Nogo receptor antagonizes p75NTR-dependent motor neuron death Luc Dupuis*†, Mariana Pehar‡, Patricia Cassina§, Fre´ de´ rique Rene*†, Raquel Castellanos§, Caroline Rouaux*†, Mandi Gandelman‡, Leda Dimou¶, Martin E. Schwab¶, Jean-Philippe Loeffler*†, Luis Barbeito‡, and Jose-Luis Gonzalez de Aguilar*†ʈ *Institut National de la Sante´et de la Recherche Me´dicale, U692, Laboratoire de Signalisations Mole´culaires et Neurode´ge´ne´ rescence, Strasbourg, F-67085 France; †Universite´Louis Pasteur, Faculte´deMe´ decine, UMRS692, Strasbourg, F-67085 France; ‡Departamento de Neurobiologı´aCelular, Institut Pasteur, Montevideo, 11600 Uruguay; §Faculdad de Medicina, Universidad de la Repu´blica, Montevideo, 11800 Uruguay; and ¶Brain Research Institute, University of Zurich and Department of Biology, Eidgeno¨ssiche Technische Hochschule, Zurich, 8057 Switzerland Edited by Don W. Cleveland, University of California at San Diego, La Jolla, CA, and approved November 28, 2007 (received for review April 26, 2007) The Nogo-66 receptor (NgR) plays a critical role in restricting axon All of the above studies clearly indicate that p75NTR can regeneration in the central nervous system. This inhibitory action trigger either neuronal death, when stimulated by NGF, or is in part mediated by a neuronal receptor complex containing neurite outgrowth inhibition, when bound to NgR. The cross- p75NTR, a multifunctional receptor also well known to trigger cell talk between these two pathways has not been explored so far. death upon binding to neurotrophins such as NGF. In the present In the present study, we asked whether NgR could regulate study, we show that Pep4 and NEP1–40, which are two peptides motor neuron survival by modulating p75NTR-induced cell derived from the Nogo-66 sequence that modulate NgR-mediated death under conditions in which both receptor partners should neurite outgrowth inhibition, prevent NGF-stimulated p75NTR- appear coexpressed. The results presented herein provide com- dependent death of cultured embryonic motor neurons. They also pelling evidence that two peptides derived from the Nogo-66 confer protection on spinal cord motor neurons after neonatal sequence can prevent p75NTR-dependent cell death of embry- sciatic nerve axotomy. These findings demonstrate an as-yet- onic cultured motor neurons. These peptides also conferred unknown function of NgR in maintaining neuronal survival that neuroprotection after neonate sciatic nerve axotomy. may be relevant for motor neuron development and degeneration. Results The NgR Pathway Is Expressed and Functional in Motor Neurons. As NGF ͉ NgR ͉ amyotrophic lateral sclerosis ͉ axotomy ͉ reticulon a first step toward understanding the interactions between NgR and p75NTR pathways, we determined whether both receptors he glycosylphosphatidylinositol (GPI)-anchored Nogo-66 re- were coexpressed in our experimental paradigm. In embryonic Tceptor (NgR) binds various structurally unrelated proteins: day 16 (E16) mouse embryos, choline acetyl transferase (ChAT) Nogo-A, myelin-associated glycoprotein, and oligodendrocyte- immunoreactive motor neurons in the ventral horns of the spinal myelin glycoprotein. These myelin-derived proteins inhibit neu- cord were also positive for NgR (Fig. 1A), and this staining rite outgrowth in vitro, and Nogo-A also prevents axonal regen- persisted in neonate and adult spinal cord motor neurons eration in vivo after injury to the adult mammalian CNS (1). NgR [supporting information (SI) Fig. 6]. We also checked which forms a ternary receptor complex with the leucine-rich repeat components of the NgR receptor complex were present in transmembrane protein LINGO-1 (2) and either the neurotro- cultures of purified embryonic motor neurons and NSC34 cells, phin receptor p75NTR (3, 4) or the orphan TNF receptor family a motor neuron-like cell line. NgR, LINGO-1, and p75NTR member TAJ/TROY (5, 6). Downstream effectors of this recep- mRNAs were all expressed in these cultures; in turn, TAJ/TROY tor complex have not been completely elucidated but may mRNA, present in whole spinal cord extracts, appeared unde- include RhoA/Rho-kinase (7), PKC (8), and transactivation of tectable in vitro (Fig. 1B). Moreover, all cultured motor neurons the EGF receptor (9). Although the role of NgR in restricting the presented with both p75NTR and NgR immunoreactivity (Fig. growth and repair of CNS axons has been clearly demonstrated 1C; two independent experiments were analyzed), thus indicat- (10–12), knockout mice for NgR display only subtle regeneration ing that NgR activation could involve p75NTR in this paradigm. of tracts upon spinal cord trauma (13, 14), most probably because To ascertain whether NgR/LINGO-1/p75NTR signaling is of compensatory mechanisms. functionally active in cultured embryonic motor neurons, we p75NTR is a versatile protein known to potentiate the trophic measured neurite outgrowth in response to