Pro-neurotrophins secreted from retinal ganglion cell axons are necessary for ephrinA-p75NTR-mediated axon guidance
© Marler et al; licensee BioMed Central Ltd. 2010
Received: 30 June 2010
Accepted: 2 November 2010
Published: 2 November 2010
Retinotectal map formation develops via topographically specific guidance and branching of retinal axons in their target area. This process is controlled, in part, by reverse signalling of ephrinAs expressed on retinal axons. As glycosylphosphatidylinositol-anchored molecules, ephrinAs require transmembrane co-receptors to exert this function, for which the two neurotrophin receptors, p75NTR and TrkB, were recently proposed.
We show here that the ligands for these receptors, the brain-derived neurotrophic factor precursor (proBDNF) and its processed form, BDNF, respectively, control the branching of retinal axons antagonistically, which they mediate by inducing the corresponding neurotrophin receptor-ephrinA complexes. Moreover, scavenging proneurotrophins, by adding antibodies specific for the pro-domain of proBNDF or a soluble extracellular domain of p75NTR, abolish repellent ephrinA reverse signalling in the stripe assay.
This indicates that retinal cells secrete proneurotrophins, inducing the ephrinA-p75NTR interaction and enabling repellent axon guidance. The antagonistic functions of proBDNF and BDNF raise the possibility that topographic branching is controlled by local control of processing of proneurotrophins.
The retino-tectal projection is a well suited model system to investigate the formation of topographic maps and the control of local axon branching. In this projection, retinal ganglion cell (RGC) axons grow into the tectum in a non-topographic manner and initially overshoot their future termination zones. Termination zones are formed through interstitial branching, with branching of axons from nasal retina in the caudal tectum and axons from temporal retina in rostral tectum. The map is refined by arborisations and pruning of overshoot axon segments. The final map is a product of both activity-independent and activity-dependent processes [1–3].
Some aspects of this mapping process are controlled by retinally expressed ephrinA molecules, with higher expression on nasal than on temporal retinal axons. This differential expression mediates a repulsion of nasal axons from parts of the target area expressing high(er) amounts of EphA molecules, that is, the anterior tectum . Recently, the neurotrophin receptors p75NTR and tropomyosin-related kinase (Trk)B were proposed as co-receptors for ephrinAs, which are glycosylphosphatidylinositol-anchored and therefore have no direct contact with the cytosol [5, 6].
Ligands for these receptors are the brain-derived neurotrophic factor precursor (proBDNF) and its processed form, BDNF. proBDNF binds with high affinity to p75NTR, while BDNF binds with high affinity to TrkB [7–9]. Both the pro-form and the processed form are secreted from neurons, and their processing is controlled on various levels [10–17]. This control of processing is crucial, as the activation of either p75NTR or TrkB leads often to opposing biological effects ; for example, activation of TrkB results in cell survival, while activation of p75NTR leads to cell death . Similarly, in synapse function, TrkB and p75NTR are involved antagonistically in long-term plasticity versus long-term depression [20, 21].
This study investigates whether the interaction of ephrinAs with either TrkB or p75NTR also results in antagonistic effects on axon guidance and branching of retinal axons . We have approached this by using various in vitro assays. Our findings on the antagonistic functions of proBDNF versus BDNF on axon guidance and branching fit well to data showing that a (conditional) inactivation of p75NTR results in a disturbance of the retinocollicular map with shifted and ectopic termination zones and an increase in non-topographic branching anterior to the termination zones .
Results and discussion
Ligand-promoted interaction of ephrinA5 with p75NTR
Proneurotrophin secretion is necessary for repellent ephrinA reverse signalling
Interestingly, these experiments (as well as comparable experiments with retinae from wild-type and p75NTR knockout mice ) were performed in the absence of added pro-neurotrophins, which is surprising in view of our data showing a ligand-induced/promoted interaction of ephrinA5 with p75NTR (see above; Figure 1).
The ligand inducibility of this repulsion is of particular interest as it appears at first glance surprising that RGC axons can invade the tectum from the high end of the repellent EphA gradient (Additional file 3). In light of our findings, retinal axons conceivably become sensitive to the repellent gradient only after their ingrowth into the tectum, as possibly only then proBDNF is secreted from retinal axons leading to an induction of the ephrinA-p75NTR complex. This switch from storage to secretion might be developmentally regulated and/or linked to the fact that proBDNF secretion is regulated by neural activity [13, 15]. Thus, a change, for example, in the pattern of spontaneous neural activity might lead to proBDNF secretion only after RGC axons have invaded the tectum, that is, after they have surmounted the high end of the repellent EphA gradient.
