Necdin Protects Embryonic Motoneurons from Programmed Cell Death Julianne Aebischer1,2, Rachel Sturny2,3, David Andrieu2,4, Anne Rieusset2,4, Fabienne Schaller2,4, Sandrine Geib2,4, Ce´ dric Raoul1,2, Franc¸ oise Muscatelli2,4* 1 Inserm-Avenir, Mediterranean Institute of Neurobiology, INMED, Marseille, France, 2 Universite´ d’Aix-Marseille, Faculte´ des Sciences, Marseille, France, 3 Developmental Biology Institute of Marseille Luminy, IBDML, Marseille, France, 4 Inserm U901, Mediterranean Institute of Neurobiology, INMED, Campus scientifique de Luminy, Marseille, France Abstract NECDIN belongs to the type II Melanoma Associated Antigen Gene Expression gene family and is located in the Prader-Willi Syndrome (PWS) critical region. Necdin-deficient mice develop symptoms of PWS, including a sensory and motor deficit. However, the mechanisms underlying the motor deficit remain elusive. Here, we show that the genetic ablation of Necdin, whose expression is restricted to post-mitotic neurons in the spinal cord during development, leads to a loss of 31% of specified motoneurons. The increased neuronal loss occurs during the period of naturally-occurring cell death and is not confined to specific pools of motoneurons. To better understand the role of Necdin during the period of programmed cell death of motoneurons we used embryonic spinal cord explants and primary motoneuron cultures from Necdin-deficient mice. Interestingly, while Necdin-deficient motoneurons present the same survival response to neurotrophic factors, we demonstrate that deletion of Necdin leads to an increased susceptibility of motoneurons to neurotrophic factor deprivation. We show that by neutralizing TNFa this increased susceptibility of Necdin-deficient motoneurons to trophic factor deprivation can be reduced to the normal level. We propose that Necdin is implicated through the TNF-receptor 1 pathway in the developmental death of motoneurons. Citation: Aebischer J, Sturny R, Andrieu D, Rieusset A, Schaller F, et al. (2011) Necdin Protects Embryonic Motoneurons from Programmed Cell Death. PLoS ONE 6(9): e23764. doi:10.1371/journal.pone.0023764 Editor: Eric J. Kremer, French National Centre for Scientific Research, France Received May 26, 2011; Accepted July 25, 2011; Published September 2, 2011 Copyright: 2011 Aebischer et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Association Franc¸aise contre les Myopathies (AFM, grant no. 14884), and the European Community (grant no. 512136 PWS). JA is a recipient of a Ph.D fellowship from PACA conseil regional, Trophos SA and AFM. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: muscatelli@inmed.univ-mrs.fr Introduction The human NECDIN gene is one of the five genes that have been associated with the Prader-Willi syndrome (PWS) [1,2,3], a rare genetic neurodevelopmental disease characterized by a variety of physical, cognitive, and behavioral defects. The most significant characteristics at birth are feeding problems, severe hypotonia, breathing alterations and hypogonadism. Later, PW infants become obese, present a short stature, a motor delay and cognitive deficits [4]. A potential role of NECDIN in the etiology of PWS is supported by studies of three mouse models [5,6,7] in which the Necdin gene has been inactivated resulting in alterations of breathing and behavior, defects of the sensory system and in the hypothalamic nuclei. Similar alterations are described in Prader- Willi patients suggesting that NECDIN is responsible for specific Prader-Willi symptoms. Necdin belongs to the type II Melanoma-Associated antigen Gene Expression (MAGE) family that shares a MAGE homology domain [8], which has recently been shown to bind RING proteins to form active E3 Ubiquitin Ligases [9]. Necdin locates in the nucleus and/or cytoplasm and has been reported to interact with cell cycle proteins (p53, E2F1, E2F2) [10,11,12,13,14], transmembrane proteins (p75NTR, TrkA, Nogo) [7,15,16,17], and cytoplasmic interactors (MAGED1, FEZ1, BBS4, NEFA) [18,19]. Through these interactions, Necdin has been proposed to participate in a broad range of biological activities including cell growth, migration, differentiation and cell death/survival (see for review [20]). However the precise molecular function as well as the physiological relevance of Necdin in those processes remains largely unknown. In the mouse nervous system, in vivo studies demonstrated Necdin involvement in cellular migration, axonal outgrowth and fasciculation processes [19,21,22,23,24,25] as well as in neuronal apoptosis [14,23,26,27]. We and others documented an anti- apoptotic function of Necdin in developing sensory neurons of the dorsal root ganglia (DRG) [7,26,27]. Interestingly, we showed that its anti-apoptotic function is restricted to a subpopulation of sensory neurons. Indeed, we showed that the abrogation of Necdin, in the lumbar region, triggered a 40% increase of post- mitotic apoptosis during the embryonic wave of naturally occurring cell death. This additional cell death resulted in a 30% loss of specified TrkA (nociception) and TrkC (propriocep- tion) sensory neurons although the TrkB population (mechanore- ception) was not modified. Beside the sensory deficits previously described in mutant mice, our study suggested that motor functions might also be deficient [27]. Here, we have further investigated the contribution of Necdin in the development of spinal motoneurons. We found that the genetic PLoS ONE | www.plosone.org 1 September 2011 | Volume 6 | Issue 9 | e23764