Brief Communications

ProBDNF and Mature BDNF as Punishment and Reward Signals for Synapse Elimination at Mouse Neuromuscular Junctions

H. Shawn Je, Feng Yang, Yuanyuan Ji, Srilatha Potluri, Xiu-Qing Fu, Zhen-Ge Luo, Guhan Nagappan, Jia Pei Chan, Barbara Hempstead, Young-Jin Son and Bai Lu
Journal of Neuroscience 12 June 2013, 33 (24) 9957-9962; DOI: https://doi.org/10.1523/JNEUROSCI.0163-13.2013

Abstract

During development, mammalian neuromuscular junctions (NMJs) transit from multiple-innervation to single-innervation through axonal competition via unknown molecular mechanisms. Previously, using an in vitro model system, we demonstrated that the postsynaptic secretion of pro-brain-derived neurotrophic factor (proBDNF) stabilizes or eliminates presynaptic axon terminals, depending on its proteolytic conversion at synapses. Here, using developing mouse NMJs, we obtained in vivo evidence that proBDNF and mature BDNF (mBDNF) play roles in synapse elimination. We observed that exogenous proBDNF promoted synapse elimination, whereas mBDNF infusion substantially delayed synapse elimination. In addition, pharmacological inhibition of the proteolytic conversion of proBDNF to mBDNF accelerated synapse elimination via activation of p75 neurotrophin receptor (p75NTR). Furthermore, the inhibition of both p75NTR and sortilin signaling attenuated synapse elimination. We propose a model in which proBDNF and mBDNF serve as potential “punishment” and “reward” signals for inactive and active terminals, respectively, in vivo.

Introduction

Activity-dependent synaptic competition plays a critical role in shaping patterns of synaptic connections in the nervous system. At the rodent neuromuscular junction (NMJ), multiple axons compete with one another for the same postsynaptic muscle cell during early postnatal days. The most active terminal gets stabilized, whereas the less active ones withdraw. By the end of the second postnatal week, all axons but one are eliminated, resulting in canonical elimination of polyneuronal innervation (Nguyen and Lichtman, 1996). This synapse elimination is thought to be mediated by an activity-dependent process that involves both “punishment” and “reward” signals from postsynaptic muscle cells (Wyatt and Balice-Gordon, 2003). Despite significant efforts over two decades, the molecular identities of these prospective punishment and/or reward signals remain unknown (Lichtman and Colman, 2000).

Brain-derived neurotrophic factor (BDNF), a neurotrophic factor known to regulate synapse development and plasticity, is secreted from target cells in an activity-dependent manner (Misgeld et al., 2002). BDNF is initially synthesized as a precursor (proBDNF), which is proteolytically processed into mature BDNF (mBDNF) (Lu, 2003). Intriguingly, these two forms of BDNF bind to two different types of cell-surface receptors in motor nerve terminals: proBDNF binds to the pan-neurotrophin receptor p75 (p75NTR), whereas mBDNF preferentially binds to the tyrosine receptor kinase B (TrkB). By binding to distinct receptor systems, proBDNF and mBDNF elicit seemingly opposite biological effects at developing neuromuscular synapses (Lu, 2003; Wu et al., 2010; Je et al., 2012). For example, exogenous application of mBDNF triggers synaptic potentiation and maturation of developing NMJs through TrkB, whereas application of proBDNF suppresses synaptic transmission and causes axonal retraction by activating presynaptic p75NTR(Yang et al., 2009a). Furthermore, using a triplet culture system that allows gene manipulation in one of two distinctly labeled axons, which innervates a single myocyte, we previously demonstrated that proBDNF serves as a punishment signal that causes axon terminals expressing p75NTR to retract. Neuronal activity converts proBDNF to mBDNF, which serves as a reward signal to stabilize and maintain the axon terminal during synaptic competition (Je et al., 2012).

Although these data strongly support a model in which postsynaptic secretion of a single molecule, proBDNF, regulates synapse elimination and stabilization, it remains unclear whether proBDNF and mBDNF play a role during synapse elimination in vivo. Here, we used both pharmacological and genetic manipulations to study the roles of endogenous proBDNF and mBDNF in synapse elimination using mouse Levator auris longus (LAL) neuromuscular synapses.

Materials and Methods

Animals.

BDNF−/− mice were bred and genotyped as previously described (Ernfors et al., 1994). TrkB knock-in (TrkBF616A) mice, in which endogenous TrkB activity can be inhibited pharmacologically by in vivo administration of 1NMPP1, were used (Chen et al., 2005). Neonatal pups of either sex were obtained from C57BL/6 mice and the date of birth was designated postnatal day 0 (P0). p75NTR knock-out mice (p75NTR KO) were purchased from The Jackson Laboratory and were backcrossed onto a C57BL/6 background for more than five generations. All animal procedures conformed to guidelines of the National Institutes of Health.

Drug treatment.

