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Regulation of Dendritic Development by BDNF Requires Activation of CRTC1 by Glutamate

2010; Elsevier BV; Volume: 285; Issue: 37 Linguagem: Inglês

10.1074/jbc.m110.125740

1083-351X

Charles Finsterwald, Hubert Fiumelli, Jean‐René Cardinaux, Jean‐Luc Martin,

Neuroscience and Neuropharmacology Research

Dendritic growth is essential for the establishment of a functional nervous system. Among extrinsic signals that control dendritic development, substantial evidence indicates that BDNF regulates dendritic morphology. However, little is known about the underlying mechanisms by which BDNF controls dendritic growth. In this study, we show that the MAPK signaling pathway and the transcription factor cAMP response element-binding protein (CREB) mediate the effects of BDNF on dendritic length and complexity. However, phosphorylation of CREB alone is not sufficient for the stimulation of dendritic growth by BDNF. Thus, using a mutant form of CREB unable to bind CREB-regulated transcription coactivator (CRTC1), we demonstrate that this effect also requires a functional interaction between CREB and CRTC1. Moreover, inhibition of CRTC1 expression by shRNA-mediated knockdown abolished BDNF-induced dendritic growth of cortical neurons. Interestingly, we found that nuclear translocation of CRTC1 results from activation of NMDA receptors by glutamate, a process that is essential for the effects of BDNF on dendritic development. Together, these data identify a previously unrecognized mechanism by which CREB and the coactivator CRTC1 mediate the effects of BDNF on dendritic growth. Dendritic growth is essential for the establishment of a functional nervous system. Among extrinsic signals that control dendritic development, substantial evidence indicates that BDNF regulates dendritic morphology. However, little is known about the underlying mechanisms by which BDNF controls dendritic growth. In this study, we show that the MAPK signaling pathway and the transcription factor cAMP response element-binding protein (CREB) mediate the effects of BDNF on dendritic length and complexity. However, phosphorylation of CREB alone is not sufficient for the stimulation of dendritic growth by BDNF. Thus, using a mutant form of CREB unable to bind CREB-regulated transcription coactivator (CRTC1), we demonstrate that this effect also requires a functional interaction between CREB and CRTC1. Moreover, inhibition of CRTC1 expression by shRNA-mediated knockdown abolished BDNF-induced dendritic growth of cortical neurons. Interestingly, we found that nuclear translocation of CRTC1 results from activation of NMDA receptors by glutamate, a process that is essential for the effects of BDNF on dendritic development. Together, these data identify a previously unrecognized mechanism by which CREB and the coactivator CRTC1 mediate the effects of BDNF on dendritic growth. IntroductionDendrites are the primary sites where neurons receive and integrate information from a vast number of synaptic inputs. The specific branching pattern of dendrites determines the number and type of synapses received by a neuron (1Whitford K.L. Dijkhuizen P. Polleux F. Ghosh A. Annu. Rev. Neurosci. 2002; 25: 127-149Crossref PubMed Scopus (232) Google Scholar). Dendritic development is essential for the formation of neuronal circuits. Indeed, defects in dendritic growth are associated with some forms of mental retardation, including Down syndrome and fragile X syndrome (2Benavides-Piccione R. Ballesteros-Yáñez I. de Lagrán M.M. Elston G. Estivill X. Fillat C. Defelipe J. Dierssen M. Prog. Neurobiol. 2004; 74: 111-126Crossref PubMed Scopus (102) Google Scholar, 3O'Donnell W.T. Warren S.T. Annu. Rev. Neurosci. 2002; 25: 315-338Crossref PubMed Scopus (390) Google Scholar). Dendritic arbor development is characterized by extension and retraction of dendritic branches, followed by stabilization and growth of these branches (4Cline H.T. Curr. Opin. Neurobiol. 