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Insulin-like growth factor-I enhances the biological activity of brain-derived neurotrophic factor on cerebrocortical neurons
Journal of Neuroimmunology 179 (2006) 186 – 190 www.elsevier.com/locate/jneuroim
Short communication
Insulin-like growth factor-I enhances the biological activity of brain-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 and plasticity. 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 the ability of BDNF to induce ERK1/2 phosphorylation. This IGF-I-induced increase in BDNF responsiveness describes a new interaction between 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 nervous system (CNS), being best characterized for its role in learning and memory (Mizuno and Giese, 2005). Its expression is associated with learning recovery following kainate administration (Duan et al., 2001) and BDNF is required for normal contextual learning (Barrientos et al., 2004). BDNF promotes neurogenesis (Zigova et al., 1998; Pencea et al., 2001) and BDNF has been recently discovered to exhibit anti-depressant-like activity (Hoshaw et al., 2005). IGF-I shares similar properties since it promotes neurogenesis (Trejo et al., 2001; Malberg and Blendy, 2005) and reduces depressive-like behaviors (Hoshaw et al., 2005). We have shown that intracerebroventricular administration of IGF-I reduces sickness behavior caused by i.c.v. injection of lipopolysaccharide or 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: [email protected] (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 treated with kainate in the absence of hippocampal neurodegeneration (Bluthe et al., 2005). The mechanism responsible for the improvement of memory by IGF-I is unknown. BDNF and IGF-I act on neurons via tyrosine kinase receptors, Trk-B and IGF-1R, respectively. Both receptors are present on cortical and hippocampal neurons (Broad et al., 2002; Chung et al., 2002; Romano, 2003) and activate MAPK/ERK or PI-3K/Akt signaling pathways in a variety of cells. BDNF utilizes both pathways to prevent neurodegeneration and glutamate-induced excitotoxicity (Zheng and Quirion, 2004; Almeida et al., 2005; Zhu et al., 2005), but acts via MAPK/ERK to promote neuronal synaptic structure and long-term potentiation (LTP) (Ying et al., 2002; Kato et al., 2003). Consequently, agedependent impairment in LTP is related to reduction in BDNF-stimulated ERK-1/2 activation (Gooney et al., 2004), emphasizing the importance of MAPK/ERK in memory and learning. However, IGF-I induces only transient or no activation of the MAPK/ERK pathway in neurons (Zheng and Quirion, 2004; Johnson-Farley et al., 2006), explaining the paucity of reports for IGF-I directly
R.H. McCusker et al. / Journal of Neuroimmunology 179 (2006) 186–190
modulating neuronal plasticity. Lack of evidence for a direct role of IGF-I on neuronal plasticity indicates that the in vivo improvement in behavior and learning by IGF-I might 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 et al., 2004), which could mediate the memory-enhancing actions of IGF-I (Bluthe et al., 2005). However, the possibility has never been reported that IGF-I regulates BDNF responsiveness. This possibility is supported by recent data showing that acute exposure of hippocampal neurons to IGF-I plus BDNF is necessary to maximally induce Akt phosphorylation (Johnson-Farley et al., 2006). Although it is clear that these neurotrophins act together to regulate neuronal homeostasis and that they share many biologic properties within the CNS (Mattson et al., 2004), the possibility that they interact via changes in neurotrophin responsiveness remains to be explored. We provide evidence to support this hypothesis by demonstrating synergism between IGF-I and BDNF at the levels of neuronal Ca 2+ homeostasis, receptor expression and signaling. 2. Materials and methods 2.1. Reagents Neurobasal-A medium (NB-A) was from Invitrogen (Carlsbad, CA), minimal essential medium (MEM) from BioWhittaker (Walkersville, MD) and IGF-I from Intergen (Purchase, NY). Chemicals were bought from Sigma (St. Louis, MO) and cultureware from Fisher Scientific (Hanover
187
Table 1 IGF-I plus BDNF depress Ca2+ in cortical neurons
Control IGF-I BDNF IGF-I + BDNF
Peak response
Area under the curve
100 ± 16 76 ± 12 93 ± 4 38 ± 17⁎
100 ± 8 89 ± 6 103 ± 7 72 ± 14⁎
Quantification of the peak Fluo-4 (points shaded in Fig. 1) or area under the curve confirm that IGF-I plus BDNF depressed initial peak Ca2+ and depressed net intracellular calcium levels over the 20 min exposure to glutamate (⁎p < 0.05 compared to control; n = 4).
