The Journal of Cell Biology, Volume 150, Number 6, September 18, 2000 1385–1397
The Rockefeller University Press, 0021-9525/2000/09/1385/13 $5.00
Synapses Deficient in Utrophin
-Syntrophin Leads to Structurally Aberrant Neuromuscular
Marvin E. Adams,* Neal Kramarcy,* Stuart P. Krall,* Susana G. Rossi,
Robert Sealock,* and Stanley C. Froehner*
*Department of Cell and Molecular Physiology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina
Department of Cell Biology and Anatomy, University of Miami School of Medicine, Miami, Florida 33143
Richard L. Rotundo,
related proteins that contain multiple protein interac-
tion motifs. Syntrophins associate directly with dystro-
phin, the product of the Duchenne muscular dystrophy
locus, and its homologues. We have generated
phin null mice by targeted gene disruption to test the
function of this association. The
evidence of myopathy, despite reduced levels of
trobrevin–2. Neuronal nitric oxide synthase, a compo-
nent of the dystrophin protein complex, is absent from
the sarcolemma of the
syntrophin isoforms are present.
lar junctions have undetectable levels of postsynaptic
The syntrophins are a family of structurally
mice show no
mice, even where other
utrophin and reduced levels of acetylcholine receptor
and acetylcholinesterase. The mutant junctions have
shallow nerve gutters, abnormal distributions of acetyl-
choline receptors, and postjunctional folds that are gen-
erally less organized and have fewer openings to the
synaptic cleft than controls. Thus,
important role in synapse formation and in the organi-
zation of utrophin, acetylcholine receptor, and acetyl-
cholinesterase at the neuromuscular synapse.
-syntrophin has an
synthase • acetylcholine receptor • acetylcholinesterase
dystrophin • dystrobrevin • nitric oxide
Syntrophins are a family of modular, signal transduction
proteins that share a common domain structure (Adams
et al., 1993; Yang et al., 1994; Ahn et al., 1996; Peters et al.,
1997). Each of the three characterized syntrophins con-
tains two pleckstrin homology (PH)
main (domain found in postsynaptic density protein-95,
discs large, and zonula occludens-1 proteins), and a COOH-
terminal syntrophin unique (SU) region. Syntrophins bind
directly to members of the dystrophin protein family (dys-
trophin, utrophin, and dystrobrevin; Ahn and Kunkel, 1995;
Dwyer and Froehner, 1995; Yang et al., 1995), an interac-
domains, a PDZ do-
tion mediated by the second PH domain and the SU region
together (Kachinsky et al., 1999). The first PH domain of
-syntrophin can bind phophotidylinositol lipids, thus pro-
viding an additional mode of membrane interaction
(Chockalingam et al., 1999). The PDZ domain of syntro-
phin binds to a variety of signaling molecules, including so-
dium channels (Gee et al., 1998a; Schultz et al., 1998), neu-
ronal nitric oxide synthase (nNOS; Brenman et al., 1996;
Hashida-Okumura et al., 1999; Hillier et al., 1999), and
serine/threonine kinases (Hasegawa et al., 1999; Lumeng et
al., 1999). Thus, syntrophin links signaling proteins to the ac-
tin cytoskeleton and the extracellular matrix via dystrophin.
In skeletal muscle, all three syntrophins are found at the
neuromuscular junction (NMJ), although in different dis-
tributions (Peters et al., 1997; Kramarcy and Sealock,
-Syntrophin is found on both the acetylcholine re-
ceptor (AChR)-rich crests and in the bottoms of the postsyn-
aptic folds, where sodium channels are localized (Kramarcy
and Sealock, 2000).
1-Syntrophin is also concentrated at
neuromuscular synapses, but this is in part due to its pres-
2-Syntrophin is the isoform most
highly localized at the synapse where it is predominantly
in the bottoms of the folds (Peters et al., 1997; Kramarcy
and Sealock, 2000). The syntrophins also differ in their de-
velopmental regulation, with
-syntrophin being the first
Address correspondence to Stanley C. Froehner, Department of Physiol-
ogy and Biophysics, Box 357290, University of Washington, Seattle, WA
98195-7290. Tel.: (206) 543-0950. Fax: (206) 685-0619. E-mail: froehner@
u.washington.edu; or to Robert Sealock, Department of Cell and Molecu-
lar Physiology, CB 7545, University of North Carolina, Chapel Hill, NC
27599. Tel.: (919) 966-1272. Fax: (919) 966-6927. E-mail: sealock@med.
Current address for Marvin E. Adams and Stanley C. Froehner is De-
partment of Physiology and Biophysics, Box 357290, University of Wash-
ington, Seattle, WA 98195-7290.
Abbreviations used in this paper:
AChE, acetylcholinesterase; AChR,
acetylcholine receptor; Bgtx,
-bungarotoxin; ES, embryonic stem; NMJ,
neuromuscular junction; nNOS, neuronal nitric oxide synthase; PDZ, do-
main found in postsynaptic density protein-95, discs large, and zonula oc-
cludens-1 proteins; PH, pleckstrin homology.
