A synaptic nidogen: Developmental regulation and role of nidogen-2 at the neuromuscular junction
© Fox et al; licensee BioMed Central Ltd. 2008
Received: 05 June 2008
Accepted: 25 September 2008
Published: 25 September 2008
The skeletal neuromuscular junction is a useful model for elucidating mechanisms that regulate synaptogenesis. Developmentally important intercellular interactions at the neuromuscular junction are mediated by the synaptic portion of a basal lamina that completely ensheaths each muscle fiber. Basal laminas in general are composed of four main types of glycosylated proteins: laminins, collagens IV, heparan sulfate proteoglycans and nidogens (entactins). The portion of the muscle fiber basal lamina that passes between the motor nerve terminal and postsynaptic membrane has been shown to bear distinct isoforms of the first three of these. For laminins and collagens IV, the proteins are deposited by the muscle; a synaptic proteoglycan, z-agrin, is deposited by the nerve. In each case, the synaptic isoform plays key roles in organizing the neuromuscular junction. Here, we analyze the fourth family, composed of nidogen-1 and -2.
In adult muscle, nidogen-1 is present throughout muscle fiber basal lamina, while nidogen-2 is concentrated at synapses. Nidogen-2 is initially present throughout muscle basal lamina, but is lost from extrasynaptic regions during the first three postnatal weeks. Neuromuscular junctions in mutant mice lacking nidogen-2 appear normal at birth, but become topologically abnormal as they mature. Synaptic laminins, collagens IV and heparan sulfate proteoglycans persist in the absence of nidogen-2, suggesting the phenotype is not secondary to a general defect in the integrity of synaptic basal lamina. Further genetic studies suggest that synaptic localization of each of the four families of synaptic basal lamina components is independent of the other three.
All four core components of the basal lamina have synaptically enriched isoforms. Together, they form a highly specialized synaptic cleft material. Individually, they play distinct roles in the formation, maturation and maintenance of the neuromuscular junction.
The formation, maturation and maintenance of chemical synapses require multiple interactions between pre- and postsynaptic elements. Many of these interactions are mediated by membrane- or matrix-associated proteins that occupy the narrow cleft separating the pre- and postsynaptic membranes [1–3]. At the skeletal neuromuscular junction (NMJ), where such interactions have been analyzed in detail, a basal lamina (BL) passing between the motor nerve terminal and the postsynaptic membrane comprises the cleft material of this synapse. As expected from this arrangement, several of the molecules required for the formation, maturation and maintenance of the NMJ are BL components [4, 5].
Members of four families of proteins are present in BLs throughout the body: laminins, collagens IV, heparan sulfate proteoglycans (HSPGs), and nidogens/entactins (referred to here as nidogens) . Synaptic and extrasynaptic portions of the muscle fiber BL are known to bear distinct isoforms of the first three of these [4, 5]. Laminins are large heterotrimers composed of α,β, and γ subunits. The major laminin in extrasynaptic BL is the α2β1γ1 heterotrimer, called laminin 211. In contrast, synaptic BL is rich in β2 but poor in β1 laminins, and contains, along with α2, the α4 and α5 subunits, both present at low levels extrasynaptically. Thus, synaptic BL contains laminins 221, 421, and 521 [7–10]. Collagens IV are trimers assembled from a set of six α chains. All muscle BL contains the α1 and α2 chains, likely in an [α1(IV)]2[α2(IV)]1 trimer, whereas the α3–6 chains are selectively associated with synaptic BL, presumably in [α3(IV)][α4(IV)][α5(IV)] and [α5(IV)]2[α6(IV)]1 trimers [8, 11, 12]. The HSPG perlecan is present in both synaptic and extrasynaptic BL, whereas another HSPG, agrin, is concentrated in the synaptic BL [13–15]. Importantly, studies of targeted mutant mice have shown that synaptic isoforms of all three families act as muscle-derived (laminins and collagens IV) or nerve-derived (agrin) synaptic organizers in vivo. Laminin β2 promotes the maturation of motor nerve terminals [16, 17], laminin α4 regulates the precise apposition of pre- and postsynaptic specializations , and together laminins α4 and α5 promote the maturation of postsynaptic specializations . Synaptic collagen chains are required for nerve terminal maintenance , and agrin is a critical stabilizer of postsynaptic differentiation [20–25].