Park, IL). BDNF, antibodies against ERK-1/2, actin and TrkB were purchased from Santa Cruz (Santa Cruz, CA), while horseradish conjugated secondary antibodies were obtained from Amersham (Piscataway, NJ). Low endotoxin fetal bovine serum (FBS) and horse serum (HS) were from Hyclone (Logan, UT). 2.2. Cell culture Primary cultures of neurons (> 95% pure) from the cerebral cortex were prepared from Balb/cJ mice (< 48 h of age). Handling of mice followed strict adherence to the guidelines for experimentation with animals and procedures were approved by the Institutional Animal Care and Use Committee. Cortices were digested with papain (20 U/ml) at 37 °C. Tissue was centrifuged at 128 ×g, decanted and pelleted in MEM containing 10% heat-inactivated FBS and HS. 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
Fig. 1. IGF-I plus BDNF depress the glutamate-induced increase in intracellular Ca2+. Neurons responded to glutamate (100 μM) with an increase in Ca2+ which was not significantly affected by pretreatment with either IGF-I or BDNF alone. However, IGF-I and BDNF together decreased Ca2+ (n = 4).
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R.H. McCusker et al. / Journal of Neuroimmunology 179 (2006) 186–190
2.3. Western blots Neurons (7 d) were treated with IGF-I (50 ng/ml) for 24 h, after which BDNF (10 ng/ml) was added for 0, 5, 10, 15 or 30 min. Protein was then extracted and electrophoresed through 10% SDS–PAGE gels. Proteins were transferred to PVDF membranes, probed with antibodies and band intensities quantified using NIH Image J (Broussard et al., 2003). 2.4. Intracellular calcium
Fig. 2. Treatment of cortical neurons with IGF-I increased the expression of Trk-B. Neurons were exposed to IGF-I for 24 h before total cell extracts were subjected to SDS–PAGE. Trk-B expression was increased (p < 0.01) by IGF-I (n = 3).
penicillin and 100 μg/ml streptomycin), treated with 500 U/ ml DNAse, triturated and filtered through a 40 μm nylon mesh. Cells were plated (2.5 × 105 cells/cm 2) on polyornithine coated plates. After 24 h, cells were washed then fed NB-A-B27−; NB-A plus glutamine, antibiotics and insulin-free B27 supplement (Brewer et al., 1993).
Neurons (7–8 d) were treated with IGF-I (50 ng/ml), BDNF (10 ng/ml) or both peptides in NB-A-B27−. After 24 h, cells were washed and incubated in NB-A plus the Ca2+ indicator Fura-4 (2 μM) for 30 min at 37 °C. Neurons were washed to remove extracellular Fluo-4 and incubated for 30 min to de-esterify internalized Fura-4. A background reading for each well (excitation 485 μm, emission 530 μm) was obtained prior to addition of 100 μM glutamate followed by 60 measurements, once every 20 s, to quantify temporal changes in Ca2+. Treatments did not affect basal Ca2+ (fluorescence before glutamate) or dye loading (fluorescence following permeabilization with 0.2% Triton-X100 in the presence of 10 mM Ca2+; not shown). 2.5. Statistical analyses Data were analyzed as a completely randomized design by ANOVA. Treatment differences were detected with Duncan's multiple range tests using Statistical Analysis System for Windows. Data are presented as means ± SEM.
Fig. 3. Treatment of cortical neurons with IGF-I increased BDNF responsiveness. BDNF-induced phosphorylation of ERK (both 42 and 44 kDa proteins) (top) and IGF-I further increased the ability of BDNF to activate ERK (BDNF × IGF-I interaction, p < 0.01). Band intensity was quantified and expressed as a ratio of pErk to total Erk (bottom, n = 3).
