Neuroscience Letters 737 (2020) 135304
Contents lists available at ScienceDirect
Neuroscience Letters
journal homepage: www.elsevier.com/locate/neulet
Review article
Alterations of neuromuscular junctions in Duchenne muscular dystrophy
Richard M. Lovering a, b, *, Shama R. Iyer a, Benjamin Edwards c, Kay E. Davies c
a
Departments of Orthopaedics and Physiology, University of Maryland School of Medicine, Baltimore, MD, USA
University of Maryland School of Medicine, Baltimore, MD, USA
c
MDUK Oxford Neuromuscular Centre, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK
b
A R T I C L E I N F O
A B S T R A C T
Keywords:
mdx
NMJ
Duchenne muscular dystrophy
Eccentric injury
The focus of this review is on Duchenne muscular dystrophy (DMD), which is caused by the absence of the
protein dystrophin and is characterized as a neuromuscular disease in which muscle weakness, increased sus­
ceptibility to muscle injury, and inadequate repair appear to underlie the pathology. Considerable attention has
been dedicated to studying muscle fiber damage, but data show that both human patients and animal models for
DMD present with fragmented neuromuscular junction (NMJ) morphology. In addition to pre- and post-synaptic
abnormalities, studies indicate increased susceptibility of the NMJ to contraction-induced injury, with corre­
sponding functional changes in neuromuscular transmission and nerve-evoked electromyographic activity. Such
findings suggest that alterations in the NMJ of dystrophic muscle may play a role in muscle weakness via
impairment of neuromuscular transmission. Further work is needed to fully understand the role of the NMJ in the
weakness, susceptibility to injury, and progressive wasting associated with DMD.
1. Introduction
The area of synaptic contact between motor neurons and their target
muscle fibers is the neuromuscular junction (NMJ). This synapse occurs
at a specialized area of the sarcolemma called the end-plate. The
“pretzel-shape” of a typical, healthy end-plate in mammalian muscle
(Fig. 1) results from the several twisting branches of the motor neuron.
The distal aspect of each branch is enlarged and these expansions form
the terminal synaptic boutons, which contain synaptic vesicles filled
with the neurotransmitter, acetylcholine (ACh). Boutons are located
over invaginations of the sarcolemma called junctional folds [89], at the
top of which high-density clusters of acetylcholine receptors (AChRs)
reside. When released into the synaptic cleft, ACh binds to its
post-synaptic receptors, causing an end-plate potential (EPP), a local
depolarization spreading along the muscle fiber as a conducted action
potential.
Accumulating evidence has made it clear that the NMJ in mature
skeletal muscle is not a fixed permanent structure [27,28], but instead is
continually remodeling, thereby possessing a large degree of functional
plasticity [30]. The morphology and physiology of the NMJ can display
alterations in synaptic organization due to exercise [26,89], inactivity
[66,67,83], denervation [49,92], aging [24,41,76], crushing of the
nerve/muscle [44,80], or the absence of associated proteins [1,3,16,45,
48]. Ultrastructural changes in the NMJ have been documented
following muscle disuse and nerve injury or denervation [9,49].
The motor end-plate (the post-synaptic membrane of NMJ) is a
specialized area of the sarcolemma that rapidly and consistently re­
sponds to release of a neurotransmitter from the overlying nerve ter­
minal. Neuromuscular transmission is normally highly reliable, as each
nerve impulse results in the release of more neurotransmitter (acetyl­
choline) than is required for evoking an action potential in the muscle
fiber. This release of surplus transmitter and consequent excess depo­
larization of the postsynaptic membrane via AChRs is often referred as
the ‘safety factor’ [91], which ensures that a post-synaptic action po­
tential will occur in response to each nerve impulse, at least in healthy
tissue. Proper development and organization at the NMJ is necessary for
effective neuromuscular transmission [79,90], but a number of patho­
logical conditions affecting the distribution of AChRs can lead to a
reduction in the safety factor and impairment of neuromuscular trans­
mission [63,91]. Although there are diseases caused by defective syn­
aptic transmission at the NMJ (congential myasthenic syndromes)[61],
this review focuses on muscular dystrophies, specifically Duchenne
muscular dystrophy.
2. NMJ morphology is altered in muscular dystrophy
Duchenne muscular dystrophy (DMD), the most common and severe
form of muscular dystrophy, is caused by the absence of dystrophin, a
* Corresponding author at: University of Maryland School of Medicine, Department of Orthopaedics, 100 Penn St., AHB, Room 540, Baltimore, MD21201USA.
E-mail address: rlovering@som.umaryland.edu (R.M. Lovering).
https://doi.org/10.1016/j.neulet.2020.135304
Received 30 April 2020; Received in revised form 9 August 2020; Accepted 11 August 2020
Available online 17 August 2020
0304-3940/© 2020 Elsevier B.V. All rights reserved.
R.M. Lovering et al.
Neuroscience Letters 737 (2020) 135304
structural protein found on the cytoplasmic surface of the sarcolemma.
Dystrophin is the central component of a molecular link that connects
the contractile apparatus inside the muscle fiber to the extracellular
matrix outside the muscle fiber, and binds directly or indirectly to a
group of proteins at the sarcolemma collectively known as the
dystrophin-associated protein complex (DAPC or DPC) or the
dystrophin-glycoprotein complex (DGC).
