cer cor11 franco-cercor-bhr199

Cerebral Cortex

doi:10.1093/cercor/bhr199

WIP Is a Negative Regulator of Neuronal Maturation and Synaptic Activity

A. 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. Anton1,4

1Centro Nacional de Biotecnologıa (CNB-CSIC), 28049 Madrid, Spain, 2Instituto Cajal (CSIC), 28002 Madrid, Spain, 3Centro de

Biologıa 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 work

Address correspondence to Dr Ines M. Anton, Cellular and Molecular Department, Centro Nacional de Biotecnologıa (CNB-CSIC), Darwin 3, 28049

Madrid, Spain. Email: [email protected].

Wiskott--Aldrich syndrome protein (WASP) --interacting protein(WIP) is an actin-binding protein involved in the regulation of actinpolymerization in cells, such as fibroblasts and lymphocytes.Despite its recognized function in non-neuronal cells, the role ofWIP in the central nervous system has not been examinedpreviously. We used WIP-deficient mice to examine WIP functionboth in vivo and in vitro. We report here that WIP2/2 hippocampalneurons exhibit enlargement of somas as well as overgrowth ofneuritic and dendritic branches that are more evident in earlydevelopmental stages. Dendritic arborization and synaptogenesis,which includes generation of postsynaptic dendritic spines, areactin-dependent processes that occur in parallel at later stages.WIP deficiency also increases the amplitude and frequency ofminiature excitatory postsynaptic currents, suggesting that WIP2/2

neurons have more mature synapses than wild-type neurons. Thesefindings reveal WIP as a previously unreported regulator ofneuronal maturation and synaptic activity.

Keywords: dendritic spine, electrophysiology, neuritogenesis, N-WASP,synapse

Introduction

Neuronal cytoarchitecture is first established through neurito-

genesis, a process in which neurons extend their neurites to

form a functional network during neuronal development (de

Curtis 2007). Neuron morphology greatly determines the final

complexity of the nervous system and is essential for the signal

flow that underlies information integration and processing. It is

therefore important that neuritogenesis occurs at the right

place and time for correct establishment of synaptic contacts

with proper targets (de Curtis 2007). Several environmental

cues converge on common coordinated intracellular pathways to

modulate neuritogenesis. Such intracellular events involve sig-

naling transduction, exocytic and endocytic mechanisms related

to membrane trafficking and cytoskeletal rearrangements.

Neurite initiation and outgrowth are based on the capacity

of the neuronal cytoskeleton, constituted mainly of actin

microfilaments (MF) and tubulin microtubules (MT), to

assemble and disassemble in response to extracellular signals

(Luo 2002; Conde and Caceres 2009). The polarized growth of

neurites requires the initial depolymerization of actin MF

(Bradke and Dotti 1999), stabilization of MT (Ferreira and

Caceres 1989), and accumulation of a number of specific

proteins (Wiggin et al. 2005). Actin polymerization is con-

trolled by the actin-related protein (Arp2/3) complex and by

the action of actin-binding proteins and nucleation-promoting

factors (NPF), such as neural Wiskott-Aldrich syndrome protein

(N-WASP). The Arp2/3 complex nucleates actin, inducing

branching and elongation, and with N-WASP, it mediates

neurite elongation (Suetsugu, Hattori, et al. 2002; Pinyol et al.

2007) and neurite branching (Kakimoto et al. 2004). N-WASP

interacts with WASP-interacting protein (WIP), a broadly

expressed proline-rich protein that regulates N-WASP function

as NPF and whose deficiency modifies actin polymerization

kinetics and the density of the subcortical actin network

(Anton et al. 2007). Through WASP/N-WASP--dependent or

--independent mechanisms, WIP participates in a wide variety

of cellular functions, including signaling, endocytosis, and actin

cytoskeleton remodeling (Anton et al. 2007). WIP deficiency in

mice alters the immune response, reducing T and mast cell

activity and increasing B cell function (Anton et al. 2002;

Kettner et al. 2004). Moreover, WIP null mice have a pro-

gressive immunological disorder of autoimmune nature, with

evident ulcerative colitis, interstitial pneumonitis, glomerular

nephropathy with IgA deposits, autoantibodies, and joint

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; Peterson

et al. 2007), few data are available on its functional impact and

even fewer regarding the central nervous system, where the

role of WIP has not been previously addressed.

Using the WIP knockout mouse as a tool, here, we describe

that loss of this protein impacts neurite and dendrite dynamics

and morphology, both in early and in late developmental stages,

in vitro and in vivo. Gross examination of WIP–/– brain revealed

changes in forebrain and hippocampal size. Extensive analysis

of WIP–/– hippocampal neuron development showed premature

neuritogenesis. Finally, electrophysiological and immunocyto-

chemical analyses demonstrated modified synaptic activity of

WIP–/– mature neurons. These studies show that WIP is an

essential negative regulator in the control of the cytoskeletal

events that underlie neuronal and synaptic development.