Drugs, recombinant proteins, peptides, and antibodies were administered by subcutaneous injection over the right LAL muscle of neonatal mice. Litters were culled to a maximum of 10 pups to minimize variation in growth due to differences in feeding. Factors were diluted to appropriate concentrations in saline containing 0.1% BSA (v/v), and 30–50 μl of was injected twice daily from P1. Doses used for various factors were as follows: IgG (Santa Cruz Biotechnology), 3 μg/ml; affinity-purified anti-p75NTR IgG, 3 μg/ml; purified proBDNF, 0.6 μg/ml; recombinant mBDNF (Regeneron), 0.5 μg/ml; pan metalloproteinase (MMP) inhibitor, 60 μm; general protease inhibitor cocktail, 50 μm; sortilin propeptide (a gift from Dr Peder Madsen, Aarhus University, Aarhus, Denmark) or GST, 10 μm. 1NMPP1 dissolved in saline was administered by intraperitoneal injection to TrkBF616A mice 2–3 times daily from P1 to P7 (5 mm in 20 μl). TrkBF616A mice that received injections of DMSO diluted in the same vehicle solution and wild-type mice that received 1NMPP1, a small molecule of the protein kinase inhibitor protein phosphatase 1 (selective antagonist of TrkB F616), were used as controls.

Immunocytochemistry and morphological analysis.

Whole mounts of LAL muscles were processed for immunostaining as previously described (Burns et al., 2007). The following primary antibodies were used: antineurofilaments (SMI312, Sternberger Monoclonals); anti-SV2 (SYP, Developmental Studies Hybridoma Bank); α-bungarotoxin (Invitrogen); anti-sortilin (R&D Systems); anti-p75NTR was a gift from Phil Barker (McGill University, Montreal, Canada); the antibody against the cytoplasmic domain of TrkB was from Santa Cruz Biotechnology and the antibody against the extracellular domain of TrkB was kindly provided by Louis Reichardt (University of California, San Francisco, CA). Secondary antibodies included fluorescein-conjugated goat anti-mouse IgG (Roche) and Alexa Fluor 647-conjugated goat anti-rabbit, anti-chick, or anti-mouse IgG (Invitrogen). Images were taken with a Zeiss LSM510 confocal microscope.

Western blot analysis.

LAL muscles of neonatal mice (from P5 to P16) were carefully dissected out, homogenized in ice-cold RIPA lysis buffer. We used anti-proBDNF (generated by the Lu Laboratory) and mouse anti-Tubulin (Abcam) as primary antibodies.

Results

Expression of p75NTR, TrkB, and proBDNF at developing mouse NMJs

We tested whether p75NTR, TrkB, and proBDNF are expressed at mouse NMJs during the period of synapse elimination. Previous studies have reported high levels of p75NTR expression in spinal motor neurons, particularly during early development (Yan and Johnson, 1988; Koliatsos et al., 1993; Garcia et al., 2011). We performed whole-mount immunohistochemistry and observed intense p75NTR immunoreactivity associated with motor nerve terminals innervating sternomastoid muscles at P5 (Fig. 1A) and P10 (data not shown). In contrast, we observed very little immunoreactivity associated with Schwann cells, indicating that neonatal Schwann cells in association with axons are not expressing p75NTR (data not shown). Next, to determine whether endogenous TrkB protein is expressed in motor nerve terminals at P5 NMJs, we performed whole-mount immunohistochemistry and observed that TrkB immunoreactivity was primarily associated with motor axons and their terminals (Fig. 1B).

Figure 1.
Figure 1.

Expression of proBDNF, p75NTR and TrkB at the developing NMJs, and the role of TrkB in synapse elimination. A, Photomicrographs of immunostaining of NMJs showing association of p75NTR immunoreactivity in neonatal axons and nerve terminals. Whole-mount preparations of sternomastoid muscles from P5 mice were stained with specific antibodies against neurofilament (NF) and the synaptic vesicle protein SV2 to label axons (NF/SV2; green), against p75NTR (white), and against postsynaptic acetylcholine receptors (AChRs; red). B, Photomicrographs of immunostaining of NMJs showing association of TrkB immunoreactivity in neonatal axons and nerve terminals. P5 sternomastoid muscles were stained as in A, except that TrkB (white) replaces p75NTR. Scale bars: A, B, D, E; 10 μm. C, Expression of proBDNF in the developing muscle. LAL muscles of neonatal mice were dissected, homogenized, and processed for Western blot using a specific antibody against proBDNF (Yang et al., 2009b). D, Elimination of axon terminals from endplates triggered by the inhibition of TrkB signaling using the TrkBF616A knock-in-1NMPP1 system. To inhibit TrkB signaling in developing NMJs in vivo, 1NMPP1 was injected intraperitoneally into TrkBF616A knock-in mice for 5 d (P2–P7). We observed that many endplates, identified by postsynaptic clusters of AChRs, were unoccupied by terminal arbors (white arrowheads). E, Normal synaptic elimination in BDNF KO. In both WT and BDNF KO mice, nearly all of the junctions were singly innervated by P14. F, Quantification of synapse elimination at mouse NMJs. Data represent mean ± SEM. for > 500 endplates from at least 3 mice per group. *Values are significantly different from control, with p < 0.05 by Fisher's test.

