- Research article
- Open Access
Time-dependent biphasic modulation of human BDNF by antidepressants in neuroblastoma cells
- Lorena Donnici†1, 2,
- Ettore Tiraboschi†1, 3,
- Daniela Tardito1,
- Laura Musazzi1,
- Giorgio Racagni1 and
- Maurizio Popoli1Email author
© Donnici et al; licensee BioMed Central Ltd. 2008
Received: 15 February 2008
Accepted: 05 July 2008
Published: 05 July 2008
Recent rodent studies reported that antidepressant treatments affect the expression of brain-derived neurotrophic factor (BDNF) mRNA in a way that is dependent on treatment duration, by selective modulation of different BDNF transcripts. However, no data are available for the human BDNF gene. We studied the effect of different antidepressants on BDNF mRNA expression in human neuroblastoma SH-SY5Y cells.
Cultured cells were treated with the antidepressants fluoxetine, reboxetine and desipramine for different time lengths (6, 24, 48 hours). Expression of total BDNF mRNA was analyzed by reverse transcription PCR and levels of different BDNF transcripts were detected by hemi-nested PCR with specific primers.
Short-term treatment (6 hours) with reboxetine or desipramine reduced total BDNF, whereas long-term treatment (48 hours) significantly increased total BDNF mRNA levels. These changes were accounted for by differential regulation of BDNF IV and VIa/b transcripts. Fluoxetine showed no significant effects.
This is the first study showing biphasic changes in the expression of total and specific BDNF transcripts in human cells following antidepressant treatments. These findings suggest that biphasic induction of BDNF by antidepressants could be a feature common to rodents and humans and encourage the use of SH-SY5Y cells as a tool for investigation of drug effects on human genes.
Brain-derived neurotrophic factor (BDNF) has been implicated in both the pathophysiology and pharmacotherapy of depression [1–3]. It has been shown that BDNF expression and/or function is impaired in major depression or following stress paradigms, while it is up-regulated by physical exercise and antidepressants. However, different and sometimes conflicting findings have been reported , showing that antidepressants change total BDNF expression level depending on length of the treatment and time interval following administration. Indeed, independent investigations showed that short-term antidepressant treatment decreased BDNF expression in rodents, whereas long-term treatment increased BDNF [4–6].
The increasing knowledge of BDNF gene in rodents [7, 8] has encouraged the research on the modulation of different BDNF transcripts by pharmacological treatments. Recent studies showed that different drugs, lengths of treatment and drug/physical exercise combination, as well as stress paradigms, may selectively influence the transcription of specific BDNF transcripts in rodents [6, 9–12].
Cell culture and pharmacological treatment
Human neuroblastoma SH-SY5Y cells were obtained from Interlab Cell Line Collection (Genova, Italy), at passage P14; only cells between passages P16 to P25 were used. Cells were grown in Minimum Essential Medium (Invitrogen, Carlsbad, CA), containing 10% foetal bovine serum, 2 mM glutamine and non-essential aminoacids (1 mg/l) in a humidified incubator (95% air, 5% CO2) at 37°C. Cells were treated for 6, 24 and 48 hours before harvesting (all cells were kept in culture for 3 days total) with different antidepressants: the selective serotonin reuptake inhibitor fluoxetine (FLX), the selective norepinephrine reuptake inhibitor reboxetine (RBX) and the tricyclic antidepressant desipramine (DMI), predominantly inhibiting norepinephrine reuptake (all 10 μM, final concentration).
RNA isolation, cDNA synthesis and reverse transcription-PCR for total BDNF expression
Sequences of primers used for RT-PCR.
RNA isolation, cDNA synthesis and reverse transcription-PCR for BDNF isoforms
Human BDNF transcripts and specific primers used for RT-PCR.
exI fwd/exIX rev1
EF674517.1; EF674518.1; EF674519.1
exII fwd/exIX rev1
exIII fwd/exIX rev1
exIV fwd/exIX rev1
exVIa/b fwd/exIX rev1
exVIb-IXbd fwd/exIX rev1
exVII fwd/exIX rev1
In order to characterize the effects of different antidepressants on total human BDNF mRNA expression, SH-SY5Y neuroblastoma cells were treated with FLX, RBX or DMI for 6 h (to assess the effects of short-term treatments), 24 or 48 hours, as in recent studies assessing the long-term effects of antidepressants in cultured cells [16, 17]. With regard to the antidepressant concentration in the cell medium, several studies have found that therapeutic plasma levels of these drugs in humans under chronic treatment are in the low micromolar range. However, it has been also shown that their concentrations in brain tissue can be as much as 20 times higher compared to plasma levels [18, 19]. Moreover, it has been reported that brain concentrations of different antidepressants administered systemically at different doses were similar, without a clear correlation with either treatment dose or treatment duration . Therefore, in order to expose the neuroblastoma cells to a drug concentration as close as possible to those reached in brain tissue, we chose to treat cultured SH-SY5Y cells with 10 μM FLX, RBX or DMI in our experiments.
