Download mechanisms regulating neuromuscular junction development and

Physiol Rev 95: 809 – 852, 2015
Published June 24, 2015; doi:10.1152/physrev.00033.2014
MECHANISMS REGULATING NEUROMUSCULAR
JUNCTION DEVELOPMENT AND FUNCTION AND
CAUSES OF MUSCLE WASTING
Lionel A. Tintignac, Hans-Rudolf Brenner, and Markus A. Rüegg
Biozentrum, University of Basel, Basel, Switzerland; Department of Biomedicine, University of Basel, Basel,
Switzerland; and INRA, UMR866 Dynamique Musculaire et Métabolisme, Montpellier, France
L
I.
II.
III.
IV.
V.
VI.
VII.
VIII.
INTRODUCTION
PHYSIOLOGY OF NEUROMUSCULAR...
DEVELOPMENT OF THE RODENT...
MOLECULES IMPORTANT FOR NMJ...
REGULATION OF SYNAPTIC AChR...
MECHANISMS OF MUSCLE WASTING
LOSS OF MUSCLE MASS IN AGING...
CONCLUSIONS AND PERSPECTIVE
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I. INTRODUCTION
The contractile activity of skeletal muscle is regulated by the
central nervous system through the transmission of action
potentials from motor neurons to muscle fibers. Transmission occurs at a highly specialized chemical synapse, the
neuromuscular junction (NMJ) or motor endplate. Accordingly, impairment of NMJ function results in muscle weakness or paralysis. Muscle disuse impairs existing, and triggers new, signaling pathways in skeletal muscle, leading
ultimately to severe muscle wasting.
Diseases that affect NMJ function are congenital myasthenic syndromes (CMS); autoimmune diseases, such as
myasthenia gravis (MG) and Lambert-Eaton myasthenic
syndrome (for review, see Refs. 124, 348); and various
forms of intoxication, such as botulism. Other neuromuscular diseases are due to the death of motor neurons and
thus loss of presynaptic input [e.g., spinal muscular atrophy
(SMA) or amyotrophic lateral sclerosis (ALS)], to impaired
myelination of the peripheral nerve [e.g., Charcot-Marie
Tooth (CMT)], or to malfunctioning of the postsynaptic
skeletal muscle fiber (e.g., muscular dystrophies). Severe
forms of all these diseases are life-threatening, but fortunately, they are rare.
This review summarizes the current view of how the NMJ
forms and is maintained, and how impairments in the neuromuscular system result in loss of muscle mass and function. The reader should keep in mind, however, that most of
the mechanistic data available on this topic stem from experiments with rodents. Thus they should be adopted with
caution to the human situation; for example, the clinical
manifestation of a defect may be different for rodents and
humans with their different loads on muscles associated
with the different postures. Furthermore, it should be noted
that we will not discuss all the pathways that have been
implicated in neuromuscular disorders by genetic experiments, as the function of many of the respective gene products remains obscure. Thus current information may not go
beyond the conclusion that those genes are “important” for
a certain aspect of NMJ development or function without
0031-9333/15 Copyright © 2015 the American Physiological Society
809
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Tintignac LA, Brenner H-R, Rüegg MA. Mechanisms Regulating Neuromuscular
Junction Development and Function and Causes of Muscle Wasting. Physiol Rev 95:
809 – 852, 2015. Published June 24, 2015; doi:10.1152/physrev.00033.2014.—
The neuromuscular junction is the chemical synapse between motor neurons and skeletal
muscle fibers. It is designed to reliably convert the action potential from the presynaptic
motor neuron into the contraction of the postsynaptic muscle fiber. Diseases that affect the neuromuscular junction may cause failure of this conversion and result in loss of ambulation and respiration.
The loss of motor input also causes muscle wasting as muscle mass is constantly adapted to contractile needs by the balancing of protein synthesis and protein degradation. Finally, neuromuscular activity
and muscle mass have a major impact on metabolic properties of the organisms. This review discusses
the mechanisms involved in the development and maintenance of the neuromuscular junction, the
consequences of and the mechanisms involved in its dysfunction, and its role in maintaining muscle
mass during aging. As life expectancy is increasing, loss of muscle mass during aging, called sarcopenia, has emerged as a field of high medical need. Interestingly, aging is also accompanied by structural
changes at the neuromuscular junction, suggesting that the mechanisms involved in neuromuscular
junction maintenance might be disturbed during aging. In addition, there is now evidence that behavioral
paradigms and signaling pathways that are involved in longevity also affect neuromuscular junction
stability and sarcopenia.
TINTIGNAC ET AL.
providing mechanistic insights. In other cases, the evidence
might solely be based on experiments in cultured cells
whose relevance in vivo remains to be examined. Because of
these uncertainties, we will largely focus on mechanisms
whose significance has been validated under physiological
conditions in vivo. The reader is thus referred to recent,
more in-depth reviews that discuss possible mechanisms
inferred from cell biological and molecular experiments on
nonmuscle cells (e.g., Refs. 44, 396, 399, 467).
mouse NMJ), which results in a local depolarization of the
muscle fiber by ⬃30 – 40 mV, called the endplate potential
(EPP). The EPP is severalfold higher than required to reach
the threshold for generating an action potential in the muscle fiber. This ratio is termed the “safety factor” of neuromuscular transmission (465).
II. PHYSIOLOGY OF NEUROMUSCULAR
TRANSMISSION
1) The size of the nerve terminal, which determines the
number of active zones.
For neuromuscular transmission of impulses, calcium influx associated with nerve action potentials in the motor
nerve terminal elicits the fusion of synaptic vesicles with the
terminal membrane at specialized sites, called active zones;
vesicle fusion releases ACh into the synaptic cleft whose
narrow width ensures its rapid (⬍1 ms) diffusion and binding to the AChRs in the subsynaptic muscle membrane to
open their ion channels. AChRs are mainly permeable to
sodium and potassium and, to a lesser extent, to calcium
ions. The ACh content of one synaptic vesicle causes a net
inward current of 3-4 nA into the muscle fiber, called quantal current or miniature endplate current (mEPC). A nerve
action potential, by causing the simultaneous fusion of tens
of vesicles, elicits a current of several hundred nA (at the
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2) The high density of voltage-gated calcium channels associated with the active zones that mediate calcium influx into
the nerve terminal upon the arrival of the action potential
and thereby trigger vesicle fusion.
3) The high concentration of ACh in a synaptic vesicle,
which was estimated in the frog to be ⬃10,000 molecules/
vesicle (239).
4) The number and density of the AChRs in the postsynaptic muscle membrane. While, in principle, the amplitude of the quantal current correlates with AChR density
in the postsynaptic membrane, the relationship is not
linear, if the AChR density is higher than needed for
efficient capture of the ACh released (which is normally
the case). As a consequence, a certain level of pharmacological blockade of AChRs reduces equilibrium currents
in response to stable, nondesensitizing concentrations of
exogenous ACh more strongly than mEPC amplitudes;
for example, blockade or removal of ⬃80% of receptors
is required to reduce the mEPC amplitude by 50% (340).
Therefore, care must be taken in linearly extrapolating
from data on receptor number and density to consequences for synaptic function.
5) The presence and depth of the postsynaptic folds with the
high density of the AChRs at their crests and the high density of voltage-gated Na channels (Nav1.4) in their troughs
(142). This geometry and the high concentration of Nav1.4
are important to ensure that the synaptic current generated
through the AChR channels at the crests reliably triggers an
action potential (401).
6) The high activity of acetylcholine esterase (AChE) associated with the synaptic portion of the muscle fibers basal
lamina, which removes ACh rapidly from the synaptic cleft.
This prevents repeated activation of individual AChR channels in response to a single action potential in the nerve.
Each of these parameters can be affected in CMS or MG
(124, 348) and lower the safety factor, leading to weakness
of muscle contraction.
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The function of the vertebrate NMJ is to transmit nerve
impulses in a 1:1 ratio from motor neurons to muscle fibers,
thus subjecting the contraction of skeletal muscle to the
control by the central nervous system. Both the presynaptic
motor axon and the postsynaptic skeletal muscle fiber are
highly specialized at the NMJ to ensure efficient transmission of action potentials (FIGURE 1). The NMJ has the characteristic structural features of other chemical synapses: the
motor nerve terminal, packed with synaptic vesicles containing the transmitter acetylcholine (ACh), is separated
from the postsynaptic muscle membrane by a narrow, 50to 80-nm-wide gap, the synaptic cleft (FIGURE 1B). The
postsynaptic membrane of the muscle fiber is deeply folded,
with the crests of the folds carrying acetylcholine receptors
(AChRs) at high density, and the troughs are equipped with
high density of voltage-gated sodium channels (142; FIGURE
1B). The muscle fibers are tightly wrapped along their entire
length by a basal lamina (also called basement membrane)
containing extracellular matrix material originating from
the muscle fibers. The basal lamina in the synaptic cleft
differs in its molecular composition from that outside the
synapse as it contains molecules secreted by both nerve and
muscle (339). The nerve terminal is capped by specialized
glial cells of the peripheral nerve, called terminal Schwann
cells. Schwann cells also produce a basal lamina, which
fuses with that of the muscle fiber at the edge of the NMJ. In
addition, the poorly characterized kranocytes form a loose
cover over the NMJ (103; FIGURE 1A).
The morphological and physiological properties of the
NMJ that contribute to this safety factor are as follows:
NEUROMUSCULAR JUNCTION DEVELOPMENT AND FUNCTION
III. DEVELOPMENT OF THE RODENT
NEUROMUSCULAR SYSTEM
solved during NMJ development. Therefore, we start out
with a description of NMJ formation and the roles of the
molecules involved. We will focus on the rodent NMJ,
since most of the concepts to be presented below are
derived from experiments using either mice or rats. While
numerous differences to human NMJs exist in their morphology and the time course of their development, molecular concepts derived from rodent NMJs have often
been validated by the identification of the same genes
being causative for acquired and congenital neuromuscular diseases in humans.
Neuromuscular transmission requires that pre- and postsynaptic components develop in tight register with one
another, implying reciprocal interactions between the
two. In this section we discuss the feedback between
motor neurons and skeletal muscle fibers with an emphasis on how presynaptic factors can affect postsynaptic
differentiation, and vice versa. The role of molecular
components for NMJ maintenance is most readily re-
A
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AChR
Nav1.4
fundamental myonuclei
non-synaptic myonuclei
mitochondria
motor neuron
myelin
terminal Schwann cell
kranocyte
B
skeletal muscle fiber
basal laminae
C
active zone
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TINTIGNAC ET AL.
A. Development of Skeletal Muscle
The formation of normal numbers of secondary myotubes
is dependent on innervation from the earliest stages of their
development (13, 175). Moreover, the expression pattern
of some muscle-specific genes is dependent on innervation
(449, 450). For example, expression of AChR subunit genes
and of distinct myosin isoforms that specify the contractile
properties of the muscle (slow versus fast twitch) is strongly
influenced by the pattern of impulse activity delivered by the
nerve (388). Thus changes in presynaptic development affect innervation, which in turn can affect the development
of the postsynaptic partner.
B. Development of the Diaphragm
Much of the work on the molecular mechanisms of synapse
formation has studied NMJ development in the rodent diaphragm. Like other muscles, diaphragm in mice develops
between E10.5 and about P10. The diaphragm forms from
progenitors that are located within the pleuroperitoneal
fold (PPF; Ref. 17). Primary fibers form between E11 to E12
(102), and as in other muscles, their formation precedes the
pioneering phrenic axons from the cervical and brachial
plexus, which arrive at the PPF at E12.5 in mice (54) and
E13.5 in rat (3, 4). The axons, which later branch out from
the main intramuscular nerve trunk, lag behind the first
After the transient, multiple innervation of muscle fibers (FIGURE 2Bb), each muscle fiber is innervated by a single motor
neuron, and the highly specialized NMJ is established with the
one-to-one match of pre- and postsynaptic specializations (FIGURE 2C; see also FIGURE 1B). The majority of muscle fibers in
the adult muscle are derived from secondary myotubes. For
example, in the adult rat diaphragm, they are the source of up
to 80% of the muscle fibers (175). Importantly, their generation and maturation are strongly influenced by innervation
and electrical activity. For example, in rat extensor digitorum
longus (EDL), secondary myotubes require the presence of the
nerve to develop their full complements at later stages (13) and
to prevent atrophy and eventual degeneration (456). Moreover, in mutants in which ACh synthesis (ChAT deficiency;
Ref. 304) or its impulse-evoked secretion (Munc-13 deficiency; Ref. 439) is lost, the muscle fibers of the diaphragm are
severely atrophic, do not align, and mature, with many myotubes still containing centralized nuclei at E18.5. Thus the
normal development of muscle requires functional innerva-
FIGURE 1. Organization of the neuromuscular system. A: motor neurons whose soma and dendrites are located in the spinal cord send their
axons to the periphery and form neuromuscular junctions to innervate skeletal muscle fibers. Axons are wrapped by myelin sheaths formed by
Schwann cells. At the site of neuromuscular contact, axons ramify into branches and form presynaptic nerve terminals that are capped by
terminal Schwann cells and covered by kranocytes. Note the accumulation of fundamental myonuclei (black) and postsynaptic folds at the site
of the nerve-muscle contact. B: high magnification of the neuromuscular junction. In addition to ACh-filled vesicles, local specializations in the
presynaptic motor nerve terminal include active zones (where vesicles fuse with the terminal membrane) and a high number of mitochondria
(brown). Postsynaptic specializations in the skeletal muscle fibers include folds that form opposite the active zones, aggregates of AChRs (red)
at the crest, and high concentrations of NaV1.4 (green) in the troughs of the folds. The localization and high concentration of AChR and NaV1.4
are important for efficient neuromuscular transmission. Three distinct basal laminae surround the neuromuscular junction: 1) basal lamina
secreted by the terminal Schwann cells (dark gray); 2) basal lamina secreted by skeletal muscle fibers (brown); and 3) synaptic basal lamina (light
blue/purple), which contains components synthesized by the motor nerve terminal and skeletal muscle fiber. C: whole mount view of the synaptic
band of a mouse diaphragm. Motor axons are labeled with antibodies to growth-associated protein 43 (GAP-43; green), and postsynaptic AChRs
are visualized by ␣-BTX (red). Scale bar ⫽ 200 ␮m.
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Muscle fibers develop from progenitor cells in the paraxial
mesoderm. In the trunk, paraxial mesoderm is segmented
into somites that lie on either side of the neural tube. Myogenesis involves two waves of precursor proliferation: the
first wave [embryonic day (E) 9.5 to E14.5 in mice] involves
muscle progenitors that proliferate in the somites and migrate to their final location (e.g., limb) where most of them
differentiate and fuse to form immature embryonic muscle
fibers, called primary myotubes. A fraction of these progenitors, however, do not fuse and give rise to fetal myoblasts
that continue to proliferate and either fuse with primary
myotubes or, using them as a scaffold, begin to fuse among
each other to form a new population of secondary myotubes between E15 and E17 (102).
wave of muscle progenitors that migrate to either end of the
diaphragm (FIGURE 2Aa). Myoblasts fuse to form primary
myotubes only after they reach the full extent of the diaphragm and are subsequently innervated by multiple motor
axons (115). Motor axons contact the developing hemidiaphragms at their midlines. Muscle progenitors destined
for secondary myotube formation proliferate largely in the
central region of the muscle, i.e., in the vicinity of the site of
contact with the motor axons (FIGURE 2Ab). The enrichment of myogenic cells in the center of the diaphragm is
reflected by a central, dense band of nuclei that express high
levels of myogenin (17), a transcription factor essential for
skeletal muscle development (177, 317). These secondary
myotubes form beneath the basal lamina of the primary
myotubes (FIGURE 2Ba). Around E14, motor nerves that
innervate the primary myotubes progressively start also to
innervate the secondary fibers (122; FIGURE 2Ba). At this
stage, the primary and the secondary myotubes are electrically coupled (FIGURE 2Ba). As the secondary myotubes
begin to elongate and separate from the primaries, they
acquire their own basal lamina (FIGURE 2Bb; Refs. 222,
335).
NEUROMUSCULAR JUNCTION DEVELOPMENT AND FUNCTION
A
a
b
B
a
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b
C
AChR clusters
basal lamina
myonuclei with high Musk expression
fundamental myonuclei
electrical activity
FIGURE 2. Development of skeletal muscle and innervation by motor neurons. A–C: overview of the different
stages of development. The red-colored area around the center of the muscle indicates regions with high
expression of myogenic factors and postsynaptic molecules, such as AChRs or MuSK. A: at early embryonic
stages (E11 to E13.5), primary myotubes are formed, which largely precedes innervation by the motor nerve.
A central zone of AChR clusters (red) without the contact to motor axons is formed. Aa: more detailed view of
the initial phase of innervation. Primary myotubes contain some AChR clusters (red) and some myonuclei (gray)
in their center express higher levels of Musk than those in the periphery (white). Ab: beginning of motor
innervation of primary myotubes. The number of myonuclei with high levels of Musk (gray) increases, and
myoblasts proliferate in the center inside of the basal laminae (brown) of the primary myotubes. The myonuclei
of the proliferating myoblasts also express high levels of Musk and other myogenic factors. B: at late embryonic
development (from E14.5 to early postnatal stages), muscle size increases by the formation of secondary
myotubes. They form in the center of the developing muscle near the site of innervation. Ba: secondary
myotubes are formed by the fusion of the proliferating myoblasts (shown in Ab) and become innervated.
Innervation initiates transcription of synaptic genes in myonuclei (black) underlying the neuromuscular
contact. Initially, the secondary myotubes are electrically coupled to the primary myotubes via gap
junctions (indicated by the indentation of their sarcolemma). The developing secondary myotubes are still
located within the basal lamina of the primary myotubes. Bb: secondary myotubes segregate from
the primary myotubes and synthesize their own basal lamina (brown). Like the primary myotubes, they become
multiply innervated, and electrical activity (flashes) leads to the restriction and condensation of AChRs to the site of
innervation. At this stage, expression of synaptic genes is suppressed in nonsynaptic and selectively stimulated in
the fundamental myonuclei. C: in mature muscle, each muscle fiber is innervated by one motor neuron.
tion. As a consequence, genetic manipulations that alter normal action potential activity in motor neurons or myotubes are
likely to affect muscle development. Therefore, the defects in
NMJ development in such mutants may be secondary to
changes in normal muscle development rather than to a direct
and specific effect on NMJ formation, making it difficult to
interpret unequivocally the neuromuscular phenotype. Given
this difficulty, it is surprising how little consideration has been
given to this fundamental interdependence between nerve and
muscle.
