JBC Papers in Press. Published on April 19, 2010 as Manuscript M110.103028        The latest version is at http://www.jbc....
machinery involved in vesicle exocytosis in the              which contains 12 elements in response topresynaptic terminal...
Calcium experiments- Hippocampal neurons of 12-            emission was collected with a 620 nm filter.13 DIV were loaded ...
low colocalization and 1 indicating high                   greater number of repetitive events in Wnt3a-colocalization.   ...
competitive antagonist of NMDA receptors (0.9 ±             effect of the Wnt3a protein we next examined0.1 Hz; N=5), sugg...
agreement with the above presented results,               antagonist, and 2 µg/ml Dickkopf 1 (DKK1), as anfluorescent     ...
Wnt/β-catenin or canonical signaling as opposed to         there was not complete inhibition, no significantthe β-catenin ...
could take place in central neurons, is further            which we have previously found expressed in thesupported by the...
16. Beaumont, V., Thompson, S. A., Choudhry, F., Nuthall, H., Glantschnig, H., Lipfert, L., David, G.    R., Swain, C. J.,...
FOOTNOTES*We acknowledge the efforts of M. Cuevas, J. Fuentealba and P. Cardenas in the initial steps of this study.This w...
release from pre-synaptic terminals induced by Wnt3a. C. Destaining associated to FM1-43 (depletedfraction: ∆F/Fi) in the ...
Table 1. Electrical parameters (action potentials and input resistance) recorded in hippocampal neurons inthe absence or p...
Avila et al 2010 wnt 3
Avila et al 2010 wnt 3
Avila et al 2010 wnt 3
Avila et al 2010 wnt 3
Avila et al 2010 wnt 3
Avila et al 2010 wnt 3
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Avila et al 2010 wnt 3

  1. 1. JBC Papers in Press. Published on April 19, 2010 as Manuscript M110.103028 The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M110.103028 CANONICAL WNT3A MODULATES INTRACELLULAR CALCIUM AND ENHANCES EXCITATORY NEUROTRANSMISSION IN HIPPOCAMPAL NEURONS Miguel E. Avila1, Fernando J. Sepúlveda2, Carlos F. Burgos1, Gustavo Moraga-Cid2, Jorge Parodi2, Randall T. Moon3, Luis G. Aguayo2, Carlos Opazo2, and Giancarlo V. De Ferrari1,4. From Department of Biochemistry and Molecular Biology1 and Physiology2, Faculty of Biological Sciences, Universidad de Concepción, Chile; HHMI, Department of Pharmacology and Institute for Stem Cell and Regenerative Medicine3, University of Washington School of Medicine, Seattle, WA 98195, USA; Center for Biomedical Research, Faculty of Biological Sciences and Faculty of Medicine, Universidad Andres Bello, Santiago, Chile4. Running head: Wnt3a, calcium and neurotransmissionAddress correspondence to: Giancarlo V. De Ferrari, Center for Biomedical Research, Faculty ofBiological Sciences and Faculty of Medicine, Universidad Andres Bello, Santiago, Chile. Email:gdeferrari@unab.cl A role for Wnt signal transduction in the excitatory synaptic transmission (15-18). For Downloaded from www.jbc.org at PONTIFICIA UNIVERSIDAD, on April 19, 2010development and maintenance of brain instance, activation of N-methyl-d-aspartatestructures is widely acknowledged. Recent (NMDA) receptors, after tetanic stimulation instudies have suggested that Wnt signaling may rodent hippocampal slices, induces the release of abe essential for synaptic plasticity and pool of vesicles containing Wnt3a in the synapticneurotransmission. However, the direct effect of terminal modulating long term potentiation eventsa Wnt protein on synaptic transmission had not (18). Similarly, double-mutant mice for Wnt7a andbeen demonstrated. Here we show that Dishevelled-1, which exhibit a decrease in thenanomolar (nM) concentrations of purified number of synapses between mossy fibers of theWnt3a protein rapidly increase the frequency of cerebellum and grainy cells, showed markedminiature excitatory synaptic currents in deterioration in the release of neurotransmittersembryonic rat hippocampal neurons, through a and in the recycling of vesicles in the existingmechanism involving a fast influx of calcium synapses (15). Furthermore, pharmacological(Ca2+) from the extracellular space, induction of analysis with drugs modulating Wnt signaling inpost-translational modifications on the hippocampal neurons showed an acute increase ofmachinery involved in vesicle exocytosis in the neurotransmitters released from the presynapticpresynaptic terminal leading to spontaneous component resulting in enhanced evoked basalCa2+ transients. Our results identify the Wnt3a activity and frequency of spontaneous andprotein and a member of its complex-receptor miniature excitatory currents (16). Finally, it hasat the membrane, the low-density lipoprotein been reported that conditioned medium containingreceptor-related protein 6 (LRP6) co-receptor, canonical Wnt7a and to a lesser extent Wn3a, butas key molecules in neurotransmission not conditioned medium containing the non-modulation and suggest a crosstalk between canonical Wnt5a, which signals mainly through thecanonical and Wnt/Ca2+ signaling in central Wnt/calcium (Wnt/Ca2+) pathway (19,20), wereneurons. found to increase synaptic transmission in CA3- CA1 slices in adult rat hippocampus and to induce Throughout mammalian brain development the recycling and exocytosis of synaptic vesicles inWnt signaling seems to be spatially confined to hippocampal neurons in culture (17).specialized regions such as the olfactory bulb, Although these results indicate that Wntfrontal cortex, hippocampal formation and the signaling probably modulates neuronalcerebellum (1-5). In these brain domains Wnt transmission, however, the direct effect of a Wntsignaling has essential roles in diverse biological protein on synaptic transmission had not beenprocesses including neurogenesis (6), axonal demonstrated. Here we show that a purified Wntremodeling (7), synapse formation and ligand, the canonical Wnt/β-catenin Wnt3a protein,maintenance of pre- and post-synaptic terminals rapidly increased miniature synaptic currents(8-14). Indeed, several studies have begun to show through a mechanism involving Ca2+ mobilizationthat Wnt signaling may also be involved in and post-translational modifications on the 1 Copyright 2010 by The American Society for Biochemistry and Molecular Biology, Inc.