Antagonistic actions of proBDNF and BDNF on axonal branching
We have shown recently that a BDNF-promoted interaction between ephrinAs and TrkB results in an increase in retinal axon branching and that increasing the level of ephrinAs on retinal axons further increases the level of branching .
Thus, while BDNF application (via TrkB) promotes retinal branching, proBDNF application (via p75NTR) suppresses it. The map disturbance seen in (conditional) p75NTR knockout mice with increased axonal branching anterior to the termination zone  is in good agreement with the data shown here, offering a molecular explanation for the observed p75NTR phenotypes. Our data suggest that the local ratio between proBDNF and BDNF (and with that a differential activation of TrkB versus p75NTR) in the tectum/superior colliculus contributes to topographically specific branching. Thus, during map formation, proBDNF might be processed to BDNF only in those areas of the tectum where branching is topographically appropriate [21, 29], leading to increased branching. Secretion and processing of proBNDF/BDNF from tectal cells might be involved here too.
Our data on the antagonism of BDNF and proBDNF in retinotectal (nasal) axon guidance strengthens the general model of neurotrophin receptor function that proposes that antagonistic functions of Trks and p75NTR control numerous biological processes [9, 18]. Our data agree well, for example, with studies in the peripheral nervous system analysing the pruning of sympathetic axons after their projection to eye muscles. Here, correctly targeted ('winning') branches are maintained because of prevalent Trk signalling, while p75NTR activation in 'losing' axons causes axonal degeneration by suppressing TrkA-mediated signalling [30, 31].
Materials and methods
EphA7-Fc was from R&D Systems, Fc control from Calbiochem, BDNF from Promega, wild-type proBDNF and proNGF from Alomone Lab (Jerusalem, Israel). PanTrk203 antibody was a gift from Joan Comella, Lleida (Spain), and the antibody specific for the pro-domain of proBDNF (mAb287 ) was from Genecopoeia, Inc. (Rockville, MD, USA) Anti-FLAG and NGF was from Sigma and αHA from Abcam (Sigma: St. Louis, MO, USA; Abcam UK: Cambridge, U.K.). αHA and αFLAG agarose beads for immunoprecipitation of HA-tagged constructs were obtained from Sigma. The source of all other reagents is described in Marler et al. .
Outgrowth/branching assays and stripe assays
Outgrowth/branching assays and stripe assays were performed as described in Marler et al. . RGCs for both the stripe assay and the axon branching assay were grown in Neurobasal media with 2% B27 supplement, 1 mM glutamine, 5 μM forskolin and 1% penicillin/streptomycin; thus, in synthetic media containing, per se, no neurotrophins. For the axon branching assay the media were supplemented with proneurotrophins or neurotrophins as described in the respective figure legends. RGCs were identified by staining for markers expressed on retinal axons, such as TrkB, Brn3A and Thy-I. Routinely, those cells with the longest axons were positive for these markers. Scoring was performed after 3 days for the branching assay using proneurotrophin/neurotrophin treatment (Figure 5). For the standard stripe assay, E6 retinae were used and scoring was performed after 2 days in culture. Here the cells were plated on a substrate of alternating lanes of EphA7-Fc and Fc, or Fc and Fc as a control. Protocols are described in Marler et al.  and Rashid et al. . To investigate axon guidance in the stripe assay, concentrations of 30 μg/ml each were used. Axons were scored as '0' if the majority of axons showed no preference for either stripe. They were scored as '1' if they showed some avoidance of the EphA7-Fc* stripe (or Fc*), and as '2' for a strong preference for growing on Fc stripes, that is, almost completely or fully avoided the EphA7-Fc* (or Fc*) stripes. The evaluation of all branching and stripe assays was done blind to the constructs transfected and ligands added. Stripe assay data shown are the result of three independently performed experiments.
For the proBDNF stripe assay experiments, single cells from E6 chick retina were electroporated with an eGFP expression plasmid and plated on alternating lanes of EphA7-Fc and Fc, or Fc and Fc. Experiments were performed in the presence of a proBDNF antibody (1:200)  or a control antibody (mouse monoclonal antibody for placental alkaline phosphatase; 1:200). The quantification of axon growth preferences shows an abolishment of repellent guidance in the presence of the proBDNF antibody. The number of axons analysed for each experimental condition was > 40.
