Bdnf gene consists of nine exons, and promoters are located upstream of each exon [11]. Because of these multiple promoters and alternative splicing, a number of Bdnf transcripts are synthesized [11]. We examined which 5’ exon-specific Bdnf mRNAs were increased by dipyrone treatment in cultured rat cortical neurons. As shown in Fig. 2C, expression levels of exon I-, exon II-, exon IV-, exon VI-, and exon IXA-containing Bdnf mRNAs were significantly increased by dipyrone. Expression levels of exon III-, exon V-, and exon VIII-containing mRNAs were low and those of exon VII-containing mRNA were barely detectable (Fig. S1). Among these transcripts, the expression levels of exon IV-containing Bdnf mRNA were highly induced by dipyrone (Fig. 2C). Bdnf promoter IV, which controls transcription of exon IVcontaining Bdnf mRNA, is a main promoter regulating neuronal
Please cite this article as: M. Fukuchi, Identifying inducers of BDNF gene expression from pharmacologically validated compounds; antipyretic drug dipyrone increases BDNF mRNA in neurons, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2020.02.019
M. Fukuchi / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
3
Fig. 1. Screening for inducers of neuronal Bdnf expression from a pharmacologically validated compound library. (A) After culture for 13 days, primary cultures of cortical neurons prepared from Bdnf-Luc mice were treated with each compound at a final concentration of 10 mM for 6 h. Luciferase activity in each well was then measured. Compared to luciferase activity of vehicle (DMSO)-treated neurons, compounds that increased luciferase activity by more than 2-fold (shown as black circle) were considered active compounds. Indicated numbers correspond to those in Fig. 1B. (B) Active compounds screened from a validated compound library that induce Bdnf expression.
activity-dependent Bdnf expression [12,13]; therefore, we predicted that dipyrone would activate Bdnf expression mediated by Ca2þ signals. To address this, we examined effects of nicardipine, an Ltype voltage-dependent Ca2þ channel (L-VDCC) blocker and APV, an NMDAR antagonist, on the dipyrone-induced Bdnf expression. As shown in Fig. 2D, dipyrone-induced Bdnf expression was significantly suppressed by nicardipine and APV. In particular, APV completely blocked the induction. These results indicated that dipyrone activates Ca2þ signaling pathways evoked via L-VDCC and NMDAR, resulting in the induction of Bdnf expression.
3.3. Neither COX inhibition nor active dipyrone metabolites contribute to the induction of Bdnf expression We next investigated how dipyrone affects Bdnf induction. Dipyrone mainly inhibits COX-3 and COX-1, but not COX-2 [14]. To examine whether the effect of dipyrone on Bdnf expression results from inhibition of COXs, we used several COX inhibitors: aspirin (a COX-1 and COX-2 inhibitor), ibuprofen (a COX-1, COX-2, and COX-3 inhibitor), and phenacetin (a COX-3 inhibitor) [14]. Although dipyrone increased levels of Bdnf mRNA in a dipyrone dose-dependent
Please cite this article as: M. Fukuchi, Identifying inducers of BDNF gene expression from pharmacologically validated compounds; antipyretic drug dipyrone increases BDNF mRNA in neurons, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2020.02.019
4
M. Fukuchi / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
Fig. 2. Upregulation of Bdnf expression by dipyrone in cultured cortical neurons. (A) Dipyrone increased Bdnf mRNA levels in a dose-dependent manner. Neurons were treated with dipyrone at the indicated concentrations for 3 h, and changes in Bdnf mRNA levels were examined by RT-PCR. Means ± SEM (n ¼ 3), ***p < 0.001 and ****p < 0.0001 vs. 0 mM (one-way ANOVA with Dunnett’s multiple comparisons test). (B) Time-course of changes in Bdnf mRNA levels after dipyrone treatment. Neurons were treated with 50 mM dipyrone, and total RNA was prepared at the indicated time points. Means ± SEM (n ¼ 3), *p < 0.05 and ****p < 0.0001 vs. DMSO at the same time point (two-way ANOVA with Tukey’s multiple comparisons test). (C) Effect of dipyrone on expression of 50 exon-specific Bdnf mRNAs. Neurons were treated with 50 mM dipyrone, and total RNA was prepared 3 h after the treatment. Means ± SEM (n ¼ 3), *p < 0.05, **p < 0.01, and ***p < 0.001 vs. DMSO (unpaired t-test). (D) Effects of nicardipine and APV on dipyrone-induced Bdnf expression. Nicardipine (Nica, 5 mM) or APV (200 mM) was added 10 min before dipyrone treatment. Total RNA was prepared 3 h after dipyrone treatment for RT-PCR. Means ± SEM (n ¼ 6), ****p < 0.0001 vs. DMSO/Vehicle, yyyyp < 0.0001 vs. Dipyrone/Vehicle (two-way ANOVA with Tukey’s multiple comparisons test).