The Journal of Cell Biology, Volume 150, 2000
to appear on the postsynaptic membrane (Kramarcy and
Sealock, 2000). This complex distribution and develop-
mental regulation of the syntrophins suggests that they
each play unique roles in synapse formation and mainte-
Mice rendered deficient in synaptic proteins through tar-
geted gene disruption or other approaches have provided
important evidence leading to the current models of neuro-
muscular synapse formation. Agrin, rapsyn, and a muscle-
specific kinase (MuSk) are each essential for neuromuscular
synapse formation (Gautum et al., 1995, 1996; DeChiara et
al., 1996). The absence of any of these proteins prevents
the formation of nicotinic AChR clusters, leading to death
of the mutant mice at birth. In contrast, disruption of TrkB
signaling using a dominant-negative approach causes disas-
sembly of mature NMJs (Gonzalez et al., 1999).
Similar studies have identified components of the dys-
trophin complex and dystrophin homologues as key pro-
teins in synaptic structure. Loss of dystroglycan, which
links dystrophin and utrophin to extracellular matrix pro-
teins, such as agrin and laminin, leads to early embryonic
death, preventing conventional studies of its role in syn-
apse formation (Williamson et al., 1997). Recently, how-
ever, a study of chimeric mice, produced from embryonic
stem (ES) cells in which both copies of the dystroglycan
gene had been removed, revealed muscle degeneration
and abnormalities in the gross structure of the neuromus-
cular synapse (Cote et al., 1999). Regions of the chimeric
mice derived from the dystroglycan null ES cells have
NMJs with reduced levels of AChRs, acetylcholinesterase
(AChE), and utrophin. Utrophin-null mice are healthy,
but have fewer postsynaptic folds and a modest decrease
in AChR density (Deconinck et al., 1997; Grady et al.,
1997). Finally, mice lacking most forms of
display moderate muscle degeneration (Grady et al., 1999)
and have structurally abnormal muscle synapses (Grady et
al., 2000). AChRs in
-dystrobrevin null myotubes are
clustered by agrin, but in contrast to normal myotubes, the
clusters disperse soon after agrin removal (Grady et al.,
2000). These results provide clear evidence in support of a
stabilization or maintenance function for the dystrophin
complex at neuromuscular synapses.
-syntrophin, the predominant form in
skeletal muscle, do not have a dystrophic phenotype, but
nNOS is absent from the sarcolemma (Kameya et al.,
1999). However, effects of
-syntrophin deficiency on neu-
romuscular synapses have not been examined. In this
study of an independently generated
mouse, we show that the postsynaptic membrane is grossly
abnormal and that the level of AChRs and AChE is signif-
icantly decreased. Furthermore, nNOS is absent from the
postsynaptic membrane and the sarcolemma, despite a sig-
nificant upregulation of
the most surprising result is that expression of utrophin at
postsynaptic sites is dependent on the presence of
Materials and Methods
-Syntrophin Null Mice
Previously, we characterized the gene encoding mouse
a genomic library derived from 129Sv DNA (Adams et al., 1995). A tar-
geting vector was constructed by cloning a 7.4-kb NotI/XbaI restriction
fragment from the region 5
of exon 1 and a 1.5-kb XbaI fragment from in-
tron 1 into the plasmid JNS2 (Dombrowicz et al., 1993; Fig. 1). The result-
ing recombinant gene is missing the entire first exon (amino acids 1–97,
which encode half of the first PH domain and part of the PDZ domain),
0.8 kb of 5
flanking sequence, and 1.7 kb of the first intron. The targeting
vector was linearized with NotI and transferred to E14 ES cells by elec-
troporation. After selection with G418 and gancyclovir (a gift from Roche
Biosciences), homologous recombinant ES cells were identified by South-
ern blotting (Fig. 1) and used for C57Bl6 blastocyst injection. Injections
were performed by the UNC-CH Embryonic Stem Cell Facility. Chimeras
were bred with C57Bl6 females and germline transmission was confirmed
by Southern blot analysis and PCR (primers: neo-CAAATTAAGGGC-
-syntrophin first intron-ACAGGAGCCCA-
GTCTTCAATCCAGG). The mice used in this study were adults
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.
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 incu-
bated with radiolabeled probe as described previously (Adams et al.,
1995). For RNA blot analysis, adult skeletal muscle RNA was isolated
-syntrophin wild-type, heterozygous, and syntrophin null mice.
RNA was purified (PolyAtract, Promega) and separated on a 1%
formaldehyde gel, transferred to GeneScreen, and probed with a
labeled full-length cDNA probe as described (Adams et al., 1993).
