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) 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 (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 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 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 (NGF) and -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 (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 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). Data are These data indicate that proNGF causes RGC death indirectly, expressed as RGC densities (RGCs/mm2; mean ± SEM; ANOVA, *P < 0.001). by stimulating production of TNFα by Müller glial cells. We The number of animals used in each experiment is shown above the corre- therefore tested whether p75NTR and sortilin activated an sponding graph bar. NRAGE-dependent pathway that resulted in TNFα production. For this purpose, TNFα protein levels were compared in retinas derived from wild-type mice vs. mice lacking p75NTR, sortilin, or vation of downstream apoptotic pathways (5), and we previously NRAGE following proNGF or vehicle injection. In wild-type demonstrated that NRAGE, a p75NTR adaptor protein, plays an NTR retinas, robust TNFα protein production was detected within 48 h obligatory role in p75 -dependent death (6). We therefore of proNGF administration, wheras PBS injection had no effect on asked whether sortilin or NRAGE was required for proNGF- TNFα levels (Fig. 4C). In contrast, proNGF-induced TNFα up- induced RGC loss in vivo. Our data show that RGCs within sor- regulation did not occur in p75NTR, sortilin, and NRAGE null tilin or NRAGE null mice retinas were protected from proNGF- mice (Fig. 4C), correlating with the failure of proNGF to induce C D NTR induced death (Fig. 2 and ). We conclude that p75 ,sortilin, RGC death in these null lines (Fig. 2 B–D). Importantly, intra- and NRAGE are each required for proNGF-induced death of ocular injection of TNFα caused RGC death in both wild-type and RGCs in the adult retina. p75NTR null strains, confirming that TNFα is the actual factor that kills these neurons (Fig. S1). Together, these data indicate that ProNGF Kills Retinal Ganglion Cells Through Glia-Mediated Production sortilin and NRAGE cooperate with p75NTR to transduce α of TNF . A simple model to explain these results would have proNGF-induced signaling events required for TNFα production NTR– proNGF binding to the p75 sortilin complex on the surface of by Müller glial cells and that the TNFα kills RGCs through a RGCs that in turn activates a cell-autonomous, NRAGE- p75NTR-independent mechanism. dependent proapoptotic pathway. However, it has been reported that Müller glia are the only cells that express p75NTR in the adult Discussion retina (9, 12–15), implying that proNGF killing of RGCs may This study reports three major findings. First, proNGF leads to NTR involve a more complex mechanism. To confirm the p75 striking neuronal death in the adult rodent retina in vivo. Sec- expression pattern in the adult mouse retina, RGCs were retro- ond, proNGF mediates RGC death indirectly via a non-cell- gradely labeled with fluorogold and then stained with antibodies autonomous mechanism that involves TNFα production by specific for p75NTR. In both naive and proNGF-treated animals, Müller glia. Third, proNGF-induced death requires p75NTR, RGCs were uniformly negative for p75NTR whereas neighboring sortilin, and NRAGE, which cooperate to stimulate TNFα pro- cells and processes typical of Müller glia invariably expressed duction by Müller cells.

3818 | www.pnas.org/cgi/doi/10.1073/pnas.0909276107 Lebrun-Julien et al. Downloaded by guest on September 29, 2021 Fig. 2. p75NTR, sortilin, and NRAGE are required for proNGF-induced death of RGCs. (A) Treatment with REX, a p75NTR function-blocking antibody, promoted RGC survival in the presence of proNGF whereas coadministration of a nonspecific, control Ig did not exert a neuroprotective effect (ANOVA, *P < 0.001). NTR RGCs from mice eyes deficient in p75 (B), sortilin (C), or NRAGE (D) were resistant to proNGF-induced death (Student’s t test, P > 0.05). Values are expressed NEUROSCIENCE as RGC densities (RGCs/mm2; mean ± SEM).