Materials and Methods

MiceWild-type (WT) and WIP KO SV129/BL6 mice (Anton et al. 2002) were

housed in specific pathogen-free conditions at the animal facility of the

Centro de Biologıa Molecular ‘‘Severo Ochoa,’’ Madrid, Spain. The

mouse colony was maintained by continuous mating of heterozygous

females with heterozygous males for more than 20 generations. To

obtain control or WIP–/– embryos/litters, we mate control male and

female or WIP–/– male and female mice. Handling of mice and all

manipulations were carried out in accordance with national and

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European Community guidelines and were reviewed and approved by

the institutional committee for animal welfare. All quantification was

conducted in a genotype-blind manner.

Brain Lysates and Western BlotControl or WIP

–/– brains were homogenized in lysis buffer (20 mM 4-(2-

hydroxyethyl)-1-piperazineethanesulfonic acid, pH 7.4, 100 mM NaCl, 5

mM ethylenediaminetetraacetic acid, 1% Triton X-100, 100 mM NaF, 1

mM Na3VO4, and the Complete Protease Inhibitor Cocktail, Roche

Diagnostics), and soluble extracts were resolved by sodium dodecyl

sulfate--polyacrylamide gel electrophoresis after determination of

protein concentration by Bradford analysis (BioRad). Proteins were

then transferred to nitrocellulose filters, which were blocked and

incubated with a mouse monoclonal antibody (mAb) specific to WIP

(1/1000, 3D10; a generous gift of Prof. R. Geha, Children’s Hospital,

Boston, MA). After exposure to a specific secondary antibody, antibody

binding was visualized by enhanced chemiluminiscence substrate

(Amersham Biosciences).

Primary Hippocampal Cultures

Neurons

Primary hippocampal cultures were prepared as described (Dotti et al.

1988; Kaech and Banker 2006). Briefly, hippocampi from E18 mouse

embryos (WT and WIP–/–) were washed and digested with 0.25% trypsin

(15 min, 37 �C). The tissue was then dissociated, resuspended in

minimun essential medium with 10% horse serum, and plated on poly-

L-lysine--coated coverslips (1 mg/ml) at a density of 6 3 103 cells/cm2

for imaging at early times (up to 24 h after plating) and at a density of 4 3

103 cells/cm2 for electrophysiological recordings. In some experiments,

neurons were allowed to adhere to the substrate (1 h) and then

incubated with 2 or 5 lM wiskostatin (BIOMOL International) in

dimethyl sulfoxide (DMSO). In all cases, after 3 h, plating medium was

replaced with neurobasal medium supplemented with B27 (Gibco). For

long-term culture, at this time, neuron-including coverslips were

transferred into dishes containing an astrocyte monolayer, with

neurons oriented facing the glia but without contacting them.

DNA Constructs and Transient Transfection

pLVWIP-GFP was obtained by digestion of pcDNA3WIP-GFP (kindly

provided by Prof. N. Ramesh, Children’s Hospital, Boston, MA) and by

cloning the insert into pLV; control pLVGFP was generated in a similar

manner. Suspended WT (cortical and hippocampal) or WIP–/– (cortical)

neurons were nucleofected using a 6 lg DNA/100 ll suspension

(Amaxa pulser; Lonza, Germany). Cells were maintained in suspension

(4 h) to permit exogenous gene expression and then plated for 24 h

before fixation for immunofluorescence analysis.

Immunofluorescence

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

min, room temperature). Cells were then permeablilized with 0.1%

Triton X-100 and labeled with phalloidin-tetramethylrhodamine iso-

thiocyanate (Sigma) and antibodies to tyrosinated a-tubulin (1/400;

T9028, Sigma), MAP2 (MT-associated protein, 1/400; 514, Sanchez

Martin et al. 1998), PSD-95 (5 lg/ml; 75-028, NeuroMab), or GFP (1/

100, 11814460; Roche); secondary fluo-rescent antibody or biotinylated

antibody and labeled streptavidin were then added. Cells were

excluded from analysis if they showed obvious features of toxicity,

such as neurite fragmentation/blebbing or vacuoles in the cell body.

Imaging

Confocal images were acquired digitally on a confocal LSM510 Meta

microscope (Zeiss) coupled to an inverted Axiovert 200 microscope

(Zeiss). Image stacks (logical size 1024 3 1024 pixels) consisted of 10

image planes acquired through a 403 (numerical aperture (NA), 1.3) or

a 633 oil-immersion lens (NA 1.4).

Time-lapse images of phase-contrast fields were captured on an

inverted Axiovert 200 microscope (Zeiss) equipped with a mono-

chrome CCD camera and ultrafast filter change. Image stacks (logical

size 1024 3 1024 pixels) consisted of 6 image planes acquired through

a 403 oil-immersion lens (NA, 1.3). Metamorph 6.2r6 (Universal

Imaging) software was used to process the time-lapse captured images.