For the axonal branching assays single cells were prepared from the nasal third of E8 chick retinae and electroporated with an eGFP expression construct to facilitate later analysis of neurite outgrowth. Cultures were plated on laminin (10 μg/ml) and merosin (2 μg/ml) coated dishes and cultured for 4 days.
Co-immunoprecipitation experiments were performed as described in Marler et al. . In brief, after transfection, cells were lysed in 25 mM Tris, pH 7.4, 150 mM NaCl, 1% (v/v) Triton X100, 1 mM EDTA pH 8.0, 10% (v/v) glycerol, 1 mM phenylmethyl-sulfonyl fluoride (PMSF), 1 mM sodium fluoride (NaF), 1 mM sodium vanadate, 1 mM sodium pyrophosphate, 1 mM glycerol pyrophosphate and Roche Complete Protease Inhibitor. (Roche UK: Hertfordshire, U.K.). After passing through a 26G needle, cell lysates were cleared by centrifugation for 20 minutes at 13,000 g. Approximately 1 mg of total protein from each sample was incubated with 20 μl of respective affinity gel overnight at 4°C on an orbital shaker. Immunocomplexes were washed four times with lysis buffer, suspended in 20 μl of Laemmli buffer, boiled and loaded onto a 10% SDS-polyacrylamide gel, and transferred onto nitrocellulose. Blots were immunodetected with suitable antibodies and developed using Western Lightning' Chemoluminescence Reagent from Perkin Elmer according to the manufacturer's instructions and it was exposed to an X-ray film as long as required (Perkin Elmer: Waltham, MA, USA).
RNAi knockdown experiments were performed as described in Marler et al. . Target sequences for p75NTR RNAi(2) were ACCTCCGATGCTGAGTGCAGA and for p75NTR RNAi(3) were CTACGGCTACTTCCAGGATG.
Cloning and expression of soluble p75NTR, sol, FLAG DNA
The extracellular domain of p75NTR was obtained by PCR using the forward primer CC TGC CTG GAC AGT GTG, and reverse primer TCA GGT GCC ACG GCT CAC, covering the amino acids PCLDS.....VSRGT, using the chick p75NTR cDNA as a template. The resultant PCR fragment was cloned into a CMV promoter-containing expression vector with 5' sequences encoding a signal peptide and a triplicate FLAG tag. Clones were sequence-verified. For stripe assay experiments involving p75NTR, sol, FLAG, two 6 cm2 plates with 5 × 105 CHO cells each were transfected per construct. Two days later the media were harvested and 500 μl of transfected or mock transfected media was added to each of the stripe assay cover slips to give a 1:1 ratio. A further 1 ml of the media was reserved and analysed for the presence of p75NTR, sol, FLAG.
brain derived neurotrophic factor
Chinese hamster ovary
enhanced green fluorescent protein
nerve growth factor
retinal ganglion cells
We would like to thank Helen Smith and Milan Makwana for help with experiments, and Ivo Lieberam and Phillip Gordon-Weeks for valuable comments on the manuscript. This work was supported by grants from the Wellcome Trust and the BBSRC.
