FER-1/Dysferlin promotes cholinergic signaling at the
neuromuscular junction in C. elegans and mice
Predrag Krajacic1,2,*, Emidio E. Pistilli1,2,*,`, Jessica E. Tanis1,2,*, Tejvir S. Khurana1,2,§and S. Todd Lamitina1,2,§
1Department of Physiology, Richards Research Building A702, University of Pennsylvania, Philadelphia, PA 19104, USA
2Pennsylvania Muscle Institute, 700A Clinical Research Building, University of Pennsylvania, Philadelphia, PA 19104, USA
*These authors contributed equally to this work
`Present address: West Virginia University, Center for Cardiovascular and Respiratory Sciences, PO Box 9105, Morgantown, WV 26506, USA
§Authors for correspondence (email@example.com; firstname.lastname@example.org)
Biology Open 2, 1245–1252
Received 30th May 2013
Accepted 25th September 2013
Dysferlin is a member of the evolutionarily conserved ferlin
gene family. Mutations in Dysferlin lead to Limb Girdle
progressive and incurable muscle disorder. However, the
molecular mechanisms underlying disease pathogenesis are
not fully understood. We found that both loss-of-function
mutations and muscle-specific overexpression of C. elegans
fer-1, the founding member of the Dysferlin gene family,
caused defects in muscle cholinergic signaling. To determine
if Dysferlin-dependent regulation of cholinergic signaling
is evolutionarily conserved, we examined the in vivo
signaling in a mouse model of Dysferlin-deficiency. In
addition to a loss in muscle strength, Dysferlin 2/2 mice
also exhibiteda cholinergic
compound muscle action potentials following repetitive
nerve stimulation, which was observed in another Dysferlin
mousemodel butnot in
mouse model of muscular dystrophy. Oral administration
acetylcholinesterase inhibitor (AchE.I) known to increase
synaptic efficacy, reversed the action potential defect and
restored in vivo muscle strength to Dysferlin 2/2 mice
withoutaltering muscle pathophysiology.
demonstrate a previously unappreciated role for Dysferlin
in the regulation of cholinergic signaling and suggest that
such regulation may play a significant pathophysiological role
in LGMD2B disease.
Pyridostigmine bromide,a clinicallyused
? 2013. Published by The Company of Biologists Ltd. This
is an Open Access article distributed under the terms of
the CreativeCommons Attribution
unrestricted use, distribution and reproduction in any
medium provided that the original work is properly
Key words: Dysferlin, Muscular dystrophy, LGMD2B, Limb-girdle,
Limb-Girdle Muscular Dystrophy 2B, or Dysferlinopathy, is an
incurable muscle disorder in which patients usually present in the
second or third decade of life with proximal and/or distal muscle
weakness, elevated serum creatine kinase (CK) levels, and
generally slow disease progression (Amato and Brown, 2011).
Both LGMD2B and a related disorder, Miyoshi Myopathy (MM)
are caused by loss-of-function mutations in the Dysferlin gene
product (Amato and Brown, 2011). Analysis of muscles lacking
Dysferlin function reveals a sub-sarcolemmal accumulation of
membrane vesicles, suggesting defects in vesicle turnover
(Bansal et al., 2003; Ho et al., 2004). Additionally, the loss of
Dysferlin leads to immunity defects, such as increased levels of
phagocytic macrophages and susceptibility to complement attack
(Wenzel et al., 2005; Nagaraju et al., 2008). Recent studies
demonstrate that restoration of Dyferlin gene expression solely in
the skeletal muscle is sufficient to rescue all disease phenotypes
(Millay et al., 2009), suggesting that the immune functions of
Dysferlin play minor roles in disease pathogenesis and that the
pathophysiological defect(s) associated with the loss of Dysferlin
is based within the muscle. Defining these muscle-specific roles
of Dysferlin could provide insight into LGMD2B pathogenesis
and may suggest therapeutic opportunities for this untreatable
Dysferlin is part of the ferlin-1 like protein family, which also
include otoferlin (Fer1L2) and myoferlin (Fer1L3), Fer1L4,
Fer1L5, and Fer1L6 (Han and Campbell, 2007). Like the other
ferlins, Dysferlin encodes a large (230 kD) protein with multiple
calcium and phospholipid-binding C2 domains and a carboxy-
terminal transmembrane domain. Dysferlin expression is highly
enriched in skeletal muscle and is also present in other tissues,
including brain and heart (Bashir et al., 1998; Liu et al., 1998). In
skeletal muscle, Dysferlin is thought to promote damage-induced
membrane repair in a Ca2+dependent manner and Dysferlin
mutants are deficient in this process (Bansal et al., 2003). Until
recently, the current model for pathogenesis of LGMD2B
suggested that mutations in Dysferlin inhibit the active repair
of muscle membranes, resulting in the progressive accumulation
of damaged muscle fibers and eventual manifestation of the
dystrophic phenotype (Han and Campbell, 2007). However, a
Research Article 1245
new study suggests that defective membrane repair may not be
the cause of the disease (Lostal et al., 2012). Whether Dysferlin
regulates other aspects of muscle physiology and whether these
processes play a significant role in disease pathogenesis has not
The C. elegans gene fer-1 encodes the founding member of
the ferlin gene family (Bashir et al., 1998; Liu et al., 1998) and
is homologous to all six human ferlin-1-like proteins (Lek et al.,
2010). Loss-of-function fer-1 mutants are sterile due to defects
in spermatogenesis. While the original characterization of C.
