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Wnt-7a Modulates the Synaptic Vesicle Cycle and Synaptic Transmission in Hippocampal Neurons

2007; Elsevier BV; Volume: 283; Issue: 9 Linguagem: Inglês

10.1074/jbc.m705943200

ISSN

1083-351X

Autores

Waldo Cerpa, Juan A. Godoy, Iván E. Alfaro, Ginny G. Farı́as, Mariajose Metcalfe, Rodrigo A. Fuentealba, Christian Bonansco, Nibaldo C. Inestrosa,

Tópico(s)

Nerve injury and regeneration

Resumo

Wnt signaling is essential for neuronal development and the maintenance of the developing nervous system. Recent studies indicated that Wnt signaling modulates long term potentiation in adult hippocampal slices. We report here that different Wnt ligands are present in hippocampal neurons of rat embryo and adult rat, including Wnt-4, -5a, -7a, and -11. Wnt-7a acts as a canonical Wnt ligand in rat hippocampal neurons, stimulates clustering of presynaptic proteins, and induces recycling and exocytosis of synaptic vesicles as studied by FM dyes. Wnt-3a has a moderate effect on recycling of synaptic vesicles, and no effect of Wnt-1 and Wnt-5a was detected. Electrophysiological analysis on adult rat hippocampal slices indicates that Wnt-7a, but not Wnt-5a, increases neurotransmitter release in CA3-CA1 synapses by decreasing paired pulse facilitation and increasing the miniature excitatory post-synaptic currents frequency. These results indicate that the presynaptic function of rat hippocampal neurons is modulated by the canonical Wnt signaling. Wnt signaling is essential for neuronal development and the maintenance of the developing nervous system. Recent studies indicated that Wnt signaling modulates long term potentiation in adult hippocampal slices. We report here that different Wnt ligands are present in hippocampal neurons of rat embryo and adult rat, including Wnt-4, -5a, -7a, and -11. Wnt-7a acts as a canonical Wnt ligand in rat hippocampal neurons, stimulates clustering of presynaptic proteins, and induces recycling and exocytosis of synaptic vesicles as studied by FM dyes. Wnt-3a has a moderate effect on recycling of synaptic vesicles, and no effect of Wnt-1 and Wnt-5a was detected. Electrophysiological analysis on adult rat hippocampal slices indicates that Wnt-7a, but not Wnt-5a, increases neurotransmitter release in CA3-CA1 synapses by decreasing paired pulse facilitation and increasing the miniature excitatory post-synaptic currents frequency. These results indicate that the presynaptic function of rat hippocampal neurons is modulated by the canonical Wnt signaling. Wnt signaling regulates crucial processes in all multicellular organisms, including cell proliferation, differentiation, migration, and morphogenesis. Since its discovery about 25 years ago, Wnt signaling has been extensively studied for its diverse roles in embryogenesis and cancer (1Logan C.Y. Nusse R. Annu. Rev. Cell Dev. Biol. 2004; 20: 781-810Crossref PubMed Scopus (4351) Google Scholar) and, more recently, in neural development and synaptic plasticity (2Patapoutian A. Reichardt L.F. Curr. Opin. Neurobiol. 2000; 10: 392-399Crossref PubMed Scopus (273) Google Scholar, 3Fradkin L.G. Garriga G. Salinas P.C. Thomas J.B. Yu X. Zou Y. J. Neurosci. 2005; 25: 10376-10378Crossref PubMed Scopus (26) Google Scholar, 4Ciani L. Salinas P.C. Nat. Rev. Neurosci. 2005; 6: 351-362Crossref PubMed Scopus (533) Google Scholar, 5Chen J. Park C.S. Tang S.J. J. Biol. Chem. 2006; 281: 11910-11916Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar). Several studies suggest that Wnt factors play a role in the formation of neuronal connections, and other reports indicate a specific effect on synapse assembly; for example, in Drosophila embryos overexpression of the Wnt gene DWnt-3, encoding a protein localized in axonal processes, disrupted the formation of commissural tracts (6Fradkin L.G. Noordermeer J.N. Nusse R. Dev. Biol. 1995; 168: 202-213Crossref PubMed Scopus (60) Google Scholar). Wnt-3 also regulates terminal arborization of neurotrophin-3-responsive spinal sensory neurons before the formation of sensory motoneuron synapses (7Krylova O. Herreros J. Cleverley K.E. Ehler E. Henriquez J.P. Hughes S.M. Salinas P.C. Neuron. 