Previously, we characterized the following antibodies: pan-specific syntro-
phin mAb SYN1351 (Froehner et al., 1987); isoform-specific syntrophin
Abs SYN17 (
-syntrophin), SYN28 (
trophin; Peters et al., 1997); Ab 1862 (utrophin; Kramarcy et al., 1994); Ab
2723 (COOH terminus of dystrophin; Kramarcy et al., 1994); dystrobrevin
Abs 638 (
-dystrobrevin–1); and D
1998). The sodium channel Ab was a gift from S. Rock Levinson (Univer-
sity of Colorado Health Sciences Center, Denver, CO). The polyclonal an-
tibody to nNOS was purchased from Diasorin Inc. Antibodies against
ankyrin G and synaptophysin were kind gifts of Vann Bennett (Duke Uni-
versity, Durham, NC) and Lian Li (University of North Carolina, Chapel
Hill, NC), respectively.
2-syntrophin) and SYN37 (
-dystrobrevin–2; Peters et al.,
Muscle protein extracts were prepared as previously described (Peters
et al., 1997). Syntrophin complex was isolated by incubating the extract
with the pan syntrophin mAb 1351, followed by precipitation with protein
G coated beads (Sigma-Aldrich). The resulting pellets were resuspended
in SDS-PAGE sample buffer, subjected to electrophoresis on a 10% poly-
acrylamide tricine-buffered gel, transferred to Immobilon-P membrane
(Millipore Corp.), and incubated with the syntrophin isoform-specific an-
tibodies SYN17 and SYN37, as previously described (Peters et al., 1997).
Immunofluorescence-labeling of unfixed muscle (see Figs. 2–5 A) was
done on 8-
m cryosections of quadriceps muscle as described (Peters et al.,
1997). For high resolution images in Fig. 6, 100–150 nm sternomastoid
muscle sections were prepared as described (Peters et al., 1998; Kramarcy
and Sealock, 2000). The two labels in Fig. 6 A were biotinylated Con A
(Molecular Probes), followed by Alexa 488-streptavidin (all Alexa-conju-
gated fluophores were obtained from Molecular Probes) and unlabeled
-bungarotoxin (Bgtx), followed by rabbit antitoxin (Jackson ImmunoRe-
search) and Alexa 594 goat anti–rabbit 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–
followed by Alexa 594 goat anti–rabbit IgG.
For en face views (see Fig. 7), thick (40
on AChR with Texas red-Bgtx or with toxin/antitoxin as above, with fluo-
rescein-conjugated VVAB4 lectin or biotinylated lectin VV
-dystrobrevin–2 or antiankyrin,
m) cryosections were labeled
Adams et al.
Neuromuscular Junction Defects of
-Syntrophin Null Mice
Aldrich), followed by Alexa-488-streptavidin; and with rabbit antisynap-
tophysin antibody, followed by Cy5-conjugated donkey anti–rabbit IgG
(Jackson ImmunoResearch). For each NMJ, the final image was obtained
by combining 10–15 images taken every 0.3
gle two-dimensional view.
AChE was labeled with a fluorescent conjugate (Oregon green) of fas-
ciculin 2, a snake alpha toxin that binds to the catalytic subunit, prepared
and imaged as described (Peng et al., 1999).
All other fluorescence microscopy was done using a Leica TCS NT
confocal microscope. For Figs. 2–5 and 9, all microscope settings for
-Syn were identical for direct comparison of fluores-
cence intensity. For Figs. 6 and 7, microscope settings were adjusted so
-Syn samples could be shown at similar intensity.
m along the z-axis into a sin-
Quantitation of AChR and AChE
To measure relative levels of AChR in NMJs at University of North Caro-
lina, Chapel Hill, thick cryostat sections (40
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 Univer-
sity of Miami, the fixed sternomastoid muscles were labeled for AChR
and AChE and teased into single fibers. Images of the NMJs were cap-
tured using a digital camera. In both cases, wild-type and
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 be-
tween the average pixel intensity in the circumscribed area and average
background pixel intensity of the corresponding muscle fiber was deter-
mined. The product of this difference and the number of circumscribed
pixels gave a total AChR- or AChE-specific intensity measure for each
For quantitation of AChE enzyme activity, tibialis anterior muscle was
homogenized in 10 vol 20 mM borate buffer, pH 9.0, containing 1% Tri-
tion X-100, 5 mM EDTA, 1 M NaCl, 0.5% BSA, and a protease inhibitor
cocktail as previously described (Rossi and Rotundo, 1993). The samples
were analyzed by velocity sedimentation on 5–20% sucrose gradients in
an SW41 rotor and the fractions assayed for catalytic activity using a ra-
m) of sternomastoid muscles
Sternomastoid muscles were pinned out under fixative (4% glutaralde-
hyde, 4% paraformaldehyde, 0.1 M sodium cacodylate, pH 7.4), fixed for
2 to 3 h, dissected into junction-containing pieces, incubated in 1% os-
mium 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 (Simionescu and Simionescu,
1976). Silver sections were poststained with uranyl acetate and lead (Sato,
1968). The typical ultrathin section through an NMJ contained 2–5 nerve-
muscle contacts. All contacts encountered were photographed in only one
section, chosen without regard to the characteristics of the contacts. Each
contact was analyzed by counting the number of junctional fold openings
to the synaptic cleft and dividing by the total length of presynaptic mem-
brane immediately apposed to the muscle cell (openings/
We established a line of mice lacking
dard targeted gene disruption methods. The entire first
exon (corresponding to amino acids 1–97, which encodes
part of the first PH domain and
PDZ domain), as well as 0.8 kb of 5
and 1.7 kb of intron 1 of the
leted 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 ex-
pected 1:2:1 ratio for wild-type (
-syntrophin by stan-
15 amino acids of the
-syntrophin gene were de-
on both alleles (
skeletal muscle of these mice showed the presence of the
expected 2.4-kb transcript (Adams et al., 1993) encoding
-syntrophin in the
-Syn mice contained low levels of a slightly
smaller transcript and a slightly larger transcript, the latter
also being present in the
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
generated by deletion of exon 2 (Kameya et al., 1999).