elegans fer-1 noted expression outside of sperm (Achanzar and
Ward, 1997) and we recently demonstrated expression of fer-1
mRNA in C. elegans muscle (Krajacic et al., 2009), a functional
role for fer-1 in C. elegans muscle has not been explored. Here,
we present evidence that, in addition to its well described role in
spermatogenesis, fer-1 is also expressed in the C. elegans body
wall muscle cells, which are the functional and anatomical
equivalent of mammalian skeletal muscles. Surprisingly, we find
that multiple fer-1 mutants, as well as animals overexpressing
cholinergic signaling, a function not previously ascribed to
Dysferlin in any system. We also show that mice carrying loss-
of-function mutations in Dysferlin exhibit defects in muscle
cholinergic signaling, suggesting that the synaptic function of
Dysferlin is evolutionarily conserved. Treatment of Dysferlin
mutant mice with the FDA-approved acetylcholinesterase
inhibitor (AchE.I) Pyridostigmine bromide reversed Dysferlin-
dependent cholinergic defects and restored muscle strength. Our
findings suggest that decreased efficiency of post-synaptic
cholinergic signaling may also contribute to the pathogenesis
of LGMD2B and could represent a novel target for disease
To gain more insights into the role of Dysferlin, we re-examined
the function of the founding member of the Dysferlin gene
family, C. elegans fer-1. In addition to its previously well
described expression in sperm (Achanzar and Ward, 1997), we
also found that a fer-1 promoter::gfp reporter showed expression
in body wall muscle cells (Fig. 1a), consistent with previous
mRNA expression analysis of purified muscle cells (Krajacic et
al., 2009). fer-1 mutants exhibit normal sarcomere structure
(Krajacic et al.,2009) and
(supplementary material Fig. S1), suggesting that fer-1 is not
required for muscle development or for the maintenance of
Although fer-1 mutants do not exhibit signs of muscle damage,
we considered the possibility that they might show defects in
muscle functional properties. Given that other ferlin family
members are known to regulate synaptic transmission (Roux et
al., 2006), we explored whether fer-1 might also play a role in
signaling at the neuromuscular junction (NMJ). In C. elegans,
NMJ signaling is reciprocally regulated by a single inhibitory
GABA receptor and two distinct excitatory acetylcholine
receptors (AchRs) (Richmond and Jorgensen, 1999). The
balance between GABA and AchR signaling can be probed
with the acetylcholinesterase inhibitor aldicarb and the AchR
agonist levamisole. Both drugs cause excitation of post-synaptic
AchRs, muscle hypercontraction, and time-dependent paralysis
(Mahoney et al., 2006). In animals treated with aldicarb the time
to paralysis is indicative of either the rate of pre-synaptic Ach/
GABA release or post-synaptic Ach/GABA signaling. In
contrast, the time to paralysis for animals treated with
levamisole can be used to identify defects in post-synaptic
signaling (Mahoney et al., 2006). We found that multiple fer-1
Fig. 1. fer-1 mutants exhibit defects in cholinergic synaptic function. (a) GFP fluorescence in animals carrying a fer-1 promoter::gfp::unc-54 39 UTR reporter
transgene shows expression in the body-wall muscles. Scale bar520 mm. (b) Loss of fer-1 causes resistance to paralysis induced by the cholinesterase inhibitor
aldicarb; P,0.005. (c) fer-1 mutants exhibit increased resistance to the paralytic effects of the L-AchR agonist levamisole; P,0.001. (d) Loss of GABA signaling in
the unc-49 mutant causes hypersensitivity to levamisole (P,0.0001) that is suppressed by the loss of fer-1 (P,0.0001). (e) Overexpression of fer-1 in either the
sperm or neurons is not sufficient to alter levamisole sensitivity; P50.90. (f) Overexpression of fer-1 in the body-wall muscles causes levamisole resistance;
P,0.005. For (b–f), each Kaplan–Meyer graph shows data from one representative experiment, n530 animals/genotype.