  2. 2. machinery involved in vesicle exocytosis in the which contains 12 elements in response topresynaptic terminal, suggesting a crosstalk TCF/LEF transcription factors (24). pBARL-HT22between canonical and Wnt/Ca2+ signaling in neurons were incubated with different treatmentscentral neurons. and after 24 h luciferase activity was measured with the Dual-Luminiscence kit (Promega Experimental Procedures Madison, Wi, USA), as previously described (25), in a Victor3 Multilabel Counter (Perkin-Elmer).Wnt3a purification- The Wnt3a purification was Recombinant secreted Frizzled related protein 1carried out following the protocol previously (sFRP1) and Dickkopf 1 (DKK1) were obtaineddescribed (21), which has been implemented in our from R&D Systems (Minneapolis, MN, USA).lab considering the changes suggested by Kishida Electrophysiology- Whole-cell patch-clampand co-workers (22). The presence of the Wnt3a recordings were performed in 12-13 DIVprotein was detected with an anti-Wnt3a antibody hippocampal neurons as previously described(R&D Systems, Minneapolis, MN, USA) and the (26,27). Culture medium was changed to anpositive fractions were pooled and used in the next external solution (ES) containing (in mM): 150 Downloaded from www.jbc.org at PONTIFICIA UNIVERSIDAD, on April 19, 2010column. Purity was analyzed by SDS-PAGE (8 %) NaCl, 5.0 KCl, 3.0 CaCl 2 , 1.0 MgCl 2 , 10 HEPESand stained with Coomassie Blue G250 and and 10 glucose (pH 7.4, 330 mOsm). The patchanalysed through densitometry by using software pipette solution contained (in mM): 120 CsCl, 4.0ImageJ. MgCl 2 , 10 BAPTA, 10 HEPES and 2 ATP (pHCultured hippocampal neurons- The animals were 7.35, 310 mOsm) and a holding potential of -60treated and handled according to NIH guidelines mV was used. Stock recordings were obtained(NIH, Maryland, USA). Hippocampal neurons using an Axopatch-1D amplifier (Axonwere dissociated and maintained as described Instruments, Inc., Burlingame, CA, USA).before (23). Briefly, cells were taken from 18-day Electrodes were pulled from borosilicate capillarypregnant Sprague-Dawley rats and maintained for glass (WPI, Sarasota, FL, USA) using a horizontal12-13 days in vitro (DIV) on 35 mm tissue culture puller (Sutter Instruments, Novato, CA, USA). Thedishes with glass coverslips (350,000 cells per current signal was filtered at 2-5 kHz and storeddish) coated with poly-L-lysine (Sigma Chemical for off line analysis using PC interfaced with aCo., St, Louis, MO, USA). The neuronal feeding Digidata 1200 acquisition board. In the records ofmedium consisted of 80% minimal essential action potentials, the current was set at 0 A. 100medium (MEM, GIBCO, Rockville, MD, USA), nM tetrodotoxin (TTX) were added when10% heat-inactivated horse serum (GIBCO, miniature synaptic currents were recorded (2 minRockville, MD, USA), 10% heat-inactivated segments). Glutamatergic transmission wasbovine fetal serum (GIBCO, Rockville, MD, USA) pharmacologically isolated during the recording ofand a mixture of nutrient supplements. The culture miniature synaptic currents through perfusion ofwas placed on a shelf in a 37º C humidified CO 2 Wnt3a in the presence of either 6-cyano-7-incubator, and the medium was changed every 3 nitroquinoxaline-2,3-dione (CNQX; 4µM) or d-2-days. amino-5-phosphonovaleric acid (APV; 50 µM),Wnt3a functional assays- Purified Wnt3a protein which were diluted in the ES without Mg2+ for itswas assessed for its ability to stabilize β-catenin in pre-application at RT (20–24 °C; see Fig. 3). Inhippocampal neurons (12-13 DIV), which were order to obtain mean average of either cumulativeincubated with 10 nM of Wnt3a for 0, 15, 30 min or peak amplitude and frequency, the data wasand 2 hr. Lithium chloride (LiCl 10 mM; 2 hr; analyzed with Mini Analysis 6.0 programSigma, St Louis M.O, USA) was used as a control. (Synaptosoft, Inc., Leonia, NY, USA) as describedThen, cells were lysed and 30 µl of lysates were previously (26). Decay time histograms weresubjected to SDS-PAGE and Western blotting. plotted with a bin of 1 pA and the frequency (%Similarly, Wnt3a activity was evaluated in total events) was calculated and expressed overpBARL-HT22 cells, a mouse hippocampal cell line control neurons. All reagents were obtained fromstably transfected with the pBARL(Beta-catenin Sigma (Sigma, St Louis M.O, USA).Activated firefly Luciferase) reporter plasmid, 2
  3. 3. Calcium experiments- Hippocampal neurons of 12- emission was collected with a 620 nm filter.13 DIV were loaded with 5 µM Fluo-4AM Finally, a similar approach was used when loading(Molecular Probes, Invitrogen, USA) in external for 1 min neurons with 50µM of the fixable probesolution for 30 min at 37ºC, washed three times AM1-43 (Molecular Probes, USA) at 37ºC, afterwith external solution for 5 min and mounted in a which cells were washed with ES for 5 min at RTperfusion chamber placed on the stage of an and mounted on a perfusion chamber forinverted fluorescent microscope (Eclipse TE, immunocytochemistry.Nikon). The microscope had a 12 bit CCD camera Immunocytochemistry- Treated and controlattached (SensiCam, PCO, Germany). After being hippocampal neurons (10-13 DIV) were washedincubated for 15 min with different treatments (see with ES and loaded with 50 µM AM1-43Results), neurons were located, selected their (Molecular Probes, USA) for 1 min at 37ºC,somatic region and illuminated (< 0.266 s) by washed