In contrast to this wealth of knowledge about laminins, collagens IV and HSPGs, little is known about localization or roles of nidogens at the neuromuscular synapse. In Caenorhabditis elegans, the lone nidogen gene is necessary for the formation of the NMJ, although the protein is not synaptically enriched . Unlike invertebrate genomes, two nidogen genes are present in mammals, encoding nidogen-1 and nidogen-2 [27–30]. In many tissues, mammalian nidogens are colocalized in BLs [31–33], and genetic studies suggest that they play largely redundant roles. Few phenotypes have been observed in targeted mutants lacking either nidogen-1 or nidogen-2, whereas double mutants lacking both nidogens die perinatally with defects in lung, heart and limb development [32–36]. Moreover, expression of nidogen-2 is dramatically increased in nidogen-1 mutants , supporting the notion that nidogens are capable of compensating for each other. In contrast, we show here that nidogen-2 is selectively associated with synaptic BL in muscle and is required for the maturation and maintenance of the adult NMJ. Synaptic laminins, collagens IV and heparan sulfate proteoglycans persist in the absence of nidogen-2, suggesting that its role extends beyond maintaining the integrity of the BL. Finally, we provide genetic evidence that synaptic localization of each of the four families of BL components is independent of the other three. Taken together with the work cited above, these results show that all four families of BL components have synapse-specific isoforms and synapse-specific functions.
Results and discussion
Selective association of nidogen-2 with neuromuscular synapses
As shown previously , nidogen-1 was associated with surfaces of adult muscle fibers whereas little if any nidogen-2 was detectable on most of the muscle fiber surface (Figure 1C, D). Nidogen-2 was, however, present on many small structures associated with muscle fibers. Most of these were capillaries that lie between muscle fibers (see below) but a minority resembled synaptic sites in size, shape and position. We therefore double-labelled muscle sections with nidogen antibodies plus a fluorescent derivative of α-bungarotoxin, which binds specifically to the acetylcholine receptors (AChRs) concentrated in the postsynaptic membrane at the NMJ. This demonstrated that synaptic sites did indeed contain nidogen-2 (Figure 1E).
On the basis of these findings, we investigated the relationship between nidogen-2 and the antigen recognized by the monoclonal antibody, 9H6, described by Chiu and Ko . 9H6 selectively stains NMJs in rat and binds a carbohydrate-dependent epitope on a nidogen-1-like antigen. This antibody was described prior to the discovery of nidogen-2, so it seemed possible that it recognized nidogen-2. Because 9H6 fails to cross-react with mouse tissue, we stained rat muscle with anti-nidogen-2. In rats, as in mice, nidogen-2 was present at NMJs and in capillaries (data not shown), whereas 9H6 stained only NMJs . Other differences in staining pattern are discussed below. Moreover, on immunoblots, 9H6 recognizes a protein with a molecular weight of 150 kDa , which is the expected size of mammalian nidogen-1 and considerably smaller than nidogen-2 (Figure 1A, B). These differences argue that the 9H6-antigen is not nidogen-2. Thus, synaptic BL may contain both a synapse-specific isoform of nidogen (nidogen-2) and a unique, glycosylated form of nidogen-1.
Synaptic BL can be deposited by muscles fibers, motor nerve terminals or Schwann cells [4, 5, 39]. Both muscle and Schwann cells have been reported to express nidogen-2 [40, 41]. To determine whether muscle cells can contribute nidogen-2 to the synaptic cleft, we used a muscle cell line, C2C12. These cells were fused into myotubes on laminin substrata to promote AChR clustering . Nidogen-2 was enriched at these AChR-rich sites even though no non-muscle cells were present in the cultures (Figure 2E). Thus, muscle cells are capable of nidogen-2 synthesis, secretion, and accumulation at synaptic sites.
As noted in the introduction, synaptic isoforms have been demonstrated for three of the families of core BL components – laminins, collagens IV and HSPGs [4, 5]. Our results add the fourth, nidogen, to this list. The presence of synapse-specific isoforms of common BL components provides a molecular explanation for the observations that synaptic and extrasynaptic BLs are structurally similar and physically continuous but functionally distinct.
