Molecular and Cellular Neuroscience 45 (2010) 226–233

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Molecular and Cellular Neuroscience

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Stimulated neuronal expression of brain-derived neurotrophic factor by Neurotropin

Yu Fukuda a,b,e, Thomas L. Berry b, Matthew Nelson c, Christopher L. Hunter c, Koki Fukuhara b,d,e,Hideki Imai e, Shinji Ito a, Ann-Charlotte Granholm-Bentley c, Allen P. Kaplan b, Tatsuro Mutoh a,⁎a Department of Neurology, Fujita Health University School of Medicine, Kutsukake-cho, Toyoake 470-1192, Japanb Konishi-MUSC Institute for Inflammation Research, and Division of Pulmonary and Critical Care Medicine, Allergy and Clinical Immunology, Department of Medicine,Medical University of South Carolina, 96 Jonathan Lucas Street, Charleston, SC 29425, USAc Center on Aging, Department of Neurosciences, Medical University of South Carolina, 173 Ashley Avenue, Charleston, SC 29425, USAd The National Institute of Nursing Research, National Institutes of Health, Bethesda, MD 20892, USAe Nippon Zoki Pharmaceutical Co., Ltd., 2-1-2 Hiranomachi, Chuo-ku, Osaka 541-0046, Japan

Abbreviations: BDNF, brain-derived neurotrophic facAMP responsive element binding protein; DMEM,medium; EIA, enzyme immunoassay; ELISA, enzyme-lGAPDH, glyceraldehyde-3-phosphate dehydrogenase;piperazineethanesulfonic acid; HRP, horseradish peroxactivated protein kinase; MEK, MAP kinase kinase (Mgrowth factor; NT-3, neurotrophin-3; PI 3-kinase, phosppolyvinylidene fluoride; RT-PCR, reverse transcription-ptropomyosin-related kinase; WRAM, water radial-arm m⁎ Corresponding author.

E-mail address: tmutoh@fujita-hu.ac.jp (T. Mutoh).

1044-7431/$ – see front matter © 2010 Elsevier Inc. Adoi:10.1016/j.mcn.2010.06.013

a b s t r a c t

a r t i c l e i n f o

Article history:Received 24 February 2010Revised 2 June 2010Accepted 18 June 2010Available online 28 June 2010

Keywords:NeurotropinBrain-derived neurotrophic factorCREB pathwayTrkBTs65Dn miceDown's syndromeRadial-arm maze

Expression of brain-derived neurotrophic factor (BDNF) was stimulated in human neuroblastoma SH-SY5Ycells by a nonprotein extract of inflamed rabbit skin inoculated with vaccinia virus (Neurotropin®), ananalgesic widely used in Japan for treatment of disorders associated with chronic pain, with the optimaldosage at 10 mNU/mL. This stimulation was accompanied by activations of p42/44 MAP kinase, CREB and c-Fos expression. Inhibitors of MAP kinases or PI 3-kinase prevented the stimulatory action of Neurotropin,indicating that neuronal TrkB/CREB pathway mediates the action. Repetitive oral administration ofNeurotropin (200 NU/kg/day, 3 months) prevented the age-dependent decline in hippocampal BDNFexpression in Ts65Dn mice, a model of Down's syndrome. This effect was associated with the improvementof spatial cognition of themice. These results open an intriguing new strategy inwhich Neurotropinmay provebeneficial treatment for neurodegenerative disorders.

ctor; cAMP, cyclic AMP; CREB,Dulbecco's modified Eagle'sinked immuno-solvent assay;HEPES, 4-(2-hydroxyethyl)-1-idase; MAP kinase, mitogen-APK/ERK kinase); NGF, nervehatidylinositol 3-kinase; PVDF,olymerase chain reaction; Trk,aze.

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© 2010 Elsevier Inc. All rights reserved.

Introduction

Brain-derived neurotrophic factor (BDNF) is a member of theneurotrophin family and plays important roles in many developmen-tally regulated processes, such as cell survival, differentiation andsynaptic plasticity of neurons as well as neurogenesis. Clinical andbasic evidence supports the idea that abnormalities in brain neuronalregeneration assisted by BDNF are associated with a wide range ofdisorders such as neurodegenerative diseases (e.g. Alzheimer'sdisease, Parkinson's disease, Down's syndrome) and psychiatric orstress-related conditions (e.g. bipolar disorder, schizophrenia, post-traumatic stress disorder) (reviewed by Kozisek et al., 2008; Pezet andMalcangio, 2004).