The DGC of the sarcolemma accumulates at the post-synaptic
membrane (motor end-plate) of the NMJ, and the absence of associ­
ated proteins can cause changes in NMJ structure and function [6]. The
NMJ is noticeably disrupted in muscles from mdx and other DMD mouse
models [3,43,55,82]. Dystrophin appears to be involved in synaptic
homeostasis [64]; it is not required for NMJ formation, but is required
for endplate maintenance [45] and likely for endplate remodeling in
regenerating fibers. dx mice show NMJ fragmentation in adult muscle
fibers and excessive nerve sprouting compared to wild-type mice [56],
and ultrastructural studies using electron microscopy indicate a loss in
the number and depth of synaptic folds of the motor end-plate in mdx
muscles [3,55].
Patients with DMD and mdx mice have muscles with weakness and
increased susceptibility to injury compared to their non-dystrophic
counterparts. Over time, this damage/degeneration exceeds the ability
to repair/regenerate muscle, leading to irreversible muscle wasting
throughout life. The increased force loss after contraction-induced
injury is typically attributed to structural weakness of the muscle fiber
cytoskeleton and changes in signaling within muscle fibers secondary to
the loss of dystrophin [53]. However, studies have reported that loss in
whole muscle function after injury is also associated with alterations in
NMJ end-plate morphology in mdx mice [68–70]. Some studies report
strong evidence showing that disrupted NMJ morphology is the conse­
quence of muscle fiber degeneration and regeneration [37,50,51,59],
but others have suggested that NMJ fragmentation in mdx muscle is
independent of degeneration and regeneration [3,46]. In support of the
latter, NMJ post-synaptic morphology, including AChR area, is normal
in fibers of muscle subjected to toxin-induced degeneration [75,88]. It is
thought that the motor neuron in DMD and mdx mice is unaffected,
except for axonal sprouting near the NMJ and changes in the terminal
bouton. However, recent data suggest that the persistent, chronic NMJ
remodeling that occurs over the lifespan of the mdx mouse can adversely
affect the motor neuron [47].
Utrophin, short for “ubiquitous dystrophin,” is a protein highly ho­
mologous to dystrophin [52]. During fetal muscle development, utro­
phin is found along the entire muscle sarcolemma. Once dystrophin is
expressed, however, utrophin disappears from most of the sarcolemmal
membrane so that, in normal adult muscle, it is located only at the
neuromuscular and myotendinous junctions [85]. One reason mdx mice
do not display pathology equivalent to that seen in DMD may be that
utrophin is up-regulated to levels sufficient to compensate, in part, for
Fig. 1. Top panels show representative images of neuromuscular junctions (NMJ) stained with α-bungarotoxin conjugated to Alexa-594 in muscles from WT (wild
type), mdx (lacking dystrophin), dko (double knockout, lacking both dystrophin and utrophin), and Fiona (lacking dystrophin but upregulation of utrophin) mice.
Bottom panels show representative skeletonized images of the corresponding NMJ. Total stained area, total stained perimeter, total area, and total perimeter were
calculated using binarized images using Image J. Dispersion index was calculated by total stained area/total area, and is a measure of density of acetylcholine
receptors. Discontinuity and number of branching were calculated using the skeletonized images. Number of clusters was calculated by counting the number of
separate acetylcholine receptor aggregates. * indicates significant difference from WT, # indicated significant difference from Fiona, p < 0.05; one-way ANOVA,
approximately 10 NMJs in each genotype, with 5 muscles analyzed).
2
R.M. Lovering et al.
Neuroscience Letters 737 (2020) 135304
the lack of dystrophin. Although studies have suggested that NMJ
disruption is not dependent on utrophin [88], NMJ morphology appears
more disrupted when dystrophin and utrophin are absent [74], and the
upregulation of utrophin (through artificial transcription factors) ap­
pears to rescue NMJ morphology [65]. In the Fiona mouse, a transgenic
mdx mouse in which utrophin is upregulated in skeletal muscles, using
established methods [68–71] (detailed methods in supplemental material)
we have found a partial rescue of NMJ morphology (Fig. 1).
Muscles from mice that lack genes for other components of the DGC,
such as α-dystrobrevin, dystroglycan, or syntrophin, have altered NMJ
morphology [1,6,33,35,39], even though absence of other DGC com­
ponents, such as α- or ϒ-sarcoglycan, does not appear to affect NMJ
morphology [23,36]. Aberrations in NMJ morphology have also been
observed in laminopathies, which are typically caused by mutations in
the LMNA gene, resulting in cardiac and skeletal disorders. Patients
often have abnormal EMG, indicating myopathy [8,58]. In mouse
models with LMNA mutations, NMJ morphology is disorganized and
fragmented, with mislocalized synaptic nuclei [58]. Furthermore, the
NMJ disorganization was observed before myopathic phenotype,
furthering the notion of NMJ aberrations as a driver of disease
phenotype.
proximal muscles have attributed reduced muscle excitability to changes
in the NMJ [68,69].
Synaptic transmission becomes more variable with age in the mdx
mouse model of DMD [12,44], which could provide one explanation
why, despite the consistent lack of dystrophin, mdx skeletal muscle
generates less specific force and becomes more susceptible to damage
with age [15]. EMG changes are measurable in patients with various
muscular dystrophies, including DMD [18,31,72]. Some animal studies
report changes in EMG activity between WT and mdx mice [38,68],
while others do not [13]. The study of different muscles might be one
reason for such conflicting data; for example, proximal muscles are
affected earlier and to a greater extent in DMD [17,57] and a similar
pattern of increased damage in more proximal muscles has been docu­
mented in young mdx mice [60].