2002; 35: 1043-1056Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). In developing cerebellum cortex it has been found that conditioned medium from granule cells increases the diameter of mossy fiber axons and growth cone complexity, a result mimicked by Wnt-7a (8Lucas F.R. Salinas P.C. Dev. Biol. 1997; 192: 31-44Crossref PubMed Scopus (246) Google Scholar, 9Hall A.C. Lucas F.R. Salinas P.C. Cell. 2000; 100: 525-535Abstract Full Text Full Text PDF PubMed Scopus (599) Google Scholar). Wingless, the prototypical Drosophila Wnt, and its receptor are localized at the larval neuromuscular junction (10Packard M. Koo E.S. Gorczyca M. Sharpe J. Cumberledge S. Budnik V. Cell. 2002; 111: 319-330Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar). Wingless is secreted by motoneurons and accumulates at both the pre- and postsynaptic terminals. The loss of Wingless leads to reduction in target-dependent synapse formation (10Packard M. Koo E.S. Gorczyca M. Sharpe J. Cumberledge S. Budnik V. Cell. 2002; 111: 319-330Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar). The expression of Wnt ligands and proteins of the Wnt signaling machinery in the mature nervous system (11Shimogori T. VanSant J. Paik E. Grove E.A. J. Comp. Neurol. 2004; 473: 496-510Crossref PubMed Scopus (126) Google Scholar, 12Tissir F. Goffinet A.M. Eur. J. Neurosci. 2006; 23: 597-607Crossref PubMed Scopus (90) Google Scholar) suggests that Wnt signaling plays a role in neuroprotection and synaptic plasticity in addition to its role in neurite patterning in the developing nervous system (3Fradkin L.G. Garriga G. Salinas P.C. Thomas J.B. Yu X. Zou Y. J. Neurosci. 2005; 25: 10376-10378Crossref PubMed Scopus (26) Google Scholar, 5Chen J. Park C.S. Tang S.J. J. Biol. Chem. 2006; 281: 11910-11916Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar, 13Fuentealba R.A. Farias G. Scheu J. Bronfman M. Marzolo M.P. Inestrosa N.C. Brain Res. Brain Res. Rev. 2004; 47: 275-289Crossref PubMed Scopus (122) Google Scholar). Indeed, Wnt ligands can act locally to regulate changes in neuronal cell shape and pre- and postsynaptic terminals, which are thought to underlie changes in synaptic function and learning. Thus, Wnt ligands would appear to be particularly well suited as mediators of synaptic plasticity (5Chen J. Park C.S. Tang S.J. J. Biol. Chem. 2006; 281: 11910-11916Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar, 14Scheiffele P. Annu Rev. Neurosci. 2003; 26: 485-508Crossref PubMed Scopus (263) Google Scholar, 15Dabdoub A. Donohue M.J. Brennan A. Wolf V. Montcouquiol M. Sassoon D.A. Hseih J.C. Rubin J.S. Salinas P.C. Kelley M.W. Development. 2003; 130: 2375-2384Crossref PubMed Scopus (178) Google Scholar). In the present study we report that Wnt-7a, a canonical ligand that stimulates vesicle clustering, induces recycling and exocytosis of synaptic vesicles in hippocampal neurons in culture and enhances synaptic transmission in adult hippocampal slices by a presynaptic mechanism. These results are consistent with the idea that the canonical Wnt pathway controls presynaptic function in rat hippocampal neurons. Detection of Wnt mRNAs in Neurons and Hippocampus—Degenerate oligonucleotides were designed for the identification of rat Wnt genes expressed at the mRNA level: TGA ACC TNC ACA ACA AYG AGG CGG G (I), GYC ACY GGG TGT