ProNGF induces TNFα-dependent death of retinal ganglion cells through a p75NTR non-cell-autonomous signaling pathway Frédéric Lebrun-Juliena,1, Mathieu J. Bertrandb,1, Olivier De Backerc, David Stellwagend, Carlos R. Moralese, Adriana Di Polo a,2,3, and Philip A. Barker b,2 aDepartment of Pathology and Cell Biology and Groupe de Recherche sur le Système Nerveux Central, Université de Montréal, Montreal, Quebec H3C 3J7, Canada; bCentre for Neuronal Survival, Montreal Neurological Institute, Montreal, Quebec H3A 2B4, Canada; cFacultés Universitaires Notre-Dame de la Paix School of Medicine, University of Namur, Namur B-5000, Belgium; dCentre for Research in Neuroscience, Montreal, Quebec H3G 1A4; and eDepartment of Anatomy and Cell Biology McGill University, Montreal, Quebec H3A 2B2, Canada Edited by Moses V. Chao, Skirball Institute of Biomolecular Medicine, New York, New York, and accepted by the Editorial Board December 30, 2009 (received for review August 17, 2009) Neurotrophin binding to the p75 neurotrophin receptor (p75NTR) Müller glial cells. Therefore, proNGF-induced neuronal loss in the activates neuronal apoptosis following adult central nervous sys- adult retina occurs through a non-cell-autonomous mechanism. tem injury, but the underlying cellular mechanisms remain poorly defined. In this study, we show that the proform of nerve growth Results factor (proNGF) induces death of retinal ganglion cells in adult ProNGF Induces Death of Retinal Ganglion Cells in Adult Rodents. To rodents via a p75NTR-dependent signaling mechanism. Expression investigate whether proNGF promotes neuronal death in vivo, we of p75NTR in the adult retina is confined to Müller glial cells; there- first retrogradely labeled RGCs of adult rats by applying fluo- fore we tested the hypothesis that proNGF activates a non-cell- rogold to the surface of the superior colliculus and then provided autonomous signaling pathway to induce retinal ganglion cell a single intraocular injection of proNGF or vehicle. A week later, (RGC) death. Consistent with this, we show that proNGF induced retinal whole mounts were prepared and RGC densities were robust expression of tumor necrosis factor alpha (TNFα) in Müller quantified. ProNGF caused a profound loss of adult rat RGCs, NEUROSCIENCE cells and that genetic or biochemical ablation of TNFα blocked whereas vehicle injection had no effect on cell death (Fig. 1A). To proNGF-induced death of retinal neurons. Mice rendered null for determine if the effect of proNGF on neuronal survival was spe- p75NTR, its coreceptor sortilin, or the adaptor protein NRAGE were cific to the proform of this neurotrophin, we asked whether defective in proNGF-induced glial TNFα production and did not mature NGF could similarly promote RGC death and found that undergo proNGF-induced retinal ganglion cell death. We conclude neuronal density was not altered by mature NGF treatment (Fig. that proNGF activates a non-cell-autonomous signaling pathway 1A). The effect of proNGF was not species specific as proNGF that causes TNFα-dependent death of retinal neurons in vivo. also caused a marked loss of RGCs in mice subjected to intra- ocular proNGF injection (Fig. 1B). We conclude that elevation of he four mammalian neurotrophins comprise a family of rela- proNGF levels within the retina promotes neuronal loss and used Tted secreted factors that are required for differentiation, sur- this system as a model for examining the cellular details of vival, development, and death of specific populations of neurons proNGF-induced cell death in vivo. and nonneuronal cells. Neurotrophins are produced as proforms NTR of ∼240 amino acids that are cleaved by furins and proconvertases p75 , Sortilin, and NRAGE Are Required for proNGF-Induced Death NTR to yield products of ∼120 amino acids. Recent studies have indi- of Retinal Ganglion Cells. To determine if p75 was required for fi cated that nerve growth factor (NGF) and brain-derived neuro- the loss of RGCs evoked by exogenous proNGF, we rst asked NTR trophic factor (BDNF) can be secreted as proforms in the central whether coinjection of the p75 function-blocking antibody nervous system (CNS) (1–3) and demonstrated that proneuro- REX (11) antagonized proNGF-induced neuronal death. Coad- fi trophins can function as potent apoptosis-inducing ligands both in ministration of proNGF and REX resulted in signi cant rescue of fi vitro and in vivo (4). However, the precise mechanisms by which RGCs in mice, whereas combined proNGF and nonspeci c Ig did A proneurotrophins lead to neuronal death are poorly defined. not exert a protective effect (Fig. 2 ). As an alternative approach, fi The biological effects of neurotrophins are mediated by binding we assessed the apoptotic effect of proNGF in mice de cient for p75NTR and showed that proNGF-induced loss of RGCs did not to TrkA, TrkB, and TrkC receptor