Biglycan is an extracellular MuSK binding protein important for synapse stability

Abstract

The receptor tyrosine kinase MuSK is indispensable for nerve-muscle synapse formation and maintenance. MuSK is necessary for prepatterning of the endplate zone anlage and as a signaling receptor for agrin-mediated postsynaptic differentiation. MuSK-associated proteins such as Dok7, LRP4, and Wnt11r are involved in these early events in neuromuscular junction formation. However, the mechanisms regulating synapse stability are poorly understood. Here we examine a novel role for the extracellular matrix protein biglycan in synapse stability. Synaptic development in fetal and early postnatal biglycan null (bgn(-/o)) muscle is indistinguishable from wild-type controls. However, by 5 weeks after birth, nerve-muscle synapses in bgn(-/o) mice are abnormal as judged by the presence of perijunctional folds, increased segmentation, and focal misalignment of acetylcholinesterase and AChRs. These observations indicate that previously occupied presynaptic and postsynaptic territory has been vacated. Biglycan binds MuSK and the levels of this receptor tyrosine kinase are selectively reduced at bgn(-/o) synapses. In bgn(-/o) myotubes, the initial stages of agrin-induced MuSK phosphorylation and AChR clustering are normal, but the AChR clusters are unstable. This stability defect can be substantially rescued by the addition of purified biglycan. Together, these results indicate that biglycan is an extracellular ligand for MuSK that is important for synapse stability.

Figure 1.

Synapses in mature biglycan null mice are abnormal. A, Biglycan is expressed at the neuromuscular junction of wild-type mice. Frozen sections of quadriceps femoris (5 weeks old) were labeled with α-bungarotoxin (red) and anti-biglycan (green). Biglycan is expressed at both the neuromuscular junctions and the noninnervated regions of the sarcolemma. No immunoreactivity is observed when sections from biglycan null muscle are stained in the same fashion (data not shown). Scale bar, 10 μm. B, C, The NMJs of biglycan null mice are hypersegmented. Sternomastoid muscle was dissected from perfusion-fixed 5-week-old wild-type (B) and biglycan null (C) littermates and then double-labeled (left) with rh-α-bungarotoxin to visualize the AChRs (red) and anti-SV/anti-neurofilament (green) to show the nerve. Merged images, left; AChR distribution only, right. Note the segmentation (arrowheads) of the biglycan null endplate in contrast to the continuous postsynaptic domains observed in the wild-type. Scale bar, 10 μm. D, The number of segments per endplate from muscles fixed and imaged as described above were scored. Endplates in biglycan null mice were significantly more segmented than those of their littermate controls (n = 60 endplates from 4 animals for each condition; Mann–Whitney U test; p < 0.01).

Figure 2.

Synapses in developing biglycan null mice are similar to wild-type. A, Postsynaptic specializations in fetal biglycan null mice. Diaphragms were dissected from E16 biglycan null mice and labeled with rh-α-bungarotoxin to visualize the AChRs. Discrete AChR clusters are arrayed in a central band across the muscle. This morphology and localization are indistinguishable from that observed in wild-type muscle at this age (data not shown). Scale bar, 20 μm. B, Diaphragms were dissected from P0 and P14 heterozygous (top) and biglycan null (bottom) littermates and double labeled with rh-α-bungarotoxin to visualize the AChRs (red) and anti-synaptophysin/anti-neurofilament (green) to show the nerve. In each set the merged and AChR-only images are shown at left and right, respectively. No structural differences were observed in the synapses from these developing biglycan null mice compared with their normal littermates. Scale bar, 10 μm.

Figure 3.

Focal misalignment of AChR and AChE at synapses in biglycan null mice. A, Hindlimb muscles from 5-week-old wild-type (top) and biglycan null (bottom) littermates were dissected, fixed, and labeled with rh-α-bungarotoxin (red) and OG-fasciculin2 (green) to visualize AChR and AChE, respectively. The AChR and AChE distributions are superimposable in the WT. However, AChE domains lacking subadjacent AChR (arrows) were observed in the biglycan null junctions. Scale bar, 10 μm. B, Quantification of the number of synapses with aligned or misaligned AChR and AChE in wild-type and biglycan null muscles. In wild-type muscle complete alignment was observed at 100% (90/90) of synapses. However, only 20% of the mutant synapses (30/150) showed complete alignment. n = number of synapses scored for each genotype.