CAG GCW CCT G (II), RTR GCC GCG GCC ACA GCA CA (III), and GCA GCA CCA GTG GAA CTT GCA (IV). Total RNA from 10 DIV 2The abbreviations used are:DIVdays in vitroHEK-293human embryonic kidney 293 cellssFRP-1soluble Frizzled receptor proteinACSFartificial cerebrospinal fluidfEPSPfield excitatory postsynaptic potentialsEPSCfield excitatory postsynaptic currentmEPSCminiature EPSCDvlDishevelledα7-nAChRα7-nicotinic acetylcholine receptorTRITCtetramethylrhodamine isothiocyanatePSD-95postsynaptic density protein-95GSKglycogen synthase kinaseJNKc-Jun NH2-terminal kinase. hippocampal neurons were extracted, pooled, and treated with DNase I. cDNA was then synthesized using oligo(dT) and Moloney murine leukemia virus reverse transcriptase, and nested PCR amplicons were subcloned in pCRII (Invitrogen). Identity of individual Wnt amplicons was revealed by its unique HaeIII and RsaI restriction endonuclease pattern after PCR reamplification and further confirmed by sequencing. This procedure cannot rule out minor expression of low transcribed Wnt genes. For Wnt-4, Wnt-5a, Wnt-7a, or Wnt-11 determinations, RNA from either adult Sprague-Dawley rat hippocampal tissue or treated hippocampal neurons were subjected to reverse transcription-PCR analysis using primer I and antisense primers ATC TGT ATG TGG CTT GAA CTG, GAA GCG GCT GTT GAC CTG TAC, GCT TCT TGA TCT TCT TCA GAA AGG, and CAA GTG CTT GCG GGT GCC CAT, respectively. β-Actin primers for normalization of cDNA loading were TCT ACA ATG AGC TGC CAG AG and TAC ATG GCT GGG GTG ATG AA. days in vitro human embryonic kidney 293 cells soluble Frizzled receptor protein artificial cerebrospinal fluid field excitatory postsynaptic potentials field excitatory postsynaptic current miniature EPSC Dishevelled α7-nicotinic acetylcholine receptor tetramethylrhodamine isothiocyanate postsynaptic density protein-95 glycogen synthase kinase c-Jun NH2-terminal kinase. Constructs—The different hemagglutinin-Wnts constructs were a kind gift of several individuals and made this work possible. Wnt-7a was a gift of Dr. Patricia Salinas, University College London, London, UK. Wnt-3a was a gift of Dr. Roel Nusse, Stanford University, Palo Alto, CA. Wnt-5a was a gift of Dr. Randall T. Moon, University of Washington, Seattle, WA, and soluble Frizzled receptor protein (sFRP-1) was a gift of Dr. Jeremy Nathans, Johns Hopkins University School of Medicine, Baltimore, MD. Conditioned Medium Containing Wnt Ligands—To generate secreting Wnt ligands, human embryonic kidney 293 cells (HEK-293) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (Invitrogen) and 100 μg/ml streptomycin and 100 units/ml penicillin. Then HEK-293 cells were transiently transfected by calcium phosphate precipitation (16Conroy W.G. Berg D.K. Mol. Pharmacol. 1998; 53: 392-401Crossref PubMed Scopus (99) Google Scholar) with constant and equal amounts of empty vector pcDNA or pcDNA-containing sequences encoding Wnt-1, Wnt-3a, Wnt-5a, and Wnt-7a constructs. Transiently transfected HEK-293 cells also were used to produce sFRP-1 coupled to the sequence encoding a hemagglutinin tag. Transiently transfected HEK-293 cells were maintained in Neurobasal medium supplemented with 100 units/ml penicillin and 100 μg/ml streptomycin medium for 60 h. Wnt-conditioned or control media or media containing sFRP-1 were prepared as described (9Hall A.C. Lucas F.R. Salinas P.C. Cell. 2000; 100: 525-535Abstract Full Text Full Text PDF PubMed Scopus (599) Google Scholar, 17Alvarez A.R. Godoy J.A. Mullendorff K. Olivares G.H. Bronfman M. Inestrosa N.C. Exp. Cell Res. 2004; 297: 186-196Crossref PubMed Scopus (204) Google Scholar, 18Quintanilla R.A. Munoz F.J. Metcalfe M.J. Hitschfeld M. Olivares G. Godoy J.A. Inestrosa N.C. J. Biol. Chem. 2005; 280: 