Although previous studies have demonstrated the expression of BDNF mRNA and protein in developing muscle cells (Funakoshi et al., 1995; Ip et al., 2001), no study has addressed whether proBDNF is expressed in muscles. Using a specific antibody against proBDNF (Yang et al., 2009b), we observed proBDNF expression in muscles during the period of synapse elimination (Fig. 1C). These data confirmed the expression of p75NTR, TrkB, and proBDNF at developing mouse NMJs in vivo.

Role of TrkB in synapse elimination at mouse NMJs

We next tested whether removal of TrkB signaling, which mediates the mBDNF signal, could accelerate synapse elimination in vivo. Because TrkB homozygous knock-out mice die shortly after birth (Klein et al., 1993), we took advantage of TrkBF616A knock-in mice, in which the endogenous TrkB is replaced with a mutated TrkBF616A (Chen et al., 2005). Upon administration of 1NMPP1, which selectively binds to the mutated TrkB receptors, TrkB autophosphorylation and its signaling are blocked in these knock-in mice. Using this chemical-genetic approach, we sought to inhibit TrkB signaling during postnatal NMJ development.

We performed a series of experiments using mouse LAL neuromuscular junction (Garcia et al., 2011). The LAL muscle has been extensively used for pharmacological studies of synapse elimination in vivo (Angaut-Petit et al., 1987). At P6, most of the LAL NMJs are polyinnervated, and at P14, nearly all junctions are singly innervated. To test whether removal of TrkB signaling accelerates synapse elimination in vivo, we injected both wild-type and TrkBF616A knock-in mice with 1NMPP1 intraperitoneally twice per day from P2 to P7. We found that 1NMPP1 dramatically accelerated synapse elimination at the NMJs of TrkBF616A knock-in mice. At P7, polyneuronal innervation was reduced from 90% in vehicle-treated to <47% in 1NMPP1-treated junctions in the LAL muscles in TrkBF616A mice (Fig. 1D; p < 0.05, Fisher's test; n > 700 NMJs, N = 3 mice per condition). Strikingly, the terminal arbors of many NMJs (>34%) appear to have shifted in TrkBF616A mice, leaving some or all postsynaptic AChRs unoccupied (Fig. 1D). No sign of sprouting or axonal degeneration was observed in these muscles (Fig. 1D). As an additional control, treatment of wild-type LAL muscles with 1NMPP1 had no effect on synaptic stabilization or elimination, suggesting that 1NMPP1 does not elicit toxic side-effects (Fig. 1D,F). These results highlight the importance of TrkB signaling in the stabilization or maintenance of axon arbors during synaptic elimination.

We next tested whether the removal of both the punishment signal (proBDNF) and the reward signal (mBDNF) could affect synapse elimination in vivo. To this end, we analyzed NMJs of BDNF knock-out mice (BDNF KO). We observed no changes in synapse elimination at P14 in BDNF KO mice (Fig. 1E,F; n > 500 NMJs, N = 4 mice). The lack of phenotype in BDNF KO mice may be due to the removal of both punishment (proBDNF) and reward (mBDNF) signals, suggesting that polyneuronal to mononeuronal synapse elimination occurs by default.

Roles for proBDNF and mBDNF in synapse elimination at mouse NMJs

Next, we performed a series of pharmacological experiments using mouse LAL neuromuscular synapses by infusing recombinant proBDNF and mBDNF (Garcia et al., 2011). At control (BSA-injected) NMJs at P6, AChR-rich postsynaptic sites were completely occupied by multiple axon terminals (Fig. 2A). More than 95% of NMJs were multiply innervated (Fig. 2D; n > 500 NMJs, N = 4 mice). In contrast, the proBDNF-treated muscles exhibited dramatic retraction of axon terminals from postsynaptic sites (Fig. 2A). Most NMJs were either innervated by a single axon or were even devoid of any axon (Fig. 2D; 34% NMJs with no innervation, 26% with single innervation, n = 800, N = 4 mice). Following the proBDNF treatment, postsynaptic AChR clusters appeared more elongated and larger, compared with controls. These AChR clusters were observed at the junctions on thin muscle fibers that lack innervation, suggesting that the phenomenon is likely to be secondary to denervation or muscle fiber atrophy-associated remodeling of AChR clusters (Fig. 2A) (Misgeld et al., 2002). Conversely, in P14 LAL muscles treated with mBDNF, ∼23% of the NMJs remained innervated by multiple axons (Fig. 2B, n > 500 NMJs, N = 4 mice). In comparison, nearly all (98%) NMJs were singly innervated by P14 in the BSA-treated muscles (Fig. 2B,D; p < 0.05, Fisher's test, n > 650 NMJs, N = 4 mice). Together, exogenous proBDNF promoted the elimination of nerve terminals, whereas mBDNF led to persistence of multiple innervations at many NMJs.

Figure 2.
Figure 2.