The BDNF transcript expression pattern in SH-SY5Y cells is similar to that of human cortical tissue : according to a recent nomenclature of BDNF gene , BDNF IV and VI are the most highly expressed transcripts, representing over 80% of the total BDNF. Therefore, in order to evaluate if the modifications induced in total BDNF mRNA levels by antidepressants were due to alterations in the main BDNF transcripts, the expression profile of BDNF IV and VIa/b transcripts were analyzed. Cells were treated with FLX, DMI or RBX for 6 hours or 48 hours, times at which expression of total BDNF mRNA was changed after antidepressants (Fig. 2A), and BDNF transcript expression level was detected with semi-quantitative hemi-nested PCR. Two way ANOVA showed significant effects of time (F2,144 = 37,42; p < .0001), drug (F2,144 = 23,39; p < .0001) and time/drug interaction (F4,144 = 26,94; p < .0001) for BDNF IV transcript: BDNF IV was significantly reduced after 6 hours and significantly increased after 48 h of RBX or DMI (Fig. 2B). FLX showed no effect. Two way ANOVA of BDNF VIa/b (Fig. 2C) showed a significant effect of time (F2,107 = 7,19; p < .05) and of time/drug interaction (F4,107 = 7,53; p < .0001). Post-hoc comparisons showed a significant reduction of BDNF VIa/b after 6 hours of RBX and no effects after 48 hours (Fig. 2C). In addition, we analyzed the expression of the low-level BDNF transcripts, BDNF I, II, VIb-IXbd and VIIa/b; all drug treatments did not change the expression of the transcripts (data not shown). BDNF III was undetectable; expression of exons V, Vh, VIII and VIIIh was not measured because, when the present work was completed, the new nomenclature was not yet published .
To the best of our knowledge, this is the first study analyzing the effect of antidepressant treatments on the expression of BDNF transcripts in cells of human origin. Previous studies found time-dependent biphasic changes in the expression of total BDNF induced by antidepressants in rat brain [4–6]. Similarly, we found that in SH-SY5Y cells RBX and DMI induced biphasic changes in total BDNF mRNA expression. Indeed, short-term treatment with both antidepressants significantly reduced, whereas long-term treatment increased total BDNF mRNA. At the intermediate time BDNF mRNA levels were similar to control. FLX did not significantly affect BDNF expression, likely owing to the absence of the serotonin transporter in SH-SY5Y cells, which instead express the norepinephrine transporter [21, 22], and the 5-HT2b serotonergic receptor and the α2c adrenoreceptor [23–26]. Indeed, we could confirm the absence of any signal for serotonin transporter mRNA in SH-SY5Y cells by PCR (not shown). An alternative explanation could be found in the lack of changes in BDNF expression after fluoxetine treatment in rodents, as reported by several studies [9, 27–30].
The present results suggest that early reduction of total BDNF mRNA in SH-SY5Y cells was mainly accounted for by a reduction of BDNF IV by RBX and DMI administration, and of BDNF VIa/b by RBX. On the other hand, the increase in total BDNF mRNA after 48 hours of treatment appeared to be mainly accounted for by the induction of BDNF IV by both drugs.
Interestingly, previous studies in rodents have shown a differential regulation of distinct BDNF transcripts by antidepressant treatments [6, 9, 11]. In particular, it has been shown that chronic defeat stress, a model of depression, selectively down-regulated in mouse hippocampus the expression of BDNF III and BDNF IV transcripts (corresponding in the present study to BDNF IV and BDNF VIa/b respectively, according to the new nomenclature of BDNF gene), while chronic imipramine treatment reversed this down-regulation .