Additionally, there is growing evidence using animal models tosuggest roles for BDNF in chronic pain, which seem to be locationdependent. In the dorsal root ganglion, spinal dorsal horn (reviewed byPezet et al., 2002; Malcangio and Lessmann, 2003) and in supra-spinalneurons (Guo et al., 2006), an elevated expression of BDNF is believed tobe crucial for establishing chronic pain (central sensitization). Mean-while, BDNF has been reported to be reduced in higher centers such asthe hippocampus and cortex (Duric and McCarson, 2005; 2007). Thus,the contribution of BDNF in chronic pain is still unclear.

Neurotropin is a nonprotein extract of inflamed rabbit skin inoculatedwithvaccinia virus,whichhasbeen reported tobeeffectiveonchronic painconditions such as low back pain (Ono et al., 1982), neck–shoulder–armsyndrome (Ono et al., 1987), post-herpetic neuralgia (Yamamura et al.,1988), complex regional pain syndrome (Muneshige and Toda, 1996)andfibromyalgia (Nagaoka et al., 2004).Moreover, clinical effectivenessof Neurotropin has been reported for a wide variety of neuropathicsymptoms associated with subacute myelo-opticoneuropathy (SMON)(Sobue et al., 1992), diabetic neuropathy (Orimoet al., 1989), infarction-associated ischemic brain damage (de Reuck et al., 1994) andAlzheimer's disease (Kimura et al., 1987). At least, the analgesic actionof Neurotropin has been shown in rodent experiments to be mediatedthrough activation of the descending monoaminergic pain regulatorysystems in stress-related chronic pain conditions induced by the“specific alternation of rhythm in environmental temperature” (SART)

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(Kawamura et al., 1998; Ohara et al., 1991), peripheral inflammation(Miura et al., 2005), spinal nerve ligation (Suzuki et al., 2005), or chronicconstriction injury (Toda et al., 1998; Saleh et al., 1998). However,precise molecular mechanisms underlying these various pharmacolog-ical actions of Neurotropin remain to be elucidated.

In the present study, we demonstrate that Neurotropin stimulatesthe expression of BDNF in the human neuroblastoma cell line, SH-SY5Y,and mouse hippocampus in vivo. Neurotropin clearly augmented theTrkB-dependent intracellular signaling pathways associated withactivations of PI 3-kinase, MAP kinases and CREB, leading to increasedBDNF expression. Furthermore, Neurotropin enhanced the expressionof BDNF in the hippocampus of the Ts65Dn mice, a model of Down'ssyndrome, associated with age-dependant decline in hippocampalneurotrophin expressions (Hunter et al., 2004), along with functionalrecovery of cognition.

Results

Expression of BDNF in SH-SY5Y cells

Human neuroblastoma cell line SH-SY5Y expresses BDNF and itshigh-affinity receptor, TrkB (Kaplan et al., 1993). Incubation of the cellswith Neurotropin resulted in enhanced expression of BDNF in a time-dependent fashion (Fig. 1A). The strength of BDNF induction was

Fig. 1. Neurotropin upregulated BDNF expression in SH-SY5Y cells. A. Time dependency ofretinoic acid (RA, 1 μM) for the indicated period of time (0, 1, 3, 6, 12, 18, and 24 h) in serumin Experimental methods. B, C. Dose dependency of neurotrophin expressions by Neurotropiconcentrations of Neurotropin (0, 2, 10, 50 and 250 mNU/mL). Then the extracted total RNassay, the data represent averaged ratio of copy numbers of BDNF to GAPDH transcripts ofstandard errors (SEs) indicated by error bars. The indicated conditions are statistically dexpression. Cells (1×106 cells) were incubatedwith indicated concentrations of Neurotropinto ELISA assay and protein quantitation as described in Experimental methods. Results arindicated conditions are statistically different from the untreated control (*Pb0.05; **Pb0.0

comparable to that by retinoic acid (1 μM) at 24 h. A similar enhance-ment was observed in expressions of other neurotrophins such as NGFandNT-3, but not for high-affinity neurotrophin receptors TrkA andTrkB(Fig. 1B). The effect onBDNFexpressionwasdose-dependently observedwith an optimal concentration at 10 mNU/mL (Fig. 1C). The effect wasreduced at higher concentrations (50 and 250 mNU/mL), suggesting abi-modal effect of Neurotropin on BDNF expression. No morphologicalchange was observed in each Neurotropin-treated culture within 48 h(data not shown). Trypan blue extrusion assay indicates that there wasno significant loss in cell viability even at the highest concentration ofNeurotropin (250 mNU/mL).

We failed to detect BDNF in culture supernatants since the amountof secreted free BDNF was lower than the detection limit (3.9 pg/mL)of our ELISA method (data not shown). This is thought to be due toimmediate sequestration of free BDNF by TrkB as previously reported(Balkowiec and Katz, 2000). Alternatively, we measured intracellularBDNF levels. Consistent with the elevated mRNA expression,intracellular expression of mature BDNF was enhanced by Neuro-tropin at concentrations greater than 10 mNU/mL (Fig. 1D).