Pharmacological manipulation of synapse elimination. The LAL muscles were immunostained to visualize preterminal and terminal portions of axons, with specific antibodies against NF, the synaptic vesicle protein SV2 (NF/SV2; green) and postsynaptic clusters of AChRs (red). A, Retraction of axon terminals induced by administration of proBDNF. Many endplates show no (white arrowhead) or only rudimentary (white arrows) arbors of nerve terminals. B, Persistence of multiple innervations due to exogenous application of mBDNF. Endplates innervated by multiple axons (yellow arrowheads) were abundant in P14 NMJs treated with mBDNF, compared with NMJs treated with BSA. C, Retraction of terminal arbors induced by treatment of pan-MMP inhibitors. Subcutaneous application of pan-MMP inhibitors (panMMP In) into LAL muscles induced partial elimination of terminal arbors at many endplates (white arrows), as indicated by the presence of AChR-rich patches (arrowheads), that were not juxtaposed with axon termini. D, Quantification of synapse elimination at mouse NMJs following various treatments. *Values differ from control groups (either P6 or P14, treated for the same period with BSA) at p < 0.05 by Fisher's test. In, Inhibitors. Scale bar, 10 μm.

Previously, we reported that metalloproteases mediate conversion of proBDNF to mBDNF at developing neuromuscular synapses (Yang et al., 2009a). Therefore, we tested whether the inhibition of proteases that convert proBDNF to mBDNF results in accumulation of endogenous proBDNF at the NMJs in vivo, leading to accelerated elimination of axon terminals. Intriguingly, we observed the significant reduction of polyneuronal innervation at P6 NMJs when LAL muscles were treated with pan MMP inhibitors for 6 d (P1–P6) (Fig. 2C,D). Approximately 22% of NMJs were singly innervated in pan MMP-treated muscles, compared with 2% in vehicle-treated muscles (Fig. 2D; p < 0.05, Fisher's test, n > 800 NMJs, N = 4 mice).

Role of p75NTR in synapse elimination at mouse NMJs in vivo

If p75NTR mediates the punishment signal, ablation of p75NTR might allow retention of polyneuronal innervations at NMJs later in development. To our surprise, p75NTR knock-out (p75NTR KO) mice showed normal synapse elimination at P14 (Fig. 3A,B; n > 500 NMJs, N = 4 mice). More detailed analysis of the developmental time course revealed that the patterns of polyneuronal innervation in p75NTR KO mice were analogous to those in wild-type littermate controls at P7, and P14 (Fig. 3B). The lack of phenotype at NMJs of p75NTR KO mice led us to hypothesize that proBDNF-mediated synaptic retraction requires simultaneous activation of p75NTR and a complementary receptor sortilin, a coreceptor that binds to pro-neurotrophins (Nykjaer et al., 2004; Teng et al., 2005; Jansen et al., 2007). Consistent with this hypothesis, we observed that sortilin is expressed in both motor neurons and postsynaptic LAL muscles (Fig. 3C).

Figure 3.
Figure 3.

Normal synapse elimination in p75NTR knock-out mice and expression of sortilin at NMJs. A, Normal synaptic elimination in p75NTR knock-out (p75NTR KO) mice. In both WT and p75NTR KO mice, almost all junctions were singly innervated by P14. Scale bar: A, C; 20 μm. B, Quantification of synapse elimination in P14 muscles from WT and p75NTR KO mice. Data represent mean ± SEM. for >500 endplates from at least 4 mice per group. C, Immunofluorescence staining showing the expression of endogenous sortilin at NMJs. Whole-mount preparations of P5 muscles were immunostained using specific antibodies against sortilin (green), synaptophysin (yellow), and acetylcholine receptors (red). DAPI (blue) was used to label nuclei. The region in the white box is magnified at the top right.

To test whether blockade of sortilin signaling in p75NTR KO mice prevents synapse elimination, LAL muscles from either p75NTR KO mice or their WT littermates were subcutaneously injected with recombinant GST-tagged sortilin propeptides (Fig. 4A) from P1 to P14. Sortilin propeptides are known to inhibit binding of sortilin ligands including neurotensin and proBDNF, but not proNGF (Munck Petersen et al., 1999; Quistgaard et al., 2009). At P14, AChR-rich postsynaptic sites were completely innervated by a single axon in two control groups (98%, p75NTR KO mice injected with GST alone, n > 300 NMJs, N = 2 mice, 99%, WT mice injected with sortilin propeptide, n > 600 NMJs, N = 4 mice) (Fig. 4B,D). Intriguingly, many NMJs were innervated by multiple axons in P14 muscles from p75NTR KO mice injected with sortilin propeptides (19%, n > 1000 NMJs, N = 6 mice; p < 0.05, ANOVA), indicating that inhibition of both p75NTR and sortilin attenuated synapse elimination in vivo (Fig. 4B,D).

Figure 4.
Figure 4.