with ES for 5 min at RT and mounted on ausing a Lamba 10-2 filter wheel (Sutter perfusion chamber. Cells were fixed with 4 %Instruments, Novato, CA, USA) computer- paraformaldehyde–PBS (1X, pH 7.4), blocked forcontrolled by Axon Instruments Work-Bench 2.2 1 h with horse fetal serum (1:10 Hyclone, USA) Downloaded from www.jbc.org at PONTIFICIA UNIVERSIDAD, on April 19, 2010software (Axon Instrument, Inc., Burlingame, CA, and permeabilized with PBS1X-Triton X-100 (0.1USA). The images at 480 nm were obtained at 2 s %) and incubated for 12 h at 4°C with theintervals during a continuous 5 min period and the following primary antibodies: p-Synapsin Ifrequency of Ca2+ transients was determined. Since antibody (Ser 553; 1:200; Santa Cruzcalcium transients are dependent on neuronal Biotechnology, USA), LRP6 (1:100; R&Dexcitability and synaptic transmission, and thus Systems, USA), Synaptophysin (1:500; Zymed,blocked by application of TTX (27,28), Ca2+ USA) and PSD95 (1:500; Affinity Bioreagent,transient records were performed in the absence of USA). Then, neurons were washed 3 times andthis inhibitor. Finally, a similar protocol was used incubated with appropriate secondary antibodieswhen examining intracellular Ca2+ concentration, conjugated to Alexa488 (1:500), Cy3 (1:400) andwith the sole exception that this time neurons were Alexa633 (1:500) (InmunoResearch Jacksonperfused with different treatments containing Laboratories, USA). After mounting the samples inWnt3a in the presence of inhibitors, and the images Dako (DAKO Corp, USA), fluorescent microscopyat 480 nm were obtained at 1 s intervals for a total was performed (60 X oil immersion objective 1.45recording period of 160 s. NA) using a TE2000U confocal microscopeRelease of synaptic vesicles- Treated and control (Nikon, Japon). Images were acquired with thehippocampal neurons (10-13 DIV) were washed Nikon software (EZ-C1 V. 3.5).with ES and incubated for 5 min in high-K+ Colocalization analysis- Fluorescent images forsolution (30 mM) at 37º C and then loaded with 15 each antibody were acquired sequentially on theµM probe FM1-43 (Molecular Probes, USA) for 5 confocal microscope. We selected ROIs (neuronalmin at 37º C, washed with ES for 5 min at RT and process) adjusted to a window/level of 300 pixelsmounted on a perfusion chamber. Different and separated the fluorescence channels associatedtreatments were applied by perfusion (see Results) with LRP6, Syp and PSD95. The images wereand regions of interest (ROIs) were selected and deconvoluted and examined for the degree ofthe decay of fluorescence associated with FM1-43 colocalization between the different channelswas continuously measured during 20 min. The through qualitative analysis of antibodyrecordings were collected with an inverted colocalization using ImageJ (pluggin JacoP).epifluorescence microscope (Eclipse TE, Nikon, Overlap of the green-red (LRP6-Syp) and green-Melville, NY, USA) equipped with a Xenon lamp, blue (LRP6-PSD95) channels were visualized in40X and 100X objectives, and a Lambda 10-2 merged images and then overlapping areas werefilter wheel (Sutter Instruments, Novato, CA, considered as colocalized. The extent ofUSA). The microscope had a SensiCam CCD colocalization was further analyzed using thecamera (PCO, Kelheim, Germany) and the FM1- Manders (M1) and Pearsons coefficients (29). Both43 fluorescence intensity was measured using a 2 x coefficients range from 0 to 1, with 0 indicating2 pixels area. FM1-43 was excited at 560 nm and 3
  4. 4. low colocalization and 1 indicating high greater number of repetitive events in Wnt3a-colocalization. treated neurons (Fig. 2B, bottom trace), reflectingWestern blot- Proteins were separated in a SDS- an increase of the neural network excitability andPage 12% gel and transferred to a nitrocellulose further indicating that the effect of the Wnt3amembrane (BioRad, CA, USA). Membranes were protein ought to be mainly on synapticblocked with 5% milk in PBS 1X and 0,1 % transmission (increased synaptic potential).Tween 20 for 1 h in agitation, then incubated with Moreover, there were no significant differences inprimary β-catenin or p-Synapsin I (Ser 553) input resistance in control and Wnt3a-treatedantibodies (both from Santa Cruz Biotechnology, neurons (Table 1). Thus, we decided to use 10 nMSanta Cruz, CA, USA) for 12 h at 4°C, washed and of Wnt3a as the effective concentration forincubated with appropriate secondary antibody subsequent recordings of synaptic potentialsconjugated to HRP (Santa Cruz Biotechnology, (miniature synaptic activity), examined in theSanta Cruz, CA, USA) for 1 h at 4°C. The presence of 100 nM of the Na+ channel blockerimmunoreactivity of reactive protein was detected tetrodotoxin (TTX).using chemoluminescence reagents (Promega, Local application of Wnt3a to neurons showed Downloaded from www.jbc.org at PONTIFICIA UNIVERSIDAD, on April 19, 2010Madison, Wi, USA). a time-dependent enhancement of miniatureData Analysis- Data are shown as mean ± s.e.m. synaptic activity compared to control neuronsand test ANOVA (*p < 0.05, **p < 0.01, ***p < treated with the Wnt3a elution buffer (Fig. 2C).0.005) was implemented. All analyses were Analysis of individual traces (i.e. mean averageperformed using the Origin 6.0 software (Microcal, peak) revealed that purified Wnt3a affectedInc. Northampton, MA, USA). significantly the frequency, but not the amplitude