Mechanism for BDNF upregulation

BDNF expression is known to be upregulated through phosphor-ylation of the cyclic AMP responsive element binding protein (CREB).

BDNF expression. Cells (5×105 cells) were treated with Neurotropin (30 mNU/mL) or-free DMEM. BDNF expression was measured by semi-quantitative RT-PCR as describedn. Cells (approx. 5×105 cells) were incubated for 24 h in serum-free DMEM at indicatedA was subjected to semi-quantitative (B) and quantitative (C) RT-PCR. For quantitativeeight experiments expressed as fold increase over control treatment (=1.0), with theifferent from the saline-treated controls (*Pb0.05; **Pb0.01). D. Intracellular BDNF(0, 2, 10, 50 mNU/mL) for 24 h. Cells were lysed and the resultant extract was subjectede expressed as means and standard deviations (SDs) of triplicate measurements. The1).

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Expression of c-Fos, a proto-oncogene also driven by CREB activation,was stimulated similarly by Neurotropin at 24 h (Fig. 2A). Neurotropinenhanced phosphorylations of CREB at Ser133 and an upstreammodulator, p42/44 MAP kinase by 10 min in SH-SY5Y cells (Fig. 2B).Exogenous BDNF stimulated these processes in a similar manner.However, it is unlikely that the effects of Neurotropin were mediatedindirectly through the activatedBDNF release, because BDNF expressionby this drug was a relatively time consuming process (Fig. 1A).Consistently, inhibitors of PI 3-kinase (LY294002, 15 μM), MAP kinasekinase (MEK) (PD98059, 10 μM)orp38MAPkinase (SB203580, 0.5 μM)prevented Neurotropin-stimulated upregulation of the BDNF geneexpression (Fig. 2C). In contrast, intracellular levels of cyclic AMP(cAMP), another major modulator of CREB, were not elevated byNeurotropin, but rather suppressed at 50 mNU/mL (Fig. 2D). These datastrongly suggest that Neurotropin upregulates CREB-dependent tran-scriptions by activating theMAP kinase pathway rather than the cAMP-dependent protein kinase cascade.

Contribution of TrkB in Neurotropin action

Since neuronal activation of MAP kinase/CREB pathway is knownto be regulated coordinately through TrkB activation (Fukumoto et al.,2000), we next examined the role for TrkB in Neurotropin action.Stimulated expression of BDNF by Neurotropin was almost complete-ly blocked in the presence of Trk tyrosine kinase inhibitor K252a(500 nM) or a TrkB-specific antibody (20 μg/mL) (Fig. 3A). Our datastrongly indicates that the action of Neurotropin involves TrkB

Fig. 2. Mechanism of Neurotropin action on neuronal BDNF expression. A. Expression ofNeurotropin (0, 2, 10, 50 mNU/mL) for 24 h. Isolated RNAwas analyzed in semi-quantitative(1×106 cells) were treated with BDNF (50 ng/ml) or Neurotropin (0, 2, 10, 50 mNU/mL) foronto PVDF membranes, c-Fos, phosphorylated and total forms of CREB or p42/44MAP kinaseintracellular signaling inhibitors on BDNF expression. Cells (5×105 cells) were incubated wiincubation, cells were treated for 30 min with PD98059 (MEK inhibitor, 10 μM), LY294002 (Precovered was reverse transcribed and subjected to semi-quantitative RT-PCR as describedCells (3.75×105 cells) were incubated with indicated concentrations of Neurotropin (0, 2, 1lysed in 0.1 M HCl and cAMP content was quantified by EIA kit. Data represents means and

activation. Any quantitative increase was not observed in transcrip-tion (Fig. 1B) or cell surface expression (Fig. 3B) of TrkB. To thecontrary, the cell surface TrkB expression was significantly reduced at48 h of Neurotropin treatment.