To determine if
-syntrophin protein is produced from
these transcripts, we immunoprecipitated total syntro-
phins 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, un-
published results) and would capture NH
cated syntrophin potentially expressed by the disrupted
gene. The antibody SYN17, produced against a peptide se-
quence encoded by exon 3 of the
ters et al., 1994), detected the expected 58-kD protein
(syntrophin) in skeletal muscle from the
but detected no protein in the muscle from
), and mice with the
-syntrophin gene disrupted
). Analysis of RNA isolated from
mice (Fig. 1 C).
mice. The top band
-syntrophin gene (Pe-
Figure 1. Generation and characterization of ?-syntrophin null
mice. A, A targeting vector was constructed using a 7.4-kb Not I
(N)/XbaI (X) restriction fragment long arm and a 1.5-kb XbaI
fragment short arm. Homologous recombination resulted in the
deletion of 2.8-kb of the ?-syntrophin gene including all of exon
1. B, Southern blot analysis was performed using genomic DNA
(isolated from ES cells or from mice) digested with EcoRI (E).
Hybridization using the 500-bp probe shown in A, detected a 6.2-
kb wild-type band and a 10-kb recombinant band. C, RNA blot
analysis of poly A? RNA from the mice indicated. The positions
of RNA standards are indicated. D, Immunoblot of muscle pro-
teins partially purified with mAb 1351 and detected with ?-syn-
trophin and ?1-syntrophin isoform specific antibodies. The posi-
tion of a 59-kD protein standard is indicated on the left.
The Journal of Cell Biology, Volume 150, 2000
(Fig. 1 D). In contrast, a blot of identical samples showed
1-syntrophin levels are moderately increased in skel-
etal muscle of
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 exer-
cise wheels. The distances run and average speed were sta-
tistically 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 (Kameya et al., 1999). Histologically, the
skeletal muscle also appears normal (Fig. 2), with little fi-
brosis, few centralized nuclei, and a normal size distribu-
tion of muscle fibers.
To determine whether elimination of ?-syntrophin af-
fected the distribution of other members of the dystrophin
protein complex, we examined quadriceps muscles by im-
munofluorescence microscopy (Fig. 2). As expected, we
detected no ?-syntrophin staining in this or any other mus-
cle examined. As in wild-type quadriceps muscle (Peters
et al., 1997), ?1-syntrophin was found in only a subset of fi-
bers in the ?-Syn?/? mice. However, in the ?-Syn?/? mus-
cle, fibers that do express ?1-syntrophin show a modest in-
crease 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 ?1-syntrophin staining in the adult (Kramarcy
and Sealock, 2000). Immunofluorescence of adult ?-Syn?/?
sternomastoid showed no detectable upregulation of ?1-
syntrophin (data not shown). In contrast, we observed a
decrease in the intensity of sarcolemmal-labeling for
?-dystrobrevin–2 in all muscle fibers. The intensity of sar-
colemmal dystrophin-labeling in the ?-Syn?/? was indistin-
guishable from littermate control ?-Syn?/? muscle.
Previously, we and others have shown that ?-syntrophin
binds nNOS in vitro via a PDZ–PDZ interaction (Bren-
Figure 2. Analysis of mouse quadriceps muscle. Hematoxylin
and eosin (H&E, top) stained quadriceps muscle from wild-type
(?-Syn?/?) and ?-syntrophin null (?-Syn?/?) mice. The bottom
panels show immunofluorescence microscopy using the indicated
antibody. Bar, 50 ?m.
Figure 3. nNOS distribution in wild-type and ?-syntrophin null
mouse quadriceps muscle. A, Immunofluorescent labeling of
nNOS shows sarcolemmal staining in wild-type that is lost in the
?-syntrophin null muscle. B, Immunofluorescence of serial sec-
tions of ?-syntrophin null muscle shows nNOS is lost from ?1-
syntrophin containing fibers. Bar, 50 ?m.