of the synaptic currents (Fig. 2C-E), effect which RESULTS was further confirmed by analyses of the cumulative frequency and amplitude between Purified Wnt3a enhances excitatory control and Wnt3a-treated neurons (Fig. S1). Theneurotransmission. We purified Wnt3a protein enhancing effect on the frequency was apparentfrom stable mouse L-cells following standards after 5 min and peaked at 15 min of incubation (1.1protocols (21,22) and consistently recovered a ± 0.1 Hz in control condition to 3.4 ± 0.3 Hz in thefully functional Wnt3a ligand that induced Wnt3a treatment; N=9), and contrary to Wnt3aaccumulation of its β-catenin target in primary treated neurons, the application of either denaturedcultures of embryonic rat hippocampal neurons Wnt3a (1.5 ± 0.2 Hz; N=8), or the co-application(12-13 DIV) (Fig. 1A-D). Then, to analyze the of 2 µg/ml of anti-Wnt3a antibody (1.1 ± 0.01 Hz;functional activity of the Wnt3a protein in mature N=8), at a dose that blocks Wnt3a activity insynapses in hippocampal neurons we carried out Wnt/β-catenin reporter-based luciferase assayselectrophysiological analysis using the whole-cell (Fig. 1E), were without effects on synapticpatch-clamp technique to record spontaneous activity. Conversely, the inhibitory effect of thesynaptic activity in the presence of different anti-Wnt3a antibody was not observed when thisconcentrations of the purified protein. We found antibody was previously denatured (3.6 ± 0.4 Hz;that Wnt3a produced a concentration dependent N=5). Moreover, the effect of Wnt3a wasincrease in the frequency of spontaneous synaptic reversible as demonstrated after its removal fromcurrents (Fig. 2A), which at the cellular level is the bath solution (washout; 1.2 ± 0.5 Hz; N=9; Fig.likely associated to an increase in the neuronal 2).network’s activity, reflecting the sum of action Wnt3a-mediated enhancement in the frequencypotentials and synaptic potentials. Analyses of of miniature synaptic currents was completelycharacteristic parameters of action potentials (AP; blocked in the presence of 6-cyano-7-i.e. threshold, amplitude and duration) showed that nitroquinoxaline-2,3-dione (CNQX; 4 µM), anno significant differences were found in control inhibitor of AMPA receptors (0.7 ± 0.1 Hz; N=5),versus treated neurons with 10 nM Wnt3a for 15 and partially silenced in the presence of d-2-min (Fig. 2B and Table 1). Interestingly, while amino-5-phosphonovaleric acid (APV; 50 µM), aindividual AP remained similar, there was a 4
  5. 5. competitive antagonist of NMDA receptors (0.9 ± effect of the Wnt3a protein we next examined0.1 Hz; N=5), suggesting that modulation of whether acute perfusion of Wnt3a alteredexcitatory glutamatergic neurotransmission play a intracellular Ca2+ levels using the fluorescent probefundamental role in the synaptic effect induced by Fluo4-AM (Fig. 4B). Consistent with our previousWnt3a (Fig. 3A,B). As previously observed, the results, perfusion of Wnt3a acutely augmented theamplitude of synaptic currents did not differ intracellular Ca2+ concentration of neurons loadedamong treatments (Fig. 3C). Furthermore, analysis with Fluo4-AM, an effect which was partiallyof time decay kinetics of synaptic events (26,27) reversed when Wnt3a was co-incubated with 2showed that records in the presence of Wnt3a were µg/ml of the anti-Wnt3a antibody (decreased to 46easily distinguishable as glutamatergic, specifically % of the Wnt3a normalized fluorescence signal,AMPAergics (93 %; τ = 0.1-10 ms; N=10), in N=15), or silenced when a denatured fraction ofcontrast to NMDA/GABAergic kinetics (7 %; τ = purified Wnt3a was used after being heated for 1011-40 ms; N=9) (Fig. 3D-F). Therefore, we min at 96° C (decreased to 8 % of normalizedconclude that low concentrations of purified fluorescence, N=6). Remarkably, Wnt3a effect onWnt3a rapidly enhance the frequency of miniature intracellular Ca2+ was dramatically diminished in Downloaded from www.jbc.org at PONTIFICIA UNIVERSIDAD, on April 19, 2010excitatory synaptic currents and that such effect is the presence of 20 µM Cd2+, (12.2 ± 0.8 % of thespecific to the treatments with the purified Wnt3a Wnt3a normalized signal, N=15), which acts as aprotein. non-selective Ca2+ channel blocker, or nearly Wnt3a induces intracellular Ca2+ influx and the abolished in the absence of extracellular calcium inrelease of presynaptic vesicles. Changes in the the working solution (zero nominal calcium; 3.7 ±frequency of miniature synaptic currents could be 1.1 % of the remaining signal, N=18), thusinterpreted by alterations in the probability of indicating that the extracellular space is the mainneurotransmitter release and it has been suggested source of the Ca2+ mobilized by the Wnt3a ligand.that such release depends on the concentration of It has been proposed that intracellular Ca2+Ca2+ within the presynaptic terminal (30). concentration is the focal point controlling theInterestingly, the effect of Wnt3a on the frequency exocytosis and trafficking of synaptic vesicles atof miniature synaptic activity was completely the presynaptic active zone of nerve terminals (31).abolished (0.5 ± 0.05 Hz; N=5) when an external Therefore, to examine whether the effect of Wnt3asolution with zero nominal Ca2+ was used in the on the intracellular Ca2+ concentration wasexperiments (Fig. 3A-C). Therefore, we next sufficient to allow the release of presynapticinvestigated whether there