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C. Innervation of Muscle by Motor Neurons:
Phenomenology
1. Embryonic to perinatal stages
Motor nerves reach the diaphragm at E12.5 in the mouse
and form an intramuscular nerve trunk in the center of the
muscle. At this time, AChRs form only a few, or no AChR
clusters in the primary myotubes in the vicinity of the nerve
fibers (136, 260), but they do not colocalize with them,
similar to what has been observed in nerve-free myotube
cultures (140; see also FIGURE 2A). One to two days later,
neuromuscular contacts form in the center of the muscle, as
indicated by the close apposition of nerve terminals with
clusters of AChRs and AChE (260, 262, 471). However,
this central band (⬃200 ␮m wide) contains many AChR
clusters that are not contacted by the nerve (FIGURE 2A);
conspicuously, AChR clusters also form in mouse mutants
lacking phrenic nerves (262, 471, 472), suggesting that receptor clusters are formed by a nerve-independent patterning mechanism. Hence, these aneural AChR clusters have
been termed “prepatterned.” Between E13.5 and E15.5
(FIGURE 2, A AND B), the fraction of AChR clusters contacted by the nerve increases from ⬍10% to close to 50%
(260). By E18.5, only innervated AChR clusters remain and
have grown in size, while aneural clusters have disappeared
(FIGURE 2Bb; Refs. 262, 471). In this early phase, a single
postsynaptic AChR cluster may be contacted by up to 10
motor axons at birth (420). Importantly, the majority of
AChRs are of the “fetal” subtype at these stages. Fetal
AChR ion channels are composed of four different subunits, termed ␣, ␤, ␥, and ␦ in the stoichiometry ␣2␤␥␦; they
have an open burst duration of ⬃4 –5 ms (306).
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2. Postnatal stages
The final steps of NMJ formation are accomplished in the
first 2–3 wk after birth. During this time, multiple innervation of individual synaptic sites is reduced to a single innervating motor axon (60, 352, 400, 420). Moreover, expression of the fetal AChR channels along the fibers is largely
lost. Beginning around the time of birth, fetal AChRs at the
NMJ are gradually replaced by the adult subtype of AChRs
over a period of ⬃10 days and are further concentrated to
high density (207, 288, 373). The adult AChRs contain the
AChR ␧-subunit instead of the ␥-subunit, which gives rise to
channels consisting of ␣2␤␧␦ and results in shorter open
burst durations (⬃1 ms) but increased conductance for
Na⫹, K⫹, and Ca2⫹ (306, 441). The physiological significance of this developmental switch remains largely unknown. A plausible, but unproven role of fetal AChRs for
neuromuscular transmission in developing fibers could be
that the average transfer of electric charge per open burst is
larger and longer than for adult AChRs; as the time course
with which fetal fibers polarize in response to the injection
of step currents is longer than for postnatal fibers, spreading
the current in time leads to greater depolarization (401).
The AChR subunit switch does contribute, however, to the
restriction of the endplate band to the central muscle region,
but does not affect normal development of individual NMJs
(231).
An analogous molecular switch occurs to the NaV1 channels in the postsynaptic membrane (272). NaV1 channels
accumulate in the vicinity of immature rat NMJs at the time
of birth (18). At this time, they are mostly of the NaV1.5
(tetrodotoxin-resistant) isoform (16). During the first two
postnatal weeks, channels of the NaV1.4 (tetrodotoxin-sensitive) isoform become concentrated within the troughs of
the developing postsynaptic folds. In contrast to the situation with AChRs, a detectable level of NaV1.4 channels
persists in the extrajunctional membrane of adults to mediate the propagation of muscle fiber action potentials.
Other postnatal changes include the metabolic stabilization
of synaptic AChRs (reviewed in Refs. 2, 374; see sect. VB2);
the formation of subsynaptic infoldings of the membrane
(280, 288); the recruitment of muscle nuclei, called fundamental myonuclei (up to 6 in adult mouse muscle; Ref.
238), to the cytoplasmic region below the synaptic membrane; and an increase in size and density of synaptic AChR
clusters accompanied by changes of their shape from
plaques to pretzels (400, 407). The breaking up of a plaqueshaped synaptic AChR cluster into a pretzel of the same
area may be important for lowering the distance for which
the synaptic current needs to flow across the high electrical
resistance along the synaptic cleft, thus maintaining high
driving force for the synaptic inward current. Finally, as the
synaptic folds mature, AChRs become concentrated at their
crests (139) and Nav1.4 in their troughs (142, 410).
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Because of this problem, important concepts have also been
derived from the study of so-called ectopic endplates
formed in the adult animal. In this paradigm, proximal
motor nerve stumps are surgically placed on the extrasynaptic region of adult, i.e., fully differentiated muscle, with
motor axons subsequently growing in among the superficial
muscle fibers without making synapses. When, 2–3 wk
later, the muscle’s own nerve is cut, new, ectopic NMJs
begin to form on the previously extrasynaptic membranes
within 2.5–3 days, by a process that recapitulates all aspects
of embryonic and fetal NMJ formation (for review, see Ref.
268). This experimental model offers the advantage 1) that
the muscle fibers are fully differentiated and 2) that action
potential activity in the nerve can be controlled separately
from that in the muscle by selective pharmacological block
or by electrical stimulation via implanted electrodes. Their
respective effects on, e.g., postsynaptic differentiation or on
synapse elimination can thus be investigated in a controlled
way. An obvious disadvantage of this paradigm is that it is
limited to genetic mutations that are not lethal.
NEUROMUSCULAR JUNCTION DEVELOPMENT AND FUNCTION
Synaptic growth is functionally important for neuromuscular action potential transmission in older fibers. Because the
amplitude of the synaptic potential evoked by the quantal
current (mEPC) decreases as the fibers grow in size, muscle
fiber growth requires compensation by an increase in synaptic current to maintain impulse transmission functional.
Interestingly, consistent with the complementary roles of
synaptic fold depth and density, and of synaptic growth for
maintaining a high safety factor of neuromuscular transmission, these two parameters are inversely correlated in
different species (465).
axons to increase the speed of impulse conduction, and they
also cover the terminal arborizations of the motor axons at
the adult NMJ (see FIGURE 1A). Schwann cell precursors
formed at E12/13 from migrating neural crest cells give rise
to immature Schwann cells at E13/15, which persist until
the time of birth. Immature Schwann cells differentiate into
myelinating and nonmyelinating Schwann cells, depending
on whether they associate with large or small axons or with
nerve terminals (terminal Schwann cells), respectively. The
latter do not begin to myelinate the most distal presynaptic
nerve branches until after synapse elimination has occurred
(400).
3. Synapse elimination
4. Role of Schwann cells
Schwann cells, which originate from neural crest cells, are
the glial cells in peripheral nerves. They myelinate motor
The presence of Schwann cells is indispensable for normal
innervation of muscle: mouse mutants lacking Schwann
cells die perinatally, with defasciculating nerves showing
aberrant muscle innervation patterns (263, 312, 353, 461,
462). Acute removal and pharmacological manipulation of
terminal Schwann cells in frogs showed that terminal
Schwann cells not only promote NMJ development, but are
also required to maintain NMJ structure in the adult as well
as its pre- but not postsynaptic function. Whether this effect
is long-term (351) or acute (15, 357) remains unclear.
Terminal Schwann cells are also involved in synapse elimination. They not only destroy retracting, “losing” axon
branches (45; see sect. IIIC3), but also attack winners and
losers indiscriminately (402). They may thus be involved in
the proposed random retraction of axons initiating synapse
elimination (432), and they may drive synaptic turnover.
Finally, terminal Schwann cells play essential roles during reinnervation of NMJs after nerve injury. By extending processes from denervated synapses they guide regenerating motor axons to vacant synaptic sites; in partially
denervated muscle, Schwann cell processes extend from
denervated to nearby intact synapses where they induce
the surviving nerve terminals to sprout and grow back to
denervated synaptic sites (404, 405). For more detailed
accounts on the role of Schwann cells in NMJ development and function, the reader is referred to detailed reviews by others (110, 137, 413).
IV. MOLECULES IMPORTANT FOR NMJ
DEVELOPMENT AND FUNCTION
Developing muscles express several molecules as part of
their developmental program that are essential for synapse
formation and function and later end up in the postsynaptic
membranes. For clarity of the argument, we will group the
molecules affecting NMJ development and function into
two classes.
1) Proteins important for the reliable transmission of nerve
impulses at the NMJ. These include the subunits of the
AChR (␣,␤,␥,␦,␧), AChE, and the voltage-gated sodium
channels (Nav1.4 and Nav1.5), which convert the EPPs into
action potentials propagated along the muscle fiber.
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At birth, multiple (up to 10) motor axons connect to the
same AChR plaque in every muscle fiber (60, 352, 420); in
fact, in some muscles every motor neuron projecting to that
particular muscle contacts each of its fibers (420). Within
the first 2 wk of postnatal life, all but one of these inputs are
lost, with a single axon per synapse remaining, a process
called synapse elimination (400). For elimination, axons
converging on the same synaptic site appear to compete
with one another, and synaptic territory vacated by a loser
is filled by a prevailing competitor (445). However, both the
signals making some axons retract and those driving remaining axons to fill the vacated synaptic space are still
debated. Based on the reversibility of “losing axons” in
response to experimental ablation of prospective “winners,” some postulate that the primary withdrawal may
occur at random (432), whereas others, using reinnervation
paradigms in adult rodent muscle, have invoked synaptic
activity and its timing in the competition process (see Ref.
134). Specifically, synchronous stimulation of motor axons
converging on a synaptic site inhibits synapse elimination,
both in the ectopic innervation paradigm (72) and at
original synapses reinnervated upon nerve crush by original axons (132). Similarly, when a muscle receiving dual
innervation via separate motor nerves (thus allowing
their independent stimulation) is reinnervated upon complete denervation, elimination of supernumerary reinnervation from both nerves is inhibited if both nerves are
stimulated synchronously (132). In contrast, elimination
is promoted if the nerves are stimulated asynchronously
(133). Consistent with a similar influence of impulse timing in newborn animals, motor neurons switch from synchronous to asynchronous firing around birth (64), probably as a consequence of reduced electrical coupling
between them (341). Interestingly, it is at this stage when
synapse elimination begins. It should be noted, however,
that the molecular signals mediating axon retraction,
whether spontaneous random or activity-dependent, remain elusive.
TINTIGNAC ET AL.
2) Proteins that organize these components into a functional postsynaptic membrane. Here, we distinguish between “core proteins,” which are required for the development and maintenance of the NMJ, and “auxiliary proteins,” which contribute in different ways to full NMJ
maturation and maintenance and thus can also affect the
safety factor.
All molecules discussed are schematically represented and
functionally grouped in FIGURE 3.
A. Molecules Involved in the Neuromuscular
Transmission of Action Potentials
1. AChR
A
During neuromuscular development, the muscle AChR
subunit (Chrn) genes are first expressed when myoblasts
fuse to form myotubes. Their expression is subsequently
differentially modified by signals from the nerve both
through molecular signals and by the electrical activity elicited in the muscle fiber. AChRs are localized at the crest of
the synaptic folds (FIGURE 1B) through interactions with the
DGC (FIGURE 3B).
B
laminins
C
agrin
AChE
perlecan
Lrp4
AChR
Wnts?
Wnts
sarcoglycans
MuSK
P
Lrp5/6
αDG
MuSK
P
neuregulins
laminins
agrin
ColQ
βDG
γ
α
β δ
Nav1.4
ss
Dok7
P
P
rapsyn
frizzled
αDB
syn
ErbBs
utrophin
f-actin
FIGURE 3. Schematic presentation of molecules involved in NMJ development. A: the agrin-Lrp4-MuSKDok7 complex is essential for the formation of the NMJ. Neural agrin with the proper amino acid inserts at the
B/z site (indicated by the bulge in the most COOH-terminal domain) binds to the first YWTD repeat-containing
␤-propeller of Lrp4. Red double arrows indicate interactions mediating activation of MuSK. Other interactions
are indicated by red arrows. Sites of phosphorylation in MuSK are shown (red dots). B: schematic of the
structural components involved in the function of the NMJ. AChE is localized to the synaptic basal lamina and
is essential to inactivate acetylcholine. The homotetrameric subunits, encoded by the Ache gene, coassemble
with the triple helical collagen tail, termed ColQ, which tethers the entire enzyme to synaptic basal lamina. The
dystrophin-glycoprotein complex (DGC) contains dystroglycan (DG), which is posttranslationally cleaved into
␣-dystroglycan (␣DG) and the transmembrane component ␤-dystroglycan (␤DG), the sarcoglycans (␣ through
␦) and sarcospan (ss). DG associates with rapsyn to link AChRs to the DGC and connects to ␣-dystrobrevin
(␣DB), to ␣-syntrophin (syn) and to utrophin. Utrophin links the entire complex to the F-actin cytoskeleton. The
voltage-gated sodium channels (Nav1.4) are localized to the troughs of the synaptic cleft (see also FIGURE 1).
Interacting proteins (italics) and sites of interaction are indicated by red arrows. C: schematic presentation of
the auxiliary components including neuregulins and their ErbB receptors, and the Wnts with their receptors
Lrp5/6 and frizzled. For details on the nomenclature and the structure of the individual proteins, please see
text and previous work and reviews (44, 66, 179, 293, 487).
816
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The “core proteins” include agrin, the signal derived from
the innervating motor axon that is required for NMJ formation and maintenance, and proteins expressed in muscle
fibers, such as muscle-specific kinase (MuSK), low-density
lipoprotein receptor-related protein 4 (Lrp4), downstream
of tyrosine kinases-7 (Dok-7), and 43 kDa receptor-associated protein of the synapse (rapsyn). The “auxiliary proteins” include neuregulins, AChRs, members of the epidermal growth factor receptor (ErbB) family, Wnts, and components of the dystrophin-glycoprotein complex (DGC).
NEUROMUSCULAR JUNCTION DEVELOPMENT AND FUNCTION
In late fetal and early postnatal development, the Chrn
mRNA-rich band becomes increasingly condensed to the
sites of innervation. Condensation of the Chrn subunit
mRNAs to the synaptic sites may be the result of two simultaneous, but independent processes: the fusion of myoblasts
with secondary myotubes, which comes to its completion
around postnatal day 7 (FIGURE 2Bb), and the onset of
nerve-induced action potential activity in the muscle fibers,
which suppresses expression of ␣-, ␤-, ␦-, and ␥-Chrn subunits in extrasynaptic nuclei of the muscle fibers (160, 269).
As mentioned above, the basis of the developmental switch
in AChR subtypes from “fetal” to “adult” at the synapse is
a switch from the ␥- to the ␧-subunit, resulting in a pentameric AChR with ␣2␤␧␦ stoichiometry. In contrast to the
␣-, ␤-, ␦-, and ␥-subunit mRNAs expressed in a band
around the newly formed synapses, the ␧-subunit transcripts, from their earliest appearance just before birth, are
always tightly associated with the nuclei directly underlying
the synapse (57, 237). On a per nucleus basis, synaptic
␧-subunit mRNA levels increase strongly over a period of
several days (57, 237). Over the same time period, the ␣-,
␤-, and ␦-subunit mRNAs originally enriched in a perisynaptic band, become confined to the subsynaptic (fundamental) nuclei, where Chrn gene expression is resistant to nerveinduced muscle activity (237, 458). The fundamental nuclei
in adult muscle thus express all the subunits required to
form the adult AChR and thereby maintain functional impulse transmission in electrically active muscle. The association of activity-resistant Chrn subunit mRNA expression
underneath the synapse strongly indicates that the innervat-
ing nerve provides the signal necessary to express those
Chrn subunits in the fundamental nuclei (57, 458). Consistent with this idea, the Chrne (␧-subunit) is also expressed
by the nuclei accumulated at ectopic endplates formed upon
foreign nerve transplantation (56).
2. AChE
AChE hydrolyzes ACh released from the motor nerve terminal and thus terminates its action. It is highly enriched in
the synaptic cleft through binding to the synaptic basal
lamina and to MuSK. The elimination of ACh from the
synaptic cleft is a very rapid process. This is indicated by the
fact that the decay time constant of the mEPCs is equal to or
only slightly longer than the mean open burst duration of
the synaptic AChR channels (99). This, in turn, suggests
that following the release of an ACh quantum, AChR channels are only activated once, i.e., that ACh is hydrolyzed
before multiple AChR openings by lingering ACh can occur. Accordingly, impairment of AChE activity will tend to
increase the amplitude of the mEPSCs and to prolong their
decay time. This principle is used therapeutically to ameliorate neuromuscular disorders caused by deficiency in synaptic AChRs, such as certain forms of CMS and MG. On
the other hand, adequate AChE activity is essential for the
maintenance of normal NMJs. Sustained activation of
AChRs due to AChE deficiency causes excessive calcium
influx through AChR channels and results in the activation
of intracellular proteases such as calpains, which damages
the postsynaptic membrane (251).
AChE at the NMJ is a very large protein (FIGURE 3B) consisting of three catalytic tetramers; each AChE module is
derived from the same gene (Ache) which is expressed at
elevated levels by the subsynaptic nuclei (209). Synaptic
expression levels are further increased by action potential
activity in the muscle; specifically, at ectopic endplates denervated at early stages of development exogenous muscle
stimulation increases synaptic AChE (270).