Differential distribution of nidogens-1 and -2 in peripheral nerve
A few structures within the nerve fascicle were rich in nidogen-2 (arrowheads in Figure 3B). Although some of these may be capillaries, most were not associated with CD31/PECAM-1, an endothelial cell marker ( and data not shown). Instead, they are likely to be the BL surrounding non-myelinating Schwann cells and their associated bundle of small-caliber, non-myelinated axons . This localization is consistent with the observation that nidogen-2 is present in the BL of terminal Schwann cells (Figure 2C), which are non-myelinating.
Nidogen-1 and -2 were also differentially distributed in BLs of muscle spindles, sensory organs that respond to muscle stretch. Each spindle contains several specialized intrafusal muscle fibers, all of which are surrounded by a capsule BL. Intrafusal fiber BL was rich in nidogen-1, but poor in nidogen-2, whereas spindle capsule BL was rich in nidogen-2 but poor in nidogen-1 (Figure 3C, D). In contrast, BLs of intramuscular capillaries, venules, and arterioles all contained both nidogen proteins (Figure 3E–H and data not shown). To our knowledge, the spindle capsule is the only BL found to date that bears nidogen-2 but not nidogen-1.
Developmental regulation of nidogens at the synaptic BL
We asked whether the time course with which nidogen-2 became restricted to the NMJ was similar to that of laminins α4 and α5, which are present extrasynaptically at birth but restricted to synaptic BL in adults . In fact, the α4 and α5 laminin chains, like nidogen-2, were present throughout muscle fiber BL in neonates, were present at markedly higher levels synaptically than extrasynaptically by P14, and were largely synapse-specific by P21 (Figure 4G–L). Agrin is lost from extrasynaptic BL with a similar time course . Thus, there may be a coordinated alteration in the composition of muscle fiber BL as development proceeds, similar to, but later than, the initial broad distribution and eventual synaptic concentration of AChRs . In contrast, laminin β2 and synaptic collagen IV chains are synaptically concentrated from their initial appearance during embryogenesis and during the third postnatal week, respectively [9, 12]. Thus, whereas the maturation of extrasynaptic BL may occur in a concerted fashion, the distinctions between synaptic and extrasynaptic BLs arise in a series of multiple steps.
Nidogen-2 is necessary for maturation and maintenance of the NMJ
To test whether defects in NMJ morphology were secondary to muscle damage, we searched for muscle fiber degeneration in nidogen-2-deficient muscle. In healthy muscle, myonuclei are concentrated in the periphery of muscle fibers. In fibers that have undergone degeneration and regeneration, however, myonuclei are centrally located . Less than 4% of mutant muscle fibers contained central nuclei (data not shown), whereas up to 80% of NMJs were abnormal (see below). Thus, defects in synaptic structure in nidogen-2-deficient mice were not the result of degenerating muscle fibers.
How might nidogen-2 promote maturation or enhance maintenance of the NMJ? Nidogens are capable of binding various cell surface receptors that are both present in pre- or postsynaptic membranes and necessary for synaptogenesis; these include integrins and leukocyte-common antigen related (LAR) receptor tyrosine phosphatase [3, 37, 39, 47]. Therefore, one possible explanation for the defects in nidogen-2-deficient NMJs is that nidogen-2 exerts direct effects by binding receptors on either the motor nerve terminal or the postsynaptic apparatus. To test this idea, we used assays previously applied to identify and characterize other synaptic organizing molecules. For presynaptic differentiation, we applied soluble recombinant nidogen-1 or -2 to cultured chick motor neurons, and grew motoneurons on substrates coated with a mixture of recombinant nidogen and laminin-111. In both cases, we assayed the ability of nidogen to promote clustering of synaptic vesicles into aggregates such as those found in nerve terminals [48, 49]. We also asked whether nidogens affected the length or branching of motor neurites. Neither nidogen-1 nor -2 detectably affected motor neurons under these conditions. For postsynaptic differentiation, we assayed the aggregation of AChRs in cultured myotubes [14, 20, 42]. In these assays, nidogen appeared to be detrimental to the health of the myotubes, so it was not possible to gauge their synaptic effects. Thus, these in vitro studies provide no evidence for a direct effect of nidogen on nerve or muscle, although we cannot draw definitive conclusions from them.
