Journal of Neuroimmunology 179 (2006) 186–190www.elsevier.com/locate/jneuroim

Short communication

Insulin-like growth factor-I enhances the biological activity ofbrain-derived neurotrophic factor on cerebrocortical neurons

Robert H. McCusker ⁎, Katherine McCrea, Samantha Zunich, Robert Dantzer,Suzanne R. Broussard, Rodney W. Johnson, Keith W. Kelley

250 Edward R. Madigan Laboratory, 1201 W. Gregory Dr. Urbana, IL 61801-3873, USA

Received 24 April 2006; received in revised form 14 June 2006; accepted 14 June 2006

Abstract

Insulin-like growth factor (IGF)-I and brain-derived neurotrophic factor (BDNF) act within the brain to enhance neuronal survival andplasticity. We extend these findings by showing that the presence of both neurotrophins is required to depress the rise in intracellular Ca2+

caused by glutamate in primary cultures of cerebrocortical neurons. IGF-I enhanced expression of BDNF receptors (Trk-B) and increased theability of BDNF to induce ERK1/2 phosphorylation. This IGF-I-induced increase in BDNF responsiveness describes a new interactionbetween these peptides in the brain.© 2006 Elsevier B.V. All rights reserved.

Keywords: Trk-B; Calcium; ERK; MAPK

1. Introduction

BDNF has diverse actions within the central nervoussystem (CNS), being best characterized for its role inlearning and memory (Mizuno and Giese, 2005). Itsexpression is associated with learning recovery followingkainate administration (Duan et al., 2001) and BDNF isrequired for normal contextual learning (Barrientos et al.,2004). BDNF promotes neurogenesis (Zigova et al., 1998;Pencea et al., 2001) and BDNF has been recentlydiscovered to exhibit anti-depressant-like activity (Hoshawet al., 2005). IGF-I shares similar properties since itpromotes neurogenesis (Trejo et al., 2001; Malberg andBlendy, 2005) and reduces depressive-like behaviors(Hoshaw et al., 2005). We have shown that intracerebro-ventricular administration of IGF-I reduces sicknessbehavior caused by i.c.v. injection of lipopolysaccharideor tumor necrosis factor-α (Dantzer et al., 1999; Bluthe et

⁎ Corresponding author. Tel.: +1 217 333 5142; fax: +1 217 244 5617.E-mail address: rmccuske@uiuc.edu (R.H. McCusker).

0165-5728/$ - see front matter © 2006 Elsevier B.V. All rights reserved.doi:10.1016/j.jneuroim.2006.06.014

al., 2006) and improves spatial memory of mice treatedwith kainate in the absence of hippocampal neurodegenera-tion (Bluthe et al., 2005). The mechanism responsible forthe improvement of memory by IGF-I is unknown.

BDNF and IGF-I act on neurons via tyrosine kinasereceptors, Trk-B and IGF-1R, respectively. Both receptorsare present on cortical and hippocampal neurons (Broad etal., 2002; Chung et al., 2002; Romano, 2003) and activateMAPK/ERK or PI-3K/Akt signaling pathways in a varietyof cells. BDNF utilizes both pathways to preventneurodegeneration and glutamate-induced excitotoxicity(Zheng and Quirion, 2004; Almeida et al., 2005; Zhu etal., 2005), but acts via MAPK/ERK to promote neuronalsynaptic structure and long-term potentiation (LTP) (Yinget al., 2002; Kato et al., 2003). Consequently, age-dependent impairment in LTP is related to reduction inBDNF-stimulated ERK-1/2 activation (Gooney et al.,2004), emphasizing the importance of MAPK/ERK inmemory and learning. However, IGF-I induces onlytransient or no activation of the MAPK/ERK pathway inneurons (Zheng and Quirion, 2004; Johnson-Farley et al.,2006), explaining the paucity of reports for IGF-I directly

Table 1IGF-I plus BDNF depress Ca2+ in cortical neurons

Peak response Area under the curve

Control 100±16 100±8IGF-I 76±12 89±6BDNF 93±4 103±7IGF-I+BDNF 38±17⁎ 72±14⁎

Quantification of the peak Fluo-4 (points shaded in Fig. 1) or area under thecurve confirm that IGF-I plus BDNF depressed initial peak Ca2+ anddepressed net intracellular calcium levels over the 20 min exposure toglutamate (⁎p<0.05 compared to control; n=4).

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modulating neuronal plasticity. Lack of evidence for adirect role of IGF-I on neuronal plasticity indicates that thein vivo improvement in behavior and learning by IGF-Imight involve an intermediate such as BDNF.