Patients with DMD have similar intramuscular fatigability, neuro­
muscular transmission, and central activation as controls [81]. Such
observations might argue against a significant role for NMJ dysfunction
in DMD in the absence of injury, however increased sensitivity to
neuromuscular blocking drugs (and a slower recovery from them) in­
dicates NMJ is vulnerable in DMD, with more obvious findings in
older-aged and ambulatory patients with DMD [88]. Furthermore,
although the reduced safety factor and miniature end-plate potentials
might not be sufficient to result in overt changes to the compound
muscle action potentials (CMAPs), they could render the NMJ
dysfunctional under certain intense activity [88].
3. Disrupted NMJ morphology in dystrophic muscle contributes
to impairment of nerve-evoked muscle contraction
Structure is clearly a major determinant of function: as the devel­
opment of force relies on the controlled overlap of actin and myosin, so
does effective neuromuscular transmission on the apposition of the
nerve terminal and the underlying motor end-plate [79,90]. Moreover,
maintenance of the neuromuscular apparatus relies on bidirectional
communication between muscle and nerve [34]. However, several
pathological conditions affecting the distribution of AChRs can lead to a
reduction in the safety factor and impairment of neuromuscular trans­
mission [63].
The increased muscle weakness and susceptibility to injury (exag­
gerated loss in force to a mechanical insult) in patients with DMD and in
the mdx murine model, is hypothesized to be due to structural weakness
of the cytoskeleton or changes in signaling secondary to the loss of
dystrophin [53], but additional mechanisms are still being clarified. A
mechanical model [4,7] suggests that the absence of dystrophin results
in structural fragility of the cytoskeleton [53]. In an attempt to under­
stand mechanisms underlying muscle weakness, much of the focus has
been on structural damage within the myofiber. The NMJ is clearly
disrupted in muscles from DMD mouse models and associated deficits in
neuromuscular function have now also been identified [3,14]. We have
reported that loss in whole muscle contractile force after muscle injury
in mdx mice is also associated with alterations in NMJ morphology, such
as increased discontinuity and branching, and corresponding aberrant
synaptic transmission, such as decreased EMG amplitudes and increased
neuromuscular transmission failure [68–70]. Other groups have also
shown disruption at the NMJ in dystrophic mouse muscles with corre­
sponding changes in function, such as reduced amplitude of miniature
endplate potentials, reduction of postsynaptic sensitivity for the
neurotransmitter acetylcholine and exhaustion of presynaptic acetyl­
choline release during intense synaptic activity [88]. After
contraction-induced injury, there is a loss of force in both the WT and
mdx muscles, but only mdx mice show significant additional changes in
NMJ morphology, neuromuscular transmission, and EMG activity [68,
69]. Although other factors, such as sarcolemma damage could be a
factor, these findings suggest that NMJ structural and functional
changes may contribute to the greater force loss seen after injury in
dystrophic muscle. While some studies attribute changes in myofiber
action potential conductivity as the primary reason for reduced muscle
excitability in mdx muscles after injury, these studies typically utilize a
low-strain, high-repetition injury protocol in distal muscles [5,11,77];
other studies utilizing a high-strain, lower repetition injury protocol in
4. Therapeutic approaches to affect NMJ form and function
Several approaches of gene therapy are being examined to amelio­
rate the pathophysiology that results from the absence of dystrophin.
Although transgenic expression of a short isoform of dystrophin in mdx
muscle can prevent muscle degeneration, NMJ morphology remains
fragmented [3]. Indeed, the threshold of dystrophin lies between 19%
and 50% for correction of NMJ morphology in muscles of mdx mice [87].
Muscle-specific Kinase (MuSK) is a transmembrane tyrosine kinase
crucial for forming and maintaining the NMJ, and activation of the
MuSK complex that drives AChR clustering [10,32]. MuSK inactivation
at the NMJ of adult muscle is known to cause a reduction in AChR
density and a change in the gross synaptic arborization of the endplate,
which can lead to the complete loss of AChRs and disappearance of the
synaptic structure [40]. MuSK levels differ between various adult skel­
etal muscles, which may correlate with muscle specific differences in the
response to agrin [25,73]. In our previous work, we assessed transcripts
for the multi-protein MuSK signaling complex responsible for AChR
clustering in WT and mdx mice [69]; interestingly, the only significant
difference between WT and mdx muscleswas a decrease in MuSK inthe
latter. Since MuSK plays a critical role in the aggregation, or clustering,
of AChRs, it is conceivable that this reduction directly contributes to the
altered morphology of mdx NMJs and is therefore a potential therapeutic
target. Indeed, increasing expression of MuSK or rapsyn (a cytoplasmic
MuSK effector protein) with adeno-associated viral vectors protected
mdx muscles from contraction-induced injury [86].
Utrophin upregulation results in a positive effect on NMJ postsynaptic morphology, including an increase in the number of AChRs
and improved NMJ morphology [65]. This is accompanied by an
improvement in muscle contractility, but it is difficult to tease apart the
contribution of the NMJ versus other changes in the cell (e.g.,
improvement in sarcolemma stability, mechanotransduction of force,
etc.). Interestingly, drugs commonly used for myasthenia gravis and
myasthenic syndromes (autoimmune disease and inherited conditions,
respectively, affecting the NMJ) do not seem to have a significant benefit
for muscular dystrophies.
