Correspondence to: Stanley C. Froehner, Department of Physiology and Biophysics, Box 357290, University of Washington, Seattle, WA 98195-7290. Tel:(206) 543-0950 Fax:(206) 685-0619
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Abstract |
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The syntrophins are a family of structurally related proteins that contain multiple protein interaction motifs. Syntrophins associate directly with dystrophin, the product of the Duchenne muscular dystrophy locus, and its homologues. We have generated -syntrophin null mice by targeted gene disruption to test the function of this association. The
-Syn-/- mice show no evidence of myopathy, despite reduced levels of
-dystrobrevin2. Neuronal nitric oxide synthase, a component of the dystrophin protein complex, is absent from the sarcolemma of the
-Syn-/- mice, even where other syntrophin isoforms are present.
-Syn-/- neuromuscular junctions have undetectable levels of postsynaptic utrophin and reduced levels of acetylcholine receptor and acetylcholinesterase. The mutant junctions have shallow nerve gutters, abnormal distributions of acetylcholine receptors, and postjunctional folds that are generally less organized and have fewer openings to the synaptic cleft than controls. Thus,
-syntrophin has an important role in synapse formation and in the organization of utrophin, acetylcholine receptor, and acetylcholinesterase at the neuromuscular synapse.
Key Words: dystrophin, dystrobrevin, nitric oxide synthase, acetylcholine receptor, acetylcholinesterase
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Introduction |
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Syntrophins are a family of modular, signal transduction proteins that share a common domain structure (-syntrophin can bind phophotidylinositol lipids, thus providing an additional mode of membrane interaction (
In skeletal muscle, all three syntrophins are found at the neuromuscular junction (NMJ), although in different distributions (-Syntrophin is found on both the acetylcholine receptor (AChR)-rich crests and in the bottoms of the postsynaptic folds, where sodium channels are localized (
-syntrophin being the first to appear on the postsynaptic membrane (
Mice rendered deficient in synaptic proteins through targeted gene disruption or other approaches have provided important evidence leading to the current models of neuromuscular synapse formation. Agrin, rapsyn, and a muscle-specific kinase (MuSk) are each essential for neuromuscular synapse formation (
Similar studies have identified components of the dystrophin complex and dystrophin homologues as key proteins in synaptic structure. Loss of dystroglycan, which links dystrophin and utrophin to extracellular matrix proteins, such as agrin and laminin, leads to early embryonic death, preventing conventional studies of its role in synapse formation (-dystrobrevin display moderate muscle degeneration (
-dystrobrevin null myotubes are clustered by agrin, but in contrast to normal myotubes, the clusters disperse soon after agrin removal (
Mice lacking -syntrophin, the predominant form in skeletal muscle, do not have a dystrophic phenotype, but nNOS is absent from the sarcolemma (
-syntrophin deficiency on neuromuscular synapses have not been examined. In this study of an independently generated
-syntrophin null mouse, we show that the postsynaptic membrane is grossly abnormal and that the level of AChRs and AChE is significantly decreased. Furthermore, nNOS is absent from the postsynaptic membrane and the sarcolemma, despite a significant upregulation of ß1- and ß2-syntrophin. Perhaps the most surprising result is that expression of utrophin at postsynaptic sites is dependent on the presence of
-syntrophin.
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Materials and Methods |
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Generation of -Syntrophin Null Mice
Previously, we characterized the gene encoding mouse -syntrophin using a genomic library derived from 129Sv DNA (
-syntrophin first intron-ACAGGAGCCCAGTCTTCAATCCAGG). The mice used in this study were adults >10-wk old and either first generation with a mixed 129Sv/C57Bl6 background or had been bred back against C57Bl6 for 3 generations. Mice with either background show similar phenotypes.