Direct injections of IGF-I, as well as moderate exercise,increase BDNF in the brain (Carro et al., 2000; Kazanis etal., 2004), which could mediate the memory-enhancingactions of IGF-I (Bluthe et al., 2005). However, thepossibility has never been reported that IGF-I regulatesBDNF responsiveness. This possibility is supported byrecent data showing that acute exposure of hippocampalneurons to IGF-I plus BDNF is necessary to maximallyinduce Akt phosphorylation (Johnson-Farley et al., 2006).Although it is clear that these neurotrophins act together toregulate neuronal homeostasis and that they share manybiologic properties within the CNS (Mattson et al., 2004),the possibility that they interact via changes in neuro-trophin responsiveness remains to be explored. We provideevidence to support this hypothesis by demonstratingsynergism between IGF-I and BDNF at the levels ofneuronal Ca2+ homeostasis, receptor expression andsignaling.

2. Materials and methods

2.1. Reagents

Neurobasal-A medium (NB-A) was from Invitrogen(Carlsbad, CA), minimal essential medium (MEM) fromBioWhittaker (Walkersville, MD) and IGF-I from Intergen(Purchase, NY). Chemicals were bought from Sigma (St.Louis, MO) and cultureware from Fisher Scientific (Hanover

Fig. 1. IGF-I plus BDNF depress the glutamate-induced increase in intracellular Ca2

was not significantly affected by pretreatment with either IGF-I or BDNF alone. H

Park, IL). BDNF, antibodies against ERK-1/2, actin and Trk-B were purchased from Santa Cruz (Santa Cruz, CA), whilehorseradish conjugated secondary antibodies were obtainedfrom Amersham (Piscataway, NJ). Low endotoxin fetalbovine serum (FBS) and horse serum (HS) were fromHyclone (Logan, UT).

2.2. Cell culture

Primary cultures of neurons (>95% pure) from thecerebral cortex were prepared from Balb/cJ mice (<48 h ofage). Handling of mice followed strict adherence to theguidelines for experimentation with animals and procedureswere approved by the Institutional Animal Care and UseCommittee. Cortices were digested with papain (20 U/ml) at37 °C. Tissue was centrifuged at 128 ×g, decanted andpelleted in MEM containing 10% heat-inactivated FBS andHS. Cells were resuspended in MEMhi; MEM plus 10%heat-inactivated FBS, high glucose (25 mg/L), glutamine(2 mM) and antibiotics (10 μg/ml gentamicin, 100 U/ml

+. Neurons responded to glutamate (100 μM) with an increase in Ca2+ whichowever, IGF-I and BDNF together decreased Ca2+ (n=4).

Fig. 2. Treatment of cortical neurons with IGF-I increased the expression ofTrk-B. Neurons were exposed to IGF-I for 24 h before total cell extractswere subjected to SDS–PAGE. Trk-B expression was increased (p<0.01) byIGF-I (n=3).

188 R.H. McCusker et al. / Journal of Neuroimmunology 179 (2006) 186–190

penicillin and 100 μg/ml streptomycin), treated with 500 U/ml DNAse, triturated and filtered through a 40 μm nylonmesh. Cells were plated (2.5×105 cells/cm2) on poly-ornithine coated plates. After 24 h, cells were washed thenfed NB-A-B27−; NB-A plus glutamine, antibiotics andinsulin-free B27 supplement (Brewer et al., 1993).

Fig. 3. Treatment of cortical neurons with IGF-I increased BDNF responsiveness. Band IGF-I further increased the ability of BDNF to activate ERK (BDNF×IGF-I inpErk to total Erk (bottom, n=3).

2.3. Western blots

Neurons (7 d) were treated with IGF-I (50 ng/ml) for24 h, after which BDNF (10 ng/ml) was added for 0, 5,10, 15 or 30 min. Protein was then extracted andelectrophoresed through 10% SDS–PAGE gels. Proteinswere transferred to PVDF membranes, probed withantibodies and band intensities quantified using NIHImage J (Broussard et al., 2003).

2.4. Intracellular calcium

Neurons (7–8 d) were treated with IGF-I (50 ng/ml),BDNF (10 ng/ml) or both peptides in NB-A-B27−. After24 h, cells were washed and incubated in NB-A plus theCa2+ indicator Fura-4 (2 μM) for 30 min at 37 °C. Neuronswere washed to remove extracellular Fluo-4 and incubatedfor 30 min to de-esterify internalized Fura-4. A backgroundreading for each well (excitation 485 μm, emission530 μm) was obtained prior to addition of 100 μMglutamate followed by 60 measurements, once every 20 s,to quantify temporal changes in Ca2+. Treatments did notaffect basal Ca2+ (fluorescence before glutamate) or dyeloading (fluorescence following permeabilization with0.2% Triton-X100 in the presence of 10 mM Ca2+; notshown).