The underlying molecular mechanisms are at present unknown; however, recent evidence from several groups suggest that different signaling mechanisms may be responsible for the regulation of BDNF transcription . As an example, it has been shown that different BDNF promoters contain multiple sites for various transcription factors (i.e. CREB, CaRF), responding to different signaling pathways mainly activated by calcium fluxes (CaM kinase cascades), by cyclic AMP (cAMP-PKA cascade) and by neurotrophic factors (MAP-Erk pathway). In this regard, we have recently reported that chronic administration of different antidepressants in rats exerts distinct actions on CREB activation that seem to depend on a differential modulation of CaM kinase IV and MAPK-Erk1/2 cascades . Furthermore, we found that the CaM kinase IV cascade is also involved in the regulation of CREB activation by the mood stabilizer lithium .
Overall, although further studies are needed to clarify the upstream mechanisms, these results suggest that time-dependent biphasic changes in the expression of BDNF IV and VIa/b account for the observed changes induced by antidepressants in total BDNF mRNA of SH-SY5Y cells. Validation by quantitative Real-Time PCR is warranted. A limitation of the present work is that BDNF protein was not measured; because often mRNA and protein expression do not correlate, measurement of BDNF protein in SH-SY5Y after antidepressant treatments is warranted. Also considering recent reports on the different structure and regulation between rodent and human BDNF gene [7, 13], SH-SY5Y cells could provide a useful tool to screen the effects of different psychotropics on human BDNF. Furthermore, it will be interesting to investigate whether similar effects are also found in peripheral cells of patients treated with antidepressants. In this regard, it is noteworthy that changes in serum concentrations of BDNF after antidepressant treatments have been measured in a number of works (as an example, see ref ).
The present findings show biphasic changes in the expression of total and specific BDNF transcripts following antidepressant treatments in human cells and encourage the use of SH-SY5Y cells as a tool for research of psychotropic drug effects on human genes.
LD, DT, GR and MP were founded by a grant from Ministry of University, PRIN # 2005054953 (Italy).
ET was founded by the PhD Program in Clinical Physiology and Pharmacology and Therapy of Metabolic Diseases, University of Milano, Medical School (Italy).
LM was founded by the PhD Program in Pharmacotoxicological and Pharmacognostic Sciences and Pharmacological Biotechnologies, University of Milano (Italy).
- Pittenger C, Duman RS: Stress, depression, and neuroplasticity: a convergence of mechanisms. Neuropsychopharmacol. 2008, 33: 88-109. 10.1038/sj.npp.1301574.View ArticleGoogle Scholar
- Tardito D, Perez J, Tiraboschi E, Musazzi L, Racagni G, Popoli M: Signaling pathways regulating gene expression, neuroplasticity, and neurotrophic mechanisms in the action of antidepressants: a critical overview. Pharmacol Rev. 2006, 58: 115-134. 10.1124/pr.58.1.7.View ArticlePubMedGoogle Scholar
- Kozisek ME, Middlemas D, Bylund DB: Brain-derived neurotrophic factor and its receptor tropomyosin-related kinase B in the mechanism of action of antidepressant therapies. Pharmacol Ther. 2008, 117: 30-51. 10.1016/j.pharmthera.2007.07.001.View ArticlePubMedGoogle Scholar
- Coppell AL, Pei Q, Zetterström TS: Bi-phasic change in BDNF gene expression following antidepressant drug treatment. Neuropharmacol. 2003, 44: 903-910. 10.1016/S0028-3908(03)00077-7.View ArticleGoogle Scholar
- De Foubert G, Carney SL, Robinson CS, Destexhe EJ, Tomlinson R, Hicks CA, Murray TK, Gaillard JP, Deville C, Xhenseval V, Thomas CE, O'Neill MJ, Zetterstrom TS: Fluoxetine-induced change in rat brain expression of brain-derived neurotrophic factor varies depending on length of treatment. Neuroscience. 2004, 128: 597-604. 10.1016/j.neuroscience.2004.06.054.View ArticlePubMedGoogle Scholar