11615-11625Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 19Garrido J.L. Godoy J.A. Alvarez A. Bronfman M. Inestrosa N.C. FASEB J. 2002; 16: 1982-1984Crossref PubMed Scopus (150) Google Scholar). Wnt secretion was verified by Western blot using an anti-hemagglutinin antibody (Upstate Biotechnology, Lake Placid, NY). Primary Rat Embryo Hippocampal Neuron Cultures and Treatments—Rat primary hippocampal neurons were prepared as previously described (17Alvarez A.R. Godoy J.A. Mullendorff K. Olivares G.H. Bronfman M. Inestrosa N.C. Exp. Cell Res. 2004; 297: 186-196Crossref PubMed Scopus (204) Google Scholar, 19Garrido J.L. Godoy J.A. Alvarez A. Bronfman M. Inestrosa N.C. FASEB J. 2002; 16: 1982-1984Crossref PubMed Scopus (150) Google Scholar, 20Farias G.G. Valles A.S. Colombres M. Godoy J.A. Toledo E.M. Lukas R.J. Barrantes F.J. Inestrosa N.C. J. Neurosci. 2007; 27: 5313-5325Crossref PubMed Scopus (102) Google Scholar, 21Banker G.A. Cowan W.M. Brain Res. 1977; 126: 397-442Crossref PubMed Scopus (978) Google Scholar, 22Hamill O.P. Marty A. Neher E. Sakmann B. Sigworth F.J. Pfluegers Arch. Eur. J. Physiol. 1981; 391: 85-100Crossref PubMed Scopus (15502) Google Scholar). Hippocampal neurons were obtained from Sprague-Dawley rat embryos E18. On 3 DIV, hippocampal neurons were treated with 1 μm 1-β-d-ar-abinofuranosylcytosine for 24 h to reduce the number of proliferating non-neuronal cells (21Banker G.A. Cowan W.M. Brain Res. 1977; 126: 397-442Crossref PubMed Scopus (978) Google Scholar, 22Hamill O.P. Marty A. Neher E. Sakmann B. Sigworth F.J. Pfluegers Arch. Eur. J. Physiol. 1981; 391: 85-100Crossref PubMed Scopus (15502) Google Scholar). The treatment were performed in 14-21 DIV. Neurons were first depleted of B-27 with Neurobasal media for 2 h and treated with Wnt and/or sFRP-1 and pcDNA control-conditioned media for different times. Immunofluorescence for Synaptic Proteins—Immunostaining was carried out using polyclonal anti-postsynaptic density protein-95 (PSD-95), synaptophysin, SV-2, synaptotagmin antibodies, and secondary antibody labeled with 488Alexa, 543Alexa, or 633Alexa (Affinity Bio Reagents Inc., Golden, CO). To study neuronal morphology, phalloidin labeled with TRITC from Molecular Probes (Leiden, The Netherlands) was used. The number of clusters/length neurite was quantified by the software Sigma Scan Pro., and the number of neurites was evaluated using Image-Pro Plus Software Media Cybernetic (Silver Spring, MD). For quantification of cluster size, the area of the clusters was measured using Image J software (National Institutes of Health). Western Blot for Wnt Components and Synaptic Protein in Hippocampal Neurons—Total extract of hippocampal neurons were used by immunodetection of synaptophysin (1:1000), β-catenin (1:1000), Dvl-3 (1:500), Engrailed-1 (1:500), cyclin-D1 (1:1000), c-Jun (1:1000), β-tubulin (1:1000) (Santa Cruz Biotechnology), phospho-JNK and JNK (1:2000) (Cell Signaling). Spontaneous Recycling of Synaptic Vesicles Using FM1-43fx and the SV-2 Marker—21-DIV hippocampal neurons were exposed to a 1 μm concentration of steryl dye FM1-43 (Molecular Probes, Eugene, OR) in Neurobasal media. The control conditioned media and the conditioned media with different Wnt ligands were added into the culture at 37 °C at different times. Following the time course, the cells were washed, fixed, and immunostained against SV2 protein. Representative photographs were taken, and the FM1-43fx positive puncta which co-localized with SV-2 were quantified with the Image J program. Imaging of FM-1-43 Destaining in Presynaptic Terminals of Cultured Hippocampal Neurons—21-DIV hippocampal neurons were incubated for 3 h with Wnt-7a or pcDNA at 37 °C. Neurons on coverslips were then washed with Tyrode modified solution, mounted