Hippocampal expression of BDNF

BDNF is largely distributed in and is required for healthy hippo-campus. We next assessed the ability of Neurotropin to maintainhippocampal BDNF expression in Ts65Dn mice, an animal model ofearly-onset Alzheimer's disease and Down's syndrome, which areassociated with an age-dependent decline in hippocampal neurotro-phins and cognitive spatial memories (Granholm et al., 2003; Bimonteet al., 2003; Hunter et al., 2003a, 2004). Apparent cognitive dys-function was observed in the water radial-arm maze (WRAM) forTs65Dn mice at 4 months of age (Fig. 4A). Repeated oral administra-tions of Neurotropin (200 NU/kg/day, 3 months) improved thelearning behavior (reference memory) of the mice, whereas placebo(saline) treatment had no significant effect (Fig. 4B). Concurrently,Neurotropin treatment prevented reduction in hippocampal BDNFlevels in Ts65Dn mice (Fig. 5A). Regression analysis revealed thathippocampal BDNF levels and averaged behavioral scores weresignificantly correlated with each other in Ts65Dn mice (N=16, r=−0.613, P=0.012) (Fig. 5B). Thus, Neurotropin prevented age-dependent decline of hippocampal BDNF and spatial memory inTs65Dn mice.

c-Fos. SH-SY5Y cells (5×105 cells) were treated with the indicated concentrations ofRT-PCR as described in Experimental methods. B. Effect on CREB pathway. SH-SY5Y cells10 min or 30 min. Cell extracts were prepared and subjected to SDS-PAGE. After blottingwere detected by specific antibodies as described in Experimental methods. C. Effect of

th 10 mNU/mL of Neurotropin for 24 h in serum-free DMEM. Prior to termination of theI 3-kinase inhibitor, 15 μM), or SB203580 (p38MAP kinase inhibitor, 0.5 μM). Total RNAin Experimental methods. Representative results are shown. D. Cellular cAMP content.0, 50 mNU/mL) or with forskolin (10 μM) for 30 min in serum-free DMEM. Cells wereSDs of 4 experiments. *Pb0.05 vs. saline-treated controls (Control).

Fig. 3. Involvement of TrkB in Neurotropin action. A. Effects of TrkB inhibition. Cells(5×105 cells) were incubated with 10 mNU/mL of Neurotropin for 24 h in serum-freeDMEM. Prior to termination of the incubation, cells were treated for 60 min with K252a(Trk inhibitor, 500 nM) or rabbit IgG specific to human TrkB (αTrkB, 20 μg/mL).Isolated total RNA was reverse transcribed and subjected to semi-quantitative RT-PCRas described in Experimental methods. Representative results are shown. B. TrkBinternalization in Neurotropin-treated cells. SH-SY5Y cells (1×106 cells) were treatedwith Neurotropin (0, 2, 10, 50 mNU/mL) as indicated for 48 h. Cell surface proteinswere labeled with membrane non-permeable biotinylation reagent, captured onstreptavidin-coated plates, and detected with anti-Trk antibody (clone C-14) asdescribed in Experimental methods. Data represents means and SDs of triplicatemeasurements, expressed as a percentage of controls. *Pb0.05 vs. saline-treatedcontrols.

Fig. 4. Behavioral improvement of Ts65Dn mice. Spatial cognition (reference memory) was efor 3 months as described in Experimental methods. Data represents means and SEs. ***PbA. Behavioral abnormalities in Ts65Dn mice (4 months of age). Cognitive functions of Ts6compared between initial (days 2–3) and outcome (days 11–15) behavioral errors. DisaNeurotropin treatment on behaviors of Ts65Dn mice (7 months of age). Initial (days 2–3) ansaline (placebo) (gray columns, N=8) and with Neurotropin (closed columns, N=8). Neuimprovement of cognitive impairment in learning task.

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Discussion

In the present study, we first present evidence of modulation ofBDNF expression in cultured neuronal cells by Neurotropin. Upregu-lation was observed not only for BDNF but also for other neuro-trophins as shown in Fig. 1B, suggesting that this observation mayrelate to previously described “autocrine loops” of neurotrophins(Davis and Wright, 1995). A reduced expression of cell surface Trkmolecules by prolonged Neurotropin treatment (48 h, Fig. 3B)indicates the cellular release of functional BDNF and succeedingactivation of autocrine/paracrine circuits through TrkB which resultsin receptor internalization, analogous to that documented in TrkA-expressing PC12 cells treated with NGF (York et al., 2000).Neurotropin had been reported to promote neurite outgrowth(Morita et al., 1988) and to protect cells from morphological damageinduced by paclitaxel, an anti-cancer drug, (Kawashiri et al., 2009) inrat pheochromocytoma PC12 cells. Since differentiation of PC12 cellsis mediated through the TrkA pathway (Kaplan et al., 1991), activatedmodulation of the NGF–TrkA autocrine loop may explain themolecular basis of these neuroprotective actions by Neurotropin.