Adams et al. Neuromuscular Junction Defects of ?-Syntrophin Null Mice
man et al., 1996; Gee et al., 1998a; Hashida-Okumura et al.,
1999). Kameya et al. (1999) found that nNOS is no longer
concentrated at the sarcolemma in ?-Syn?/? mice and we
have confirmed that levels of sarcolemmal nNOS are re-
duced to nearly undetectable amounts (Fig. 3). Interest-
ingly, this loss of nNOS occurs even in those fibers that
express sarcolemmal ?1-syntrophin (Fig. 3 B). Thus, ?1-
syntrophin is not able to compensate for the loss of ?-syn-
trophin in recruiting nNOS to the membrane, even though
it binds nNOS in vitro (Gee et al., 1998b).
Dystrophin Complex Proteins at
?-Syntrophin is present on the sarcolemma, but is en-
riched 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 as-
sociated 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. Al-
though ?1-syntrophin was originally characterized as be-
ing enriched at the NMJ, this enrichment has been shown
to be due, at least in part, to the presence of ?1-syntrophin
in presynaptic structures (Peters et al., 1997; Kramarcy
and Sealock, 2000). At ?-Syn?/? synapses, we observed no
increase in postsynaptic ?1-syntrophin in both fibers ex-
pressing and not expressing ?1-syntrophin on the sarco-
lemma. ?2-Syntrophin, the isoform that is most tightly
confined to the NMJ in adult muscle (Kramarcy and Seal-
ock, 2000), appeared to be somewhat upregulated in the
?-Syn?/? mice, although this increase was not measured
quantitatively. Despite the presence of ?2-syntrophin,
nNOS is absent from junctions lacking ?-syntrophin estab-
lishing that ?2-syntrophin, like ?1-syntrophin, does not re-
cruit nNOS to the membrane to compensate for the loss of
?-Syntrophin has also been shown to bind the muscle
sodium channels, SkM1 and SkM2, via their COOH-ter-
minal sequences (Gee et al., 1998a; Schultz et al., 1998).
Using a polyclonal antibody that recognizes both isoforms,
we found that sodium channels remain concentrated at the
NMJ and on the perisynaptic membrane of ?-Syn?/? mus-
cle, a distribution similar to that found in control muscle.
Thus, ?-syntrophin is not required for expression or syn-
aptic localization of sodium channels in skeletal muscle.
?-Dystrobrevin shares homology with dystrophin (Wag-
ner et al., 1993; Blake et al., 1996; Sadoulet-Puccio et al.,
1996) and is directly associated with it (Peters et al., 1997;
Sadoulet-Puccio et al., 1997). Two isoforms, ?-dystrobre-
vin–1 and –2, both bind syntrophin, but are differentially
localized in skeletal muscle. ?-Dystrobrevin–1 is largely
synaptic, whereas ?-dystrobrevin–2 is found on both the
sarcolemma and at the synapse (Peters et al., 1998). Both
?-dystrobrevin–1 and –2 are present at ?-Syn?/? synapses,
but at slightly lower levels than wild-type synapses.
The distribution of dystrophin (Byers et al., 1991; Seal-
ock et al., 1991) appears unaltered at the ?-Syn?/? junc-
tion, 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 (Be-
wick et al., 1992) and at much lower levels on presynaptic
elements, is dramatically reduced in the postsynaptic
membrane of the ?-Syn?/? mice. Images of 8-?m sections
show low levels of utrophin at the ?-Syn?/? NMJ (Figs. 4
and 5 A), but at high resolution (see Materials and Meth-
ods), even when the confocal microscope is adjusted to
give a strong image of the weak presynaptic staining, utro-
phin is essentially undetectable on the postsynaptic mem-
brane (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 ob-
served 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 indi-
cated 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 junc-
tions 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 reso-
lution by double-labeling muscle sections with Bgtx and
concanavalin A. The lectin labels extracellular glycopro-
teins throughout muscle tissue, particularly highlighting the
synaptic cleft and junctional folds. It also labels the mate-
rial overlying junctional nerve terminals, but not the ter-
minals 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 con-
trast, 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 nerve–
muscle 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 ?-dystrobrevin–2 (Fig. 6 B, a
and b), ankyrin G (Fig. 6 B, c and d), ?2-syntrophin (Fig.
4), and dystrophin (Fig. 4) retained these distributions in
?-Syn?/? muscle (Fig. 6 B, b and d). The perisynaptic dis-
tribution of ankyrin G did not overlap, but rather interdig-
itated 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
The Journal of Cell Biology, Volume 150, 2000
Figure 4. Distribution of syntrophins and associated proteins at the NMJ. Mouse quadriceps muscle sections were double-labeled with
the indicated antibody and bodipy-labeled Bgtx. The two images were combined (merged) to show the relative positions of the indi-
cated proteins and AChRs. All wild-type (?-Syn?/?) and ?-syntrophin null (?-Syn?/?) images were collected under identical conditions.
Bar, 5 ?m.