in a microscope perfusion chamber, and incubated for 30 s with 10 μm FM1-43 (Molecular Probes) followed by 1 min of loading with mild depolarization with 30 mm KCl. Nonspecific and non-synaptic FM-1-43 staining was diminished by washing with 10 min of continue perfusion of Tyrode solution at 1-2 ml/min controlled with a peristaltic pump (Cole Palmer, Vernon Hills, IL). The chamber was adapted at the stage of a Zeiss Axiovert 200M microscope coupled to Pascal LSM5 confocal laser scanning system. Neurons were imaged with a 63 × 1.4 NA oil objective at 512 × 512 full-frame resolution using a 488-nm argon laser to excite the FM1-43 probe, and the fluorescence signals were collected over 505 nm. Then, after a period of 50 s of basal fluorescence acquisition, neurons were depolarized with 60 mm KCl and imaged by 300 s at 1-s intervals. Images from presynaptic loaded puncta were selected for measuring fluorescence intensities using region of interest areas of 1.5 × 1.5 μm. Images of Wnt-7a-treated neurons and control neurons were obtained using identical settings for laser power, confocal thickness, and detector sensitivity. All measures were carried at room temperature (25 °C). Slice Preparation and Electrophysiology—Hippocampal slices were prepared according to standard procedures from 22- to 30-day-old Sprague-Dawley rats. Transverse slices (250-300 μm) from the dorsal hippocampus were cut under cold artificial cerebrospinal fluid (ACSF) using a Vibroslice microtome (World Precision Instruments) and incubated in ACSF for more than 1 h at room temperature. In all experiments picrotoxin (10 μm) was added to ACSF perfusion media to suppress inhibitory γ-aminobutyric acid, type A transmission. Then slices were transferred to an experimental chamber (2 ml), superfused (3 ml/min, at 22-26 °C) with gassed ACSF, and visualized by transillumination with a binocular stereo microscope (MSZ-10, Nikon). The experiments were carried out at room temperature (21 °C-22 °C), measured at the recording chamber. Two recording methods were used; that is, path clamp (22Hamill O.P. Marty A. Neher E. Sakmann B. Sigworth F.J. Pfluegers Arch. Eur. J. Physiol. 1981; 391: 85-100Crossref PubMed Scopus (15502) Google Scholar, 23Bonansco C. Buno W. Hippocampus. 2003; 13: 150-163Crossref PubMed Scopus (31) Google Scholar) and extracellular field potentials recording (24Hu H. Shao L.R. Chavoshy S. Gu N. Trieb M. Behrens R. Laake P. Pongs O. Knaus H.G. Ottersen O.P. Storm J.F. J. Neurosci. 2001; 21: 9585-9597Crossref PubMed Google Scholar). Single cell recording were made in the whole-cell configuration with fire-polished pipettes (3-5 megaohms) filled with intracellular solution (see below) and connected to a tight seal (>1 gigaohm), and whole-cell a recordings were obtained from the cell body of neurons in the CA1 pyramidal layer. Patch electrodes were made from borosilicate glass and had a resistance of 2-5 megaohms when filled with 97.5 mm potassium gluconate, 32.5 mm KCl, 10.0 mm HEPES, 1.0 mm MgCl2, 5.0 EGTA, and 4.0 mm sodium salt (Na-ATP), pH 7.2 (289 mosm). Neurons were voltage-clamped with an EPC-7 amplifier (Heka Instruments), and the experiments started after a 5-10-min stabilization period after access to the intracellular compartment with patch electrodes. The access resistance (10-25 megaohms) was monitored, and cells were rejected if it changed more than 20% during the experiment. Extracellular field potentials recording (25Andersen P. Silfvenius H. Sundberg S.H. Sveen O. Wigstrom H. Brain Res. 1978; 144: 11-18Crossref PubMed Scopus (158) Google Scholar) were made with a glass pipette (2-4 megaohms, filled with the perfusion medium), connected to an AC amplifier (P-5 Series, Grass), with gain 10,000×, low pass filter 3.0 