WIP Is a Negative Regulator of Neuronal Maturation and Synaptic Activity

A. Franco 2 3 S. Knafo 0 1 2 I. Banon-Rodriguez 2 3 P. Merino-Serrais 1 2 I. Fernaud-Espinosa 1 2 M. Nieto 2 3 J.J. Garrido 0 1 2 4 J.A. Esteban 0 2 F. Wandosell 0 2 4 I.M. Anton 2 3 4 0 Centro de Biolog a Molecular ''Severo Ochoa'' (CSIC-UAM), Universidad Auto noma de Madrid , 28049 Madrid, Spain 1 Instituto Cajal (CSIC) , 28002 Madrid, Spain 2 The Author 2011. Published by Oxford University Press. All rights reserved. For permissions , please 3 Centro Nacional de Biotecnolog a (CNB-CSIC) , 28049 Madrid, Spain 4 CIBERNED, Centro Investigacio n Biome dica en Red de Enfermedades Neurodegenerativas , 28031 Spain Wiskott--Aldrich syndrome protein (WASP) --interacting protein (WIP) is an actin-binding protein involved in the regulation of actin polymerization in cells, such as fibroblasts and lymphocytes. Despite its recognized function in non-neuronal cells, the role of WIP in the central nervous system has not been examined previously. We used WIP-deficient mice to examine WIP function both in vivo and in vitro. We report here that WIP2/2 hippocampal neurons exhibit enlargement of somas as well as overgrowth of neuritic and dendritic branches that are more evident in early developmental stages. Dendritic arborization and synaptogenesis, which includes generation of postsynaptic dendritic spines, are actin-dependent processes that occur in parallel at later stages. WIP deficiency also increases the amplitude and frequency of miniature excitatory postsynaptic currents, suggesting that WIP2/2 neurons have more mature synapses than wild-type neurons. These findings reveal WIP as a previously unreported regulator of neuronal maturation and synaptic activity. Introduction Neuronal cytoarchitecture is first established through neuritogenesis, 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 signaling 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 controlled 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 progressive 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 WIPWASP/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 immunocytochemical 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 Mice Wild-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 Biolog a 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 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 Blot Control or WIP/ brains were homogenized in lysis buffer (20 mM 4-(2hydroxyethyl)-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, Childrens 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 w (...truncated)