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Cer cor11 franco-cercor-bhr199

  1. 1. Cerebral Cortex Advance Access published August 1, 2011Cerebral Cortexdoi:10.1093/cercor/bhr199WIP Is a Negative Regulator of Neuronal Maturation and Synaptic ActivityA. Franco1, S. Knafo2,3, I. Banon-Rodriguez1, P. Merino-Serrais2, I. Fernaud-Espinosa2, M. Nieto1, J.J. Garrido2,3,4, J.A. Esteban3,F. Wandosell3,4 and I.M. Anton1,41 Centro Nacional de Biotecnologı´ a (CNB-CSIC), 28049 Madrid, Spain, 2Instituto Cajal (CSIC), 28002 Madrid, Spain, 3Centro deBiologı´ a Molecular ‘‘Severo Ochoa’’ (CSIC-UAM), Universidad Autonoma de Madrid, 28049 Madrid, Spain and 4CIBERNED, Centro ´Investigacion Biomedica en Red de Enfermedades Neurodegenerativas, 28031 Spain ´ ´A. Franco and S. Knafo contributed equally to this workAddress correspondence to Dr Ines M. Anton, Cellular and Molecular Department, Centro Nacional de Biotecnologı´ a (CNB-CSIC), Darwin 3, 28049 ´ ´Madrid, Spain. Email: ianton@cnb.csic.es.Wiskott--Aldrich syndrome protein (WASP) --interacting protein factors (NPF), such as neural Wiskott-Aldrich syndrome protein(WIP) is an actin-binding protein involved in the regulation of actin (N-WASP). The Arp2/3 complex nucleates actin, inducingpolymerization in cells, such as fibroblasts and lymphocytes. branching and elongation, and with N-WASP, it mediatesDespite its recognized function in non-neuronal cells, the role of neurite elongation (Suetsugu, Hattori, et al. 2002; Pinyol et al.WIP in the central nervous system has not been examined 2007) and neurite branching (Kakimoto et al. 2004). N-WASP Downloaded from cercor.oxfordjournals.org at Universidad Autonoma de Madrid. Facultad de Medicina on August 2, 2011previously. We used WIP-deficient mice to examine WIP function interacts with WASP-interacting protein (WIP), a broadlyboth in vivo and in vitro. We report here that WIP2/2 hippocampal expressed proline-rich protein that regulates N-WASP functionneurons exhibit enlargement of somas as well as overgrowth of as NPF and whose deficiency modifies actin polymerizationneuritic and dendritic branches that are more evident in early kinetics and the density of the subcortical actin networkdevelopmental stages. Dendritic arborization and synaptogenesis, (Anton et al. 2007). Through WASP/N-WASP--dependent orwhich includes generation of postsynaptic dendritic spines, are --independent mechanisms, WIP participates in a wide varietyactin-dependent processes that occur in parallel at later stages. of cellular functions, including signaling, endocytosis, and actinWIP deficiency also increases the amplitude and frequency of cytoskeleton remodeling (Anton et al. 2007). WIP deficiency inminiature excitatory postsynaptic currents, suggesting that WIP2/2 mice alters the immune response, reducing T and mast cellneurons have more mature synapses than wild-type neurons. These activity and increasing B cell function (Anton et al. 2002;findings reveal WIP as a previously unreported regulator of Kettner et al. 2004). Moreover, WIP null mice have a pro-neuronal maturation and synaptic activity. gressive immunological disorder of autoimmune nature, with evident ulcerative colitis, interstitial pneumonitis, glomerularKeywords: dendritic spine, electrophysiology, neuritogenesis, N-WASP, nephropathy with IgA deposits, autoantibodies, and jointsynapse inflammation that lead, all together, to premature death (Curcio et al. 2007). Although molecular details of WIP- WASP/N-WASP inter-action have been studied extensively (Volkman et al. 2002; Ho et al. 2004; Dong et al. 2007; PetersonIntroduction et al. 2007), few data are available on its functional impact andNeuronal cytoarchitecture is first established through neurito- even fewer regarding the central nervous system, where thegenesis, a process in which neurons extend their neurites to role of WIP has not been previously addressed.form a functional network during neuronal development (de Using the WIP knockout mouse as a tool, here, we describeCurtis 2007). Neuron morphology greatly determines the final that loss of this protein impacts neurite and dendrite dynamicscomplexity of the nervous system and is essential for the signal and morphology, both in early and in late developmental stages,flow that underlies information integration and processing. It is in vitro and in vivo. Gross examination of WIP–/– brain revealedtherefore important that neuritogenesis occurs at the right changes in forebrain and hippocampal size. Extensive analysisplace and time for correct establishment of synaptic contacts of WIP–/– hippocampal neuron development showed prematurewith proper targets (de Curtis 2007). Several environmental neuritogenesis. Finally, electrophysiological and immunocyto-cues converge on common coordinated intracellular pathways to chemical analyses demonstrated modified synaptic activity ofmodulate neuritogenesis. Such intracellular events involve sig- WIP–/– mature neurons. These studies show that WIP is annaling transduction, exocytic and endocytic mechanisms related essential negative regulator in the control of the cytoskeletalto membrane trafficking and cytoskeletal rearrangements. events that underlie neuronal and synaptic development. Neurite initiation and outgrowth are based on the capacityof the neuronal cytoskeleton, constituted mainly of actin Materials and Methodsmicrofilaments (MF) and tubulin microtubules (MT), toassemble and disassemble in response to extracellular signals Mice(Luo 2002; Conde and Caceres 2009). The polarized growth of Wild-type (WT) and WIP KO SV129/BL6 mice (Anton et al. 2002) wereneurites requires the initial depolymerization of actin MF housed in specific pathogen-free conditions at the animal facility of the(Bradke and Dotti 1999), stabilization of MT (Ferreira and Centro de Biologı´ a Molecular ‘‘Severo Ochoa,’’ Madrid, Spain. The mouse colony was maintained by continuous mating of heterozygousCaceres 1989), and accumulation of a number of specific females with heterozygous males for more than 20 generations. Toproteins (Wiggin et al. 2005). Actin polymerization is con- obtain control or WIP–/– embryos/litters, we mate control male andtrolled by the actin-related protein (Arp2/3) complex and by female or WIP–/– male and female mice. Handling of mice and allthe action of actin-binding proteins and nucleation-promoting manipulations were carried out in accordance with national andÓ The Author 2011. Published by Oxford University Press. All rights reserved.For permissions, please e-mail: journals.permissions@oup.com