- Clandinin TR, Feldheim DA: Making a visual map: mechanisms and molecules. Curr Opin Neurobiol. 2009, 19: 174-180. 10.1016/j.conb.2009.04.011.PubMed CentralView ArticlePubMedGoogle Scholar
- Huberman AD, Feller MB, Chapman B: Mechanisms underlying development of visual maps and receptive fields. Annu Rev Neurosci. 2008, 31: 479-509. 10.1146/annurev.neuro.31.060407.125533.PubMed CentralView ArticlePubMedGoogle Scholar
- McLaughlin T, O'Leary DD: Molecular gradients and development of retinotopic maps. Annu Rev Neurosci. 2005, 28: 327-355. 10.1146/annurev.neuro.28.061604.135714.View ArticlePubMedGoogle Scholar
- Rashid T, Upton AL, Blentic A, Ciossek T, Knoll B, Thompson ID, Drescher U: Opposing gradients of ephrin-As and EphA7 in the superior colliculus are essential for topographic mapping in the mammalian visual system. Neuron. 2005, 47: 57-69. 10.1016/j.neuron.2005.05.030.View ArticlePubMedGoogle Scholar
- Lim YS, McLaughlin T, Sung TC, Santiago A, Lee KF, O'Leary DD: p75(NTR) mediates ephrin-A reverse signaling required for axon repulsion and mapping. Neuron. 2008, 59: 746-758. 10.1016/j.neuron.2008.07.032.PubMed CentralView ArticlePubMedGoogle Scholar
- Marler KJ, Becker-Barroso E, Martinez A, Llovera M, Wentzel C, Poopalasundaram S, Hindges R, Soriano E, Comella J, Drescher U: A TrkB/EphrinA interaction controls retinal axon branching and synaptogenesis. J Neurosci. 2008, 28: 12700-12712. 10.1523/JNEUROSCI.1915-08.2008.PubMed CentralView ArticlePubMedGoogle Scholar
- Reichardt LF: Neurotrophin-regulated signalling pathways. Philos Trans R Soc Lond B Biol Sci. 2006, 361: 1545-1564. 10.1098/rstb.2006.1894.PubMed CentralView ArticlePubMedGoogle Scholar
- Chao MV: Neurotrophins and their receptors: a convergence point for many signalling pathways. Nat Rev Neurosci. 2003, 4: 299-309. 10.1038/nrn1078.View ArticlePubMedGoogle Scholar
- Greenberg ME, Xu B, Lu B, Hempstead BL: New insights in the biology of BDNF synthesis and release: implications in CNS function. J Neurosci. 2009, 29: 12764-12767. 10.1523/JNEUROSCI.3566-09.2009.PubMed CentralView ArticlePubMedGoogle Scholar
- Barnes P, Thomas KL: Proteolysis of proBDNF is a key regulator in the formation of memory. PLoS One. 2008, 3: e3248-10.1371/journal.pone.0003248.PubMed CentralView ArticlePubMedGoogle Scholar
- Bergami M, Santi S, Formaggio E, Cagnoli C, Verderio C, Blum R, Berninger B, Matteoli M, Canossa M: Uptake and recycling of pro-BDNF for transmitter-induced secretion by cortical astrocytes. J Cell Biol. 2008, 183: 213-221. 10.1083/jcb.200806137.PubMed CentralView ArticlePubMedGoogle Scholar
- Domeniconi M, Hempstead BL, Chao MV: Pro-NGF secreted by astrocytes promotes motor neuron cell death. Mol Cell Neurosci. 2007, 34: 271-279. 10.1016/j.mcn.2006.11.005.PubMed CentralView ArticlePubMedGoogle Scholar
- Nagappan G, Zaitsev E, Senatorov VV, Yang J, Hempstead BL, Lu B: Control of extracellular cleavage of ProBDNF by high frequency neuronal activity. Proc Natl Acad Sci USA. 2009, 106: 1267-1272. 10.1073/pnas.0807322106.PubMed CentralView ArticlePubMedGoogle Scholar
- Yang J, Siao CJ, Nagappan G, Marinic T, Jing D, McGrath K, Chen ZY, Mark W, Tessarollo L, Lee FS, Lu B, Hempstead BL: Neuronal release of proBDNF. Nat Neurosci. 2009, 12: 113-115. 10.1038/nn.2244.PubMed CentralView ArticlePubMedGoogle Scholar
- Lou H, Kim SK, Zaitsev E, Snell CR, Lu B, Loh YP: Sorting and activity-dependent secretion of BDNF require interaction of a specific motif with the sorting receptor carboxypeptidase e. Neuron. 2005, 45: 245-255. 10.1016/j.neuron.2004.12.037.View ArticlePubMedGoogle Scholar
- Matsumoto T, Rauskolb S, Polack M, Klose J, Kolbeck R, Korte M, Barde YA: Biosynthesis and processing of endogenous BDNF: CNS neurons store and secrete BDNF, not pro-BDNF. Nat Neurosci. 2008, 11: 131-133. 10.1038/nn2038.View ArticlePubMedGoogle Scholar