p75NTR is an important neuronal signaling protein that inter- with p75NTR and sortilin on Müller cells must induce death of acts with numerous ligands and coreceptors to exert a wide range RGCs through an indirect, non-cell-autonomous pathway. of functions. The role of p75NTR as an apoptotic receptor is well Pharmacological or genetic blockade of TNFα dramatically established as it has been shown to facilitate developmental cell reduced proNGF-induced RGC death, indicating that Müller death of peripheral sympathetic neurons and early retinal neurons cell-derived TNFα plays a crucial role in neuronal loss. There are (8, 24–26). p75NTR has also been implicated in cell loss following several direct and indirect mechanisms by which TNFα produced various forms of CNS injury and promotes apoptosis of cortical by Müller cells could kill RGCs but two predominate. First, pre- and hippocampal neurons, basal forebrain neurons, oligoden- vious work established that TNFα can directly kill primary cortical drocytes, and photoreceptor cells (8, 27–31). Overall, the func- neurons via a caspase 8-dependent mechanism in models of tional role of p75NTR in neuronal death is best understood in excitotoxicity (32, 33). It is thus possible that direct caspase acti- neurons that endogenously express this receptor. In this study, vation occurs in RGCs. However, caspase-8 inhibitors that we however, we identify a unique mechanism by which p75NTR in glial validated in vivo and in vitro did not block proNGF-induced death cells can profoundly influence neuronal death by activating pro- of RGCs. Thus, we favor a second possibility, which involves the duction of neurotoxic TNFα in the adult retina in vivo. recent discovery that TNFα promotes the selective insertion of Recent studies have indicated that proNGF, but not NGF, Ca2+-permeable AMPA receptors (AMPARs) into the neuronal functions as a potent proapoptotic ligand that binds to a cell sur- cell surface (34, 35). Under physiological circumstances, TNFα- face complex of p75NTR and sortilin (5). Consistent with this, we mediated increase in Ca2+-permeable AMPARs plays a role in showed that fully processed NGF does not share the apoptotic synaptic scaling (36) but during injury, TNFα-mediated increase effect of proNGF and that proNGF is unable to induce retinal cell in these channels can facilitate death of primary hippocampal and death in mice rendered null for sortilin. NRAGE is a crucial neurons (37, 38). Importantly, this mechanism of cell adaptor protein required for p75NTR-dependent apoptosis in cell death may play a role in RGC loss after injury. For example, lines, in primary cells, and in vivo (6). Consistent with this, we RGCs lacking TNFR1 are protected following optic nerve crush found that NRAGE is also necessary for proNGF-induced death of (39), and we recently showed that TNFα-mediated increase in cell RGCs within the adult retina. Previous studies have established surface Ca2+-permeable AMPARs leads to RGC loss in vivo (15). that RGCs express abundant p75NTR during retinal development Glaucoma is a group of diseases characterized by progressive (25) and that p75NTR and sortilin collaborate to induce NGF- optic nerve degeneration leading to visual field loss and irrever- dependent apoptosis of a subset of these neurons at embryonic day sible blindness. A common characteristic of all forms of glaucoma (E)15 (8, 25, 26). Therefore, it is possible that proNGF induces cell is the death of RGCs (40). Of significant interest, TNFα and death in the adult retina by directly binding to a p75NTR–sortilin TNFR1 are up-regulated in human donor eyes with glaucoma (19, complex on adult RGCs. This is very unlikely, however, because 41, 42), TNFα levels are increased in the aqueous humor of examination of p75NTR expression in the adult rodent retina using glaucoma patients (43), and TNFα polymorphisms have been light and electron microscopy has established that retinal p75NTR correlated with primary open angle glaucoma (44, 45). Therefore, expression is confined to Müller glial cells (9, 12–15). A recent an important priority in future studies will be to determine if the study reported that sortilin is expressed by Müller glia in the adult proNGF, p75NTR, and TNFα cascade described here contributes retina (16), and the data shown here indicate that NRAGE is also to retinal degeneration and optic neuropathy in preclinical models expressed in these glial cells. Therefore, association of proNGF of disease and in humans.