Morphometry

Image stacks (physical size 76.9 3 76.9 lm) were imported to the

confocal module of Neurolucida 7.1 (MicroBrightfield, Inc., Williston,

VT), and neuronal dendritic trees were traced by drawing the dendrites

and the bifurcation points. Sholl analysis was performed for each traced

neuron by automatically calculating the number of dendritic intersec-

tions and the dendritic length at 10-lm interval starting from the soma.

Total dendritic length and total number of intersections and branches for

each neuron were also calculated as an index of dendritic complexity.

Soma area was determined by drawing soma contours while tracing cells.

Miniature Excitatory Postsynaptic Currents

Miniature excitatory postsynaptic currents (mEPSC) were recorded

from dissociated hippocampal neurons bathed in artificial cerebro-

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).Spontaneous activity was recorded for 3 min for each cell. mEPSC were

identified using pClamp software and corrected by eye on the basis of

their kinetics.

Morphology and Unbiased Stereology

Perfusion

Three-month-old male mice (WT, n = 6; WIP–/–, n = 6) were

anesthetized with pentobarbital (0.04 mg/kg) and perfused trans-

cardially with PBS (20 ml) followed by 100 ml 4% PFA (pH 7.4) in the

same buffer. Brains were postfixed in the same fixative (24 h).

Volumetric Analyses

Brains were sectioned coronally at a thickness of 50 lm to facilitate

measurements. Strict morphological criteria were used in all mice to

determine the boundaries of these brain regions. Briefly, hippocampal

outlines encompassed the CA1--3 fields of Ammon’s horn and the

subiculum but not the presubiculum or fimbria hippocampus. The

dentate gyrus was measured separately. Starting with one of the

sections, randomly selected across brains, 1 of every 6 sections was

analyzed through the extent of a hemisphere of the brain. One

hemisphere of each brain was analyzed. Using this sampling strategy,

7--10 histological sections per brain were analyzed. All volumetric

quantifications were performed with an Olympus BX51 with a 1.253

objective, a motorized XYZ axis computer-controlled stage (Prior

Scientific, Houston, TX), a digital video camera (JVC), and Stereo-

Investigator, a stereology software package (version 8.03, MicroBright-

Field). When calculating hippocampus volume, the boundaries were

defined and the volumes determined with Stereo-Investigator software.

Intracellular Injection of Lucifer Yellow

Coronal sections (150 lm) were cut on a vibratome. Sections were

prelabeled with 4,6-diamidino-2-phenylindole, and a continuous cur-

rent used to inject individual cells with lucifer yellow (8% in 0.1 M Tris

buffer, pH 7.4). Dye was injected into neurons in the dentate gyrus until

the individual dendrites of each cell could be traced to an abrupt end at

the distal tips and the dendritic spines were readily visible, indicating

that the dendrites were completely filled. The sections were then

processed, first with rabbit anti-lucifer yellow (Cajal Institute,

1/400 000 in stock solution of 2% Bovine serum albumin, 1% Triton

X-100, 5% sucrose in PBS) and then with Alexa 488-conjugated

secondary antibody (Molecular Probes, 1/1000, 4 h). Sections were

mounted on a glass slide in fresh ProLong Gold antifade reagent

(Invitrogen, Eugene, OR; 24 h, room temperature in the dark) and

sealed with nail polish.

Confocal Microscopy

Imaging was performed on a Leica laser scanning multispectral

confocal microscope (TCS SP5) using an argon laser. Image stacks

(physical size 76.9 3 76.9 lm, logical size 1024 3 1024 pixels) consisted

of 100--350 image planes acquired through a 633 glycerol-immersion

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lens (NA, 1.3; working distance, 280 lm; refraction index, 1.45) with

a calculated optimal zoom factor of 3.2 and a z-step of 0.14 lm (voxel

size, 75.1 3 75.1 3 136.4 nm). These settings and optics represent the

highest resolution currently possible with confocal microscopy. For

each neuron (5 neurons per mouse, 55 neurons total), 1--5 randomly

selected dendrites were scanned from soma to tip, and stacks were

processed with a 3D blind deconvolution algorithm (Autodeblur;

Autoquant, Media Cybernetics) for 10 iterations to reduce the out-of-

focus light.

Statistical AnalysisQuantification was performed in a blind fashion by independent

researchers. For statistical analyses, we used the two-tailed Student t-

test to compare means, the chi-square test to compare nominal

variables, and the two-way analysis of variance to compare Sholl analysis

or the Kolmogorov--Smirnov test to compare cumulative frequency

analysis. Results are shown as mean ± standard error of the mean.