2001; 11: 118-126Crossref PubMed Scopus (414) Google Scholar, 5Jan Y.N. Jan L.Y. Genes Dev. 2001; 15: 2627-2641Crossref PubMed Scopus (65) Google Scholar). This multistep process is regulated both by intrinsic genetic programs that are capable of generating a basic dendritic arborization and by external signals such as neuronal activity, guidance molecules, and growth factors that are essential for sculpting dendrites to their final form (1Whitford K.L. Dijkhuizen P. Polleux F. Ghosh A. Annu. Rev. Neurosci. 2002; 25: 127-149Crossref PubMed Scopus (232) Google Scholar, 6Dijkhuizen P.A. Ghosh A. J. Neurobiol. 2005; 62: 278-288Crossref PubMed Scopus (130) Google Scholar, 7Miller F.D. Kaplan D.R. Curr. Opin. Neurobiol. 2003; 13: 391-398Crossref PubMed Scopus (137) Google Scholar). There is compelling in vitro and in vivo evidence that BDNF, a member of the neurotrophin family, regulates dendritic morphology. In particular, BDNF plays an important role in controlling the dendritic length and branching of pyramidal neurons in the developing visual cortex (8McAllister A.K. Lo D.C. Katz L.C. Neuron. 1995; 15: 791-803Abstract Full Text PDF PubMed Scopus (799) Google Scholar, 9McAllister A.K. Katz L.C. Lo D.C. Neuron. 1996; 17: 1057-1064Abstract Full Text Full Text PDF PubMed Scopus (419) Google Scholar). In addition, BDNF overexpression in pyramidal neurons induces sprouting of basal dendrites (10Horch H.W. Krüttgen A. Portbury S.D. Katz L.C. Neuron. 1999; 23: 353-364Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar), and release of BDNF from single cells elicits local dendritic growth in nearby neurons (11Horch H.W. Katz L.C. Nat. Neurosci. 2002; 5: 1177-1184Crossref PubMed Scopus (317) Google Scholar). Despite these findings, the underlying mechanisms by which BDNF exerts its effects on dendritic growth remain largely unknown.Neurotrophins trigger a variety of biological responses by activating Trk receptor tyrosine kinases (12Reichardt L.F. Philos. Trans. R. Soc. Lond. B Biol. Sci. 2006; 361: 1545-1564Crossref PubMed Scopus (1571) Google Scholar). Binding of neurotrophins to Trk receptors leads to the activation of three major intracellular signaling pathways, including MAPK, PI3K, and phospholipase Cγ1 (PLCγ1) 2The abbreviations used are: PLCγ1phospholipase Cγ1CREBcAMP response element-binding proteinCRTCCREB-regulated transcription coactivator. (12Reichardt L.F. Philos. Trans. R. Soc. Lond. B Biol. Sci. 2006; 361: 1545-1564Crossref PubMed Scopus (1571) Google Scholar). Neurotrophin signaling through the MAPK, PI3K, and PLCγ1 pathways regulates neuronal differentiation, neuronal survival, and synaptic plasticity, respectively (12Reichardt L.F. Philos. Trans. R. Soc. Lond. B Biol. Sci. 2006; 361: 1545-1564Crossref PubMed Scopus (1571) Google Scholar). Trk-mediated signaling can propagate to the nucleus to regulate gene transcription through the activation of several transcription factors (12Reichardt L.F. Philos. Trans. R. Soc. Lond. B Biol. Sci. 2006; 361: 1545-1564Crossref PubMed Scopus (1571) Google Scholar). Of particular interest, the transcription factor cAMP response element-binding protein (CREB) is activated by BDNF (13Finkbeiner S. Tavazoie S.F. Maloratsky A. Jacobs K.M. Harris K.M. Greenberg M.E. Neuron. 1997; 19: 1031-1047Abstract Full Text Full Text PDF PubMed Scopus (765) Google Scholar) and plays a key role in mediating dendritic development in response to neuronal activity (14Redmond L. Ghosh A. Cell Calcium. 2005; 37: 411-416Crossref PubMed Scopus (85) Google Scholar). Although CREB activation requires phosphorylation of serine 133, there is evidence that phosphorylation of CREB is not always sufficient to initiate gene transcription (15Bonni A. Ginty D.D. Dudek H. Greenberg M.E. Mol. Cell. Neurosci. 