Lebrun-Julien et al. PNAS | February 23, 2010 | vol. 107 | no. 8 | 3819 Downloaded by guest on September 29, 2021 downstream of these p75NTR-dependent events will be an inter- esting topic for future work. In summary, our work demonstrates that proNGF can induce neuronal death in the retina through a non-cell-autonomous mechanism that involves the activation of p75NTR and production of TNFα by Müller glial cells. These findings raise the possibility that non-cell-autonomous events may be a general feature of p75NTR-dependent cell apoptosis in vivo. Materials and Methods Experimental Animals. Procedures were carried out in adult C57BL/6 transgenic or wild-type littermate control mice, with the exception of results shown in Fig. 1A that were carried out in adult Sprague-Dawley rats. All animal procedures were performed in accordance with the policies on the Use of Animals in Neuroscience Research and the Canadian Council on Animal Care guidelines (49). p75NTR (50), TNFα (51), and NRAGE (6) null mice have been previously described. To inactivate the sortilin gene in ES cells we used the recombination cloning vector pML. A 4.6-kb fragment of the 5′-flanking genomic sequence and a 3.2-kb fragment of the 3′-flanking region of sortilin were subcloned upstream and downstream, respectively, of the neomycin resistance gene within the vector. The Neomycin/G418 in the pML vector was used for positive selection. This vector contains a thymidine kinase gene (TK) that in combina- tion with gancyclovir was used for negative selection. The targeting construct was linearized by PmeI restriction digestion and electroporated into ES cells. These G418 and gancyclovir-resistant ES cell clones were screened by Southern blot after digestion of the ES genomic DNA with HindIII. The homologous recombination resulted in the replacement of a segment between 2 and intron 3 of the sortilin gene with the neomycin resistance cassette. Chimeric cells were injected into C57BL/6 blastocysts giving rise to chimeric mice, which were then backcrossed to C57BL/6 mice to identify if the germ-line trans- mission of the mutant allele had taken place (52). These heterozygotes were backcrossed with C57BL/6 mice for a minimum of seven generations before they were used in this study. All genotypes were verified by PCR using pfu- Turbo (Stratagene). The number of animals used in each experiment (n)is shown in each corresponding graph.

Intraocular Injections. A mutant form of proNGF that is resistant to cleavage by proteases(proNGFmut-m,25ng/μL,Alomone Labs) wasinjectedintothe vitreous chamber of the left eye using a 10-μL Hamilton syringe adapted with a 32-gauge glass microneedle (totalvolume: 2 μL). The average vitreous fluid volume inadult mice is estimated to be ∼10 μL(53–55); therefore, the concentration of proNGF that reached RGCs was 0.1 μM. ProNGF was injected alone or in combination with Etanercept (Enbrel, 25 μg/μL, Wyeth), the p75NTR antibody REX (10 μg/μL), or an Fc control (25 μg/μL, Sigma). Control eyes were injected with mature NGF (0.1 μg/μL, human recombinant NGF, PeproTech), TNFα (200 ng/mL, murine Fig. 3. p75NTR and p75NTR-induced TNFα are expressed by Müller cells in the recombinant TNFα, R&D Systems), or vehicle (PBS). Intraocular injections were adult mouse retina. (A) Confocal microscopy images show absence of p75NTR performed under general anesthesia (2% Isoflurane/oxygen mixture, 0.8 L/min). within Fluorogold-positive RGCs, demonstrating that adult RGCs are devoid The needle tip was inserted into the superior hemisphere of the eye, at a of p75NTR.