was a relationship vesicles from mature hippocampal neurons, webetween the Wnt3a-induced enhancement in applied a depolarizing stimulus with a high K+excitatory synaptic transmission and intracellular concentration (30 mM), loaded neurons with FM1-Ca2+ levels. In order to do so, neurons were loaded 43 and then treatments (i.e. Wnt3a) were appliedwith Fluo4-AM and the fluorescent signal related by perfusion throughout the entire record (Fig.to the frequency of spontaneous calcium transients 4C). Indeed, time-course experiments revealed thatwas recorded using fluorescence microscopy. Wnt3a treatment induced a fast release of synapticNotably, as shown in Fig. 4A, neurons incubated vesicles seen as the decay in the fluorescencewith a concentration of 10 nM Wnt3a for 15 min associated with FM1-43 after starting theand in the absence of TTX significantly augmented incubation, which subsequently reached a plateauthe frequency of spontaneous Ca2+ transients (13.9 at 12-15 min of stimulation (Fig. 4C). The value of± 0.5 x 10-2 Hz; N=50) when compared to control the depleted fraction (∆F/Fi) found after 20cells (3.6 ± 0.1 x 10-2 Hz; N=45, p < 0.005). minutes of recording was 0.1 ± 0.04 in the controlConversely, neurons treated either with denatured condition and 0.8 ± 0.02 in neurons treated withWnt3a (boiled) or co-incubated with Wnt3a and Wnt3a. Similarly, such effect was specific toanti-Wnt3a (2 µg/ml) did not show the above Wnt3a since no significant differences werementioned effect (4.1 ± 0.2 x 10-2 and 4.7 ± 0.6 x observed when boiled Wnt3a or co-incubation with10-2 Hz, respectively). As a way to distinguish the anti-Wnt3a antibody were used as treatmentsamong the Ca2+ source required for the synaptic (0.42 ± 0.03 and 0.6 ± 0.03, respectively). In 5
  6. 6. agreement with the above presented results, antagonist, and 2 µg/ml Dickkopf 1 (DKK1), as anfluorescent immunochemistry analysis in antagonist of the Low Density Receptor Relatedhippocampal neurons with the fluorescent probe Protein 6 (LRP6), which acts as a co-receptor (32).AM1-43 (equivalent dye to FM1-43 but with the Remarkably, we observed blockade of the Wnt3aadditional property of being fixable) showed that effect by both sFRP1 and DKK1 on the frequencythe signal associated to synaptic vesicles strongly of miniature synaptic currents (1.6 ± 0.14 Hz; N=6decreased after 15 min of treatment with purified and 1.2 ± 0.04 Hz; n = 5, respectively; Fig. 5A)Wnt3a (Fig. 4D, upper panel). Collectively, these and Ca2+ transients (3.8 ± 0.4 x 10-2 Hz, N=40 andexperiments, which were made in the absence of 6.8 ± 0.09 x 10-2 Hz, N=50, respectively; Fig. 5B),TTX, indicate that acute incubations with purified suggesting that the Wnt/β-catenin complexWnt3a allows the influx of Ca2+ from the receptor is functionally active at the synapticextracellular space, augmenting the intracellular terminal. No significant increase was observedconcentration of Ca2+ and enhancing the release of when sFRP1 and DKK1 proteins were appliedsynaptic vesicles from the presynaptic terminal. alone onto the hippocampal neurons. Previous studies have shown that Wnt-7a DKK1 interferes directly with Wnt3a Downloaded from www.jbc.org at PONTIFICIA UNIVERSIDAD, on April 19, 2010induced the clustering at remodeled areas of mossy presentation to the LRP6 receptor itself, instead offibers of the neuron-specific phosphoprotein being sequestered in the extracellular space as it isSynapsin I as a preliminary step in synaptogenesis the case for the action of sFRP1. Given that we(11). Therefore, we further investigated whether have previously shown that the LRP6 protein isWnt3a treatment had any effect on such post- expressed in the human hippocampus (25),translational modification in hippocampal neurons. therefore we wanted to study whether LRP6 wasIndeed, purified Wnt3a induced a substantial present in the synaptic terminal. In order to do so,increase in the signal of phosphorylated Synapsin I the subcellular localization of the LRP6, as well as(shown in green) when compared with control pre- and post-synaptic markers synaptophysinneurons (Fig. 4D, lower panel). Finally, Western (Syp) and PSD95, respectively, was examined inblot analysis revealed that induction of Synapsin I 14-15 DIV hippocampal neurons by confocalphosphorylation was specific for the treatment microscopy. The results obtained are summarizedwith purified Wnt3a (Fig. 4E), suggesting that this in Fig. 6, which shows that LRP6 is widelypost-translational modification may be an essential distributed both in the soma and in the neuronalstep towards the release of synaptic vesicles from processes of hippocampal neurons, where is co-presynaptic terminals induced by Wnt3a in localized and/or closely apposed to the signalhippocampal neurons. corresponding to synaptophysin and PSD95. Involvement of the Wnt/β-catenin complex Further quantitative analysis of co-localization forreceptor at the membrane in Wnt3a LRP6-Syp and LRP6-PSD95 proteins (Fig. 6B &neurotransmission. As shown previously, purified C) revealed that there were no significantWnt3a induced β-catenin accumulation in differences in the co-localization coefficientshippocampal neurons (Fig. 1C & D). Then, would Manders (0.34 ± 0.042 and 0.38 ± 0.044,the molecular machinery normally transducing respectively; N=15) or Person (0.54 ± 0.029 andcanonical Wnt3a be the one responsible for the 0.52 ± 