BDNF expression is regulated by intracellular signaling cascadessuch as the Ras–MAP kinase cascade and phosphorylation of CREB,which are downstream of Trk receptors (Patapoutian and Reichardt,2001). Hoshino et al. (2007) recently reported that Neurotropinprotected oxidant-exposed lung A549 cells from apoptotic celldamage through elevated expression of a redox-regulating molecule,thioredoxin-1. The thioredoxin-1 induction profile in A594 cells intheir report (optimum expression: 9 h, 10 mNU/mL) was close to ourobservation regarding neuronal BDNF expression. Thioredoxin-1 isnow recognized as a neurotrophic co-factor which mediates theaction of NGF (reviewed by Masutani et al., 2004). Since expressionsof thioredoxin-1 (Hirota et al., 2000) and BDNF (Finkbeiner et al.,1997) are both regulated by a cyclic AMP responsive element (cis-acting transcriptional enhancer element controlled by phosphorylat-ed CREB), effects on these factors may share a molecular mechanismtargeted by Neurotropin, as implied by our observation on CREBphosphorylation and c-Fos mRNA expression (Figs. 2A and B). Theinhibition study (Fig. 2C) suggests the presence of a Neurotropin

valuated in WRAM tests prior to (A) and after completion of (B) Neurotropin treatment0.001; *Pb0.05; ns, not significant vs. initial scores (averaged scores on days 2 and 3).5Dn (gray columns, N=16) and normosomic control (open columns, N=16) werebility in learning task (decrease in errors) was evident in Ts65Dn mice. B. Effect ofd outcome (days 11–15) behavioral errors were compared in Ts65Dn mice treated withrotropin administration gave Ts65Dn mice a significant decrease in errors, indicating

Fig. 5. Hippocampal BDNF levels in Ts65Dn mice. A. Effect of Neurotropin on hippocampal levels of BDNF. BDNF contents in hippocampal homogenates of Ts65Dn and normosomicmice were measured by ELISA as described in Experimental methods. Data represent averages and SDs (N=8 for each group). BDNF levels were significantly deceased in thehippocampus of Ts65Dn mice (gray column). With Neurotropin treatment, Ts65Dn mice had higher levels of BDNF comparable to those in normosomic controls. Crl, controls withsaline; Ntp, Neurotropin-treated animals. *Pb0.05; ns, not significant. B. Correlation between hippocampal BDNF and cognitive task in Ts65Dnmice. A linear regression analysis wasperformed on hippocampal levels of BDNF and averaged cognitive errors between days 11 and 15 in Ts65Dn mice (N=16). A statistically significant negative correlation wasdetected each other with a Pearson correlation coefficient (r) being −0.613 (Pb0.05). Closed symbols, individuals treated with Neurotropin; gray symbols, saline-treated animals.

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target in the upstreammodulators of signaling cascades including the PI3-kinase/MAP kinase pathways and the Ras–MAP kinase cascades.Although the same signaling pathways are known to be activated byneurotrophin-3 (Soiampornkul et al., 2008), neural cell adhesion mole-cule (Jessen et al., 2001), fibroblast growth factor-2 (Ditlevsen et al.,2008) or norepinephrine (Chen et al., 2007), Neurotropin presumablytargets TrkB directly to activate intrinsic tyrosine kinase, since BDNFinduction by the drug was affected by Trk tyrosine kinase inhibitorK252a or anti-TrkB antibody (Fig. 3A). Additional recent studiesdemonstrate that Neurotropin controls efficiency of the neurotrophinsignaling through Trk receptors probably by organizing lipid raft micro-domain (manuscript in preparation).

In order to assess the capacity of Neurotropin for induction ofBDNF in vivo, we employed Ts65Dn mice which are characterized bythe segmental trisomy of chromosome 16 (Davisson et al., 1990). Miceunder a trisomic background demonstrate behavioral abnormalities at4 months of age, followed by biochemical abnormalities in the brainsuch as enhanced accumulation in amyloid β peptide and decreaseddensity of cholinergic neurons in the cortex and basal forebrain, aswell as progressive loss of hippocampal neurotrophins (Hunter et al.,2003a,b). We started Neurotropin administration when behavioralabnormalities typically appear, in an attempt to relate BDNF functionto maintenance of brain cognitive function. As expected, Ts65Dn micereceiving Neurotropinmaintained the hippocampal BDNF expressionsas well as improved performance in the memory task (Figs. 4 and 5),confirming our previous data that BDNF is essential for learning(Bimonte-Nelson et al., 2003).

In the present study, we observed a sustained expression of BDNF inthe limbic system by an analgesic, Neurotropin. Similarly, severalantidepressive therapies have been shown to enhance brain BDNFexpressions (reviewed by Schmidt and Duman, 2007; Angelucci et al.,2004), and are clinically effective for certain chronic pain conditions(reviewed by Tsai, 2005; Verdu et al., 2008). In a previous study,Neurotropin ameliorated depressive symptoms in subjects with seniledementia (Kimura et al., 1987), further implying a similarity ofNeurotropin and antidepressants. However, the inabilities of variousantidepressants to induce neuronal BDNF directly in SH-SY5Y cells(Henkel et al., 2008) indicate that Neurotropin and antidepressantshave distinct mechanisms of action in the hippocampus. Nevertheless,the precisemolecularmechanism for hippocampal BDNF induction andits relation to the pharmacological effects of Neurotropin including thedescending pain modulatory system remain to be elucidated.