(B) Double immunolabeling with antibodies against p75NTR (REX) 45° angle through the sclera into the vitreous body. This route of administration and the Müller cell marker cellular retinaldehyde-binding protein (CRALBP) avoided retinal detachment or injury to eye structures, including the iris and lens, shows strong expression of p75NTR in Müller cell processes surrounding RGCs. which release factors that induce neuronal survival (56, 57). (C) Fluorescent microscopy images show that NRAGE protein colocalizes with Müller cell soma and processes, visualized with CRALBP. (D) Robust TNFα Retrograde Labeling and Quantification of Neuronal Survival. Retrograde labeling protein induction was observed in retinas exposed to proNGF compared to of RGCs was performed using Fluorogold (2%, Fluorochrome) in 0.9% NaCl, low, basal levels in eyes injected with vehicle or recombinant, mature NGF. which was applied to the superior colliculus as described (58). Subsequent sur- (E) Confocal microscopy images show that TNFα colocalizes with CRALBP- gical procedures were performed at 1 week after Fluorogold application (59– positive Müller cells. GCL, ganglion cell layer; INL, inner nuclear layer; IPL, 62). Mice were perfused with 4% paraformaldehyde; eyes were dissected and inner plexiform layer; ONL, outer nuclear layer; OPL, outer plexiform layer. flat mounted vitreal side up on glass sides. Fluorogold-positive RGCs were (Scale bars: A and B,10μm; C–E,50μm.) counted in 12 retinal zones: Three areas in each eye quadrant (dorsal, ventral, nasal, and temporal) located at 0.5, 1.0, and 1.5 mm from the optic nerve head were examined (58), corresponding to a total area of 0.5 mm2. Statistical Several recent studies have highlighted the important role that analyses were performed using one-way analysis of variance (ANOVA) followed non-cell-autonomous mechanisms play in neuronal cell death, by a nonparametric test (Bonferroni’s multiple-comparison test) or by a Stu- ’ fi notably in models of amyotrophic lateral sclerosis, spinocerebellar dent s t test, as indicated in the gure legends. ataxia, and Huntington disease (46). TNFα is a hallmark of acute fl Retinal Immunohistochemistry. Mice were perfused with 4% paraformalde- and chronic neuroin ammation and has emerged as an important hyde and retinal sections were prepared as above. Tissue sections were candidate for mediating non-cell-autonomous effects in these incubated in 3% BSA and 0.3% Triton X-100 (Sigma) to block nonspecific conditions (47). p75NTR expressed by cholinergic neurons is binding and then incubated with primary antibodies (see list below) overnight required for production of an unknown factor that facilitates at 4 °C, followed by incubation with secondary antibodies at room temper- NTR ature. Slides were mounted with SlowFade (Molecular Probes) and visualized GABAergic neuron development (48), and p75 expressed on on a Zeiss Axioskop 2 Plus microscope (Carl Zeiss Canada) or a confocal Müller glial cells mediates light-induced photoreceptor death via a microscope (Leica Microsystems). Primary antibodies used were anti-cellular non-cell-autonomous pathway (9). The potential role of TNFα retinaldehyde-binding protein (1:1,000, gift from J. C. Saari, University of

3820 | www.pnas.org/cgi/doi/10.1073/pnas.0909276107 Lebrun-Julien et al. Downloaded by guest on September 29, 2021 Fig. 4. Sortilin and NRAGE cooperate with p75NTR to transduce proNGF-induced signaling events required for TNFα production. (A) Coadministration of proNGF with the TNFα inhibitor Etanercept leads to striking RGC neuroprotection, but RGCs did not survive in Fc-treated control retinas (ANOVA, *P < 0.001). (B) RGCs from TNFα null mice were resistant to proNGF-induced death (Student’s t test, P > 0.05). (C) Western blot analyses of retinal extracts show that α α

whereas TNF protein levels are below the detection limit in wild-type retinas, exposure to proNGF elicits robust TNF protein up-regulation within 48 h of NEUROSCIENCE proNGF injection. ProNGF-induced TNFα production is completely lost in p75NTR null, sortilin null, and NRAGE null animals (n = 5 mice/group). Lower blots were probed with antibodies that recognize p75NTR, sortilin, or NRAGE, respectively, and actin was used as a control for equal protein loading.