0.027, respectively; N=15) (29), suggestingenhancement of intracellular calcium and the fast that the LRP6 could be simultaneously presentrelease of synaptic vesicles from the pre-synaptic both in pre- and post-synaptic terminals.terminals? We began to approach this question byinterfering Wnt3a signal presentation at the DISCUSSIONmembrane using recombinant proteins that inhibitthe activity of its complex receptor Frizzled- In recent years it has become clear that theLRP5/6. Therefore, the synaptic activity of signaling cascade activated by any Wnt isoform ishippocampal neurons exposed to Wnt3a was highly dependent on cellular context or therecorded either in the presence of 50 nM secreted complementation between cell surface WntFrizzled Related Protein 1 (sFRP1), a Wnt receptors (33). Wnt3a belongs to the so called 6
  7. 7. Wnt/β-catenin or canonical signaling as opposed to there was not complete inhibition, no significantthe β-catenin independent or non-canonical differences were observed between APV-treatedpathways, which transduce their signals to control and control neurons. Finally, considering that theasymmetric cell division and morphogenetic blockade of GABA A receptors with bicuculline ormovements during vertebrate gastrulation, strychnine induced an increase in the frequency ofincluding the Wnt/Ca2+ pathway (19,20,32). Here miniature AMPA receptor-mediated EPSCs (34)we demonstrate that synaptic transmission activity and a change in the network activity ofwas modulated by direct application of a canonical hippocampal neurons (27,34), which were withinWnt3a protein to mature hippocampal neurons, the time-frame for the Wnt3a effect described herewhich is in general agreement with the effect of a (i.e. > 15 min), therefore, as an indirect way torecombinant Wnt3a preparation used to study LTP assess the effects on inhibitory activity, we carriedevents on hippocampal slices (18) and the use of out analyses of time decay kinetics (τ) events. Ineither conditioned medium containing Wnt ligands agreement with previous observations (15-18), our(15,17) or Wnt/β-catenin small molecule results clearly distinguished the effect of purifiedmodulators (16). Moreover, the effect was Wnt3a as excitatory and not inhibitory and further Downloaded from www.jbc.org at PONTIFICIA UNIVERSIDAD, on April 19, 2010dependent on the incubation with the purified implicated AMPA receptors as the main effectorsprotein (Fig. 2-4), and not observed in the presence controlling the action of the Wnt3a protein at theof recombinant DKK1 and sFRP1 proteins, synapse (Fig. 3D-F).classical inhibitors of the Wnt/β-catenin pathway It is well known that synaptic transmission is(Fig. 5), suggesting that the canonical complex affected by changes in presynaptic Ca2+ level (31).receptor at the membrane is functionally active at Notably, the Wnt3a effect on neurotransmissionthe synaptic terminal. seems to be mediated through a fast influx of Ca2+ Although we initially observed that purified which subsequently induced the phosphorylationWnt3a induced an increase both in the frequency of synapsin I and the release of synaptic vesiclesand the amplitude of spontaneous activity in from the presynaptic terminal (Fig. 4).Wnt3a-treated neurons (Fig. 2A), which could Interestingly, the source of this Ca2+ influxreflect enhanced neuronal excitability, we appeared to be mainly extracellular. Indeed, thesubsequently observed that the Wnt3a dependent- Wnt3a effect was nearly abolished when using aneffect was not due to changes in neuronal electrical external solution with zero nominal calcium orparameters such as action potentials or input silenced in experiments in the presence of Cd2+,resistance (Fig. 2B and Table 1). Moreover, which suggest that influx through voltage-analyses of miniature post-synaptic currents in dependent Ca2+ channels (VDCCs) or transientTTX-treated neurons indicate that the Wnt3a effect receptor potential (TRP) ion channels, which arewas directly on synaptic transmission, via both Cd2+-sensitive (35-37), could be involved inaugmenting the frequency of neurotransmitters the Wnt3a evoked effect. Supporting this idea, itrelease, and not related to the number of activated has been recently observed that a Wnt/Ca2+receptors in the post-synaptic region (Fig. 2C-E; pathway ligand, the Wnt5a protein (19,20),see also Fig. S1). modulates cortical axonal guidance/repulsion To investigate whether excitatory or inhibitory processes via augmenting the concentration ofneurotransmission is the target of purified Wnt3a, intracellular Ca2+ through activation of TRP ionwe first evaluated the effects of excitatory channels (38). Nevertheless, activation ofAMPA/kainate and NMDA receptors. Our results presynaptic neurotransmitter receptors that areshow that when neurons were treated with 4 µM permeable to Ca2+, and which enable a sufficientCNQX (AMPA/kainate antagonist) the Wnt3a rise in intracellular Ca2+ to trigger neurotransmittereffect on the release of synaptic vesicles was release, should be also examined in further detaildramatically decreased (i.e. similar to that when explaining the remaining 12 % in theobserved in the control neurons) (Fig. 3A-C). fluorescent signal associated with the Wnt3a effectSimilarly, in the case of the NMDA receptor in the presence of Cd2+ (Fig. 4B).antagonist APV, the results show that although The data, which suggest that a cross-talk between Wnt/β-catenin and Wnt/Ca2+ signaling 7
