Muscle-derived collagen XIII regulates maturation of the skeletal neuromuscular junction

Abstract

Formation, maturation, stabilization, and functional efficacy of the neuromuscular junction (NMJ) are orchestrated by transsynaptic and autocrine signals embedded within the synaptic cleft. Here, we demonstrate that collagen XIII, a nonfibrillar transmembrane collagen, is another such signal. We show that collagen XIII is expressed by muscle and its ectodomain can be proteolytically shed into the extracellular matrix. The collagen XIII protein was found present in the postsynaptic membrane and synaptic basement membrane. To identify a role for collagen XIII at the NMJ, mice were generated lacking this collagen. Morphological and ultrastructural analysis of the NMJ revealed incomplete adhesion of presynaptic and postsynaptic specializations in collagen XIII-deficient mice of both genders. Strikingly, Schwann cells erroneously enwrapped nerve terminals and invaginated into the synaptic cleft, resulting in a decreased contact surface for neurotransmission. Consistent with morphological findings, electrophysiological studies indicated both postsynaptic and presynaptic defects in Col13a1(-/-) mice, such as decreased amplitude of postsynaptic potentials, diminished probabilities of spontaneous release and reduced readily releasable neurotransmitter pool. To identify the role of collagen XIII at the NMJ, shed ectodomain of collagen XIII was applied to cultured myotubes, and it was found to advance acetylcholine receptor (AChR) cluster maturation. Together with the delay in AChR cluster development observed in collagen XIII-deficient mutants in vivo, these results suggest that collagen XIII plays an autocrine role in postsynaptic maturation of the NMJ. Altogether, the results presented here reveal that collagen XIII is a novel muscle-derived cue necessary for the maturation and function of the vertebrate NMJ.

Figure 1.

Collagen XIII is concentrated at the NMJ. A, Wild-type mouse muscle sections are stained using an anti-human collagen XIII/NC3 antibody (Col XIII; green) and BTX (red). B, C, Immunoelectron microscopy of P28 mouse diaphragm muscles with the anti-mouse collagen XIII/NC3 antibody (B) and of P56 quadriceps muscles with the anti-human collagen XIII/NC3 antibody (C). Muscle is indicated by transparent green and nerve terminal by transparent purple. Staining at the synaptic sarcolemma and in the adjacent BM is shown by red circles (B, C) and staining in the muscle beneath the NMJ by arrows (B).

Figure 2.

Collagen XIII at the NMJ is expressed by muscle and subjected to shedding. A, Schematic presentation of the primary structure of mouse collagen XIII, with collagenous domains (COL1–COL3) indicated by yellow triple helices, non-collagenous domains (NC1–NC4) in pink, and the membrane-spanning region by a green box. Below is the fusion protein expressed in the Col13a1^LacZ reporter mice, the blue box indicating β-galactosidase. Residues encoded by exon 2 are indicated by a pink striped box. Size is indicated in residues (amino acids) and the furin cleavage site by an arrowhead, this being absent from the fusion protein. B, Quadriceps muscles from Col13a1^LacZ/LacZ mice are stained as whole mounts for β-galactosidase enzyme activity (arrows). C, Cross-sections of whole-mount stained muscles are poststained with eosin. D, E, Muscle sections from Col13a1^LacZ/+ mice are costained with BTX (green, D) or anti-neurofilament antibody (NF; green, E) together with anti-β-galactosidase antibody (β-gal; red). F, Muscle whole mounts are stained for β-galactosidase enzyme activity and studied by electron microscopy, showing crystals (arrows) in the Col13a1^LacZ/LacZ samples (nerve terminal, transparent purple; Schwann cell, transparent blue). G, Both transmembrane (TM) and shed forms of collagen XIII are detected in Western blotting with the anti-human collagen XIII/NC3 antibody in the conditioned medium (M_T) and cell lysate from differentiated C2C12 myotubes (C_T) but not from C2C12 myoblasts (C_B). Proteins from differentiated myotubes are fractioned and subjected to Western blotting (Nu, nuclear proteins; Cy, cytoplasmic proteins; Ma, membrane-associated proteins; Tm, transmembrane proteins). Proteins representing 4% of the proteins in whole-cell lysates, conditioned medium, or in each fraction on a 75 cm² plate are run per line. In the membrane-associated and membrane fractions, the collagen XIII protein is present as one major and one minor band, which are likely to represent a major and a minor splice form of the collagen XIII transcripts, as reported previously for a number of other tissues (Peltonen et al., 1997). A large amount of serum proteins in the medium sample (M_T) may affect the mobility of the collagen XIII protein on SDS-PAGE, resulting in a higher molecular weight than expected.

Figure 3.