Synapse elimination mediated by endogenous proBDNF via both sortilin and p75NTR. A, The 3D structure of sortilin. Top, Schematic diagram of sortilin. Sortilin-pro, Sortilin-propeptides. Bottom, 3D representation of sortilin (purple) and putative binding sites of sortilin propeptides (yellow). The 3D image was constructed using PDB (PDBID: 3F6K; http://www.rcsb.org/pdb/explore/explore.do?pdbId=3F6K) (Quistgaard et al., 2009). B, Substantial polyinnervation in P14 NMJ of p75NTR KO mice injected with sortilin propeptides. Note that synaptic elimination proceeds normally in WT mice injected with sortilin propeptides and also in p75NTR KO mice injected with GST peptides, which served as a negative control. Yellow arrowheads indicate endplates innervated by multiple axon terminals. Scale bar: B, C; 20 μm. C, Persistence of polyneuronal innervation upon exogenous application of antibodies against p75NTR (anti-p75NTR IgG). LAL muscles were treated with anti-p75NTR IgG twice daily for 14 d (P1–P14). Endplates innervated by multiple axons (yellow arrowheads) increased in the anti-p75NTR IgG-treated muscles, but not increased muscles treated with the control IgG. D, Quantification of synapse elimination at mouse NMJs following various treatments in B and C. *Values differ from control groups (WT, IgG-treat or GST-treated, p < 0.05, ANOVA).

The effectiveness of postnatal injection of sortilin propeptide suggests the importance of inhibiting the punishment signal during the first two postnatal weeks, when synaptic competition occurs. To test this idea, we locally injected an affinity purified anti-p75NTR antibody to the LAL muscle of wild-type animals twice daily from P1 to P14. Because p75NTR and sortilin cooperate to promote pro-neurotrophin binding (Nykjaer et al., 2004), it is likely that the anti-p75NTR antibody blocks the proBDNF-sortilin interaction. Remarkably, anti-p75NTR antibody treatment resulted in many NMJs of the LAL muscles retaining polyneuronal innervation at P14 (Fig. 4C,D; ranging from 11% to 22% NMJs, n = 825 NMJs, N = 8 mice). In comparison, <3% of NMJs were innervated by multiple axons at P14 in the LAL muscles treated with control IgG (p < 0.05, Fisher's test, n = 425 NMJs, N = 4 mice). Together, blockade of p75NTR signaling during the period of synapse elimination attenuates synapse elimination at developing mouse NMJs.

Discussion

There are two hypotheses for activity-dependent synaptic elimination: the “synaptotoxin” hypothesis proposes that the postsynaptic cell secretes a destabilizing factor that remove presynaptic terminals, and the “synaptotrophin” hypothesis proposes that axons compete against one another for a trophic factor derived from the postsynaptic cell (Snider and Lichtman, 1996). The in vivo results presented in this study, together with the in vitro data published earlier (Yang et al., 2009a; Je et al., 2012), suggest a new model in which a single molecule, BDNF, can be either the punishment or the “reward signal”, depending on proteolytic conversion. Thus, proBDNF from postsynaptic muscle cells serves as a “punishment signal” that induces retraction of nerve terminals through p75NTR. In parallel, neuronal activity drives secretion and/or activation of metalloproteases to convert proBDNF to mBDNF, a reward signal for which all terminals compete.

Although our “gain-of-function” pharmacological studies demonstrated that the conversion of the punishment signal (proBDNF) to the reward signal (mBDNF) is critical for synapse elimination, results from the “loss-of-function” experiments using knock-out mice were not so straightforward. For example, BDNF KO NMJs showed normal synapse elimination (Fig. 1E,F). This result is not completely unexpected, because genetic compensation is quite common in neurotrophin knock-out mice (Conover et al., 1995; Liu et al., 1995). Furthermore, deletion of the p75NTR gene did not attenuate synapse elimination. This may due to that the punishment signal (proBDNF) at the p75NTR−/− NMJ may be mediated or compensated for by other proBDNF receptors such as sortilin. In support for this hypothesis, the injection of a sortilin antagonist to the LAL muscle in the p75NTR−/− significantly increased polyinnervation (Fig. 4A), whereas blockade of sortilin signal alone in the wild-type muscle did not affect synapse elimination (Fig. 4A,B).

The other possibility to explain the lack of phenotype in p75NTR−/− NMJ is that the effect of inhibiting p75NTR is only relevant during the period of synapse elimination. In this model, deletion of the p75NTR gene in early embryos may eliminate the need for competition, leading to a default single innervation at the postnatal NMJ. Notably, similar “default” mechanisms have been reported. For example, although agrin is required for AChR clustering later in development, the AChR clusters form normally by default in early embryos of agrin knock-out mice (Lin et al., 2001). Additionally, in the visual cortex, complete inhibition of neuronal activity early in development does not prevent the formation of ocular dominance columns, although postnatal manipulation of visual activity drastically alters ocular dominance (Crowley and Katz, 1999, 2000). In this study, we show that infusion of the p75NTR blocking antibody to wild-type NMJs during the process of synapse competition markedly attenuated synapse elimination (Fig. 4A,B). Similarly, inhibition of TrkB signaling by daily injection of 1NMPP1 to TrkBF616A knock-in mice accelerated synapse elimination (Fig. 1D).