kHz, and high pass filter 0.30 Hz, which was placed in the middle of stratum radiatum of CA1. The electric pulses (50 μs, 0.3 Hz, 20-100 μA) were applied on Schaeffer collaterals eliciting compound action potentials from the presynaptic axons (fiber volley) followed by field excitatory postsynaptic potentials (fEPSPs). To evoke excitatory postsynaptic currents (EPSCs) and fEPSPs, Schaeffer collaterals fibers were activated by bipolar cathodic stimulation, generated by a stimulator (Master 8, AMPI) connected to an isolation unit (Isoflex, AMPI). The bipolar concentric electrodes (platinum/iridium, 125 μm outer diameter, FHC Inc.) were placed in the stratum radiatum within 100-200 μm from the recording site. Miniature EPSCs (mEPSCs) were recorded in presence of tetrodotoxin (0.5 μm), and the Ca2+/Mg2+ ratio was elevated to increase the probability of neurotransmitter release (4.0 mm Ca2+ and 0.5 mm Mg2+) (26Hsia A.Y. Malenka R.C. Nicoll R.A. J. Neurophysiol. 1998; 79: 2013-2024Crossref PubMed Scopus (224) Google Scholar). The presynaptic origin of the regulation of EPSCs amplitude by Wnt-7a was evaluated with conventional stimulation estimating changes in the paired pulse facilitation, which are considered to be of presynaptic origin (27Clark K.A. Randall A.D. Collingridge G.L. Exp. Brain Res. 1994; 101: 272-278Crossref PubMed Scopus (75) Google Scholar, 28Kuhnt U. Voronin L.L. Neuroscience. 1994; 62: 391-397Crossref PubMed Scopus (74) Google Scholar). The paired pulse facilitation index was calculated by ((R2-R1)/R1), where R1 and R2 were the peak amplitudes of the first and second EPSCs, respectively. Additionally, because the changes in the frequency of mEPSCs are considered to be of presynaptic origin (29Manabe T. Renner P. Nicoll R.A. Nature. 1992; 355: 50-55Crossref PubMed Scopus (282) Google Scholar, 30Wyllie D.J. Manabe T. Nicoll R.A. Neuron. 1994; 12: 127-138Abstract Full Text PDF PubMed Scopus (100) Google Scholar), we tested the mean frequency of mEPSCs as an indicator of changes in the probability of the presynaptic transmitter release. Recordings were filtered at 2.0-3.0 kHz, sampled at 4.0 kHz using an A/D converter (ITC-16, Intrutech), and stored with Pulse FIT software (Heka Instruments). Both evoked postsynaptic responses, and mEPSCs were analyzed off-line using an analysis software (Minianalysis, Synaptosoft) which allowed visual detection of events, computing only those events that exceeded and arbitrary threshold. Data are expressed as the means ± S.E. (number of cells) unless otherwise noted. The Student's t test or Kolmogorov-Smirnov analysis at p < 0.05 determined significant differences between control and mutant cells. Statistical Analysis—Data were expressed as the mean ± S.E. of the values from the number of experiments as indicated in the corresponding figures. Data were evaluated statistically by using Student's t test, with p < 0.05 considered significant. Analysis of variance was used to compare n differences between experiments. Several Wnts Ligands Are Expressed in Embryonic Hippocampal Neurons and in the Adult Rat Hippocampus—Wnt ligands are secreted glycoproteins encoded by a family of about 19 conserved genes in humans and other mammals (1Logan C.Y. Nusse R. Annu. Rev. Cell Dev. Biol. 2004; 20: 781-810Crossref PubMed Scopus (4351) Google Scholar, 31Moon R.T. Bowerman B. Boutros M. Perrimon N. Science. 