  2. 2. European Community guidelines and were reviewed and approved by a 403 oil-immersion lens (NA, 1.3). Metamorph 6.2r6 (Universalthe institutional committee for animal welfare. All quantification was Imaging) software was used to process the time-lapse captured images.conducted in a genotype-blind manner. Morphometry Image stacks (physical size 76.9 3 76.9 lm) were imported to theBrain Lysates and Western Blot confocal module of Neurolucida 7.1 (MicroBrightfield, Inc., Williston,Control or WIP–/– brains were homogenized in lysis buffer (20 mM 4-(2- VT), and neuronal dendritic trees were traced by drawing the dendriteshydroxyethyl)-1-piperazineethanesulfonic acid, pH 7.4, 100 mM NaCl, 5 and the bifurcation points. Sholl analysis was performed for each tracedmM ethylenediaminetetraacetic acid, 1% Triton X-100, 100 mM NaF, 1 neuron by automatically calculating the number of dendritic intersec-mM Na3VO4, and the Complete Protease Inhibitor Cocktail, Roche tions and the dendritic length at 10-lm interval starting from the soma.Diagnostics), and soluble extracts were resolved by sodium dodecyl Total dendritic length and total number of intersections and branches forsulfate--polyacrylamide gel electrophoresis after determination of each neuron were also calculated as an index of dendritic complexity.protein concentration by Bradford analysis (BioRad). Proteins were Soma area was determined by drawing soma contours while tracing cells.then transferred to nitrocellulose filters, which were blocked andincubated with a mouse monoclonal antibody (mAb) specific to WIP(1/1000, 3D10; a generous gift of Prof. R. Geha, Children’s Hospital, Miniature Excitatory Postsynaptic CurrentsBoston, MA). After exposure to a specific secondary antibody, antibody Miniature excitatory postsynaptic currents (mEPSC) were recordedbinding was visualized by enhanced chemiluminiscence substrate from dissociated hippocampal neurons bathed in artificial cerebro-(Amersham Biosciences). spinal fluid (containing 119 mM NaCl, 2.5 mM KCl, 1 mM NaH2PO4, 11 mM glucose, 26 mM NaHCO3, 2.5 mM CaCl2, and 1.3 mM MgCl2) in the presence of 1 lM tetrodotoxin and 100 lM picrotoxin (at 29 °C).Primary Hippocampal Cultures Spontaneous activity was recorded for 3 min for each cell. mEPSC were Downloaded from cercor.oxfordjournals.org at Universidad Autonoma de Madrid. Facultad de Medicina on August 2, 2011 identified using pClamp software and corrected by eye on the basis ofNeurons their kinetics.Primary hippocampal cultures were prepared as described (Dotti et al.1988; Kaech and Banker 2006). Briefly, hippocampi from E18 mouseembryos (WT and WIP–/–) were washed and digested with 0.25% trypsin Morphology and Unbiased Stereology(15 min, 37 °C). The tissue was then dissociated, resuspended inminimun essential medium with 10% horse serum, and plated on poly- PerfusionL-lysine--coated coverslips (1 mg/ml) at a density of 6 3 103 cells/cm2 Three-month-old male mice (WT, n = 6; WIP–/–, n = 6) werefor imaging at early times (up to 24 h after plating) and at a density of 4 3 anesthetized with pentobarbital (0.04 mg/kg) and perfused trans-103 cells/cm2 for electrophysiological recordings. In some experiments, cardially with PBS (20 ml) followed by 100 ml 4% PFA (pH 7.4) in theneurons were allowed to adhere to the substrate (1 h) and then same buffer. Brains were postfixed in the same fixative (24 h).incubated with 2 or 5 lM wiskostatin (BIOMOL International) indimethyl sulfoxide (DMSO). In all cases, after 3 h, plating medium was Volumetric Analysesreplaced with neurobasal medium supplemented with B27 (Gibco). For Brains were sectioned coronally at a thickness of 50 lm to facilitatelong-term culture, at this time, neuron-including coverslips were measurements. Strict morphological criteria were used in all mice totransferred into dishes containing an astrocyte monolayer, with determine the boundaries of these brain regions. Briefly, hippocampalneurons oriented facing the glia but without contacting them. outlines encompassed the CA1--3 fields of Ammon’s horn and the subiculum but not the presubiculum or fimbria hippocampus. TheDNA Constructs and Transient Transfection dentate gyrus was measured separately. Starting with one of thepLVWIP-GFP was obtained by digestion of pcDNA3WIP-GFP (kindly sections, randomly selected across brains, 1 of every 6 sections wasprovided by Prof. N. Ramesh, Children’s Hospital, Boston, MA) and by analyzed through the extent of a hemisphere of the brain. Onecloning the insert into pLV; control pLVGFP was generated in a similar hemisphere of each brain was analyzed. Using this sampling strategy,manner. Suspended WT (cortical and hippocampal) or WIP–/– (cortical) 7--10 histological sections per brain were analyzed. All volumetricneurons were nucleofected using a 6 lg DNA/100 ll suspension quantifications were performed with an Olympus BX51 with a 1.253(Amaxa pulser; Lonza, Germany). Cells were maintained in suspension objective, a motorized XYZ axis computer-controlled stage (Prior(4 h) to permit exogenous gene expression and then plated for 24 h Scientific, Houston, TX), a digital video camera (JVC), and Stereo-before fixation for immunofluorescence analysis. Investigator, a stereology software package (version 8.03, MicroBright- Field). When calculating hippocampus volume, the boundaries wereImmunofluorescence defined and the volumes determined with Stereo-Investigator software.At 3 h, 1 and 22 DIV (days in vitro) postplating, cells were fixed in 4%paraformaldehyde (PFA) in phosphate-buffered saline (PBS) (pH 7.4; 20 Intracellular Injection of Lucifer Yellowmin, room temperature). Cells were then permeablilized with 0.1% Coronal sections (150 lm) were cut on a vibratome. Sections wereTriton X-100 and labeled with phalloidin-tetramethylrhodamine iso- prelabeled with 4,6-diamidino-2-phenylindole, and a continuous cur-thiocyanate (Sigma) and antibodies to tyrosinated a-tubulin (1/400; rent used to inject individual cells with lucifer yellow (8% in 0.1 M TrisT9028, Sigma), MAP2 (MT-associated protein, 1/400; 514, Sanchez buffer, pH 7.4). Dye was injected into neurons in the dentate gyrus untilMartin et al. 1998), PSD-95 (5 lg/ml; 75-028, NeuroMab), or GFP (1/ the individual dendrites of each cell could be traced to an abrupt end at100, 11814460; Roche); secondary fluo-rescent antibody or biotinylated the distal tips and the dendritic spines were readily visible, indicatingantibody and labeled streptavidin were then added. Cells were that the dendrites were completely filled. The sections were thenexcluded from analysis if they showed obvious features of toxicity, processed, first with rabbit anti-lucifer yellow (Cajal Institute,such as neurite fragmentation/blebbing or vacuoles in the