Adams et al. Neuromuscular Junction Defects of ?-Syntrophin Null Mice
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 ?m in length extended beyond the gut-
ters (see examples in Fig. 7 A). The edges of gutters were
often little brighter that the center (Fig. 7, A, 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 pat-
tern 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, VV?-B4. This lectin labels only the NMJ in muscle
(Scott et al., 1988), with much stronger labeling in the
troughs of the folds than on the AChR-rich crests (Kra-
marcy, N., and R. Sealock, unpublished), thereby providing
a measure of the extent of junctional folding. In ?-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?). Interest-
ingly, even areas that had folds, indicative of morphologi-
cal maturity, could be devoid of labeling by antisynapto-
physin (Fig. 7 B, b and b??), suggesting the absence of a
functional nerve terminal and making participation in syn-
aptic 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 re-
sults contrast with ?-Syn?/? junctions, in which essentially
the entire AChR-rich area was labeled by VVA-B4 and by
antisynaptophysin (not shown).
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 mus-
cles 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 nerve–muscle 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 junc-
tional 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 num-
ber of openings per micrometer of presynaptic membrane
was reduced 59 and 77% in two ?-Syn?/? mice from differ-
ent 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
Secondly, the junctional folds in ?-Syn?/? mice gener-
ally 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 indi-
cating that they open to the surface at some point.
AChE in Mutant Neuromuscular Synapses
Despite the fact that the NMJs in ?-Syn?/? mice are struc-
turally 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 oc-
curs 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 fascic-
ulin 2 (a toxin that specifically labels AChE; Peng et al.,
1999), and then imaged as described in Materials and
Methods. The distribution of AChE is altered in muscle
lacking ?-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. 9,
b and b?, arrowhead), as would be expected from the ab-
sence of folds in such areas (Fig. 7 B). Measurement of the
Figure 5. Loss of utrophin from the ?-Syn?/? postsynaptic mem-
brane. A, Mouse quadriceps (8-?m sections) were labeled with
antiutrophin antibody. NMJs are intensely labeled in ?-Syn?/?
mice and weakly labeled in ?-Syn?/? mice, whereas blood vessels
are labeled with similar intensity. Bars, 10?m. B, High resolution
images of 150-nm sections show that ?-Syn?/? NMJ utrophin la-
beling is largely presynaptic. Bar, 2 ?m.
The Journal of Cell Biology, Volume 150, 2000
Figure 6. Distribution of AChR in ?-Syn?/? and ? -Syn?/? NMJs. Sternomastoid NMJs from littermate ?-Syn?/? and ?-Syn?/? mice
were labeled as indicated for AChR, concanavalin A receptors (basal lamina and other extracellular matrix materials), and postsynap-
tic/sarcolemmal proteins. Inset drawing in B, b, is a schematic of normal junctional folds with the utrophin/AChR-rich membrane in
green, the sodium channel/ankyrin-G/dystrophin-rich membrane in red. A, Nerve terminals are unlabeled areas bounded by synaptic
gutters and the overlying matrix. Arrow shows a contact with sparse junctional folds. A similar contact lies to the left, and a contact ap-
parently without folds, to the right (arrowhead). AChR is organized in clusters at all three contacts, and clusters of AChR extend be-
yond nerve-muscle contacts (double arrowhead). B, ?-Dystrobrevin-2 was present mainly in the troughs of the junctional folds in
?-Syn?/? NMJs (b, arrows), as in wild-type mice (a; see also Peters et al., 1998). Ankyrin G appeared to be present exclusively in the
troughs in ?-Syn?/? NMJs (d), as in the wild type (c; Flucher and Daniels, 1989). Synaptic AChR fields were smooth and continuous in
the ?-Syn?/? NMJs (a, inset), but broken into clusters in the ?-Syn?/? NMJs (d, color inset). Perisynaptically, these proteins interdigi-
tated with clusters of AChR (grayscale insets). Microscope settings were separately optimized for each image. Bar: (A, ?-Syn?/?) 10.6
?m; (A, ?-Syn?/?) 8.9 ?m; (B, a) 6.3 ?m; (B, a, inset) 2.2 ?m; (B, b) 6.2 ?m; (B, c) 7.2 ?m; (B, d) 7.7 ?m, (B, d, color inset) 4.0 ?m; (B,
d, grayscale insets) 10.3 ?m.
Adams et al. Neuromuscular Junction Defects of ?-Syntrophin Null Mice
levels of AChE in 24 junctions showed a significant reduc-
tion 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 nor-
mal and mutant mice by sucrose gradient analysis (Rossi
and Rotundo, 1993). The total amount of soluble, catalyti-
cally active AChE, as well as the relative amounts of the
monomeric (G1), tetrameric (G4), and collagen-tailed syn-
aptic (A12) forms were indistinguishable between ?-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 ?-syntro-
phin has no detectable effect on synthesis, assembly, or
availability of the AChE forms in muscle. Thus, the abnor-
mality must lie in localization and/or retention of the en-
zyme at the synapse.
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. Tis-
sue immunofluorescence and protein blot analyses dem-
onstrate that these mice do not produce ?-syntrophin. De-
spite the loss of ?-syntrophin, these mice are mobile, fertile,
and show no signs of a dystrophic phenotype. These obser-
vations are consistent with those of Kameya et al. (1999)
who have reported an ?-syntrophin null mouse generated
by deleting exon 2. A weak band hybridizing to the ?-syn-
trophin cDNA is present on Northern blots of ?-Syn?/?