2002; 296: 1644-1646Crossref PubMed Scopus (899) Google Scholar). Therefore, we first screenED for Wnt genes expressed in hippocampal neurons using a prototype mammalian Wnt gene (Fig. 1A) and we found several Wnt ligands including Wnt-4, Wnt-5a, Wnt-7a, and Wnt-11 (Fig. 1B); moreover, all four Wnt mRNAs detected in primary hippocampal cultures are also present in the hippocampus of adult rats (Fig. 1C). Analysis of mRNA shows that in particular, Wnt-7a and Wnt-5a are modulated by cell density in hippocampal neuronal cultures (Fig. 1, D and E). These results suggest that the Wnt signaling pathway may play a role in the mammalian central nervous system throughout the organism lifespan. Wnt-7a Increases the Clusters of Synaptic Vesicles in Rat Hippocampal Cultures—Because different Wnt ligands were observed in embryonic and adult hippocampal neurons, we assessed the capacity of two Wnt ligands, Wnt-7a and Wnt-5a, to affect the clustering of synaptic proteins in mature hippocampal neurons. We examined the localization of synaptophysin, a presynaptic vesicle protein (32Fletcher T.L. Cameron P. De Camilli P. Banker G. J. Neurosci. 1991; 11: 1617-1626Crossref PubMed Google Scholar). Hippocampal neurons exposed to Wnt-7a for 1 h showed a different pattern of synaptophysin localization in comparison with control neurons (Fig. 2A). Wnt-7a-treated neurons present more synaptophysin clusters (Fig. 2Ac) than both Wnt-5a-treated neurons and pcDNA (control) cultures (Fig. 2A, a and b; more details are shown in supplemental Fig. S1A. We quantified the effect of Wnt-7a in the hippocampal cultures, counting the number of synaptophysin clusters in 100 μm of neurite length. The results show that Wnt-7a increases the clustering of synaptophysin (Fig. 2Ad). Moreover, we found that Wnt-7a increases the number of clusters of different presynaptic proteins as synaptotagmin and SV-2 (data not shown). In all cases the maximal effect was obtained after 1 h of incubation with the Wnt-conditioned medium (Fig. 3B; data not shown). The increased number of synaptic vesicle clusters is due to a redistribution of synaptophysin because the amount of synaptophysin protein did not change under the different experimental conditions used as evaluated by Western blots (Fig. 2B).FIGURE 3Wnt-7a ligand stabilizes β-catenin but induces the clustering of presynaptic proteins independent of GSK-3β activity and β-catenin stabilization. A, representative Western blot of hippocampal neurons stimulated with Wnt-5a, Wnt-7a, or pcDNA (control) for 1 h show that only Wnt-7a ligand stabilized β-catenin (cat) levels. The densitometric analysis of β-catenin was normalized against β-tubulin (tub; n = 4). Hippocampal neurons were exposed to Wnt-7a for different times to study the clustering of synaptophysin (B) or β-catenin stabilization (C). Wnt-7a significantly increases synaptophysin clustering (n = 2) and stabilization of β-catenin (n = 4) in a similar time-course dependent form. D-F, Western blot of hippocampal neurons exposed to control (pcDNA), Wnt-7a, or 20 mm LiCl (GSK-3β inhibitor) for 1 h were tested for different component of Wnt pathways. D, Wnt-7a and LiCl induce the β-catenin levels as showed in the densitometric analysis (n = 4); however, Dvl, an upstream component in the Wnt pathway, is only activated by Wnt-7a as indicated by the increase in the phosphorylated state. E, Wnt target genes as Eng-1, cyclin-D1, and c-Jun are not affected by Wnt-7a or LiCl at 1 h of treatment (n = 2). F, JNK, a non-canonical Wnt component, is not induced by Wnt-7a at 1 h of treatment, suggesting that Wnt-7a acts mainly as a canonical Wnt ligand. p-, phospho-. G, immunofluorescence labeling for synaptophysin in neurons subjected to treatments with control, Wnt-7a, or LiCl for 1 h indicates that Wnt-7a but not LiCl is able to induce the clusters number of synaptophysin as shown in representative photographs and in quantification (n = 2). H, fluorescence labeling of processes by phalloidin and immunofluorescence labeling for PSD-95 and for synaptophysin are shown in neurons exposed to control conditions or in the presence of wortmannin (WT; 100 nm) or bisindolamide-X (BSD-X;1 μm) (GSK-3β activators) for 1 h. Representative photographs and the quantification of the Syp clusters and PSD-95 clusters show not changes in the number of clusters by Wnt-7a (n = 3). The bar represents the mean ± S.E. (*, p < 0.01 Student's t test). Eng-1, Engrailed-1; Dvl, Dishevelled; Syp, synaptophysin.