The AChE tetramers are covalently linked in the Golgi apparatus to a noncatalytic AChE subunit, a three-stranded
collagen-like tail, called ColQ (286, 361). Together with the
synaptic expression pattern of Ache, ColQ mediates synaptic AChE enrichment both through binding to the proteoglycan perlecan, which is concentrated in the synaptic
basal lamina (10), and to MuSK (82, 398). Accordingly,
numerous mutations in the respective interaction domains
of the COLQ gene have been identified in which AChE fails
to localize to the NMJ, causing CMS with AChE deficiency
(320, 333), and antibodies directed to the site in MuSK that
binds to ColQ cause MG (221). These data clearly show the
necessity of AChE and ColQ for NMJ function, yet they
suggest that NMJs can form in their absence. Indeed, mice
deficient for ColQ are born and breathe but fail to thrive
and remain small. Death occurs at different time points in
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The genes coding for the subunits composing the fetal
AChRs (␣,␤,␥,␦) are first expressed in muscle precursors,
and their expression is elevated when myoblasts fuse to
myotubes. Transcripts encoding the AChR subunits accumulate in a central band of the developing muscle near the
future region of innervation; conspicuously, centrally elevated transcripts are also seen in mutants that lack innervation (262, 471). Transcript enrichment is in register with the
central muscle region where prepatterned, aneural AChR
clusters form. As the formation of secondary myotubes is
also initiated in the central region of the muscle (see also
sect. IIIB), the spatial distribution of the Chrn mRNAs
might be based on the high density of muscle precursors in
this region (FIGURE 2, Ab AND Ba), which express high levels
of myogenin (17), a myogenic transcription factor also required for the constitutive expression of the fetal Chrn subunit genes (39, 40, 121, 298, 300, 346). Consistent with
this, in neonatal leg muscle, Chrna1 (encoding the muscle
AChR ␣-subunit) mRNA is expressed by many nuclei in a
band around the newly formed synapses (57). Alternatively,
centrally patterned Chrn mRNA may reflect myotubes with
compartments of higher Chrn mRNA expression at their
center than at their ends, perhaps regulated through Wnt
signaling (see sect. IVB).
TINTIGNAC ET AL.
the first few weeks with a very low percentage of mice
surviving until adulthood (135).
3. Voltage-gated sodium channel (Nav1.4)
In addition to the AChRs, voltage-gated sodium channels
(FIGURE 3B) are a second class of ion channels that contribute to the safety factor of neuromuscular transmission.
Their high density at the synapse combined with their localization in the troughs of the folds (FIGURE 1B) lowers the
threshold for converting the depolarization of the EPPs into
a propagating action potential in the muscle fiber (281,
464).
B. Core Set of Proteins Required for the
Formation of the NMJ
1. Molecules expressed by the motor neuron
A) AGRIN. Agrin is the signal from motor neurons that organizes all aspects of the NMJ. Agrin is a heparan sulfate
proteoglycan expressed by cells in different organs in different isoforms generated by tissue-specific, alternative mRNA
splicing (FIGURE 3A; Ref. 44).
More than three decades ago, experiments on regenerating
frog muscle demonstrated that the synaptic portion of the
muscle fiber’s basal lamina contains biological activities
that are sufficient to trigger the formation of a new postsynaptic apparatus in regenerating muscle fibers deprived of
innervation (65, 291). Analogous experiments subsequently showed that synaptic basal lamina can also induce
818
Subsequent cloning of the cDNA encoding agrin (369, 370,
431) and generation of mice with a targeted deletion of the
Agrn gene (151) support the predictions of the agrin hypothesis (but see “myocentric model” in sect. VA). For example, agrin-deficient mice die at birth because functional
NMJs do not form (68, 151). Conversely, forced expression
of agrin or injection of recombinant agrin in nonsynaptic
regions of a muscle is sufficient to create an ectopic and fully
differentiated postsynaptic apparatus including folds that
otherwise is only found at the NMJ (42, 94, 214, 294, 355,
410), as well as the synapse-specific expression of AChR
subunit genes (214). These findings strongly support the
notion that motor axons, by secreting agrin and contacting
a muscle fiber responsive to agrin, initiate the formation of
the NMJ (neurocentric model of NMJ formation).
The Agrn gene is expressed in several isoforms as a result of
alternative mRNA splicing (138, 369). The core protein has
a predicted molecular mass of 225 kDa. Agrin is extensively
N- and O-linked glycosylated at the NH2-terminal half,
which lets the protein migrate on SDS-PAGE with an apparent molecular mass between 400 and 600 kDa (117,
430). Alternative mRNA splicing results in two different
NH2-terminal ends; one isoform is expressed as a type II
transmembrane protein (67, 69, 321), whereas the other
isoform is secreted and encodes a binding site for laminins
to allow its attachment to basal lamina (116, 218). While
the transmembrane form of agrin is mainly expressed in
neurons of the brain, motor neurons express mainly the
secreted, laminin-binding form of agrin (44, 69, 321). Alternative mRNA splicing at the 3’ end of the transcripts
generates protein isoforms that differ in the presence or
absence of two small exons that encode 8 or 11 amino acids
(138, 369). Thus splicing at this site (called B- or z-site)
results in agrin isoforms with 0, 8, 11, or 19 (8 ⫹ 11) amino
acid inserts. Importantly, isoforms lacking any amino acids
at the B/z-site do not induce AChR clustering, do not activate MuSK, and do not bind to Lrp4, while those with
amino acid inserts can exert those functions with various
potency (138, 155, 158, 227, 369, 392, 477, 487). Consistent with the agrin hypothesis, motor neurons express agrin
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Skeletal muscle fibers express two types of sodium channels,
Nav1.5 and Nav1.4, with patterns revealing differential spatiotemporal regulation (85). Nav1.5, which requires higher
concentrations of tetrodotoxin (TTX) for blockade (therefore called TTX-resistant), are expressed constitutively in
developing muscle cells, are downregulated upon innervation through the onset of muscle activity, and are reexpressed along their entire length following denervation. In
contrast, (TTX-sensitive) Nav1.4 protein increases greatly
around birth primarily at the NMJ, but once induced, its
expression pattern becomes independent of innervation.
These patterns are regulated at the level of the respective
mRNAs and are consistent with increased transcriptional
activation of the gene encoding Nav1.4 (Scn4a) from subsynaptic nuclei by the nerve (16), apparently through the
secretion of agrin (410). Unlike the localization of the
AChRs at the crests of the folds, which arises through their
interaction with the DGC (7; FIGURE 3B), that of Nav1.4
protein in the troughs may be conferred, or stabilized,
through interactions with ␤-spectrin and ankyrinG (41,
463), similar to their suspected role in localizing voltagegated sodium channels to nodes of Ranvier in myelinated
axons (36).
presynaptic differentiation in the absence of muscle (381).
The component that induces the formation of the postsynaptic apparatus was subsequently isolated from basal laminae of the electric organ of Torpedo californica, a giant
homolog of the NMJ (325, 444). Based on its AChR clustering activity, the purified protein was termed agrin (from
the Greek word “␣␥␧␫␳␧␫␯” ⫽ to accumulate). Based on its
AChR-aggregating activity on cultured myotubes and the
presence of agrin-like immunoreactivity and AChR-clustering activity in motor neurons (274), McMahan (290) formulated the “agrin hypothesis.” It states that agrin is the
motor neuron-released factor that interacts with a receptor
in the muscle membrane and thereby triggers the formation
of postsynaptic structures during NMJ development.
NEUROMUSCULAR JUNCTION DEVELOPMENT AND FUNCTION
isoforms that include the amino acid inserts at the B/z-site,
and skeletal muscle expresses the isoform lacking this insert
(192, 369, 403, 431).
2. Molecules expressed in muscle
A) MUSCLE-SPECIFIC KINASE. Muscle-specific kinase (MuSK) is a
single transmembrane receptor tyrosine kinase that is involved in all aspects of NMJ development (FIGURE 3A).
MuSK is the signaling component in the Lrp4-MuSK receptor complex necessary for triggering postsynaptic differentiation upon binding to neural agrin (227, 477). MuSK is
required for the formation of aneural AChR clusters in
embryonic myotubes (262) and for the formation of the
postsynaptic apparatus, which clusters and anchors AChRs
in the adult postsynaptic membrane (112). In addition,
MuSK can recruit the fundamental myonuclei to the synapse and induce their transcriptional specialization (127,
215). Finally, it can, at least in part, induce a retrograde
differentiation signal to the presynaptic motor axon (226).
Musk is expressed throughout muscle development, starting from early myotome (226). Upon innervation, Musk
expression is downregulated in nonsynaptic muscle nuclei,
but remains high in the fundamental nuclei, and is reinduced in nonsynaptic nuclei upon denervation (52, 437).
Thus Musk mRNA expression follows the same pattern as
Chrn subunit mRNAs. For example, Musk transcripts also
The most decisive evidence for the key role of MuSK in the
formation of the NMJ came from knockout studies in mice
(112). No AChR clusters are visible in any of the mutant
muscles, and the mice die at birth because of respiratory
failure. In addition, motor axons grow beyond the central
muscle region along the entire myotube (112, 262, 471).
Similarly, myotubes cultured from MuSK-deficient mice do
not accumulate any spontaneous AChR clusters, nor do
they respond to exogenous agrin (158).
MuSK activation is not only necessary, but also sufficient to
induce the entire postsynaptic apparatus. This has been
demonstrated by inducing dimerization and thereby selfactivation of MuSK by overexpression (347, 376), by antibody-mediated crosslinking (195, 469) or by using a
dimerization mutant in the transmembrane domain (215).
In all those conditions, activated MuSK causes the formation of AChR clusters in cultured myotubes or, when expressed in nonsynaptic regions of the innervated skeletal
muscle, it leads to the formation of nerve-free postsynapselike structures. Importantly, it also includes the accumulation of fundamental myonuclei that express genes encoding
MuSK itself, ErbBs, and AChRs (215, 308, 376).
Self-activation of MuSK by overexpression in skeletal muscle is also able to induce some synaptic differentiation of
motor axons in mice that are deficient for agrin (226). However, the mice are runted and survive only for a few weeks
(226). The effect of MuSK on the motor axons may be
indirect through the secretion of muscle factors acting retrogradely on the nerve terminal. For example, forced expression
of constitutively active MuSK induces, at nerve-free ectopic
postsynapses, the accumulation of the laminin-␤2 chain
(215), a laminin isoform that has been shown to regulate
active zone formation during nerve terminal differentiation
(327). Other components of the synaptic basal lamina that
affect nerve terminal differentiation, such as fibroblast
growth factors (FGFs) and type IV collagens (143), might be
regulated similarly.
B) LOW-DENSITY LIPOPROTEIN RECEPTOR-RELATED PROTEIN 4.
Biochemical and genetic experiments provide strong evidence
that low-density lipoprotein receptor-related protein 4
(Lrp4) associates with MuSK to form a receptor complex
necessary for the binding of agrin (FIGURE 3A; Refs. 227,
466, 475, 477, 487). Specifically, Lrp4 has been shown to
bind to agrin and to MuSK, thereby mediating tyrosine
phosphorylation of MuSK by agrin (227, 477). The existence of such a coreceptor, at the time called muscleassociated specificity component (MASC), was postulated almost two decades ago (158) as agrin, when added
to cultured myotubes, induced MuSK phosphorylation,
but did not detectably bind to the MuSK ectodomain;
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Deletion of Agrn prevents NMJ formation (151), and the
mice are unable to breathe and thus die at birth. Recent
evidence has implicated agrin also in the formation of muscle spindles (481). In early fetal diaphragm of agrin-deficient mice, a few dispersed AChR clusters are seen that are
or are not contacted by nerve fibers (262, 471). This is
similar to the AChR clusters transiently expressed in the
central diaphragm region in nerve-free mouse mutants lacking the phrenic nerve (262, 471, 472). Agrn⫺/⫺ mutants at
E18.5 also lack a central band of elevated Chrna1 mRNA,
which in wild-type mice colocalizes with the innervation
region, consistent with a direct or indirect requirement of
agrin for synapse-specific Chrn gene expression (151). Axons of the phrenic nerve in Agrn⫺/⫺ mutants sprout excessively over the entire muscle, consistent with the lack of a
stop signal provided by agrin itself (76, 77) and/or because
of the lack of a postsynaptic specialization, which presents
a retrograde signal to cause presynaptic differentiation. Interestingly, muscle-specific, transgenic expression of a miniaturized form of agrin that comprises only the NH2-terminal, laminin-binding region and the COOH-terminal region
essential for AChR clustering is sufficient to rescue the
Agrn⫺/⫺ mice (261), arguing that it is not agrin itself that
feeds back to the motor axon but a signal that is assembled
during postsynaptic differentiation. Recent data have argued that Lrp4 can serve as such retrograde signal (see sect.
IVB2).
accumulate in a prepattern (226), and it is thus likely regulated in a similar way as discussed above for Chrn subunits.
TINTIGNAC ET AL.
likewise, agrin did not induce MuSK phosphorylation in
myoblasts or (upon forced expression) in nonmuscle cells
(158).
The function of Lrp4 as the essential binding receptor for
neural agrin has been corroborated by the cocrystallization
of the first YWTD repeat-containing ␤-propeller of Lrp4
and the COOH-terminal laminin G-like domain (LG3) of
neural agrin (containing an 8-amino acid-long insert at the
B/z-site; see definitions in Ref. 44). This structure, resolved
to 2.8 Å, revealed that the 8-amino acid insert (E-L-T-N-EI-P-A) becomes highly structured in the cocrystal (487). In
contrast, the structure of amino acid insert could not be
resolved in a crystal of the LG3 domain of chick neural
agrin with even higher overall resolution of up to 1.3 Å
(408). These results therefore indicate an “induced-fit”
binding between agrin and Lrp4. The interaction interface
between the 8-amino acid insert and Lrp4 involves the middle portion of the insert (in particular asparagine and isoleucine in E-L-T-N-E-I-P-A) by forming hydrogen bonds
with amino acids in Lrp4 (487). The structure data are in
perfect agreement with previous site-directed mutagenesis
of chick agrin LG3 showing that MuSK phosphorylation in
cultured myotubes requires these amino acids (392). Most
interestingly, a construct in which all, except the middle
three amino acids of the insert, were mutated to alanine
(A-A-A-N-E-I-A-A) has the same MuSK-activating potency
as wild-type neural chick agrin (392), arguing that flanking
amino acid solely serve as spacer to allow the “induced-fit”
binding to Lrp4. The cocrystal between agrin and Lrp4 also
contains agrin-agrin and Lrp4-Lrp4 interfaces, strongly
suggesting that the functional agrin-Lrp4-MuSK complex is
a dimer (FIGURE 3A).
At the NMJ, Lrp4 is expressed not only in skeletal muscle
fibers but also in motor neurons, and this differential expression affects NMJ development in a distinct way. Selective deletion of Lrp4 in both skeletal muscle and motor
820
The fact that mice deficient for Lrp4 in skeletal muscle show
a presynaptic phenotype suggested that Lrp4 may also act
as a retrograde signal for the motor axon. Indeed, presentation of Lrp4 to motor neurons in culture (either by forced
expression of Lrp4 in nonmuscle cells or by immobilizing
recombinant Lrp4 on polystyrene beads) is sufficient to induce differentiation of motor neuron growth cones into a
presynapse-like structure (466, 475). Although those observations are intriguing, it is important to note that
agrin and several additional components of the synaptic
basal lamina membrane also induce the formation of
presynapse-like structures in culture when presented on
the surface of nonmuscle cells or when immobilized in the
substrate (76, 77, 143, 199). Moreover, at the adult NMJ
of the frog, synaptic basal lamina is alone sufficient to
induce presynaptic differentiation (381). Thus it remains
to be examined whether the Lrp4 ectodomain is found in
synaptic basal lamina.
C) DOWNSTREAM OF TYROSINE KINASES-7. Downstream of tyrosine kinases-7 (Dok-7) belongs to a family of seven adaptor-like proteins (Dok-1 to Dok-7) that are characterized by
NH2-terminal pleckstrin homology (PH) and phosphotyrosine-binding (PTB) domains followed by src homology 2
(SH2) target motifs (284). Several lines of evidence indicate
that the Dok family members act as promiscuous cytosolic
adapter molecules for tyrosine kinases. In some cases, binding of the Dok protein inhibits tyrosine kinase activation,
whereas in others (as is the case for Dok-7), binding
strongly enhances tyrosine kinase activity (284). Similar to
the discovery of Lrp4 being the coreceptor of MuSK, the
identification of Dok-7 as an adapter necessary for the full
activation of MuSK was based on the observation that mice
deficient for Dok-7 die perinatally because of respiratory
failure due to the lack of NMJs (334). Similar to agrin-,
MuSK-, or Lrp4-deficient mice, motor axons overshoot and
grow over the entire muscle fiber. In contrast to Lrp4, expression of Dok-7 is confined to skeletal and heart muscle
(334), and thus the lack of Dok-7 may explain the failure of
agrin to activate MuSK in nonmuscle cells (158).
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The identification of Lrp4 as the agrin-MuSK coreceptor
was largely guided by the serendipitous finding that mouse
null mutants of Lrp4, generated chemically by using Nethyl-N-nitrosourea (ENU), showed a NMJ phenotype
with no AChR clusters and a failure of motor axons to stop
at the presumptive synaptic band (451). In addition, cultured myotubes from Lrp4-deficient mice do not form
AChR clusters upon addition of agrin. Thus Lrp4 deficiency
largely phenocopies the neuromuscular defects observed in
MuSK- and agrin-deficient mice. Biochemical studies later
showed that 1) Lrp4, when coexpressed with MuSK in heterologous cells, confers agrin-induced MuSK phosphorylation; 2) the ectodomain of Lrp4 binds to the agrin isoforms
expressed by motor neurons (neural agrin) but not to those
expressed by nonneuronal cells (see details in sect. IVB1);
and 3) Lrp4 binds to MuSK and this binding is enhanced by
agrin (227, 477, 487).
neurons results in the complete abrogation of NMJ formation as observed after germline deletion of Lrp4 (466). In
contrast, deletion of Lrp4 selectively in motor neurons does
not result in any neuromuscular phenotype, and deletion in
skeletal muscle alone still allows for the formation of NMJs
so that the mice survive for weeks (466). These results are
consistent with a model where the extracellular domain of
Lrp4 is released from the cell surface of motor neurons
and/or skeletal muscle fibers to form a functional agrinLrp4-MuSK complex (see FIGURE 3A ). Indeed, Lrp4
knock-in mice that express only the extracellular domain of
Lrp4 are indistinguishable from wild-type mice (118, 164),
suggesting that transmembrane and intracellular domains
of Lrp4 are not important.
NEUROMUSCULAR JUNCTION DEVELOPMENT AND FUNCTION
Another rather unusual aspect of Dok-7 signaling is the
finding that forced overexpression of Dok-7 is sufficient to
form AChR clusters in cultured myotubes and to allow mice
to survive for several weeks in the absence of agrin (334,
422). Similarly, expression of Dok-7 via AAV vectors in
skeletal muscles enlarges the NMJ and thereby ameliorates
the disease in a mouse model for Emery-Dreifuss muscular
dystrophy (11). These findings show that Dok-7 can also
mediate inside-out signaling, a phenomenon that is well
documented for integrins, a family of heterodimeric (with
one ␣ and one ␤ subunit) receptors that bind to a wide
variety of extracellular matrix proteins (278). Interestingly,
the Dok-7 homolog Dok-1 binds to the NPXY motif of
integrin ␤-subunits and inhibits the inside-out activation of
the integrins (6). Thus MuSK and integrin activation could
be a result of convergent evolution.