Exercise can exert beneficial effects not only on muscle, but also to
the NMJ morphology and function [22,78]. Endurance training affects
the morphology of NMJs in young adults, and has been studied as a
measure to counter changes in the NMJ with aging [2]. Specific
3
R.M. Lovering et al.
Neuroscience Letters 737 (2020) 135304
adaptations to exercise training include increases in the length and
number of nerve terminal branches, a higher number of pre-synaptic
vesicles, and increase number and distribution of AChRs [20,22,78,
89]. Thus, the benefits attained from endurance training are likely
beyond just muscle fiber remodeling and extend to the NMJ. Exercise
can induce activation of neurotrophic factors and other molecules,
which have a positive impact on NMJ morphology [29,62]. Further­
more, alterations of structure induced by endurance training are asso­
ciated with significant NMJ functional changes, such as synaptic
transmission. Resistance exercise appears to yield similar benefits for the
NMJ, but to a lesser degree [19,21].
It is established that heavy resistance training has deleterious effects
on dystrophic skeletal muscle, particularly if it involves eccentric con­
tractions [54]; in addition to the risk of further muscle damage, there is
no evidence of beneficial adaptation to heavy resistance training in
dystrophic animals, or in humans with muscular dystrophy. Although
historically exercise has been used sparingly in the treatment of
muscular dystrophies, evidence suggests beneficial impact from mod­
erate exercise [42]. Several well-controlled studies indicate that light to
moderate exercise can have beneficial effects in patients with muscular
dystrophies, such as increased strength. Unfortunately, there are rela­
tively few controlled studies available that are easily translated to the
human population. There is therefore a great need for careful studies to
determine the forms of exercise that are most beneficial to patients with
different types of muscle diseases, and the effects of exercise on the NMJ
in dystrophic muscle.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.neulet.2020.135304.
References
[1] M.E. Adams, N. Kramarcy, S.P. Krall, S.G. Rossi, R.L. Rotundo, R. Sealock, S.
C. Froehner, Absence of alpha-syntrophin leads to structurally aberrant
neuromuscular synapses deficient in utrophin, J.Cell Biol. 150 (2000) 1385–1398.
[2] M.H. Andonian, M.A. Fahim, Effects of endurance exercise on the morphology of
mouse neuromuscular junctions during ageing, J. Neurocytol. 16 (1987) 589–599.
[3] G.B. Banks, J.S. Chamberlain, S.C. Froehner, Truncated dystrophins can influence
neuromuscular synapse structure 1, Mol.Cell Neurosci. 40 (2009) 433–441.
[4] C.L. Batchelor, S.J. Winder, Sparks, signals and shock absorbers: how dystrophin
loss causes muscular dystrophy, Trends Cell Biol. 16 (2006) 198–205.
[5] C.W. Baumann, G.L. Warren, D.A. Lowe, Plasmalemma function Is rapidlyrestored
in mdxmuscle after eccentriccontractions, Med. Sci. Sports Exerc. 52 (2020)
354–361.
[6] D.C. Belhasan, M. Akaaboune, The role of the dystrophin glycoprotein complex on
the neuromuscular system, Neurosci. Lett. 722 (2020) 134833.
[7] R.J. Bloch, H. Gonzalez-Serratos, Lateral force transmission across costameres in
skeletal muscle, Exerc. Sport Sci. Rev. 31 (2003) 73–78.
[8] G. Bonne, S. Quijano-Roy, Emery-Dreifuss muscular dystrophy, laminopathies, and
other nuclear envelopathies, Handb. Clin. Neurol. 113 (2013) 1367–1376.
[9] M.C. Brown, W.G. Hopkins, R.J. Keynes, Importance of pathway formation for
nodal sprout production in partly denervated muscles 15, Brain Res. 243 (1982)
345–349.
[10] S.J. Burden, SnapShot: neuromuscular Junction 7, Cell 144 (2011), 826-826.
[11] J.A. Call, G.L. Warren, M. Verma, D.A. Lowe, Acute failure of action potential
conduction in mdx muscle reveals new mechanism of contraction-induced force
loss 1, J. Physiol 591 (2013) 3765–3776.
[12] C.G. Carlson, D.M. Roshek, Adult dystrophic (mdx) endplates exhibit reduced
quantal size and enhanced quantal variation 2, Pflugers Arch. 442 (2001) 369–375.
[13] G.T. Carter, K.J. Longley, R.K. Entrikin, Electromyographic and nerve conduction
studies in the mdx mouse 2, Am. J. Phys. Med. Rehabil. 71 (1992) 2–5.
[14] J.S. Chamberlain, J. Metzger, M. Reyes, D. Townsend, J.A. Faulkner, Dystrophindeficient mdx mice display a reduced life span and are susceptible to spontaneous
rhabdomyosarcoma 3, FASEB J. 21 (2007) 2195–2204.
[15] S. Chan, S.I. Head, J.W. Morley, Branched fibers in dystrophic mdx muscle are
associated with a loss of force following lengthening contractions, Am. J. Physiol
Cell Physiol. 293 (2007) C985–C992.