2.5. Statistical analyses

Data were analyzed as a completely randomized designby ANOVA. Treatment differences were detected withDuncan's multiple range tests using Statistical AnalysisSystem for Windows. Data are presented as means±SEM.

DNF-induced phosphorylation of ERK (both 42 and 44 kDa proteins) (top)teraction, p<0.01). Band intensity was quantified and expressed as a ratio of

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3. Results and discussion

Calcium plays an important role in neuronal survivaland plasticity (Bliss and Collingridge, 1993; Sattler andTymianski, 2001). Chronic IGF-I suppresses the glutamate-receptor dependent elevation of intracellular Ca2+ andsubsequent death of hippocampal neurons followingglucose deprivation (Cheng and Mattson, 1992); albeit itis unknown if IGF-I depresses Ca2+ following directglutamate-receptor activation. Similarly, lowered Ca2+ ispart of BDNF-induced neuroprotection (Tremblay et al.,1999). We now have identified a novel interaction betweenthese two neurotrophins by showing that IGF-I and BDNFsynergistically attenuate glutamate-induced increases inCa2+ (Fig. 1). We selected a dose of each neurotrophinthat, when applied for 24 h pretreatment, did not affectglutamate-induced increases in intracellular Ca2+. How-ever, BDNF plus IGF-I reduced by 40 to 50% theglutamate-induced increase in both peak (p<0.05) andsustained (area under the curve, p<0.05) Fluo-4 activation(Table 1). These data are in accord with the recentobservation that BDNF plus IGF-I is more effective thaneither neurotrophin alone in activating Akt or promotingsurvival of hippocampal neurons (Johnson-Farley et al.,2006). These reductions in Ca2+ contrast with the increasein intracellular Ca2+ caused by acute exposure of neuronsto either BDNF (Kume et al., 1997; Sakai et al., 1997;Kafitz et al., 1999; Climent et al., 2000; Mizoguchi andNabekura, 2003; He et al., 2005; Yang and Gu, 2005) orIGF-I (Marshall et al., 2003; Shan et al., 2003). However,both IGF-I and BDNF continuously bathe neurons withinthe brain, as in our in vitro model. Since agents thatdepress glutamate-induced Ca2+ also depress excitotoxicity(Schurr, 2004), the chronic exposure to BDNF plus IGF-Iin vivo may synergistically depress the response of neuronsto excitotoxic concentrations of glutamate.

We next searched for a potential mechanism to explainthe synergistic effect of BDNF plus IGF-I. Treatment ofcortical neurons with IGF-I for 24 h increased Trk-Bexpression (Fig. 2, top). When normalized to actin, IGF-Iinduced a significant increase in Trk-B (Fig. 2, bottom).This is the first report describing the ability of IGF-I toincrease Trk-B expression. It therefore appears that IGF-Iaugments both BDNF secretion (Carro et al., 2000;Kazanis et al., 2004) and expression of its receptor.

Increased Trk-B may not necessarily translate into anincreased BDNF activity. Thus, cortical neurons werepretreated with or without IGF-I prior to BDNF addition.In accordance with previous results (Ying et al., 2002; Katoet al., 2003; Zheng and Quirion, 2004; Almeida et al., 2005;Zhu et al., 2005), acute exposure to BDNF activated(p<0.01) the MAPK/ERK pathway, as determined byincreased phosphorylation of ERK at 5, 10 15 and 30 minwithout changing total ERK (Fig. 3, top). This BDNF-induced increase in ERK signaling was significantlyenhanced (p<0.01) when neurons were pretreated with

IGF-I (Fig. 3, bottom). Similar to the results of others (Zhengand Quirion, 2004; Johnson-Farley et al., 2006), we foundthat IGF-I alone (p>0.05) did not activate ERK in neurons.The significant interaction between BDNF and IGF-I(p<0.01) led us to conclude that IGF-I caused an increasein BDNF responsiveness and that this response was not asimple additive effect of the neurotrophins.

Synthesis of BDNF is known to be increased by IGF-I,but the possibility that IGF-I also increases BDNFresponsiveness has not been reported. The present experi-ments establish that BDNF activity (as measured by ERKphosphorylation) is enhanced by chronic exposure to IGF-I, an effect accompanied by increased expression of Trk-B.That IGF-I synergizes with BDNF to alter Ca2+ metabo-lism while increasing BDNF responsiveness sheds directinsights into their well-described similarities in vivo(Mattson et al., 2004). This synergism may begin toexplain how IGF-I regulates behavior, memory andlearning in the absence of known direct effects onneuronal plasticity or LTP.

Acknowledgements

Supported by the NIH to KWK (MH 51569), RWJ(AG023580) and RD (MH71349) and the USDA to RHM(AG 2004-35206-14144).

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