Together, our study supports a model that single innervation of a NMJ may be a default mechanism during early development, and the punishment (proBDNF)-reward (mBDNF) system acts postnatally to ensure the precision of the motorneuron-muscle connections in vivo.

Footnotes

  • The study was supported by NIMH and NICHD intramural research programs (B.L.), NIH, MDA, and Shriners Hospitals grants (B.L.H. and Y.-J.S.), and by grants from the Singapore National Medical Research Council and the Singapore Ministry of Education (H.S.J.). We thank Drs Eugene Zaitsev, Phillip Nelson, Neil Shneider, Keri Martinowitch, Jay Chang, Newton Woo, and Albert Chen for their thoughtful comments and suggestions, and Regeneron Pharmaceuticals for providing recombinant BDNF. We express our gratitude to Drs Bruce Carter, Mark Bothwell, Moses Chao, and Phil Barker for antibodies to p75NTR, and Louis Reichardt and Moses Chao for antibodies to TrkB. We are particularly grateful to Dr David Ginty and Xi Chen, who provided us the TrkBF616A knock-in mice and also helped with the injection of 1NMPP1. We thank Dr Peder Madsen for the construct to express sortilin propeptide.

  • The authors declare no competing financial interests.

  • Correspondence should be addressed to one of the following: Dr. Bai Lu, GlaxoSmithKline, R&D China, Shanghai, 201203, China, Bai.b.lu{at}gsk.com; Young-Kin Son, Shriners Hospitals Pediatric Research Center, Temple University School of Medicine, Philadelphia, PA 19140, yson{at}temple.edu; or Program in Neuroscience and Behavioral Disorders, DUKE-NUS, 8 College Road, 169857 Singapore, shawn.je{at}duke-nus.edu.sg