Over the last decade, many studies have shown that new neuronsare continually generated in two specific regions in the mammalianbrain; namely, the subventicullar zone (SVZ) of the lateral ventricle,and the subgranular zone (SGZ) of the hippocampal dentate gyrus(Gould and McEwen, 1993). Involvement of BDNF in adult hippo-campal neurogenesis has been intensively studied (reviewed by Leeand Son, 2009), and this neurotrophic factor is implicated in a broadspectrum of human conditions, such as neurodegenerative diseases,psychological, stress-related disorders, and aging (reviewed byKozisek et al., 2008). Many animal studies suggest that delivery ofBDNF itself or BDNF expressing vectors could represent an effectiveclinical approach to several disorders. However, difficulties intargeting specific brain regions and potential side reactions associatedwith the BDNF delivery needed to be solved and further preclinicalvalidation is required. Towards this extent, small nonprotein mole-cules which are permeable across the blood–brain barrier and boostBDNF expression could be a valid therapeutic option.

Experimental methods

Chemicals and reagents

Neurotropin was provided as sterile solutions at 10 and 20Neurotropin units (NU) per mL from Nippon Zoki PharmaceuticalCo. Ltd. (Osaka, Japan). The analgesic activity of Neurotropin isstandardized by a behavioral test in animals loaded with the SARTstress, a repeated cold stress by which hypersensitivity to noxiousstimuli is produced (Kita et al., 1979; Yoneda, 1992). For signalingstudies, LY294002 (a PI-3 kinase inhibitor) and PD98059 (a MEKinhibitor) were purchased from Enzo Life Sciences (PA, USA). K252a(a Trk tyrosine kinase inhibitor) and SB203580 (a p38 MAP kinaseinhibitor) were purchased from Biomol (PA, USA) and LC Laboratories(MA, USA), respectively. Rabbit polyclonal IgG specific to human TrkB(clone H-181) was purchased from Santa Cruz Biotechnology (CA,USA).

Animals

Three month-old male Ts65Dn mice (weighing 20–32 g) and con-trol littermates (weighing 22–37 g) were developed by and obtainedfrom the Jackson Laboratory (ME, USA). Karyotype of Ts65Dn strain isa partial trisomy for chromosome 16 close to the gene of amyloid

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precursor protein. Animals were singly housed with free access tofood and water, and were maintained on a 12-h light/dark cycle(7:00 AM/7:00 PM). All experiments were approved by the Institu-tional Animal Care and Use Committee of the Medical University ofSouth Carolina according to the National Institutes of Healthguidelines.

Drug administration and behavioral testing

The subjects in this study consisted of 3 month-old male trisomymice (Ts65Dn, N=16) and their normosomic littermates (N=16).Animals were divided into 2 Ts65Dn and 2 Normosomy groupsconsisting of 8 animals per group. After one-month of acclimation tothe vivarium, one group each of Ts65Dn and Normosomy groupsreceived daily administration of Neurotropin at 200 NU/kg/day for3 months till 7 months of age using oral gavage delivery. Saline (0.9%NaCl) was administered for remaining animal groups as a placebocontrol. Between groups received Neurotropin and placebo, there wasno significant difference in body weight (mean±SD, PN0.5 byStudent's t-test): prior to administration, 27.1±3.8 g and 26.5±3.1 g respectively for trisomy, 32.0±5.0 g and 33.4±3.7 g respec-tively for normosomic littermates; at the end of the behavioral testing,29.6±4.9 g and 28.8±2.7 g respectively for trisomy, 34.9±5.7 g and34.9±5.4 g respectively for normosomic littermates. Each individualdid not show any apparent abnormality in locomotive activity.

Behavior of the mice was tested twice on the WRAM as describedby Hunter et al. (2003b), at the onset and at the completion of drugadministration (4 and 7 months of age, respectively). Spatial cognitivememory (reference memory) was evaluated as the error numbers offirst entries into any arm that never contained a platform. Thebehavioral task was administered by continuous daily sessions for15 days. Data from Day 1 was excluded from the analysis because itwas considered a training session. Data from the sessions in Days 2–3and Days 11–15 were averaged as initial and outcome scores,respectively.

