Necdin Protects Motoneurons from Death Figure 2. Loss of lumbar specified motoneurons during embryonic development. Transverse sections of E11.5 (A) and E17.5 (C) lumbar spinal cords were immunolabeled with anti-Islet-1/-2 antibodies. (A) and (B) At E11.5, when pools of post-mitotic motoneurons are generated, no significant difference in the number of Islet-1/-2 positive cells/section is detected between the ventral horn spinal cords of mutant (Necdin+/2) and control (Necdin+/+) mice. (C) and (D) At E17.5, when motoneurons are specified, a significant difference in the number of Islet-1/-2 positive cells/ section is observed between both genotypes, with a loss of 31% of cells in Necdin-deficient mice (Necdin+/2). Scale bar, 50 mm. doi:10.1371/journal.pone.0023764.g002 death receptors, which can be activated following NTF depriva- tion [31]. In the first hypothesis, we should observe, in the presence of trophic factors, an increased motoneuron death in Necdin-deficient explants compared to wildtype explants, whereas in the absence of NTFs, no significant difference in neuron survival between mutant and wildtype explants should be found. In the second hypothesis, the presence of trophic factors should lead to a similar survival rate of motoneurons in both mutant and wildtype explants, whereas in the absence of trophic factors, we should observe an increased susceptibility to endogenous death factors in Necdin-deficient explants. We therefore cultured ventral lumbar spinal cord explants from E12.5 mutant or wildtype embryos in the presence or in the absence of a cocktail of NTFs (Glial cell line Derived Neurotrophic Factor, GDNF; Brain- derived neurotrophic factor, BDNF and Ciliary Neurotrophic Factor, CNTF) and determined motoneuron survival by counting Islet-1/-2 positive cells (Figure 4A). We found that in the presence of NTFs, the number of surviving motoneurons was not significantly different between wildtype and Necdin-deficient explants (Necdin+/+: 71 (59, 72), n = 5; Necdin+/2: 57 (56, 62), n = 5, n.s) (Figure 4B). However, in the absence of NTFs the number of surviving motoneurons appeared significantly different with a decrease of 43% of surviving cells in Necdin- deficient explants compared with wildtype explants (Necdin+/+: 26 (25, 29), n = 8; Necdin+/2: 17 (15, 21), n = 8; P,0.01) (Figure 4C). We therefore propose that Necdin acts by counteracting death signals rather than by potentiating the action of NTFs in motoneurons. TNFR1 expression correlates with Necdin expression in the lumbar spinal cord We next focused on the identity of the potential death receptor involved in the increased death of motoneurons in Necdin-deficient mice. A first potential candidate was the Unc5A receptor, since it has been shown to interact with MageD1, a MAGE gene with homology to Necdin, and to promote motoneuron death in the cervical spinal cord during PCD [32]. Since MageD1 is also expressed in lumbar motoneurons at E13.5 where, in contrast to Necdin, it plays a pro-apoptotic role [33], we hypothesized that Necdin might interact with Unc5A to interfere with the pro- apoptotic signal. Using an antibody specific to Unc5A, we analyzed its expression pattern, at E12.5, along the rostro-caudal axis. We detected a strong Unc5A immunoreactivity in the ventral part of the brachial spinal cord, which was reduced in the thoracic region. However, we were not able to detect any Unc5A expression in the lumbar region of the spinal cord (Figure 5A), indicating that Unc5A unlikely acts as a death receptor involved in the increase of PCD in Necdin mutants. A second potential candidate was TNFR1. Indeed, it has been shown that, in the brachial region of the spinal cord, TNFa via TNFR1 commits developing motoneurons to cell death before PCD [34]. In addition, Necdin has been proposed to intervene in the TNFa pathway in myoblasts [35]. Interestingly, we found that TNFR1 was expressed in the ventral part of E12.5 spinal cords (Figure 5B) and showed that TNFR1-positive cells were Necdin- positive neurons (Figure 5C). Using mice expressing the green fluorescent protein (GFP) under the control of the motoneuron- PLoS ONE | www.plosone.org 4 September 2011 | Volume 6 | Issue 9 | e23764