A third possibility is that nidogen-2 selectively binds and presents a matrix-associated synaptic organizing molecule to nerve or muscle. Indeed, several bioactive matrix molecules have been reported to bind with much higher affinity to nidogen-2 than nidogen-1, including tropoelastin, collagen XIII and collagen XVIII/endostatin [37, 51–53]. Interestingly, we have found that mice with a targeted mutation of the collagen XIII gene have neuromuscular defects , and recent studies have shown that collagen XVIII is critical for motor axon growth and NMJ formation in C. elegans and zebrafish [26, 55, 56]. We therefore used immunohistochemical methods to ask whether synaptic localization of collagens XIII and XVIII are perturbed in nid2-/- mice. Collagen XIII was concentrated at NMJs in control muscle, as reported previously [54, 57] and this concentration persisted in the absence of nidogen-2, although we cannot rule out the possibility that its level may be modestly affected (data not shown). In both control and nid2-/- muscles, collagen XVIII was undetectable in synaptic BL, although it was associated with the BL of terminal Schwann cells (data not shown). Thus, the mechanisms by which nidogen-2 contributes to synaptic maturation and maintenance remain to be determined.
Intermuscular differences in the role of nidogen-2
Muscles, and the NMJs within them, differ from each other in many respects  and it is not clear whether any documented factors explain the intermuscular differences we have observed. It is intriguing that NMJs more dependent on the presence of nidogen-2 are found in constantly active muscles (that is, diaphragm muscle controls respiration and soleus muscle controls lower limb posture), whereas fewer defects are observed in young, phasically active muscles (tibialis anterior and extensor digitorum longus). Another distinction is between two categories of muscles described by Pun et al. . Called 'fast synapsing' or 'fasyn' and 'delayed synapsing' or 'desyn,' they were initially distinguished by the tempo and pattern of NMJ formation within them during embryogenesis. They were subsequently shown to differ in their sensitivity to nerve injury and neurological disease [58, 61, 62]. Interestingly, the two muscles we examined that were most severely affected, diaphragm and soleus, are both 'delayed synapsing' muscles, whereas the two muscles less affected in nid2-/- mice, tibialis anterior and extensor digitorum longus, are 'fast synapsing' muscles .
Independent localization of synaptic BL components
Biochemical studies have shown that interactions among three main structural components, laminins, collagens IV, and nidogens, are involved in BL assembly [63, 64]. Moreover, all three of these components bind to HSPGs [4, 63]. Our results have shown that specific isoforms of all four components are colocalized in synaptic BL. One might therefore presume that interactions among synaptic isoforms would mediate assembly of this specialized domain. Yet, we found that nidogen-2 is dispensable for the synaptic localization of synaptic laminins, collagens IV and agrin (Figure 7). We therefore considered the alternative possibility, that other synaptic BL components might be required for synaptic localization of nidogen-2. To test this idea, we examined the distribution of nidogen-2 in four mutants lacking synaptic laminin chains (β2, α4, α5, or both α4 and α5) and in collagen α5(IV) mutants, which lack all four synaptic collagen IV chains (α3–6) . It was not possible to assess nidogen-2 in agrin mutants since these mice die at birth , prior to the synaptic restriction of nidogen-2.
Enrichment of laminins, collagens IV, nidogen 2 and agrin in synaptic basal lamina of mutant mice
Enrichment in synaptic basal lamina
lama4-/-; lama5M/M; HSA-Cre
We report here that nidogen-2 is selectively associated with synaptic BL at the NMJ and required for maturation or maintenance of this synapse. Combined with previous studies on laminins, collagens IV and HSPGs, these findings establish that each of the four major families of BL components has isoforms that are enriched in synaptic BL and required for synaptogenesis: laminins α4, α5, and β2, collagens α3–6(IV), agrin [7–9, 11, 12, 15] (reviewed in [4, 5, 39], and nidogen-2. It remains unclear whether nidogen-2 interacts directly with receptors embedded in synaptic membranes or whether it acts by concentrating and presenting other synaptogenic factors. In C. elegans it has been proposed that nidogen exerts it synaptic organizing activity through a receptor protein tyrosine phosphatase present in presynaptic terminals . We are currently testing the possibility that one or more members of this large family may be present at the NMJ. Importantly, synaptic functions of nidogen-2 cannot be fully compensated for by nidogen-1, so receptors or ligands mediating this effect are likely to selectively bind nidogen-2.