Results

WIP Is Expressed in the WT Mouse Brain

The expression pattern of WIP messenger RNA (mRNA) suggests

that WIP is ubiquitously expressed in all mouse tissues, including

brain (Ramesh et al. 1997; Tsuboi 2006). To date, however, the

levels of endogenous WIP protein have only been analyzed in

fibroblasts, myoblasts, and hematopoietic-derived cells (reviewed

in Anton et al. 2007). To studyWIP expression in brain, we probed

westernblotsofcell lysates from3regionsof the adultmousebrain

(cortex, hippocampus, and olfactory bulb), using theWIP-specific

mAb 3D10 (Koduru et al. 2007). This mAb recognized 2 protein

bands in cell lysates from each of the regions examined (Fig. 1A),

which could represent 2 WIP isoforms and/or be the result of

posttranslational modifications of WIP. Neither band was present

inWIP-deficient tissue. Thesedata demonstrateWIP expression in

murine brain and corroborate previous mRNA studies (Ramesh

et al. 1997; Tsuboi 2006) as well as the data included in the Allen

atlas (http://www.brain-map.org/).

Brain Hypertrophy in WIP-Deficient Mice

To assess general effects of WIP deficiency on the mouse brain,

we determined volumes of the hippocampus and of the rest of

the murine forebrain (3-month-old male mice). Contours of the

structures of interest were drawn on Nissl-stained serial sec-

tions with the aid of Stereo-Investigator software to yield the

volume of each. There was a significant increase (30%) in

forebrain volume in WIP-deficient brains (n = 5) compared

with WT littermates (n = 6, Fig. 1B--D and Supplementary Fig.

1). The hippocampus proper (without the dentate gyrus) and

the dentate gyrus itself were also hypertrophied in WIP–/– brain

(Fig. 1E,F). WIP deficiency thus causes general macrocephaly

that includes hippocampal hypertrophy.

Enhanced Early Neuronal Development in WIP –/– Neurons

The enlarged brain observed in WIP–/– mice relative to their WT

littermates raised the possibility that the greater brain volume

could be due to increased neuritic branching since dendrites

and axon collaterals account for most of the brain volume

(Acebes and Ferrus 2000). To test this hypothesis at the cellular

level, we examined the effect of WIP deficiency on early

neuronal development. Primary hippocampal neurons from

control and WIP–/– embryos were grown at very low density so

that their neurite arbors did not overlap to avoid confusion

about which neurites protruded from each cell body. Cells

were labeled with anti-tyrosinated a-tubulin and fluorescent

phalloidin (Fig. 2A,B). As a measure of neuronal development,

we quantified the fraction of neurons at each developmental

stage at 3 h postplating. At stage 1, the MT-containing soma is

Figure 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 WIP�/� mouse brain. Glyceraldehyde-3-phosphate dehydrogenase labeling confirmed equivalent protein loading in cortical samples. (B) Representative WT (left)and WIP�/� brain (right) at 3 months of age. Note: the enlargement of the WIP�/� brain. Scale bar, 4 mm. (C) Representative Nissl staining showing hippocampus enlargementin WIP�/� mice. Scale bar, 500 lm. (D--F) Average volumes of forebrain (D), hippocampus (E), and dentate gyrus (F) are increased in WIP�/� mice. P values were determinedwith Student’s t-test; *P\ 0.05; **P\ 0.01.

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Figure 2. WIP deficiency accelerates development of dissociated hippocampal neurons. (A) Representative confocal images of WT- and WIP�/�-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 WIP�/� 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 WIP�/� at developmental stage 1 compared with WT


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surrounded by flattened actin-rich lamellipodia, while in stage

2, the lamellipodia are transformed into equidistant neurites

(Dotti et al. 1988). Stage 1+ is defined as cells with incipient

tubulin-rich small protrusions. We found a clear difference in

the distribution of these populations (Fig. 2C); there was

a significant decrease (35%) in the frequency of WIP–/–

neurons classified as developmental stage 1 compared with

WT neurons (n = 4 cultures and n = 150 neurons/genotype,

Fig. 2C). Conversely, there was an increase (45%) in the

percentage of WIP-deficient neurons at stage 2. As another

measure of neuronal development, we quantified the number

of neurites arising from each neuron at 3 h postplating and

found a significant increase (36%) in the average number of

neurites arising from WIP–/– (n = 172) compared with WT

neurons (n = 151, Fig. 2D). This finding was confirmed in time-

lapse images made with phase-contrast microscopy for 3 h

after plating (Fig. 2E and Supplementary Videos 1 and 2).

These data suggest that, at 3 h after plating, WIP–/– neurons are

in a more advanced developmental stage than WT neurons.

This implies enhanced early neuronal development in the

absence of WIP.

N-WASP Activity and Fine-Tuned WIP Levels Regulate NeuronalDevelopment

To test whether this accelerated neuronal development

continues at later developmental times, we quantified the

fraction of neurons that remains at developmental stage 1 at 24 h

postplating. Cultured hippocampal neurons were fixed and

labeled with anti-tyrosinated a-tubulin antibody and fluores-

cent phalloidin and imaged by confocal microscopy. As

observed at 3 h postplating, we found a decrease of 48% in

the frequency of WIP–/– neurons at developmental stage 1

(Fig. 3A left and Fig. 3B), implying that WIP–/– neurons still

show accelerated development at this time.