cell body. 1/400 000 in stock solution of 2% Bovine serum albumin, 1% Triton X-100, 5% sucrose in PBS) and then with Alexa 488-conjugatedImaging secondary antibody (Molecular Probes, 1/1000, 4 h). Sections wereConfocal images were acquired digitally on a confocal LSM510 Meta mounted on a glass slide in fresh ProLong Gold antifade reagentmicroscope (Zeiss) coupled to an inverted Axiovert 200 microscope (Invitrogen, Eugene, OR; 24 h, room temperature in the dark) and(Zeiss). Image stacks (logical size 1024 3 1024 pixels) consisted of 10 sealed with nail polish.image planes acquired through a 403 (numerical aperture (NA), 1.3) ora 633 oil-immersion lens (NA 1.4). Confocal Microscopy Time-lapse images of phase-contrast fields were captured on an Imaging was performed on a Leica laser scanning multispectralinverted Axiovert 200 microscope (Zeiss) equipped with a mono- confocal microscope (TCS SP5) using an argon laser. Image stackschrome CCD camera and ultrafast filter change. Image stacks (logical (physical size 76.9 3 76.9 lm, logical size 1024 3 1024 pixels) consistedsize 1024 3 1024 pixels) consisted of 6 image planes acquired through of 100--350 image planes acquired through a 633 glycerol-immersionPage 2 of 12 WIP Regulates Neuronal Maturation d Franco et al.
  3. 3. lens (NA, 1.3; working distance, 280 lm; refraction index, 1.45) with murine brain and corroborate previous mRNA studies (Ramesha calculated optimal zoom factor of 3.2 and a z-step of 0.14 lm (voxel et al. 1997; Tsuboi 2006) as well as the data included in the Allensize, 75.1 3 75.1 3 136.4 nm). These settings and optics represent the atlas (http://www.brain-map.org/).highest resolution currently possible with confocal microscopy. Foreach neuron (5 neurons per mouse, 55 neurons total), 1--5 randomlyselected dendrites were scanned from soma to tip, and stacks were Brain Hypertrophy in WIP-Deficient Miceprocessed with a 3D blind deconvolution algorithm (Autodeblur; To assess general effects of WIP deficiency on the mouse brain,Autoquant, Media Cybernetics) for 10 iterations to reduce the out-of- we determined volumes of the hippocampus and of the rest offocus light. the murine forebrain (3-month-old male mice). Contours of the structures of interest were drawn on Nissl-stained serial sec-Statistical Analysis tions with the aid of Stereo-Investigator software to yield theQuantification was performed in a blind fashion by independent volume of each. There was a significant increase (30%) inresearchers. For statistical analyses, we used the two-tailed Student t- forebrain volume in WIP-deficient brains (n = 5) comparedtest to compare means, the chi-square test to compare nominal with WT littermates (n = 6, Fig. 1B--D and Supplementary Fig.variables, and the two-way analysis of variance to compare Sholl analysis 1). The hippocampus proper (without the dentate gyrus) andor the Kolmogorov--Smirnov test to compare cumulative frequencyanalysis. Results are shown as mean ± standard error of the mean. the dentate gyrus itself were also hypertrophied in WIP–/– brain (Fig. 1E,F). WIP deficiency thus causes general macrocephaly that includes hippocampal hypertrophy.Results Downloaded from cercor.oxfordjournals.org at Universidad Autonoma de Madrid. Facultad de Medicina on August 2, 2011 Enhanced Early Neuronal Development in WIP –/– NeuronsWIP Is Expressed in the WT Mouse Brain The enlarged brain observed in WIP–/– mice relative to their WTThe expression pattern of WIP messenger RNA (mRNA) suggests littermates raised the possibility that the greater brain volumethat WIP is ubiquitously expressed in all mouse tissues, including could be due to increased neuritic branching since dendritesbrain (Ramesh et al. 1997; Tsuboi 2006). To date, however, the and axon collaterals account for most of the brain volumelevels of endogenous WIP protein have only been analyzed in (Acebes and Ferrus 2000). To test this hypothesis at the cellular ´fibroblasts, myoblasts, and hematopoietic-derived cells (reviewed level, we examined the effect of WIP deficiency on earlyin Anton et al. 2007). To study WIP expression in brain, we probed neuronal development. Primary hippocampal neurons fromwestern blots of cell lysates from 3 regions of the adult mouse brain control and WIP–/– embryos were grown at very low density so(cortex, hippocampus, and olfactory bulb), using the WIP-specific that their neurite arbors did not overlap to avoid confusionmAb 3D10 (Koduru et al. 2007). This mAb recognized 2 protein about which neurites protruded from each cell body. Cellsbands in cell lysates from each of the regions examined (Fig. 1A), were labeled with anti-tyrosinated a-tubulin and fluorescentwhich could represent 2 WIP isoforms and/or be the result of phalloidin (Fig. 2A,B). As a measure of neuronal development,posttranslational modifications of WIP. Neither band was present we quantified the fraction of neurons at each developmentalin WIP-deficient tissue. These data demonstrate WIP expression in stage at 3 h postplating. At stage 1, the MT-containing soma isFigure 1. Brain hypertrophy in WIP-deficient mice. (A) Western blot indicates WIP expression in WT mouse brain (cortex, hippocampus, and olfactory bulb; 80 lg total protein/lane) but not in WIPÀ/À mouse brain. Glyceraldehyde-3-phosphate dehydrogenase labeling confirmed equivalent protein loading in cortical samples. (B) Representative WT (left)and WIPÀ/À brain (right) at 3 months of age. Note: the enlargement of the WIPÀ/À brain. Scale bar, 4 mm. (C) Representative Nissl staining showing hippocampus enlargementin WIPÀ/À mice. Scale bar, 500 lm. (D--F) Average volumes of forebrain (D), hippocampus (E), and dentate gyrus (F) are increased in WIPÀ/À mice. P values were determinedwith Student’s t-test; *P 0.05; **P 0.01. Cerebral Cortex Page 3 of 12
  4. 4. Downloaded from cercor.oxfordjournals.org at Universidad Autonoma de Madrid. Facultad de Medicina on August 2, 2011Figure 2. WIP deficiency accelerates development of dissociated hippocampal neurons. (A) Representative confocal images of WT- and WIPÀ/À-dissociated hippocampalneurons fixed at 3 h postplating and stained for F-actin (phalloidin-tetramethylrhodamine isothiocyanate, red) and MT (Alexa488-anti-tyrosinated tubulin [TT], green). At this timepoint, the WIPÀ/À neuron population showed more advanced development compared with WT neurons. Scale bar, 10 lm. (B) High magnification images of the inset in A. Scalebar, 5 lm. (C) Neuron classification by developmental stage (3 h postplating) showed significantly lower frequency of WIPÀ/À at developmental stage 1 compared with WTPage 4 of 12 WIP Regulates Neuronal Maturation d Franco et al.