manner (Fig. 3A), other COX inhibitors did not significantly affect Bdnf expression at the same concentrations (Fig. 3B), indicating that COX inhibition did not increase Bdnf expression. 4-aminoantipyrine and 4-methylaminoantipyrine are active metabolites of dipyrone [15]. Therefore, we also examined whether these active metabolites of dipyrone affect Bdnf expression. However, these metabolites did not significantly affect levels of Bdnf mRNA (Fig. 3C). Taken together, dipyrone-induced Bdnf expression is independent of COX inhibition and active dipyrone metabolites. Previously, it has been reported that endocannabinoid systems, CB1 receptors in particular, are involved in the effects of dipyrone
[16]. Therefore, we next investigated whether activation of CB1 affects Bdnf expression in neurons. As shown in Fig. 4, CB1 agonist WIN 55,212-2 significantly decreased Bdnf mRNA expression in a dose-dependent manner. Because activation of CB1 negatively regulates Bdnf expression in neurons, it is suggested that CB1 did not contribute to the dipyrone-induced Bdnf expression. 4. Discussion In this study, we identified several pharmacologically validated compounds that upregulate Bdnf expression using a recently
Please cite this article as: M. Fukuchi, Identifying inducers of BDNF gene expression from pharmacologically validated compounds; antipyretic drug dipyrone increases BDNF mRNA in neurons, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2020.02.019
M. Fukuchi / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
5
Fig. 3. Effects of cyclooxygenase inhibitors or active dipyrone metabolites on expression levels of Bdnf in cultured cortical neurons. After 13 days in culture, dipyrone (A), COX inhibitors [B, COXi (aspirin, ibuprofen, and phenacetin)], or active dipyrone metabolites (C, 4-aminoantipyrine and 4methylaminoantipyrine), were added at the indicated concentrations. Total RNA was prepared 3 h after the addition, and changes in Bdnf mRNA were examined by RT-PCR. Means ± SEM (n ¼ 3), **p < 0.01 and ****p < 0.0001 vs. 0 mM (one-way ANOVA with Dunnett’s multiple comparisons test).
Fig. 4. Effects of canncabinoid CB1 agonist on expression levels of Bdnf in cultured cortical neurons. After 13 days in culture, WIN 55,212-2, cannabinoid CB1 agonist, was added at the indicated concentrations. Total RNA was prepared 3 h after the addition, and changes in Bdnf mRNA were examined by RT-PCR. Means ± SEM (n ¼ 3), *p < 0.05 and **p < 0.01 vs. 0 mM (one-way ANOVA with Dunnett’s multiple comparisons test).