  8. 8. could take place in central neurons, is further which we have previously found expressed in thesupported by the following observations: First, we human hippocampus and genetically linked tohave shown here that purified Wnt3a protein, prevalent neurological conditions (25), is seen herewhich is strongly expressed in the hippocampus localized within pre-and post-synaptic regions(39), along with inducing the influx of Ca2+ (Fig. 6), being essential for inducing themaintains its activity in the canonical pathway in intracellular increase of Ca2+ in order to trigger thestabilizing β-catenin and activating a Wnt/β- Wnt3a-dependent effect on neurotransmissioncatenin luciferase-associated gene reporter (Fig. 1) (Fig. 5).(21,22). Second, it has been observed that in the Finally, the experimental data reported in thisproliferation of PC12 cells the canonical Wnt1 study does not necessarily imply that the Wnt3aprotein activates intracellular components of the effect on neurotransmission would be independentWnt/Ca2+ pathway, including the protein kinase C of the activity mediated by β-catenin/TCF-LEF(PKC) enzyme (40), which has been previously complexes, which have been previously involvedinvolved in the regulation of neurotransmitter in LTP events (16,18), but rather suggest that suchrelease (41,42). Third, the Wnt-responsive a rapid effect on the neuronal network (i.e. min) Downloaded from www.jbc.org at PONTIFICIA UNIVERSIDAD, on April 19, 2010Dishevelled (Dvl) protein, which acts as a involves the cross-talk between receptors andbranching point between canonical and non- kinases acting as canonical or Wnt/Ca2+canonical Wnt signalling pathways, binds to components, which is the molecular machinerySynaptotagmin and thus participates in the process associated with or immediately downstream of itsof endo- and exocytosis of neurotransmitter- complex receptor at the membrane in the Wnt3acontaining vesicles in differentiated PC12 cells effect on synaptic transmission in mature(43). Fourth, the canonical LRP6 co-receptor, hippocampal neurons. REFERENCES1. Grove, E. A., Tole, S., Limon, J., Yip, L., and Ragsdale, C. W. (1998) Development 125, 2315-23252. Houart, C., Caneparo, L., Heisenberg, C., Barth, K., Take-Uchi, M., and Wilson, S. (2002) Neuron 35, 255-2653. Lee, S. M., Tole, S., Grove, E., and McMahon, A. P. (2000) Development 127, 457-4674. Maretto, S., Cordenonsi, M., Dupont, S., Braghetta, P., Broccoli, V., Hassan, A. B., Volpin, D., Bressan, G. M., and Piccolo, S. (2003) Proc Natl Acad Sci U S A 100, 3299-33045. Zhou, C. J., Zhao, C., and Pleasure, S. J. (2004) J Neurosci 24, 121-1266. Lie, D. C., Colamarino, S. A., Song, H. J., Desire, L., Mira, H., Consiglio, A., Lein, E. S., Jessberger, S., Lansford, H., Dearie, A. R., and Gage, F. H. (2005) Nature 437, 1370-13757. Takeichi, M., and Abe, K. (2005) Trends Cell Biol 15, 216-2218. Bamji, S. X., Shimazu, K., Kimes, N., Huelsken, J., Birchmeier, W., Lu, B., and Reichardt, L. F. (2003) Neuron 40, 719-7319. Ciani, L., and Salinas, P. C. (2005) Nat Rev Neurosci 6, 351-36210. Farias, G. G., Valles, A. S., Colombres, M., Godoy, J. A., Toledo, E. M., Lukas, R. J., Barrantes, F. J., and Inestrosa, N. C. (2007) J Neurosci 27, 5313-532511. Hall, A. C., Lucas, F. R., and Salinas, P. C. (2000) Cell 100, 525-53512. Packard, M., Koo, E. S., Gorczyca, M., Sharpe, J., Cumberledge, S., and Budnik, V. (2002) Cell 111, 319-33013. Speese, S. D., and Budnik, V. (2007) Trends Neurosci 30, 268-27514. Wang, J., Jing, Z., Zhang, L., Zhou, G., Braun, J., Yao, Y., and Wang, Z. Z. (2003) Nat Neurosci 6, 1017-101815. Ahmad-Annuar, A., Ciani, L., Simeonidis, I., Herreros, J., Fredj, N. B., Rosso, S. B., Hall, A., Brickley, S., and Salinas, P. C. (2006) J Cell Biol 174, 127-139 8
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  10. 10. FOOTNOTES*We acknowledge the efforts of M. Cuevas, J. Fuentealba and P. Cardenas in the initial steps of this study.This work was supported by an associative CONICYT Grant PBCT ACT-04 from the Chileangovernment to G.V.D., C.O. and L.G.A. R.T.M. is supported as an Investigator of the Howard HughesMedical Institute. FIGURE LEGENDSFig. 1. Purification and functional assays of Wnt3a activity. A. Coomassie Blue staining of an SDS-PAGEgel which was loaded with Wnt3a-L-cells conditioned medium (CM) and purified Wnt3a, resulting from a3-step chromatographic purification (BS: Blue Sepharose; HiL: High Load 16-60 Superdex 200; Hep:Heparin). B & C. Western blotting analysis of purified Wnt3a and time-dependent effect on β-cateninstabilization in hippocampal neurons following different periods of Wnt3a application (15, 30 min and 2hr); LiCl (10 mM) for 2 hr was used as a control. D. Summary of data as shown in (C). E. β-catenin Downloaded from www.jbc.org at PONTIFICIA UNIVERSIDAD, on April 19, 2010reporter activity in pBARL-HT22 cells treated with 10 nM Wnt3a, Wnt3a vehicle (buffer), boiled Wnt3a(denatured 10 min at 96 ºC), 10 nM Wnt3a plus 50 nM recombinant secreted Frizzled related protein 1(sFRP1), 50 nM sFRP1, Wnt3a plus 70 nM recombinant Dickkopf 1 (DKK1) and 70 nM DKK1. Control:basal reporter activity in this cell line. Data from 3 independent experiments is shown as mean ± s.e.m.and test ANOVA (*p < 0.05, ***p < 0.005) was implemented.Fig. 2. Enhancement of miniature synaptic activity by purified Wnt3a in primary cultures of