D) 43 KDA RECEPTOR-ASSOCIATED PROTEIN OF THE SYNAPSE
(RAPSYN). Rapsyn is a cytoplasmic scaffolding protein expressed constitutively in myotubes; it is present at the NMJ
from the earliest stages of development, and in adult muscle,
expression is largely restricted to the synaptic region. Rapsyn
binds tightly to AChRs to form a high-density network of the
two proteins (434). Rapsyn also binds to dystroglycan (26)
and is thus thought to link AChRs to the postsynaptic actin
cytoskeleton (FIGURE 3B). The molecular signaling pathways
that lead from MuSK activation to the clustering of the AChRrapsyn network are not well understood.
Rapsyn is required for AChR cluster formation in cultured
myotubes and for NMJ formation in vivo. Accordingly,
mice deficient for Rapsn fail to form postsynaptic specializations (152). Interestingly, Rapsn knockout mice have a
less severe phenotype than MuSK-, Lrp4-, or Dok-7-deficient mice as they are able to breathe weakly and therefore
survive for a few hours after birth. Examination of the
neuromuscular contact sites revealed lack of any discrete
AChR clusters and cytoskeletal specializations. However,
components of the synaptic basal lamina, such as AChE and
laminin-␤2, still accumulate at the synapse. Interestingly,
MuSK is still aggregated underneath the nerve terminal (8),
and Chrna1 mRNA is highly expressed in the fundamental
myonuclei (152). These experiments show that rapsyn is
essential for the tethering of AChRs to the postsynaptic
apparatus but not for synaptic gene transcription; furthermore, they imply that at early postnatal stages even nonclustered AChRs at the neural contact site are sufficient to
support some low-level muscle contraction.
E) ACHR.
In addition to its role in neuromuscular transmission, AChRs play an active role in the assembly of the
postsynaptic apparatus. Specifically, unlike what has been
described for nonmuscle cells where rapsyn becomes clustered when overexpressed (e.g., Ref. 343), the formation of
such clusters in muscle cells requires AChRs. For example,
antibody-mediated depletion of AChRs from the cell surface
of cultured myotubes or low-power laser-induced removal of
AChRs results in the concomitant loss of rapsyn clusters (62,
276). Moreover, rapsyn has been found to assemble with
AChRs already in the secretory pathway (277, 310). The active role of AChRs in the formation of the postsynaptic scaffold is also supported by the finding that deletion of the gene
encoding the AChR ␥-subunit (Chrng), which largely prevents
the assembly of functional AChRs during embryogenesis, results in continued growth of motor axons beyond the synaptic
band and in aberrant nerve branching (264). Interestingly,
MuSK is still clustered in the middle of the diaphragm in those
mice, whereas rapsyn remains unclustered. In summary,
AChRs seem to contribute to postsynaptic differentiation, and
functional AChRs are important for the delivery of rapsyn to
the postsynaptic apparatus.
C. Auxiliary Proteins That Contribute to
NMJ Formation
1. The dystrophin-glycoprotein complex
The structural specializations of the postsynaptic membrane, including its high AChR density, are maintained by
the subsynaptic apparatus, an elaborate, specialized network of the cortical actin cytoskeleton and comprising, in
addition to MuSK and rapsyn, the dystrophin-glycoprotein
complex (DGC; FIGURE 3B). The DGC is a transmembrane
complex of proteins linking the actin cytoskeleton of the
muscle fiber to the basal lamina. It includes dystrophin or its
homolog at the synapse, utrophin, linked to cytoskeletal
actin, three groups of transmembrane proteins dystroglycan (DG), sarcoglycans and sarcospan, and two groups of
cytoplasmic proteins, the dystrobrevins and syntrophins
(399). In nonsynaptic muscle regions, the DGC is required
to prevent muscle fiber damage caused by contraction, as
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The binding of Dok-7 to the cytoplasmic region of MuSK
resides in the NPXY sequence motif at the juxtamembrane
region whose phosphorylation at the tyrosine residue is necessary and sufficient for MuSK activity (FIGURE 3A; Refs.
184, 185). Cultured myotubes from Dok-7 knockout mice
do not form AChR clusters upon addition of agrin, nor is
MuSK activated, indicating that Dok-7 is required for
MuSK function. Activation of Lrp4-MuSK by agrin also
causes phosphorylation of Dok-7, which in turn, creates
binding sites for v-crk sarcoma virus CT10 oncogene homolog (Crk) and Crk-like (Crk-L) (172). Skeletal musclespecific deletion of Crk and Crk-L results in smaller NMJs
and aberrant innervation and mice die around birth (172).
Crk and Crk-L are widely expressed and interact with many
intracellular kinases, including c-abl oncogene 1 (Abl).
Moreover, germline deletion of Crk alone is embryonic lethal mainly because of vascular issues resulting in bleeding
and malformations of the heart (337). Thus it remains an
open question of how specific the interaction of Crk and
Crk-L is for NMJ signaling.
TINTIGNAC ET AL.
shown in muscular dystrophies, which often result from
mutations in components of the DGC (111). Dystroglycan
is posttranslationally cleaved into the transmembrane
␤-dystroglycan and the extracellular, sarcolemma-associated ␣-dystroglycan (202). ␣-Dystroglycan is heavily glycosylated, and this glycosylation is essential for the binding to
its ligands agrin, laminins, perlecan, neurexin, and pikachurin (51, 126, 385, 412, 416). Accordingly, mutations of the
enzymes that are involved in the glycosylation of these unusual sugar side chains cause underglycosylation of DG and
can result in very severe, early-onset congenital muscular
dystrophies (309).
2. Neuregulins
The neuregulins (Nrgs) (FIGURE 3C) have a domain in common with epidermal growth factor (EGF). There are six
closely related neuregulin genes Nrg1 to Nrg6 (293). Isoforms of Nrg-1 and Nrg-2 are expressed by motor neurons,
muscle cells, and/or terminal Schwann cells and are recruited to the postsynaptic muscle membrane (295, 313,
355, 356), but their functions in NMJ development in vivo
are poorly understood.
Nrgs activate the receptor tyrosine kinases ErbB2, ErbB3,
and ErbB4, all of which are present at the NMJ (see below).
822
3. ErbB receptors
The founding member of the ErbB family of receptors is the
epidermal growth factor receptor (EGFR; also called
ErbB1). ErbB proteins are receptor tyrosine kinases comprising ErbB2, ErbB3, and ErbB4 isoforms, which are encoded by distinct genes. They share high similarity with
the EGF receptor and are stimulated by Nrgs through
dimerization. However, ErbB2 lacks the binding site for
Nrg-1, and ErbB3 lacks a kinase domain; thus signaling
occurs through homo- (ErbB4) or heterodimerization in the
combinations ErbB2/3, ErbB2/4, or ErbB4/EGFR (452).
Dimerization leads to auto- and trans-phosphorylation of
tyrosine residues in their intracellular domains, which serve
as docking sites for adaptor proteins and enzymes initiating
downstream signaling cascades, including the Raf-MEKERK and JNK pathways.
ErbB2, -3, and -4 are also expressed in the postsynaptic
muscle membrane (429). Like Musk, Chrn subunits, and
Rapsn, Erbb2 and -3 are expressed at increased levels from
fundamental nuclei in response to MuSK activation (308).
Selective depletion of ErbB2 in skeletal muscle destabilizes
synaptic AChRs and lowers their density in the postsynaptic
muscle membrane, apparently through impairment of
␣-dystrobrevin function (389; see also below). Also, it impairs normal muscle spindle development (252). In contrast, germline deletion of Erbb2 in mice, in which the malformation of the heart was prevented by transgenic expression of Erbb2, causes aberrant development of motor axons
and NMJs, apparently through impairment of normal
Schwann cell development (263, 461; see also sect. IIIC4).
4. Wnts
Wnts are secreted glycoproteins expressed as 15 isoforms in
zebrafish and as 19 isoforms in mouse and humans. They
are involved in multiple aspects of development (179, 438),
including axon pathfinding and synaptogenesis. Their function during the formation of the NMJ has been best de-
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At the NMJ, the function of ␣-dystroglycan as a binding
receptor for agrin is not well established. Initial reports that
suggested ␣-dystroglycan being the agrin receptor important for AChR aggregation (51, 78, 153) proved to be incorrect as ␣-dystroglycan binds to the agrin isoforms that
lack amino acid inserts at the B/z site much more strongly
than those that carry an insert at this site (155, 414). Moreover, the agrin deletion fragment that is sufficient to induce
AChR aggregation (156) and rescues the NMJ phenotype of
agrin knockout mice (261) does not bind to ␣-dystroglycan
at all (155). However, recent genetic manipulations of the
extent of glycosylation of ␣-dystroglycan have shown that
underglycosylation causes fragmentation of the NMJ (159,
174), indicating that dystroglycan has a stabilizing role for
the NMJ. In addition, deletion of intracellular components
of the DGC impairs postnatal (but not neonatal) synaptic
development, including AChR stability (AChR half-life),
AChR density, and fold formation (2, 167). For example, deletion of ␣-dystrobrevin or synthrophin, in addition to impairing NMJ structure, reduces AChR density and lowers AChR
half-life by reducing AChR recycling (see sect. VB2; Refs. 165,
282). Similar, although weaker, effects are generated by mutations in ␣-dystrobrevin, which prevent COOH-terminal tyrosine phosphorylation (165), or in ␣-syntrophin (282). A severe fragmentation and loss of the NMJ is only observed in
triple mutant mice that lack utrophin, dystrophin, and ␣-dystrobrevin (167), whereas single deletions have only mild effects (113, 166, 167, 273). Thus it is difficult to attribute
specific functions to ␣-dystrobrevin, dystroglycan, or syntrophin for the stability of the NMJ.
Among the Nrg-1 isoforms, Nrg-1␤, originally identified as
acetylcholine receptor inducing activity (ARIA), was isolated from chicken brain based on its ability to increase
expression of Chrna1 mRNA in cultured chick myotubes
(130, 211). It is expressed in motor neurons and was proposed to be the motor neuron released factor to control expression of Chrn subunit genes in fundamental myonuclei (see
sect. VB1). Deletion of Nrg1 is lethal due to heart malformation (302). However, while Nrg1 mutants can be rescued by
transgenic expression of Nrg-1 in the heart, neuromuscular
development remains impaired, apparently through impairment of Schwann cell development and survival (250, 428; see
also sect. IIIC4) rather than through impaired signaling in
skeletal muscle (127). However, Nrg1 deletion abolishes normal muscle spindle development (191).
NEUROMUSCULAR JUNCTION DEVELOPMENT AND FUNCTION
scribed in invertebrates (Drosophila melanogaster and Caenorhabditis elegans), and involvement of Wnt signaling at
the vertebrate NMJ has also been revealed (reviewed in
Refs. 24, 232). Wnts in mammals signal through 11 different Frizzled (Fzd) receptors to regulate, via at least five
different transduction pathways involving Dishevelled
(Dvl), various cellular processes (FIGURE 3C).
The mechanisms by which Wnt signaling affects prepatterning at rodent NMJs is far from understood. In addition,
given the involvement of Wnts in myogenic proliferation
and differentiation (443), Wnts may also affect the innervation pattern indirectly by altering muscle differentiation
(see sect. IIIB). For example, Wnts might affect the elongation of secondary myotubes in developing muscle, which
according to the model presented in FIGURE 2 (see sects. IIC
and IVA), would affect the width of the prepatterned muscle region. Alternatively, Wnts might affect expression of
molecules involved in synapse formation, such as MuSK
(224) or rapsyn (447), thus modulating agrin responsiveness. Experiments in mouse diaphragm are consistent with
several of these possibilities. For example, genetic deletion
of Wnt4 widens the central band of prepatterned AChR
clusters in developing muscle, reduces their numbers, and
causes nerve overgrowth (411); a function similar to that
described for Wnt11r in zebrafish (212).
A role of Wnts in NMJ formation and maintenance is further
corroborated by the findings that components of the Wnt
pathway, as for example Dvl1, bind to the intracellular domain of MuSK (271) and that deletion of the Wnt components
␤-catenin (253) or adenomatous polyposis coli (APC; Ref.
446) perturb aspects of NMJ formation in rodents. In addition, mice deficient for the gene encoding caspase-3, which
cleaves Dvl1, show a transient increase in the number of aneural AChR clusters in the developing mouse diaphragm
(448). However, the mice do not show any major NMJ phenotype postnatally, arguing that the interaction of MuSK with
Dvl1 is of minor importance for NMJ function. Finally and
most importantly, knock-in mice in which full-length MuSK
is replaced by a mutant that lacks the CRD domain
(MuSK⌬CRD) show an aberrant prepatterning during development and exert a CMS-like phenotype including weakness
and fatigability (301). In summary, Wnts appear to have a role
at the NMJ, and some of those effects seem to be mediated by
their interaction with MuSK. However, the detailed mechanisms involved are still not understood; one difficulty in resolving them could be the redundancy and promiscuity of the
different Wnt isoforms.
V. REGULATION OF SYNAPTIC AChR
CLUSTER FORMATION
A. Regulation of AChR Cluster Formation in
Fetal Muscle
As mentioned above, starting at E12, constitutively expressed AChRs begin to cluster in a central band of fetal
diaphragm independently of innervation (262, 471, 472).
Aneural AChR cluster formation is dependent on the expression of MuSK and of some signaling molecules down-
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The strongest evidence for the physiological function of
Wnt signaling in the neuromuscular system in vivo stems
from work in zebrafish, which indicates a role in the patterning of muscle innervation. As described above (sect.
IIIC1; FIGURE 2), AChRs cluster in the presumptive innervation zone in the center of the developing muscle independently and prior to the appearance of motor axons. In
developing zebrafish, deletion of the unplugged gene, the
ortholog of mammalian Musk, abolishes AChR prepatterning, impairs axon guidance, possibly due to modifications in
the extracellular matrix, and NMJ formation (391, 480).
Deletion of SV1, which is one of the three unplugged splice
variants that lacks the Ig domains required for MuSK to be
phosphorylated by agrin (484), abolished AChR prepatterning and results in aberrant axon branching of the motor
neurons (480). Interestingly, however, the formation of
NMJs is still possible when expression of full-length unplugged is again restored after the initial loss of the prepattern (212). Thus prepatterned AChRs are dispensable for
NMJ formation, consistent with the observation in mouse
diaphragm that NMJs can also form outside the zone of
prepatterned AChR clusters (260). The guidance of motor
axons to the central muscle zone and the prepatterning of
AChRs acts in zebrafish via the binding of unplugged/
MuSK to Wnt11r (212). Taken together, these findings indicate that unplugged/Musk, activated by Wnt11r, confines
motor axon outgrowth and promotes AChR prepatterning
in the central muscle region. This region, in mouse diaphragm, is also most sensitive to agrin (260).
Experiments aimed at better understanding the molecular
mechanisms of how Wnts affect prepattening have focused
on 1) testing binding of different Wnts to MuSK and 2)
examining their effect on AChR clustering in cultured myotubes. These experiments, while failing to yield a conclusive
picture, have indeed provided evidence that several Wnts bind
to MuSK and affect AChR clustering. For example, most Wnts
tested bind to the extracellular, frizzled-related, cysteine-rich
domain (CRD) of MuSK (411, 476; see also FIGURE 3A), a site
that is distinct from the Ig domains, which have been implicated in mediating agrin-induced activation of MuSK (484).
Moreover, autoantibodies directed towards MuSK that cause
MG can also be directed to the CRD domain of MuSK (415).
Finally, AChR clustering assays on cultured myotubes indicate
that Wnts promote MuSK phosphorylation in the absence of
agrin (411, 476) or enhance AChR clustering induced by agrin
(183). The Wnts implicated in those activities are Wnt-3,
Wnt-4, Wnt-9a, and Wnt-11 (24). However, other Wnts have
recently been shown to inhibit agrin-induced AChR clustering
in culture (24).
TINTIGNAC ET AL.
stream of MuSK, such as Dok-7. Given that MuSK and
Dok-7 can induce AChR clustering in the absence of agrin
by forced overexpression, the prepatterning of AChRs may
reflect autoactivation of MuSK signaling in the oldest, central region of the developing muscle.
While the two models each represent extreme cases, the true
mechanism of how NMJs form may in fact include both
views. For example, in zebrafish larval muscle, where NMJ
formation can be observed in real time, a majority of motor
axons grow and extend their filopodia preferentially towards preexisting AChR clusters, where they begin to accumulate nerve-terminal-specific material, as predicted by
the myocentric model (336). In contrast, other axons induce
new AChR clusters and may also incorporate preexisting
AChR clusters (141). Interestingly, the formation of presynaptic specialization in the central region of the muscle was
still observed when AChRs were blocked by ␣-bungarotoxin (␣-BTX) or when the formation of AChR clusters was
abolished by genetic ablation of the ␦-AChR subunit (336).
Thus muscle-derived cues that are colocalized with, but do
not represent, the prepatterned AChR clusters themselves
determine motor axon outgrowth, branching, and initial
neuromuscular synaptogenesis. Support of the neurocentric
model is provided by observations in mouse diaphragm and
in zebrafish (212, 260) where NMJs also form outside of the
central band of prepatterned AChR clusters. Furthermore,
although denervated adult muscle fibers lack extrajunctional AChR clusters, they are receptive to ectopic innervation by transplanted foreign nerves (see sect. III). In summary, prepatterned AChR clusters may represent a zone
with a high expression of MuSK and other synaptic genes
(226) to render this region highly sensitive to agrin signaling
(260) and thereby delineate a region of preferred innervation. Consistent with this view, agrin alone is not sufficient
to induce ectopic postsynaptic structures in muscles expressing low extrasynaptic levels of MuSK (347).