Washington, Seattle, WA), anti-p75NTR (REX, 2 ng/μL) (63), anti-NRAGE (5 ng/μL, (1:1,000) (64), Sortilin (1:1,000) (BD Biosciences), TNFα (0.2 μg/mL, Santa Cruz Orbigen), and anti-TNFα (0.4 μg/mL, Chemicon). The secondary antibodies used Biotechnology), and β-actin (1:30,000) (Sigma). Secondary HRP-coupled anti- were sheep anti-mouse IgG (1 μg/mL, FITC conjugate, Sigma) or anti-rabbit IgG bodies were obtained from Jackson ImmunoResearch Laboratories. Protein (1 μg/mL, Cy3, Jackson ImmunoResearch Laboratories). signals were detected using a chemiluminescence reagent (ECL, Amersham Biosciences) followed by exposure of blots to X-Omat (Kodak) imaging film. Immunoblot Analysis. Retinas were homogenized in lysis buffer [20 mM Tris (pH 8.0), 135 mM NaCl, 1% SDS, and 10% glycerol supplemented with protease ACKNOWLEDGMENTS. This work was supported by independent grants inhibitors] and centrifuged at 14,000 rpm for 5 min. The supernatants were from the Canadian Institutes of Health Research (to A.D.P. and P.A.B.). F.L.-J. collected, diluted in Laemmli sample buffer (4% SDS, 10% glycerol, 0.004% is recipient of a Fonds de Recherche en Santé du Québec doctoral fellowship, bromophenol blue, 0.1 M DTT, and 0.125 M Tris, pH 6.8), and analyzed by SDS– A.D.P. holds a Fonds de Recherche en Santé du Québec Chercheur Senior polyacrylamide gel electrophoresis and immunoblotting following standard Scholarship, and P.A.B. holds a Fonds de Recherche en Santé du Québec protocols. Primary antibodies were against p75NTR (REX, 1:1,000) (64), NRAGE Chercheur National and is a McGill Dawson Scholar.

1. Bruno MA, Cuello AC (2006) Activity-dependent release of precursor nerve growth 12. Ding J, Hu B, Tang LS, Yip HK (2001) Study of the role of the low-affinity neurotrophin factor, conversion to mature nerve growth factor, and its degradation by a protease receptor p75 in naturally occurring cell death during development of the rat retina. cascade. Proc Natl Acad Sci USA 103:6735–6740. Dev Neurosci 23:390–398. 2. Fahnestock M, Michalski B, Xu B, Coughlin MD (2001) The precursor pro-nerve growth 13. Hu B, Yip HK, So K-F (1998) Localization of p75 neurotrophin receptor in the retina of factor is the predominant form of nerve growth factor in brain and is increased in the adult SD rat: an immunocytochemical study at light and electron microscopic Alzheimer’s disease. Mol Cell Neurosci 18:210–220. levels. Glia 24:187–197. 3. Lee R, Kermani P, Teng KK, Hempstead BL (2001) Regulation of cell survival by 14. Hu B, Yip HK, So KF (1999) Expression of p75 neurotrophin receptor in the injured and secreted proneurotrophins. Science 294:1945–1948. regenerating rat retina. Neuroreport 10:1293–1297. 4. Hempstead BL (2006) Dissecting the diverse actions of pro- and mature neurotrophins. 15. Lebrun-Julien F, et al. (2009) Excitotoxic death of retinal neurons in vivo occurs via a Curr Alzheimer Res 3:19–24. non-cell-autonomous mechanism. J Neurosci 29:5536–5545. 5. Nykjaer A, et al. (2004) Sortilin is essential for proNGF-induced neuronal cell death. 16. Xu F, et al. (2009) Immunohistochemical localization of sortilin and p75(NTR) in Nature 427:843–848. normal and ischemic rat retina. Neurosci Lett 454:81–85. 