AChR, folds, and synaptic vesicles
within ?-Syn?/? NMJs. A, Sterno-
mastoid NMJs from wild-type and
?-Syn?/? mice were labeled for
AChR and imaged en face. In wild-
type NMJs (a), AChR was distributed
smoothly throughout the synaptic
gutter and the gutter edges appeared
bright. In ?-Syn?/? NMJs (b–e),
AChR in the gutters was distributed
in streaks and clusters (b–e) and
could even be absent in places (b).
The gutter edges often showed little
additional intensity, indicating shal-
low gutters. Most ?-Syn?/? NMJs
contained lines of AChR extending
beyond the gutters (b–e). Some NMJs
contained areas of near normal ap-
pearance (c), whereas others were ab-
normal throughout (e). Bar: (a) 5.0
?m; (b) 4.8 ?m; (c) 14.7 ?m; (d) 11.4
?m; (e) 13.1 ?m. B, Sternomastoid
NMJs from ?-Syn?/? mice were dou-
ble- or triple-labeled as indicated. Re-
gions of highly altered AChR distri-
bution (a) could be largely devoid of
VVA-B4 labeling (a?), suggesting ab-
sence of junctional folds. In other
NMJs, virtually the entire AChR field
(b) was well labeled by VVA-B4 (b?).
Regions that gave strong signals for
both AChR (b) and junctional folds
(b?), which suggests maturity, could
nevertheless be devoid of synapto-
physin labeling (b??), suggesting ab-
sence of a functional nerve terminal.
Bar: (a and a?) 12.1 ?m; (b and b??)
Global distribution of
The Journal of Cell Biology, Volume 150, 2000
muscle RNA performed by us and by Kameya et al.
(1999). This may represent a form of ?-syntrophin pro-
duced 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 de-
tects no protein. Alternatively, since ?2-syntrophin can be
encoded by a 10-, 5-, or 2-kb mRNA (Adams et al., 1993),
it is possible that this band is a result of cross-hybridization
to the 2-kb ?2-syntrophin message.
In previous work, we and others have shown that ?-syn-
trophin binds nNOS via a PDZ–PDZ interaction, thus
providing a role for the dystrophin complex in targeting
this signaling protein to the membrane. In agreement with
Kameya et al. (1999), nNOS is absent from the sarco-
lemma of our mice lacking ?-syntrophin. This defect oc-
curs even in fibers that contain abnormally high amounts
of ?1-syntrophin, and at the NMJ where ?2-syntrophin is
concentrated. Thus, despite the highly conserved se-
quences of syntrophin PDZ domains, only ?-syntrophin is
able to bind nNOS PDZ in vivo. Sarcolemmal nNOS is im-
portant for maintenance of adequate blood flow to exer-
cising muscles by counteracting adrenergically mediated
vasoconstriction (Thomas et al., 1998). ?-Syn?/? mice are
not detectably impaired in their ability to exercise, since
they run voluntarily for similar times and distances as con-
trols. However, the voluntary exercise test may not reveal
abnormalities in this system. Experiments examining ad-
renergic mediation of vasoconstriction in mice lacking
?-syntrophin are underway.
The absence of ?-syntrophin selectively affects the ex-
pression of other members of the dystrophin protein com-
plex, although dystrophin itself appears to be unaltered.
The levels of ?1-syntrophin (in some fibers) and ?2-syn-
trophin are increased at the sarcolemma and the NMJ,
respectively. Despite the increase in ?1-syntrophin, ?-dys-
trobrevin–2 levels are significantly reduced on the ?-Syn?/?
sarcolemma. The reduced levels of ?-dystrobrevin–2 are
not sufficient, however, to induce the mild dystrophy seen
in the ?-dystrobrevin null mice (Grady et al., 1999). The
most dramatic change observed in the ?-Syn?/? mouse is
the loss of utrophin from the postsynaptic membrane. This
loss occurs despite the increased levels of ?2-syntrophin at
the NMJ. This is surprising since ?2- and ?-syntrophin have
similar ability to bind utrophin in vitro (Ahn and Kunkel,
1995) and ?2-syntrophin is the isoform that is colocalized
with utrophin in many nonmuscle tissues (Kachinsky et al.,
1999). Thus, ?2-syntrophin must perform a different syn-
aptic role than ?-syntrophin in vivo. These alterations sug-
Figure 8. Ultrastructural analysis of
?-Syn?/? NMJs. Nerve-muscle con-
tacts in null (a and b) and wild-type
(d) sternomastoid muscles treated
with tannic acid to highlight synaptic
clefts and junctional folds (synaptic
basal lamina). Folds in ?-Syn?/?
NMJs varied from near normal in ap-
pearance (straight, oriented toward
the membrane; b) to moderately de-
ranged (curved elements, short folds,
folds parallel to the membrane; a).
The number of junctional fold open-
ings to the synaptic cleft was reduced
in ?-Syn?/? NMJs. Arrows in a and b
indicate the only folds that open to
the cleft in the views shown. In con-
trast, 12 such folds are apparent in
the view of the wild-type NMJ (d).
