Phencyclidine rapidly decreases neuronal mRNA of brain-derived neurotrophic factor

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<ul><li><p>Phencyclidine Rapidly Decreases NeuronalmRNA of Brain-Derived Neurotrophic</p><p>FactorYUSUKE KATANUMA,1,2 TADAHIRO NUMAKAWA,1,3* NAOKI ADACHI,1,3 NORIKO YAMAMOTO,1</p><p>YOSHIKO OOSHIMA,1 HARUKI ODAKA,1,2 TAKAFUMI INOUE,2 AND HIROSHI KUNUGI1,31Department of Mental Disorder Research, National Institute of Neuroscience, National Center of Neurology and</p><p>Psychiatry, Tokyo, Japan2Department of Life Science and Medical Bioscience, School of Advanced Science and Engineering,</p><p>Waseda University, Tokyo, Japan3Core Research for Evolution Science and Technology Program (CREST), Japan Science and Technology Agency</p><p>(JST), Tokyo, Japan</p><p>KEY WORDS phencyclidine; BDNF; NMDA receptor; schizophrenia</p><p>ABSTRACT Downregulation of brain-derived neurotrophic factor (BDNF), a mem-ber of neurotrophin family, has been implicated in psychiatric diseases including schiz-ophrenia. However, detailed mechanisms of its reduction in patients withschizophrenia remain unclear. Here, using cultured cortical neurons, we monitoredBDNF mRNA levels following acute application of phencyclidine [PCP; an N-methyl-D-aspartate (NMDA) receptor blocker], which is known to produce schizophrenia-likesymptoms. We found that PCP rapidly caused a reduction in total amount of BDNFtranscripts without effect on cell viability, while mRNA levels of nerve growth factorwas intact. Actinomycin-D (ActD), an RNA synthesis inhibitor, decreased total BDNFmRNA levels similar to PCP, and coapplication of ActD with PCP did not show furtherreduction in BDNF mRNA compared with solo application of each drug. Among BDNFexons I, IV, and VI, the exon IV, which is positively regulated by neuronal activity, washighly sensitive to PCP. Furthermore, PCP inactivated cAMP response element-binding protein (CREB; a regulator of transcriptional activity of exon IV). The inacti-vation of CREB was also achieved by an inhibitor for Ca21/calmodulin kinase II (CaM-KII), although coapplication with PCP induced no further inhibition on the CREBactivity. It is possible that PCP decreases BDNF transcription via blocking the NMDAreceptor/CaMKII/CREB signaling. Synapse 68:257265, 2014. VC 2014 Wiley Periodicals, Inc.</p><p>INTRODUCTION</p><p>Schizophrenia affects about 1% of the populationand is characterized by hallucinations, delusions (pos-itive-symptoms), emotional dullness (negative-symp-toms), and cognitive deficits (Favalli et al., 2012).Importantly, it has been reported that dysfunction ofthe prefrontal cortex (PFC) is closely related to theschizophrenia-like behaviors. Cognitive performancedeficits in schizophrenia patients result, at least inpart, from impaired function of the PFC (Weinbergeret al., 1986). Abnormal glutamatergic neurotransmis-sion in the PFC has been suggested to be involved inthe pathology of schizophrenia (Frankle et al., 2003).</p><p>Phencyclidine (PCP), a noncompetitive antagonistfor N-methyl-D-aspartate (NMDA) receptor, caninduce positive and negative symptoms in humans(Domino and Luby, 2012). Because PCP-treatedrodents also show schizophrenia-like behaviors</p><p>(Domino and Luby, 2012; Noda et al., 1995), they arewidely used as an animal model of schizophrenia.Notably, adverse effects of PCP on neuronal functionas well as behavior are evident (Domino and Luby,</p><p>Contract grant sponsor: Health and Labor Sciences Research Grants (Com-prehensive Research on Disability, Health, and Welfare H21-kokoro-002, H.K.); Contract grant sponsor: Core Research for Evolutional Science and Tech-nology Program and CREST, Japan Science and Technology Agency (JST, T.N.,N.A. and H.K.); Contract grant sponsor: Takeda Science Foundation (T. N.);Contract grant sponsor: Grant-in-Aid for Scientific Research (B); Contractgrant number: JSPS KAKENHI 24300139 (T. N.); Contract grant sponsor:Grant-in-Aid for Challenging Exploratory Research; Contract grant number:JSPS KAKENHI 25640019 (T. N.); Contract grant sponsor: Ministry of Educa-tion, Culture, Sports, Science, and Technology of Japan.