Dysferlin regulation of cholinergic signaling 1246
loss of function mutants, including the putative null allele hc47
(supplementary material Fig. S2) were weakly resistant to both
aldicarb (Fig. 1b) and levamisole (Fig. 1c). As has been
described for fer-1 spermatogenesis defective phenotypes, the
levamisole resistance for all non-null fer-1 alleles was also
temperature-sensitive, suggesting that the genetic basis of the fer-
1 synaptic phenotype is loss-of-function, as has been previously
demonstrated for fer-1 sperm phenotypes (Ward and Miwa,
1978). Along with our previous observations demonstrating fer-1
mRNA expression in purified cultured muscle cells but not in
purified neuronal cells (Krajacic et al., 2009), these findings
suggest that FER-1 acts in post-synaptic body wall muscle cells
(Mahoney et al., 2006). Resistance to cholinergic stimulation was
also observed in fer-1 mutants lacking a germline, but not in
another related fertility mutant, spe-5, demonstrating that
resistance was due to fer-1 somatic functions and not to fer-1
sterility (supplementary material Fig. S3).
Levamisole resistance could result from either enhanced
excitatory signaling (via Ach receptors) at the NMJ (Richmond
and Jorgensen, 1999). To distinguish between these possibilities,
we examined the levamisole resistance of fer-1 mutants in an
unc-49 mutant background, which encodes the sole C. elegans
ionotropic GABA receptor. Compared to the unc-49 single
mutant, levamisole resistance was still observed in the fer-1;unc-
49 double mutant (Fig. 1d), suggesting that loss of fer-1 does not
cause levamisole resistance through enhanced GABA signaling.
The mechanism by which loss of fer-1 disrupted cholinergic
signaling did not appear to involve the steady-state clustering of
L-AchRs since the localization of an UNC-63::YFP fusion
protein (Gendrel et al., 2009) appeared normal in multiple fer-1
mutants (supplementary material Fig. S2). Together, these data
suggest that fer-1 reduces cholinergic signaling at the C. elegans
In mice, muscle-specific overexpression of Dysferlin gives rise
to a progressive muscular dystrophy, although this phenotype is
distinct from that caused by loss of Dysferlin since there is no
evidence of sarcolemmal membrane damage (Glover et al.,
2010). Given that fer-1 is expressed in C. elegans muscle, we
hypothesized that muscle-specific overexpression of fer-1 might
also cause muscle defects, possibly by disrupting AchR signaling.
To test this hypothesis, we overexpressed the fer-1 genomic
coding sequence under the control of the muscle specific myo-3
promoter, the neuron-specific unc-119 promoter, or the sperm-
specific spe-11 promoter using the single-copy insertion method
(Frøkjær-Jensen et al., 2008). The fer-1 single-copy expression
clone was functional since the spe-11p::fer-1 transgene was able
to rescue the sterility of fer-1(hc47) animals (data not shown).
Animals overexpressing fer-1 in either the sperm or the neurons
exhibited normal levamisole response (Fig. 1e). However,
levamisole resistance equivalent to that seen in fer-1 loss-of-
function mutants (Fig. 1f). Introduction of the myo-3p::fer-1
transgene into the fer-1(hc24) background did not rescue or
enhance fer-1 mutant levamisole resistance (Fig. 1f), suggesting
that fer-1 overexpression disrupts a similar pathway as that
affected by fer-1 loss-of-function. These data suggest that
overexpression of fer-1 in C. elegans muscle, but not in
neurons orsperm, can phenocopy
mutants and reduce cholinergic signaling in C. elegans.
However, it remains possible that the mechanism by which
in the muscleexhibited
fer-1 overexpression reduces cholinergic signaling is distinct
from that caused by loss of fer-1.