Once the motor axon has established an initial contact with
postsynaptic AChR clusters, aneural clusters are eliminated. Several lines of evidence indicate that ACh is the
824
An alternative interpretation of the genetic data from
ChAT/agrin double mutants would be that the lack of ACh,
by blocking neuromuscular transmission, impairs muscle
development and upregulates synaptic genes, such as AChR
and MuSK, thus increasing the agrin responsiveness of muscle regions outside the normal endplate band. Indeed, at
E12.5, representing the time when nerves make first contacts with the diaphragm, the phrenic nerve of ChAT⫺/⫺
mice already shows exuberant branching compared with
control mice (54). As development proceeds, both aneural
and nerve-associated AChR clusters are observed, but the
“synaptic” band is much wider and the number of AChR
clusters is higher than in control mice (54, 304). Moreover,
ChAT deficiency also affects motor neuron survival (many
more motor neurons are detected in the spinal cord) and
myogenesis (muscle remains much thinner), consistent with
the observation that in culture, ACh expressed by muscle
cells is required for fusion of myoblasts to form myotubes
(125, 236).
B. Stabilization of NMJs in Postnatal and
Adult Muscle
The fact that several synaptic components remain expressed
in the fundamental myonuclei after the assembly of the
NMJ while they are gradually downregulated in extrasynaptic regions by electrical muscle activity implies that their
synapse-specific expression is maintained in an activity-resistant manner by signals from the nerve. Indeed, in situ
hybridization shows that mRNA levels encoding these and
several other components are higher at the NMJ than outside. The mechanisms important for the maintenance of
expression in the fundamental nuclei have been most extensively studied for the Chrn subunit genes.
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It has been proposed that the muscle-autonomous, aneural
AChR clusters serve as potential platforms for the nerve to
form NMJs (241). In this model, secretion of agrin by the
nerve selectively stabilizes the preexisting AChR clusters it
contacts while AChR clusters remaining without neural
contact would be dispersed through the secretion of ACh
from the nerve terminals (241). Importantly, in this model,
it would be the muscle, through the generation of prepatterned AChR clusters, that determines the exact location for
a new NMJ to be formed; hence, this model is known as the
myocentric model of NMJ formation. The myocentric
model thus abandons the agrin hypothesis where the nerve,
by secretion of agrin, determines the site of a synapse (neurocentric model; Ref. 290).
factor that causes the dispersal of such aneural AChRs.
Such role of ACh as a signal for the dispersal of aneural
AChR clusters was invoked from genetic experiments with
agrin and choline acetyltransferase (ChAT) single and double mutants. Specifically, concomitant removal of ACh
(through ChAT-knockout) and of agrin synthesis in vivo
increases the total number, size, and percentage of innervated AChR clusters in embryonic diaphragm compared
with ablation of Agrn alone, and the endplate band width is
increased (305). Consistent with a dispersing action of
ACh, cholinergic agonists reduce the number of spontaneous AChR clusters in cultured myotubes (46), at least in
part by promoting AChR endocytosis (406). However, for
the nerve to eliminate distant, aneural AChR clusters
through the secretion of ACh, it seems unlikely that the
ACh concentrations required for ACh-mediated dispersal
(⬃10 ␮M) in cultured muscle cells would be reached at
aneural AChR clusters over the entire central band, i.e., tens
of microns away.
NEUROMUSCULAR JUNCTION DEVELOPMENT AND FUNCTION
1. Regulation of Chrn gene expression at the
synapse
Several molecules from the nerve have been implicated in
mediating the neural regulation of synapse-specific Chrn
and Musk gene expression in fundamental nuclei. Among
them, Nrg-1␤ (ARIA; Refs. 130, 211) and agrin have commanded most interest.
Instead, synaptic genes appear to be regulated by agrin.
Specifically, ectopic expression of agrin or of a constitutively active form of MuSK in muscle in vivo induces ectopic, nerve-free postsynapses, including the transcription of
the ␧-subunit of the Chrn and of Musk genes; this occurs
even in the absence of Nrg/ErbB signaling (127, 242), indicating that agrin-MuSK signaling not only triggers aggregation of AChRs to the site of nerve-muscle contact but also
directs the transcription of genes encoding AChRs and, in a
positive-feedback loop, the gene encoding MuSK (308).
How is agrin-induced transcription of these genes mediated? Several of the synaptic genes, including Chrne, Chrnd,
Ache, utr (utrophin), and Musk, share a regulatory, sixbase-pair-long sequence, termed N-box, that targets their
expression to subsynaptic nuclei (see review in Ref. 386;
FIGURE 4A). Specifically, when introduced in vivo into muscle fibers in the context of the respective promoter-reporter
fragments, N-boxes confer reporter expression to the synaptic region, but with lower spatial specificity compared
with the expression of the endogenous gene product; single
point mutations of the N-box abolish synapse-specific transcription, but not upregulation in extrasynaptic nuclei upon
denervation (144). Combined, these data strongly suggest
that the N-box is required for synapse-specific transcription, perhaps in cooperation with other promoter sites. Independent confirmation of its physiological relevance
comes from patients who suffer from severe AChR deficiency (a form of congenital myasthenia) that is associated
with point mutations in the N-box of the Chrne promoter
(322, 332).
The pathways linking agrin-Lrp4-MuSK and N-box-dependent activation of synaptic genes remain uncertain. Factors
proposed to mediate the transcriptional response of Chrn
and Musk genes from subsynaptic nuclei include GA-binding protein GABP␣/GABP␤ dimers (58, 144, 387) and Erm
In summary, the signaling pathways discussed above maintain expression of Chrn subunit genes from the fundamental nuclei at the synapse against their repression by electrical
muscle activity, as occurs in extrasynaptic nuclei. The latter
is mediated by the increase in cytosolic calcium through the
activation of L-type calcium channels associated with muscle stimulation. For details of activity signaling in Chrn
subunit expression, the reader is referred to the review by
Sanes and Lichtman (380).
2. Regulation of synaptic AChR density through AChR
insertion and removal
In fetal muscle AChRs become clustered by agrin-Lrp4MuSK signaling (154, 296). Experiments examining AChR
clustering induced by the addition of neural agrin in cultured muscle cells indicate that synaptic components (including AChRs, rapsyn, and MuSK) are delivered to the cell
surface via exocytic vesicles (277). While AChR, MuSK,
and rapsyn appear to be constitutively associated with lipid
rafts, stimulation with agrin and subsequent activation of
MuSK results in their coalescence first into AChR microclusters and then into larger clusters (75, 338, 485). As a
consequence, disruption of lipid rafts inhibits agrin-induced
AChR clustering. Microcluster coalescence is thought to be
driven by actin-based raft movements promoted by local
actin polymerization induced in turn through agrin-Lrp4MuSK-induced activation of small GTPases Rac, Cdc42,
and Rho (455) as well as the actin nucleation factors NWASP and the Arp2/3 complex (81). Indeed, Rac is involved in the formation of AChR microclusters, whereas
Rho seems important for their coalescence to large aggregates (454). Finally, recent studies described Coronin 6 as
being associated in AChR clusters and mediating their association with the actin cytoskeleton (87). Although in vivo
knockdown of Coronin 6 by electroporation of shRNA
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Nrg-1␤ is present at the NMJ as well as at agrin-induced
ectopic postsynaptic membranes (295, 354), implicating its
recruitment also from muscle. Its receptors, the ErbBs, are
enriched in the postsynaptic muscle membrane (486). However, in adult mice in which Nrg signaling to muscle was
abolished by muscle-selective deletion of ErbB2 and ErbB4
receptors, the NMJs are only marginally affected, and synaptic transcription of the Chrne, Chrnd, and Musk genes at
NMJs is grossly normal (127, 242). Thus Nrgs are dispensable for synapse-specific Chrn gene transcription in vivo.
(190), both members of the Ets family of transcription factors (FIGURE 4A). Of these, GABP␣/GABP␤ indeed binds to
the N-box upon Nrg-1-induced phosphorylation of GABP␣
in cultured muscle cells (144), and forced expression of a
dominant-negative mutant of GABP␤ in muscle fibers inhibits agrin-induced postsynaptic differentiation in vivo
(58). However, disruption of GABP␣ in vivo does not or
only marginally affects NMJ function, consistent with
GABP␤ binding to the N-box, but arguing against a physiological role in synapse-specific transcription (210, 328). In
contrast, deletion of the Ets transcription factor Erm
strongly impairs normal NMJ formation and function in
vivo, and is associated with downregulation of many synaptic genes; however, binding of Erm to the N-box sequences has not been tested directly, and its disruption is
not lethal, indicating that Erm boosts, but does not regulate
synaptic genes in an all-or-none manner (190). Finally, experiments in cultured muscle cells suggest that agrin-Lrp4MuSK signaling can activate N-box-dependent gene expression via ERK and JNK pathways (242).
TINTIGNAC ET AL.
A
innervated
agrin
Lrp4-MuSK
?
}
Erm
GABPα/GABPβ
N
Chrn
N
Musk
B
denervated
initial response
HDAC4/5
Trim63
Dach2
Fbxo32
Myog
HDAC9
E
Chrn
C
denervated
prolonged response
Trim63
Fbxo32
P-Akt
mTORC1
protein
synthesis
proteasomal
degradation
P-FoxO
S6K
P-Ulk1
autophagy
FIGURE 4. Molecular mechanisms involved in muscle atrophy triggered by denervation. A: action potential
activity (red flashes) in innervated muscles suppresses synaptic genes in nonsynaptic myonuclei (white), while
their expression is maintained in the fundamental nuclei (black) by agrin-Lrp4-MuSK signaling and the subsequent activation of transcription factors Erm and GABP␣/GABP␤, which drive expression of AChR-encoding
genes (Chrn) and Musk (see text for details). B: denervation results in the immediate loss of electrical activity
and strong upregulation of Hdac4 and Hdac5 and downregulation of Hdac9. HDAC4, HDAC5, and HDAC9
induce, via Dach2 and Myog, expression of the E3 ligases Trim63 and Fbxo32 and increase proteasomal
degradation. In addition, AChR levels, including expression of Chrng, are increased in nonsynaptic regions. C:
during prolonged denervation, mTORC1 becomes activated and causes an increase in protein synthesis.
Feedback inhibition of phosphorylation of Akt/PKB (p-Akt) by S6K enhances expression of Trim63 and
Fbxo32 via FoxO transcription factors. mTORC1 also inhibits autophagy via phosphorylating Ulk1. See text
for details.
results in the fragmentation of NMJs, it remains to be
shown how Coronin 6 mediates the interaction with the
actin cytoskeleton and whether there is any connection to
the DGC, which has also been implicated in the linkage of
AChRs to the actin cytoskeleton (see sect. IVC). In summary, these data indicate that lipid rafts may serve as signaling platforms driving the formation and growth of the
synaptic AChR clusters in the initial phase of NMJ development.
In adult muscle, AChRs are highly concentrated at the synaptic membrane, with a receptor density of ⬃10,000 –
20,000 AChRs/␮m2, and falling off within a few microns to
⬃1–10/␮m2 (139). Given this low extrasynaptic AChR concentration, the synaptic AChR cluster is maintained not by
trapping of receptors diffusing laterally in the surface mem-
826
brane, but rather through their focal delivery to the postsynaptic membrane. In principle, the synaptic AChR density is determined by the rates of their insertion into and
their removal from the postsynaptic membrane.
Like the accumulation of AChRs described above, this focal
insertion of AChRs into the adult synaptic membrane is also
regulated by the cytoskeleton but requires, at least in part, a
network of microtubules (MTs) associated with the synaptic membrane (208). This network is organized through
agrin-induced capturing of MTs at the synaptic membrane
and serves to focally transport AChRs to the synaptic membrane (390). MT capturing is mediated by the MT plus-end
protein cytoplasmic linker associated protein 2 (CLASP2)
interacting with PH-like domain family B member 2
(Phldb2; also called LL5␤), enriched at the synaptic mem-
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proteasomal
degradation
NEUROMUSCULAR JUNCTION DEVELOPMENT AND FUNCTION
The metabolic stability of AChRs is measured by pulselabeling them irreversibly with ␣-BTX and monitoring the
bound ␣-BTX over time. In myotubes and in extrasynaptic
membrane of denervated muscle, the metabolic stability of
the AChRs is low with a half-life of ⬃1 day. In contrast,
AChRs at the mature NMJ have a half-life of 10 –14 days.
The metabolic stability of the synaptic AChR is not determined by the rate of their internalization alone. Rather,
upon their internalization, a fraction of the synaptic AChRs
is targeted to degradation, whereas another fraction is recycled to the synaptic membrane (61). Thus the measured
metabolic AChR half-life is determined by the net result of
the rates of AChR internalization, the fractions of AChRs
that are recycled versus degraded after internalization.
As outlined above, AChR anchoring and metabolic stabilization (increase in half-life) of the synaptic AChRs is
thought to be achieved by linking them, through the DGC,
to the cortical actin cytoskeleton (108). Accordingly, deletion of DGC components impairs metabolic stabilization of
synaptic AChRs during synapse maturation. The function
of the DGC to stabilize synaptic AChRs is, furthermore,
strongly affected by 1) ErbB-dependent phosphorylation of
␣-dystrobrevin-1 (165, 389) and 2) action potential activity
in the muscle as revealed at ectopic endplates denervated
early in development, where full metabolic AChR stabilization requires exogenous muscle stimulation (362). Likewise, at mature endplates, where chronic denervation reduces the half-life of synaptic AChRs from ⬃14 to ⬃4 days,
AChRs are restabilized upon chronic electrical stimulation
of the muscle fibers via implanted electrodes (146). This
restabilization is mediated by calcium influx through voltage-gated (L-type) calcium channels and is dependent on
protein phosphorylation (80). Finally, application of recombinant neural agrin to extrasynaptic regions of the muscle in vivo showed that agrin alone can metabolically stabilize AChRs at ectopic clusters in a dose-dependent fashion
(43), a process that is generated by agrin’s action to promote AChR recycling (55).
All treatments affecting synaptic AChR stability examined
so far, i.e., removing muscle activity through denervation,
blocking of calcium influx, and impairment of DGC function have been found to either shift the fraction of internalized AChRs towards the degradation pathway (calcium,
action potentials, deletion of ␣-dystrobrevin-1 or ␣-syntrophin) or by selectively accelerating the internalization of the
recycled AChR pool (389). However, the molecular mechanisms of how agrin-Lrp4-MuSK signaling assembles the
DGC, how the DGC aggregates and stabilizes synaptic
AChR clusters, and how muscle activity affects DGC function remain unknown.
VI. MECHANISMS OF MUSCLE WASTING
A. Maintenance of the NMJ and
Myasthenias
Many of the molecules involved in the formation of the
NMJ have also an essential role in its maintenance. In particular, conditional and/or inducible knockout techniques
in mice reveal that NMJs disassemble and become functionally impaired upon loss of agrin, laminin-␤2 (375), Lrp4
(23), and MuSK (188, 234). In humans, mutations in genes
involved in NMJ function, such as those encoding AChR
subunits, ColQ (resulting in the loss of AChE from the
NMJ), Nav1.4, or rapsyn are the most frequent cause of
CMS (reviewed in Ref. 124). While this is not surprising
based on their necessity in neurotransmission, more recent
mapping studies have shown that molecules regulating
NMJ development can also cause CMS. These include agrin
(200, 283, 323), MuSK (89), Lrp4 (331), and Dok-7 (32).
Likewise, sporadic MG, where the majority is caused by
autoantibodies to AChRs, can also be caused by antibodies
directed against MuSK (193), Lrp4 (189, 342, 479), and
agrin (150, 478). Interestingly, there are still many CMS
and MS patients where the cause of the disease is not
known. Current estimates are that between 30 and 50% of
all CMS patients do not have a genetic diagnosis, and in a
recent study on 680 patients, this number was even higher
than 50% (1). Recent mapping studies and next-generation
sequencing approaches have revealed CMS-causing mutations in enzymes involved in glycosylation (34, 101, 394).
There is evidence that these mutations can affect glycosylation of AChR subunits and thus impair intracellular trafficking of functional AChRs to the sarcolemma (34). However, it is highly likely that these glycosylation enzymes have
additional targets that could participate in the disease phenotype. Thus future research may reveal additional genes
involved in the function of the NMJ and may help to further
resolve the mechanisms involved in NMJ maintenance.
B. Denervation Causes Muscle Atrophy
NMJ maintenance is important to maintain muscle mass,
and thus there is an emerging interest in studying the mo-
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brane by synapse-specific expression (28, 230) and recruited by synaptic phosphatidylinositol-3,4,5-trisphosphate (PIP3; Ref. 390). Remarkably, synaptic MT capture
and focal AChR vesicle delivery also require an intact actin
cytoskeleton, consistent with AFD/cofilin-dependent actin
reorganization at and AChR delivery to nascent synaptic
sites (248). Abolishment of this CLASP2 pathway in vivo
lowers synaptic AChR insertion as well as the density and
size of synaptic AChR clusters by ⬃30% each. However,
CLASP2-dependent MTs are only one mechanism for focal
AChR delivery, as Clasp2⫺/⫺ mice retain some synaptic MT
network (perhaps through its related MT plus-end protein
CLASP1), and synaptic AChR density, though lowered, remains sufficient for neuromuscular action potential transmission (390).
TINTIGNAC ET AL.
lecular mechanisms involved in the loss of muscle mass
upon loss of innervation. As highlighted in section VB1,
gene expression of synaptic genes in fundamental myonuclei (e.g., Chrn, Musk) is maintained in innervated muscle
by sustained activation of agrin-Lrp4-MuSK signaling,
while electrical activity suppresses those genes in nonsynaptic myonuclei (FIGURE 4A). Denervation or malfunctioning of NMJ transmission (e.g., in MuSK-positive myasthenia) results in muscle wasting, which is based on activation
or inhibition of specific signaling pathways.
Changes in class II HDACs and their activation of proteasomal degradation drive muscle atrophy mainly in the initial phase of denervation. If muscle remains denervated for
a few days, additional pathways become activated or inhibited, respectively. One of these is the mammalian target of
rapamycin complex 1 (mTORC1) pathway, which is a key
activator of protein synthesis and inhibitor of autophagy
(see sect. VID1; Refs. 246, 468). There is agreement that
proteasomal degradation remains increased throughout denervation (FIGURE 4C). In contrast, the activation state of
autophagy during prolonged denervation remains controversial. While it has been proposed that expression of autophagy genes (275, 482) and autophagy flux are increased
(285), one recent report indicated that autophagy is inhibited rather than activated upon denervation and that this
828
C. Muscle Atrophy and Muscle Wasting Can
Be Triggered by Many Factors
Muscle atrophy can also be triggered by mechanical unloading (e.g., by hindlimb suspension) or immobilization
(e.g., casting or as a consequence of spinal cord transection). In these latter conditions, innervation of the muscle
remains intact, but muscle activity is decreased. Muscle atrophy can also be observed upon prolonged fasting, a phenomenon that is particularly prominent in mice. Fastinginduced muscle atrophy is based on the requirement to generate free amino acids for protein synthesis in the absence of
amino acids supplied by the food. Thus proteins of the
skeletal muscle are degraded by autophagy and the proteasomal pathway. Muscle loss (measured as loss of lean mass)
can also be the consequence of a primary disease, such as
cancer, AIDS, sepsis, congestive heart failure, or chronic
obstructive pulmonary disease (COPD), a phenomenon
termed cachexia. Although cachexia is secondary to a disease, loss of lean mass is an important prognostic factor in
cancer as the higher the extent of weight loss, the shorter the
survival time. Finally, muscle loss is also seen as consequence of normal aging. This condition, called sarcopenia,
is the main contributor to progressive muscle weakness and
frailty and thus contributes substantially to the loss of quality of life in the elderly (364). “Atrogenes” are regulated in
a common pattern in all those paradigms (47, 247, 371).