  5. 5. surrounded by flattened actin-rich lamellipodia, while in stage (pLVGFP; Fig. 3C). At 24 h postplating, while 31% of GFP-2, the lamellipodia are transformed into equidistant neurites expressing neurons were in stage 1, the frequency of stage 1(Dotti et al. 1988). Stage 1+ is defined as cells with incipient WIP-GFP--expressing neurons was 80% (Fig. 3D). The de-tubulin-rich small protrusions. We found a clear difference in velopmental delay at 24 h induced by WIP overexpression wasthe distribution of these populations (Fig. 2C); there was confirmed in cortical embryonic neurons (Supplementary Fig.a significant decrease (35%) in the frequency of WIP–/– 2). This effect was sustained over time, since 48 h after plating,neurons classified as developmental stage 1 compared with 22.6% of neurons with increased WIP levels remained at stage 1WT neurons (n = 4 cultures and n = 150 neurons/genotype, compared with none of the GFP-expressing neurons.Fig. 2C). Conversely, there was an increase (45%) in the We used rescue experiments to confirm the WIP contributionpercentage of WIP-deficient neurons at stage 2. As another to neuron development. WIP–/– neurons were nucleofected withmeasure of neuronal development, we quantified the number a lentiviral-based vector coding for WIP-GFP or control GFP, andof neurites arising from each neuron at 3 h postplating and 24 h after plating, the fraction of cells in stage 1 or 2 wasfound a significant increase (36%) in the average number of quantified (Fig. 3E,F; WIP-GFP--expressing WIP–/– neurons). Theneurites arising from WIP–/– (n = 172) compared with WT population distribution of rescued neurons was indistinguishableneurons (n = 151, Fig. 2D). This finding was confirmed in time- from that of WT neurons and differed significantly from that oflapse images made with phase-contrast microscopy for 3 h GFP-expressing WIP–/– neurons. WIP-GFP expression in WIP –/–after plating (Fig. 2E and Supplementary Videos 1 and 2). neurons also reversed the phenotype, as the mean number ofThese data suggest that, at 3 h after plating, WIP–/– neurons are primary neurites (Fig. 3G and Supplementary Fig. 3) and neuritic Downloaded from cercor.oxfordjournals.org at Universidad Autonoma de Madrid. Facultad de Medicina on August 2, 2011in a more advanced developmental stage than WT neurons. bifurcations (Supplementary Fig. 3) were similar to those of GFP-This implies enhanced early neuronal development in the positive WT neurons.absence of WIP. These findings indicate that WIP acts as a negative regulator of neuronal differentiation and that neuronal development can be modulated bidirectionally by WIP levels.N-WASP Activity and Fine-Tuned WIP Levels Regulate NeuronalDevelopmentTo test whether this accelerated neuronal development Enhanced Dendritic Maturation in WIP –/– Neuronscontinues at later developmental times, we quantified the To test whether the WIP regulatory role persists in differen-fraction of neurons that remains at developmental stage 1 at 24 h tiated neurons, we also imaged cultured hippocampal neuronspostplating. Cultured hippocampal neurons were fixed and at 22 DIV (Fig. 4A). For quantitative analysis of the branchinglabeled with anti-tyrosinated a-tubulin antibody and fluores- pattern of the neuritic or dendritic tree at the distinct neu-cent phalloidin and imaged by confocal microscopy. As ronal developmental stages, we traced neurons (1 and 22 DIV)observed at 3 h postplating, we found a decrease of 48% in with Neurolucida software. Sholl analysis was used to measurethe frequency of WIP–/– neurons at developmental stage 1 the number of neurites/dendrites crossing circles at various(Fig. 3A left and Fig. 3B), implying that WIP–/– neurons still radial distances from the soma (Sholl 1953). Both neuronalshow accelerated development at this time. types showed addition of dendritic branches over time (Fig. To determine whether this phenotype depends on the 4B,D), although the number of crossings was significantlyactivity of N-WASP, an actin NPF with WIP-binding capacity, higher in WIP–/– neurons compared with WT neurons at bothwe incubated cultures (23 h) with wiskostatin, a selective time points. This finding implies that WIP–/– neurons showreversible N-WASP inhibitor (Peterson et al. 2004). Wiskosta- higher ramification of their neuritic (1 DIV) and dendritictin is a cell-permeable N-alkylated carbazole derivative that arbor (22 DIV, Fig. 4D). Nevertheless, the difference in theselectively blocks actin filament assembly by binding to N- number of crossings between phenotypes, which was 82% atWASP, stabilizing the autoinhibited conformation, and pre- 1 DIV, decreased to 45% at 22 DIV (Supplementary Fig. 4A).venting activation of the Arp2/3 complex (Wegner et al. The increased ramification was reflected in greater total2008). Wiskostatin produced a dose-dependent increase in neuritic or dendritic length of WIP–/– neurons at both time pointsthe fraction of stage 1 neurons, indicating impairment in (Fig. 4C,E), a difference that was also reduced in mature neuronsneuronal