R.H. McCusker et al. / Journal of Neuroimmunology 179 (2006) 186–190
3. Results and discussion Calcium plays an important role in neuronal survival and plasticity (Bliss and Collingridge, 1993; Sattler and Tymianski, 2001). Chronic IGF-I suppresses the glutamatereceptor dependent elevation of intracellular Ca2+ and subsequent death of hippocampal neurons following glucose deprivation (Cheng and Mattson, 1992); albeit it is unknown if IGF-I depresses Ca2+ following direct glutamate-receptor activation. Similarly, lowered Ca2+ is part of BDNF-induced neuroprotection (Tremblay et al., 1999). We now have identified a novel interaction between these two neurotrophins by showing that IGF-I and BDNF synergistically attenuate glutamate-induced increases in Ca2+ (Fig. 1). We selected a dose of each neurotrophin that, when applied for 24 h pretreatment, did not affect glutamate-induced increases in intracellular Ca2+. However, BDNF plus IGF-I reduced by 40 to 50% the glutamate-induced increase in both peak (p < 0.05) and sustained (area under the curve, p < 0.05) Fluo-4 activation (Table 1). These data are in accord with the recent observation that BDNF plus IGF-I is more effective than either neurotrophin alone in activating Akt or promoting survival of hippocampal neurons (Johnson-Farley et al., 2006). These reductions in Ca2+ contrast with the increase in intracellular Ca2+ caused by acute exposure of neurons to either BDNF (Kume et al., 1997; Sakai et al., 1997; Kafitz et al., 1999; Climent et al., 2000; Mizoguchi and Nabekura, 2003; He et al., 2005; Yang and Gu, 2005) or IGF-I (Marshall et al., 2003; Shan et al., 2003). However, both IGF-I and BDNF continuously bathe neurons within the brain, as in our in vitro model. Since agents that depress glutamate-induced Ca2+ also depress excitotoxicity (Schurr, 2004), the chronic exposure to BDNF plus IGF-I in vivo may synergistically depress the response of neurons to excitotoxic concentrations of glutamate. We next searched for a potential mechanism to explain the synergistic effect of BDNF plus IGF-I. Treatment of cortical neurons with IGF-I for 24 h increased Trk-B expression (Fig. 2, top). When normalized to actin, IGF-I induced a significant increase in Trk-B (Fig. 2, bottom). This is the first report describing the ability of IGF-I to increase Trk-B expression. It therefore appears that IGF-I augments both BDNF secretion (Carro et al., 2000; Kazanis et al., 2004) and expression of its receptor. Increased Trk-B may not necessarily translate into an increased BDNF activity. Thus, cortical neurons were pretreated with or without IGF-I prior to BDNF addition. In accordance with previous results (Ying et al., 2002; Kato et 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 by increased phosphorylation of ERK at 5, 10 15 and 30 min without changing total ERK (Fig. 3, top). This BDNFinduced increase in ERK signaling was significantly enhanced (p < 0.01) when neurons were pretreated with
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IGF-I (Fig. 3, bottom). Similar to the results of others (Zheng and Quirion, 2004; Johnson-Farley et al., 2006), we found that 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 increase in BDNF responsiveness and that this response was not a simple additive effect of the neurotrophins. Synthesis of BDNF is known to be increased by IGF-I, but the possibility that IGF-I also increases BDNF responsiveness has not been reported. The present experiments establish that BDNF activity (as measured by ERK phosphorylation) is enhanced by chronic exposure to IGFI, an effect accompanied by increased expression of Trk-B. That IGF-I synergizes with BDNF to alter Ca2+ metabolism while increasing BDNF responsiveness sheds direct insights into their well-described similarities in vivo (Mattson et al., 2004). This synergism may begin to explain how IGF-I regulates behavior, memory and learning in the absence of known direct effects on neuronal 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). References Almeida, R.D., Manadas, B.J., Melo, C.V., Gomes, J.R., Mendes, C.S., Graos, M.M., Carvalho, R.F., Carvalho, A.P., Duarte, C.B., 2005. Neuroprotection by BDNF against glutamate-induced apoptotic cell death is mediated by ERK and PI3-kinase pathways. Cell Death Differ. 12, 1329–1343. Barrientos, R.M., Sprunger, D.B., Campeau, S., Watkins, L.R., Rudy, J.W., Maier, S.F., 2004. BDNF mRNA expression in rat hippocampus following contextual learning is blocked by intrahippocampal IL-1beta administration. J. Neuroimmunol. 155, 119–126. Bliss, T.V., Collingridge, G.L., 1993. A synaptic model of memory: longterm potentiation in the hippocampus. Nature 361, 31–39. Bluthe, R.M., Frenois, F., Kelley, K.W., Dantzer, R., 2005. Pentoxifylline and insulin-like growth factor-I (IGF-I) abrogate kainic acid-induced cognitive impairment in mice. J. Neuroimmunol. 169, 50–58. Bluthe, R.M., Kelley, K.W., Dantzer, R., 2006. Effects of insulin-like growth factor-I on cytokine-induced sickness behavior in mice. Brain Behav. Immun. 20, 57–63. Brewer, G.J., Torricelli, J.R., Evege, E.K., Price, P.J., 1993. Optimized survival of hippocampal neurons in B27-supplemented Neurobasal, a new serum-free medium combination. J. Neurosci. Res. 35, 567–576. Broad, K.D., Mimmack, M.L., Keverne, E.B., Kendrick, K.M., 2002. Increased BDNF and Trk-B mRNA expression in cortical and limbic regions following formation of a social recognition memory. Eur. J. Neurosci. 16, 2166–2174. Broussard, S.R., McCusker, R.H., Novakofski, J.E., Strle, K., Shen, W.H., Johnson, R.W., Freund, G.G., Dantzer, R., Kelley, K.W., 2003. Cytokine-hormone interactions: tumor necrosis factor alpha impairs biologic activity and downstream activation signals of the insulin-like growth factor I receptor in myoblasts. Endocrinology 144, 2988–2996. Carro, E., Nunez, A., Busiguina, S., Torres-Aleman, I., 2000. Circulating insulin-like growth factor I mediates effects of exercise on the brain. J. Neurosci. 20, 2926–2933.