[16] P.H. Chipman, C.K. Franz, A. Nelson, M. Schachner, V.F. Rafuse, Neural cell
adhesion molecule is required for stability of reinnervated neuromuscular
junctions 1, Eur. J. Neurosci. 31 (2010) 238–249.
[17] D. Cros, P. Harnden, J.F. Pellissier, G. Serratrice, Muscle hypertrophy in duchenne
muscular dystrophy. a pathological and morphometric study 1, J. Neurol. 236
(1989) 43–47.
[18] K.L. Derry, S.L. Venance, T.J. Doherty, Decomposition-based quantitative
electromyography in the evaluation of muscular dystrophy severity 1, Muscle
Nerve 45 (2012) 507–513.
[19] M.R. Deschenes, D.A. Judelson, W.J. Kraemer, V.J. Meskaitis, J.S. Volek, B.
C. Nindl, F.S. Harman, D.R. Deaver, Effects of resistance training on neuromuscular
junction morphology, Muscle Nerve 23 (2000) 1576–1581.
[20] M.R. Deschenes, C.M. Maresh, L.E. Armstrong, J. Covault, W.J. Kraemer, J.
F. Crivello, Endurance and resistance exercise induce muscle fiber type specific
responses in androgen binding capacity, J. Steroid Biochem. Mol. Biol. 50 (1994)
175–179.
[21] M.R. Deschenes, C.M. Maresh, J.F. Crivello, L.E. Armstrong, W.J. Kraemer,
J. Covault, The effects of exercise training of different intensities on neuromuscular
junction morphology, J. Neurocytol. 22 (1993) 603–615.
[22] M.R. Deschenes, M.A. Roby, E.K. Glass, Aging influences adaptations of the
neuromuscular junction to endurance training, Neuroscience 190 (2011) 56–66.
[23] F. Duclos, V. Straub, S.A. Moore, D.P. Venzke, R.F. Hrstka, R.H. Crosbie,
M. Durbeej, C.S. Lebakken, A.J. Ettinger, M.J. van der, K.H. Holt, L.E. Lim, J.
R. Sanes, B.L. Davidson, J.A. Faulkner, R. Williamson, K.P. Campbell, Progressive
muscular dystrophy in alpha-sarcoglycan-deficient mice, J. Cell Biol. 142 (1998)
1461–1471.
[24] M.K. Elkerdany, M.A. Fahim, Age changes in neuromuscular junctions of masseter
muscle 9, Anat. Rec. 237 (1993) 291–295.
[25] A. Eusebio, F. Oliveri, P. Barzaghi, M.A. Ruegg, Expression of mouse agrin in
normal, denervated and dystrophic muscle 1, Neuromuscul. Disord. 13 (2003)
408–415.
[26] M.A. Fahim, Endurance exercise modulates neuromuscular junction of C57BL/
6NNia aging mice 1, J. Appl. Physiol. 83 (1997) 59–66.
[27] D.M. Fambrough, Control of acetylcholine receptors in skeletal muscle 7, Physiol.
Rev. 59 (1979) 165–227.
[28] D.M. Fambrough, P.N. Devreotes, J.M. Gardner, D.J. Card, The life history of
acetylcholine receptors 6, Prog. Brain Res. 49 (1979) 325–334.
[29] L. Ferraiuolo, J.P. De Bono, P.R. Heath, H. Holden, P. Kasher, K.M. Channon,
J. Kirby, P.J. Shaw, Transcriptional response of the neuromuscular system to
exercise training and potential implications for ALS, J. Neurochem. 109 (2009)
1714–1724.
5. Conclusions
Knowledge of NMJ dysfunction in DMD animal models is incom­
plete, and sometimes conflicting, but findings suggest that, in addition to
mechanical damage to the myofiber, structural and functional changes
at the NMJ may be another contributor to the greater force loss seen
after injury in dystrophic muscle. In terms of functional impact, the
relative roles of muscle fiber injury, degeneration, and denervation that
contribute to the changes seen in NMJ morphology of dystrophic mus­
cles are not yet known, but will be important to determine, as NMJ
dysfunction is fundamental to understanding impairment of muscle. The
notion that structure determines function is a key tenet in biology, yet it
is possible that disruption of NMJ morphology in dystrophic muscle
represents an effective repair process that maintains efficacy; there is
still no clear evidence showing correlation between the degree of NMJ
fragmentation and the efficacy of transmission [84]. Continued experi­
mentation is paramount for elucidating mechanisms underlying the
dystrophic progression. The specific role of the NMJ in neuromuscular
health and its relationship to dystrophy is still being defined, but the
NMJ might play a role in the exacerbated response to injury.
The electrophysiological NMJ features of one muscle might not
necessarily be identical in all skeletal muscles [88], but there is now
sound evidence indicating a role for the NMJ in muscle weakness, sus­
ceptibility to damage, and loss of functional performance in dystrophic
muscle. Future work is needed to systematically study constituents of
the MuSK signaling complex, compare various muscles, and to follow
the morphology and function of the NMJ at various time points after
injury and throughout the lifespan.
Funding
This work was supported by grants to RML from the National In­
stitutes of Health (R56AR073193), to SRI from the National Institutes of
Health (K01AR074048) and the Muscular Dystrophy Association
development grant (MDA 577897), and to KD from the Medical
Research Council, UK
4
R.M. Lovering et al.
Neuroscience Letters 737 (2020) 135304
[59] E. Minatel, N.H. Santo, M.J. Marques, Acetylcholine receptors and neuronal nitric
oxide synthase distribution at the neuromuscular junction of regenerated muscle
fibers 1, Muscle Nerve 24 (2001) 410–416.