To determine whether this phenotype depends on the

activity of N-WASP, an actin NPF with WIP-binding capacity,

we incubated cultures (23 h) with wiskostatin, a selective

reversible N-WASP inhibitor (Peterson et al. 2004). Wiskosta-

tin is a cell-permeable N-alkylated carbazole derivative that

selectively blocks actin filament assembly by binding to N-

WASP, stabilizing the autoinhibited conformation, and pre-

venting activation of the Arp2/3 complex (Wegner et al.

2008). Wiskostatin produced a dose-dependent increase in

the fraction of stage 1 neurons, indicating impairment in

neuronal development after blockade of N-WASP-dependent

filament assembly (Fig. 3A,B) (da Silva and Dotti 2002;

Dent et al. 2007). Inhibition of N-WASP activity with 2 lMwiskostatin induced a phenotype in WIP

–/– neurons resem-

bling that of untreated control neurons, and with 5 lMwiskostatin, WIP

–/– neurons no longer showed a lower

frequency of stage 1 neurons (Fig. 3A,B). This finding suggests

that N-WASP activity is essential in mediating the accelerated

development of WIP–/– neurons.

To further evaluate the role of WIP in neurite sprouting, WT

neurons were nucleofected with lentiviral constructs for over-

expression of WIP-GFP (pLVWIP-GFP) or GFP as control

(pLVGFP; Fig. 3C). At 24 h postplating, while 31% of GFP-

expressing neurons were in stage 1, the frequency of stage 1

WIP-GFP--expressing neurons was 80% (Fig. 3D). The de-

velopmental delay at 24 h induced by WIP overexpression was

confirmed in cortical embryonic neurons (Supplementary Fig.

2). This effect was sustained over time, since 48 h after plating,

22.6% of neurons with increased WIP levels remained at stage 1

compared with none of the GFP-expressing neurons.

We used rescue experiments to confirm the WIP contribution

to neuron development. WIP–/– neurons were nucleofected with

a lentiviral-based vector coding for WIP-GFP or control GFP, and

24 h after plating, the fraction of cells in stage 1 or 2 was

quantified (Fig. 3E,F; WIP-GFP--expressing WIP–/– neurons). The

population distribution of rescued neurons was indistinguishable

from that of WT neurons and differed significantly from that of

GFP-expressing WIP–/– neurons. WIP-GFP expression in WIP

–/–

neurons also reversed the phenotype, as the mean number of

primary neurites (Fig. 3G and Supplementary Fig. 3) and neuritic

bifurcations (Supplementary Fig. 3) were similar to those of GFP-

positive WT neurons.

These findings indicate that WIP acts as a negative regulator

of neuronal differentiation and that neuronal development can

be modulated bidirectionally by WIP levels.

Enhanced Dendritic Maturation in WIP –/– Neurons

To test whether the WIP regulatory role persists in differen-

tiated neurons, we also imaged cultured hippocampal neurons

at 22 DIV (Fig. 4A). For quantitative analysis of the branching

pattern of the neuritic or dendritic tree at the distinct neu-

ronal developmental stages, we traced neurons (1 and 22 DIV)

with Neurolucida software. Sholl analysis was used to measure

the number of neurites/dendrites crossing circles at various

radial distances from the soma (Sholl 1953). Both neuronal

types showed addition of dendritic branches over time (Fig.

4B,D), although the number of crossings was significantly

higher in WIP–/– neurons compared with WT neurons at both

time points. This finding implies that WIP–/– neurons show

higher ramification of their neuritic (1 DIV) and dendritic

arbor (22 DIV, Fig. 4D). Nevertheless, the difference in the

number of crossings between phenotypes, which was 82% at

1 DIV, decreased to 45% at 22 DIV (Supplementary Fig. 4A).

The increased ramification was reflected in greater total

neuritic or dendritic length of WIP–/– neurons at both time points

(Fig. 4C,E), a difference that was also reduced in mature neurons

(from 109% to 40%; Supplementary Fig. 4B). This difference was

even smaller in neurons of adult mice (see below).

Soma size determines the number of dendrites and associated

branches in neurons (Kernell and Zwaagstra 1989; Kollins and

Davenport 2005). To analyze whether WIP deficiency alters

soma area, we quantified this parameter in WT and WIP–/–

neurons. Similar to our observations for the dendritic arbor,

soma area was significantly larger in WIP–/– neurons (Fig. 4F,G),

but this difference attenuated from 40% to 27% with time

(Supplementary Fig. 4C). The effect of WIP deficiency on

promoting neuritic or dendritic arborization and on soma size is

thus greater in early developmental stages.

neurons, implying an average more advanced developmental stage for WIP�/� neurons. Chi-square test, *P 5 0.0194. (D) The average number of primary neurites arising fromWIP�/� somas was significantly larger compared with WT neurons (3 h postplating), again indicating the more advanced developmental stage of WIP�/� 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 (WIP�/�; Supplementary Video2) hippocampal embryonic neurons cultured on poly-L-lysineover a 3-h period.