References

  1. ↵
    1. Angaut-Petit D,
    2. Molgo J,
    3. Connold AL,
    4. Faille L
    (1987) The levator auris longus muscle of the mouse: a convenient preparation for studies of short- and long-term presynaptic effects of drugs or toxins. Neurosci Lett 82:83–88, doi:10.1016/0304-3940(87)90175-3, pmid:2962016.
    OpenUrlCrossRefPubMed
  2. ↵
    1. Burns AS,
    2. Jawaid S,
    3. Zhong H,
    4. Yoshihara H,
    5. Bhagat S,
    6. Murray M,
    7. Roy RR,
    8. Tessler A,
    9. Son YJ
    (2007) Paralysis elicited by spinal cord injury evokes selective disassembly of neuromuscular synapses with and without terminal sprouting in ankle flexors of the adult rat. J Comp Neurol 500:116–133, doi:10.1002/cne.21143, pmid:17099885.
    OpenUrlCrossRefPubMed
  3. ↵
    1. Chen X,
    2. Ye H,
    3. Kuruvilla R,
    4. Ramanan N,
    5. Scangos KW,
    6. Zhang C,
    7. Johnson NM,
    8. England PM,
    9. Shokat KM,
    10. Ginty DD
    (2005) A chemical-genetic approach to studying neurotrophin signaling. Neuron 46:13–21, doi:10.1016/j.neuron.2005.03.009, pmid:15820690.
    OpenUrlCrossRefPubMed
  4. ↵
    1. Conover JC,
    2. Erickson JT,
    3. Katz DM,
    4. Bianchi LM,
    5. Poueymirou WT,
    6. McClain J,
    7. Pan L,
    8. Helgren M,
    9. Ip NY,
    10. Boland P
    (1995) Neuronal deficits, not involving motor neurons, in mice lacking BDNF and/or NT4. Nature 375:235–238, doi:10.1038/375235a0, pmid:7746324.
    OpenUrlCrossRefPubMed
  5. ↵
    1. Crowley JC,
    2. Katz LC
    (1999) Development of ocular dominance columns in the absence of retinal input. Nat Neurosci 2:1125–1130, doi:10.1038/16051, pmid:10570491.
    OpenUrlCrossRefPubMed
  6. ↵
    1. Crowley JC,
    2. Katz LC
    (2000) Early development of ocular dominance columns. Science 290:1321–1324, doi:10.1126/science.290.5495.1321, pmid:11082053.
    OpenUrlAbstract/FREE Full Text
  7. ↵
    1. Ernfors P,
    2. Lee KF,
    3. Jaenisch R
    (1994) Mice lacking brain-derived neurotrophic factor develop with sensory deficits. Nature 368:147–150, doi:10.1038/368147a0, pmid:8139657.
    OpenUrlCrossRefPubMed
  8. ↵
    1. Funakoshi H,
    2. Belluardo N,
    3. Arenas E,
    4. Yamamoto Y,
    5. Casabona A,
    6. Persson H,
    7. Ibáñez CF
    (1995) Muscle-derived neurotrophin-4 as an activity-dependent trophic signal for adult motor neurons. Science 268:1495–1499, doi:10.1126/science.7770776, pmid:7770776.
    OpenUrlAbstract/FREE Full Text
  9. ↵
    1. Garcia N,
    2. Tomas M,
    3. Santafe MM,
    4. Lanuza MA,
    5. Besalduch N,
    6. Tomas J
    (2011) Blocking p75 (NTR) receptors alters polyinnervationz of neuromuscular synapses during development. J Neurosci Res 89:1331–1341, doi:10.1002/jnr.22620, pmid:21674565.
    OpenUrlCrossRefPubMed
  10. ↵
    1. Ip FC,
    2. Cheung J,
    3. Ip NY
    (2001) The expression profiles of neurotrophins and their receptors in rat and chicken tissues during development. Neurosci Lett 301:107–110, doi:10.1016/S0304-3940(01)01603-2, pmid:11248434.
    OpenUrlCrossRefPubMed
  11. ↵
    1. Jansen P,
    2. Giehl K,
    3. Nyengaard JR,
    4. Teng K,
    5. Lioubinski O,
    6. Sjoegaard SS,
    7. Breiderhoff T,
    8. Gotthardt M,
    9. Lin F,
    10. Eilers A,
    11. Petersen CM,
    12. Lewin GR,
    13. Hempstead BL,
    14. Willnow TE,
    15. Nykjaer A
    (2007) Roles for the pro-neurotrophin receptor sortilin in neuronal development, aging and brain injury. Nat Neurosci 10:1449–1457, doi:10.1038/nn2000, pmid:17934455.
    OpenUrlCrossRefPubMed
  12. ↵
    1. Je HS,
    2. Yang F,
    3. Ji Y,
    4. Nagappan G,
    5. Hempstead BL,
    6. Lu B
    (2012) Role of pro-brain-derived neurotrophic factor (proBDNF) to mature BDNF conversion in activity-dependent competition at developing neuromuscular synapses. Proc Natl Acad Sci U S A 109:15924–15929, doi:10.1073/pnas.1207767109, pmid:23019376.
    OpenUrlAbstract/FREE Full Text
  13. ↵
    1. Klein R,
    2. Smeyne RJ,
    3. Wurst W,
    4. Long LK,
    5. Auerbach BA,
    6. Joyner AL,
    7. Barbacid M
    (1993) Targeted disruption of the trkB neurotrophin receptor gene results in nervous system lesions and neonatal death. Cell 75:113–122, doi:10.1016/S0092-8674(05)80088-1, pmid:8402890.
    OpenUrlCrossRefPubMed
  14. ↵
    1. Koliatsos VE,
    2. Clatterbuck RE,
    3. Winslow JW,
    4. Cayouette MH,
    5. Price DL
    (1993) Evidence that brain-derived neurotrophic factor is a trophic factor for motor neurons in vivo. Neuron 10:359–367, doi:10.1016/0896-6273(93)90326-M, pmid:8080464.
    OpenUrlCrossRefPubMed
  15. ↵
    1. Lichtman JW,
    2. Colman H
    (2000) Synapse elimination and indelible memory. Neuron 25:269–278, doi:10.1016/S0896-6273(00)80893-4, pmid:10719884.
    OpenUrlCrossRefPubMed
  16. ↵
    1. Lin W,
    2. Burgess RW,
    3. Dominguez B,
    4. Pfaff SL,
    5. Sanes JR,