[60] F. Muntoni, A. Mateddu, F. Marchei, A. Clerk, G. Serra, Muscular weakness in the
mdx mouse 2, J. Neurol.Sci. 120 (1993) 71–77.
[61] S. Nicole, Y. Azuma, S. Bauche, B. Eymard, H. Lochmuller, C. Slater, Congenital
myasthenicsyndromes or inheriteddisorders of neuromusculartransmission:
recentdiscoveries and Open questions, J. Neuromuscul. Dis. 4 (2017) 269–284.
[62] H. Nishimune, J.A. Stanford, Y. Mori, Role of exercise in maintaining the integrity
of the neuromuscular junction, Muscle Nerve 49 (2014) 315–324.
[63] K.E. Personius, R.P. Sawyer, Variability and failure of neurotransmission in the
diaphragm of mdx mice, Neuromuscul. Disord. 16 (2006) 168–177.
[64] G.S. Pilgram, S. Potikanond, R.A. Baines, L.G. Fradkin, J.N. Noordermeer, The roles
of the dystrophin-associated glycoprotein complex at the synapse, Mol. Neurobiol.
41 (2010) 1–21.
[65] C. Pisani, G. Strimpakos, F. Gabanella, M.G. Di Certo, A. Onori, C. Severini,
S. Luvisetto, S. Farioli-Vecchioli, I. Carrozzo, A. Esposito, T. Canu, E. Mattei,
N. Corbi, C. Passananti, Utrophin up-regulation by artificial transcription factors
induces muscle rescue and impacts the neuromuscular junction in mdx mice,
Biochim. Biophys. Acta Mol. Basis Dis. 1864 (2018) 1172–1182.
[66] Y.S. Prakash, H. Miyata, W.Z. Zhan, G.C. Sieck, Inactivity-induced remodeling of
neuromuscular junctions in rat diaphragmatic muscle 1, Muscle Nerve 22 (1999)
307–319.
[67] Y.S. Prakash, W.Z. Zhan, H. Miyata, G.C. Sieck, Adaptations of diaphragm
neuromuscular junction following inactivity 4, Acta Anat. (Basel) 154 (1995)
147–161.
[68] S.J. Pratt, S.B. Shah, C.W. Ward, M.P. Inacio, J.P. Stains, R.M. Lovering, Effects of
in vivo injury on the neuromuscular junction in healthy and dystrophic muscles 1,
J. Physiol. 591 (2013) 559–570.
[69] S.J. Pratt, S.B. Shah, C.W. Ward, J.P. Kerr, J.P. Stains, R.M. Lovering, Recovery of
altered neuromuscular junction morphology and muscle function in mdx mice after
injury, Cell Mol. Life Sci. (2014).
[70] S.J. Pratt, A.P. Valencia, G.K. Le, S.B. Shah, R.M. Lovering, Pre- and postsynaptic
changes in the neuromuscular junction in dystrophic mice, Front. Physiol. 6 (2015)
252.
[71] S.J.P. Pratt, S.R. Iyer, S.B. Shah, R.M. Lovering, Imaging analysis of the
neuromuscular Junction in dystrophicmuscle, Methods Mol.Biol. 1687 (2018)
57–72.
[72] A. Priez, J. Duchene, F. Goubel, Duchenne muscular dystrophy quantification: a
multivariate analysis of surface EMG 1, Med. Biol. Eng. Comput. 30 (1992)
283–291.
[73] A.R. Punga, M. Maj, S. Lin, S. Meinen, M.A. Ruegg, MuSK levels differ between
adult skeletal muscles and influence postsynaptic plasticity 3, Eur. J. Neurosci. 33
(2011) 890–898.
[74] J.A. Rafael, E.R. Townsend, S.E. Squire, A.C. Potter, J.S. Chamberlain, K.E. Davies,
Dystrophin and utrophin influence fiber type composition and post-synaptic
membrane structure, Hum. Mol. Genet. 9 (2000) 1357–1367.
[75] M. Rich, J.W. Lichtman, Motor nerve terminal loss from degenerating muscle
fibers, Neuron 3 (1989) 677–688.
[76] N. Robbins, Compensatory plasticity of aging at the neuromuscular junction 10,
Exp. Gerontol. 27 (1992) 75–81.
[77] P. Roy, F. Rau, J. Ochala, J. Messeant, B. Fraysse, J. Laine, O. Agbulut, G. ButlerBrowne, D. Furling, A. Ferry, Dystrophin restoration therapy improves both the
reduced excitability and the force drop induced by lengthening contractions in
dystrophic mdx skeletal muscle, Skelet Muscle 6 (2016) 23.
[78] R. Rudolf, M.M. Khan, S. Labeit, M.R. Deschenes, Degeneration of neuromuscular
junction in age and dystrophy, Front. Aging Neurosci. 6 (2014) 99.
[79] J.R. Sanes, J.W. Lichtman, Development of the vertebrate neuromuscular junction,
Annu. Rev. Neurosci. 22 (1999) 389–442.
[80] N.H. Santo, A.J. Martins, E. Minatel, M.J. Marques, Axonal sprouting in mdx mice
and its relevance to cell and gene mediated therapies for Duchenne muscular
dystrophy 1, Neurosci. Lett. 343 (2003) 67–69.
