indicate that the BH3-only members bind to the pro-survival Bcl-2 proteins and neutralize them, thereby allowing Bax-like proteins to initiate apoptosis [12]. The different BH3-only members respond to different forms of cellular stress and are subject to regulation at both transcriptional and posttranslation levels. During ano¨ıkis, activation of MAP kinase, PI-3 kinase/Akt and JNK pathways regulate Bcl-2 family members such as the anti-apoptotic Bcl-2 and Bcl-xL proteins or the pro-apoptotic proteins Bad or Bim [10]. Bim primary transcripts undergo alternative splicing to generate three major isoforms: Bim-S, Bim-L and Bim-EL, the latter representing the predominant isoform in most tissues [13]. Bim- EL is up-regulated during ano¨ıkis while its down-regulation by RNA interference inhibits ano¨ıkis [14]. It has been established that Bim acts as a critical mediator of neuronal apoptosis [13,15,16,17]. Indeed, Putcha et al., have reported that Nerve Growth Factor (NGF) deprivation in sympathetic neurons rapidly induced expression of Bim-EL [16]. Conversely, Bim deletion conferred protection against cytochrome c release and neuronal apoptosis upon NGF deprivation. Similar protective effect of Bim deletion against apoptosis was observed in cerebellar granule neurons subjected to K+ withdrawal [16]. N-cadherin is the predominant form of cadherin expressed ubiquitously in the early neural tube [18]. N-cadherin participates to the development and functional organization of the adult neural tissue [19]. This receptor is necessary for the polarisation of the neuroepithelial sheets, the radial organization of developing cortical plate and likely the migration of neuronal precursors [20]. It is also implicated in neurite outgrowth, dendritic arborization, axon guidance, and in the early stages of synaptogenesis [21]. Later in development, N-cadherin localizes at synapses [22], where it not only plays an adhesive role but also participates to the regulation of synaptic function and plasticity [23,24,25]. In this study, we assessed whether cadherin-mediated cell-cell adhesion provides a survival signal for neuronal cells and thereby focused our study on the contribution of N-cadherin. For that purpose, we used an immobilized N-cadherin (N-cad) recombinant protein coated on culture plates mimicking cell-cell contact formation to tightly control cadherin engagement [26,27]. Controlled mobilization of N-cadherin was shown to induce cell cycle exit and myogenic differentiation [28] and to promote neurite outgrowth [29,30]. Our work demonstrates that N- cadherin-mediated adhesion provides neuronal cells with pro- survival signal, protecting them from apoptosis in the absence of growth factors and extracellular matrix stimuli. In addition, we describe a molecular pathway by which cadherin regulates cell survival. Indeed, we report that the engagement of N-cadherin results in the phosphorylation of Erk1/2 and the down-regulation of the pro-apoptotic protein Bim, whereas the PI3-kinase/Akt survival pathway and Bcl-2 protein are not affected. Materials and Methods Antibodies and reagents The following antibodies were used for western blotting : rabbit anti-Akt (1:1000; Cell Signaling Technology, Beverly, MA), rabbit anti-phospho-Akt (1:1000; Cell Signaling Technology), rabbit anti- Erk1/2 (1/10000; Upstate Biotechnology, Inc. Lake Placid, NY), mouse anti-phospho-Erk1/2 (1/10000; Sigma), mouse anti-Bcl-2 (1/500; Transduction Laboratories, Becton Dickinson Europe, Le Pont de Claix, France), rabbit anti-Bcl-xL (1/1000; Cell Signaling Technology), rabbit anti-Bim (1/1000; Pharmingen, San Diego, CA), rabbit anti-phospho-Bad (1/1000; Cell Signaling Technol- ogy), rabbit anti-PARP (1/1000; Cell signalling Technology), mouse anti-b-actin (1/20000; clone AC-15; Sigma), mouse anti-a- N-Cadherin Anti-Apoptotic Signaling tubulin (1:20000, clone E7, Developmental Studies Hybridoma Bank). Mouse anti-cytochrome c (1/200, clone 6H2.B4; Pharmin- gen) anti-bIII-tubulin (1/1000, clone Tuj1; Covance, CA) and anti-paxillin antibodies (1/100, Upstate Biotechnologies) mono- clonal antibodies were used for immunostaining. 