Our studies also provide the foundation for analyzing assembly of the synaptic cleft. Biochemical evidence has suggested that laminins, collagens IV, and nidogens are all necessary for BL assembly: laminins for initial BL formation; collagens IV for BL stabilization and maintenance; and nidogens for cross-linking laminin and collagen IV networks [36, 66–68]. At the NMJ, however, the synaptic localization of each class of BL component appears to be independent of the others. Although it is possible that multiple, redundant interactions stabilize the synaptic cleft when a single component is removed, a more attractive idea is that synaptic laminins, collagens IV, HSPGs and nidogen are all localized by interactions with components of pre- or postsynaptic membranes. Previous studies have defined sites important for synaptic localization of laminin β2 and suggested that the receptor tyrosine kinase MuSK may be a localizing receptor for laminins or acetylcholinesterase [69–71]. Further analysis of how synaptic BL components are localized may provide a good model for understanding how the much less accessible synaptic cleft of central synapses is organized.
Materials and methods
Targeted mutant and transgenic mice used in this study have been described previously. They are: nidogen-2 mutants (nid2-/-) , laminin β2 mutants (lamb2-/-) , laminin α4 mutants (lama4-/-) , collagen α5(IV) mutants (col4a5-/Y) , obtained from Jackson Laboratories, Bar Harbor, ME, USA), conditional laminin α5 mutants (lama5flox/flox) , and mice that express Cre selectively in skeletal muscle (HSA-Cre) . Mutants lacking laminin α5 selectively in skeletal muscle were generated by crossing lama5flox/floxand HAS-Cre mice; we refer to the lama5flox/flox;HSA-Cre mice as lama5M/M. Mice lacking both laminin α4 and α5 (lama4-/-; lama5M/M) were generated by crossing lama4-/-;lama5flox/+; HSA-Cre males and females. All mutants and transgenics were maintained on a C57/B6 background. In most cases, littermates of mutants were used as controls. CD1 mice were obtained from Charles River Laboratories, Inc. (Wilmington, MA, USA). All analyses conformed to NIH guidelines and were carried out under an animal protocol approved by the Harvard University Standing Committee on the Use of Animals in Research and Teaching.
Antibodies used in this study
znp-1 (Synaptotagmin 2)
Zebrafish International Resource Center
Gift of Y Sado (Shigei Medical Research Institute, Okayuma)
Gift of Y Sado
Generated in our lab
Gift of Y Sado
Gift of T Sasaki and R Timpl (Max Plank Institute, Munich)
Kind gift of T Sasaki and R Timpl
Generated in our lab
Gift of Z Hall (UCSF)
Full-length human nidogen-1 and -2 fusion proteins were purchased from R&D Systems (Minneapolis, MN, USA). Recombinant proteins (250 ng) were denatured by boiling in Laemmli buffer and separated by SDS-PAGE. Electrophoretically separated proteins were transferred to Immuno-Blot PVDF membrane (Bio-Rad, Hercules, CA, USA) in Tris-Glycine buffer (25 mM Tris, 190 mM glycine, pH 8.4)/20% methanol at 300 mA for 2 hours. Immunoblotted proteins were detected as previously described .
Mice were perfused with phosphate-buffered saline (PBS) and tibialis muscles were dissected. Tissue was immediately frozen in OCT on dry ice and 4 μm sections were cut on a cryostat. Sections were collected on gelatin-coated slides and allowed to air-dry for 15 minutes before tissue was fixed by incubating in ice-cold acetone for 10 minutes. For collagen IV antibodies, tissue was treated for 10 minutes in a 1:1 mix of 1 M KCl and 1 M HCl following fixation . Sections were washed several times in PBS to remove any residual acid. After fixation, tissue was incubated with blocking buffer (5% non-fat milk in PBS with 0.2% Triton-X100 in PBS) for 30 minutes. Primary antibodies, diluted in blocking buffer, were incubated on the sections for 12 hours at 4°C, then sections were washed several times with PBS. Secondary antibodies, diluted in blocking buffer, were then incubated on the slides for 1 hour at room temperature. For controls, primary antibody incubation was omitted from the immunostaining protocol described above. Sections were washed thoroughly with PBS, cover-slipped with VectaShield, and visualized on an Olympus FV1000 scanning confocal microscope (Olympus America Inc., Melville, NY, USA).
C2C12 cells (ATCC, Manassas, VA, USA) were cultured as previously described by Kummer et al. . Myotubes were fixed with ice-cold acetone and stained as described above.
heparan sulfate proteoglycan
We thank members of the Sanes' lab for comments regarding experimental design. This work was supported by NIH/NINDS (JRS).
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