Cell cultures

Human neuroblastoma SH-SY5Y cells were cultured as describedby Hamano et al. (2005). The culture was maintained in Dulbecco'smodified Eagle's medium (Gibco DMEM; Invitrogen, CA, USA)supplemented with 10% fetal calf serum, 1 mM pyruvate, 0.37%glucose, and 25 mIU/mL penicillin/streptomycin. The cells werecultured in a humidified incubator at 37 °C and 5% CO2. Cell viabilitywas determined by extrusion of Trypan Blue dye (0.4%; Sigma, MO,USA).

Isolation of RNA and RT-PCR

SH-SY5Y cells (5×105 cells) were cultured in the presence ofNeurotropin or saline as a control in serum-free DMEM for 24 h. Forinhibition of cellular signaling molecules (Figs. 2C and 3A), inhibitorsdissolved in culture medium were added to the culture 30 or 60 minprior to termination of the incubation. Following removal of culturesupernatant, total RNA was isolated by TriZol reagent (Invitrogen,USA). Recovery and purity of the RNA preparations were evaluated byUV absorption at 260 nm and 280 nm (GeneQuant Pro, GE Health-care), and an equal amount of RNA was reverse transcribed into first-strand DNA with random primers by Superscript III first-strand DNAsynthesis kit (Invitrogen, USA), followed by polymerase chainreaction (PCR) by Taq DNA polymerase (Invitrogen, USA). For semi-quantitative assays, PCR primers (Invitrogen, Tokyo) for NGF, BDNF,NT-3 and cyclophilin A were designed as described by Yamamotoet al. (1996). PCR primers (Sigma Genosys, Hokkaido, Japan) for TrkA,TrkBandc-fosweredesignedoriginally as the followingsequences: TrkA(178 bp), 5′-tggacaaccctttcgagttc-3′ and 5′-cgtccacatttgttgagcac-3′;

TrkB (188 bp), 5′-ccgagattggagcctaacag-3′ and 5′-tgcaggttgctgtttttcag-3′;c-Fos (178 bp), 5′-gcttcccttgatctgactgg-3′ and 5′-atgatgctgggaacaggaag-3′. PCR products were visualized by ethidium bromide (0.1 μg/mL)staining in 3% agarose gel (NuSieve3:1Agarose, TakaraBio, Shiga, Japan)after electrophoretic separation. Template amount–product amountlinearity in PCR was checked separately.

Quantitation of mRNA was performed by real-time PCR system(Prism 7900HT, Applied Biosystems) with target cDNA templates ofknown concentrations. PCR primers (Sigma Genosys, Japan) consistedof the following nucleotide sequences: BDNF, 5′-gctgcaaacatgtccatgag-3′, 5′-atgggattgcacttggtctc-3′; GAPDH, 5′-atcactgccacccagaagac-3′, 5′-tttctagacggcaggtcagg-3′.

Immunoblotting of signaling molecules

Intracellular signaling molecules were detected using specificantibodies raised against CREB (MAB5432; Millipore, MA, USA),phosphorylated CREB (clone 10E9;Millipore, USA), p42/44MAP kinase(Cell Signaling Technology, MA, USA), phosphorylated p42/44 MAPkinase (Tyr202/Tyr204; Cell Signaling Technology, USA), and c-Fos(Gene Tex, CA, USA). SH-SY5Y cells (1×106 cells) were cultured inserum-free DMEM for 6 h and treated with human recombinant BDNF(50 ng/mL; Peprotech, NJ, USA) or Neurotropin for 10 or 30 min. Then,cells were scraped off with cell scrapers (BD Biosciences, CA, USA) andimmediately washed in ice-cold phosphate-buffered saline (PBS),followed by the lysis as described previously (Mutoh et al., 1995). Thelysates were subjected to SDS-PAGE in 4%–20% gels (ePAGEL; AttoChemicals, Tokyo, Japan), then blotted onto a PVDF membrane(Immobilon-P; Millipore, USA). Membranes were blocked for 1 h in20 mMTris–HCl, pH 7.5, 0.15 MNaCl (TBS)with 0.1% Tween 20 (TBS-T)with 3% nonfat milk. Incubation with primary as well as HRP-coupledsecondary antibodies was performed for 1 h at room temperature inTBS-T. Immunoreactive bands were visualized by an enhancedchemiluminescence detection system (ECL; GE Healthcare, Buckin-ghamshire, UK).

Intracellular cAMP levels

SH-SY5Y cells, plated at 3.75×105 cells per well on a 24-wellculture dish, were treated with Neurotropin (2, 10, 50 mNU/mL) orforskolin (10 μM; Sigma, USA) for 30 min in serum-free DMEM. Afterremoval of culture supernatant, cells were immediately lysed at 4 °Cin 300 μL/well of 0.1 M HCl. Cellular cAMP content of the lysate wasdirectly measured by EIA kit (Cayman Chemicals, MI, USA) accordingto the manufacturer's instruction. Protein concentration was deter-mined with BCA protocol (Thermo Fisher Scientific, MA, USA) withstandard BSA solution (Thermo Fisher Scientific, USA).