[81] K.R. Sharma, M.A. Mynhier, R.G. Miller, Muscular fatigue in duchenne muscular
dystrophy, Neurology 45 (1995) 306–310.
[82] T. Shiao, A. Fond, B. Deng, M. Wehling-Henricks, M.E. Adams, S.C. Froehner, J.
G. Tidball, Defects in neuromuscular junction structure in dystrophic muscle are
corrected by expression of a NOS transgene in dystrophin-deficient muscles, but
not in muscles lacking alpha- and beta1-syntrophins 1, Hum. Mol.Genet. 13 (2004)
1873–1884.
[83] D.C. Sieck, W.Z. Zhan, Y.H. Fang, L.G. Ermilov, G.C. Sieck, C.B. Mantilla, Structureactivity relationships in rodent diaphragm muscle fibers vs. Neuromuscular
junctions 1, Respir. Physiol. Neurobiol. 180 (2012) 88–96.
[84] C.R. Slater, ’Fragmentation’ of NMJs: a sign of degeneration or regeneration? A
long journey with many junctions, Neuroscience 439 (2020) 28–40.
[85] J. Tinsley, N. Deconinck, R. Fisher, D. Kahn, S. Phelps, J.M. Gillis, K. Davies,
Expression of full-length utrophin prevents muscular dystrophy in mdx mice, Nat.
Med. 4 (1998) 1441–1444.
[86] S. Trajanovska, J. Ban, J. Huang, P. Gregorevic, M. Morsch, D.G. Allen, W.
D. Phillips, Muscle specific kinase protects dystrophic mdx mouse muscles from
eccentric contraction-induced loss of force-producing capacity, J. Physiol. 597
(2019) 4831–4850.
[87] E.M. van der Pijl, M. van Putten, E.H. Niks, J. Verschuuren, A. Aartsma-Rus, J.
J. Plomp, Low dystrophin levels are insufficient to normalize the neuromuscular
synaptic abnormalities of mdx mice, Neuromuscul. Disord. 28 (2018) 427–442.
[88] E.M. van der Pijl, M. van Putten, E.H. Niks, J.J. Verschuuren, A. Aartsma-Rus, J.
J. Plomp, Characterization of neuromuscular synapse function abnormalities in
[30] J. Ferre, E. Mayayo, R. Brunet, Morphometric study of the neuromuscular synapses
in the adult rat with special reference to the remodelling concept 1, Biol. Cell 60
(1987) 133–144.
[31] M. Frascarelli, L. Rocchi, I. Feola, EMG computerized analysis of localized fatigue
in Duchenne muscular dystrophy 3, Muscle Nerve 11 (1988) 757–761.
[32] N. Ghazanfari, K.J. Fernandez, Y. Murata, M. Morsch, S.T. Ngo, S.W. Reddel, P.
G. Noakes, W.D. Phillips, Muscle specific kinase: organiser of synaptic membrane
domains 1, Int. J. Biochem. Cell Biol. 43 (2011) 295–298.
[33] R.M. Grady, H. Zhou, J.M. Cunningham, M.D. Henry, K.P. Campbell, J.R. Sanes,
Maturation and maintenance of the neuromuscular synapse: genetic evidence for
roles of the dystrophin–glycoprotein complex, Neuron 25 (2000) 279–293.
[34] A.D. Grinnell, Dynamics of nerve-muscle interaction in developing and mature
neuromuscular junctions 1, Physiol. Rev. 75 (1995) 789–834.
[35] J.D. Gumerson, C.S. Davis, Z.T. Kabaeva, J.M. Hayes, S.V. Brooks, D.E. Michele,
Muscle-specific expression of LARGE restores neuromuscular transmission deficits
in dystrophic LARGE(myd) mice, Hum. Mol. Genet. 22 (2013) 757–768.
[36] A.A. Hack, C.T. Ly, F. Jiang, C.J. Clendenin, K.S. Sigrist, R.L. Wollmann, E.
M. McNally, Gamma-sarcoglycan deficiency leads to muscle membrane defects and
apoptosis independent of dystrophin, J.Cell Biol. 142 (1998) 1279–1287.
[37] S.G. Haddix, Y.I. Lee, J.N. Kornegay, W.J. Thompson, Cycles of myofiber
degeneration and regeneration lead to remodeling of the neuromuscular junction
in two mammalian models of Duchenne muscular dystrophy, PLoS One 13 (2018)
e0205926.
[38] J.J. Han, G.T. Carter, J.J. Ra, R.T. Abresch, J.S. Chamberlain, L.R. Robinson,
Electromyographic studies in mdx and wild-type C57 mice 3, Muscle Nerve 33
(2006) 208–214.
[39] R. Herbst, T. Iskratsch, E. Unger, R.E. Bittner, Aberrant development of
neuromuscular junctions in glycosylation-defective large(myd) mice,
Neuromuscul. Disord. 19 (2009) 366–378.
[40] B.A. Hesser, O. Henschel, V. Witzemann, Synapse disassembly and formation of
new synapses in postnatal muscle upon conditional inactivation of MuSK 1, Mol.
Cell Neurosci. 31 (2006) 470–480.
[41] Y.C. Jang, H. Van Remmen, Age-associated alterations of the neuromuscular
junction 2, Exp. Gerontol. 46 (2011) 193–198.