D. Proteostasis and Muscle Homeostasis
Muscle is the largest organ in humans, comprising up to
50% of the total body weight. Because of its high energy
demand and its secretory function, muscle is a major con-
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Genome-wide searches for genes whose expression is altered upon denervation led to the identification of a set of
transcripts (47, 163, 247). As expression of several of those
transcripts is also altered in other muscle atrophy-inducing
paradigms, they were termed atrophy-related genes or
“atrogenes” (163, 247). Among those atrogenes, tripartite
motif containing 63 (Trim63), also called muscle ring-finger
1 (MuRF1), and F-box protein 32 (Fbxo32), also called
muscle atrophy F-box (MAFbx) or atrogin-1, are two muscle-specific E3 ubiquitin ligases that control substrate specificity of the proteasome. As described below, both E3 ligases have been shown to contribute to the increase in protein degradation and thus the loss of muscle mass during
muscle atrophy. Several lines of evidence suggest that this
denervation-induced upregulation of Trim63 and Fbxo32
is based on the altered expression of class II histone deacetylases (HDACs), in particular HDAC4 (97, 311, 418) and
HDAC5 (311). The increase in the expression of Hdac4
and Hdac5 after denervation triggers a signaling cascade
that involves changes in the MAP kinase (93) and the
Dach2/myogenin pathways (311, 418; FIGURE 4B). In
addition, denervation causes the downregulation of
Hdac9 in nonsynaptic regions (FIGURE 4B). Forced overexpression of HDAC9 blunts denervation-induced expression of Chrn subunits and of Myog while HDAC9
knockout mice are supersensitive to ACh in response to
denervation (297). Thus several HDACs seem to contribute strongly to the changes in gene expression in denervated muscle (FIGURE 4B).
inhibition is paralleled by the activation of mTORC1 (349).
Activation of mTORC1 is not a compensatory response to
the activation of proteasomal degradation (and hence protein loss) during denervation, as genetically engineered mice
lacking the mTORC1 inhibitor tuberous sclerosis complex
1 (Tsc1) cause accelerated muscle atrophy upon denervation rather than sparing (417). This effect is, at least in part,
based on feedback inhibition of S6 kinase (S6K) onto insulin receptor substrate 1 (Irs1; Ref. 433) and thus pronounced inhibition of protein kinase B (PKB, also called
Akt; FIGURE 4C, see also FIGURE 5A). This, in turn, activates
Forkhead box O (FoxO) transcription factors and results in
increased expression of Trim63 and Fbxo32 and thus increased protein degradation by the proteasome (FIGURE
4C). Consistent with this notion, depletion of FoxO1,
FoxO3, and FoxO4 protects skeletal muscle from denervation-induced muscle atrophy (417). Finally, there is recent
evidence that the mTORC1 inhibitor rapamycin mitigates
muscle weight loss and muscle fiber atrophy upon denervation (417), while others have reported an enhancement of
muscle atrophy by rapamycin (349).
NEUROMUSCULAR JUNCTION DEVELOPMENT AND FUNCTION
A
P
P
Akt
CoFoxO activators
P
“Atrogenes”: Gabarapl1,
Bnip3, Map1lc3b, Fbxo32,
Trim63, Ub, proteasome subunits...
FKBP12
Rapamycin
Nucleus
FoxO
P
FoxO3
P
P
P
mTORC1
Raptor
14.3.3
Translocation
P
P
Akt
P
P
P
PDK1
SGK1
P
P
P
P
mTORC2
Rictor
RagA/B
RagC/D
P P
P
PKC
PTEN
P
P
IGF1
insulin
Cytoskeletal
reorganization
P
Akt
P
1
Irs1
P
S6K1
TSC1 Rheb
TSC2 GDP
Amino acids
P
PI3K
p85
P
TSC2
P
RTK
Growth
factors
P
P
P
P
p110
v-ATPase
GTP
IGF1-R
P
Ragulator
complex
Rheb
P
P
PIP2
Downloaded from http://physrev.physiology.org/ by 10.220.32.247 on June 18, 2017
PIP3
P
2
Lysosome
14.3.3
P
B
2
4EBP
4EBP
Pre-initiation
complex
eIF4E
eIF2
mRNA
eIF4E
P P P P
eIF3f
P
1
eIF2
m7-GTP
P
Protein synthesis
TOP and TOP-like
translation
Ub
SQSTM1
P
P
P
P
mTORC1
Raptor
S6K1
P
Ulk1
β
AMPK
Aggregated
protein
P
PDK1
α
P
P
P
γ
S6
P
P
26S proteasome
LC3-PE
Ulk1
complex
Processed
protein
Autophagy
initiation
LAMP2
Lysosome
Phagophore
recruitment
Beclin 1/Vps34
complex
Autophagosome
Autolysosome
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829
TINTIGNAC ET AL.
tributor to the overall metabolism, and it is also the main
storage for glucose and amino acids. As a consequence,
muscle size is dynamically adapted to the needs of the body.
For example, an increase in muscle load causes the muscle
to become hypertrophic, unloading results in muscle atrophy. Similarly, high energy demand can cause some loss of
lean body mass during ultra-endurance races, such as an
Ironman triathlon (314). Thus muscle contains molecular
sensors that allow the adjustment of the balance between
protein synthesis and protein breakdown (157, 368, 377).
1. Protein synthesis
A) TARGET OF RAPAMYCIN.
In yeast, TOR1 and TOR2 associate with particular proteins to form two distinct complexes TOR complex 1
(TORC1) and TOR complex 2 (TORC2) (266). Although
there is only one Mtor gene in mammals, the associated
components are conserved and also assemble into two distinct protein complexes mTORC1 and mTORC2. Besides
mTOR, six additional components constitute mTORC1
and seven components mTORC2 (FIGURE 5A). While some
of those components are shared between the two complexes, the proteins regulatory associated protein of mTOR
(raptor) and rapamycin-insensitive companion of mTOR
(rictor) are specific for mTORC1 and mTORC2, respectively (FIGURE 5A; Refs. 205, 382). Importantly, raptor and
Experiments in yeast and cultured mammalian cells have
indicated that mTORC2 affects the organization of the actin cytoskeleton (205, 266, 382). While it is not known in
detail how mTORC2 affects the actin cytoskeleton, biochemical evidence shows clearly that mTORC2 controls
phosphorylation of the hydrophobic motif of different
AGC kinases (128, 149, 203, 383). These include Akt/PKB,
protein kinase C (PKC) and the serum- and glucocorticoidinduced kinase 1 (SGK1; FIGURE 5A). AGC kinases are involved in diverse cellular processes including cell growth
and actin cytoskeletal regulation and are thought to be deregulated in several disease states including cancer (for recent review on AGC kinases, see Ref. 9).
Germline deletion of Rictor and thus inactivation of
mTORC2 in mice causes embryonic death (169, 397).
However, tissue-specific deletion of Rictor in several tissues
including skeletal muscle results in only minor phenotypes
(38, 240). Strikingly, such rictor-deficient skeletal muscle
fibers do not show an obvious change in the actin cytoskeleton (38). However, changes in the cytoskeleton and thus in
the cell morphology are observed in mice where rictor was
depleted in the entire central nervous system or in the forebrain (197, 423). Interestingly, rictor depletion in motor
neurons does not affect the NMJ (S. Lin and M. A. Rüegg,
unpublished observation). These results indicate that the
functions of mTOR in skeletal muscle are mainly carried by
mTORC1.
B) ACTIVATORS OF MTORC1. mTORC1 is a central platform
involved in cell growth. Activation of mTORC1 results in
an increase in protein synthesis. Stimuli that activate
mTORC1 can be nutrient availability, the energy state of a
cell, and growth factor signaling. For example, a high ATP/
AMP ratio inhibits AMP-activated kinase (AMPK) thereby
releasing its inhibition on mTORC1 (FIGURE 5B).
Amino acids, in particular branched-chain amino acids
(e.g., leucine or arginine), activate the lysosome-attached
vesicular ATPase (v-ATPase) and acidify the lysosomes.
The v-ATPase controls Ragulator and the guanine nucleo-
FIGURE 5. mTORC1 and mTORC2 signaling in skeletal muscle. A: cross talks between mTORC1 and mTORC2 complexes in the maintenance
of muscle homeostasis. Activation of the mTORC1 complex in skeletal muscle requires 1) amino acid-dependent activation of the Rag-GTPase,
docked at the lysosomal surface by the Ragulator complex, and 2) the insulin/IGF1/growth factor-dependent activation of the PI3K-Akt/PKB
pathway. Active Akt/PKB induces phosphorylation (step 1) and dissociation (step 2) of the TSC1-TSC2 complex from the lysosomal Rheb
GTPase. Rheb-GTP recruits and activates mTORC1 at the lysosomal surface. Activation of Akt/PKB or SGK1, which involves mTORC2, also
prevents the nuclear accumulation of FoxO transcription factors, thereby preventing expression of the “atrogenes.” Importantly, the mTORC1
target S6K initiates a feedback loop and inhibits Irs1, thereby dampening activation of Akt/PKB and preventing hyperactivation of mTORC1. B:
MTORC1 orchestrates protein synthesis and degradation. The phosphorylation of 4EBP by mTORC1 on multiple sites (step 1) causes the
dissociation of 4EBP from eIF4E (step 2) and induces the assembly of the 43S preinitiation complex (PIC) and the initiation of 5’TOP mRNA (5’
terminal oligopyrimidine) translation. On the other hand, mTORC1-dependent phosphorylation of Ulk1 prevents the induction of autophagy. For
further details see text.
830
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Among the key regulators of protein synthesis is the target of rapamycin (TOR). TOR was
initially identified by genetic screens in yeast that yielded
two mutants that were resistant to the growth-inhibitory
properties of the immunophilin-immunosuppressant complex consisting of 12 kDa FK506-binding protein 12
(FKBP12) and rapamycin. Rapamycin (also known as
Sirolimus) is a macrocyclic lactone discovered in a soil sample from the Easter Islands as a metabolite of the bacteria
Streptomyces hygroscopicus. The two mutants resistant to
the FKBP12-rapamycin complex were defective in TOR1 or
TOR2 (181). Vertebrates have only one gene that encodes
the protein mTOR (for mammalian TOR; also called mechanistic TOR). Rapamycin and its synthetic analogs have
been on the market as immunosuppressive drugs for several
years; they are in clinical trials also for additional indications including cancer (35).
rictor are necessary for the function of the respective complex (see below).
NEUROMUSCULAR JUNCTION DEVELOPMENT AND FUNCTION
C) TARGETS OF MTORC1. The two main downstream signaling
components of mTORC1 are S6K and eukaryotic translation initiation factor 4E (eIF4E) binding protein (4EBP),
which together control protein synthesis (FIGURE 5B). Phosphorylation of 4EBP induces its release from eIF4E, which
enables assembly of the 40S ribosome with the ternary complex of eIF2, M7-tRNA, and GTP at the 5’-cap of mRNA
and thus the formation of the preinitiation complex to initiate cap-dependent translation. Phosphorylation of S6K
also participates in the assembly of the preinitiation complex (194) by activation of eIF2, and it phosphorylates S6
on several sites (FIGURE 5B). Taken together, activation of
mTORC1 controls diverse steps in protein synthesis by its
effect on 4EBP and S6K (FIGURE 5B).
2. The insulin/IGF1-PI3K-Akt/PKB pathway
Another important input that results in the activation of
mTORC1 is growth factors. In contrast to simple organisms, growth factor availability is the main determinant of
organ size in mammals. The best-characterized pathway
that affects mTORC1 activation is initiated by insulin/insulin-like growth factor 1 (IGF1). Specifically, binding of insulin/IGF1 induces autophosphorylation of its receptor,
which creates docking sites for Irs1 and other scaffolding
proteins (FIGURE 5A). These scaffolding proteins, in turn,
become phosphorylated by the receptor and activate the
p85 regulatory subunit of class I phosphoinositide 3-kinase
(PI3K). Activated PI3K converts phosphatidylinositol-4,5bisphosphate (PIP2) into PIP3 to recruit and activate Akt/
PKB via 3-phosphoinositide-dependent protein kinase 1
(PDK1) and the participation of mTORC2 (FIGURE 5A).
Activation of Akt/PKB causes phosphorylation of TSC2
upon dissociation of TSC2 from the lysosome (see above),
which then results in the activation of mTORC1 via Rheb
(see above).
Akt/PKB has many additional downstream targets (see
overview in Ref. 182). One such target that also plays a role
in skeletal muscle atrophy is glycogen synthase kinase-3
(GSK-3), which becomes phosphorylated by Akt/PKB. Interestingly, GSK-3 affects multiple pathways such as glycogen synthesis, Wnt/␤-catenin signaling, and protein synthesis, which all have been shown to play a role in skeletal
muscle (157, 368). Recent evidence also indicates that
GSK-3 directly phosphorylates neighbor of BRCA gene 1
(NBR1) to prevent aggregation of ubiquitinated proteins
and to enhance their autophagosomal degradation (324). In
addition to the above activities of Akt/PKB, phosphorylation and thereby inhibition of FoxO transcription factors
are most significant for the control of muscle mass (FIGURE
5A). Specifically, phosphorylation of FoxOs exports them
from the myonuclei back to the cytoplasm and thus prevents the expression of the FoxO targets (379, 409). Among
those targets in skeletal muscle are the “atrogenes,” which
include the E3 ligases Trim63 and Fbox32 and genes involved in autophagy (FIGURE 5A; see details below). Another upstream kinase that phosphorylates FoxOs is SGK1.
Like Akt/PKB, SGK1 inhibits FoxO3 by phosphorylation
(63). Interestingly, SGK1, but not Akt/PKB, is activated
during hibernation in squirrels to protect muscles from atrophy (5). Thus activation of Akt/PKB increases protein
synthesis via mTORC1-dependent activation of translation
and inhibits protein degradation via FoxO-dependent inhibition of proteasomes (FIGURE 5). This dual function of
Akt/PKB probably explains the profound effect of the overexpression of a constitutively active form of this kinase on
muscle size (204, 244).
3. Protein degradation
The two most important protein degradation pathways
during muscle atrophy are the autophagy/lysosomal pathway and the ubiquitin-proteasome system (UPS). We will
not discuss caspases, whose activation is required to trigger
apoptosis and the calcium-activated calpains, which participate in various calcium-mediated intracellular signal transduction pathways (reviewed in Refs. 161, 265).
A) AUTOPHAGY.
mTORC1 is also a main regulator of autophagy (“self-eating”), a catabolic process that degrades
cellular components via the lysosomal pathway. Autophagy
can be grouped into macroautophagy (usually referred to as
autophagy), microautophagy, and chaperone-mediated autophagy (307). Autophagy is active at low levels in all cells
and is highly induced by situations of stress where it can act
as a survival mechanism. Dysregulation of autophagy has
been implicated in many diseases (see review in Ref. 91).
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tide loading of the two ras-related GTPases (Rags) RagA
and RagB (21). RagA/B and RagC/D heterodimers directly
bind to raptor, if they are in the proper nucleotide-bound
state (22; FIGURE 5A). This recruits mTORC1 to lysosomes
and causes its activation by the small GTPase ras homolog
enriched in brain (Rheb), which also resides at the lysosomal surface (FIGURE 5A). Recent observations have, in
addition, shown that TSC1 and TSC2 also participate in the
activation/inhibition of mTORC1 by amino acids. TSC2
(also known as tuberin) forms a complex with TSC1 (also
known as hamartin). Phosphorylation of TSC2 by Akt/PKB
(see below) downregulates the GTPase-activating protein
(GAP) activity of the TSC1/TSC2 complex and thereby activates Rheb (FIGURE 5A). In the absence of amino acids,
TSC2 is attached to the lysosome and inhibits Rheb (114).
Growth factor stimulation in the presence of amino acids
dissociates TSC2 from the lysosome and releases inhibition
of Rheb, which, in turn, leads to activation of mTORC1
(299). The release of TSC2 from the lysosome involves its
phosphorylation by Akt/PKB (299). These findings raise the
possibility that subcellular localization of TSC2 is a general
mechanism for regulating the TSC1/TSC2 complex by
amino acids and growth factors, and they suggest a crosstalk between amino acid availability and growth factor signaling.
TINTIGNAC ET AL.
For example, excessive autophagy is thought to contribute
to neurodegenerative diseases including Alzheimer’s disease
(470), whereas autophagy is inhibited in Huntington’s disease (326). Similarly, autophagy flux is perturbed in mouse
models for different muscular dystrophies (79, 168).
In contrast to many tissues, autophagy in skeletal muscle
has been proposed to be mainly controlled by FoxO3 signaling (FIGURE 5A) and not by mTORC1 (275, 482). This
view is based on the inability of the mTORC1 inhibitor
rapamycin to increase autophagy flux in skeletal muscle
(275). For example, although rapamycin was shown to increase autophagy flux in the heart, autophagy in skeletal
muscle was not affected (92, 350). However, the failure of
rapamycin to affect autophagy is more likely based on its
inability to sufficiently penetrate skeletal muscle, as sustained activation of mTORC1 by the conditional deletion
of Tsc1 in skeletal muscle results in a complete blockade of
autophagy induction (83). As a consequence of this inhibition, mutant mice suffer from a late-onset myopathy (83),
which is reminiscent of that caused by the skeletal musclespecific deletion of autophagy-related protein 7 (Atg7; Ref.
285), one of the “core” Atg proteins (307).