6. Bertrand MJ, et al. (2008) NRAGE, a p75NTR adaptor protein, is required for 17. Berger S, et al. (2008) Deleterious role of TNF-alpha in retinal ischemia-reperfusion developmental apoptosis in vivo. Cell Death Differ 15:1921–1929. injury. Invest Ophthalmol Vis Sci 49:3605–3610. 7. Linggi MS, et al. (2005) Neurotrophin receptor interacting factor (NRIF) is an essential 18. Nakazawa T, et al. (2006) Tumor necrosis factor-alpha mediates oligodendrocyte mediator of apoptotic signaling by the p75 neurotrophin receptor. J Biol Chem 280: death and delayed retinal ganglion cell loss in a mouse model of glaucoma. J Neurosci 13801–13808. 26:12633–12641. 8. Frade JM, Barde YA (1999) Genetic evidence for cell death mediated by nerve growth 19. Tezel G, Li LY, Patil RV, Wax MB (2001) TNF-alpha and TNF-alpha receptor-1 in the factor and the neurotrophin receptor p75 in the developing mouse retina and spinal retina of normal and glaucomatous eyes. Invest Ophthalmol Vis Sci 42:1787–1794. cord. Development 126:683–690. 20. Hiscott J, et al. (1993) Characterization of a functional NF-kappa B site in the human 9. Harada T, et al. (2000) Modification of glial-neuronal cell interactions prevents interleukin 1 beta promoter: Evidence for a positive autoregulatory loop. Mol Cell photoreceptor apoptosis during light-induced retinal degeneration. Neuron 26: Biol 13:6231–6240. 533–541. 21. Mori N, Prager D (1996) Transactivation of the interleukin-1alpha promoter by human 10. Srinivasan B, Roque CH, Hempstead BL, Al-Ubaidi MR, Roque RS (2004) Microglia- T-cell leukemia virus type I and type II Tax proteins. Blood 87:3410–3417. derived pronerve growth factor promotes photoreceptor cell death via p75 22. Shakhov AN, Collart MA, Vassalli P, Nedospasov SA, Jongeneel CV (1990) Kappa B- neurotrophin receptor. J Biol Chem 279:41839–41845. type enhancers are involved in lipopolysaccharide-mediated transcriptional activation 11. Weskamp G, Reichardt LF (1991) Evidence that biological activity of NGF is mediated of the tumor necrosis factor alpha gene in primary macrophages. J Exp Med 171: through a novel subclass of high affinity receptors. Neuron 6:649–663. 35–47.

Lebrun-Julien et al. PNAS | February 23, 2010 | vol. 107 | no. 8 | 3821 Downloaded by guest on September 29, 2021 23. Fantuzzi F, Del Giglio M, Gisondi P, Girolomoni G (2008) Targeting tumor necrosis 44. Funayama T, et al. (2004) Variants in optineurin gene and their association with factor alpha in psoriasis and psoriatic arthritis. Expert Opin Ther Targets 12: tumor necrosis factor-alpha polymorphisms in Japanese patients with glaucoma. 1085–1096. Invest Ophthalmol Vis Sci 45:4359–4367. 24. Majdan M, et al. (1997) Transgenic mice expressing the intracellular domain of the 45. Lin HJ, et al. (2003) Association of tumour necrosis factor alpha -308 gene p75 neurotrophin receptor undergo neuronal apoptosis. J Neurosci 17:6988–6998. polymorphism with primary open-angle glaucoma in Chinese. Eye (Lond) 17:31–34. 25. Harada C, et al. (2006) Effect of p75NTR on the regulation of naturally occurring cell 46. Lobsiger CS, Cleveland DW (2007) Glial cells as intrinsic components of non-cell- death and retinal ganglion cell number in the mouse eye. Dev Biol 290:57–65. autonomous neurodegenerative disease. Nat Neurosci 10:1355–1360. 