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Cheng, B., Mattson, M.P., 1992. IGF-I and IGF-II protect cultured hippocampal and septal neurons against calcium-mediated hypoglycemic damage. J. Neurosci. 12, 1558–1566. Chung, Y.H., Shin, C.M., Joo, K.M., Kim, M.J., Cha, C.I., 2002. Regionspecific alterations in insulin-like growth factor receptor type I in the cerebral cortex and hippocampus of aged rats. Brain Res. 946, 307–313. Climent, E., Sancho-Tello, M., Minana, R., Barettino, D., Guerri, C., 2000. Astrocytes in culture express the full-length Trk-B receptor and respond to brain derived neurotrophic factor by changing intracellular calcium levels: effect of ethanol exposure in rats. Neurosci. Lett. 288, 53–56. Dantzer, R., Gheusi, G., Johnson, R.W., Kelley, K.W., 1999. Central administration of insulin-like growth factor-1 inhibits lipopolysaccharide-induced sickness behavior in mice. NeuroReport 10, 289–292. Duan, W., Lee, J., Guo, Z., Mattson, M.P., 2001. Dietary restriction stimulates BDNF production in the brain and thereby protects neurons against excitotoxic injury. J. Mol. Neurosci. 16, 1–12. Gooney, M., Messaoudi, E., Maher, F.O., Bramham, C.R., Lynch, M.A., 2004. BDNF-induced LTP in dentate gyrus is impaired with age: analysis of changes in cell signaling events. Neurobiol. Aging 25, 1323–1331. He, J., Gong, H., Luo, Q., 2005. BDNF acutely modulates synaptic transmission and calcium signalling in developing cortical neurons. Cell. Physiol. Biochem. 16, 69–76. Hoshaw, B.A., Malberg, J.E., Lucki, I., 2005. Central administration of IGFI and BDNF leads to long-lasting antidepressant-like effects. Brain Res. 1037, 204–208. Johnson-Farley, N.N., Travkina, T., Cowen, D.S., 2006. Cumulative activation of Akt, and consequent inhibition of glycogen synthase kinase-3, by brain-derived neurotrophic factor and insulin-like growth factor-1 in cultured hippocampal neurons. J. Pharmacol. Exp. Ther. 316, 1062–1069. Kafitz, K.W., Rose, C.R., Thoenen, H., Konnerth, A., 1999. Neurotrophinevoked rapid excitation through TrkB receptors. Nature 401, 918–921. Kato, A., Fukazawa, Y., Ozawa, F., Inokuchi, K., Sugiyama, H., 2003. Activation of ERK cascade promotes accumulation of Vesl-1S/Homer-1a immunoreactivity at synapses. Brain Res. Mol. Brain Res. 118, 33–44. Kazanis, I., Giannakopoulou, M., Philippidis, H., Stylianopoulou, F., 2004. Alterations in IGF-I, BDNF and NT-3 levels following experimental brain trauma and the effect of IGF-I administration. Exp. Neurol. 186, 221–234. Kume, T., Kouchiyama, H., Kaneko, S., Maeda, T., Kaneko, S., Akaike, A., Shimohama, S., Kihara, T., Kimura, J., Wada, K., Koizumi, S., 1997. BDNF prevents NO mediated glutamate cytotoxicity in cultured cortical neurons. Brain Res. 756, 200–204. Malberg, J.E., Blendy, J.A., 2005. Antidepressant action: to the nucleus and beyond. Trends Pharmacol. Sci. 26, 631–638. Marshall, J., Dolan, B.M., Garcia, E.P., Sathe, S., Tang, X., Mao, Z., Blair, L. A., 2003. Calcium channel and NMDA receptor activities differentially regulate nuclear C/EBP beta levels to control neuronal survival. Neuron 39, 625–639. Mattson, M.P., Maudsley, S., Martin, B., 2004. A neural signaling triumvirate that influences ageing and age-related disease: insulin/IGF1, BDNF and serotonin. Ageing Res. Rev. 3, 445–464.