1995; 6: 168-183Crossref PubMed Scopus (262) Google Scholar, 16Ravnskjaer K. Kester H. Liu Y. Zhang X. Lee D. Yates 3rd, J.R. Montminy M. EMBO J. 2007; 26: 2880-2889Crossref PubMed Scopus (138) Google Scholar). These observations suggest that additional factors such as CREB-regulated transcription coactivators (CRTCs), also known as transducers of regulated CREB activity (17Conkright M.D. Canettieri G. Screaton R. Guzman E. Miraglia L. Hogenesch J.B. Montminy M. Mol. Cell. 2003; 12: 413-423Abstract Full Text Full Text PDF PubMed Scopus (492) Google Scholar), may control CREB-mediated gene transcription. CRTCs are latent cytoplasmic coactivators that shuttle to the nucleus in response to increased levels of calcium and cAMP (18Bittinger M.A. McWhinnie E. Meltzer J. Iourgenko V. Latario B. Liu X. Chen C.H. Song C. Garza D. Labow M. Curr. Biol. 2004; 14: 2156-2161Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 19Screaton R.A. Conkright M.D. Katoh Y. Best J.L. Canettieri G. Jeffries S. Guzman E. Niessen S. Yates 3rd, J.R. Takemori H. Okamoto M. Montminy M. Cell. 2004; 119: 61-74Abstract Full Text Full Text PDF PubMed Scopus (505) Google Scholar). After translocation into the nucleus, CRTCs associate with the basic leucine zipper domain of CREB independently of its phosphorylation status and increase CREB transcriptional activity (17Conkright M.D. Canettieri G. Screaton R. Guzman E. Miraglia L. Hogenesch J.B. Montminy M. Mol. Cell. 2003; 12: 413-423Abstract Full Text Full Text PDF PubMed Scopus (492) Google Scholar, 20Iourgenko V. Zhang W. Mickanin C. Daly I. Jiang C. Hexham J.M. Orth A.P. Miraglia L. Meltzer J. Garza D. Chirn G.W. McWhinnie E. Cohen D. Skelton J. Terry R. Yu Y. Bodian D. Buxton F.P. Zhu J. Song C. Labow M.A. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 12147-12152Crossref PubMed Scopus (310) Google Scholar). Among CRTC family members, CRTC1 is primarily expressed in the brain and is involved in activity-dependent transcription of BDNF and in late-phase long-term potentiation (21Kovács K.A. Steullet P. Steinmann M. Do K.Q. Magistretti P.J. Halfon O. Cardinaux J.R. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 4700-4705Crossref PubMed Scopus (148) Google Scholar, 22Zhou Y. Wu H. Li S. Chen Q. Cheng X.W. Zheng J. Takemori H. Xiong Z.Q. PLoS ONE. 2006; 1: e16Crossref PubMed Scopus (92) Google Scholar). Although there is compelling evidence supporting a critical role of BDNF in regulating dendritic morphology, the signaling pathways and downstream effectors necessary for BDNF to promote dendritic development of cortical neurons remain to be identified.In this study, we show that activation of MAPK, CREB, and CRTC1 mediates BDNF-induced changes in cortical dendritic morphology. We provide evidence that nuclear translocation of CRTC1 results from NMDA receptor-mediated activation of calcineurin and is essential for the regulation of cortical dendritic development by BDNF.DISCUSSIONThere is considerable evidence that BDNF regulates dendritic growth during development, but the underlying mechanisms remain unclear. In this study, we have shown that activation of MAPK, CREB, and CRTC1 plays a critical role in mediating the effects of BDNF on dendritic growth. In addition, our data revealed that nuclear translocation of CRTC1 results from NMDA receptor-mediated activation of calcineurin. Finally, we found that activation of CRTC1 by glutamate via stimulation of NMDA receptors and calcineurin is essential for the effects of BDNF on dendritic development.Characterization of the signaling cascade by which BDNF regulates dendritic development revealed that activation of the MAPK signaling pathway mediates BDNF-induced dendritic growth and branching (Fig. 1A). In line with these findings, activation of MAPK was found to be sufficient to increase the total dendritic length and branch point number of cortical neurons (Fig. 2, A and B). Interestingly, previous studies have provided evidence that BDNF induces primary dendrite formation in cortical neurons via activation of the PI3K and MAPK signaling pathways (6Dijkhuizen P.A. Ghosh A. J. Neurobiol. 