development after blockade of N-WASP-dependent (from 109% to 40%; Supplementary Fig. 4B). This difference wasfilament assembly (Fig. 3A,B) (da Silva and Dotti 2002; even smaller in neurons of adult mice (see below).Dent et al. 2007). Inhibition of N-WASP activity with 2 lM Soma size determines the number of dendrites and associatedwiskostatin induced a phenotype in WIP–/– neurons resem- branches in neurons (Kernell and Zwaagstra 1989; Kollins andbling that of untreated control neurons, and with 5 lM Davenport 2005). To analyze whether WIP deficiency alterswiskostatin, WIP–/– neurons no longer showed a lower soma area, we quantified this parameter in WT and WIP–/–frequency of stage 1 neurons (Fig. 3A,B). This finding suggests neurons. Similar to our observations for the dendritic arbor,that N-WASP activity is essential in mediating the accelerated soma area was significantly larger in WIP–/– neurons (Fig. 4F,G),development of WIP–/– neurons. but this difference attenuated from 40% to 27% with time To further evaluate the role of WIP in neurite sprouting, WT (Supplementary Fig. 4C). The effect of WIP deficiency onneurons were nucleofected with lentiviral constructs for over- promoting neuritic or dendritic arborization and on soma size isexpression of WIP-GFP (pLVWIP-GFP) or GFP as control thus greater in early developmental stages.neurons, implying an average more advanced developmental stage for WIPÀ/À neurons. Chi-square test, *P 5 0.0194. (D) The average number of primary neurites arising fromWIPÀ/À somas was significantly larger compared with WT neurons (3 h postplating), again indicating the more advanced developmental stage of WIPÀ/À neurons at this time.Data derived from quantification of 150 neurons in each experimental group (from 4 separate experiments). Student’s t-test; ***P 0.001. (E) Selected images from a phase-contrast time-lapse series of control (WT; Supplementary Video1) and WIP-deficient (WIPÀ/À; Supplementary Video2) hippocampal embryonic neurons cultured on poly-L-lysineover a 3-h period. Cerebral Cortex Page 5 of 12
  6. 6. Downloaded from cercor.oxfordjournals.org at Universidad Autonoma de Madrid. Facultad de Medicina on August 2, 2011Figure 3. Inhibition of N-WASP prevents WIPÀ/À phenotype, WIP overexpression delays neurite protrusion, and WIP reexpression reverts the phenotype. (A) Representative imagesof dissociated WT and WIPÀ/À hippocampal neurons, alone or wiskostatin-treated, fixed at 24 h postplating and stained for F-actin (phalloidin-tetramethylrhodamine isothiocyanate,red) and tyrosinated MT (tyrosinated tubulin [TT], green). As at 3 h postplating (Fig. 2), a typical 24-h WIPÀ/À neuron showed more advanced development than a WT neuron. Scalebar, 10 lm. (B) Percentage of cells at developmental stage 1, alone or wiskostatin-treated. Cells were dissociated and cultured without additives (control), with DMSO, or withwiskostatin (2 or 5 lM). Wiskostatin (2 lM) caused a significant increase in the frequency of WIPÀ/À neurons at stage 1, equivalent to the frequency for untreated WT neurons. Ahigher wiskostatin concentration (5 lM) prevented differentiation of both WT and WIPÀ/À neurons. Student’s t-test; *P 0.05; **P 0.01. (C) Images of phalloidin-stained (red)hippocampal neurons expressing GFP (left) or GFP-WIP (right) at 24 h postplating. (D) The percentage of cells at developmental stage 1 was greater in WIP-overexpressing cells,implying that WIP overexpression delays neurite formation. Student’s t-test; *P 0.05. (E) Representative images of cortical control WT neurons nucleofected to express GFP and ofWIPÀ/À neurons nucleofected to express GFP or WIP-GFP. Cortical neurons were fixed at 24 h postplating and stained for F-actin (phalloidin-tetramethylrhodamine isothiocyanate, red)and anti-GFP (green). A typical WIPÀ/À neuron--expressing WIP-GFP showed similar development to that of a WT neuron. Scale bar, 10 lm. (F) Percentage of cells at developmentalstage 1 after WIP reexpression. The fraction of cells at stage 1 was quantified at 24 h postplating. Distribution of rescued WIPÀ/À neurons was similar to that of WT neurons,confirming the role of WIP in neuronal development. Student’s t-test; **P 0.01. (G) Mean number of primary neurites per GFP-expressing WT neuron is similar to that of WIP-GFP--expressing WIPÀ/À neurons and differs significantly from that of WIPÀ/ÀGFP-expressing neurons. Student’s t-test; ***P 0.001; ns, not significant.Enhanced Neuronal Ramification in Adult WIP –/– Mice (Fig. 5A) and studied the structure of granule cells in theTo test whether WIP deficiency produces a long-lasting effect dentate gyrus. Sholl analysis showed significantly more cross-on neuronal maturation in vivo, we evaluated dendritic archi- ings in the distal part of the dendrite in WIP–/– mice (n = 59tecture in adult (3-month-old) mice after injection of lucifer neurons from 6 mice) compared with WT mice (n = 39yellow into hippocampal neurons of fixed brain sections neurons from 5 mice, Fig. 5B). This increased number ofPage 6 of 12 WIP Regulates Neuronal Maturation d Franco et al.
