Necdin Protects Motoneurons from Death Figure 4. Role of trophic factors in motoneuron survival of Necdin-deficient E12.5 embryos. (A) Dissections of the lumbar spinal cord regions were performed at E12.5 and the explants were cultured in the presence or in the absence of NTFs for 2 days. Cryostat transverse sections of explants were then immunolabeled using Islet-1/-2 antibodies (green) in order to define the number of surviving motoneurons per explant. (B) In the presence of NTFs (+NTFs), the number of Islet-1/-2 positive cells is not significantly higher in mutant (Necdin+/2) explants (n = 5) compared with wildtype (Necdin+/+) explants (n = 5). (C) In the absence of NTFs (2NTFs), a significant 35% decrease of Islet-1/-2 positive cells is observed in Necdin- deficient (Necdin+/2) explants (n = 8) compared to wildtype (Necdin+/+) explants (n = 8), suggesting that the increase of cell death in Necdin-deficient motoneurons is not dependant on NTFs. Scale bar: 100 mm. doi:10.1371/journal.pone.0023764.g004 for 24 h and then treated for 48 hrs with TNFa at a concentration that has previously been demonstrated to efficiently kill motoneu- rons [36]. We observed that Necdin-deficient and wildtype motoneurons were killed at a similar rate (about 60%; data not shown). Since we proposed that Necdin acts by interfering with extrinsic death signals in the absence of NTFs, we performed a competition assay against endogenous TNFa in motoneuron cultures maintained in the presence or absence of NTFs, using a soluble chimeric protein (TNFR1-Fc) that impedes TNFa-TNFR1 interaction. In wildtype motoneurons, we observed that TNFR1- Fc did not influence motoneuron survival either in the presence or the absence of NTFs. In Necdin-deficient motoneurons, we observed that, in the absence of NTFs, TNFR1-Fc rescued motoneurons to the level observed in wildtype motoneurons, cultured under the same conditions (none: 31.5 (21, 34), n = 3 and TNFR1-Fc: 45.6 (41.3, 57.2), n = 3; P,0.05) (Figure 7B). By interfering with TNFa-TNFR1 signaling, we can restore a normal rate of death in mutant motoneurons, suggesting a role for Necdin in the TNFR1 signaling pathway. Altogether, our results provide evidence that Necdin contributes to motoneuron survival and suggest that Necdin tunes sensitivity to TNFR1 signaling in motoneurons. Discussion This study aimed at investigating the contribution of Necdin in establishing and/or maintaining motor functions. Having previ- ously observed that Necdin-deficient pups have a motor deficit that impairs hindlimb movement, we show here that Necdin plays an important role during the development of the motor system, Necdin-deficient mice lacking approximately 30% of their lumbar motoneurons. Our first observation was that Necdin is expressed in all post- mitotic motoneurons along the rostro-caudal axis as early as E10.5 and that its expression is maintained until postnatal ages. At E17.5, which corresponds to the end of the period of naturally-occurring cell death in the lumbar spinal cords, we observed a loss of 31% of specified motoneurons in Necdin-deficient mice. However, at E11.5, prior to the period of PCD, the pools of post-mitotic motoneurons were similar between both mutant and wildtype mice. During the wave of PCD, we further revealed a significant 40% increase in cell death in the lumbar region of Necdin-deficient spinal cords (Figure 8). It is intriguing that although there is an expression of Necdin in all postmitotic spinal motoneurons, an increased developmental death is observed only at the lumbar level in Necdin-deficient mice. Indeed, at E12.5, quantification of the number of cleaved caspase-3 positive motoneurons in the brachial and thoracic spinal cord gave similar results between wildtype and mutant mice. Significantly, we previously reported an increase in PCD of developing sensory neurons specifically in the lumbar DRGs. Although, a concomitant sensory-motor pathology has already been described in spinal and bulbar muscular atrophy, distal spinal muscular atrophy type V or Charcot-Marie-Tooth disease type 2D [37,38], restriction of the Necdin-associated pathology to the lumbar region remains unreported. Longitudinal gene profiling or proteomic analyses on different regions of the spinal cord during development would help us to identify factors governing such specificity. Necdin and the developmental cell death of motoneurons Motoneurons are generated in excess in the developing spinal cord and about 50% of them are eliminated during a phase of PLoS ONE | www.plosone.org 6 September 2011 | Volume 6 | Issue 9 | e23764