Mizoguchi, Y., Nabekura, J., 2003. Sustained intracellular Ca2+ elevation induced by a brief BDNF application in rat visual cortex neurons. NeuroReport 14, 1481–1483. Mizuno, K., Giese, K.P., 2005. Hippocampus-dependent memory formation: do memory type-specific mechanisms exist? J. Pharmacol. Sci. 98, 191–197. Pencea, V., Bingaman, K.D., Wiegand, S.J., Luskin, M.B., 2001. Infusion of brain-derived neurotrophic factor into the lateral ventricle of the adult rat leads to new neurons in the parenchyma of the striatum, septum, thalamus, and hypothalamus. J. Neurosci. 21, 6706–6717. Romano, G., 2003. The complex biology of the receptor for the insulin-like growth factor-1. Drug News Perspect. 16, 525–531. Sakai, N., Yamada, M., Numakawa, T., Ogura, A., Hatanaka, H., 1997. BDNF potentiates spontaneous Ca2+ oscillations in cultured hippocampal neurons. Brain Res. 778, 318–328. Sattler, R., Tymianski, M., 2001. Molecular mechanisms of glutamate receptor-mediated excitotoxic neuronal cell death. Mol. Neurobiol. 24, 107–129. Schurr, A., 2004. Neuroprotection against ischemic/hypoxic brain damage: blockers of ionotropic glutamate receptor and voltage sensitive calcium channels. Curr. Drug Targets 5, 603–618. Shan, H., Messi, M.L., Zheng, Z., Wang, Z.M., Delbono, O., 2003. Preservation of motor neuron Ca2+ channel sensitivity to insulin-like growth factor-1 in brain motor cortex from senescent rat. J. Physiol. 553, 49–63. Trejo, J.L., Carro, E., Torres-Aleman, I., 2001. Circulating insulin-like growth factor I mediates exercise-induced increases in the number of new neurons in the adult hippocampus. J. Neurosci. 21, 1628–1634. Tremblay, R., Hewitt, K., Lesiuk, H., Mealing, G., Morley, P., Durkin, J.P., 1999. Evidence that brain-derived neurotrophic factor neuroprotection is linked to its ability to reverse the NMDA-induced inactivation of protein kinase C in cortical neurons. J. Neurochem. 72, 102–111. Yang, B., Gu, Q., 2005. Contribution of glutamate receptors to brain-derived neurotrophic factor-induced elevation of intracellular Ca2+ levels. NeuroReport 16, 977–980. Ying, S.W., Futter, M., Rosenblum, K., Webber, M.J., Hunt, S.P., Bliss, T.V., Bramham, C.R., 2002. Brain-derived neurotrophic factor induces longterm potentiation in intact adult hippocampus: requirement for ERK activation coupled to CREB and upregulation of Arc synthesis. J. Neurosci. 22, 1532–1540. Zheng, W.H., Quirion, R., 2004. Comparative signaling pathways of insulinlike growth factor-1 and brain-derived neurotrophic factor in hippocampal neurons and the role of the PI3 kinase pathway in cell survival. J. Neurochem. 89, 844–852. Zhu, D., Wu, X., Strauss, K.I., Lipsky, R.H., Qureshi, Z., Terhakopian, A., Novelli, A., Banaudha, K., Marini, A.M., 2005. N-methyl-D-aspartate and TrkB receptors protect neurons against glutamate excitotoxicity through an extracellular signal-regulated kinase pathway. J. Neurosci. Res. 80, 104–113. Zigova, T., Pencea, V., Wiegand, S.J., Luskin, M.B., 1998. Intraventricular administration of BDNF increases the number of newly generated neurons in the adult olfactory bulb. Mol. Cell. Neurosci. 11, 234–245.