2005; 62: 278-288Crossref PubMed Scopus (130) Google Scholar). However, it is important to note that the effects of BDNF on primary dendrite formation were independent of new protein synthesis (6Dijkhuizen P.A. Ghosh A. J. Neurobiol. 2005; 62: 278-288Crossref PubMed Scopus (130) Google Scholar), whereas increases in dendritic length and complexity by BDNF, described in the present study, were mediated by transcription-dependent mechanisms, as shown by the complete inhibition of BDNF-induced dendritic growth by actinomycin D (Fig. 4A). Inhibition of CREB phosphorylation by expression of CREB S133A suppressed BDNF-induced increases in dendritic length and branching (Fig. 4C), indicating that phosphorylation of CREB is required for the stimulation of dendritic growth by BDNF. In line with these observations, BDNF induced a sustained phosphorylation of CREB that was abolished by inhibition of the MAPK pathway (Fig. 3, A and B). Although CREB phosphorylation is critical for the regulation of cAMP response element-mediated gene expression (28Ginty D.D. Bonni A. Greenberg M.E. Cell. 1994; 77: 713-725Abstract Full Text PDF PubMed Scopus (674) Google Scholar), there is evidence that phosphorylation of CREB at serine 133 is not sufficient to activate gene transcription (15Bonni A. Ginty D.D. Dudek H. Greenberg M.E. Mol. Cell. Neurosci. 1995; 6: 168-183Crossref PubMed Scopus (262) Google Scholar, 16Ravnskjaer K. Kester H. Liu Y. Zhang X. Lee D. Yates 3rd, J.R. Montminy M. EMBO J. 2007; 26: 2880-2889Crossref PubMed Scopus (138) Google Scholar). In this context, members of the CRTC family potently enhance CREB-dependent transcription via a phosphorylation-independent interaction with the basic leucine zipper DNA-binding/dimerization domain of CREB (17Conkright M.D. Canettieri G. Screaton R. Guzman E. Miraglia L. Hogenesch J.B. Montminy M. Mol. Cell. 2003; 12: 413-423Abstract Full Text Full Text PDF PubMed Scopus (492) Google Scholar, 20Iourgenko V. Zhang W. Mickanin C. Daly I. Jiang C. Hexham J.M. Orth A.P. Miraglia L. Meltzer J. Garza D. Chirn G.W. McWhinnie E. Cohen D. Skelton J. Terry R. Yu Y. Bodian D. Buxton F.P. Zhu J. Song C. Labow M.A. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 12147-12152Crossref PubMed Scopus (310) Google Scholar). Among CRTC family members, CRTC1 is abundantly expressed in the brain and is involved in activity-dependent dendritic growth and hippocampal long-term potentiation (21Kovács K.A. Steullet P. Steinmann M. Do K.Q. Magistretti P.J. Halfon O. Cardinaux J.R. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 4700-4705Crossref PubMed Scopus (148) Google Scholar, 22Zhou Y. Wu H. Li S. Chen Q. Cheng X.W. Zheng J. Takemori H. Xiong Z.Q. PLoS ONE. 2006; 1: e16Crossref PubMed Scopus (92) Google Scholar, 32Li S. Zhang C. Takemori H. Zhou Y. Xiong Z.Q. J. Neurosci. 2009; 29: 2334-2343Crossref PubMed Scopus (101) Google Scholar). Expression of a CREB construct bearing a point mutation in the CRTC1-binding domain, as well as depletion of CRTC1 expression by shRNA-mediated knockdown, abolished BDNF-induced increases in dendritic length and branching (Figs. 4D and 5C), indicating that the interaction of CREB with CRTC1 is required for the effects of BDNF on dendritic growth.Under resting conditions, CRTC1 is sequestered in the cytoplasm through a phosphorylation-dependent interaction with 14-3-3 proteins (19Screaton R.A. Conkright M.D. Katoh Y. Best J.L. Canettieri G. Jeffries S. Guzman E. Niessen S. Yates 3rd, J.R. Takemori H. Okamoto M. Montminy M. Cell. 2004; 119: 61-74Abstract Full Text Full Text PDF PubMed Scopus (505) Google Scholar). Increases in intracellular calcium levels have been shown to trigger the dephosphorylation and nuclear translocation of CRTC1 via activation of the calcium/calmodulin- dependent protein phosphatase calcineurin (18Bittinger M.A. McWhinnie E. Meltzer J. Iourgenko V. Latario B. Liu