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Southern Analysis
Genomic DNA was isolated from ES cells or mouse tail biopsies using a QIAmp tissue kit (Qiagen), digested with EcoRI, and resolved on a 1% agarose gel. GenScreen (NEN Life Science Products) replicas were incubated with radiolabeled probe as described previously (-syntrophin wild-type, heterozygous, and syntrophin null mice. Poly A+ RNA was purified (PolyAtract, Promega) and separated on a 1% formaldehyde gel, transferred to GeneScreen, and probed with a 32P-labeled full-length cDNA probe as described (
Antibodies
Previously, we characterized the following antibodies: pan-specific syntrophin mAb SYN1351 (-syntrophin), SYN28 (ß2-syntrophin) and SYN37 (ß1-syntrophin;
-dystrobrevin1); and DB2 (
-dystrobrevin2;
Immunoblotting
Muscle protein extracts were prepared as previously described (
Fluorescence Microscopy
Immunofluorescence-labeling of unfixed muscle (see Fig 2 Fig 3 Fig 4 Fig 5 A) was done on 8-µm cryosections of quadriceps muscle as described (-bungarotoxin (Bgtx), followed by rabbit antitoxin (Jackson ImmunoResearch) and Alexa 594 goat antirabbit IgG. The red and green channels in Fig 6 A were digitally switched to conform to the other images (AChR label in green). The labels in Fig 6 B were biotinylated Bgtx followed by Alexa 488-streptavidin and rabbit anti
-dystrobrevin2 or antiankyrin, followed by Alexa 594 goat antirabbit IgG.
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For en face views (see Fig 7), thick (40 µm) cryosections were labeled on AChR with Texas red-Bgtx or with toxin/antitoxin as above, with fluorescein-conjugated VVAB4 lectin or biotinylated lectin VVA-B4 (Sigma-Aldrich), followed by Alexa-488-streptavidin; and with rabbit antisynaptophysin antibody, followed by Cy5-conjugated donkey antirabbit IgG (Jackson ImmunoResearch). For each NMJ, the final image was obtained by combining 1015 images taken every 0.3 µm along the z-axis into a single two-dimensional view.
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AChE was labeled with a fluorescent conjugate (Oregon green) of fasciculin 2, a snake alpha toxin that binds to the catalytic subunit, prepared and imaged as described (
All other fluorescence microscopy was done using a Leica TCS NT confocal microscope. For Fig 2 Fig 3 Fig 4 Fig 5 and Fig 9, all microscope settings for -Syn+/+ and
-Syn-/- were identical for direct comparison of fluorescence intensity. For Fig 6 and Fig 7, microscope settings were adjusted so that
-Syn+/+ and
-Syn-/- samples could be shown at similar intensity.
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Quantitation of AChR and AChE
To measure relative levels of AChR in NMJs at University of North Carolina, Chapel Hill, thick cryostat sections (40 µm) of sternomastoid muscles of wild-type and -syntrophin null mice were labeled with Texas red-Bgtx. NMJs lying fully in the section were imaged in the confocal microscope with the pinhole fully open to eliminate optical sectioning. At the University of Miami, the fixed sternomastoid muscles were labeled for AChR and AChE and teased into single fibers. Images of the NMJs were captured using a digital camera. In both cases, wild-type and
-syntrophin null NMJs were imaged under identical conditions. After circumscribing the digital image of each NMJ using Adobe Photoshop (University of North Carolina) or Metamorph (University of Miami), the difference between the average pixel intensity in the circumscribed area and average background pixel intensity of the corresponding muscle fiber was determined. The product of this difference and the number of circumscribed pixels gave a total AChR- or AChE-specific intensity measure for each NMJ.