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Enhanced Neuronal Ramification in Adult WIP –/– Mice

To test whether WIP deficiency produces a long-lasting effect

on neuronal maturation in vivo, we evaluated dendritic archi-

tecture in adult (3-month-old) mice after injection of lucifer

yellow into hippocampal neurons of fixed brain sections

(Fig. 5A) and studied the structure of granule cells in the

dentate gyrus. Sholl analysis showed significantly more cross-

ings in the distal part of the dendrite in WIP–/– mice (n = 59

neurons from 6 mice) compared with WT mice (n = 39

neurons from 5 mice, Fig. 5B). This increased number of

Figure 3. Inhibition of N-WASP prevents WIP�/� phenotype, WIP overexpression delays neurite protrusion, and WIP reexpression reverts the phenotype. (A) Representative imagesof dissociated WT and WIP�/� 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 WIP�/� 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 WIP�/� neurons at stage 1, equivalent to the frequency for untreated WT neurons. Ahigher wiskostatin concentration (5 lM) prevented differentiation of both WT and WIP�/� 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 ofWIP�/� 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 WIP�/� 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 WIP�/� 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 WIP�/� neurons and differs significantly from that of WIP�/�GFP-expressing neurons. Student’s t-test; ***P\ 0.001; ns, not significant.


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Figure 4. Increased ramification in WIP�/� neurons in vitro. (A) Representative images of dissociated WT and WIP�/� 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 WIP�/� neurons at both 1 and 22 DIV. (B and C) Sholl analysis of tracedWT and WIP�/� neurons showed more ramification in WIP�/� 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 WIP�/� somasdemonstrating increased soma surface in WIP�/� neurons. (G) WIP�/� 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.


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crossings reflects a larger number of ramifications at the outer

molecular layer of the dentate gyrus. As a result of the higher

dendritic ramification, the dendritic length in this layer is also

increased in WIP–/– mice (Fig. 5C ), leading to greater total

dendritic length (18%) in WIP–/– mice (Fig. 5D). Cumulative

frequency analysis indicated that WIP–/– mice have a sub-

population of highly ramified neurons not detected in WT

mice (Fig. 5E ). WIP deficiency thus leads to enhanced

dendritic ramification in the adult mouse. Nonetheless, the

magnitude of change in total dendritic length of the adult

WIP–/– mouse is smaller than in early developmental stages.

Enhanced Synaptic Maturation in WIP –/– Neurons

During later stages of neuronal development, dendritic

arborization and synaptogenesis occur in parallel (Cantallops

et al. 2000; Cline 2001). The actin cytoskeleton has a pivotal

role in both processes. In the context of synaptogenesis, actin

dynamics control the morphogenesis and function of the den-

dritic spines, small actin-rich protrusions from dendritic shafts

(Ethell and Pasquale 2005). As we found that WIP modulates

dendritic arborization, we examined the possibility that WIP

deficiency fosters synaptic maturation. We measured the effects

of WIP deficiency on spontaneous miniature (action-potential

independent) postsynaptic currents (mEPSC) in dissociated

hippocampal neurons (22 DIV) in the presence of 1 lMtetrodotoxin (Fig. 6A). Both the amplitude and the frequency

of mEPSC were significantly increased in WIP–/– compared with

WT neurons (n = 17 and 15 neurons, respectively; Fig. 6B,C).

mEPSC amplitude is indicative of the postsynaptic strength of

individual functional synapses, whereas mEPSC frequency

depends on synapse number and presynaptic properties

(Turrigiano et al. 1998; Han and Stevens 2009). WIP deficiency

functionally increases both the strength and possibly the

number of individual synapses, suggesting that at this time,

WIP–/– neurons show either a more mature phenotype or more

abundant and/or enlarged dendritic spines.

To examine whether WIP deficiency resulted in an increased

number of structural synapses, we stained dissociated hippo-

campal neurons at the same developmental stage (22 DIV) for the

postsynaptic marker PSD-95, known to drive maturation of

glutamatergic synapses (El-Husseini et al. 2000). WIP–/– neurons

showed increased fluorescence intensity of individual PSD-95

puncta, indicating greater PSD-95 accumulation in spines in the

absence of WIP (WT, n = 117 spines from 6 neurons; WIP–/–, n =

168 spines from 7 neurons, Fig. 6D,E). Frequency analysis

showed that in WIP–/– neurons, staining of some PSD-95--positive

spines is particularly intense, whereas such a population was not

found in WT neurons (Fig. 6F ). Positive spine density was

nonetheless similar inWIP–/– andWTneurons (Fig. 6G ). AsWIP

–/–

neurons are substantially more branched than WT neurons (and

their total dendritic length is therefore increased), the total

number of PSD-95--positive spines is significantly increased in

WIP–/– neurons (Fig. 6H). These neuronal cultures were

maintained over WT astrocytes, excluding possible astrocyte

involvement in the synaptic modulation derived from the lack of

WIP. These results indicate that WIP deficiency increases the

number and size of structural and functional synapses.