832
B) THE UPS. While protein synthesis is largely under the control of mTORC1, muscle atrophy involves primarily the
activation of the UPS, which is also called UPP for ubiquitin
proteasome pathway (358, 372). Pioneering work has established that muscle atrophy induced by different paradigms involves increased transcription of components of
the proteasome and a two- to threefold increase in proteolytic activity (292).
The ATP-dependent proteasome activity largely depends on
protein ubiquitination. The proteolytic activity of this oligomeric protease resides in the inner, barrel-shaped structure of the 20S core particle, which assembles with the 19S
regulatory particle to the 26S proteasome (315), where the
substrate proteins are hydrolyzed (FIGURE 6A). The ubiquitination process sequentially involves 1) activation of the
free ubiquitin moiety by a unique and ubiquitously expressed ubiquitin-activating enzyme E1 (UBE1); 2) conjugation of activated ubiquitin to the ubiquitin carrier protein
(Ubc) or E2 protein; and 3) recognition of the substrate and
transfer of the activated ubiquitin to the substrate by a
ubiquitin ligase or E3(-ligase). The addition of a polyubiquitin chain to the ␧-amino group of the lysine of the substrate determines the fate of the target protein (FIGURE 6A;
for review, see Ref. 186). According to the “ubiquitin
code,” the ubiquitin monomers can be polymerized at seven
possible lysines (Lys 11, 27, 29, 33, 48, or 63), and the side
chain of lysine 48 is responsible for the targeting of the
protein substrate to the 26S proteasome (187, 474). Other
modifications such as polyubiquitination of lysine 63 are
thought to modify activity, trafficking, and/or localization
of the modified substrate.
Although the E2 enzymes may display some ubiquitin conjugation specificity, the E3 ligases are thought to control
specificity (FIGURE 6A; Refs. 186, 424). More than 600
E3-like genes are found in the human genome that belong to
two major classes; the homologous to E6-AP COOH-ter-
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Autophagy is a highly dynamic process that is controlled in
many tissues by mTORC1 via its inhibitory function on
unc-51-like kinase 1 (Ulk1). Ulk1 is part of the large Ulk1
complex (FIGURE 5B; reviewed in Ref. 245). The inhibitory
function of mTORC1 is mediated by phosphorylation of
Ser757 in Ulk1, which prevents activation of the Ulk1 complex by AMPK (225). Activation of the Ulk1 complex and,
equally important, activation of a protein complex containing the core components Beclin1 and vacuolar protein sorting 34 (Vps34), called Beclin 1/Vps43 complex (148, 366),
results in the formation of phagophores (FIGURE 5B). This
nucleation step is followed by the recruitment of microtubule-associated protein 1 light chain 3 (MAP1LC3), more
commonly referred to LC3, and of target proteins, which in
turn are recruited via adaptor proteins such as sequestome 1
(SQSTM1/p62). Phosphatidylethanolamine (PE) is conjugated to LC3 at its COOH terminus (LC3-PE) to associate
LC3 to membranes, which then results in the formation of
the autophagosomes, intermediate structures that are characterized by their double membrane (FIGURE 5B). With the
assistance of lysosome-associated membrane protein 2
(LAMP2), the autophagsome then fuses with lysosomes,
resulting in the formation of autolysosomes where the proteins are degraded and recycled (FIGURE 5B). The tight control of the autophagy process by mTORC1 is a mechanism
to couple sensing of amino acids to autophagy. In the presence of amino acids (i.e., fed state; mTORC1 active), autophagy is inhibited, while in the absence of amino acids
(i.e., starved state; mTORC1 inhibited) autophagy is activated. Thus activation of autophagy by the inhibition of
mTORC1 allows the release of free amino acids to maintain
a basal level of protein synthesis (307).
Despite the now strong evidence that induction of autophagy in skeletal muscle is regulated by mTORC1, longterm maintenance of a proper autophagy flux requires increased expression of autophagy genes. In skeletal muscle,
FoxOs have been shown to affect transcription of several
autophagy genes including Map1lc3b, Gabarapl1, and
Bnip3. Some of those genes are direct targets of FoxO3 as
demonstrated by chromatin immunoprecipitation (275,
482). Thus the role of FoxO3 is mainly in the regulation of
protein degradation by transactivation of genes involved in
the UPS and the autophagosomal/lysosomal pathway (FIGURE 5A). Accumulating evidence suggests the existence of a
complex crosstalk between these two processes. It has recently been reported that upon denervation, accumulation
of Trim63 at the NMJ regulates both the recycling of
AChRs as well as the trafficking of autophagic vacuoles
(367).
NEUROMUSCULAR JUNCTION DEVELOPMENT AND FUNCTION
A
K
Free ubiquitin
K
ATP
K
Substrate
Recognition and
deubiquitination
Thioester
bond
E3
complex
S
S
E2
Chaperone-mediated
transport (UBDs)
E1
Conjugation
Opening
Activation
SH
G
G
SH
E2
Protease
Base
Release
G
G
K
E1
K
ATP
Peptides
Homotypic or
mixed Ub chain
S
Substrate-binding specificity
(motif-PTM-degron) and
ubiquitin chain transfer
Lid
α
β
β
α
Cleavage
AMP + PPi
Ligation
Bortezomib
MG132
ADP Opening
Unfolding
Cytosolic peptidase
Polyubiquitinated substrate
B
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Amino acids
E3 ligase
HECT
RING finger
Multi-subunits
CRL2, 3, 4A, 4B, 5, 7
K
K
K
K
S
S
Fbxo25
Nedd4-1
Fbxo
S
Skp1
Substrate
K
K
K
Fbxo32
Fbxo30
Fbxo40
S
E2
Rbx1
RING
CUL
CUL7
finger
(or CUL2,
N8 Nedd8
3, 4A, 4B, 5)
Single-subunit
CRL1/SCF
K
K
K
Fbxo
S
S
E2
CUL
CUL1
K
Trim63
Mul1
cbl-c
E2
Rbx1
Skp1
K
S
K
S
RING finger
N8
C
Mitochondrion cycle
Mgn
HDAC4
NFκB
Fusion
Transactivation
FoxO
ER
TNFα, Fbxo32, Trim63,
Fbxo29, Fbxo40, Ub.
proteasome subunits...
Fission
Mitophagy
Nucleus
Smad
2/3/4
p65 p50
Mfn2
Mul1
Myogenic
program
MyoD
CK
Trim63
RING finger
CRL1/SCF
PIC
Protein
synthesis
Skp1
Myosin
eIF3f
CUL
N8
Fbxo32
Fbxo40
mTORC2
Rictor
Z disk
M-band
Sarcomere
P
Fbxo29
S6K1
P
mTORC1
Raptor
P
P
PI3K
P
Z disk
Akt
P
P
P
Irs1 p110 p85
P
P
PDK1
α-actinin Actin
Rbx1
Cytosol
Plasma membrane
IGF1-R
IGF1 insulin
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TINTIGNAC ET AL.
The E3 ligases so far identified in skeletal muscle belong to
the RING-finger domain family except neural precursor cell
expressed developmentally downregulated protein 4 isoform 1 (Nedd4-1), whose expression is induced during muscle atrophy (29). Although the detailed mechanisms are not
known, muscle-specific Nedd4-1 knockout mice display
some protection of fast-twitch fibers to denervation-induced atrophy (318). The protein of the RING-finger domain family that is induced by denervation the most is
Fbxo32. Fbxo32 transcripts are also increased during fasting, hindlimb suspension, immobilization, oxidative stress,
or sepsis (47, 163, 372, 409), which assigns Fbxo32 to the
group of the “atrogenes” (sect. VIB). It is well established
that the FoxO transcription factors, via the insulin/IGF1PI3K-Akt/PKB pathway, are important regulators of the
“atrogenes” (FIGURE 6C). However, other pathways that
not necessarily make use of FoxOs have also been implicated in the regulation of “atrogenes” and in the control of
muscle atrophy. Examples are the p38/MAP kinase (255)
and the NF-␬B pathways (74, 409).
Fbxo32 is an atypical Fbox protein because the Fbox motif
is located in the COOH-terminal part and because it lacks
the common substrate binding domain (SBD) found in
other Fbox proteins. Fbxo32 forms a multicomponent E3
ligase of the CRL1 or SCF subfamily of the RING finger E3
ligases (FIGURE 6B). In skeletal muscle, Fbxo32 has been
shown to target the degradation of the myogenic determinant (MyoD; Ref. 425), a key transcription factor driving
myoblast differentiation during myogenesis and regeneration, and the regulatory subunit f of the eukaryotic initiation factor 3 (eIF3f) of translation (106, 107, 243; FIGURE
6C). As eIF3f also participates in the activation of S6K by
mTORC1 to form the preinitiation complex of translation
(see also FIGURE 5B), Fbxo32 inhibits protein synthesis (14,
206, 247). Knockout animals for Fbxo32 are fertile and
display no obvious phenotype compared with their littermates. However, when subjected to denervation or starvation, their muscles are partially resistant to atrophy (56%
sparing), suggesting a role for Fbxo32 in regulating muscle
size (47). However, denervated muscle from Fbxo32-deficient mice becomes severely myopathic 28 days postdenervation (instead of 14 days as in Ref. 47), suggesting that
Fbxo32 is necessary for maintaining muscle integrity upon
denervation (162).
FIGURE 6. UPS function and regulation in skeletal muscle. A: principles of ubiquitin-mediated protein degradation. Protein ubiquitination
involves the sequential activation and conjugation of ubiquitin monomers by E1 and E2 enzymes. This ATP-consuming process uses the
COOH-terminal glycine residue of ubiquitin and loads it on the cysteine residues of the E2-conjugating enzyme via a thioester bond (exchanged
upon trans-esterification with E1-Ub). E3 ligases further regulate the ultimate step of ubiquitination, ensuring both the recognition of the
substrate as well as the transfer of the ubiquitin from E2 enzymes to one or several lysine residue of the target substrate (S). The polymerization
of the ubiquitin chain involves the combination of ubiquitin monomers through linkage via one of the 7 possible lysine binding sites of ubiquitin (Lys
11, 27, 29, 33, 48 and 63). The resulting ”ubiquitin code⬙ determines the fate of the substrate. Degradation of the ubiquitinated protein in the
26S proteasome requires the release of the ubiquitin chain by deubiquitinating enzymes (DUBs) and activates the opening of the proteasome lid.
The proteolytic activity of the proteasome is located in the ␤-subunits (as showed by colored stars corresponding to chymotrypsin-like, trypsin-like,
and caspase-like activity) and generates short polypeptides (6 – 8 amino acids long) that are further processed by cellular peptidases and
contribute to the replenishment of the amino acid pool in a cell. B: identity and structure of the muscle E3 ligases involved in muscle wasting.
The RING finger E3 ligases involved in muscle wasting are functional complexes, called cullin/RING-finger ubiquitin ligases (CRL), that lack
enzymatic activity (except for the member of the HECT family, Nedd4-1) and function as scaffolding platform that specifically load their substrate
in close proximity with the E2-ub intermediates. Fbox proteins are essential to build a functional E3 ligase and their diversity, in association with
the different members of the cullin family, accounts for the substrate specificity of the CRL. Neddylation (N8 stands for Nedd8) of the members
of the cullin family is required to activate the E3 ligase complex. C: proposed model for E3 ligase-mediated muscle breakdown. Activation, during
muscle wasting, of catabolic pathways leads to the transcriptional upregulation of several components of the UPS, such as the E3 ligases
Fbxo32/29/40, Mul1, and Trim63. Although the molecular mechanisms responsible for the activation and the specificity of the corresponding
newly assembled E3 ligase complexes are largely unknown, they control the targeted degradation of substrates involved in transcription,
translation initiation, and growth signaling via the formation of functional CRL complexes. In addition, structural components (sarcomere) and
metabolic organelles (mitochondria) are degraded via single subunit ring finger protein Trim63 and Mul1, respectively. For details see text.
834
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minal (HECT) domain-containing enzymes that transfer
ubiquitin from the E2-loaded enzyme to the substrate via
a specific E3-ubiquitin thioester intermediate (196, 198)
and the cullin/RING-finger ubiquitin ligases (CRL),
which are mainly nonenzymatic recognition factors that
scaffold the substrate in close proximity (FIGURE 6B). The
functionality of such complexes relies on the ability of the
cullins to link the polyubiquitin-loaded E2 with the specific substrate to be degraded (483). This assembly requires the binding of a RING-finger adapter Ring box-1
(Rbx 1) at the COOH-terminal end of cullin, which then
recruits the E2 enzyme loaded with ubiquitin (FIGURE
6B). At the NH2 terminus, cullin binds to the substratespecificity module composed of the S-phase kinase adaptor-1 (Skp1) protein and the Fbox protein to form the
Skp1-Cul1-Fbox complex or SCF (FIGURE 6B). Modification of the cullin subunit by NEDDylation has been
proposed to be important for the activation of the SCF
complex. NEDDylation is the posttranslational modification where a ubiquitin-like protein, the neuronal precursor cell expressed, developmentally downregulated
protein 8 (Nedd8) is conjugated to the lysine of the substrate (Lys 720 in Cul1). At the protein level, more than
1,300 different E2/E3-RING ligase complexes are found
in mammals, which in combination with the 69 Fbox
proteins identified so far, are thought to determine substrate specificity (33, 233, 279).
NEUROMUSCULAR JUNCTION DEVELOPMENT AND FUNCTION
In addition to Fbox32, Fbxo29 (also called F-box and WD
repeat domain containing 8; FBXW8) and Fbxo40 also affect mTORC1 signaling (FIGURE 5C). Fbxo29 has been proposed to establish a feedback regulatory loop between the
mTOR and Akt/PKB pathway by targeting the degradation
of the phosphorylated form of Irs1 (228), and Fbxo40
seems to target the degradation of activated Irs1 in muscle
cells (395, 473). Another member of the Fbxo family involved in muscle atrophy is Fbxo30, also called muscle
ubiquitin ligase of the SCF complex in atrophy 1 (MUSA1).
It becomes activated in the absence of the prohypertrophic,
transforming growth factor-␤ family member bone morphogenetic protein (BMP) (FIGURE 6C; Refs. 384, 457).
Another RING-finger protein, casitas B-lineage lymphoma
proto-oncogene (cbl-c), has also been reported to be upregulated in disused muscle (330). Cbl-c has been proposed
to mediate the degradation of Irs1 when phosphorylated at
tyrosine residue 608 in unloaded muscles (319). Consistently, unloaded Cbl-c-deficient muscles display high levels
of phosphorylated FoxO3, suggesting that the restoration
of Irs1 signaling activates Akt/PKB (319). More recently,
the RING-finger domain ligase mitochondrial E3 ubiquitin
protein ligase 1 (Mul1) has been identified as a new FoxO3/
1-regulated gene that targets the degradation of mitofusin 2
(Mfn2). Mul1 activates mitochondrial fragmentation and
loss during atrophy via a process called mitophagy (267).
Importantly, this process seems to be regulated in a fiber
type-dependent fashion.
VII. LOSS OF MUSCLE MASS IN AGING
(SARCOPENIA)
In previous sections, the development and function of the
NMJ including the molecular mechanisms involved have
been discussed. As perturbation of NMJ function has a
strong impact on muscle metabolism and performance, and
ultimately on muscle size, we then described the most important molecular pathways that are involved in the control
of muscle size. In this final section, we discuss possible
mechanisms involved in the loss of muscle mass in aging
(called sarcopenia). Although the mechanisms underlying
sarcopenia are still poorly understood, there is evidence that
the function and structure of NMJs are altered during ageing and that the mechanisms involved in the regulation of
muscle mass discussed in the previous section affect NMJs.
This section will provide insights into those mechanisms
and will discuss of how future research can help to better
understand them.
A. Sarcopenia Is a Multifactorial Syndrome
of Old Age
The term sarcopenia (360) was coined in 1988 and means
loss of flesh or muscle [from the Greek words of “␴␣␳␰
␴␣␳␬␱␵” (flesh) and “␲␧␯␫␣” (deficiency)]. While originally
defined as loss of lean body mass, it has subsequently come
to include functional parameters. According to recently established guidelines (104), sarcopenic patients need to fulfill
two criteria. The first criterion is the loss of muscle mass,
measured as “skeletal mass index” (SMI), which represents
the muscle mass in kilograms divided by the square of the
body height in meters (kg/m2). One of the challenges with
this parameter is the fact that muscle mass can be measured
with different methods. For example, if lean body mass is
measured by dual-energy X-ray absorptiometry (DXA)
scan, the mean SMI of young men is 8.6 ⫾ 1.1 (SD) kg/m2
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Nonconventional single RING-finger E3 ligases are also
involved in muscle atrophy. One of those is Trim63 (FIGURE
6B), which was coidentified with Fbxo32 to be upregulated
in muscle atrophy-inducing paradigms (47, 163). Trim63
localizes to the M line of the sarcomere, and its function in
muscle atrophy (FIGURE 6C) has been linked to its capacity
to target degradation of key structural components, such as
myosin heavy chain (MyHC), the myosin light chains
MyLC-1 and MyLC-2, and the myosin binding protein-C
(MyBP-C; Refs. 95, 96). Trim63 has also been implicated in
muscle metabolism by targeting the muscle-type creatine
kinase for degradation (235). The gastrocnemius muscle of
Trim63 knockout mice shows some resistance to atrophy
induced by denervation or starvation (36% sparing). The
muscle-sparing effect in Trim63 knockout mice seems, at
least in part, also to be attributed by the absence of HDAC4
induction (147). Trim63 shares more than 60% sequence
identity with its related isoforms Trim55/MuRF2 and
Trim54/MuRF3 (86). Despite their conserved function in
the UPS, only Trim63 has been reported to be upregulated
in response to denervation and starvation (163). Trim63
thus appears as an attractive candidate for pharmacological
intervention in response to denervation. A first compound
has recently been shown to inhibit Trim63 in a dose-dependent manner in C2C12 myoblasts (123).
Consistent with the concept that inhibition of UPS affects
muscle wasting, proteasome inhibitors, such as MG132,
prevent muscle atrophy in rats and ameliorate the disease in
mdx mice, the mouse model of Duchenne muscular dystrophy (50, 131, 421). More recently, the new peptidomimetic
inhibitors of the 20 core particle peptidases, such as Bortezomib, have been shown in vivo to be protective against
myofibrillar breakdown and are currently tested in clinical
trials (31, 213). The identification of specific E3 ligases
being involved in muscle wasting has raised the hope to find
more selective drugs that avoid the deleterious side effects of
long-term inhibition of the proteasome activity (258). To
this end, several questions remain to be answered. For example, which signals activate Fbxo32 and Trim63; what is
the nature of the E2 complex associated with these E3 ligases; and which proteins are selectively targeted by these
complexes during denervation-induced muscle wasting?