Short communication
Insulin-like growth factor-I enhances the biological activity of brain-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 and plasticity. 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 the ability of BDNF to induce ERK1/2 phosphorylation. This IGF-I-induced increase in BDNF responsiveness describes a new interaction between 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 nervous system (CNS), being best characterized for its role in learning and memory (Mizuno and Giese, 2005). Its expression is associated with learning recovery following kainate administration (Duan et al., 2001) and BDNF is required for normal contextual learning (Barrientos et al., 2004). BDNF promotes neurogenesis (Zigova et al., 1998; Pencea et al., 2001) and BDNF has been recently discovered to exhibit anti-depressant-like activity (Hoshaw et al., 2005). IGF-I shares similar properties since it promotes neurogenesis (Trejo et al., 2001; Malberg and Blendy, 2005) and reduces depressive-like behaviors (Hoshaw et al., 2005). We have shown that intracerebroventricular administration of IGF-I reduces sickness behavior caused by i.c.v. injection of lipopolysaccharide or 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: [email protected] (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 treated with kainate in the absence of hippocampal neurodegeneration (Bluthe et al., 2005). The mechanism responsible for the improvement of memory by IGF-I is unknown. BDNF and IGF-I act on neurons via tyrosine kinase receptors, Trk-B and IGF-1R, respectively. Both receptors are present on cortical and hippocampal neurons (Broad et al., 2002; Chung et al., 2002; Romano, 2003) and activate MAPK/ERK or PI-3K/Akt signaling pathways in a variety of cells. BDNF utilizes both pathways to prevent neurodegeneration and glutamate-induced excitotoxicity (Zheng and Quirion, 2004; Almeida et al., 2005; Zhu et al., 2005), but acts via MAPK/ERK to promote neuronal synaptic structure and long-term potentiation (LTP) (Ying et al., 2002; Kato et al., 2003). Consequently, agedependent impairment in LTP is related to reduction in BDNF-stimulated ERK-1/2 activation (Gooney et al., 2004), emphasizing the importance of MAPK/ERK in memory and learning. However, IGF-I induces only transient or no activation of the MAPK/ERK pathway in neurons (Zheng and Quirion, 2004; Johnson-Farley et al., 2006), explaining the paucity of reports for IGF-I directly
R.H. McCusker et al. / Journal of Neuroimmunology 179 (2006) 186–190
modulating neuronal plasticity. Lack of evidence for a direct role of IGF-I on neuronal plasticity indicates that the in vivo improvement in behavior and learning by IGF-I might 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 et al., 2004), which could mediate the memory-enhancing actions of IGF-I (Bluthe et al., 2005). However, the possibility has never been reported that IGF-I regulates BDNF responsiveness. This possibility is supported by recent data showing that acute exposure of hippocampal neurons to IGF-I plus BDNF is necessary to maximally induce Akt phosphorylation (Johnson-Farley et al., 2006). Although it is clear that these neurotrophins act together to regulate neuronal homeostasis and that they share many biologic properties within the CNS (Mattson et al., 2004), the possibility that they interact via changes in neurotrophin responsiveness remains to be explored. We provide evidence to support this hypothesis by demonstrating synergism between IGF-I and BDNF at the levels of neuronal Ca 2+ homeostasis, receptor expression and signaling. 2. Materials and methods 2.1. Reagents Neurobasal-A medium (NB-A) was from Invitrogen (Carlsbad, CA), minimal essential medium (MEM) from BioWhittaker (Walkersville, MD) and IGF-I from Intergen (Purchase, NY). Chemicals were bought from Sigma (St. Louis, MO) and cultureware from Fisher Scientific (Hanover
187
Table 1 IGF-I plus BDNF depress Ca2+ in cortical neurons
Control IGF-I BDNF IGF-I + BDNF
Peak response
Area under the curve
100 ± 16 76 ± 12 93 ± 4 38 ± 17⁎
100 ± 8 89 ± 6 103 ± 7 72 ± 14⁎
Quantification of the peak Fluo-4 (points shaded in Fig. 1) or area under the curve confirm that IGF-I plus BDNF depressed initial peak Ca2+ and depressed net intracellular calcium levels over the 20 min exposure to glutamate (⁎p < 0.05 compared to control; n = 4).
Park, IL). BDNF, antibodies against ERK-1/2, actin and TrkB were purchased from Santa Cruz (Santa Cruz, CA), while horseradish conjugated secondary antibodies were obtained from Amersham (Piscataway, NJ). Low endotoxin fetal bovine serum (FBS) and horse serum (HS) were from Hyclone (Logan, UT). 2.2. Cell culture Primary cultures of neurons (> 95% pure) from the cerebral cortex were prepared from Balb/cJ mice (< 48 h of age). Handling of mice followed strict adherence to the guidelines for experimentation with animals and procedures were approved by the Institutional Animal Care and Use Committee. Cortices were digested with papain (20 U/ml) at 37 °C. Tissue was centrifuged at 128 ×g, decanted and pelleted in MEM containing 10% heat-inactivated FBS and HS. 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
Fig. 1. IGF-I plus BDNF depress the glutamate-induced increase in intracellular Ca2+. Neurons responded to glutamate (100 μM) with an increase in Ca2+ which was not significantly affected by pretreatment with either IGF-I or BDNF alone. However, IGF-I and BDNF together decreased Ca2+ (n = 4).