X. Chen C.H. Song C. Garza D. Labow M. Curr. Biol. 2004; 14: 2156-2161Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 19Screaton R.A. Conkright M.D. Katoh Y. Best J.L. Canettieri G. Jeffries S. Guzman E. Niessen S. Yates 3rd, J.R. Takemori H. Okamoto M. Montminy M. Cell. 2004; 119: 61-74Abstract Full Text Full Text PDF PubMed Scopus (505) Google Scholar). In our study, cortical neurons exhibited a strong nuclear accumulation of CRTC1 (Fig. 6, A and B) that was not further increased by exposure of cortical neurons to BDNF (Fig. 6F). In this context, it is interesting to note that previous studies have shown that stimulation of cortical neurons by BDNF induces calcineurin activity and nuclear translocation of NFATc, a transcription factor that regulates embryonic axon outgrowth (33Graef I.A. Wang F. Charron F. Chen L. Neilson J. Tessier-Lavigne M. Crabtree G.R. Cell. 2003; 113: 657-670Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar). The marked nuclear accumulation of CRTC1 exhibited by cortical neurons in our study was prevented in the absence of glutamate or in the presence of the NMDA receptor antagonist MK-801 (Fig. 6, A–D), indicating that nuclear translocation of CRTC1 results from the activation of NMDA receptors. In agreement with these data, recent studies have provided evidence that activation of NMDA receptors by intravitreous injection of NMDA triggers nuclear accumulation of CRTC1 in retinal ganglion cells (34Deng J. Zhang X.L. Wang J.W. Teng L.L. Ge J. Takemori H. Xiong Z.Q. Zhou Y. Neuroscience. 2009; 159: 1023-1031Crossref PubMed Scopus (5) Google Scholar). Pharmacological inhibition of calcineurin prevented the dephosphorylation and nuclear accumulation of CRTC1 (Fig. 6, A, B, and E), which is consistent with previous findings (18Bittinger M.A. McWhinnie E. Meltzer J. Iourgenko V. Latario B. Liu X. Chen C.H. Song C. Garza D. Labow M. Curr. Biol. 2004; 14: 2156-2161Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 19Screaton R.A. Conkright M.D. Katoh Y. Best J.L. Canettieri G. Jeffries S. Guzman E. Niessen S. Yates 3rd, J.R. Takemori H. Okamoto M. Montminy M. Cell. 2004; 119: 61-74Abstract Full Text Full Text PDF PubMed Scopus (505) Google Scholar). Together, these data demonstrate that nuclear translocation of CRTC1 results from NMDA receptor-mediated activation of calcineurin. Finally and most importantly, nuclear translocation of CRTC1 by NMDA receptor-mediated activation of calcineurin was shown to be essential for the effects of BDNF on dendritic growth of cortical neurons. Thus, prevention of nuclear localization of CRTC1 by MK-801 or FK506 or by stimulating cortical neurons in the absence of glutamate suppressed BDNF-induced increases in dendritic length and complexity (Fig. 7, A–C), indicating that regulation of dendritic growth by BDNF requires glutamate-mediated activation of NMDA receptors and calcineurin.Previous studies in ferret cortical brain slices have demonstrated that BDNF increases dendritic growth of layer 4 pyramidal neurons (9McAllister A.K. Katz L.C. Lo D.C. Neuron. 1996; 17: 1057-1064Abstract Full Text Full Text PDF PubMed Scopus (419) Google Scholar). Interestingly, application of the competitive NMDA receptor antagonist 2-amino-5-phosphonovaleric acid prevented BDNF from increasing the complexity of basal and apical dendrites (9McAllister A.K. Katz L.C. Lo D.C. Neuron. 