- Khundakar AA, Zetterström TS: Biphasic change in BDNF gene expression following antidepressant drug treatment explained by differential transcript regulation. Brain Res. 2006, 1106: 12-20. 10.1016/j.brainres.2006.05.063.View ArticlePubMedGoogle Scholar
- Aid T, Kazantseva A, Piirsoo M, Palm K, Timmusk T: Mouse and rat BDNF gene structure and expression revisited. J Neurosci Res. 2007, 85: 525-535. 10.1002/jnr.21139.PubMed CentralView ArticlePubMedGoogle Scholar
- Liu QR, Lu L, Zhu XG, Gong JP, Shaham Y, Uhl GR: Rodent BDNF genes, novel promoters, novel splice variants, and regulation by cocaine. Brain Res. 2006, 1067: 1-12. 10.1016/j.brainres.2005.10.004.View ArticlePubMedGoogle Scholar
- Dias BG, Banerjee SB, Duman RS, Vaidya VA: Differential regulation of brain derived neurotrophic factor transcripts by antidepressant treatments in the adult rat brain. Neuropharmacol. 2003, 45: 553-563. 10.1016/S0028-3908(03)00198-9.View ArticleGoogle Scholar
- Nair A, Vadodaria KC, Banerjee SB, Benekareddy M, Dias BG, Duman RS, Vaidya VA: Stressor-Specific Regulation of Distinct Brain-Derived Neurotrophic Factor Transcripts and Cyclic AMP Response Element-Binding Protein Expression in the Postnatal and Adult Rat Hippocampus. Neuropsychopharmacol. 2007, 32: 1504-1519. 10.1038/sj.npp.1301276.View ArticleGoogle Scholar
- Russo-Neustadt AA, Alejandre H, Garcia C, Ivy AS, Chen MJ: Hippocampal brain-derived neurotrophic factor expression following treatment with reboxetine, citalopram, and physical exercise. Neuropsychopharmacol. 2004, 29: 2189-2199. 10.1038/sj.npp.1300514.View ArticleGoogle Scholar
- Tsankova NM, Berton O, Renthal W, Kumar A, Neve RL, Nestler EJ: Sustained hippocampal chromatin regulation in a mouse model of depression and antidepressant action. Nat Neurosci. 2006, 9: 519-525. 10.1038/nn1659.View ArticlePubMedGoogle Scholar
- Pruunsild P, Kazantseva A, Aid T, Palm K, Timmusk T: Dissecting the human BDNF locus: Bidirectional transcription, complex splicing, and multiple promoters. Genomics. 2007, 90: 397-406. 10.1016/j.ygeno.2007.05.004.PubMed CentralView ArticlePubMedGoogle Scholar
- Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F: Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 2002, 3: RESEARCH0034-10.1186/gb-2002-3-7-research0034.PubMed CentralView ArticlePubMedGoogle Scholar
- Montenegro SH, Gilman RH, Sheen P, Cama R, Caviedes L, Hopper T, Chambers R, Oberhelman RA: Improved detection of Mycobacterium tuberculosis in Peruvian children by use of a heminested IS6110 polymerase chain reaction assay. Clin Infect Dis. 2003, 36: 16-23. 10.1086/344900.View ArticlePubMedGoogle Scholar
- Chiou SH, Chen SJ, Peng CH, Chang YL, Ku HH, Hsu WM, Ho LL, Lee CH: Fluoxetine up-regulates expression of cellular FLICE-inhibitory protein and inhibits LPS-induced apoptosis in hippocampus-derived neural stem cell. Biochem Biophys Res Commun. 2006, 343: 391-400. 10.1016/j.bbrc.2006.02.180.View ArticlePubMedGoogle Scholar
- Cecconi D, Mion S, Astner H, Domenici E, Righetti PG, Carboni L: Proteomic analysis of rat cortical neurons after fluoxetine treatment. Brain Res. 2007, 1135: 41-51. 10.1016/j.brainres.2006.12.008.View ArticlePubMedGoogle Scholar
- Karson CN, Newton JE, Livingston R, Jolly JB, Cooper TB, Sprigg J, Komoroski RA: Human brain fluoxetine concentrations. J Neuropsychiatry Clin Neurosci. 1993, 5: 322-329.View ArticlePubMedGoogle Scholar
- Bolo NR, Hodé Y, Nèdèlec JF, Lainè E, Wagner G, Macher JP: Brain pharmacokinetics and tissue distribution in vivo of fluvoxamine and fluoxetine by fluorine magnetic resonance spectroscopy. Neuropsychopharmacol. 2000, 23: 428-438. 10.1016/S0893-133X(00)00116-0.View ArticleGoogle Scholar