developed screening assay comprising primary cultures of cortical neurons prepared from Bdnf-Luc mouse embryos [7]. Among these compounds, we focused on dipyrone, a COX inhibitor, and confirmed that dipyrone increased endogenous Bdnf expression in cultured cortical neurons. Among the active compounds identified using the screening assay, most were b adrenergic agonists. Considering previous
findings regarding Gs- or Gq-coupled GPCR signaling-induced Bdnf expression through an NMDAR/calcineurin/CRTC1/CREBdependent pathway [5], it is plausible that b adrenergic agonists increase Bdnf expression via Gs-coupled b adrenergic receptors. b adrenergic agonists reduced expression of a-synuclein, excess expression of which is thought to be a risk factor in Parkinson’s disease [17,18], and a clinically used b adrenergic agonist, salbutamol, is associated with lower risk of Parkinson’s disease [19]. In addition, reduced BDNF expression has been reported in the substantia nigra in Parkinson’s disease [20]. These findings and those of the present study indicate that b adrenergic agonists may exert protective effects in the Parkinson’s disease brain by reducing asynuclein and inducing BDNF expression. We also found that the analgesic non-opioid drug, dipyrone, increased endogenous Bdnf expression in cultured cortical neurons. We examined effects of COX inhibition and active dipyrone metabolites on Bdnf expression but detected no increase in Bdnf expression. The analgesic effects of dipyrone are mediated via cannabinoid CB1 receptors [16]. CB1 is Gi/o-copuled GPCR and widely expressed in axon terminals of the nervous systems, and activation of CB1 causes inhibition of glutamatergic and cholinergic neurotransmitter release [21]. In support of the inhibitory effects of CB1 on neurotransmissions, we found that CB1 agonist significantly decreased the levels of Bdnf mRNA in neurons. In addition, our present results strongly indicate that dipyrone increased Bdnf expression by Ca2þ signals evoked via L-VDCC and NMDAR. This supports the idea that CB1 is not involved in dipyrone-induced Bdnf expression. On the other hand, a series of previous reports suggested that activity-dependent Bdnf expression would be observed within 1 h [22,23]. Although the dipyrone-induced Bdnf expression is likely activity-dependent manner, the induction was observed after dipyrone treatment for 3 h, but not 1 h. Therefore, it is likely that dipyrone indirectly induces neuronal activity, resulting in the delayed induction of Bdnf expression via L-VDCC and NMDAR. Further investigations are necessary to uncover the molecular mechanisms underlying the dipyrone-induced Bdnf expression in neurons. Depressive behaviors in mice induced by unpredictable chronic mild stress are suppressed by chronic administration of dipyrone
Please cite this article as: M. Fukuchi, Identifying inducers of BDNF gene expression from pharmacologically validated compounds; antipyretic drug dipyrone increases BDNF mRNA in neurons, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2020.02.019
6
M. Fukuchi / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
[24], indicating that dipyrone has an anti-depressant-like effect. Levels of BDNF are reduced in the brains of depression patients, and BDNF levels increase in patients treated with antidepressant medications [25]. Furthermore, brain BDNF levels are reduced in some neurological disease cases [26]. Therefore, dipyrone may have beneficial and protective effects on neurological diseases that have reduced BDNF levels in the brain, although it should be demonstrated whether Bdnf expression in the brain is increased by administration of dipyrone in vivo. Although our screening assay is difficult to demonstrate how active compound induces Bdnf expression, this method is convenient to identify inducers of Bdnf expression in primary neuronal cells. Numerous studies support the idea that enhancing Bdnf expression could be beneficial for neurodegenerative and neuropsychiatric diseases. Our screening assay using Bdnf-Luc cortical neurons is applicable for mining inducers of Bdnf expression from pharmacologically validated compound libraries, as the first screen of drug re-positioning, as well as for finding novel candidates for ameliorating neurological disorders that have low levels of BDNF in the brain, such as dementia, depression, and so on.
[5]
[6]
[7]
[8]
[9]
[10]
Declaration of competing interest [11]
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This work was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Nos. JP25870256 [Grant-in-Aid for Young Scientists (B) to M.F.], 16K12894 (Grant-in-Aid for Challenging Exploratory Research to M.F.), and 16H05275 [Grant-in- Aid for Scientific Research (B) to M.F.], the Takeda Science Foundation (to M.F.), the Mochida Memorial Foundation for Medical and Pharmaceutical Research (to M.F.), a Hokugin Research Grant (to M.F.), and a research grant from the Toyama First Bank Foundation (to M.F.). We appreciate the contribution of members of Professor Mori’s laboratory (Department of Molecular Neuroscience, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama) in the generation of Bdnf-Luc mice. This work was supported by the Platform Project for Supporting in Drug Discovery and Life Science Research from the Japan Agency for Medical Research and Development (AMED). We thank Jeremy Allen, PhD, from Edanz Group (www.edanzediting.com/ac) for editing a draft of this manuscript.