1996; 17: 1057-1064Abstract Full Text Full Text PDF PubMed Scopus (419) Google Scholar). These observations support our findings and indicate that regulation of dendritic growth of cortical neurons by BDNF requires activation of NMDA receptors. However, the cellular mechanisms underlying the cooperative actions of BDNF and glutamate in the regulation of dendritic development were not known. Here, we identified the cellular mechanisms underlying these concerted actions by showing that activation of MAPK and phosphorylation of CREB by BDNF are necessary but not sufficient to mediate the effects of BDNF on dendritic development. Indeed, inhibition of NMDA receptors or stimulation of cortical neurons in the absence of glutamate prevented the increased dendritic growth by BDNF (Fig. 7, A and C). Further analysis revealed that activation of NMDA receptors triggered the nuclear translocation of CRTC1 (Fig. 6, A and B), whose interaction with CREB was shown to be essential for the regulation of cortical dendritic growth by BDNF (Fig. 4D).Together, these results support the conclusion that regulation of dendritic growth by BDNF requires both the stimulation of CREB phosphorylation by BDNF and the induction of CRTC1 nuclear translocation by glutamate through NMDA receptor activation. These data are consistent with previous studies demonstrating that phosphorylation of CREB at serine 133 is necessary but not always sufficient for CREB-dependent transcription (15Bonni A. Ginty D.D. Dudek H. Greenberg M.E. Mol. Cell. Neurosci. 1995; 6: 168-183Crossref PubMed Scopus (262) Google Scholar). For instance, in PC12 cells that extend neurites upon exposure to the neurotrophin NGF, it has been shown that NGF induces the phosphorylation of CREB but is unable to activate CREB-mediated transcription (15Bonni A. Ginty D.D. Dudek H. Greenberg M.E. Mol. Cell. Neurosci. 1995; 6: 168-183Crossref PubMed Scopus (262) Google Scholar). These observations suggest that, in addition to phosphorylation of CREB at serine 133, the cooperation of CREB with transcription coactivators may be required to effectively stimulate CREB-dependent transcription in response to NGF. Our data support this hypothesis by revealing a previously unrecognized mechanism by which CREB and the coactivator CRTC1 mediate the effects of BDNF on dendritic length and complexity.There is compelling evidence supporting functional and cooperative interactions between BDNF and glutamate. Thus, in developing cortical and hippocampal neurons, glutamate and BDNF co-regulate one another such that glutamate increases the expression and secretion of BDNF (35Lessmann V. Gottmann K. Malcangio M. Prog. Neurobiol. 2003; 69: 341-374Crossref PubMed Scopus (508) Google Scholar, 36Zafra F. Castrén E. Thoenen H. Lindholm D. Proc. Natl. Acad. Sci. U.S.A. 1991; 88: 10037-10041Crossref PubMed Scopus (481) Google Scholar), and conversely, BDNF enhances glutamate release (31Takei N. Numakawa T. Kozaki S. Sakai N. Endo Y. Takahashi M. Hatanaka H. J. Biol. Chem. 1998; 273: 27620-27624Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Moreover, BDNF enhances glutamatergic synaptic transmission through pre- and postsynaptic mechanisms (37Nicoll R.A. Schmitz D. Nat. Rev. Neurosci. 2005; 6: 863-876Crossref PubMed Scopus (469) Google Scholar). Data from the present study provide evidence for a novel cooperative interaction between BDNF- and glutamate-mediated signaling that converges on CREB to regulate the expression of target genes involved in dendritic development.A role for activity in shaping dendritic morphology is well established (4Cline H.T. Curr. Opin. Neurobiol. 2001; 11: 118-126Crossref PubMed Scopus (414) Google Scholar, 38Sin W.C. Haas K. Ruthazer E.S. Cline H.T. Nature. 2002; 419: 475-480Crossref PubMed Scopus (366) Google Scholar, 39Wong R.O. Ghosh A. Nat. Rev. Neurosci. 2002; 3: 803-812Crossref PubMed Scopus (509) Google Scholar). For instance, visual stimulation of Xenopus laevis tadpoles, which enhances synaptic activity on tectal neurons, promotes dendritic growth in an NMDA receptor-dependent manner (38Sin W.C. Haas K. Ruthazer E.S. Cline H.T. Nature. 