26. Jansen P, et al. (2007) Roles for the pro-neurotrophin receptor sortilin in neuronal 47. McCoy MK, Tansey MG (2008) TNF signaling inhibition in the CNS: Implications for development, aging and brain injury. Nat Neurosci 10:1449–1457. normal brain function and neurodegenerative disease. J Neuroinflammation 5:45. 27. Beattie MS, et al. (2002) ProNGF induces p75-mediated death of oligodendrocytes 48. Lin PY, Hinterneder JM, Rollor SR, Birren SJ (2007) Non-cell-autonomous regulation of following spinal cord injury. Neuron 36:375–386. GABAergic neuron development by neurotrophins and the p75 receptor. J Neurosci 28. Friedman WJ (2000) Neurotrophins induce death of hippocampal neurons via the p75 27:12787–12796. receptor. J Neurosci 20:6340–6346. 49. Olfert ED, Cross BM, McWilliams AA (1993) The Guide to the Care and Use of 29. Harrington AW, et al. (2004) Secreted proNGF is a pathophysiological death-inducing Experimental Animals (Canadian Council on Animal Care, Ottawa, ON, Canada). fi ligand after adult CNS injury. Proc Natl Acad Sci USA 101:6226–6230. 50. Lee K-F, et al. (1992) Targeted mutation of the gene encoding the low af nity NGF fi 30. Roux PP, Colicos MA, Barker PA, Kennedy TE (1999) p75 neurotrophin receptor receptor p75 leads to de cits in the peripheral sensory nervous system. Cell 69: – expression is induced in apoptotic neurons after seizure. J Neurosci 19:6887–6896. 737 749. 31. Volosin M, et al. (2006) Interaction of survival and death signaling in basal forebrain 51. Taniguchi T, Takata M, Ikeda A, Momotani E, Sekikawa K (1997) Failure of germinal neurons: Roles of neurotrophins and proneurotrophins. J Neurosci 26:7756–7766. center formation and impairment of response to endotoxin in tumor necrosis factor fi – 32. Kaushal V, Schlichter LC (2008) Mechanisms of microglia-mediated neurotoxicity in a alpha-de cient mice. Lab Invest 77:647 658. new model of the stroke penumbra. J Neurosci 28:2221–2230. 52. Zeng J, Racicott J, Morales CR (2009) The inactivation of the sortilin gene leads to a fi 33. Velier JJ, et al. (1999) Caspase-8 and caspase-3 are expressed by different populations partial disruption of prosaposin traf cking to the . Exp Cell Res 315: 3112–3124. of cortical neurons undergoing delayed cell death after focal stroke in the rat. J 53. Remtulla S, Hallett PE (1985) A schematic eye for the mouse, and comparisons with Neurosci 19:5932–5941. the rat. Vision Res 25:21–31. 34. Ogoshi F, et al. (2005) Tumor necrosis-factor-alpha (TNF-alpha) induces rapid insertion 54. Sharma S, Ball SL, Peachey NS (2005) Pharmacological studies of the mouse cone of Ca2+-permeable alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA)/ electroretinogram. Vis Neurosci 22:631–636. kainate (Ca-A/K) channels in a subset of hippocampal pyramidal neurons. Exp Neurol 55. Yu D-Y, Cringle SJ (2006) Oxygen distribution in the mouse retina. Invest Ophthalmol 193:384–393. Vis Sci 47:1109–1112. 35. Stellwagen D, Beattie EC, Seo JY, Malenka RC (2005) Differential regulation of AMPA 56. Fischer D, Heiduschka P, Thanos S (2001) Lens-injury-stimulated axonal regeneration receptor and GABA receptor trafficking by tumor necrosis factor-alpha. J Neurosci 25: throughout the optic pathway of adult rats. Exp Neurol 172:257–272. 