- Keifer J, Sabirzhanov BE, Zheng Z, Li W, Clark TG: Cleavage of proBDNF to BDNF by a tolloid-like metalloproteinase is required for acquisition of in vitro eyeblink classical conditioning. J Neurosci. 2009, 29: 14956-14964. 10.1523/JNEUROSCI.3649-09.2009.PubMed CentralView ArticlePubMedGoogle Scholar
- Lu B, Pang PT, Woo NH: The yin and yang of neurotrophin action. Nat Rev Neurosci. 2005, 6: 603-614. 10.1038/nrn1726.View ArticlePubMedGoogle Scholar
- Lee R, Kermani P, Teng KK, Hempstead BL: Regulation of cell survival by secreted proneurotrophins. Science. 2001, 294: 1945-1948. 10.1126/science.1065057.View ArticlePubMedGoogle Scholar
- Rosch H, Schweigreiter R, Bonhoeffer T, Barde YA, Korte M: The neurotrophin receptor p75NTR modulates long-term depression and regulates the expression of AMPA receptor subunits in the hippocampus. Proc Natl Acad Sci USA. 2005, 102: 7362-7367. 10.1073/pnas.0502460102.PubMed CentralView ArticlePubMedGoogle Scholar
- Pang PT, Teng HK, Zaitsev E, Woo NT, Sakata K, Zhen S, Teng KK, Yung WH, Hempstead BL, Lu B: Cleavage of proBDNF by tPA/plasmin is essential for long-term hippocampal plasticity. Science. 2004, 306: 487-491. 10.1126/science.1100135.View ArticlePubMedGoogle Scholar
- Cohen-Cory S, Lom B: Neurotrophic regulation of retinal ganglion cell synaptic connectivity: from axons and dendrites to synapses. Int J Dev Biol. 2004, 48: 947-956. 10.1387/ijdb.041883sc.View ArticlePubMedGoogle Scholar
- Herzog KH, Bailey K, Barde YA: Expression of the BDNF gene in the developing visual system of the chick. Development. 1994, 120: 1643-1649.PubMedGoogle Scholar
- Herzog KH, von Bartheld BC: Contributions of the optic tectum and the retina as sources of brain-derived neurotrophic factor for retinal gangion cells in the chick embryo. J Neurosci. 1998, 18: 2891-2906.PubMedGoogle Scholar
- Wang KC, Kim JA, Sivasankaran R, Segal R, He Z: P75 interacts with the Nogo receptor as a co-receptor for Nogo, MAG and OMgp. Nature. 2002, 420: 74-78. 10.1038/nature01176.View ArticlePubMedGoogle Scholar
- Jansen P, Giehl K, Nyengaard JR, Teng K, Lioubinski O, Sjoegaard SS, Breiderhoff T, Gotthardt M, Lin F, Eilers A, Petersen CM, Lewin GR, Hempstead BL, Willnow TE, Nykjaer A: Roles for the pro-neurotrophin receptor sortilin in neuronal development, aging and brain injury. Nat Neurosci. 2007, 10: 1449-1457. 10.1038/nn2000.View ArticlePubMedGoogle Scholar
- Naska S, Lin DC, Miller FD, Kaplan DR: p75NTR is an obligate signaling receptor required for cues that cause sympathetic neuron growth cone collapse. Mol Cell Neurosci. 2010, 45: 108-120. 10.1016/j.mcn.2010.05.015.View ArticlePubMedGoogle Scholar
- Cohen-Cory S, Fraser SE: Effects of brain-derived neurotrophic factor on optic axon branching and remodelling in vivo. Nature. 1995, 378: 192-196. 10.1038/378192a0.View ArticlePubMedGoogle Scholar
- Lochner JE, Honigman LS, Grant WF, Gessford SK, Hansen AB, Silverman MA, Scalettar BA: Activity-dependent release of tissue plasminogen activator from the dendritic spines of hippocampal neurons revealed by live-cell imaging. J Neurobiol. 2006, 66: 564-577. 10.1002/neu.20250.View ArticlePubMedGoogle Scholar
- Kohn J, Aloyz RS, Toma JG, Haak-Frendscho M, Miller FD: Functionally antagonistic interactions between the TrkA and p75 neurotrophin receptors regulate sympathetic neuron growth and target innervation. J Neurosci. 1999, 19: 5393-5408.PubMedGoogle Scholar
- Singh KK, Park KJ, Hong EJ, Kramer BM, Greenberg ME, Kaplan DR, Miller FD: Developmental axon pruning mediated by BDNF-p75NTR-dependent axon degeneration. Nat Neurosci. 2008, 11: 649-658. 10.1038/nn.2114.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.