hippocampalneurons. A. Representative traces of spontaneous synaptic currents recorded in the presence of differentWnt3a concentrations (0-10 nM). B. The upper panel shows representative traces of individual events involtage records (action potentials) obtained from control neurons and those treated with 10 nM of theWnt3a protein. The lower panel depicts a repetitive event induced by 10 nM Wnt3a. The dotted line marks0 mV. Records (N=5) were obtained in the absence of TTX and the treatments were applied by perfusion.C. Miniature synaptic activity following the application of Wnt3a (5 and 15 min) and various treatmentsby perfusion, in the presence of 100 nM TTX (holding potential of -60 mV; 2 min duration). D & E.Summary of miniature synaptic activity data for current frequency and amplitude, respectively. Control:Wnt3a vehicle; Wnt3a: 10 nM Wnt3a; Boiled: denatured Wnt3a (boiled 96 ºC); anti-Wnt3a: antibody anti-Wnt3a; Boiled anti-Wnt3a: boiled antibody anti-Wnt3a (96 ºC); washout: Wnt3a removed from externalsolution. Test ANOVA (***; p < 0.005; n = 5). Data are shown as mean ± s.e.m.Fig. 3. Inhibition of Wnt3a-induced synaptic activity by glutamatergic blockers CNQX and APV. (A) Theminiature synaptic transmission induced by 10 nM Wnt3a (15 min) in hippocampal neurons waspharmacologically blocked following application of CNQX (4 µM) or APV (50 µM) by perfusion in thepresence of ligand and TTX (100 nM) and in the absence of Mg2+. B & C. Data summary for the effectson the frequency and amplitude of the miniature currents, respectively. Control: Wnt3a vehicle; WithoutCa2+: zero nominal calcium. Data are shown as mean ± s.e.m. and test ANOVA (*p < 0.05, ***p < 0.005;N=5) was implemented. D-F. Analysis of an extended trace of miniature synaptic activity recorded in thepresence of 10 nM Wnt3a. E & F. Stacked bars plot showing event decay-time (ms) distribution histogramand the frequency of total events in Wnt3a treated neurons. Fast AMPAergic events: 0,1 – 10 ms; SlowGABAergic events: 10 – 40 ms.Fig. 4. Enhancement of intracellular Ca2+ and synaptic vesicle release by Wnt3a in hippocampal neurons.A. Representative fluorescent traces showing spontaneous enhancement of calcium transients followingapplication of Wnt3a and various treatments for 15 min. B. Strokes representing the effect of acute influxof extracellular Ca2+ following perfusion of Wnt3a and various treatments. C & D. Synaptic vesicle 10
  11. 11. release from pre-synaptic terminals induced by Wnt3a. C. Destaining associated to FM1-43 (depletedfraction: ∆F/Fi) in the presence or absence of Wnt3a. Treatments were applied by perfusion during theentire record (20 min) and the burden of hippocampal neurons was recorded (N=60–80 neurons). D.Immunocytochemical analysis showing the decay of the signal associated with the fixable AM1-43 probe(upper panel) and the enhancement of phosphorylated Synapsin I (Ser 553) after treatment with Wnt3a for15 min (lower panel). E. Western blot of phosphorylated Synapsin I after treatment for 15 min withpurified Wnt3a compared to control neurons treated either with boiled Wnt3a or co-incubated with aWnt3a-specific antibody. All records of Ca2+ changes were made in the absence of TTX. Calibration barrepresents 20 µm. Control: Wnt3a vehicle; ENS: external normal solution containing 10 nM Wnt3a; Cd2+:ENS plus 10 nM Wnt3a and 20 µM Cd2+; w/o Ca2+: 10 nM Wnt3a plus external solution without Ca2+(zero nominal Ca2+); Boiled: denatured Wnt3a (boiled 10 min to 96 °C); anti-Wnt3a: co-incubation of 10nM Wnt3a and 2 µg/ml of antibody anti-Wnt3a.Fig. 5. Evidence for the participation of the Wnt/β-catenin complex-receptor in Wnt3a-induced Downloaded from www.jbc.org at PONTIFICIA UNIVERSIDAD, on April 19, 2010neurotransmission. A & B. Representative traces showing the inhibition of the Wnt3a enhancement in thefrequency of miniature synaptic activity (test ANOVA, ***p<0.005; n=5; 100 nM TTX) and calciumtransients (test ANOVA, ***p<0.005; n=40-50), respectively, following co-incubation for 15 min of 10nM Wnt3a with either 50 nM sFRP1 or 70 nM DKK1, which act as inhibitors of the Wnt/β-cateninmembrane-associated receptors (see also Fig. 1E). C & D. Summary of miniature synaptic activityfrequency and calcium transient data, respectively. Data are shown as mean ± s.e.m.Fig. 6. LRP6 immunoreactivity in the soma and neuronal processes associated with pre- and post-synapticmarkers Synaptophysin (Syp) and PSD95. A. Confocal images were obtained from neurons of 14-15 DIVusing antibodies against LRP6 (green-Alexa488), Syp (red-Cy3) and PSD95 (blue-Alexa633). Arrowsshow LRP6-Syp and LRP6-PSD95 colocalization. Calibration bar: 20 µm. B & C. Mander’s andPearson’s correlation coefficients for the colocalization of the LRP6 and pre- and post-synaptic markersSyp and PSD95, respectively. An average of coefficients of at least 3 ROIs of 10 neurons was examined. 11
  12. 12. Table 1. Electrical parameters (action potentials and input resistance) recorded in hippocampal neurons inthe absence or presence of Wnt3a (10nM, 15 min). Action Potential IR (GΩ) AMP (mV) TH (mV) AP/2 (ms)Control 84 ± 1.4 -46 ± 0.6 0,0015 ± 5.0 x 10-5 0,65 ± 0,05Wnt3a 85 ± 1.5 -44 ± 1.0 0,0014 ± 7.3 x 10-5 0,7 ± 0,04p-value 0.56 0.13 0.45 0.32AMP: amplitude; TH: threshold; and AP/2: duration of action potentials; IR: input resistance. Downloaded from www.jbc.org at PONTIFICIA UNIVERSIDAD, on April 19, 2010 12