2002; 419: 475-480Crossref PubMed Scopus (366) Google Scholar). Because excitatory activity regulates the expression and secretion of BDNF, predominantly by glutamate signaling, this suggests that the cooperative interaction between BDNF- and glutamate-mediated signaling, described in this study, may have important implications in activity-dependent dendritic growth by facilitating the transcription of CREB target genes that contribute to the development of dendritic morphology. IntroductionDendrites are the primary sites where neurons receive and integrate information from a vast number of synaptic inputs. The specific branching pattern of dendrites determines the number and type of synapses received by a neuron (1Whitford K.L. Dijkhuizen P. Polleux F. Ghosh A. Annu. Rev. Neurosci. 2002; 25: 127-149Crossref PubMed Scopus (232) Google Scholar). Dendritic development is essential for the formation of neuronal circuits. Indeed, defects in dendritic growth are associated with some forms of mental retardation, including Down syndrome and fragile X syndrome (2Benavides-Piccione R. Ballesteros-Yáñez I. de Lagrán M.M. Elston G. Estivill X. Fillat C. Defelipe J. Dierssen M. Prog. Neurobiol. 2004; 74: 111-126Crossref PubMed Scopus (102) Google Scholar, 3O'Donnell W.T. Warren S.T. Annu. Rev. Neurosci. 2002; 25: 315-338Crossref PubMed Scopus (390) Google Scholar). Dendritic arbor development is characterized by extension and retraction of dendritic branches, followed by stabilization and growth of these branches (4Cline H.T. Curr. Opin. Neurobiol. 2001; 11: 118-126Crossref PubMed Scopus (414) Google Scholar, 5Jan Y.N. Jan L.Y. Genes Dev. 2001; 15: 2627-2641Crossref PubMed Scopus (65) Google Scholar). This multistep process is regulated both by intrinsic genetic programs that are capable of generating a basic dendritic arborization and by external signals such as neuronal activity, guidance molecules, and growth factors that are essential for sculpting dendrites to their final form (1Whitford K.L. Dijkhuizen P. Polleux F. Ghosh A. Annu. Rev. Neurosci. 2002; 25: 127-149Crossref PubMed Scopus (232) Google Scholar, 6Dijkhuizen P.A. Ghosh A. J. Neurobiol. 2005; 62: 278-288Crossref PubMed Scopus (130) Google Scholar, 7Miller F.D. Kaplan D.R. Curr. Opin. Neurobiol. 2003; 13: 391-398Crossref PubMed Scopus (137) Google Scholar). There is compelling in vitro and in vivo evidence that BDNF, a member of the neurotrophin family, regulates dendritic morphology. In particular, BDNF plays an important role in controlling the dendritic length and branching of pyramidal neurons in the developing visual cortex (8McAllister A.K. Lo D.C. Katz L.C. Neuron. 1995; 15: 791-803Abstract Full Text PDF PubMed Scopus (799) Google Scholar, 9McAllister A.K. Katz L.C. Lo D.C. Neuron. 1996; 17: 1057-1064Abstract Full Text Full Text PDF PubMed Scopus (419) Google Scholar). In addition, BDNF overexpression in pyramidal neurons induces sprouting of basal dendrites (10Horch H.W. Krüttgen A. Portbury S.D. Katz L.C. Neuron. 1999; 23: 353-364Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar), and release of BDNF from single cells elicits local dendritic growth in nearby neurons (11Horch H.W. Katz L.C. Nat. Neurosci. 2002; 5: 1177-1184Crossref PubMed Scopus (317) Google Scholar). Despite these findings, the underlying mechanisms by which BDNF exerts its effects on dendritic growth remain largely unknown.Neurotrophins trigger a variety of biological responses by activating Trk receptor tyrosine kinases (12Reichardt L.F. Philos. Trans. R. Soc. Lond. B Biol. Sci. 2006; 361: 1545-1564Crossref PubMed Scopus (1571) Google Scholar). Binding of neurotrophins to Trk receptors leads to the activation of three major intracellular signaling pathways, including MAPK, PI3K, and phospholipase Cγ1 (PLCγ1) 2The abbreviations used are: PLCγ1phospholipase Cγ1CREBcAMP response element-binding proteinCRTCCREB-regulated transcription coactivator. (12Reichardt L.F. Philos. Trans. R. Soc. Lond. B Biol. Sci. 2006; 361: 1545-1564Crossref PubMed Scopus (1571) Google Scholar). Neurotrophin signaling through the MAPK, PI3K, and PLCγ1 pathways regulates neuronal differentiation, neuronal survival, and synaptic plasticity, respectively (12Reichardt L.F. Philos. Trans. R. Soc. Lond. B Biol. Sci. 2006; 361: 1545-1564Crossref PubMed Scopus (1571) Google Scholar). Trk-mediated signaling can propagate to the nucleus to regulate gene transcription through the activation of several transcription factors (12Reichardt L.F. Philos. Trans. R. Soc. Lond. B Biol. Sci. 2006; 361: 1545-1564Crossref PubMed Scopus (1571) Google Scholar). Of particular interest, the transcription factor cAMP response element-binding protein (CREB) is activated by BDNF (13Finkbeiner S. Tavazoie S.F. Maloratsky A. Jacobs K.M. Harris K.M. Greenberg M.E. Neuron. 