</p><p>*Correspondence to: Tadahiro Numakawa, Ph.D., Department of MentalDisorder Research, National Institute of Neuroscience, National Center ofNeurology and Psychiatry, 4-1-1 Ogawa-Higashi, Kodaira, Tokyo 1878502,Japan. E-mail: numakawa@ncnp.go.jp</p><p>Received 26 September 2013; Accepted 7 February 2014</p><p>DOI: 10.1002/syn.21735</p><p>Published online 12 February 2014 in Wiley Online Library(wileyonlinelibrary.com).</p><p> 2014 WILEY PERIODICALS, INC.</p><p>SYNAPSE 68:257265 (2014)</p></li><li><p>2012; Wang et al., 2001). Continuous administrationof PCP caused neurodegeneration in the posteriorentorhinal cortex, ventral dentate gyrus, and cingu-late cortex (Ellison and Switzer, 1993). The numberof dendritic spines in the rat PFC was decreasedafter subchronic PCP administration (Hajszan et al.,2006). Consistently, a significant decrease in immuno-reactivity of synaptophysin, one of synaptic vesicleproteins, in the PFC of patients with schizophreniawas confirmed (Glantz and Lewis, 1997).</p><p>Brain-derived neurotrophic factor (BDNF) isbroadly expressed in the central nervous system(CNS) and has critical roles in neuronal survival andmaturation, neurotransmitter release, and synapticplasticity via stimulating several intracellular signal-ing pathways including phosphoinositide 3-kinase/Akt (PI3K/Akt), extracellular signal-regulated kinase(ERK), and phospholipase C-g pathways (Huang andReichardt, 2003; Minichiello et al., 2009; Numakawaet al., 2013; Russo et al., 2009). Reduced BDNF func-tion and/or expression are suggested to be involved inthe pathogenesis of a variety of brain diseases suchas major depression and schizophrenia (Angelucciet al., 2005; Favalli et al., 2012; Javitt and Zukin,1991; Karege et al., 2002). Functional magnetic reso-nance imaging and postmortem studies have reporteddecreased expression levels of BDNF mRNA and pro-tein in the PFC of schizophrenia patients (Callicottet al., 2000; Weickert et al., 2003), suggesting thatdecreased cortical BDNF function may contribute tothe development of schizophrenia, although molecu-lar mechanisms underlying such a reduction inBDNF expression have not been fully elucidated. Onestudy has shown that BDNF mRNA expression invarious brain regions including the PFC was reducedby subchronic PCP administration in female rats(Snigdha et al., 2011).</p><p>As both PCP-mediated neuronal damage andBDNF dysfunction are key concerns to investigateschizophrenia-like behaviors, it is intriguing to clarifythe relationship between the two molecules. Werecently found that a marked impairment of synapticfunction occurs due to reduced secretion of BDNFprotein after subchronic PCP treatment in culturedcortical neurons (Adachi et al., 2013). In the presentstudy, we investigated an effect of PCP on BDNFtranscription and found a rapid decrease of BDNFmRNA via suppression of neural activity-mediatedmechanisms.</p><p>MATERIALS AND METHODSPreparation of primary cortical neurons</p><p>The cerebral cortex was removed from postnatal 1-to 2-day old Wistar rats (SLC, Shizuoka, Japan), anddissociated cultures were prepared as described inour previous report (Numakawa et al., 2009). Briefly,cortical tissues were digested with papain solution,</p><p>and then the dissociated cortical neurons in culturemedia, which consists of 1:1 mixture of Dulbeccosmodified Eagles medium and Hams F-12 medium,5% fetal bovine serum (FBS), 5% heated-inactivatedhorse serum, and penicillinstreptomycin (penicillin:18 units/mL, streptomycin: 18 mg/mL) were plated onpolyethylenimine-coated 3.5 cm dishes or plates (BDFalcon, CA). All experiments were conducted accord-ing to the laboratory animals ethical guidelines of theNational Institute of Neuroscience, National Centerof Neurology and Psychiatry, Japan.