For quantitation of AChE enzyme activity, tibialis anterior muscle was homogenized in 10 vol 20 mM borate buffer, pH 9.0, containing 1% Trition X-100, 5 mM EDTA, 1 M NaCl, 0.5% BSA, and a protease inhibitor cocktail as previously described (
Electron Microscopy
Sternomastoid muscles were pinned out under fixative (4% glutaraldehyde, 4% paraformaldehyde, 0.1 M sodium cacodylate, pH 7.4), fixed for 2 to 3 h, dissected into junction-containing pieces, incubated in 1% osmium tetroxide, 0.1 M sodium cacodylate for 2 to 3 h, incubated in 3% tannic acid (Mallinckrodt, no. 1764), 0.1 M sodium cacodylate, pH 7.4, for 3 h, and were then embedded in Epon (
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Results |
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Generation of -Syn-/- Mice
We established a line of mice lacking -syntrophin by standard targeted gene disruption methods. The entire first exon (corresponding to amino acids 197, which encodes part of the first PH domain and
15 amino acids of the PDZ domain), as well as 0.8 kb of 5' flanking sequence, and 1.7 kb of intron 1 of the
-syntrophin gene were deleted by homologous recombination (Fig 1). Two ES cell lines were identified by Southern blotting and used for blastocyst injection. One of these lines produced chimeras capable of germline transmission. Subsequent breeding of the heterozygous mice produced offspring in the expected 1:2:1 ratio for wild-type (
-Syn+/+), heterozygotes (
-Syn+/-), and mice with the
-syntrophin gene disrupted on both alleles (
-Syn-/-). Analysis of RNA isolated from skeletal muscle of these mice showed the presence of the expected 2.4-kb transcript (
-syntrophin in the
-Syn+/+ and
-Syn+/- mice (Fig 1 C). The
-Syn-/- mice contained low levels of a slightly smaller transcript and a slightly larger transcript, the latter also being present in the
-Syn+/- mice. The top band most likely represents the product of transcription driven by the PGK promoter upstream of the neo gene. The source of the lower band is unknown, but is similar in size and intensity to the 1.9-kb band present in
-Syn-/- mice generated by deletion of exon 2 (
To determine if -syntrophin protein is produced from these transcripts, we immunoprecipitated total syntrophins from skeletal muscle extracts using mAb 1351 (a high affinity, pan-specific antisyntrophin antibody), and then immunoblotted the products using isoform-specific antibodies. mAb SYN1351 recognizes an epitope in exon 2 (the PDZ domain; Adams, M.E., and S.C. Froehner, unpublished results) and would capture NH2-terminal truncated syntrophin potentially expressed by the disrupted gene. The antibody SYN17, produced against a peptide sequence encoded by exon 3 of the
-syntrophin gene (
-Syn+/+ mice, but detected no protein in the muscle from
-Syn-/- mice (Fig 1 D). In contrast, a blot of identical samples showed that ß1-syntrophin levels are moderately increased in skeletal muscle of
-Syn-/- mice.
Characterization of Skeletal Muscle in -Syn-/- Mice
The -Syn-/- mice are mobile, reproduce normally, and show no overt signs of muscular dystrophy. We tested their mobility by allowing them to run voluntarily on exercise wheels. The distances run and average speed were statistically indistinguishable from C57Bl6 control mice (data not shown). This result is consistent with the finding that the contractile properties of normal and
-Syn-/- muscles are the same (
To determine whether elimination of -syntrophin affected the distribution of other members of the dystrophin protein complex, we examined quadriceps muscles by immunofluorescence microscopy (Fig 2). As expected, we detected no
-syntrophin staining in this or any other muscle examined. As in wild-type quadriceps muscle (
-Syn-/- mice. However, in the
-Syn-/- muscle, fibers that do express ß1-syntrophin show a modest increase in labeling intensity. This finding is in agreement with the increase found by immunoblotting (Fig 1). Some muscles, such as the sternomastoid, consist totally of fibers that show no ß1-syntrophin staining in the adult (
-Syn-/- sternomastoid showed no detectable upregulation of ß1-syntrophin (data not shown). In contrast, we observed a decrease in the intensity of sarcolemmal-labeling for
-dystrobrevin2 in all muscle fibers. The intensity of sarcolemmal dystrophin-labeling in the
-Syn-/- was indistinguishable from littermate control
-Syn+/+ muscle.
