Necdin Protects Motoneurons from Death Figure 8. A proposed model for the anti-apoptotic role of Necdin in developing motoneurons. During embryogenesis, the development of motoneurons starts by a proliferation step in which Necdin is not expressed. These neuronal precursors differentiate into post-mitotic neurons and start to express Necdin (E10.5). These pre-specified motoneurons become sensitive to external death factors and in a normal situation (A) half of them will die and half of them will survive thanks to NTFs. In absence of Necdin (B), motoneurons are more sensitive to the TNFa death factor and an increase in cell death is observed. doi:10.1371/journal.pone.0023764.g008 during this critical period and we also showed a TNFR1 expression at E12.5 (and not at E11.5) in lumbar motoneurons. In the absence of Necdin, a TNF-dependent process could be exacerbated. Necdin and TNFa/TNFR1 pathway Using cultures of E12.5 spinal cord explants and motoneurons, we observed that in the absence of NTFs, motoneuron survival was significantly lower (about 40%) in the absence of Necdin than in wildtype. This difference in neuron viability, which is similar to that observed in vivo, suggests that Necdin-deficient motoneurons have an increased susceptibility to death factor(s)/death receptors signaling. The first Necdin interactor to consider was the receptor p75NTR. p75NTR is expressed in rat developing motoneurons during the wave of PCD [41], and also in the spinal cord of wild- type and Necdin-deficient embryos at E13.5 (data not shown). Necdin interacts with p75NTR [15,42] and a common signaling pathway involving both proteins and allowing the internalization of p75NTR has been proposed [43]. However, mice lacking p75NTR did not shown any loss of spinal cord motoneurons at P7 [44] neither an increase of PCD at E13.5 [33]. In developing sensory neurons of dorsal root ganglia of Necdin-deficient embryos, we previously described a similar increased cell death during the wave of PCD; however, this increased cell death does not affect the number of p75NTR expressing neurons [27]. Altogether, these data indicate that Necdin might also function in a p75NTR-independent manner to prevent motoneuron developmental cell death in vivo. Based on our data, we propose that the TNFa/TNFR death signaling pathway is involved in the increased cell death observed in Necdin-deficient motoneurons. It has previously been proposed that Necdin intervenes in the TNFa pathway [35]. The authors showed that in primary myoblasts isolated from Necdin-deficient mice, the TNFa pathway inhibits, at least in part, their myogenic differentiation. Interestingly, these mutant myoblasts expressed a higher amount of TNFR1 at the plasma membrane, as measured by fluorescence-activated cell sorting (FACS) analysis, although the quantity of the Tnfr1 mRNA was not increased [35]. Taken together, these data suggest that an increase in TNFR1 receptors at the plasma membrane of motoneurons might also explain why Necdin-deficient motoneurons have an increased sensitivity to TNFa in the window of time in which PCD occurs. In our hands, primary cultured motoneurons did not survive FACS analysis and we were not able to reliably quantify the levels of TNFR1 receptor at the plasma membrane by immunolabeling. Necdin interaction with the TNFR1 signaling pathway The constitutive or inducible TNFR1 release to the extracellular compartment plays a role in the control of TNFR1 at the cell membrane and can also regulate the TNFa activity by the generation of extracellular receptors that function as TNFa- binding proteins [45]. In human vascular endothelial cells, it has been shown that extracellular TNFR1 release requires the calcium-dependent formation of a Nucleobindin-2 (NucB2)- aminopeptidase regulator of TNFR1 shedding (Arts-1) complex associated with TNFR1 [46]. Interestingly, it has previously been shown that Necdin interacts with NucB2, thereby participating in the control of Ca2+ homeostasis in neuronal cytoplasm [18]. We therefore hypothesized that, indirectly, Necdin might control the TNFR1 release via regulation of the NucB2-Arts-1 complex. First, we performed immunofluorescence confocal laser- scanning microscopy experiments in order to check whether NucB2 and Arts-1 are expressed in E12.5 developing motoneu- rons and to search for a colocalization of Necdin, NucB2, Arts-1, and TNFR1. We found that TNFR1 colocalized partially with PLoS ONE | www.plosone.org 9 September 2011 | Volume 6 | Issue 9 | e23764