Figure 4.

Increased number of perijunctional folds in biglycan null synapses. A, Electron micrographs of synapses in sternomastoid muscles from 5-week-old wild-type and congenic biglycan null mice. In the wild-type muscle junctional folds are largely restricted to the region directly under the synaptic bouton. However, in biglycan null animals an increased number of perijunctional folds (arrowheads) are observed flanking the synapse. Scale bar, 2 μm. B, Frequency histogram of perijunctional folds in synapses from wild-type and biglycan null mice. The number of perijunctional folds within 0.5–2 μm from the edge of nerve terminal was scored for two wild-type (n = 36 perijunctional regions) and two biglycan null muscles (n = 72 perijunctional regions). There are significantly more perijunctional folds in mutant muscle (Kolmogorov–Smirnov; p < 0.01).

Figure 5.

MuSK expression is decreased at synapses in biglycan null mice. A, Quadriceps femoris sections from 5-week-old wild-type and biglycan null animals were immunolabeled with a mouse anti-utrophin (green) and a rabbit anti-MuSK (affinity purified 29–31, see Materials and Methods, Antibodies; red). Utrophin is expressed at similar levels at synapses in wild-type and biglycan null muscle. However, a decrease in the intensity of MuSK staining at biglycan null neuromuscular junctions is observed. Scale bar, 10 μm. B, The mean pixel signal intensity of MuSK normalized to the mean pixel signal intensity of utrophin at the neuromuscular junctions of wild-type, 10.78 ± 3.54, and biglycan null muscle, 1.00 ± 1.32 (at least 3 synapses per condition) was determined as described in Materials and Methods. The signal intensity of MuSK was significantly higher at wild-type versus biglycan null junctions (Student's unpaired t test, *p < 0.02). C, Sternomastoid sections from 5-week-old wild-type and biglycan null mice were immunolabeled as in A. Utrophin is expressed at similar levels at the synapses in wild-type and biglycan null sternomasoid while synaptic MuSK is decreased in the biglycan null sternomastoid muscle. Scale bar, 10 μm.

Figure 6.

Biglycan binds to MuSK. A, Direct binding of biglycan and MuSK. Purified MuSK ectodomain or BSA was immobilized on plastic wells and then incubated with either BSA or with 500 ng of biotinylated recombinant biglycan followed by avidin-alkaline phosphatase. Biglycan binds to MuSK but not to BSA (0.45 ± 0.01 and 0.06 ± 0.01, respectively; n = 3, Student's unpaired t test, p < 0.01). B, Biglycan binds to MuSK expressed in heterologous cells. COS cells were transfected for 24 h with FLAG-tagged full-length wild-type MuSK (WT-MuSK), TrkA, or FLAG-tagged MuSK mutants lacking either the IgI (ΔIgI) or CRD/Fz domain (ΔCRD/Fz) as indicated. Live cells were incubated with purified recombinant biglycan polypeptide (20 nm) for 30 min at 4°C and bound biglycan was visualized with an anti-biglycan monoclonal antibody and Alexa488-conjugated secondary antibodies (green) and then fixed. The transfected constructs (red) were visualized with anti-FLAG (all MuSK constructs) or anti-trkA ectodomain. Note that biglycan binds to COS cells expressing full-length, wild-type MuSK (top), but not to cells expressing TrkA (middle), mutant MuSK ΔIgI, or ΔCRD/Fz (bottom). No binding was detected to untransfected cells (data not shown). Scale bars, 10 μm. C, Quantification of biglycan binding to MuSK or TrkA transfected cells. Data are expressed as the mean percentage of cells that bound biglycan per microscope field Little biglycan binding was detected on cells expressing heterologous TrkA (3.3 ± 3.3%; n = 10 fields). Cells expressing wild-type MuSK exhibit robust biglycan binding (86.3 ± 7.1%; n = 10 fields; *p < 0.001, Students unpaired t test). Biglycan binding to cells expressing MuSK-ΔIgI or ΔCRD/Fz was not significantly different from that to TrKA-expressing cells; p > 0.8, one-way ANOVA).