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
Appendix A. Supplementary data [21]
Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2020.02.019. References [1] M. Miranda, J.F. Morici, M.B. Zanoni, et al., Brain-derived neurotrophic factor: a key molecule for memory in the healthy and the pathological brain, Front. Cell. Neurosci. 13 (2019) 363, https://doi.org/10.3389/fncel.2019.00363. [2] Y. Du, H.T. Wu, X.Y. Qin, et al., Postmortem brain, cerebrospinal fluid, and blood neurotrophic factor levels in Alzheimer’s disease: a systematic review and meta-analysis, J. Mol. Neurosci. (2018) 289e300, https://doi.org/10.1007/ s12031-018-1100-8. [3] A.S. Buchman, L. Yu, P.A. Boyle, et al., Higher brain BDNF gene expression is associated with slower cognitive decline in older adults, Neurology 86 (2016) 735e741, https://doi.org/10.1212/WNL.0000000000002387. [4] A. Sheldrick, S. Camara, M. Ilieva, et al., Brain-derived neurotrophic factor (BDNF) and neurotrophin 3 (NT3) levels in post-mortem brain tissue from
[22]
[23]
[24]
[25]
[26]
patients with depression compared to healthy individuals - a proof of concept study, Eur. Psychiatr. 46 (2017) 65e71, https://doi.org/10.1016/ j.eurpsy.2017.06.009. M. Fukuchi, A. Tabuchi, Y. Kuwana, et al., Neuromodulatory effect of Gas- or Gaq-coupled G-protein-coupled receptor on NMDA receptor selectively activates the NMDA receptor/Ca2þ/calcineurin/cAMP response element-binding protein-regulated transcriptional coactivator 1 pathway to effectively induce brain-derived neurotrophic factor expression in neurons, J. Neurosci. 35 (2015) 5606e5624, https://doi.org/10.1523/JNEUROSCI.3650-14.2015. M. Fukuchi, H. Izumi, H. Mori, et al., Visualizing changes in brain-derived neurotrophic factor (BDNF) expression using bioluminescence imaging in living mice, Sci. Rep. 7 (2017) 4949, https://doi.org/10.1038/s41598-01705297-x. M. Fukuchi, Y. Okuno, H. Nakayama, et al., Screening inducers of neuronal BDNF gene transcription using primary cortical cell cultures from BDNFluciferase transgenic mice, Sci. Rep. 9 (2019) 11833, https://doi.org/10.1038/ s41598-019-48361-4. n, et al., Cholinergic regulation of M. da Penha Berzaghi, J. Cooper, E. Castre brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) but not neurotrophin-3 (NT-3) mRNA levels in the developing rat hippocampus, J. Neurosci. 13 (1993) 3818e3826, https://doi.org/10.1523/JNEUROSCI.13-0903818.1993. M. Fukuchi, Y. Kirikoshi, A. Mori, et al., Excitatory GABA induces BDNF transcription via CRTC1 and phosphorylated CREB-related pathways in immature cortical cells, J. Neurochem. 131 (2014) 134e146, https://doi.org/10.1111/ jnc.12801. Z.J. Wang, L. Sun, T. Heinbockel, Resibufogenin and cinobufagin activate central neurons through an ouabain-like action, PloS One 9 (2014), e113272, https://doi.org/10.1371/journal.pone.0113272. T. Aid, A. Kazantseva, M. Piirsoo, et al., Mouse and rat BDNF gene structure and expression revisited, J. Neurosci. Res. 85 (2007) 525e535, https://doi.org/ 10.1002/jnr.21139. E.J. Hong, A.E. McCord, M.E. Greenberg, A biological function for the neuronal activity-dependent component of Bdnf transcription in the development of cortical inhibition, Neuron 60 (2008) 610e624, https://doi.org/10.1016/ j.neuron.2008.09.024. K. Sakata, N.H. Woo, K. Martinowich, et