  7. 7. Downloaded from cercor.oxfordjournals.org at Universidad Autonoma de Madrid. Facultad de Medicina on August 2, 2011Figure 4. Increased ramification in WIPÀ/À neurons in vitro. (A) Representative images of dissociated WT and WIPÀ/À hippocampal neurons fixed at 1 and 22 DIV and stainedwith anti-tyrosinated-a-tubulin (1 DIV) or anti-MAP2 (22 DIV) antibodies. Note the greater ramification of WIPÀ/À neurons at both 1 and 22 DIV. (B and C) Sholl analysis of tracedWT and WIPÀ/À neurons showed more ramification in WIPÀ/À neurons (B), reflected as greater total neuritic or dendritic length for these cells (C). Two-way analysis of variance;**P 0.01; ***P 0.001. (D) Total number of crossings at 1 and 22 DIV. Note the increase with time in the total number of crossings for both genotypes. (E) Total neuriticor dendritic length at 1 and 22 DIV. Note the increase over time in the total dendritic length for both genotypes. (F) Representative images of WT and WIPÀ/À somasdemonstrating increased soma surface in WIPÀ/À neurons. (G) WIPÀ/À neuronal somas are larger than WT neuronal somas at 1 and 22 DIV. (D, E, and G) Student’s t-test;**P 0.01; ***P 0.001. Cerebral Cortex Page 7 of 12
  8. 8. crossings reflects a larger number of ramifications at the outer neuronal development. We found that, in hippocampal primarymolecular layer of the dentate gyrus. As a result of the higher neurons, loss of WIP accelerates the onset of neuritogenesis anddendritic ramification, the dendritic length in this layer is also increases neuritic and dendritic branching. Moreover, lack ofincreased in WIP–/– mice (Fig. 5C ), leading to greater total WIP enhances PSD-95 accumulation in hippocampal dendriticdendritic length (18%) in WIP–/– mice (Fig. 5D). Cumulative spines and increases neuronal synaptic activity. Conversely, WIPfrequency analysis indicated that WIP–/– mice have a sub- overexpression blocks the initiation of neuritogenesis, suggestingpopulation of highly ramified neurons not detected in WT that WIP is a bidirectional regulator of neuronal development.mice (Fig. 5E ). WIP deficiency thus leads to enhanced WIP Modulates Neuritic Branching During Neuronaldendritic ramification in the adult mouse. Nonetheless, the Developmentmagnitude of change in total dendritic length of the adult Marked dendritic elaboration normally begins in neuronalWIP–/– mouse is smaller than in early developmental stages. cultures 2--3 days after axonal outgrowth (Nowakowski and Rakic 1979; Dotti et al. 1988). Our experiments showed that immatureEnhanced Synaptic Maturation in WIP –/– Neurons WIP–/– neurons begin to emit neurites starting at the first day inDuring later stages of neuronal development, dendritic culture, much earlier than control neurons. After cell cycle exitarborization and synaptogenesis occur in parallel (Cantallops and before neuronal polarization, cortical postmitotic neuronset al. 2000; Cline 2001). The actin cytoskeleton has a pivotal make a transition through a multipolar stage, when multiplerole in both processes. In the context of synaptogenesis, actin neurites emerge rapidly from the cell body (Barnes and Polleuxdynamics control the morphogenesis and function of the den- 2009). This morphological transition is also controlled by WIP Downloaded from cercor.oxfordjournals.org at Universidad Autonoma de Madrid. Facultad de Medicina on August 2, 2011dritic spines, small actin-rich protrusions from dendritic shafts during brain development, as GFP in utero electroporation shows(Ethell and Pasquale 2005). As we found that WIP modulates a notable increase in the percentage of multipolar cells in WIP–/–dendritic arborization, we examined the possibility that WIP embryos, in parallel to a reduction in the fraction of round anddeficiency fosters synaptic maturation. We measured the effects unipolar neurons (not shown). In addition, neurites in immatureof WIP deficiency on spontaneous miniature (action-potential WIP–/– neurons are more branched than neurites of WT neurons.independent) postsynaptic currents (mEPSC) in dissociated Dendritic branching is also increased in WIP–/– mature neurons,hippocampal neurons (22 DIV) in the presence of 1 lM although to a lesser extent than at early neuronal developmentaltetrodotoxin (Fig. 6A). Both the amplitude and the frequency stages (Fig. 4 and Supplementary Fig. 4). Sholl analysis demon-of mEPSC were significantly increased in WIP–/– compared with strated that the initial morphological features acquired byWT neurons (n = 17 and 15 neurons, respectively; Fig. 6B,C ). immature WIP–/– neurons determine the pattern of dendriticmEPSC amplitude is indicative of the postsynaptic strength of branching of the more differentiated cells. We therefore suggestindividual functional synapses, whereas mEPSC frequency that WIP acts as a negative modulator that controls the precisedepends on synapse number and presynaptic properties timing of the correct onset of dendritic development. Our results(Turrigiano et al. 1998; Han and Stevens 2009). WIP deficiency point to the possibility that some pathologies detected in maturefunctionally increases both the strength and possibly the neurons might have their origin, yet unknown, in alterednumber of individual synapses, suggesting that at this time, processes that occur during early neuronal development.WIP –/– neurons show either a more mature phenotype or moreabundant and/or enlarged dendritic spines. WIP versus Other Negative Regulators of Dendritic To examine whether WIP deficiency resulted in an increased Branchingnumber of structural synapses, we stained dissociated hippo- Numerous molecules are involved in the positive control ofcampal neurons at the same developmental stage (22 DIV) for the neuritic or dendritic outgrowth and branching, many of thempostsynaptic marker PSD-95, known to drive maturation of related to the control of cytoskeleton dynamics (McAllister 2000;glutamatergic synapses (El-Husseini et al. 2000). WIP–/– neurons Urbanska et al. 2008). Only a minority of these molecules, such asshowed increased fluorescence intensity of individual PSD-95 PTEN (Kwon et al. 2001; Jaworski et al. 2005) and RhoA (Negishipuncta, indicating greater PSD-95 accumulation in spines in the and Katoh 2002; da Silva et al. 2003), have been identified asabsence of WIP (WT, n = 117 spines from 6 neurons; WIP–/–, n = negative regulators of these processes. Similarly to our findings,168 spines from 7 neurons, Fig. 6D,E ). Frequency analysis conditional adult Pten mutant mice show enlargement of cortexshowed that in WIP–/– neurons, staining of some PSD-95--positive and hippocampus associated with dendritic hypertrophy andspines is particularly intense, whereas such a population was not increased soma size (Kwon et al. 2001, 2006). At difference fromfound in WT neurons (Fig. 6F ). Positive spine density was the WIP–/– phenotype observed here, PTEN is not necessary fornonetheless similar in WIP–/– and WT neurons (Fig. 6G ). As WIP–/– initiation of neuritogenesis (Lachyankar et al. 2000). The mostneurons are substantially more branched than WT neurons (and striking difference between WIP- and PTEN-mediated negativetheir total dendritic length is therefore increased), the total regulation thus appears to depend on the temporal window ofnumber of PSD-95--positive spines is significantly increased in neuronal development in which both proteins operate. PTENWIP–/– neurons (Fig. 6H). These neuronal cultures were does not control early stages of neuritogenesis, and its influencemaintained over WT astrocytes, excluding possible astrocyte on neuronal structure increases progressively throughout theinvolvement in the synaptic modulation derived from the lack of animal’s life. In contrast, WIP modulates neuronal developmentWIP. These results indicate that WIP deficiency increases the and synaptogenesis early in postnatal life, and its effects arenumber and size of structural and functional synapses. attenuated as neuronal maturation progresses. Similarly to the absence of WIP, RhoA inhibition increases both the number of primary neurites and total neuritic lengthDiscussion in immature dissociated hippocampal neurons, whereas ex-Here, we present the first report of a role for WIP in the brain, pression of a constitutively active form of RhoA arrests cells inshowing a specific role for WIP as a negative regulator of murine stage 1 (da Silva et al. 2003). In mature neurons, transfection ofPage 8 of 12 WIP Regulates Neuronal Maturation d Franco et al.