Figure 7.

Biglycan regulates agrin-induced MuSK phosphorylation. A, Biglycan potentiates agrin-induced MuSK phosphorylation. C2C12 myotubes were treated for 1 h with 1U agrin, 1.4 nm of purified recombinant biglycan, or both as indicated. The levels of MuSK tyrosine phosphorylation were then assessed by probing MuSK immunoprecipitates with anti-phosphotyrosine antibody (P-Tyr). The blots were stripped and reprobed with anti-MuSK to verify equal loading. Agrin induces MuSK phosphorylation and this activity is potentiated by biglycan. B, Quantification. Western blots were scanned and analyzed with ImageQuant software (GE Healthcare) to compare relative changes in MuSK phosphorylation. Simultaneous treatment with 1U agrin and 1.4 nm biglycan induces a 1.5-fold increase in MuSK phosphorylation compared with 1U agrin alone (p < 0.001, n = 10). Treatment with saturating concentrations of agrin (5 U) alone induces a 1.6-fold increase in MuSK phosphorylation above 1U agrin (p < 0.02, n = 5). C, Biglycan does not modulate MuSK phosphorylation induced by saturating concentrations (max) of agrin. C2C12 cells were stimulated with 5 U agrin in the presence or absence of 1.4 nm biglycan. MuSK phosphorylation was assessed as in A. D, Quantification. Biglycan does not induce a statistically significant change in MuSK phosphorylation above that induced by 5 U agrin alone (p > 0.3, n = 3). E, Equivalent levels of MuSK are expressed in C2C12 and bgn^−/o myotubes. Cultured C2C12 and biglycan null myotubes were extracted and MuSK was incubated with anti-MuSK antisera followed by protein-A-beads. Immunoprecipitates solubilized in sample buffer, electrophoresed, transferred to nitrocellulose and probed with an anti-MuSK antibody. F, Biglycan is not required for agrin-induced MuSK phosphorylation. Biglycan null myotubes were treated for 15 min, 30 min, or 1 h with 1U agrin. MuSK activation was determined by probing MuSK immunoprecipitates with anti-tyrosine antibody (P-Tyr). Note that agrin induces MuSK phosphorylation in biglycan null cells within 15 min of treatment and phosphorylation levels increase through 1 h of treatment.

Figure 8.

Agrin-induced AChR clustering is defective in biglycan null myotubes. A, Time course of agrin-induced AChR clustering in wild-type and bgn^−/o myotubes. Myotubes of indicated genotype were made from immortalized myoblast lines and were treated with 5 U agrin for 0, 2, 4, 6, 8, or 12 h. Short duration agrin treatment (2, 4, 6 h) induces small, discrete AChR clusters in both wild-type and biglycan null cells. After 12 h of agrin treatment wild-type myotubes displayed typical compact AChR clusters (<1000 μm²). In contrast, biglycan null myotubes treated for 12 h with agrin exhibited diffuse AChR microcluster islands that were abnormally large (∼1.4 × 10³ to ∼1 × 10⁴ μm²). Scale bar, 10 μm. B, Exogenous biglycan rescues the stability defects of agrin-induced AChR clusters in bgn^−/o myotubes. Myotubes were incubated for 12 h with 5 U agrin with or without added biglycan (1.4 nm). Treatment with agrin and biglycan induced the formation of smaller, compact AChR clusters compared with agrin alone. Treatment with biglycan alone had no effect on AChR distribution (data not shown). Scale bar, 10 μm. C, Quantification of biglycan rescue. The number of AChR clusters (>4 μm in length and <1 × 10³ μm²) per myotube segment on cultures treated with agrin alone or with agrin plus biglycan was scored (n = 70 segments/condition from 3 independent experiments; Student's unpaired t test, p < 0.03).