Discussion

Here, we present the first report of a role for WIP in the brain,

showing a specific role for WIP as a negative regulator of murine

neuronal development. We found that, in hippocampal primary

neurons, loss of WIP accelerates the onset of neuritogenesis and

increases neuritic and dendritic branching. Moreover, lack of

WIP enhances PSD-95 accumulation in hippocampal dendritic

spines and increases neuronal synaptic activity. Conversely, WIP

overexpression blocks the initiation of neuritogenesis, suggesting

that WIP is a bidirectional regulator of neuronal development.

WIP Modulates Neuritic Branching During NeuronalDevelopment

Marked dendritic elaboration normally begins in neuronal

cultures 2--3 days after axonal outgrowth (Nowakowski and Rakic

1979; Dotti et al. 1988). Our experiments showed that immature

WIP–/– neurons begin to emit neurites starting at the first day in

culture, much earlier than control neurons. After cell cycle exit

and before neuronal polarization, cortical postmitotic neurons

make a transition through a multipolar stage, when multiple

neurites emerge rapidly from the cell body (Barnes and Polleux

2009). This morphological transition is also controlled by WIP

during brain development, as GFP in utero electroporation shows

a notable increase in the percentage of multipolar cells in WIP–/–

embryos, in parallel to a reduction in the fraction of round and

unipolar neurons (not shown). In addition, neurites in immature

WIP–/– neurons are more branched than neurites of WT neurons.

Dendritic branching is also increased in WIP–/– mature neurons,

although to a lesser extent than at early neuronal developmental

stages (Fig. 4 and Supplementary Fig. 4). Sholl analysis demon-

strated that the initial morphological features acquired by

immature WIP–/– neurons determine the pattern of dendritic

branching of the more differentiated cells. We therefore suggest

that WIP acts as a negative modulator that controls the precise

timing of the correct onset of dendritic development. Our results

point to the possibility that some pathologies detected in mature

neurons might have their origin, yet unknown, in altered

processes that occur during early neuronal development.

WIP versus Other Negative Regulators of DendriticBranching

Numerous molecules are involved in the positive control of

neuritic or dendritic outgrowth and branching, many of them

related to the control of cytoskeletondynamics (McAllister 2000;

Urbanska et al. 2008). Only aminority of thesemolecules, such as

PTEN (Kwon et al. 2001; Jaworski et al. 2005) and RhoA (Negishi

and Katoh 2002; da Silva et al. 2003), have been identified as

negative regulators of these processes. Similarly to our findings,

conditional adult Ptenmutant mice show enlargement of cortex

and hippocampus associated with dendritic hypertrophy and

increased soma size (Kwon et al. 2001, 2006). At difference from

the WIP–/– phenotype observed here, PTEN is not necessary for

initiation of neuritogenesis (Lachyankar et al. 2000). The most

striking difference between WIP- and PTEN-mediated negative

regulation thus appears to depend on the temporal window of

neuronal development in which both proteins operate. PTEN

does not control early stages of neuritogenesis, and its influence

on neuronal structure increases progressively throughout the

animal’s life. In contrast, WIP modulates neuronal development

and synaptogenesis early in postnatal life, and its effects are

attenuated as neuronal maturation progresses.

Similarly to the absence of WIP, RhoA inhibition increases

both the number of primary neurites and total neuritic length

in immature dissociated hippocampal neurons, whereas ex-

pression of a constitutively active form of RhoA arrests cells in

stage 1 (da Silva et al. 2003). In mature neurons, transfection of


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a dominant-negative RhoA construct does not affect dendritic

morphology (Nakayama et al. 2000), whereas WIP–/– neurons

still show enhanced dendritic arborization. The effect of WIP

on neuronal maturation thus appears to be longer lasting than

that of other negative modulators of neuritic and dendritic

outgrowth and branching.

Possible Mechanisms for WIP-Mediated Neurite Sproutingand Branching

Sprouting of primary neurites and interstitial branching from

neuritic or dendritic shafts follows the same sequence as

cortical cytoskeletal rearrangements (Wu et al. 1999; Dent et al.

2007). For the extension of a single filopodium, which is

Figure 5. Increased ramification in WIP�/� neurons in vivo. (A) Representative projection images of lucifer yellow--injected granule neurons of 3-month-old WT and WIP�/�

mice. Scale bar, 25 lm. (B and C) Sholl analysis of traced WT and WIP�/� granule neurons showed greater ramification in the distal part of WIP�/� 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 WIP�/� 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 WIP�/� mice. Kolmogorov--Smirnov test; *P\ 0.05.