Cell surface expression of TrkB

Cell surface density of TrkB molecule was evaluated by the methoddescribedbySerres andCarney(2006)with somemodifications. SH-SY5Ycells were cultured by seeding at 1×105 per mL, and treated withNeurotropin (2, 10, 50 mNU/mL) for 48 h in DMEM containing 1% FCS.Cells were washed 3 times in ice-cold PBS containing 0.1 mM CaCl2 and1 mM MgCl2 to prevent receptor internalization, then cell surfacemolecules were biotinylated for 30 min at 4 °C in 250 μg/mL EZ-Linksulfo–LC–LC–NHS–biotin (Thermo Fisher Scientific, USA) dissolved inPBS. After 2 washes with ice-cold PBS containing 10 mM glycine forquenching unreacted biotinylation reagent, cells were lysed in aminimum volume of lysis buffer (1% Nonidet P-40, 0.5% sodiumdeoxycholate, 0.1% SDS in PBS) containing protease inhibitor cocktails(Sigma,USA). Protein concentration of sampleswas adjusted to 50 μg/mLin lysis buffer.

Fivemicrograms of sample (100 μL)were incubated in streptavidin-coated plates (Thermo Fischer, USA) for 2 h at room temperature. After

232 Y. Fukuda et al. / Molecular and Cellular Neuroscience 45 (2010) 226–233

washing 3 times with PBS containing 0.2% Tween 20, rabbit anti-Trkantibody (100 μL; clone C-14; Santa Cruz Biotechnology, USA) wasadded at a dilution of 1:1000 for 1 h. After washing, anti-rabbit IgGconjugated with horseradish peroxidase (1:1000, 100 μL; AP132P;Chemicon International, CA, USA) was added for 1 h, followed byenzymatic reaction with TMB One substrate (100 μL; Promega, USA).The reactionwas terminated by the addition of 0.1 NHCl (50 μL). Boundspecific antibody was quantitatively measured by reading opticaldensity at 450 nm (Benchmark microplate reader; BioRad). This valuewas expressed as a percentage of controls.

ELISA assays for neurotrophins in cultured cells and mousehippocampus

Intracellular levels of BDNF were measured for cell lysates asfollows. SH-SY5Y cells (1×106 cells) were scraped off the plate andwashed one with ice-cold PBS. The cell pellet were treated in a lysisbuffer (20 mM HEPES, pH 7.2, 1% Nonidet P-40, 10% glycerol, 50 mMNaF, 1 mM phenylmethylsulfonyl fluoride, 1 mMNaVO4) containing aprotease inhibitor cocktail (Sigma, USA). After the removal of celldebris, the cell lysate was stored at −80 °C until ELISA assay. Proteincontent of the lysates wasmeasured by BCA protein assay kit (ThermoFisher Scientific, USA).

Five days after the conclusion of WRAM testing, brains of micewere dissected under anesthesia with Halothane to obtain thehippocampus. The hippocampal tissue was immediately frozen indry ice–ethanol bath and stored at −70 °C until analysis. The tissuewas homogenized in a lysis buffer containing 137 mM NaCl, 20 mMTris (pH 8.0), and 1% Nonidet P-40 (Sigma, USA).

BDNF were quantified using commercially available ELISA kits(Emax ELISA kits, Promega, WI, USA) according to our standardprotocol. Briefly, 96-well, polystyrene plates (Immulon 4 HBX;Thermo Fisher Scientific, USA) were coated with the correspondingantibody to capture neurotrophin. The captured neurotrophin wassubjected to the secondary antibody, followed by detection withspecies-specific antibody conjugated to horseradish peroxidase. Afterremoval of unbound conjugates, bound enzyme activity was assessedby chromogenic substrate for measurement at 450 nm by a micro-plate reader (Benchmark microplate reader, BioRad). Using these kits,BDNF can be quantified in the range of 3.9–500 pg/mL. Cross-reactivity with other neurotrophic proteins is less than 2%.

Statistics

All animal experiments were blinded including drug administra-tion, behavioral WRAM testing, and neurotrophin measurement. Alldata were analyzed after the completion of experiments by Student'st-test (SAS system version 8.2, SAS Institute, Japan). All significancetests used a level=0.05 (two-tailed).

Acknowledgment

We thank Dr. Irwin J. Kopin (NINDS, NIH) for carefully reviewingour draft manuscript.

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