</p><p>For astrocyte culture, cortical cells were plated ona noncoated 75 cm2 flask (Corning, NY). Cell mainte-nance was performed with minimum essentialmedium-based growth medium (containing 100 mg/Lepidermal growth factor, 5% FBS, and 0.5 mM gluta-mine), and astroglial cells were replated on 3.5 cmdishes before experiments.</p><p>Drug treatment</p><p>Cultured cortical neurons at 1112 days in vitro or6080% confluent astrocytes were treated with PCP(Sigma-Aldrich, MO), actinomycin D (ActD, Sigma-Aldrich, MO), D-(2)-amino-5-phosphonopentanoic acid(D-APV, Tocris Bioscience, UK), MK801 (Sigma-Aldrich, MO), KN-93 (5 mM, Calbiochem, CA),KT5720 (200 nM, Calbiochem), and K252a (200 nM,Sigma-Aldrich, MO). Except for the examination ofdose- or time-dependent effect, PCP, ActD, and D-APVwere applied for 3 h at 1, 1, and 50 mM, respectively.All drugs were applied to cultured cells after dilutionwith water or dimethyl sulfoxide and correspondingvehicle was treated as control.</p><p>Total RNA extraction, reverse transcription,and quantitative PCR</p><p>Total RNA extraction was conducted using mirVa-naTM miRNA Isolation Kit (Life Technologies, CA)according to the manufactures protocol. Reverse tran-scription reaction (25C, 10 min, 42C, 60 min, and85C, 5 min) with the same amount of RNA was per-formed using GeneAmp polymerase chain reaction(PCR) System 9700 (Applied Biosystems, CA). Theobtained complementary DNA was determined by thequantitative PCR with ABI Prism 7000 (Applied Bio-systems). After an initial denaturation step at 95C for10 min, 40 PCR cycles of denaturation at 95C for 15 sand annealing/extension at 60C for 1 min were done.Amount of each mRNA was determined by calculatingfrom the obtained threshold cycle. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was usedfor normalization. The primer and probe sets (TaqManGene Expression Assays from Applied Biosystems)were as follows: BDNF exon I: Rn01484924_m1,BDNF exon IV: Rn01484927_m1, BDNF exon VI:Rn01484928_m1, total BDNF: Rn02531967_s1,Microtubule-associated protein 2 (MAP2):</p><p>258 Y. KATANUMA ET AL.</p><p>Synapse</p></li><li><p>Rn00565046_m1, nerve growth factor (NGF):Rn01533872_m1, GAPDH: 4352338E.</p><p>MTT assay</p><p>After aspiration of culture medium, 3-(4,5-di-meth-ylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, tetra-zole (MTT) diluted in fresh medium was applied tocortical neurons and incubated for 1.52.5 h at 37C.Then lysis buffer including sodium dodecyl sulfate(SDS) (0.1 g/mL) was added for 1 h. The absorbanceat 550 nm was measured by a microplate reader (Bio-Rad Laboratories, CA).</p><p>Western blotting</p><p>Immunoblotting was performed as previouslyreported (Numakawa et al., 2009). The same amountof protein was electrophoresed in the SDS-polyacrylamide gel and transferred to a membrane. After block-ing with 5% skim milk for 12 h, anti-CREB (1:500,Cell Signaling Technology, MA), anti-pCREB (1:500,Cell Signaling Technology, MA), anti-NR2A (1:500,Sigma-Aldrich, MO), anti-NR2B (1:500, Sigma-Aldrich, MO), anti-GluR1 (1:500, Chemicon Interna-tional, CA), or anti-bactin antibody (as a control pro-tein, 1:5000, Sigma-Aldrich, MO) was incubatedovernight at 4C. Observation of immunoreactivityand data analysis was performed with Ez-capture con-trolled by ImageSaver5 software and CS Analyzer 3.0software, respectively (ATTO, Tokyo, Japan).</p><p>Statistical analysis</p><p>The experimental data were expressed as mean-6 standard deviation (SD). The statistical significancewas evaluated with Students t-test, or one- or two-way analysis of variance (ANOVA) followed by Bon-ferroni post hoc test in SPSS ver.18 (IBM, Japan) andconsidered to be significant when the probabilityvalue was less than 5%.