N-Cadherin Mediates Neuronal Cell Survival through Bim Down-Regulation Elise C. Lelie` vre1,2,3., Charlotte Plestant1,2,3., Ce´ cile Boscher1,2,3¤, Emeline Wolff1,2,3, Rene´ -Marc Me` ge1,2,3*", He´ le` ne Birbes1,2,3*" 1 INSERM, UMRS-839, Paris, France, 2 Universite´ Pierre et Marie Curie-Paris 6, Paris, France, 3 Institut du Fer a` Moulin, Paris, France Abstract N-cadherin is a major adhesion molecule involved in the development and plasticity of the nervous system. N-cadherin- mediated cell adhesion regulates neuroepithelial cell polarity, neuronal precursor migration, growth cone migration and synaptic plasticity. In vitro, it has been involved in signaling events regulating processes such as cell mobility, proliferation and differentiation. N-cadherin has also been implicated in adhesion-dependent protection against apoptosis in non- neuronal cells. In this study, we investigated if the engagement of N-cadherin participates to the control of neuronal cells survival/death balance. We observed that plating either primary mouse spinal cord neurons or primary rat hippocampal neurons on N-cadherin recombinant substrate greatly enhances their survival compared to non-specific adhesion on poly-L- lysine. We show that N-cadherin engagement, in the absence of other survival factors (cell-matrix interactions and serum), protects GT1-7 neuronal cells against apoptosis. Using this cell line, we then searched for the signaling pathways involved in the survival effect of N-cadherin engagement. The PI3-kinase/Akt survival pathway and its downstream effector Bad are not involved, as no phosphorylation of Akt or Bad proteins in response to N-cadherin engagement was observed. In contrast, N- cadherin engagement activated the Erk1/2 MAP kinase pathway. Moreover, N-cadherin ligation mediated a 2-fold decrease in the level of the pro-apoptotic protein Bim-EL whereas the level of the anti-apoptotic protein Bcl-2 was unchanged. Inhibition of Mek1/2 kinases with U0126, and the resulting inhibition of Erk1/2 phosphorylation, induced the increase of both the level of Bim-EL and apoptosis of cells seeded on the N-cadherin substrate, suggesting that Erk phosphorylation is necessary for cell survival. Finally, the overexpression of a phosphorylation defective form of Bim-EL prevented N-cadherin- engagement induced cell survival. In conclusion, our results show that N-cadherin engagement mediates neuronal cell survival by enhancing the MAP kinase pathway and down-regulating the pro-apoptotic protein Bim-EL. Citation: Lelie`vre EC, Plestant C, Boscher C, Wolff E, Me`ge R-M, et al. (2012) N-Cadherin Mediates Neuronal Cell Survival through Bim Down-Regulation. PLoS ONE 7(3): e33206. doi:10.1371/journal.pone.0033206 Editor: Masuko Ushio-Fukai, University of Illinois at Chicago, United States of America Received December 9, 2010; Accepted February 11, 2012; Published March 12, 2012 Copyright: 2012 Lelie`vre et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by institutional funding from INSERM (Institut National de la Sante´ et Recherche Me´dicale), Universite´ Pierre et Marie Curie, as well as grants from AFM (Association Franc¸aise contre les Myopathies) and ARC (Association Franc¸aise de Recherche contre le Cancer). Dr. Lelie`vre, Dr. Plestant and Dr. Boscher have been supported by French Research Ministry graduate student fellowships. ARC provided also Dr. Boscher with a PhD fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: rene-marc.mege@inserm.fr (RMM); helene.birbes@inserm.fr (HB) . These authors contributed equally to this work. " These authors also contributed equally to this work. ¤ Current address: Department of Cellular and Physiological Sciences, Life Sciences Institute, University of British Columbia, Vancouver, British Columbia, Canada Introduction Cadherins are a family of transmembrane proteins that mediate calcium-dependent homophilic cell-cell contacts. They provide anchorage in between neighboring cells by interacting with the actin cytoskeleton through catenins [1]. Cadherins play crucial roles in embryonic morphogenesis by affecting cell shape, differentiation, migration and proliferation [2]. In addition, cadherin-mediated cell-cell interactions may regulate the balance between cell survival and cell death during development and maintenance of tissue homeostasis. Indeed several studies have reported that disruption of cadherin adhesion initiates apoptosis in various epithelial [3,4,5] and tumor cells [6,7,8,9]. However, the mechanisms whereby cadherin adhesion contributes to cell fate by regulating the survival/cell death balance is poorly understood by comparison to the well-known molecular mechanism of ano¨ıkis (apoptosis induced by the loss of cell contacts with the extracellular matrix (ECM)) [10]. Depending on cell types, the initiation and execution of ano¨ıkis, which is refrained in physiological conditions by integrin engagement, is mediated by different pathways that all converge to the regulation of anti-apoptotic and pro-apoptotic Bcl- 2 family proteins, leading to activation of caspases and subsequent activation of endonucleases, DNA fragmentation and eventually cell death [10]. Proteins of the Bcl-2 family are major regulators of apoptosis [11]. Anti-apoptotic members, such as Bcl-2, Bcl-xL, and Mcl-1 contain four Bcl-2 homology (BH) domains while the pro- apoptotic proteins fall into two categories: the Bax-like proteins, such as Bax and Bak that contain multiple BH domains, and the BH3-only members, Bad, Bid, Bmf, and Bim that contain only the BH3 domain. These pro-apoptotic proteins promote the release of apoptogenic factors from the mitochondria. Several studies PLoS ONE | www.plosone.org 1 March 2012 | Volume 7 | Issue 3 | e33206