The effect of fer-1 on C. elegans AchR signaling could be due
to unique features of worm neuroanatomy/physiology or could
represent a functionally significant, but previously undescribed
role for Dysferlin. Therefore, we explored the effect of a
Dysferlin loss-of-function mutation on cholinergic signaling at
the mouse NMJ using the well-established A/J Dysferlin mutant
mouse model (Ho et al., 2004; Wenzel et al., 2007; Millay et al.,
2009). Muscle pathophysiology in A/J mice is progressive
(observable histological muscle damage not present until ,6
months of age, followed by relatively slow progression). To
determine if loss of Dysferlin altered cholinergic signaling at the
NMJ, we developed an in vivo physiological apparatus
(supplementary material Fig. S4) that allowed us to perform
repetitive nerve stimulation (RNS) of the peroneal nerve at a
defined frequency while simultaneously monitoring the Ach-
dependent compound muscle action potential (CMAP) through
electromyography (EMG) and muscle force production with a
force transducer (Fig. 2a). Aged wild type mice (15 month old A/
HeJ) could undergo RNS at either 0.1 Hz or 3 Hz without
becoming tetanic or exhibiting significant CMAP or force drops
(Table 1). However, aged A/J mice, but not young A/J mice,
exhibited a significant CMAP drop at RNS frequencies of 3.0 Hz
that was not observed at 0.1 Hz (Fig. 2b,c; Table 1). A similar
frequency-dependent CMAP drop was also observed in a
different mouse Dysferlin mutant (SJL/J model), suggesting that
this phenotype is due to loss of Dysferlin and not to other defects
associated with a specific mouse strain (supplementary material
Fig. S5). Furthermore, we did not observe these phenotypes in the
unrelated mouse muscular dystrophy mutant (mdx dystrophin
mutant, supplementary material Fig. S5) (Bittner et al., 1999;
Turk et al., 2006). Given that mdx mice exhibit similar if not
greater levels of muscle degeneration/regeneration than Dysferlin
mutant mice, these data suggest that the cholinergic signaling
defects in Dysferlin mutant mice are not due to generalized
muscle damage or regeneration. They are also not simply due to
aging since age-matched wild type controls do not exhibit similar
cholinergic signaling defects.
We also examined the ex vivo properties of electrically evoked
twitch and tetanic contractions in EDL muscles of A/HeJ, A/J and
SJL/J mice. For all genotypes and age groups except for SJL/J,
the normalized ex vivo twitch and tetanic forces were not
different between groups (supplementary material Table S1). A
small but significant decrease in normalized twitch force, but not
normalized tetanic force, was observed in SJL/J (supplementary
material Table S1). Taken together, these data suggest that loss of
Dysferlin has little, if any, effect on the contractile properties of
muscle under ex vivo conditions that bypass the neuromuscular
The CMAP defect observed in Dysferlin mutants loosely
resembles that found in Myasthenic syndromes, where a
reduction in cholinergic signaling brought about through a
variety of molecular mechanisms causes a RNS CMAP
strength (Hirsch, 2007). While our findings above suggest a
weak RNS CMAP decrement and loss of muscle strength
associated with loss of Dysferlin, LGMD2B patients are not
thought to exhibit fatigueability and are often sportive through
childhood and early adult years, which indicates that LGMD2B is
not a classical myasthenic syndrome. Still, we considered the
andloss of muscle
Dysferlin regulation of cholinergic signaling 1247
possibility that loss of Dysferlin may alter cholinergic signaling
in a way that resembles other bonafide cholinergic disease states.
These syndromes are often responsive to small molecule
inhibitors of AchE (AchE.I), the enzyme that breaks down Ach
in the synaptic cleft of the NMJ. We hypothesized that if the
CMAP defect caused by loss of Dysferlin was similar to that
found in myasthenic conditions, then the Dysferlin CMAP defect
might be improved via AchE.I therapy. Furthermore, if the
CMAP defect is a primary cause of reduced muscle performance
(rather than a secondary consequence of disease), then AchE.I
therapy should also improve in vivo muscle strength in A/J mice.
To test this hypothesis, we treated A/J mice with the AchE.I
(treatment beginning at 2 months of age) or co-pathologically
(treatment beginning at 7 months of age) and then measured their
in vivo CMAPs, muscle performance, and muscle histology. Both
treatment (Fig. 3c,d) eliminated the CMAP drop found in
Dysferlin mutants. BothAchE.I
significantly improved the in vivo muscle performance of A/J
mice (Table 1) but did not have a significant effect on either ex
vivo muscle performance or the presence of centrally-nucleated
myofibers (supplementary material Table S1), suggesting the
presence of continued regeneration in AchE.I-treated A/J mice.
Taken together, these findings show that treatment of A/J mice
with the AchE.I Pyridostigmine bromide prevents the CMAP
decrement and substantially improves the in vivo muscle
performance of muscles lacking Dysferlin.