- Garzon DJ, Fahnestock M: Oligomeric amyloid decreases basal levels of brain-derived neurotrophic factor (BDNF) mRNA via specific downregulation of BDNF transcripts IV and V in differentiated human neuroblastoma cells. J Neurosci. 2007, 27: 2628-2635. 10.1523/JNEUROSCI.5053-06.2007.View ArticlePubMedGoogle Scholar
- O'Neill CM, Ball SG, Vaughan PF: Effects of ischaemic conditions on uptake of glutamate, aspartate, and noradrenaline by cell lines derived from the human nervous system. J Neurochem. 1994, 63: 603-611.View ArticlePubMedGoogle Scholar
- Lode HN, Bruchelt G, Seitz G, Gebhardt S, Gekeler V, Niethammer D, Beck J: Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of monoamine transporters in neuroblastoma cell lines: correlations to meta-iodobenzylguanidine (MIBG) uptake and tyrosine hydroxylase gene expression. Eur J Cancer. 1995, 31A: 586-590. 10.1016/0959-8049(95)00039-L.View ArticlePubMedGoogle Scholar
- Kazmi SM, Mishra RK: Identification of alpha 2-adrenergic receptor sites in human retinoblastoma (Y-79) and neuroblastoma (SH-SY5Y) cells. Biochem Biophys Res Commun. 1989, 158: 921-928. 10.1016/0006-291X(89)92810-6.View ArticlePubMedGoogle Scholar
- Parsley S, Gazi L, Bobirnac I, Loetscher E, Schoeffter P: Functional alpha2C-adrenoceptors in human neuroblastoma SH-SY5Y cells. Eur J Pharmacol. 1999, 372: 109-115. 10.1016/S0014-2999(99)00190-9.View ArticlePubMedGoogle Scholar
- Schmuck K, Ullmer C, Engels P, Lübbert H: Cloning and functional characterization of the human 5-HT2B serotonin receptor. FEBS Lett. 1994, 342: 85-90. 10.1016/0014-5793(94)80590-3.View ArticlePubMedGoogle Scholar
- Duxon MS, Flanigan TP, Reavley AC, Baxter GS, Blackburn TP, Fone KC: Evidence for expression of the 5-hydroxytryptamine-2B receptor protein in the rat central nervous system. Neuroscience. 1997, 76: 323-329. 10.1016/S0306-4522(96)00480-0.View ArticlePubMedGoogle Scholar
- Conti AC, Cryan JF, Dalvi A, Lucki I, Blendy JA: cAMP response element-binding protein is essential for the upregulation of brain-derived neurotrophic factor transcription, but not the behavioral or endocrine responses to antidepressant drugs. J Neurosci. 2002, 22: 3262-3268.PubMedGoogle Scholar
- Miró X, Pérez-Torres S, Artigas F, Puigdomènech P, Palacios JM, Mengod G: Regulation of cAMP phosphodiesterase mRNAs expression in rat brain by acute and chronic fluoxetine treatment. An in situ hybridization study. Neuropharmacol. 2002, 43: 1148-1157. 10.1016/S0028-3908(02)00220-4.View ArticleGoogle Scholar
- Altieri M, Marini F, Arban R, Vitulli G, Jansson BO: Expression analysis of brain-derived neurotrophic factor (BDNF) mRNA isoforms after chronic and acute antidepressant treatment. Brain Res. 2004, 1000: 148-155. 10.1016/j.brainres.2003.12.028.View ArticlePubMedGoogle Scholar
- Larsen MH, Hay-Schmidt A, Rønn LC, Mikkelsen JD: Temporal expression of brain-derived neurotrophic factor (BDNF) mRNA in the rat hippocampus after treatment with selective and mixed monoaminergic antidepressants. Eur J Pharmacol. 2008, 578: 114-122. 10.1016/j.ejphar.2007.08.050.View ArticlePubMedGoogle Scholar
- Tiraboschi E, Tardito D, Kasahara J, Moraschi S, Pruneri P, Gennarelli M, Racagni G, Popoli M: Selective phosphorylation of nuclear CREB by fluoxetine is linked to activation of CaM kinase IV and MAP kinase cascades. Neuropsychopharmacol. 2004, 29: 1831-1840. 10.1038/sj.npp.1300488.View ArticleGoogle Scholar
- Tardito D, Tiraboschi E, Kasahara J, Racagni G, Popoli M: Reduced CREB phosphorylation after chronic lithium treatment is associated o downregulation of CaM kinase IV in rat hippocampus. Int. J Neuropsychopharmacol. 2007, 10: 491-496. 10.1017/S1461145706007140.View ArticlePubMedGoogle Scholar
- Hellweg R, Ziegenhorn A, Heuser I, Deuschle M: Serum concentrations of nerve growth factor and brain-derived neurotrophic factor in depressed patients before and after antidepressant treatment. Pharmacopsychiatry. 2008, 41: 66-71. 10.1055/s-2007-1004594.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.