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R.H. McCusker et al. / Journal of Neuroimmunology 179 (2006) 186–190
2.3. Western blots Neurons (7 d) were treated with IGF-I (50 ng/ml) for 24 h, after which BDNF (10 ng/ml) was added for 0, 5, 10, 15 or 30 min. Protein was then extracted and electrophoresed through 10% SDS–PAGE gels. Proteins were transferred to PVDF membranes, probed with antibodies and band intensities quantified using NIH Image J (Broussard et al., 2003). 2.4. Intracellular calcium
Fig. 2. Treatment of cortical neurons with IGF-I increased the expression of Trk-B. Neurons were exposed to IGF-I for 24 h before total cell extracts were subjected to SDS–PAGE. Trk-B expression was increased (p < 0.01) by IGF-I (n = 3).
penicillin and 100 μg/ml streptomycin), treated with 500 U/ ml DNAse, triturated and filtered through a 40 μm nylon mesh. Cells were plated (2.5 × 105 cells/cm 2) on polyornithine coated plates. After 24 h, cells were washed then fed NB-A-B27−; NB-A plus glutamine, antibiotics and insulin-free B27 supplement (Brewer et al., 1993).
Neurons (7–8 d) were treated with IGF-I (50 ng/ml), BDNF (10 ng/ml) or both peptides in NB-A-B27−. After 24 h, cells were washed and incubated in NB-A plus the Ca2+ indicator Fura-4 (2 μM) for 30 min at 37 °C. Neurons were washed to remove extracellular Fluo-4 and incubated for 30 min to de-esterify internalized Fura-4. A background reading for each well (excitation 485 μm, emission 530 μm) was obtained prior to addition of 100 μM glutamate followed by 60 measurements, once every 20 s, to quantify temporal changes in Ca2+. Treatments did not affect basal Ca2+ (fluorescence before glutamate) or dye loading (fluorescence following permeabilization with 0.2% Triton-X100 in the presence of 10 mM Ca2+; not shown). 2.5. Statistical analyses Data were analyzed as a completely randomized design by ANOVA. Treatment differences were detected with Duncan's multiple range tests using Statistical Analysis System for Windows. Data are presented as means ± SEM.
Fig. 3. Treatment of cortical neurons with IGF-I increased BDNF responsiveness. BDNF-induced phosphorylation of ERK (both 42 and 44 kDa proteins) (top) and IGF-I further increased the ability of BDNF to activate ERK (BDNF × IGF-I interaction, p < 0.01). Band intensity was quantified and expressed as a ratio of pErk to total Erk (bottom, n = 3).
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3. Results and discussion Calcium plays an important role in neuronal survival and plasticity (Bliss and Collingridge, 1993; Sattler and Tymianski, 2001). Chronic IGF-I suppresses the glutamatereceptor dependent elevation of intracellular Ca2+ and subsequent death of hippocampal neurons following glucose deprivation (Cheng and Mattson, 1992); albeit it is unknown if IGF-I depresses Ca2+ following direct glutamate-receptor activation. Similarly, lowered Ca2+ is part of BDNF-induced neuroprotection (Tremblay et al., 1999). We now have identified a novel interaction between these two neurotrophins by showing that IGF-I and BDNF synergistically attenuate glutamate-induced increases in Ca2+ (Fig. 1). We selected a dose of each neurotrophin that, when applied for 24 h pretreatment, did not affect glutamate-induced increases in intracellular Ca2+. However, BDNF plus IGF-I reduced by 40 to 50% the glutamate-induced increase in both peak (p < 0.05) and sustained (area under the curve, p < 0.05) Fluo-4 activation (Table 1). These data are in accord with the recent observation that BDNF plus IGF-I is more effective than either neurotrophin alone in activating Akt or promoting survival of hippocampal neurons (Johnson-Farley et al., 2006). These reductions in Ca2+ contrast with the increase in intracellular Ca2+ caused by acute exposure of neurons to either BDNF (Kume et al., 1997; Sakai et al., 1997; Kafitz et al., 1999; Climent et al., 2000; Mizoguchi and Nabekura, 2003; He et al., 2005; Yang and Gu, 2005) or IGF-I (Marshall et al., 2003; Shan et al., 2003). However, both IGF-I and BDNF continuously bathe neurons within the brain, as in our in vitro model. Since agents that depress glutamate-induced Ca2+ also depress excitotoxicity (Schurr, 2004), the chronic