</p><p>RESULTSPCP rapidly decreased total BDNF mRNA in</p><p>cultured cortical neurons</p><p>To examine the influence of PCP on BDNF mRNAlevels, matured cortical neurons at DIV1112 wereexposed to PCP at final concentrations of 0.110 mMfor 3 h. As shown in Figure 1A, 1 and 10 mM of PCPsignificantly reduced total BDNF mRNA levels. Atime-course analysis of the PCP effect (1 mM) alsorevealed significant reductions in the total BDNFmRNA after 1, 3, and 6 h PCP treatment (Fig. 1B).When an influence of PCP on BDNF mRNA expres-sion in pure astroglial culture was examined, no sig-nificant change was observed (Fig. 1C), indicatingthat the observed BDNF mRNA reduction by PCP isattributable to neuronal response. To determinewhether the PCP-induced reduction in total BDNFmRNA immediately affects neuronal viability or not,</p><p>the MTT assay was conducted. PCP did not alter cellviability at any dose examined (0.110 mM, 3 h; Fig.1D). To see specificity of the PCP effect in corticalneurons, MAP2 and NGF mRNA levels after PCPtreatment (1 mM, 3 h) were determined. Both MAP2and NGF mRNA expressions were unchanged byPCP while BDNF mRNA was affected (Fig. 1E).</p><p>PCP inhibited transcriptional activityin BDNF exon IV</p><p>The decreased total BDNF mRNA caused by PCPis attributable to suppression of transcriptional activ-ity and/or acceleration of degradation. Therefore,next we examined the possible effect of ActD, a tran-scription inhibitor. Importantly, ActD also decreasedBDNF mRNA expression in a similar time course asPCP (see Figs. 1B and 2A). The degree of change intotal BDNF mRNA levels after coapplication of ActDwith PCP (1 mM, 3 h) was not significantly differentfrom that after solo application of each drug(Fig. 2B). There was a significant interaction betweenPCP and ActD treatments (two-way ANOVAF(1,20)5 19.3, P&lt; 0.001). If PCP promoted degrada-tion of mRNA, an additional reduction in levels ofBDNF mRNA would have been achieved by the coap-plication. These data, therefore, suggested that PCPdiminishes the amount of BDNF mRNA mainly viarepressing transcriptional activity. To confirm contri-bution of NMDA receptor activity to the reducedBDNF mRNA levels, other inhibitors for NMDAreceptors were tested. Three-hour treatment with D-APV (a competitive antagonist) at the concentrationof 50 mM, the dose of which abolishes NMDAreceptor-mediated current (Liu et al., 2004), caused acomparable reduction in total BDNF mRNA to that ofPCP (1 mM, 3 h) or ActD (1 mM, 3 h; Fig. 2C), imply-ing that impairment in the NMDA receptor-mediatedtranscription is a major contributor to the decrease intotal BDNF mRNA. MK-801, a noncompetitiveNMDA receptor antagonist, also showed a significantinhibitory effect on BDNF mRNA levels (Fig. 2D).</p><p>The rodent BDNF gene has at least nine promoters(BDNF pIIX) and nine exons (BDNF exon IIX, Aidet al., 2007). As exon I and IV are positively regu-lated by neuronal activity (Pruunsild et al., 2011), weexamined BDNF transcripts containing these exons.In addition, we measured the exon VI transcriptwhich was assumed to be activity independent. Wefound that the basal levels of exon IV transcript weremuch higher than that of exon I or VI in cortical cul-tures (Fig. 2E). Conversely, exon VI transcript wasthe major population in astroglial cell cultures (Fig.2F). We then investigated effects of PCP, ActD, andD-APV on expression levels of exon I, IV, and VI tran-scripts. In contrast to the total BDNF mRNA, eachexon-containing transcript showed various degrees ofreduction in response to these drugs. Regarding exon</p><p>DECREASED BDNF mRNA BY PCP 259</p><p>Synapse</p></li><li><p>I and VI, ActD strongly reduced their transcript lev-els while weaker suppression was observed in PCP orD-APV application (Fig. 2G). For exon IV, by contrast,PCP, D-APV, and...</p></li></ul>

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