Collagen XIII is required for presynaptic differentiation. A, Restriction of Col13a1 in the exon 1 area, with 5′ UTR (black) and translated sequences (gray), and generation of a targeted (Col13a1^T) and a knock-out allele (Col13a1⁻). The loxP sites flanking the PGK-neo^r, the direction of which is indicated by an arrow, are shown by arrowheads (B, BamHI; S, SfiI; X, XbaI). B, Quantification of collagen XIII transcripts in wild-type (white columns; n = 4) and Col13a1^−/− (black columns; n = 5) E16 fetuses by quantitative real-time PCR with two distinct primer pairs and probes chosen from the areas of exons 2–4 and exons 26–27. The value for the wild-type fetuses is defined as 100% in artificial units as a mean ± SEM. C, Western blotting of lung and femoral tissues (20 μg/line) from wild-type and Col13a1^−/− mice with the anti-human collagen XIII/NC3 antibody. D, Selected high-magnification images of wild-type and Col13a1^−/− diaphragms at P56 labeled with BTX (arrowheads; extrajunctional protrusions). E, Selected additional muscles at P56 are labeled with BTX, and the AChR clusters are evaluated morphologically (TA, anterior tibialis, n = 92 for wild-type and 91 for Col13a1^−/−; Dia, diaphragm, n = 100 for wild-type and 600 for Col13a1^−/−, EDL, extensor digitorum longus, n = 105; Sol, soleus, n = 138). F, NMJs in P28 mouse diaphragms are colabeled with anti-Syt2 antibody (green) together with BTX (red). Axonal branches growing beyond AChR clusters are indicated as arrows, and synaptotagmin 2 staining in preterminal axons as arrowheads. G, NMJs in a P56 Col13a1^−/− mouse diaphragm are colabeled with an anti-Syt2 antibody (green) together with BTX (red; arrowheads, lack of synaptotagmin 2 staining at AChR-positive areas).

Figure 4.

Collagen XIII is required for the adhesion of presynaptic and postsynaptic partners. A–C, Electron microscopy of NMJs in the wild-type and Col13a1^−/− mouse quadriceps muscle at P28 (A) and diaphragm muscle at P56 (B-C) (muscle, transparent green; nerve terminal, transparent purple; Schwann cell, transparent blue; arrow, enwrapped nerve terminus; arrowhead, degeneration; red circle, active zone). Note the difference in scale bars between wild-type and Col13a1^−/− samples in B and C.

Figure 5.

Electrophysiological measurements indicate compromised function of the neuromuscular synapse in the lack of collagen XIII. A, B, Recording of MEPPs in diaphragm muscle in 3-month-old control and Col13a1^−/− mice indicates amplitudes of 0.33 ± 0.02 mV in control (n = 21 synapses) versus 0.28 ± 0.02 mV in Col13a1^−/− (n = 20; p = 0.04). Wild-type samples are indicated by white columns and Col13a1^−/− samples as black columns (B,C; E,F; H,I). C, The frequency of MEPPs is 0.73 ± 0.06 for wild-type (n = 21) and 0.20 ± 0.04 s⁻¹ for Col13a1^−/− (n = 20; p < 0.001) mice. D, E, Sucrose at 500 mm increases the frequency of ACh release to 13.97 ± 0.82 in wild-type (n = 31) compared with 3.99 ± 0.37 s⁻¹ in Col13a1^−/− (n = 32; p < 0.001) samples. F, Potassium at 15 mm increases the frequency of ACh release to 8.10 ± 0.94 in wild-type (n = 26) and 3.79 ± 0.61 in Col13a1^−/− (n = 37; p < 0.001) mice. G, H, The postsynaptic EPP is 14.84 ± 1.43 in wild-type (n = 42) and 9.82 ± 0.55 mV in Col13a1^−/− (n = 32; p = 0.03) mice. I, Paired-pulse stimulation with the interstimulus interval of 30 ms induces increase in amplitude to 7.10 ± 1.26 in wild-type mice (n = 42), although it leads to depression of −10.47 ± 1.88% in Col13a1^−/− mice (n = 32; showing p < 0.001 with Mann–Whitney test). J, K, The sciatic nerve of 3-month-old wild-type (n = 13) and Col13a1^−/− (n = 12) male mice is repetitively stimulated at 3 Hz in electroneuromyography. Maximum decrement (percentage) of the amplitude is determined between the second and fifth compound muscle potential after stimulation (arrows in J). Means (wild-type, 1.6; Col13a1^−/−, −0.3) are indicated by striated lines (K).

Figure 6.

Collagen XIII enhances postsynaptic maturation in vitro. A, B, C2C12 mouse myoblasts are differentiated in culture and stained for AChR clusters with BTX at 3 DPF (A) or 4 DPF (B). Medium of selected cultures is supplemented with soluble collagen XIII ectodomain (sCol XIII) at fusion. C, Differentiated C2C12 myotubes supplemented with soluble collagen XIII are stained for LL5β (green) together with BTX (red) at 3 DPF.

Figure 7.

Collagen XIII has a role in postsynaptic maturation. NMJs from wild-type and Col13a1^−/− diaphragms are labeled with BTX at P0, P14, P28, and P56.