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To determine whether Wnt-7a induces a regulation of postsynaptic proteins, we study the distribution of the PSD-95. Hippocampal neurons exposed to Wnt-7a for 1 h did not show any change in the distribution of PSD-95 (Fig. 2C, a-d). The number of PSD-95 clusters was unaffected by Wnt-7a (Fig. 2Ce). The effect of Wnt-7a in synapse was not due to change in neuronal morphology as observed by phalloidin staining (Fig. 2C, c and d). These results indicate that Wnt-7a ligand regulates the clustering of presynaptic proteins in mature hippocampal neurons cultures. Wnt-7a Is a Canonical Wnt Ligand That Increases Presynaptic Protein Clustering Independent of GSK-3β Activity and β-Catenin Stabilization—At least three different signaling pathways are activated by Wnt proteins; the canonical Wnt/β-catenin pathway and two non-canonical Wnt pathways, Wnt/JNK and Wnt/Ca2+. We assessed the capacity of Wnt-7a and Wnt-5a ligands to activate the canonical Wnt pathway. When hippocampal neurons were exposed to Wnt ligands for 1 h, Wnt-7a but not Wnt-5a was able to increase β-catenin protein levels with respect to control as observed by Western blot (Fig. 3A). Similar results were obtained by immunofluorescence (data not shown). Because Wnt-7a but not Wnt-5a increase the presynaptic protein clustering, hippocampal neurons were exposed to Wnt-7a in a time course experiment to evaluate both the clustering of the presynaptic protein synaptophysin and the stabilization of β-catenin. The clustering of synaptophysin became evident after 30 min with a peak at 60 min of Wnt-7a ligand exposure (Fig. 3B). When the stabilization of β-catenin was evaluated, a similar pattern of increase in β-catenin levels was observed; such change was similar to the increase for synaptophysin clustering. In both cases the induction was observed after 30 min and maintained at 60 min (Fig. 3C). To test the specificity of the Wnt ligand, Wnt-7a was incubated with the sFRP-1. sFRP recaptured the Wnt ligands, thereby preventing their interaction with cellular membrane-bound Frizzled receptor (33Rattner A. Hsieh J.C. Smallwood P.M. Gilbert D.J. Copeland N.G. Jenkins N.A. Nathans J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2859-2863Crossref PubMed Scopus (494) Google Scholar). Hippocampal neurons exposed to Wnt-7a in the presence of sFRP-1 showed a decrease in β-catenin levels with respect to Wnt-7a treatment, indicating the specificity of Wnt-7a to activate the Wnt pathway through its interaction with Frizzled receptors. To determine whether GSK-3β activity and β-catenin stabilization, two components of the canonical Wnt pathway, are required to induce the clustering of presynaptic proteins, we used lithium, the classical GSK-3 inhibitor (34Jope R.S. Trends Pharmacol. Sci. 2003; 24: 441-443Abstract Full Text Full Text PDF PubMed Scopus (418) Google Scholar). Lithium induces the canonical Wnt pathway through GSK-3β inactivation that leads to β-catenin stabilization. We first compared the β-catenin stabilization induced by Wnt-7a and lithium. Hippocampal neurons exposed to lithium for 1 h showed an increase in the β-catenin levels as Wnt-7a (Fig. 3D); however, Dvl, a component of the Wnt pathway upstream of the GSK-3β and β-catenin proteins, was activated by Wnt-7a but not by lithium, as evidenced by phosphoryla

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