Previously, we and others have shown that -syntrophin binds nNOS in vitro via a PDZPDZ interaction (
-Syn-/- mice and we have confirmed that levels of sarcolemmal nNOS are reduced to nearly undetectable amounts (Fig 3). Interestingly, this loss of nNOS occurs even in those fibers that express sarcolemmal ß1-syntrophin (Fig 3 B). Thus, ß1-syntrophin is not able to compensate for the loss of
-syntrophin in recruiting nNOS to the membrane, even though it binds nNOS in vitro (
Dystrophin Complex Proteins at Neuromuscular Synapses
-Syntrophin is present on the sarcolemma, but is enriched at the postsynaptic membrane. We therefore compared the morphology of NMJs from
-Syn+/+ and
-Syn-/- mice. We also studied localization of members of the dystrophin protein complex and signaling proteins associated with the complex at NMJs of the
-Syn-/- mice (Fig 4). As was the case for sarcolemmal staining, no
-syntrophin was observed at the
-Syn-/- NMJs. Although ß1-syntrophin was originally characterized as being enriched at the NMJ, this enrichment has been shown to be due, at least in part, to the presence of ß1-syntrophin in presynaptic structures (
-Syn-/- synapses, we observed no increase in postsynaptic ß1-syntrophin in both fibers expressing and not expressing ß1-syntrophin on the sarcolemma. ß2-Syntrophin, the isoform that is most tightly confined to the NMJ in adult muscle (
-Syn-/- mice, although this increase was not measured quantitatively. Despite the presence of ß2-syntrophin, nNOS is absent from junctions lacking
-syntrophin establishing that ß2-syntrophin, like ß1-syntrophin, does not recruit nNOS to the membrane to compensate for the loss of
-syntrophin.
-Syntrophin has also been shown to bind the muscle sodium channels, SkM1 and SkM2, via their COOH-terminal sequences (
-Syn-/- muscle, a distribution similar to that found in control muscle. Thus,
-syntrophin is not required for expression or synaptic localization of sodium channels in skeletal muscle.
-Dystrobrevin shares homology with dystrophin (
-dystrobrevin1 and 2, both bind syntrophin, but are differentially localized in skeletal muscle.
-Dystrobrevin1 is largely synaptic, whereas
-dystrobrevin2 is found on both the sarcolemma and at the synapse (
-dystrobrevin1 and 2 are present at
-Syn-/- synapses, but at slightly lower levels than wild-type synapses.
The distribution of dystrophin (-Syn-/- junction, remaining concentrated in the postjunctional folds in the absence of
-syntrophin (Fig 4). However, utrophin staining, which is normally at the crests of the folds (
-Syn-/- mice. Images of 8-µm sections show low levels of utrophin at the
-Syn-/- NMJ (Fig 4 and Fig 5 A), but at high resolution (see Materials and Methods), even when the confocal microscope is adjusted to give a strong image of the weak presynaptic staining, utrophin is essentially undetectable on the postsynaptic membrane (Fig 5 B). Thus, full expression and/or localization of utrophin at the postsynaptic membrane is dependent on the presence of
-syntrophin.
AChR Levels at Mutant Neuromuscular Synapses
During the immunofluorescent studies, we consistently observed that the levels of AChR were much lower in the junctions of -syntrophin null mice than in the wild-type mice. Therefore, we measured total levels of AChR in NMJs of sternomastoid muscle from two
-Syn-/- and two
-Syn+/+ mice from a single litter and pooled the data. Analyses were performed independently in two separate laboratories (see Materials and Methods). Data collected from 70 wild-type and 92
-syntrophin null junctions indicated that the average AChR content per NMJ in the null mice was reduced 60% (University of Miami lab) and 67% (University of North Carolina lab) compared with the wild-type junctions. The AChR content of the null junctions was thus only
35% of wild-type. This difference was highly significant by the two-tailed t test (P < 0.0001) for each of the two data sets.
Structure of Mutant Neuromuscular Synapses
The structure of -Syn-/- NMJs was assessed at high resolution by double-labeling muscle sections with Bgtx and concanavalin A. The lectin labels extracellular glycoproteins throughout muscle tissue, particularly highlighting the synaptic cleft and junctional folds. It also labels the material overlying junctional nerve terminals, but not the terminals themselves. Wild-type NMJs are characterized by deep synaptic gutters, plentiful junctional folds, and an AChR distribution that is continuous, bright, and tightly confined to the gutters (Fig 6 A, left, B, a and c). In contrast, nerve-muscle contacts in
-Syn-/- mice often had shallow gutters, fewer folds, synaptic AChRs separated into distinct clusters, and perisynaptic clusters of AChR (i.e., clusters that extended beyond recognizable nervemuscle contacts; Fig 6 A, right, and B, b and d). Proteins that normally occur perisynaptically and in the troughs of the junctional folds, such as
-dystrobrevin2 (Fig 6 B, a and b), ankyrin G (Fig 6 B, c and d), ß2-syntrophin (Fig 4), and dystrophin (Fig 4) retained these distributions in
-Syn-/- muscle (Fig 6 B, b and d). The perisynaptic distribution of ankyrin G did not overlap, but rather interdigitated with, the perisynaptic clusters of AChR (readily seen in grayscale insets in Fig 6 B, d).