al., Critical role of promoter IV-driven BDNF transcription in GABAergic transmission and synaptic plasticity in the prefrontal cortex, Proc. Natl. Acad. Sci. U. S. A 106 (2009) 5942e5947, https:// doi.org/10.1073/pnas.0811431106. N.V. Chandrasekharan, H. Dai, K.L. Roos, et al., COX-3, a cyclooxygenase-1 variant inhibited by acetaminophen and other analgesic/antipyretic drugs: cloning, structure, and expression, Proc. Natl. Acad. Sci. U. S. A 99 (2002) 13926e13931, https://doi.org/10.1073/pnas.162468699. A. Jasiecka, T. Maslanka, J.J. Jaroszewski, Pharmacological characteristics of metamizole, Pol. J. Vet. Sci. 17 (2014) 207e214. https://10.2478/pjvs-20140030. F. Crunfli, F.C. Vilela, A. Giusti-Paiva, Cannabinoid CB1 receptors mediate the effects of dipyrone, Clin. Exp. Pharmacol. Physiol. 42 (2015) 246e255, https:// doi.org/10.1111/1440-1681.12347. A.B. Singleton, M. Farrer, J. Johnson, et al., alpha-Synuclein locus triplication causes Parkinson’s disease, Science 302 (2003) 841, https://doi.org/10.1126/ science.1090278. M. Farrer, J. Kachergus, L. Forno, et al., Comparison of kindreds with parkinsonism and alpha-synuclein genomic multiplications, Ann. Neurol. 55 (2004) 174e179, https://doi.org/10.1002/ana.10846. S. Mittal, K. Bjørnevik, D.S. Im, et al., b2-Adrenoreceptor is a regulator of the asynuclein gene driving risk of Parkinson’s disease, Science 357 (2017) 891e898, https://doi.org/10.1126/science.aaf3934. M. Mogi, A. Togari, T. Kondo, et al., Brain-derived growth factor and nerve growth factor concentrations are decreased in the substantia nigra in Parkinson’s disease, Neurosci. Lett. 270 (1999) 45e48, https://doi.org/10.1016/ S0304-3940(99)00463-2. A. Busquets-Garcia, J. Bains, G. Marsicano, CB1 receptor signaling in the brain: extracting specificity from ubiquity, Neuropsychopharmacology 43 (2018) 4e20, https://doi.org/10.1038/npp.2017.206. X. Tao, S. Finkbeiner, D.B. Arnold, et al., Ca2þ influx regulates BDNF transcription by a CREB family transcription factor-dependent mechanism, Neuron 20 (1998) 709e726, https://doi.org/10.1016/S0896-6273(00)81010-7. X. Tao, A.E. West, W.G. Chen, et al., A calcium-responsive transcription factor, CaRF, that regulates neuronal activity-dependent expression of BDNF, Neuron 33 (2002) 383e395, https://doi.org/10.1016/S0896-6273(01)00561-X. lu, K.E. Demirkol, F. Berktas¸, et al., Dipyrone ameliorates behavioural O.E. Kirog changes induced by unpredictable chronic mild stress: gender differences, Clin. Invest. Med. 39 (2016) 27494, https://doi.org/10.25011/cim.v39i6.27494. B. Chen, D. Dowlatshahi, G.M. MacQueen, et al., Increased hippocampal BDNF immunoreactivity in subjects treated with antidepressant medication. https:// doi.org/10.1016/S0006-3223(01)01083-6, 2001. T. Numakawa, H. Odaka, N. Adachi, Actions of brain-derived neurotrophin factor in the neurogenesis and neuronal function, and its involvement in the pathophysiology of brain diseases, Int. J. Mol. Sci. 19 (2018), https://doi.org/ 10.3390/ijms19113650 pii: E3650.
Please cite this article as: M. Fukuchi, Identifying inducers of BDNF gene expression from pharmacologically validated compounds; antipyretic drug dipyrone increases BDNF mRNA in neurons, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2020.02.019