Pep4, a peptide corresponding to residues 31–55 of Nogo-66 (21), and NEP1–40, actions of Trk receptor tyrosine kinases but also, under partic- another fragment of Nogo-66 that antagonizes Nogo-66-induced ular conditions, to trigger apoptosis upon binding to neurotro- growth cone collapse (11). Total neurite length was reduced by phins such as NGF and BDNF or their related precursors (15). Ϸ40% in the presence of 100 nM Pep4, and this effect was Embryonic motor neurons, which lack TrkA receptors, express abolished by excess NEP1–40 (Fig. 1D). Pep4 was inefficient to p75NTR in vitro and undergo apoptosis when cocultured on reactive astrocytes secreting NGF or treated with exogenous NGF in the presence of low steady-state concentrations of NO Author contributions: L. Dupuis and M.P. contributed equally to this work; L. Dupuis, M.P., (16, 17). This apoptotic activity strictly depends on p75NTR, P.C., J.-P.L., L.B., and J.-L.G.d.A. designed research; L. Dupuis, M.P., P.C., F.R., R.C., C.R., and because cultured motor neurons from p75NTR knockout mice M.G. performed research; L. Dimou and M.E.S. contributed new reagents/analytic tools; L. Dupuis, M.P., P.C., J.-P.L., L.B., and J.-L.G.d.A. analyzed data; and L. Dupuis and J.-L.G.d.A. do not respond to NGF (17). In vivo spinal motor neurons also wrote the paper. express p75NTR during the embryonic period of naturally The authors declare no conflict of interest. occurring cell death, but its expression gradually ends after birth. This article is a PNAS Direct Submission. In contrast, adult motor neurons can reexpress p75NTR after ʈTo whom correspondence should be addressed. E-mail: gonzalez@neurochem. axotomy (18) or under disease conditions such as ALS (17). This u-strasbg.fr. phenomenon, together with increased NGF levels, has indeed This article contains supporting information online at www.pnas.org/cgi/content/full/ been involved in the motor neuron death characteristic of such 0703842105/DC1. conditions (17, 19, 20). © 2008 by The National Academy of Sciences of the USA 740–745 ͉ PNAS ͉ January 15, 2008 ͉ vol. 105 ͉ no. 2 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0703842105 Downloaded by guest on September 25, 2021 Fig. 2. Pep4 and NEP1–40 prevent p75NTR-dependent motor neuron death. (A) Survival rates of cultured rat embryonic (E15) motor neurons treated for 24 h with 100 nM Pep4 or NEP1–40 in the presence (GDNF) or absence (none) of GDNF. Pep4 and NEP1–40 did not modify motor neuron survival in any condition (P Ͼ 0.05; n ϭ 5 independent experiments). (B) Cultures were treated for 24 h with 100 nM Pep4 or NEP1–40 in the presence of NGF or NGF Fig. 1. The Nogo-66/NgR pathway is expressed and functional in embryonic plus NOC-18 (NO generator) (*, P Ͻ 0.05 vs. control; #, P Ͻ 0.05 vs. NGFϩNO motor neurons. (A) Photomicrographs showing ChAT (green) and NgR (red) alone; n ϭ 5 independent experiments). (C) Rat embryonic (E15) motor neu- immunoreactivity in the lumbar spinal cord of embryonic (E16) rats. Note that rons were cocultured on rat astrocyte monolayers and treated for 24 h with ChAT and NgR mostly colocalize within the same motor neurons (merge), 100 nM Pep4 or NEP1–40 in the presence (NGF) or absence (none) of NGF (*, except for some few NgR-positive cells that are not stained for ChAT (aster- P Ͻ 0.05 vs. control; #, P Ͻ 0.05 vs. NGF; n ϭ 3 independent experiments). (D) ␮ isks). (Scale bar, 25 m.) (B) RT-PCR showing the mRNA levels of the different Cultures were treated for 24 h with 100 nM Pep4 or NEP1–40 and lumbar constituents of the NgR signaling platform in cultured rat embryonic (E15) spinal cord lysates from symptomatic SOD1(G93A) or nontransgenic (WT) motor neurons (MNs), motor neuron-like NSC34 cells, and adult mouse spinal littermates in the presence of NOC-18 (NO generator) (*, P Ͻ 0.05 vs. G93A; #, cord (SC). 18S rRNA levels are shown as internal control. (C) Photomicrographs P Ͻ 0.05 vs. G93AϩNO; n ϭ 3 independent experiments). (B–D) GDNF was showing p75NTR (green) and NgR (red) immunoreactivity in cultured rat present in all experimental conditions. embryonic (E15) motor neurons. (Scale bar, 25 ␮m.) (D) Total neurite length of cultured rat embryonic (E15) motor neurons treated for 24 h with Pep4 and/or ϩ ␮ ϩϩ NEP1–40 at 100 nM ( )or1 M( ). Motor neurons were cultured in the steady-state concentrations of NO (Ͻ50 nM), triggers the death presence of GDNF. In some cases, motor neurons were pretreated with PI-PLC. Ϸ Data are presented as percentages of total neurite length in the control of 45% of cultured embryonic motor neurons (17). In the condition (GDNF alone) (**, P Ͻ 0.01; ***, P Ͻ 0.001 vs. GDNF; #, P Ͻ 0.05 vs. present study, this effect was completely prevented by Pep4 or Pep4; n ϭ 3 independent experiments). NEP1–40 administration (Fig. 2B). Identical results were ob- tained when NGF-treated motor neurons were cocultured with astrocytes as natural source of NO (17) (Fig.