To further characterize the AChR distribution, NMJs were visualized en face after labeling with Bgtx. In -Syn+/+ NMJs, the AChR labeling was consistently smooth, continuous, and confined to the synaptic gutters (part of an NMJ is shown in Fig 7 A, a). The edges of the gutters, which turn up parallel to the axis of the microscope in such samples, were intensely bright. In contrast, the
-Syn-/- NMJs were highly variable, even within single synapses. In the more extreme derangements (Fig 7 A, b), the AChR pattern in synaptic gutters consisted of streaks and dots, while thin lines of AChR
1 µm in length extended beyond the gutters (see examples in Fig 7 A). The edges of gutters were often little brighter that the center (Fig 7A, Fig b), consistent with the shallow gutters seen in cross-section. Some NMJs contained these features over their whole extent (Fig 7 A, e), whereas others contained areas of aberrant AChR pattern next to areas of more normal appearance (Fig 7 A, c).
To assess the presence of nerve terminals and junctional folds in regions of aberrant AChR distribution, sections were labeled with an antibody against the synaptic vesicle protein synaptophysin and with fluorophore-conjugated lectin, VVA-B4. This lectin labels only the NMJ in muscle (-Syn-/- NMJs, major areas of membrane containing AChR either were labeled weakly or not at all by VVA-B4 (Fig 7 B, a and a'), suggesting the absence of junctional folds. Other areas were strongly stained (Fig 7 B, b and b'). Interestingly, even areas that had folds, indicative of morphological maturity, could be devoid of labeling by antisynaptophysin (Fig 7 B, b and b''), suggesting the absence of a functional nerve terminal and making participation in synaptic transmission unlikely. This was a local phenomenon within NMJs, as the major portions of all
-Syn-/- NMJs labeled positive for synaptophysin (Fig 7 B, b''). These results contrast with
-Syn+/+ junctions, in which essentially the entire AChR-rich area was labeled by VVA-B4 and by antisynaptophysin (not shown).
Ultrastructural Analysis
Sternomastoid muscles from one pair of -Syn+/+ and
-Syn-/- littermates from each of two separate litters were examined by EM. After fixation and osmication, the muscles were treated with tannic acid to enhance heavy metal staining of extracellular elements, notably the basal lamina of the synaptic cleft and junctional folds. Examination was restricted to recognizable nervemuscle contacts, i.e., sites at which nerve terminals were closely apposed to muscle cells. Presynaptic elements (nerve terminals and overlying Schwann cells) appeared normal in all samples.
EM revealed two main abnormalities in the postsynaptic membrane of -Syn-/- NMJs. First, the number of junctional fold openings to the synaptic cleft was substantially reduced compared with
-Syn+/+ mice. Even where the folds were plentiful and oriented toward the synaptic cleft, the number that actually opened to the cleft was reduced (compare Fig 8, a and b, with d). Quantitatively, the number of openings per micrometer of presynaptic membrane was reduced 59 and 77% in two
-Syn-/- mice from different litters, each compared with an
-Syn+/+ littermate. The pooled data are shown graphically in Fig 8 c. In both pairs, the difference was highly significant (P < 0.0001; t test).
Secondly, the junctional folds in -Syn-/- mice generally appeared to be less well organized than in
-Syn+/+ mice. This most often consisted of curved elements, short folds, and folds that ran parallel to the surface membrane (Fig 8). Direct morphological examination showed that all these structures contained basal lamina, presumably indicating that they open to the surface at some point.