[42] M. Jansen, N. van Alfen, A.C. Geurts, I.J. de Groot, Assisted bicycle training delays
functional deterioration in boys with Duchenne muscular dystrophy: the
randomized controlled trial “no use is disuse”, Neurorehabil. Neural Repair 27
(2013) 816–827.
[43] F. Jerusalem, A.G. Engel, M.R. Gomez, Duchenne dystrophy. II. Morphometric
study of motor end-plate fine structure, Brain 97 (1974) 123–130.
[44] M. Kawabuchi, H. Tan, S. Wang, Age affects reciprocal cellular interactions in
neuromuscular synapses following peripheral nerve injury 1, Ageing Res. Rev. 10
(2011) 43–53.
[45] J. Kong, J.E. Anderson, Dystrophin is required for organizing large acetylcholine
receptor aggregates 1, Brain Res. 839 (1999) 298–304.
[46] J. Kong, L. Yang, Q. Li, J. Cao, J. Yang, F. Chen, Y. Wang, C. Zhang, The absence of
dystrophin rather than muscle degeneration causes acetylcholine receptor cluster
defects in dystrophic muscle, Neuroreport 23 (2012) 82–87.
[47] V.S. Krishnan, A. Aartsma-Rus, M. Overzier, C. Lutz, L. Bogdanik, M.D. Grounds,
Implications of increased S100beta and Tau5 proteins in dystrophic nerves of two
mdx mouse models for Duchenne muscular dystrophy, Mol. Cell. Neurosci. (2020)
103484.
[48] S.A. Kulakowski, S.D. Parker, K.E. Personius, Reduced TrkB expression results in
precocious age-like changes in neuromuscular structure, neurotransmission, and
muscle function 1, J. Appl. Physiol 111 (2011) 844–852.
[49] S.S. Labovitz, N. Robbins, M.A. Fahim, Endplate topography of denervated and
disused rat neuromuscular junctions: comparison by scanning and light microscopy
1, Neuroscience 11 (1984) 963–971.
[50] Y. Li, Y. Lee, W.J. Thompson, Changes in aging mouse neuromuscular junctions are
explained by degeneration and regeneration of muscle fiber segments at the
synapse 1, J. Neurosci. 31 (2011) 14910–14919.
[51] Y. Li, W.J. Thompson, Nerve terminal growth remodels neuromuscular synapses in
mice following regeneration of the postsynaptic muscle fiber 1, J. Neurosci. 31
(2011) 13191–13203.
[52] D.R. Love, D.F. Hill, G. Dickson, N.K. Spurr, B.C. Byth, R.F. Marsden, F.S. Walsh, Y.
H. Edwards, K.E. Davies, An autosomal transcript in skeletal muscle with homology
to dystrophin, Nature 339 (1989) 55–58.
[53] R.M. Lovering, L. Michaelson, C.W. Ward, Malformed mdx myofibers have normal
cytoskeletal architecture yet altered EC coupling and stress-induced Ca2+
signaling, Am. J. Physiol. Cell Physiol. 297 (2009) C571–C580.
[54] R.M. Lovering, N.C. Porter, R.J. Bloch, The muscular dystrophies: from genes to
therapies, Phys. Ther. 85 (2005) 1372–1388.
[55] P.R. Lyons, C.R. Slater, Structure and function of the neuromuscular junction in
young adult mdx mice 1, J. Neurocytol. 20 (1991) 969–981.
[56] M.J. Marques, A.P. Taniguti, E. Minatel, H.S. Neto, Nerve terminal contributes to
acetylcholine receptor organization at the dystrophic neuromuscular junction of
mdx mice 2, Anat. Rec. (Hoboken.) 290 (2007) 181–187.
[57] S. Mathur, D.J. Lott, C. Senesac, S.A. Germain, R.S. Vohra, H.L. Sweeney, G.
A. Walter, K. Vandenborne, Age-related differences in lower-limb muscle crosssectional area and torque production in boys with Duchenne muscular dystrophy 2,
Arch. Phys. Med. Rehabil. 91 (2010) 1051–1058.
[58] A. Mejat, V. Decostre, J. Li, L. Renou, A. Kesari, D. Hantai, C.L. Stewart, X. Xiao,
E. Hoffman, G. Bonne, T. Misteli, Lamin A/C-mediated neuromuscular junction
defects in emery-dreifuss muscular dystrophy, J. Cell Biol. 184 (2009) 31–44.
5
R.M. Lovering et al.
Neuroscience Letters 737 (2020) 135304
multiple duchenne muscular dystrophy mouse models, Eur. J. Neurosci. 43 (2016)
1623–1635.
[89] M.H. Wilson, M.R. Deschenes, The neuromuscular junction: anatomical features
and adaptations to various forms of increased, or decreased neuromuscular activity
2, Int. J. Neurosci. 115 (2005) 803–828.
[90] S.J. Wood, C.R. Slater, The contribution of postsynaptic folds to the safety factor
for neuromuscular transmission in rat fast- and slow-twitch muscles, J. Physiol.
500 (Pt 1) (1997) 165–176.
[91] S.J. Wood, C.R. Slater, Safety factor at the neuromuscular junction, Prog.
Neurobiol. 64 (2001) 393–429.
[92] R. Xu, M.M. Salpeter, Acetylcholine receptors in innervated muscles of dystrophic
mdx mice degrade as after denervation 2, J.Neurosci. 17 (1997) 8194–8200.
6