Fig. 2. Dysferlin deficient A/J mice show age-dependent CMAP decrement upon repetitive stimulation. (a) Representative raw data traces of simultaneously
recorded stimulation (green), CMAP (blue) and dorsiflexion force (red). Insert: one rep-stim cycle. (b,c) Normalized mean CMAP voltage on 3 Hz repetitive nerve
stimulation, expressed as a % of CMAP 1, for 2 months A/J (n58) and 14.5 months old A/J mice (n57) compared to 14.5 months old A/HeJ mice (n57).
(d) Normalized mean CMAP voltage on rep-stim 4. One way ANOVA with Bonferroni’s multiple comparison test: *P,0.05; **P,0.01; ***P,0.001.
Table 1. In vivo muscle physiology.
Contraction force (mN)
CMAP D (0.1 Hz)
CMAP D (0.5 Hz)
CMAP D (1 Hz)
CMAP D (3 Hz)
CMAP D (10 Hz)
54.35 6 28.45
102.50 6 4.24
103.38 6 5.43
101.02 6 6.86
102.68 6 5.16
99.68 6 2.46
26.52 6 5.04*
99.41 6 4.85
102.07 6 3.99
97.48 6 4.31
95.37 6 2.97*
95.36 6 8.07
55.24 6 24.58
103.52 6 6.03
99.49 6 2.17
101.84 6 3.51
102.75 6 7.13
97.98 6 3.79
57.32 6 25.80
101.50 6 5.57
101.57 6 4.57
101.86 6 3.90
100.71 6 4.72
97.91 6 4.21
74.35 6 11.52
100.61 6 4.22
101.12 6 6.40
101.00 6 3.78
106.33 6 7.53
99.35 6 2.29
77.07 6 35.61
97.47 6 14.03
97.21 6 12.78
102.55 6 18.02
96.12 6 9.92
91.62 6 7.94**
50.68 6 17.40
100.85 6 2.02
100.95 6 2.60
101.22 6 3.94
98.78 6 1.1
95.18 6 3.62
(CMAP D)5rep-stim 4 as % of rep-stim 1. Data presented as Mean 6 S.D., and analyzed using one-way ANOVA with Bonferroni’s Multiple Comparison Test.
1age: 14.5 months
2age: 2 months
3age: 10 months
4age: 9 months
5treated with 0.32 mg?ml21pyridostigmine bromide
Dysferlin regulation of cholinergic signaling1248
While Dysferlin is expressed in many cell types, muscle
expression is sufficient to rescue disease pathophysiology in
mice (Millay et al., 2009). Until recently, it was largely thought
that this muscle-specific role was related to the membrane repair
activity of Dysferlin at the muscle plasma membrane, since a
major pathological hallmark of Dysferlinopathies is membrane
damage with an accumulation of sub-sarcolemmal membrane
vesicles (Glover and Brown, 2007; Han and Campbell, 2007).
However, a recent study showed that even when a minimal
Dysferlin expression construct sufficient to restore membrane
repair in isolated muscle fibers was transgenically expressed in
mice, muscle damage and weakness continued to occur in vivo
(Lostal et al., 2012). One possible explanation for this surprising
result is that sarcolemmal repair in vivo differs from repair in
isolated fibers. Another possibility is that Dysferlin, in addition to
its role in membrane repair, is also involved in other physiological
processes in muscle and that loss of these functions plays
important roles in disease pathogenesis. Outside of its role in
muscle membrane repair, other physiological roles for Dysferlin in
muscle have not been described. Our discovery that loss of
Dysferlin affects cholinergic signaling in both worms and mice
could represent one such additional role for Dysferlin in muscle.
Our studies show that old, but not young, Dysferlin mutant
mice exhibit frequency-dependent RNS EMG defects. While
these phenotypes are consistent with the progressive nature of
disease onset in humans, they could also result from the ageing
process itself. Indeed, previous work shows that ageing does
cause progressive synaptic degeneration at morphological and
functional levels (Balice-Gordon, 1997; Li et al., 2011).
However, significant morphological changes occur primarily
after 24 months of age and most functional alterations in synaptic
activity occur within the first 6 weeks of age. Given that our
studies were carried out during a period where there are minimal
reported age-related changes in muscle structure/function (2
months to 15 months of age), it seems unlikely that our data are
simply due to physiological consequences of ageing. In support
of this, we found that when A/HeJ mice, the control strain for the
A/J mouse Dysferlin model (http://jaxmice.jax.org), were aged in
parallel to the A/J mince, they did not exhibit significant RNS
EMG rundown or alterations in ex vivo contractile properties
(Table 1; supplementary material Table S1).