AChE in Mutant Neuromuscular Synapses
Despite the fact that the NMJs in -Syn-/- mice are structurally abnormal and contain low levels of AChRs, the mutant mice show no deficiencies in mobility as assessed by voluntary running wheel experiments (see above). We considered the possibility that a compensatory change occurs in the mutant junction such that synaptic transmission is still effective. A possible candidate for compensation is AChE, which, if reduced at mutant NMJs, would enhance ACh efficacy. We therefore investigated the distributions and levels of AChE at junctions of
-Syn-/- mice. Bundles of sternomastoid muscle fibers from two normal and two mutant mice were labeled with fluorescently tagged fasciculin 2 (a toxin that specifically labels AChE;
-syntrophin and in general appears much like that of AChR (Fig 9). Areas of AChR distribution that consisted entirely of fingers did not label for AChE (Fig 9b and Fig b', arrowhead), as would be expected from the absence of folds in such areas (Fig 7 B). Measurement of the levels of AChE in 24 junctions showed a significant reduction of 55% (P < 0.02) in the
-Syn-/- NMJs. Thus, AChRs and AChE are reduced by similar amounts in muscles of the
-Syn-/- mice.
The reduction in AChE could occur by either a defect in synthesis or in localization and retention at the synapse. To discern between these two mechanisms, we compared the total amount and isoform distribution of AChE in normal and mutant mice by sucrose gradient analysis (-Syn+/+ and
-Syn-/- samples (Fig 9 C). AChE analyzed by this method is derived largely from the cytosol, the Golgi, and the rough ER, and represents the pool available for export to the surface. Synaptic AChE is not solubilized by this method. The results suggest that the absence of
-syntrophin has no detectable effect on synthesis, assembly, or availability of the AChE forms in muscle. Thus, the abnormality must lie in localization and/or retention of the enzyme at the synapse.
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Discussion |
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Syntrophins are thought to function by recruiting signaling proteins to the dystrophin/utrophin protein complex. We investigated the function of -syntrophin using targeted gene disruption to delete the first exon of the
-syntrophin gene, thereby generating mice lacking
-syntrophin. Tissue immunofluorescence and protein blot analyses demonstrate that these mice do not produce
-syntrophin. Despite the loss of
-syntrophin, these mice are mobile, fertile, and show no signs of a dystrophic phenotype. These observations are consistent with those of
-syntrophin null mouse generated by deleting exon 2. A weak band hybridizing to the
-syntrophin cDNA is present on Northern blots of
-Syn-/- muscle RNA performed by us and by
-syntrophin produced by an alternative promoter that would presumably have to be located downstream of exon 3, since our SYN17 antibody is made to an epitope encoded by exon 3, but detects no protein. Alternatively, since ß2-syntrophin can be encoded by a 10-, 5-, or 2-kb mRNA (
In previous work, we and others have shown that -syntrophin binds nNOS via a PDZPDZ interaction, thus providing a role for the dystrophin complex in targeting this signaling protein to the membrane. In agreement with
-syntrophin. This defect occurs even in fibers that contain abnormally high amounts of ß1-syntrophin, and at the NMJ where ß2-syntrophin is concentrated. Thus, despite the highly conserved sequences of syntrophin PDZ domains, only
-syntrophin is able to bind nNOS PDZ in vivo. Sarcolemmal nNOS is important for maintenance of adequate blood flow to exercising muscles by counteracting adrenergically mediated vasoconstriction (
-Syn-/- mice are not detectably impaired in their ability to exercise, since they run voluntarily for similar times and distances as controls. However, the voluntary exercise test may not reveal abnormalities in this system. Experiments examining adrenergic mediation of vasoconstriction in mice lacking
-syntrophin are underway.
The absence of -syntrophin selectively affects the expression of other members of the dystrophin protein complex, although dystrophin itself appears to be unaltered. The levels of ß1-syntrophin (in some fibers) and ß2-syntrophin are increased at the sarcolemma and the NMJ, respectively. Despite the increase in ß1-syntrophin,
-dystrobrevin2 levels are significantly reduced on the
-Syn-/- sarcolemma. The reduced levels of
-dystrobrevin2 are not sufficient, however, to induce the mild dystrophy seen in the
-dystrobrevin null mice (
-Syn-/- mouse is the loss of utrophin from the postsynaptic membrane. This loss occurs despite the increased levels of ß2-syntrophin at the NMJ. This is surprising since ß2- and
-syntrophin have similar ability to bind utrophin in vitro (
-syntrophin in vivo. These alterations suggest that
-syntrophin is a crucial component having unique activities in forming and/or maintaining the dystrophin protein complex.
