Necdin Protects Motoneurons from Death Figure 6. Necdin deficiency decreases motoneuron survival in absence of NTF but does not alter axonal growth. (A) Embryonic motoneurons isolated from Necdin+/+and Necdin+/2 mice were cultured in the presence (+NTFs) or in the absence (2NTFs) of a cocktail of NTFs. After 24 hours, surviving motoneurons were counted and for each genotype survival is expressed as the percentage of the number of motoneurons surviving in the presence of NTF. In absence of NTFs a significant decrease of surviving motoneurons is observed in mutant compared to wildtype motoneurons. (B) When cultured for 3 days in the presence of NTFs Tau immunolabeling reveals a similar morphology between both genotypes. (C) When measuring, axonal length no difference is detected between mutant (Necdin+/2) and wildtype (Necdin+/+) motoneurons. Values are means 6 S.D (in (A) or 6 standard error of the mean in (B). doi:10.1371/journal.pone.0023764.g006 death during the wave of PCD but no, or limited compensatory mechanisms take place. Our results are in agreement with a model (Figure 8), in which newly generated post-mitotic motoneurons are committed to undergo programmed cell death very early, before they innervate their target muscles. The death program would not be completed if they gain access to trophic support (around 50% of them). In the remaining proportion of motoneurons that fail to access NTFs, the death program would be completed and cells eliminated. In the light of our results, we can propose that Necdin may act as an anti- apoptotic molecule that maintains post-mitotic motoneurons competent to respond to neurotrophic signals (Figure 8). Sedel, F. and colleagues proposed that TNFa can act as an extrinsic signal that commits motoneuron to a cell death fate [34], at least at the brachial level, two days before the period of cell death begins. Interestingly, at the brachial level, TNFR1 is transiently expressed Figure 7. Increase of motoneuron cell death in absence of Necdin is linked to the TNFR1 pathway. (A) When cultured in presence of NTFs Necdin wildtype (Necdin+/+) and mutant (Necdin+/2) motoneurons are equally sensitive to death triggered by TNF alpha. Embryonic mutant and wildtype motoneurons were cultured in presence of NTFs for 24 hours and then treated or not with TNFa. After 48 hours of treatment motoneuron survival was assessed and expressed relative to non-treated cells. (B) In the absence of NTFs, TNFR1-Fc does not rescue cell death observed in wildtype motoneurons, but it restores the level of cell death in Necdin deficient motoneurons to the level observed in wild type cells. Wildtype or mutant motoneurons were cultured in presence (+) or absence (2) of NTFs and treated or not with 100 ng/ml of TNFR1-Fc 1 hours after seeding. Motoneuron survival was determined 24 hours after treatment and expressed as the percentage of the number of motoneurons surviving in the presence of NTF under same conditions. These results suggest that the increase of cell death observed in Necdin-deficient motoneurons is linked to the TNFa/TNFR1 pathway. Histograms show mean values 6 S.D of triplicate wells in at least three independent experiments. doi:10.1371/journal.pone.0023764.g007 PLoS ONE | www.plosone.org 8 September 2011 | Volume 6 | Issue 9 | e23764