We have interpreted our findings in a way that suggests the RNS
EMG defect associated with loss of Dysferlin is not related to
chronological age but rather is related to a specific role for
Dysferlin in the regulation of cholinergic signaling. Regardless of
the specific mechanism(s), we find that treatment with the AchE.I
pyridostigmine can rescue the functional declines in muscle
performance in Dysferlin mutants. Interestingly, histopathological
properties of the disease, such as the presence of centrally
nucleated fibers, were unaffected, perhaps suggesting that
cholinergic defects are a consequence rather than the underlying
cause of disease. Nevertheless, this is a highly significant and
translational finding as there are currently no established
treatments for LGMD2B. Whether pyridostigmine treatment
rescues muscle functional performance in Dysferlin mutant mice
because it enhances cholinergic signaling or because it prevents
age-induced declines in cholinergic signaling remains an open
question that warrants future study.
While our data show a small but statistically significant and
reproducible decrease in the high-frequency RNS CMAP
response of Dysferlin mutant mice (,5% at 3 Hz in A/J), the
Fig. 3. Dysferlin deficient A/J mice CMAP defect can be corrected with Acetylcholinesterase inhibitors. (a,c) Normalized mean CMAP voltage (3 Hz repetitive
nerve stimulation) expressed as a % of CMAP 1. (a) CMAP decrement in A/J mice treated with Pyridostigmine bromide (A/J pre-onset group, n59). Treatment
started before the disease onset at 2 months of age and continued until 14 months of age. Age matched control groups (A/HeJ, n57; A/J, n57) received no treatment.
(b) Normalized mean CMAP voltage on rep-stim 4. (c) CMAP decrement in A/J mice treated with Pyridostigmine bromide (A/J post-onset group, n58).
Treatment started after the disease onset at 7 months of age and continued until 14 months of age. Age matched control groups (A/HeJ, n57; A/J, n57) received no
treatment. (d) Normalized CMAP voltage on rep-stim 4. 1 way ANOVA with Bonferroni’s multiple comparison test: *P,0.05; **P,0.01; ***P,0.001.
Dysferlin regulation of cholinergic signaling1249
applicability of these findings to humans is currently unknown.
Currently, there is no evidence for NMJ functional defects in
LGMD2B patients and standard EMGs in these patients is
reportedly normal. However, standard clinical EMGs do not
always utilize a RNS protocol and whether or not LGMD2B
patients exhibit RNS-evoked defects remains an open question
that warrants more clinical study in light of our findings. While
human patients with defects in cholinergic signaling, such as
those with Myasthenia gravis, can exhibit substantial RNS
rundowns of 20% or more, some bonafide Myasthenia gravis
patients exhibit reduced or even no RNS rundown (Stalberg,
1980; Claussen et al., 1995), suggesting that even small EMG
defects can be reflective of underlying NMJ dysfunction. Further
study is needed to determine if the small synaptic defects we
observe in Dysferlin mutant mice represents an atypical
presentation of synaptic dysfunction, whether these defects are
present only in a subset of muscle groups, and how relevant
synaptic dysfunction may be to disease pathogenesis in humans.
While our findings show that Dysferlin contributes to muscle
cholinergic signaling in both worms and mice, we have not
defined the molecular or cell biological mechanism(s) through
which Dysferlin acts or whether Dysferlin exerts such action via
its localization at the NMJ or other subcellular location. Given its
role as a regulator of membrane fusion (Han and Campbell,
2007), one hypothesis is that Dysferlin controls synaptic AchR
levels via regulated insertion/retrieval of AchR-containing
vesicles. However, our studies in worms suggest that steady-
state synaptic AchR levels are not altered (supplementary
material Fig. S2). Whether Dysferlin coordinates activity-
dependent AchR turnover is an open question that will require
more sophisticated live-animal imaging approaches.
Aside from regulating vesicular insertion of AchRs at the
synapse, Dysferlin could act in additional ways to promote
cholinergic signaling. For example, Dysferlin could regulate the
activity of synaptic AchRs without altering AchR abundance or
cholinergic signaling via effects on excitation–contraction
coupling or regulation of sarcoplasmic Ca2+levels (Roche et al.,
2011). Regardless of the mechanism, our finding that treatment
appropriate synaptic function and rescues muscle strength in
Dysferlin mutant mice suggests that elevating synaptic Ach levels
can compensate for this defect. While these pharmacological
findings support the hypothesis that there is a deficit of Ach
signaling inDysferlinmutantmice,they also suggesttheintriguing
possibility that LGMD2B patients might benefit from AchE.I
therapy. Given the long clinical history and general long-term
safety of these compounds (Maggi and Mantegazza, 2011), more
pre-clinical studies to examine the potential benefit of AchE.I
therapy for LGMD2B patients are warranted. In this regard, it is
important to note that in our studies, AchE.I therapy improved
muscle physiology, but did not affect other markers of disease (i.e.