  9. 9. Downloaded from cercor.oxfordjournals.org at Universidad Autonoma de Madrid. Facultad de Medicina on August 2, 2011Figure 5. Increased ramification in WIPÀ/À neurons in vivo. (A) Representative projection images of lucifer yellow--injected granule neurons of 3-month-old WT and WIPÀ/Àmice. Scale bar, 25 lm. (B and C) Sholl analysis of traced WT and WIPÀ/À granule neurons showed greater ramification in the distal part of WIPÀ/À neurons (B). This is reflectedas greater dendritic length for these neurons (two-way analysis of variance; *P 0.05) (C). (D) Total dendritic length is greater in neurons from WIPÀ/À mice. Student’s t-test;*P 0.05. (E) Cumulative frequency curves of total dendritic length indicating a shift toward higher values in neurons from WIPÀ/À mice. Kolmogorov--Smirnov test; *P 0.05.a dominant-negative RhoA construct does not affect dendritic Possible Mechanisms for WIP-Mediated Neurite Sproutingmorphology (Nakayama et al. 2000), whereas WIP–/– neurons and Branchingstill show enhanced dendritic arborization. The effect of WIP Sprouting of primary neurites and interstitial branching fromon neuronal maturation thus appears to be longer lasting than neuritic or dendritic shafts follows the same sequence asthat of other negative modulators of neuritic and dendritic cortical cytoskeletal rearrangements (Wu et al. 1999; Dent et al.outgrowth and branching. 2007). For the extension of a single filopodium, which is Cerebral Cortex Page 9 of 12
  10. 10. Downloaded from cercor.oxfordjournals.org at Universidad Autonoma de Madrid. Facultad de Medicina on August 2, 2011Figure 6. WIP deficiency increases the strength and the number of individual synapses. (A) Representative trace (calibration: 20 pA, 500 ms) and event (calibration: 10 pA, 2ms) of mEPSC recorded from a WT and a WIPÀ/À neuron. (B and C) Cumulative frequency representation of the amplitude (B) and frequency (C) of mEPSC, showing significantshifts to higher values in WIPÀ/À neurons. Kolmogorov--Smirnov test; ***P 0.001. (D) Representative projection confocal images of WT and WIPÀ/À dendrites, stained withanti-MAP2 (blue) and -PSD-95 antibodies (green). Note that there are intense dendritic spines in WIPÀ/À but not in WT dendrites. (E) Average intensity of PSD-95--positive spinesin WT and WIPÀ/À neurons. Student’s t-test; ***P 0.001. (F) Cumulative frequency representation of the intensity of PSD-95--positive spines. Observe the subpopulation ofparticularly intense PSD-95--positive spines in WIPÀ/À but not in WT neurons. Kolmogorov--Smirnov test; ***P 0.001. (G) Sholl analysis of PSD-95--positive spine density in WTand WIPÀ/À neurons. (H) The number of PSD-95--positive spines was calculated as a function of the distance from soma (Sholl analysis) by multiplying spine density for eachdistance by total dendritic length at the same distance. Two-way analysis of variance; ***P 0.001.subsequently stabilized by MT invasion, F-actin and MT must Our data indicate that WIP modulates neuritogenesisundergo local depolymerization triggered by extracellular signals through its previously described ability to maintain N-WASP(Acebes and Ferrus 2000; da Silva and Dotti 2002; Luo 2002). ´ in its autoinhibited state (Martinez-Quiles et al. 2001). N-WASPWIP inhibits F-actin depolymerization (Martinez-Quiles et al. promotes neurite outgrowth and regulates neurite branching2001), which could explain the neuritogenesis arrest found in in hippocampal neurons (Suetsugu, Hattori, et al. 2002; AbeWIP-overexpressing neurons. et al. 2003; Pinyol et al. 2007). At the molecular level, N-WASPPage 10 of 12 WIP Regulates Neuronal Maturation d Franco et al.