The unexpected loss of utrophin from the -Syn-/- NMJs could arise by either a structural or signaling mechanism. Utrophin, like dystrophin, is thought to be bound to the membrane primarily through interactions with ß-dystroglycan.
-Syntrophin may stabilize these protein interactions, provide a second site of protein interaction, or potentially bind directly with phospholipids in the membrane (
-syntrophin is part of a signaling pathway that regulates the synaptic expression of utrophin. The recent observation that the receptor-tyrosine kinase, ErbB4, is associated via its COOH-terminal tail with the PDZ domain of syntrophins (
-syntrophin may bolster efforts to design a therapy for Duchenne muscular dystrophy based on upregulation of utrophin as a substitute for dystrophin.
The alterations seen at neuromuscular synapses of mice lacking -syntrophin are similar to changes in other genetically altered mice. Like the utrophin null mice (
-Syn-/- mice show reduced levels of AChR, fewer postjunctional fold openings, and no dystrophic muscle properties. Thus, some of the alterations seen in the
-Syn-/- mouse could be due to the loss of utrophin. However, the reduction in AChR levels is larger in
-Syn-/- muscle than in muscle lacking utrophin, indicating that additional factors must be involved. AChR number at the postsynaptic membrane could be regulated in several different ways, including synthesis, targeting and degradation. The recent observation that AChR in mdx muscle are less stable than in normal muscle suggests a role for the dystrophin complex in maintaining receptor stability (
En face views of the -Syn-/- NMJ are remarkably similar to those seen in the
-dystrobrevinnull mouse (
-dystrobrevin mutant may be secondary to the partial loss of
-syntrophin observed in these mice. However, the
-dystrobrevin null mouse suffers from a moderate level of dystrophy (
-dystrobrevin function in muscle that do not occur in the
-Syn-/- mice. Agrin induces AChR clusters on cultured myotubes lacking
-dystrobrevin, but the clusters are unstable and disperse after removal of agrin. Similar results are obtained with myotubes lacking
-dystroglycan (
-dystroglycan are structurally abnormal and contain low levels of AChR and other synaptic proteins (
-syntrophin may be especially important in the mechanisms by which stabilization occurs.
Severe reduction in AChR levels at the NMJ, due to acquired autoimmunity or congenital causes, leads to myasthenia gravis, a muscle weakness disease (reviewed in -Syn-/- mice unexpectedly show reductions in total AChR levels to about one-third of normal. This raises the possibility that
-syntrophin mutations could be the primary defect in cases of congenital myasthenia in humans in which the AChR genes are normal. Interestingly, two candidate kinships have been described that have NMJs which lack utrophin and show reduced numbers of AChRs like the
-Syn-/- mouse (
-Syn-/- mice, however, do not show overt signs of myasthenia. It may be that this level of reduction is not sufficient to nullify the large safety factor at the normal NMJ (
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Footnotes |
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Current address for Marvin E. Adams and Stanley C. Froehner is Department of Physiology and Biophysics, Box 357290, University of Washington, Seattle, WA 98195-7290.
1 Abbreviations used in this paper: AChE, acetylcholinesterase; AChR, acetylcholine receptor; Bgtx, -bungarotoxin; ES, embryonic stem; NMJ, neuromuscular junction; nNOS, neuronal nitric oxide synthase; PDZ, domain found in postsynaptic density protein-95, discs large, and zonula occludens-1 proteins; PH, pleckstrin homology.
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Acknowledgements |
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We thank S. Rock Levinson for providing sodium channel antibody, Vann Bennett for ankyrin G antibody, and Lian Li for synaptophysin antibody. We also thank Kirk McNaughton for providing histological samples, L. Gretta Gray for genotyping assistance, and our colleagues for helpful discussions.
This work was funded by the National Institutes of Health grants NS33145 (to S.C. Froehner and R. Sealock) and AG05917 (to R.L. Rotundo), and grants from the Council for Tobacco Research and the Muscular Dystrophy Association (to R.L. Rotundo and R. Sealock).
Submitted: 15 March 2000
Revised: 22 June 2000
Accepted: 12 July 2000
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References |
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