49,6-diamino-2- phenylindole, dihydrochloride (DAPI) was from Molecular Probes (Eugene, OR). U0126 and LY294002 were from Cell Signaling Technology, PMA was from Sigma, Triton X-100 was from Pierce. The expression vector encoding for the dominant negative mutant form of N-cadherin deleted of its extracellular domain (DN-Ncad), initially described in [31], has been modified by replacing the Myc tag by the DsRed protein coding sequence [32]. The expression vectors coding for wild type Bim-EL and the ERK- dependent phosphorylation site defective mutant Bim-EL (S69G) have been described in [33]. Cell culture Mouse GT1-7 cells [34] were grown at 37uC, 5%CO2, in Dulbecco’s Modified Eagle Medium with 4.5 g/l glucose (DMEM, Life Technologies) supplemented with 10% fetal bovine serum (FBS, Life Technologies) and antibiotics (100 IU/ml penicillin and 100 mg/ml streptomycin, Life Technologies). GT1-7 cells were transfected using Lipofectamine 2000 (Life Technologies). Cells were seeded at 8.104 cells/cm2 and transfected 24 hours later according to manufacturer’s instructions. Primary mouse ventral spinal cord and rat hippocampal neurons were prepared as previously described [35,36]. Briefly, ventral spinal cord of E12.5 mouse embryos from the OF1 pregnant mice were isolated and digested with trypsin for 10 min at 37uC in Ham F10 modified medium containing 0.025% trypsin. Tissues were then mechanically dissociated in L15 medium containing DNAse I and the supernatant was laid on a BSA 4% solution and centrifuged. Spinal cells were seeded on coated coverslips at 2.104 cells/cm2 in Neurobasal medium containing 0.5 mM L-glutamine and cultivated for 24 hours to 48 hours at 37uC, 5% CO2. Hippocampi from E18.5 rat embryos (Sprague Dawley pregnant rat, R. Janvier laboratories) were isolated in HBSS-20 mM Hepes and digested with trypsin 0,05% 10 min at 37uC, then mechanically dissociated. Hippocampal cells were recovered in MEM medium supplemented with 2 mM glutamine, 1 mM sodium pyruvate and seeded on coated coverslips at 2.104 cells/cm2 and cultivated for 24 hours at 37uC, 5% CO2. All experiments with animals were in accordance with the guidelines of the French Agriculture and Forestry Ministry for handling animals (decree 87849, license 75–765-Renouvellement) and approved by the ‘‘Charles Darwin ethical committee in animal experimentation, Paris’’ under permit number Ce5/2010/064. Substrates coating Adhesive substrates were prepared as described in [28]. Briefly, glass coverslips or thermosterilized bacterial 35-mm Petri dishes (Falcon, Becton Dickinson Europe) were incubated with goat anti- mouse Fcc fragment antibody (Jackson ImmunoResearch, West Grove, PA) at 1 mg/cm2 in 0.1 M borate buffer pH 8.0 overnight at 4uC. Plates were washed and then incubated with purified Ncad-Fc chimera (extracellular domain of the chicken N-cadherin fused to the mouse IgG2b Fc fragment [27]) at a concentration of 1 mg/cm2 for 2 hours at room temperature. For the control samples, glass coverslips or dishes were incubated with poly-L- 2 lysine (PL, 0.01%, Sigma) or fibronectin (FN, 10 mg/cm , Invitrogen), in water overnight at 4uC. After washes, surfaces were then saturated with 1.5% purified BSA (Sigma) in PBS for 5– 10 min at room temperature. PLoS ONE | www.plosone.org 2 March 2012 | Volume 7 | Issue 3 | e33206