N-Cadherin Anti-Apoptotic Signaling Figure 1. N-cadherin engagement sustains spinal cord and hippocampal neuronal cell survival in minimal medium conditions. Neurons were dissociated from E18.5 rat hippocampus (A) or E12.5 mouse ventral spinal cords (B) and cultured on N-cad or PL in MEM and Neurobasal basal medium, for hippocampal and spinal neurons, respectively. After 24 hours, they were fixed, and stained with anti-bIII tubulin antibody to identify neurons and with DAPI to assess the nuclear morphology. Arrows point toward cells dying with condensed nuclei while asterisks indicate cells with healthy nuclei. Scale bar: 10 mm in A; 20 mm in B. doi:10.1371/journal.pone.0033206.g001 neurons die, likely by apoptosis, in these stringent conditions when plated on PL, while they displayed a high survival rate when plated on N-cad. They suggest thus that N-cadherin engagement promotes their survival. N-cadherin engagement protects GT1-7 cells from undergoing apoptosis However, molecular and biochemical studies on primary culture models are difficult to perform due to the limited amount of biological material. Therefore to study the molecular pathway whereby N-cadherin mediates cell survival, we decided to use GT1-7 immortalized hypothalamic neurons [34], which express N-cadherin. These cells usually grow as confluent layers in serum- supplemented media which provides them cell-cell adhesion as well as diffusive (growth factors) and anchorage-dependent (fibronectin/vitronectin) survival factors. To investigate the contribution of cadherin-mediated cell-cell adhesion to neuronal cell survival, we forced serum-starved GT1-7 cells to grow in suspension by plating them on agarose-coated dishes, as previously reported [7,8]. We then manipulated extracellular calcium concentrations to allow (1.8 mM Ca2+) or inhibit (0 mM free Ca2+) calcium–dependent cell-cell adhesion. The presence of a physiological concentration of Ca2+ induced the expected cell aggregation, while GT1-7 cells only loosely aggregated in the absence of free Ca2+ consistent with a massive inhibition of cell-cell adhesion (Fig. 3A). To determine the involvement of calcium-dependent cell adhesion on cell survival, we then analyzed the cleavage of poly (ADP-ribose) polymerase (PARP) induced in apoptotic cells by activated caspases. The inhibition of calcium-dependent cell-cell adhesion specifically induced the PARP cleavage whereas PARP cleavage remained very low in cells cultured in the presence of calcium (Fig. 3B). These results show that cells forced to grow under serum-free anchorage-independent conditions survive as multicellular aggre- gates and that Ca2+-dependent cell adhesion sustain cell survival in these conditions, as previously reported in other cellular models [7,8]. Next, we set up more controlled cell culture conditions to investigate specifically the effect of N-cadherin adhesion on cell survival. GT1-7 cells were seeded either on N-cad substrate to specifically engage N-cadherin homophilic liganding or on PL to deprive them of specific adhesion, in (i) serum-free conditions to exclude cell-ECM and growth factors activated signaling, and (ii) at low density to prevent uncontrolled cell-cell contact formation (Fig. S3). In some control experiments, cells were seeded on fibronectin substrate (FN) to allow for controlled integrin mobilization, a situation known to mediate cell survival. GT1-7 cell death was first evaluated by the nuclear condensa- tion after DNA staining (Fig. 4). 72% of cells seeded on PL were positive for nuclear condensation whereas only 18% of cells seeded on N-cad exhibited condensed nuclei after 24 hours (Fig. 5A). Interestingly, a similar low proportion of condensed nuclei (,20%) was also obtained among cells seeded on fibronectin substrate (FN), where cell-matrix-anchorage was expected to induce an integrin-mediated cell survival pathway. To exclude the possibility that cell survival observed on N-cad was the consequence of integrin engagement induced by the deposition of ECM components by GT1-7 cells themselves, we evaluated integrin pathway activation by phosphorylated FAK and paxillin immu- nostaining. We did not detect paxillin (Fig. S4) nor phosphorylated forms of FAK (data not shown) accumulation at the ventral face of the cells seeded on PL or N-cad while both stainings were strongly Figure 2. N-cadherin engagement sustains spinal cord and hippocampal neuronal cell survival. (A) In 24 hours hippocampal cultures, the number of bIII tubulin-positive cells per mm2 were counted on each substrate and expressed as a percentage of that on N-cad substrates, arbitrarily fixed to 100%. (B) The numbers of bIII tubulin-positive cells with condensed (white bars) or non-condensed nuclei (dark bars) were determined in ventral spinal cords cultures after 24 hours on each substrate. The number of bIII tubulin-positive cells per mm2 was expressed as a percentage of the number of bIII tubulin-positive cells per mm2 on N-cad substrates, arbitrarily fixed to 100%. Results are expressed as the mean 6 SD of two independent experiments. doi:10.1371/journal.pone.0033206.g002 PLoS ONE | www.plosone.org 5 March 2012 | Volume 7 | Issue 3 | e33206