Pooled data for junctional fold open-
ings per micrometer of presynaptic
membrane in wild-type and null
NMJs are shown in c. The small vesi-
cle-like structures (arrowhead in a),
which appear to be caveoli budding
from the junctional folds, were plen-
tiful in all NMJs, although few are
present in the ?-Syn?/? image shown
here (d). Bar: (a) 1.1 ?m; (b) 1.5 ?m;
(d) 1.4 ?m.
Adams et al. Neuromuscular Junction Defects of ?-Syntrophin Null Mice
gest that ?-syntrophin is a crucial component having
unique activities in forming and/or maintaining the dystro-
phin 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 ?-dystro-
glycan. ?-Syntrophin may stabilize these protein interac-
tions, provide a second site of protein interaction, or po-
tentially bind directly with phospholipids in the membrane
(Chockalingam et al., 1999). A more intriguing possibility
is that ?-syntrophin is part of a signaling pathway that reg-
ulates the synaptic expression of utrophin. The recent ob-
servation that the receptor-tyrosine kinase, ErbB4, is asso-
ciated via its COOH-terminal tail with the PDZ domain of
syntrophins (Garcia et al., 2000; Huang et al., 2000) is par-
ticularly noteworthy since ErbB ligands upregulate the ex-
pression of utrophin (Gramolini et al., 1999; Khurana et al.,
1999). Further studies of the function of ?-syntrophin may
bolster efforts to design a therapy for Duchenne muscular
dystrophy based on upregulation of utrophin as a substi-
tute for dystrophin.
The alterations seen at neuromuscular synapses of mice
lacking ?-syntrophin are similar to changes in other genet-
ically altered mice. Like the utrophin null mice (Decon-
inck et al., 1997; Grady et al., 1997), the ?-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 in-
volved. AChR number at the postsynaptic membrane
could be regulated in several different ways, including syn-
thesis, 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 main-
taining receptor stability (Xu and Salpeter, 1997).
En face views of the ?-Syn?/? NMJ are remarkably sim-
ilar to those seen in the ?-dystrobrevin–null mouse (Grady
et al., 2000). These junctions have shallow nerve terminal
gutters and an abnormal pattern of AChR distribution
within and beyond synaptic gutters. These synaptic alter-
ations in the ?-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 (Grady et al., 1999), suggest-
ing there are additional alterations in ?-dystrobrevin func-
tion in muscle that do not occur in the ?-Syn?/? mice.
Agrin induces AChR clusters on cultured myotubes lack-
ing ?-dystrobrevin, but the clusters are unstable and dis-
perse after removal of agrin. Similar results are obtained
with myotubes lacking ?-dystroglycan (Grady et al., 2000).
These findings, along with the observation that NMJs in
chimeric muscle lacking ?-dystroglycan are structurally
abnormal and contain low levels of AChR and other syn-
aptic proteins (Cote et al., 1999), indicate that the dystro-
phin complex plays an important role in synaptic stabiliza-
tion. Our findings suggest that ?-syntrophin may be
especially important in the mechanisms by which stabiliza-
Severe reduction in AChR levels at the NMJ, due to ac-
quired autoimmunity or congenital causes, leads to myas-
thenia gravis, a muscle weakness disease (reviewed in
Lindstrom, 1997). Here, we have shown that ?-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 kin-
ships have been described that have NMJs which lack
Figure 9. AChE in wild-type and ?-Syn?/? NMJs. Sternomastoid
NMJs from wild-type and ?-Syn?/? mice were double-labeled for
AChR and AChE, and imaged en face under identical micro-
scope settings. The intensities of both labels are substantially re-
duced in the null NMJs (b and b?) compared with wild-type (a
and a?). The AChE distribution in null NMJs shows most of the
alterations found in the AChR distributions, except that AChE
does not extend into the thin fingers of AChR (arrowhead in b
and b?). Bar, 2 ?m. C, The muscles from ?-Syn?/? mice show no
apparent differences in the synthesis and assembly of AChE. The
AChE from sternomastoid muscles of ?-Syn?/? and ?-Syn?/?
mice was extracted and analyzed by velocity sedimentation. G1,
Monomeric AChE; G4, tetrameric AChE; A12, synaptic form of
AChE consisting of three tetramers attached to a collagen-like tail.
The Journal of Cell Biology, Volume 150, 2000
utrophin and show reduced numbers of AChRs like the
?-Syn?/? mouse (Sieb et al., 1998). The ?-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 (Wood and Slater,
1997). In addition, we have identified one probable com-
pensating factor, the reduction of AChE levels to about
one-half of normal. The mechanism leading to the reduc-
tion of AChR and AChE in the absence of syntrophin will
be the focus of future study.
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 dis-
This work was funded by the National Institutes of Health grants
NS33145 (to S.C. Froehner and R. Sealock) and AG05917 (to R.L. Ro-
tundo), and grants from the Council for Tobacco Research and the Mus-
cular 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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