CNFs). Therefore, it seems likely that, at best, a combined
therapeutic approach targeting both AchR synaptic dysfunction
and defective membrane repair processes may be needed to best
address the sequalae of LGMD2B.
Materials and Methods
All animals were handled in strict accordance with good animal practice as defined
by the relevant national and/or local animal welfare bodies, and all animal work
was approved by the appropriate committee: Institutional Animal Care and Use
Committee (IACUC), University of Pennsylvania Perelman School of Medicine,
Philadelphia, PA 19104 (Protocol no. 802799).
C. elegans strains were maintained at 20˚C under standard conditions (Brenner,
1974). The wild-type strain was Bristol N2; mutations used include: fer-1(hc1),
fer-1(hc24), fer-1(b232), fer-1(hc47), unc-49(e409), unc-119(ed3). The molecular
alterations caused by the various fer-1 mutations are as follows (positions based on
fer-1a isoforms); hc1 – G290E, hc24 – L1809F, b232 – S1486N, hc47 –
W494STOP. Since the fer-1 alleles are temperature sensitive, animals used for
assays were synchronized by the hypochlorite method then grown at the restrictive
temperature of 25˚C to generate staged young adults.
Transgenes for cell-specific fer-1 expression
Cell-specific expression constructs were created using the Gateway cloning
strategy (Invitrogen) and each contained a cell-specific promoter, the fer-1
genomic sequence, and the unc-54 39 untranslated region. The myo-3 promoter
(muscle-specific), spe-11 promoter (sperm-specific) and unc-119 promoter
(neuron-specific) transgenes drSi6, drSi16, and drSi19, respectively, were
chromosomally integrated into the chromosome II ttTi5605 Mos site using
single copy insertion (Frøkjær-Jensen et al., 2008). Germline transformation of the
fer-1p::gfp::unc-54 39 UTR PCR fusion product and him-4p::Mb::yfp plasmid was
performed using standard techniques (Mello et al., 1991).
C. elegans were immobilized with 3 mM levamisole (Sigma) on 3% agar pads.
Fluorescence Z-stack images were obtained using a Leica DMI4000B microscope
(636objective) with a Leica DFC340Fx digital camera. The width of muscle arms
from ventral left body wall muscle 11 was measured at the midpoint of each arm
(Leica advanced fluorescence 6000 software).
For the UNC-63::YFP experiments, fer-1 mutants were crossed into the unc-
63::YFP strain (Gendrel et al., 2009) using standard genetic methods. All strains
were verified to be homozygous for the unc-63::yfp marker by PCR genotyping.
Fluorescence Z-stack images of the neuromuscular junctions were acquired as
described above. All analyses were carried out on collapsed Z-stacks of the raw
images and quantified in Leica Advanced Fluorescence 6000 software.
Assay plates were prepared by adding aldicarb (Fisher) or levamisole (Sigma)
stock solutions to NGM agar at 50˚C to a final concentration of 1 mM (aldicarb) or
0.5 mM (levamisole). After pouring, plates were stored at 4˚C and used within one
week. For each assay, 30 animals/genotype were picked to drug plates (10 animals/
plate) and prodded every 15 min. (aldicarb assay) or 10 min. (levamisole assay).
Worms that failed to respond were classified as paralyzed. All experiments were
conducted by two independent researchers who were blinded to genotype and
repeated at least two times.
Male A/HeJ and A/J, mice were purchased from Jackson Laboratories. Two groups
(n510) of A/J mice were treated with Pyridostigmine bromide (Sigma–Aldrich)
dissolved in water (0.32 mg/ml water) available ad libitum. The treatment for one
group began at the age of 8 weeks (pre-disease onset) and at the age of 7 months
for the second group (co-disease onset) and continued until the age of 14 months
for both groups. Age matched control groups received no treatment.
In vivo repetitive nerve stimulation protocol
Mice were anesthetized
injection) and placed in a prone position. A 2.0 cm incision was made exposing
the biceps femoris muscle, and the artery genus descendes was used as a landmark
(Bourquin et al., 2006). The sciatic nerve was isolated by blunt dissection. The
tibial and sural nerves were cut, leaving the common peroneal nerve intact.