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subsequently stabilized by MT invasion, F-actin and MT must

undergo local depolymerization triggered by extracellular signals

(Acebes and Ferrus 2000; da Silva and Dotti 2002; Luo 2002).

WIP inhibits F-actin depolymerization (Martinez-Quiles et al.

2001), which could explain the neuritogenesis arrest found in

WIP-overexpressing neurons.

Our data indicate that WIP modulates neuritogenesis

through its previously described ability to maintain N-WASP

in its autoinhibited state (Martinez-Quiles et al. 2001). N-WASP

promotes neurite outgrowth and regulates neurite branching

in hippocampal neurons (Suetsugu, Hattori, et al. 2002; Abe

et al. 2003; Pinyol et al. 2007). At the molecular level, N-WASP

Figure 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 WIP�/� neuron. (B and C) Cumulative frequency representation of the amplitude (B) and frequency (C) of mEPSC, showing significantshifts to higher values in WIP�/� neurons. Kolmogorov--Smirnov test; ***P\ 0.001. (D) Representative projection confocal images of WT and WIP�/� dendrites, stained withanti-MAP2 (blue) and -PSD-95 antibodies (green). Note that there are intense dendritic spines in WIP�/� but not in WT dendrites. (E) Average intensity of PSD-95--positive spinesin WT and WIP�/� 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 WIP�/� but not in WT neurons. Kolmogorov--Smirnov test; ***P\ 0.001. (G) Sholl analysis of PSD-95--positive spine density in WTand WIP�/� 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.


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acts as a signal integration device that can precisely target actin

polymerization to membrane sites at which PI(4,5)P2 and

activated Src kinases and Cdc42 are located (Prehoda et al.

2000; Suetsugu, Miki, et al. 2002). Cdc42 stimulates the actin-

polymerizing activity of N-WASP, creating free barbed ends

from which actin polymerization can take place (Miki,

Suetsugu, et al. 1998), leading to filopodium formation (Miki,

Sasaki, et al. 1998). In the absence of WIP, N-WASP would be

more easily released from inhibition and be hyperactivated to

initiate premature filopodium formation. This interpretation is

supported by pharmacological inhibition of N-WASP by wiskos-

tatin, which blocked the accelerated neuritogenesis seen in WIP-

deficient neurons (Fig. 3).

WIP Modulates Synaptic Activity

We report here that mEPSC amplitude and frequency are

increased in WIP–/– neurons, suggesting both stronger AMPA

(alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)

receptor--mediated synaptic currents and more functional

synapses in these cells (Fig. 6). In addition, we describe an

increase in PSD-95 accumulation at individual spines in WIP–/–

neurons. These 2 events are probably linked since PSD-95

drives the maturation of excitatory synapses with the in-

corporation of AMPA receptors (El-Husseini et al. 2000).

Indeed, the contribution of AMPA receptors to synaptic

transmission increases gradually with neuronal maturation

(Mammen et al. 1997; Petralia et al. 1999). The increase in

PSD-95 accumulation at individual WIP–/– spines might suggest

that the PSD (and therefore the synapse) is enlarged in the

absence of WIP. Given that the synaptic area correlates

positively with the number of synaptic AMPA receptors (Nusser

et al. 1998), our electrophysiological findings are strengthened

by the morphological analysis of PSD-95 in spines.

In conclusion, this study sheds light on the molecular events

that control early neuronal development by presenting WIP as

a previously undescribed regulator that prevents premature

dendritic and synaptic maturation.

Supplementary Material

Supplementary material can be found at: http://www.cercor.

oxfordjournals.org/.

Funding

Grants from Consejo Superior de Investigaciones Cientıficas-

Comunidad de Madrid (CCG08-CSIC/SAL-3471), CSIC (PIE2-

00720I002), and the Spanish Ministry of Education and Science

(BFU2007-64144 and BFU2010-21374) to I.M.A., from Centro de

Investigacion Biomedica en Red Enfermedades Neurodegener-

ativas (Instituto de Salud Carlos III), the Plan Nacional DGCYT

(SAF2009-12249-C02-01) to F.W. and SAF2010-15676 to S.K.,

and by an institutional grant from the Fundacion Ramon Areces.

A.F. was a recipient of an FPU MEC fellowship (AP2005-3405),

I.B. held a contract from the Comunidad Autonoma de Madrid

and S.K., a Ramon y Cajal contract.

Notes

We thank Chiara Ragazzini and Sonia Perez for their excellent technical

assistance and Javier de Felipe for his contribution to the morphomet-

ric analysis with NeuroLucida Software. We are grateful to Raif Geha

and Narayanaswamy Ramesh for the 3D10 mAb and to Lola Ledesma

and Carlos Dotti for helpful advice on the manuscript. We acknowledge

Daniel Gallego for electrophysiological signal processing, Jose Ramon

Valverde for statistical assistance and Catherine Mark for editorial

assistance. Conflict of Interest : None declared.

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