3219–3228. 57. Leon S, Yin Y, Nguyen J, Irwin N, Benowitz LI (2000) Lens injury stimulates axon 36. Stellwagen D, Malenka RC (2006) Synaptic scaling mediated by glial TNF-alpha. regeneration in the mature rat optic nerve. J Neurosci 20:4615–4626. Nature 440:1054–1059. 58. Sapieha PS, et al. (2005) Receptor protein tyrosine phosphatase sigma inhibits axon 37. Ferguson AR, et al. (2008) Cell death after spinal cord injury is exacerbated by rapid regrowth in the adult injured CNS. Mol Cell Neurosci 28:625–635. TNF alpha-induced trafficking of GluR2-lacking AMPARs to the plasma membrane. J 59. Berkelaar M, Clarke DB, Wang Y-C, Bray GM, Aguayo AJ (1994) Axotomy results in – Neurosci 28:11391 11400. delayed death and apoptosis of retinal ganglion cells in adult rats. J Neurosci 14: 38. Leonoudakis D, Zhao P, Beattie EC (2008) Rapid tumor necrosis factor alpha-induced 4368–4374. exocytosis of glutamate receptor 2-lacking AMPA receptors to extrasynaptic plasma 60. Cheng L, Sapieha P, Kittlerová P, Hauswirth WW, Di Polo A (2002) TrkB gene transfer – membrane potentiates excitotoxicity. J Neurosci 28:2119 2130. protects retinal ganglion cells from axotomy-induced death in vivo. J Neurosci 22: 39. Tezel G, Yang X, Yang J, Wax MB (2004) Role of tumor necrosis factor receptor-1 in 3977–3986. the death of retinal ganglion cells following optic nerve crush injury in mice. Brain Res 61. Mansour-Robaey S, Clarke DB, Wang Y-C, Bray GM, Aguayo AJ (1994) Effects of ocular – 996:202 212. injury and administration of brain-derived neurotrophic factor on survival and 40. Quigley HA (2005) Glaucoma: Macrocosm to microcosm the Friedenwald lecture. regrowth of axotomized retinal ganglion cells. Proc Natl Acad Sci USA 91:1632–1636. Invest Ophthalmol Vis Sci 46:2662–2670. 62. Peinado-Ramón P, Salvador M, Villegas-Pérez MP, Vidal-Sanz M (1996) Effects of 41. Yan X, Tezel G, Wax MB, Edward DP (2000) Matrix metalloproteinases and tumor axotomy and intraocular administration of NT-4, NT-3, and brain-derived necrosis factor alpha in glaucomatous optic nerve head. Arch Ophthalmol 118: neurotrophic factor on the survival of adult rat retinal ganglion cells. A quantitative 666–673. in vivo study. Invest Ophthalmol Vis Sci 37:489–500. 42. Yuan L, Neufeld AH (2000) Tumor necrosis factor-alpha: A potentially 63. Bhakar AL, et al. (1999) The p75 neurotrophin receptor (p75NTR) alters tumor necrosis neurodestructive cytokine produced by glia in the human glaucomatous optic nerve factor-mediated NF-kappaB activity under physiological conditions, but direct head. Glia 32:42–50. p75NTR-mediated NF-kappaB activation requires cell stress. J Biol Chem 274: 43. Sawada H, Fukuchi T, Tanaka T, Abe H (2010) Tumor necrosis factor-{alpha} 21443–21449. concentrations in the aqueous humor of glaucoma patients. Invest Ophthalmol Vis 64. Salehi AH, etal. (2000) NRAGE, a novel MAGEprotein, interacts withthe p75 neurotrophin Sci, 10.1167/iovs.09-4247. receptor and facilitates nerve growth factor-dependent apoptosis. Neuron 2:279–288.

3822 | www.pnas.org/cgi/doi/10.1073/pnas.0909276107 Lebrun-Julien et al. Downloaded by guest on September 29, 2021