exposure to BDNF plus IGF-I in vivo may synergistically depress the response of neurons to excitotoxic concentrations of glutamate. We next searched for a potential mechanism to explain the synergistic effect of BDNF plus IGF-I. Treatment of cortical neurons with IGF-I for 24 h increased Trk-B expression (Fig. 2, top). When normalized to actin, IGF-I induced a significant increase in Trk-B (Fig. 2, bottom). This is the first report describing the ability of IGF-I to increase Trk-B expression. It therefore appears that IGF-I augments both BDNF secretion (Carro et al., 2000; Kazanis et al., 2004) and expression of its receptor. Increased Trk-B may not necessarily translate into an increased BDNF activity. Thus, cortical neurons were pretreated with or without IGF-I prior to BDNF addition. In accordance with previous results (Ying et al., 2002; Kato et 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 by increased phosphorylation of ERK at 5, 10 15 and 30 min without changing total ERK (Fig. 3, top). This BDNFinduced increase in ERK signaling was significantly enhanced (p < 0.01) when neurons were pretreated with
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IGF-I (Fig. 3, bottom). Similar to the results of others (Zheng and Quirion, 2004; Johnson-Farley et al., 2006), we found that 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 increase in BDNF responsiveness and that this response was not a simple additive effect of the neurotrophins. Synthesis of BDNF is known to be increased by IGF-I, but the possibility that IGF-I also increases BDNF responsiveness has not been reported. The present experiments establish that BDNF activity (as measured by ERK phosphorylation) is enhanced by chronic exposure to IGFI, an effect accompanied by increased expression of Trk-B. That IGF-I synergizes with BDNF to alter Ca2+ metabolism while increasing BDNF responsiveness sheds direct insights into their well-described similarities in vivo (Mattson et al., 2004). This synergism may begin to explain how IGF-I regulates behavior, memory and learning in the absence of known direct effects on neuronal 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). References Almeida, R.D., Manadas, B.J., Melo, C.V., Gomes, J.R., Mendes, C.S., Graos, M.M., Carvalho, R.F., Carvalho, A.P., Duarte, C.B., 2005. Neuroprotection by BDNF against glutamate-induced apoptotic cell death is mediated by ERK and PI3-kinase pathways. Cell Death Differ. 12, 1329–1343. Barrientos, R.M., Sprunger, D.B., Campeau, S., Watkins, L.R., Rudy, J.W., Maier, S.F., 2004. BDNF mRNA expression in rat hippocampus following contextual learning is blocked by intrahippocampal IL-1beta administration. J. Neuroimmunol. 155, 119–126. Bliss, T.V., Collingridge, G.L., 1993. A synaptic model of memory: longterm potentiation in the hippocampus. Nature 361, 31–39. Bluthe, R.M., Frenois, F., Kelley, K.W., Dantzer, R., 2005. Pentoxifylline and insulin-like growth factor-I (IGF-I) abrogate kainic acid-induced cognitive impairment in mice. J. Neuroimmunol. 169, 50–58. Bluthe, R.M., Kelley, K.W., Dantzer, R., 2006. Effects of insulin-like growth factor-I on cytokine-induced sickness behavior in mice. Brain Behav. Immun. 20, 57–63. Brewer, G.J., Torricelli, J.R., Evege, E.K., Price, P.J., 1993. Optimized survival of hippocampal neurons in B27-supplemented Neurobasal, a new serum-free medium combination. J. Neurosci. Res. 35, 567–576. Broad, K.D., Mimmack, M.L., Keverne, E.B., Kendrick, K.M., 2002. Increased BDNF and Trk-B mRNA expression in cortical and limbic regions following formation of a social recognition memory. Eur. J. Neurosci. 16, 2166–2174. Broussard, S.R., McCusker, R.H., Novakofski, J.E., Strle, K., Shen, W.H., Johnson, R.W., Freund, G.G., Dantzer, R., Kelley, K.W., 2003. Cytokine-hormone interactions: tumor necrosis factor alpha impairs biologic activity and downstream activation signals of the insulin-like growth factor I receptor in myoblasts. Endocrinology 144, 2988–2996. Carro, E., Nunez, A., Busiguina, S., Torres-Aleman, I., 2000. Circulating insulin-like growth factor I mediates effects of exercise on the brain. J. Neurosci. 20, 2926–2933.
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