tyrosine kinases and to the p75 NTR neurotrophin receptor (p75NTR). Trk receptors respond prefer- occur in p75 null retinas. Together, these data indicate that proNGF binding to p75NTR is required to induce RGC death. entially to mature neurotrophins whereas proneurotrophins exert NTR their apoptotic effect via a receptor complex that contains p75NTR p75 and sortilin have been shown to form a cell surface and sortilin (5). The precise signaling cascades evoked by occu- receptor complex for proneurotrophins that is required for acti- pancy of the p75NTR–sortilin complex remain to be elucidated, but several lines of evidence indicate that NRIF and NRAGE adaptor proteins play key roles in death signaling cascades evoked Author contributions: F.L.-J., M.J.B., A.D.P., and P.A.B. designed research; F.L.-J. and M.J.B. NTR performed research; O.D.B., D.S., and C.R.M. contributed new reagents/analytic tools; by p75 (6, 7). F.L.-J., M.J.B., A.D.P., and P.A.B. analyzed data; and F.L.-J., M.J.B., A.D.P., and P.A.B. wrote Previous studies have shown that neurotrophins induce cell death the paper. NTR NTR via p75 during early retinal development (8). p75 has also The authors declare no conflict of interest. been implicated in light-induced photoreceptor death in adult This article is a PNAS Direct Submission. M.V.C. is a guest editor invited by the Editorial NTR rodents in vivo (9) and a proNGF-p75 link has been proposed to Board. facilitate apoptosis in a retinal cell line (10). Here, we investigate the 1F.L.J. and M.J.B. contributed equally to this work. role of proNGF in the adult retina and demonstrate that proNGF 2A.D.P. and P.A.B. contributed equally to this work. promotes death of retinal ganglion cells (RGCs) in vivo. Importantly, 3To whom correspondence should be addressed. E-mail: [email protected]. NTR proNGF-induced RGC loss is indirect and requires the p75 - This article contains supporting information online at www.pnas.org/cgi/content/full/ dependent production of tumor necrosis factor-alpha (TNFα)by 0909276107/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.0909276107 PNAS | February 23, 2010 | vol. 107 | no. 8 | 3817–3822 Downloaded by guest on September 29, 2021 abundant quantities of this receptor (Fig. 3A). Costaining with the Müller cell-specific marker CRALBP confirmed that p75NTR is abundantly expressed by Müller glia, but not RGCs (Fig. 3B). Previous studies have shown that sortilin is expressed by Müller glia in the mouse retina (16), and when we examined the dis- tribution of NRAGE in this system, we found that this p75NTR adaptor protein is abundantly expressed in Müller cell soma and processes (Fig. 3C). On the basis of these results, we hypothesized that proNGF promotes RGC death through an indirect pathway by stimulating the production of a proapoptotic factor by Müller cells. A can- didate proapoptotic factor downstream of p75NTR is tumor necrosis factor alpha (TNFα) because exogenous and endogenous TNFα can induce death of retinal neurons (17–19) and because p75NTR activates NF-κB, a transcription complex that is a potent inducer of TNFα production (20–22). To address whether TNFα could play a role in proNGF-induced RGC killing, we first determined if retinal TNFα levels were increased in eyes injected with proNGF. Immunostaining showed that in eyes injected with vehicle or mature NGF, TNFα basal levels were low. In contrast, eyes injected with proNGF showed robust TNFα expression, both in cell bodies in the inner nuclear layer and within processes that extended radially across the breadth of the retina (Fig. 3D). Double immunocytochemistry using antibodies against CRALBP identified these TNFα-expressing cells as Müller glia (Fig. 3E). The finding that proNGF stimulates TNFα production by Müller cells prompted us to ask whether RGC death induced by this proneurotrophin could be blocked by Etanercept, a recombinant TNFα antagonist in which the extracellular ligand- binding domain of TNFα receptor 2 (TNFR2) is fused to an Fc fragment (23). Fig. 4A shows that intraocular injection of Eta- nercept markedly blocked RGC death induced by proNGF. To rule out the possibility that Etanercept may have off-target pharmacological effects and to further substantiate a role for TNFα in proNGF-induced killing, we also examined whether proNGF led to RGC loss in TNFα null mice. Our data show that Fig. 1. Exogenous proNGF leads to marked RGC loss in the adult rodent eye. α Quantitative analysis is shown of RGC survival in rat (A) and mouse (B)ret- proNGF administration failed to induce RGC death in TNF null inas at 1 week after intraocular injection of proNGF (solid bars), vehicle (PBS, mice (Fig. 4B), indicating that TNFα plays a crucial role in RGC shaded bars), or NGF (bars with horizontal lines). The density of RGCs in death induced by proNGF. intact, noninjected retinas is shown as reference (open bars).