Figure 4. N-cadherin engagement protects neuronal cells from nuclear condensation and cytochrome c release. Serum-starved GT1-7 cells were seeded at low density on FN, N-cad or PL substrates and grown in serum-free medium. After 24 hours, cells were fixed and immunostained for cytochrome c (right) while nuclei were stained with DAPI (left). Arrows point toward cells showing condensed nuclei and diffuse or absence of cytochrome staining while asterisks indicate cells with healthy nuclei and mitochondrial cytochrome staining. Scale bar: 20 mm. doi:10.1371/journal.pone.0033206.g004 engagement, we analyzed cell death by two complementary approaches: (i) TUNEL staining and (ii) PARP cleavage. After 4 hours of culture, the number of TUNEL positive GT1-7 cells was significantly higher on PL (8.660.9%) than on N-cad (2.160.6%) indicating that the deprivation of specific cell adhesion (PL substrate) induced apoptotic cell death (Fig. 5C). Moreover, when GT1-7 cells were seeded on PL, PARP cleavage was detected by the appearance of the characteristic 85 kDa fragment. By contrast, cells seeded on N-cad showed a reduced PARP cleavage (Fig. 5D). Altogether, these results indicate that N- cadherin engagement is sufficient to sustain GT1-7 cell survival by protecting them from apoptotic cell death. PI3-kinase/Akt pathway is not involved in N-cadherin engagement To elucidate the pathways involved in the protective effects of N-cadherin mobilization, we studied the activation of the PI3- kinase/Akt pathway, a key regulator of cell survival. First, we investigated whether N-cadherin mobilization could activate Akt kinase by phosphorylation on Ser-473. We previously showed that cells require at least 2 hours in order to spread on N-cadherin substrate; therefore, Akt phosphorylation was investigated by western blotting analysis using a phospho-specific antibody starting at 2 hours after plating. As cell death had reached a plateau at 6 hours, phosphorylation studies were conducted up to 6 hours. N-Cadherin Anti-Apoptotic Signaling Spreading of cells on N-cad did not affect Akt phosphorylation at any time point studied (Fig. 6A). To confirm these results, we used a calcium switch approach with GT1-7 grown at confluency. Although less specific than Ncad-Fc mediated engagement, calcium restoration rapidly initiates cadherin-dependent cell-cell contact formation [39] allowing study of earlier time points. However, Akt phosphorylation was not changed even at earlier time points (Fig. 6B), confirming that Akt phosphorylation was not activated by N-cadherin engagement. Alternatively, Akt could be a direct substrate for caspases. Indeed, it has been shown that Akt is cleaved by caspases leading to a reduction in Akt protein level in MDCK cells detached from the ECM. In turn this cleavage of Akt contributes to ano¨ıkis [40]. To test this hypothesis, we assessed the total amount of Akt at various time points, but cellular levels of total Akt protein were not decreased on PL substrate compared with those on N-cad (Fig. 6C). Altogether, these observations suggest that the PI3- kinase/Akt pathway is not involved in the anti-apoptotic effect of N-cadherin in GT1-7 cells. In addition, the PI3-kinase inhibitor LY294002 had no effect on cell survival, nuclear condensation and TUNEL labeling of cell seeded on N-cad (data not shown). Thus, we investigated the involvement of other pathways, such as MAP kinase signaling, which has been reported to mediate cell survival [10]. MAP kinase Erk signaling is required for cell survival induced by N-cadherin engagement To examine whether N-cadherin engagement could trigger a signal transduction pathway involving the MAP kinases, we evaluated the phosphorylation state of Erk1 and Erk2 using a phospho-specific antibody. The phosphorylation of Erk1/2 was increased in a time-dependent manner in cells seeded on N-cad substrate as compared to cells seeded on PL. Phosphorylation of Erk1 and Erk2 was maximal at 4 hours with a 2.5 fold increase compared to cells seeded on PL (Fig. 7A). Furthermore, the restoration of intercellular adhesion in the calcium switch assay induced a strong elevation of Erk1 and Erk2 phosphorylation with a 6-fold increase observed at 30 min (Fig. 7B). These results support an activation of the Erk1/2 pathway in response to N- cadherin engagement in accordance with our previous report on the activation of Erk1/2 by N-cadherin engagement in primary spinal cord neurons. To confirm the involvement of the MAP kinase pathway in relaying the anti-apoptotic signal triggered by N-cadherin engagement, we examined the effect of U0126, a specific inhibitor of the Mek1/2 kinase activity. We used this inhibitor at 20 mM. Indeed this concentration was sufficient to abolish Erk1/2 phosphorylation during the calcium switch (Fig. S7). U0126- treated cells were seeded either on PL or N-cad and apoptotic cells were scored at 6 and 24 hours using nuclear condensation and cytochrome c release markers (Fig. 8). U0126 treatment did not affect cell death on PL. However, this treatment reversed the protective effects of N-cadherin since a similar apoptosis level as for cells seeded on PL was observed on the Ncad substrate (Fig. 8B, C). These results indicate that N-cadherin protection is lost in the presence of Mek1/2 inhibitor and support the hypothesis that the Erk1/2 pathway is involved cell survival downstream of N- cadherin engagement. N-cadherin engagement does not regulate Bcl-2 or Bad protein levels We next aimed at determining the downstream targets of the MAP kinases involved in cell survival pathway induced by the N- PLoS ONE | www.plosone.org 7 March 2012 | Volume 7 | Issue 3 | e33206