Following nerve preparation, the tissue was hydrated with saline solution and
mouse body temperature maintained using a heat lamp.
A specially designed apparatus was constructed to simultaneously record the
muscle compound action potential (CMAP) and muscle contractile force following
nerve stimulation, based on previous reports (Ashton-Miller et al., 1992; Gorselink
et al., 2001). The apparatus was constructed of acetal polymer and consisted of a
lower platform, measuring 30 cm625 cm, and a secondary platform, measuring
20 cm 6 10 cm which was raised approximately 1.5 cm off the lower platform.
The mouse was supine on the lower platform with the right leg positioned on the
raised platform. A force transducer (FT03, Grass Technologies) coupled to a foot
plate was positioned on the raised platform and the foot was secured to the
footplate using 6.0 nylon suture. A micromanipulator was used to position
stimulation electrodes around the sciatic nerve. Additional micromanipulators
were used to position needle recording electrodes into the tibialis anterior muscle,
(Tribromoethanol0.4–0.75 mg/kg, intraperitoneal
Dysferlin regulation of cholinergic signaling1250
which allowed the acquisition of CMAPs. The force transducer allowed the
acquisition of muscle contractile forces resulting from dorsiflexion of the anterior
compartment of the lower leg at the ankle joint. Data were acquired using an A/D
(supplementary material Fig. S3). CMAP and force features were quantified using
custom MATLAB scripts, which were validated through comparisons with
manually compiled data.
A low to high frequency repeated stimulation protocol was performed,
consisting of a set of 10 contractions at the following stimulation frequencies:
0.1, 0.5, 1.0, 3.0, and 10 Hz. Stimulations lasted 0.1 ms at a voltage of 5 V. One
minute rest was allowed in between each set, and this protocol was repeated three
times to ensure that the synaptic decrement was not due to degeneration of the
preparation. In all cases, A/J and SJL/J mice showed normal responses to low
frequency stimulation (0.1 Hz) after exhibiting CMAP decrement at high
frequency (3 or 10 Hz) stimulation, indicating that the defect was due to the
physiological properties of the muscle and not to the loss of integrity of either the
preparation or the recording setup. Ex vivo EDL muscle contractile properties were
examined in freshly dissected EDL muscles, as previously described (Pistilli et al.,
2011; Bogdanovich et al., 2002). Upon completion of the in vivo experiments,
mice were euthanized by placing them in a 100% CO2chamber for three minutes,
followed by cervical dislocation.
Muscles were imbedded in Tissue Freezing Medium (TBS, Durham, NC), flash
frozen, and stored at 280˚C. Frozen sections (10 mm thick) of quadriceps muscle
were obtained using a cryostat at maintained at 221˚C and placed onto glass slides
(Superfrost/Plus, Fisher Scientific). Sections were fixed in ice-cold methanol for
5 min and then processed for histological examination by hematoxylin and eosin-
phloxine (H&E) staining. Digital images were acquired using an Olympus BX51
microscope at 406. The number of centrally nucleated fibers was determined by
All data are presented as means 6 S.D., unless noted otherwise. Data were
analyzed using either the Students T-test for comparisons between two groups or
one-way ANOVA analysis with Boneferroni’s correction for comparisons between
3 or more groups. For the levamisole and aldicarb resistance assays, the number of
motile animals over time was analyzed using the Logrank test as implemented in
Graphpad Prism 4. P values of ,0.05 were taken to indicate statistical
We thank Steve L’Hernault (Emory University) for providing several
fer-1 mutant strains, G. Rapti and J. L. Bessereau for providing the
him-4::Mb-YFP and the UNC-63::YFP marker and other reagents,
and Drs Janet Richmond, Rita Balice-Gordon, and Clara Franzini-
Armstrong for helpful discussions and suggestions. Some nematode
strains used in this work were provided by the Caenorhabditis
Genetics Center, which is funded by the NIH National Center for
Research Resources (NCRR).
This work was supported by grants from the National Institutes of
Health [grant number NS065936 to T.S.K. and S.T.L., grant number
F32AR060128 to J.E.T.], the Muscular Dystrophy Association (to
S.T.L.), and the Pennsylvania Muscle Institute (to S.T.L. and T.S.K).
P.K., E.E.P. and J.E.T. performed the experiments. All authors
analyzed the data and wrote the paper.
The authors have no competing interests to declare.
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