  11. 11. acts as a signal integration device that can precisely target actin Daniel Gallego for electrophysiological signal processing, Jose Ramon ´ ´polymerization to membrane sites at which PI(4,5)P2 and Valverde for statistical assistance and Catherine Mark for editorialactivated Src kinases and Cdc42 are located (Prehoda et al. assistance. Conflict of Interest : None declared.2000; Suetsugu, Miki, et al. 2002). Cdc42 stimulates the actin-polymerizing activity of N-WASP, creating free barbed ends Referencesfrom which actin polymerization can take place (Miki,Suetsugu, et al. 1998), leading to filopodium formation (Miki, Abe T, Kato M, Miki H, Takenawa T, Endo T. 2003. Small GTPase Tc10 and its homologue RhoT induce N-WASP-mediated long processSasaki, et al. 1998). In the absence of WIP, N-WASP would be formation and neurite outgrowth. J Cell Sci. 116:155--168.more easily released from inhibition and be hyperactivated to Acebes A, Ferrus A. 2000. Cellular and molecular features of axon ´initiate premature filopodium formation. This interpretation is collaterals and dendrites. Trends Neurosci. 23:557--565.supported by pharmacological inhibition of N-WASP by wiskos- Anton IM, de la Fuente MA, Sims TN, Freeman S, Ramesh N, Hartwig JH,tatin, which blocked the accelerated neuritogenesis seen in WIP- Dustin ML, Geha RS. 2002. WIP deficiency reveals a differential roledeficient neurons (Fig. 3). for WIP and the actin cytoskeleton in T and B cell activation. Immunity. 16:193--204. Anton IM, Jones GE, Wandosell F, Geha R, Ramesh N. 2007. WASP-WIP Modulates Synaptic Activity interacting protein (WIP): working in polymerisation and muchWe report here that mEPSC amplitude and frequency are more. Trends Cell Biol. 17:555--562.increased in WIP–/– neurons, suggesting both stronger AMPA Barnes AP, Polleux F. 2009. Establishment of axon-dendrite polarity in(alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) developing neurons. Annu Rev Neurosci. 32:347--381. Downloaded from cercor.oxfordjournals.org at Universidad Autonoma de Madrid. Facultad de Medicina on August 2, 2011receptor--mediated synaptic currents and more functional Bradke F, Dotti CG. 1999. The role of local actin instability in axonsynapses in these cells (Fig. 6). In addition, we describe an formation. Science. 283:1931--1934.increase in PSD-95 accumulation at individual spines in WIP–/– Cantallops I, Haas K, Cline HT. 2000. Postsynaptic CPG15 promotes synaptic maturation and presynaptic axon arbor elaboration in vivo.neurons. These 2 events are probably linked since PSD-95 Nat Neurosci. 3:1004--1011.drives the maturation of excitatory synapses with the in- Cline HT. 2001. Dendritic arbor development and synaptogenesis. Currcorporation of AMPA receptors (El-Husseini et al. 2000). Opin Neurobiol. 11:118--126.Indeed, the contribution of AMPA receptors to synaptic Conde C, Caceres A. 2009. Microtubule assembly, organization andtransmission increases gradually with neuronal maturation dynamics in axons and dendrites. Nat Rev Neurosci. 10:319--332.(Mammen et al. 1997; Petralia et al. 1999). The increase in Curcio C, Pannellini T, Lanzardo S, Forni G, Musiani P, Anton IM. 2007.PSD-95 accumulation at individual WIP–/– spines might suggest WIP null mice display a progressive immunological disorder that resembles Wiskott-Aldrich Syndrome. J Pathol. 211:67--75.that the PSD (and therefore the synapse) is enlarged in the da Silva JS, Dotti CG. 2002. Breaking the neuronal sphere: regulation ofabsence of WIP. Given that the synaptic area correlates the actin cytoskeleton in neuritogenesis. Nat Rev Neurosci.positively with the number of synaptic AMPA receptors (Nusser 3:694--704.et al. 1998), our electrophysiological findings are strengthened da Silva JS, Medina M, Zuliani C, Di Nardo A, Witke W, Dotti CG. 2003.by the morphological analysis of PSD-95 in spines. RhoA/ROCK regulation of neuritogenesis via profilin IIa-mediated In conclusion, this study sheds light on the molecular events control of actin stability. J Cell Biol. 162:1267--1279.that control early neuronal development by presenting WIP as de Curtis I. 2007. Intracellular mechanisms of neuritogenesis. New York: Springer.a previously undescribed regulator that prevents premature Dent EW, Kwiatkowski AV, Mebane LM, Philippar U, Barzik M,dendritic and synaptic maturation. Rubinson DA, Gupton S, Van Veen JE, Furman C, Zhang J, et al. 2007. Filopodia are required for cortical neurite initiation. Nat Cell Biol. 9:1347--1359.Supplementary Material Dong X, Patino-Lopez G, Candotti F, Shaw S. 2007. Structure-functionSupplementary material can be found at: http://www.cercor. analysis of the WIP role in T cell receptor-stimulated NFAToxfordjournals.org/. activation: evidence that WIP-WASP dissociation is not required and that the WIP NH2 terminus is inhibitory. J Biol Chem. 282:30303--30310.Funding Dotti CG, Sullivan CA, Banker GA. 1988. The establishment of polarityGrants from Consejo Superior de Investigaciones Cientı´ ficas- by hippocampal neurons in culture. J Neurosci. 8:1454--1468.Comunidad de Madrid (CCG08-CSIC/SAL-3471), CSIC (PIE2- El-Husseini AE, Schnell E, Chetkovich DM, Nicoll RA, Bredt DS. 2000.00720I002), and the Spanish Ministry of Education and Science PSD-95 involvement in maturation of excitatory synapses. Science. 290:1364--1368.(BFU2007-64144 and BFU2010-21374) to I.M.A., from Centro de Ethell IM, Pasquale EB. 2005. Molecular mechanisms of dendritic spineInvestigacion Biomedica en Red Enfermedades Neurodegener- ´ ´ development and remodeling. Prog Neurobiol. 75:161--205.ativas (Instituto de Salud Carlos III), the Plan Nacional DGCYT Ferreira A, Caceres A. 1989. The expression of acetylated microtubules(SAF2009-12249-C02-01) to F.W. and SAF2010-15676 to S.K., during axonal and dendritic growth in cerebellar macroneuronsand by an institutional grant from the Fundacion Ramon Areces. ´ ´ which develop in vitro. Brain Res Dev Brain Res. 49:205--213.A.F. was a recipient of an FPU MEC fellowship (AP2005-3405), Han EB, Stevens CF. 2009. Development regulates a switch betweenI.B. held a contract from the Comunidad Autonoma de Madrid ´ post- and presynaptic strengthening in response to activity deprivation. Proc Natl Acad Sci U S A. 106:10817--10822.and S.K., a Ramon y Cajal contract. ´ Ho HY, Rohatgi R, Lebensohn AM, Le M, Li J, Gygi SP, Kirschner MW. 2004. Toca-1 mediates Cdc42-dependent actin nucleation byNotes activating the N-WASP-WIP complex. Cell. 118:203--216.We thank Chiara Ragazzini and Sonia Perez for their excellent technical ´ Jaworski J, Spangler S, Seeburg DP, Hoogenraad CC, Sheng M. 2005. Controlassistance and Javier de Felipe for his contribution to the morphomet- of dendritic arborization by the phosphoinositide-3’-kinase-Akt-ric analysis with NeuroLucida Software. We are grateful to Raif Geha mammalian target of rapamycin pathway. J Neurosci. 25:11300--11312.and Narayanaswamy Ramesh for the 3D10 mAb and to Lola Ledesma Kaech S, Banker G. 2006. Culturing hippocampal neurons. Nat Protoc.and Carlos Dotti for helpful advice on the manuscript. We acknowledge 1:2406--2415. Cerebral Cortex Page 11 of 12
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