    6. Lee KF
    (2001) Distinct roles of nerve and muscle in postsynaptic differentiation of the neuromuscular synapse. Nature 410:1057–1064, doi:10.1038/35074025, pmid:11323662.
    OpenUrlCrossRefPubMed
  17. ↵
    1. Liu X,
    2. Ernfors P,
    3. Wu H,
    4. Jaenisch R
    (1995) Sensory but not motor neuron deficits in mice lacking NT4 and BDNF. Nature 375:238–241, doi:10.1038/375238a0, pmid:7746325.
    OpenUrlCrossRefPubMed
  18. ↵
    1. Lu B
    (2003) Pro-region of neurotrophins: role in synaptic modulation. Neuron 39:735–738, doi:10.1016/S0896-6273(03)00538-5, pmid:12948441.
    OpenUrlCrossRefPubMed
  19. ↵
    1. Misgeld T,
    2. Burgess RW,
    3. Lewis RM,
    4. Cunningham JM,
    5. Lichtman JW,
    6. Sanes JR
    (2002) Roles of neurotransmitter in synapse formation: development of neuromuscular junctions lacking choline acetyltransferase. Neuron 36:635–648, doi:10.1016/S0896-6273(02)01020-6, pmid:12441053.
    OpenUrlCrossRefPubMed
  20. ↵
    1. Munck Petersen C,
    2. Nielsen MS,
    3. Jacobsen C,
    4. Tauris J,
    5. Jacobsen L,
    6. Gliemann J,
    7. Moestrup SK,
    8. Madsen P
    (1999) Propeptide cleavage conditions sortilin/neurotensin receptor-3 for ligand binding. EMBO J 18:595–604, doi:10.1093/emboj/18.3.595, pmid:9927419.
    OpenUrlAbstract
  21. ↵
    1. Nguyen QT,
    2. Lichtman JW
    (1996) Mechanism of synapse disassembly at the developing neuromuscular junction. Curr Opin Neurobiol 6:104–112, doi:10.1016/S0959-4388(96)80015-8, pmid:8794054.
    OpenUrlCrossRefPubMed
  22. ↵
    1. Nykjaer A,
    2. Lee R,
    3. Teng KK,
    4. Jansen P,
    5. Madsen P,
    6. Nielsen MS,
    7. Jacobsen C,
    8. Kliemannel M,
    9. Schwarz E,
    10. Willnow TE,
    11. Hempstead BL,
    12. Petersen CM
    (2004) Sortilin is essential for proNGF-induced neuronal cell death. Nature 427:843–848, doi:10.1038/nature02319, pmid:14985763.
    OpenUrlCrossRefPubMed
  23. ↵
    1. Quistgaard EM,
    2. Madsen P,
    3. Groftehauge MK,
    4. Nissen P,
    5. Petersen CM,
    6. Thirup SS
    (2009) Ligands bind to Sortilin in the tunnel of a ten-bladed beta-propeller domain. Nat Struct Mol Biol 16:96–98, doi:10.1038/nsmb.1543, pmid:19122660.
    OpenUrlCrossRefPubMed
  24. ↵
    1. Snider WD,
    2. Lichtman JW
    (1996) Are neurotrophins synaptotrophins? Mol Cell Neurosci 7:433–442, doi:10.1006/mcne.1996.0031, pmid:8875427.
    OpenUrlCrossRefPubMed
  25. ↵
    1. Teng HK,
    2. Teng KK,
    3. Lee R,
    4. Wright S,
    5. Tevar S,
    6. Almeida RD,
    7. Kermani P,
    8. Torkin R,
    9. Chen ZY,
    10. Lee FS,
    11. Kraemer RT,
    12. Nykjaer A,
    13. Hempstead BL
    (2005) ProBDNF induces neuronal apoptosis via activation of a receptor complex of p75NTR and sortilin. J Neurosci 25:5455–5463, doi:10.1523/JNEUROSCI.5123-04.2005, pmid:15930396.
    OpenUrlAbstract/FREE Full Text
  26. ↵
    1. Wu H,
    2. Xiong WC,
    3. Mei L
    (2010) To build a synapse: signaling pathways in neuromuscular junction assembly. Development 137:1017–1033, doi:10.1242/dev.038711, pmid:20215342.
    OpenUrlAbstract/FREE Full Text
  27. ↵
    1. Wyatt RM,
    2. Balice-Gordon RJ
    (2003) Activity-dependent elimination of neuromuscular synapses. J Neurocytol 32:777–794, doi:10.1023/B:NEUR.0000020623.62043.33, pmid:15034267.
    OpenUrlCrossRefPubMed
  28. ↵
    1. Yan Q,
    2. Johnson EM Jr.
    (1988) An immunohistochemical study of the nerve growth factor receptor in developing rats. J Neurosci 8:3481–3498, pmid:2845023.
    OpenUrlAbstract
  29. ↵
    1. Yang F,
    2. Je HS,
    3. Ji Y,
    4. Nagappan G,
    5. Hempstead B,
    6. Lu B
    (2009a) Pro-BDNF-induced synaptic depression and retraction at developing neuromuscular synapses. J Cell Biol 185:727–741, doi:10.1083/jcb.200811147, pmid:19451278.
    OpenUrlAbstract/FREE Full Text
  30. ↵
    1. Yang J,
    2. Siao CJ,
    3. Nagappan G,
    4. Marinic T,
    5. Jing D,
    6. McGrath K,
    7. Chen ZY,
    8. Mark W,
    9. Tessarollo L,
    10. Lee FS,
    11. Lu B,
    12. Hempstead BL
    (2009b) Neuronal release of proBDNF. Nat Neurosci 12:113–115, doi:10.1038/nn.2244, pmid:19136973.
    OpenUrlCrossRefPubMed
Back to top

In this issue

Email
Print
View Full Page PDF
Citation Tools
Respond to this article
ProBDNF and Mature BDNF as Punishment and Reward Signals for Synapse Elimination at Mouse Neuromuscular Junctions
H. Shawn Je, Feng Yang, Yuanyuan Ji, Srilatha Potluri, Xiu-Qing Fu, Zhen-Ge Luo, Guhan Nagappan, Jia Pei Chan, Barbara Hempstead, Young-Jin Son, Bai Lu
Journal of Neuroscience 12 June 2013, 33 (24) 9957-9962; DOI: 10.1523/JNEUROSCI.0163-13.2013
del.icio.us logo Digg logo Reddit logo Twitter logo Facebook logo Google logo Mendeley logo

Jump to section

Responses to this article

Respond to this article

Jump to comment:

No eLetters have been published for this article.