1997; 19: 1031-1047Abstract Full Text Full Text PDF PubMed Scopus (765) Google Scholar) and plays a key role in mediating dendritic development in response to neuronal activity (14Redmond L. Ghosh A. Cell Calcium. 2005; 37: 411-416Crossref PubMed Scopus (85) Google Scholar). Although CREB activation requires phosphorylation of serine 133, there is evidence that phosphorylation of CREB is not always sufficient to initiate gene transcription (15Bonni A. Ginty D.D. Dudek H. Greenberg M.E. Mol. Cell. Neurosci. 1995; 6: 168-183Crossref PubMed Scopus (262) Google Scholar, 16Ravnskjaer K. Kester H. Liu Y. Zhang X. Lee D. Yates 3rd, J.R. Montminy M. EMBO J. 2007; 26: 2880-2889Crossref PubMed Scopus (138) Google Scholar). These observations suggest that additional factors such as CREB-regulated transcription coactivators (CRTCs), also known as transducers of regulated CREB activity (17Conkright M.D. Canettieri G. Screaton R. Guzman E. Miraglia L. Hogenesch J.B. Montminy M. Mol. Cell. 2003; 12: 413-423Abstract Full Text Full Text PDF PubMed Scopus (492) Google Scholar), may control CREB-mediated gene transcription. CRTCs are latent cytoplasmic coactivators that shuttle to the nucleus in response to increased levels of calcium and cAMP (18Bittinger M.A. McWhinnie E. Meltzer J. Iourgenko V. Latario B. Liu X. Chen C.H. Song C. Garza D. Labow M. Curr. Biol. 2004; 14: 2156-2161Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 19Screaton R.A. Conkright M.D. Katoh Y. Best J.L. Canettieri G. Jeffries S. Guzman E. Niessen S. Yates 3rd, J.R. Takemori H. Okamoto M. Montminy M. Cell. 2004; 119: 61-74Abstract Full Text Full Text PDF PubMed Scopus (505) Google Scholar). After translocation into the nucleus, CRTCs associate with the basic leucine zipper domain of CREB independently of its phosphorylation status and increase CREB transcriptional activity (17Conkright M.D. Canettieri G. Screaton R. Guzman E. Miraglia L. Hogenesch J.B. Montminy M. Mol. Cell. 2003; 12: 413-423Abstract Full Text Full Text PDF PubMed Scopus (492) Google Scholar, 20Iourgenko V. Zhang W. Mickanin C. Daly I. Jiang C. Hexham J.M. Orth A.P. Miraglia L. Meltzer J. Garza D. Chirn G.W. McWhinnie E. Cohen D. Skelton J. Terry R. Yu Y. Bodian D. Buxton F.P. Zhu J. Song C. Labow M.A. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 12147-12152Crossref PubMed Scopus (310) Google Scholar). Among CRTC family members, CRTC1 is primarily expressed in the brain and is involved in activity-dependent transcription of BDNF and in late-phase long-term potentiation (21Kovács K.A. Steullet P. Steinmann M. Do K.Q. Magistretti P.J. Halfon O. Cardinaux J.R. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 4700-4705Crossref PubMed Scopus (148) Google Scholar, 22Zhou Y. Wu H. Li S. Chen Q. Cheng X.W. Zheng J. Takemori H. Xiong Z.Q. PLoS ONE. 2006; 1: e16Crossref PubMed Scopus (92) Google Scholar). Although there is compelling evidence supporting a critical role of BDNF in regulating dendritic morphology, the signaling pathways and downstream effectors necessary for BDNF to promote dendritic development of cortical neurons remain to be identified.In this study, we show that activation of MAPK, CREB, and CRTC1 mediates BDNF-induced changes in cortical dendritic morphology. We provide evidence that nuclear translocation of CRTC1 results from NMDA receptor-mediated activation of calcineurin and is essential for the regulation of cortical dendritic development by BDNF.