TINTIGNAC ET AL.
and of young women 7.3 ⫾ 0.9 kg/m2 (30). Pathological
loss of muscle mass was defined as being more than two
standard deviations below the mean SMI in young adults
(104). The second criterion for sarcopenia is poor physical
performance, such as low gait speed (e.g., ⬍0.8 m/s measured in a 6-min walk test) or poor grip strength. While the
correlation between impaired physical performance and
low muscle mass is weak (as there are many reasons for
poor physical performance), the correlation between low
muscle mass and impaired physical performance is stronger
(249). Consistent with sarcopenia having a major impact on
function, it correlates highly with the incidence of falls
(419).
Sarcopenic muscle is characterized by muscle fiber atrophy
(i.e., smaller average fiber diameter) and an increase in fiber
size heterogeneity; reduction of the total number of muscle
fibers; selective loss of fast-twitch (type II) and preponderance of slow-twitch (type I) fibers; presence of “mixed”
fibers (expressing both slow and fast isoforms of myosin
heavy chain); fiber type grouping (indicative of reinnervation by nonaffected motor neurons); presence of centralized
myonuclei; and infiltration of nonmuscle cells (e.g., adipocytes and connective tissue cells). In addition, old muscle
regenerates poorly upon injury due to decreased regenerative capacity of aged satellite cells (see review by Ref. 53).
This multitude of phenotypes indicates that most likely several factors contribute to sarcopenia. Specific factors discussed are hormonal dysregulation (i.e., changes in testosterone, estrogen, growth hormone, IGF1) and muscle-specific changes in the anabolic or catabolic pathways, in
mitochondrial function, and in excitation-contraction coupling (105, 229). All these intrinsic alterations in combination with changes in daily activity and in nutritional habits
(e.g., resulting in sarcopenic obesity; Ref. 363) may contribute to the loss of muscle. As there is no particular treatment
available for sarcopenia at the moment, the most successful
“therapeutic” interventions to prevent or slow-down sarcopenia are life style based. They include changes in caloric
intake and in the composition of food, combined with an
exercise regime of resistance and aerobic training (287).
836
1. Human studies
It is not clear whether changes of the NMJ in sarcopenic
muscle are primarily triggered by changes in the motor neuron or in the muscle fiber. Indications for a presynaptic
involvement are muscle atrophy and fiber type grouping,
two clinical manifestations that are also hallmarks of ALS.
Indications for a postsynaptic origin are the increase in the
relative number of central myonuclei, characteristic of muscle fiber degeneration/regeneration, and alterations in excitation-contraction coupling, whereas changes in muscle
metabolism and mitochondrial function could be either of
pre- or postsynaptic origin. As both cells interact with each
other at the NMJ, the numerous structural alterations at
NMJs observed in the course of aging are not surprising (see
below). What is not known, however, is whether these
changes reflect NMJ dysfunction as a primary cause of sarcopenia or whether impaired NMJ function is secondary to
age-related motor neuron and/or muscle fiber dysfunction.
It is important to note that these questions have not been
unequivocally resolved so far. For example, structural alterations at the NMJ correlate with age (329, 460), but a
correlation neither with sarcopenic muscle nor with NMJ
function has been established. Specifically, age-dependent
morphological changes include an increase in the length, the
branching, and the overall area of the postsynaptic membrane and increased perijunctional AChR labeling near old
NMJs (329). Electron microscopy revealed the presence of
degenerating synaptic folds that were often not opposed by
a presynaptic nerve terminal. Finally, terminal Schwann
cells tended to invade the synaptic clefts more frequently
(460). These studies thus indicate that the number of “pathological” NMJs increases with age. However, it still needs
to be established whether these changes are more frequent
in sarcopenic compared with normally ageing muscle.
Age dependence of NMJ function has been examined
electrophysiologically by single fiber electromyography
(SFEMG), a technique to record transmission of action potentials from single muscle fibers in response to nerve stimulation or voluntary activation of skeletal muscles. SFEMG
enables analysis of the efficacy of neuromuscular transmission (216). The values for jitter, i.e., the variability in transmission time between the point at which the axon is stimulated and the position along the muscle fiber at which the
action potentials are recorded, can be used as an estimate of
the safety factor of neuromuscular transmission (216). An
increase in jitter indicates a lower safety factor. SFEMG also
allows the measurement of fiber density, which is thought to
represent the size of the motor units. Meta-analysis of the
jitter values and the fiber density in different muscles has
shown that both values increase with age (19, 59). Again,
however, SFEMG has not been measured specifically in sarcopenic patients.
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The definition of clear-cut criteria for sarcopenia is important for its recognition as a syndrome, and it is essential for
calculating the prevalence of the syndrome and for estimating the resulting health care costs. So far, most epidemiological studies did not include functional parameters
(e.g., Ref. 30), which explains the rather large difference
in the observed prevalence between studies (see Ref.
109). According to a recent study in more than 700 people from northern Italy, which used both SMI and physical performance as inclusion criteria, 31.6% of women
and 17.4% of men aged 80 years or older were sarcopenic, and the prevalence of sarcopenia increased
steeply with age (442).
B. Changes at the NMJ During Aging
NEUROMUSCULAR JUNCTION DEVELOPMENT AND FUNCTION
2. Rodent studies
Curiously, the frequency but not the amplitude of mEPPs is
decreased in 29- to 35-mo-old CBF-1 mice (20) and in 28mo-old C57/BL6 mice (129). The amplitudes of EPPs were
in both cases even increased, and few, if any, electrophysiological indications of denervation were observed (20,
129). Given that muscle fibers become smaller in aged mice,
their input resistance will increase, which may, in part, explain the increase in EPP amplitude. A change in mEPP
frequency and not amplitude has also been described in a
mouse model of a severe MuSK-based CMS (90), although
in this case, the muscle force upon nerve stimulation was
much lower than in control animals. Thus there is little
evidence for a functional impairment of neuromuscular
transmission in old mice despite the strong fragmentation of
the NMJ. This is in stark contrast to data from human and
rats, where neuromuscular transmission at high age is
clearly diminished (see above).
Gene expression analyses in sarcopenic rats have revealed
prominent signs of denervation. Molecular signatures include upregulation of Chrna1 (␣ subunit of AChRs), Musk,
and Lrp4 (25, 201). Moreover, interventions that have been
shown to slow down sarcopenia, such as caloric restriction
C. Mechanisms Involved in Sarcopenia
As mentioned above, many different factors have been implicated in the triggering of sarcopenia, and it is likely that
several of those factors also affect the NMJ. Similarly, the
fact that muscle mass is lost in sarcopenia has led to the
concept that mechanisms involved in acute muscle atrophy
(for example those induced by denervation) are also causative to sarcopenia. Whether this is indeed the case is not
resolved.
D. Connection Between Pathways That
Affect Lifespan and Sarcopenia
As sarcopenia is an age-dependent syndrome, many studies
have linked longevity and sarcopenia. One of the best-characterized experimental paradigms that prolong lifespan is
caloric or dietary restriction (CR or DR). In CR, daily caloric intake is limited to ⬃60% of that under ad libitum
conditions without causing malnutrition. This paradigm
has been shown to prolong lifespan in many species (345),
although recent work has questioned the universality of the
phenomenon. For example, in some mouse strains, CR
shortens lifespan (257), and recent long-term experiments
have not been able to repeat the initial report (98) that CR
significantly increases lifespan in rhesus monkeys (289).
While these controversial results are disturbing at first sight,
they may in fact argue that multiple pathways are involved
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As humans are not amenable to large-scale and detailed
studies of the NMJ, most molecular and interventional
studies have examined NMJs in aged rodents. Like humans,
aged mice and rats (older than 24 mo) show loss of skeletal
muscle mass. These changes are very consistent among individuals, but the extent of muscle loss may differ between
rodent strains and may also depend on the holding conditions. Close examination of the NMJs in aging mice and
rats has revealed that they undergo marked morphological
remodeling. The phenomena include a high percentage of
fragmentation and a substantial degree of partial and complete denervation concomitant with the appearance of
mixed fibers (88, 254, 365, 435). Moreover, postsynaptic
AChR staining becomes fainter (88, 254, 435). Functional
changes include a marked drop (by 45%) of the nerveevoked peak tetanic force in the EDL muscle of 24-mo-old
rats compared with 6-mo-old controls (201). Presynaptic
changes include the thinning and swelling of motor axons.
Interestingly, the extent and onset of the structural changes
differ greatly between muscles, and the differential response
of muscle correlates well with the extent of NMJ changes
observed in a mouse model of ALS (436). Whereas it is
thought that the synaptic changes are gradual and slowly
progressing over time, repeated vital imaging has revealed
that changes can occur rapidly (254). These sudden changes
correlate with the local degeneration and regeneration of
the muscle fibers. Thus one mechanism that may lead to the
fragmentation of the NMJ during aging might be local degeneration/regeneration of those muscle fiber segments underlying the NMJ (254), as is also observed in the mdx
mouse, a model for Duchenne muscular dystrophy (273).
and exercise, also normalize the structure of aged NMJs
(88, 129, 435). Finally, conditional deletion of agrin in
adult motor neurons (375) or removal of agrin from the
NMJ by overexpression of the agrin-selective protease neurotrypsin in motor neurons (49) results in the fragmentation
and denervation of the NMJ reminiscent of the changes
observed at aged synapses. Interestingly, muscles in these
transgenic mice show many of the general features of sarcopenia already at a young age (73). In summary, the findings described above and the fact that impairment of neuromuscular transmission (as observed in CMS or sporadic
MG) is usually associated with structural changes of the
NMJ suggest that impairment of synaptic function may
contribute to sarcopenia. However, it is not established
whether changes of the NMJ are the cause or the consequence of sarcopenia. Thus unequivocal demonstration of
NMJ involvement would require a more systematic examination of NMJ function and structure at clearly defined
disease states. The assessment of functional innervation of
the entire muscle could for example be done by comparing
the muscle force generated in response to stimulation
through the nerve with that to direct electrical stimulation
of the muscle as has been done in a mouse model of a severe
congenital muscular dystrophy (170). This would allow distinguishing between impairment of neuromuscular transmission and general weakness of the muscle.
TINTIGNAC ET AL.
in mediating the effect of CR on lifespan. Thus the observed
heterogeneity between strains and between experimental
paradigms could in fact be used to unravel the genetic basis
for the observed lifespan extension (see recent review in Ref.
256).
SIRT1 also deacetylates peroxisome proliferator-activated
receptor ␥ (PPAR␥) coactivator 1␣ (PGC1␣ or PPARGC1␣), a
transcriptional coactivator that has been shown to be a key
regulator of mitochondrial biogenesis (359). Endurance exercise induces expression of PPARGC1a in human skeletal
muscle (344), and transgenic overexpression of PGC1␣ in
skeletal muscle of mice increases the number of mitochondria and the oxidative properties (259). Interestingly, endurance training in mice also reduces the fragmentation of
the NMJ in aged mice (88, 435), and overexpression of
PGC1␣ in skeletal muscle improves the morphology of the
NMJ in aged mice (453). More detailed morphological and
functional studies of the NMJs in the PGC1␣ transgenic
mice at young age show that NMJs are altered to a phenotype characteristic of slow muscle fibers and that those
changes also affect the presynaptic terminals (12). It is also
interesting to note that PGC1␣ has been implicated to act as
cotranscriptional regulator of GABP␣/GABP␤ in the expression of synaptic genes at the NMJ (173). In summary,
overexpression of PGC1␣ phenocopies, at least in part, endurance training and prevents some of the detrimental effects of aging including fragmentation of the NMJ. Conversely, a decrease in mitochondrial function in the course
of aging may also cause changes in NMJ structure observed
at higher age.
Another signaling pathway that is perturbed during aging
and thus may participate in sarcopenia is the mTOR pathway. As described in section VI, mTORC1 is a central hub
that integrates growth factor signaling and the nutrient status (in particular amino acid availability) to control cell
growth. Intriguingly, inhibition of mTORC1 has been im-
838
E. mTORC1 and Sarcopenia
Although the onset of sarcopenia was not investigated in
detail in the rapamycin-treated mice, age-related loss of
locomotory activity is delayed in male mice treated with
rapamycin (303), indicating a beneficial effect on muscle. It
appears paradoxical that rapamycin would prevent sarcopenia as mTORC1 signaling is known to promote cell
growth and to be required for compensatory hypertrophy in
response to hindlimb suspension (48). Moreover, skeletal
muscle fibers deficient for raptor (i.e., inactive mTORC1)
do not become hypertrophic in functional overloading experiments (37). Activation of the mTORC1 pathway by
infection of skeletal muscle with IGF1-expressing viruses
(27) or transgenic expression of IGF1 (316) induces a strong
muscle hypertrophy in young and old mice. Finally, expression of a constitutively active form of Akt/PKB also induces
muscle hypertrophy (48, 204, 244). In humans, rapamycin
given before resistance exercise, which is a well-established
stimulus to induce muscle hypertrophy, prevents the increase in muscle protein synthesis (120). These data have
led to the hypothesis that IGF1-PI3K-Akt/PKB-mTOR signaling might be lowered at high age. Indeed, mTORC1
signaling and subsequent increase in muscle protein synthesis in response to resistance exercise is blunted in more than
70-yr-old people compared with young adults (145). These
observations have led to the suggestion that local application of IGF1 and thus activation of mTORC1 might be a
valid strategy to counteract sarcopenia (70).
In contradiction to this suggestion, there is no significant
change in the activation state of IGF1 or Akt/PKB at old
age. In fact, phosphorylation of S6, a downstream target of
mTORC1, is increased in the muscles of old humans and of
old mice compared with young adults (378). These data
indicate that mTORC1 might rather be hyper- than hypoactive at high age. Consistent with the notion that hyperactivation of mTORC1 might boost and not prevent sarcopenia, long-term activation of this pathway in skeletal muscle
in mice is detrimental (83, 378). In fact, mice where
mTORC1 is constitutively active develop a myopathy at the
age of ⬃1 yr (83). The observed phenotype is reminiscent of
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Attempts to unravel the genetics of CR have resulted in the
identification of several candidate pathways. These include
the sirtuins, which are a class of nicotinamide adenine dinucleotide (NAD)⫹-dependent enzymes (171). Most of the
sirtuins have deacetylase activity. Cellular aging is accompanied by changes in mitochondrial function, and CR has
been shown to positively affect mitochondrial function and
to increase deacetylation. As SIRT3 is required for many of
the deacetylation processes activated in mitochondria
(180), a role of sirtuins in cellular aging has been suggested.
Indeed, overexpression of sirtuins and thus increased
deacetylation promotes longevity in several species (219,
426). However, others have not been able to reproduce this
effect (71). Thus, similar to CR, differences in the experimental set-up, in the genetic background of the animals, or
in the degree of overexpression in the different tissues may
affect the outcome.
plicated in longevity. For example, mutations that render
the TOR pathway hypoactive increase lifespan in yeast and
make the mutants not responsive to the life-prolonging effect of CR (217). A similar positive effect on lifespan is seen
with mutants for Sch9, the yeast ortholog of S6K. TOR
mutants also live longer in Drosophila (220) and C. elegans
(440). Most importantly, rapamycin fed to mice has a lifeprolonging effect (176), which is even seen when treatment
starts only at the age of 9 mo (303). Finally, body-wide
deletion of the mTORC1 target S6K1 also extends lifespan
in mice (393). Thus the evidence is strong that mTORC1
affects longevity, but it remains to be determined which
age-related diseases are mitigated by mTORC1 inhibition.
NEUROMUSCULAR JUNCTION DEVELOPMENT AND FUNCTION
mTOR signaling and muscle homeostasis may well also
affect NMJ integrity.
sarcopenia in several aspects, including the lowering of
muscle mass and size (37), and the lower specific muscle
force (83). Several lines of evidence indicate that the phenotype is, at least in part, caused by the block of autophagy
induction in skeletal muscle (84), a process that is well
known to be involved in longevity (307). Consistent with
this interpretation, blockade of autophagy by muscle-specific elimination of Atg7 results in a late-onset myopathy
(285). Interestingly, autophagy is strongly induced by endurance exercise (178) and CR (459), which are both paradigms that ameliorate sarcopenia. Thus the “anti-aging”
effect of CR and endurance exercise might be based on its
dampening mTORC1 signaling and thereby increasing autophagy flux to allow clearance of nonfunctional proteins
and organelles (e.g., mitochondria). The recent finding that
rapamycin lowers rather than increases the atrophy of muscle upon denervation (417) supports the notion that sustained activation of mTORC1 might accelerate the development of sarcopenia.
VIII. CONCLUSIONS AND PERSPECTIVE
The findings that the neuromuscular system undergoes major changes during aging (see also FIGURE 7A) have led to a
new wave of molecular studies investigating the role of the
genes involved in NMJ development in the course of aging.
In our view, it will be of utmost importance to first firmly
establish that changes in neuromuscular transmission indeed contribute to sarcopenia as sarcopenia can arise from
many different causes. For example, progressive denervation of muscle can be the result of sporadic death of indi-
Although little is known whether changes in mTOR signaling also affect the NMJ directly, recent work indicates that
the turnover of the AChRs is affected by the overall metabolic state of the muscle. For example, fasting and denervation also affect the overall turnover of AChRs by increasing
the number of AChR-containing endosomes/lysosomes
(223). Thus changes in muscle metabolism that influence
3 months
2 years
AChR
Nf/Syn
A
B
motor neuron
: challenge
?
: inhibitor
NMJ
exercise
CR
sarcolemma
?
FIGURE 7. Age-dependent changes in the structure of
the NMJ and possible mechanisms involved. A: whole
mount view of the NMJ in the extensor digitorum longus of
a 3-mo-old (left) and a 2-yr-old mouse (right). AChRs are
visualized by ␣-BTX (red) and motor axons and presynaptic
nerve terminals by antibodies to neurofilament and synaptophysin (green). Note that the NMJ in the 2-yr-old mouse
is partially fragmented. Scale bar ⫽ 20 ␮m. B: age-dependent challenges (red flashes) of the neuromuscular system
affect motor neuron survival, neuromuscular junction integrity, mitochondrial function, autophagy, and stability of the
muscle fiber sarcolemma. Behavioral changes, such as
exercise and caloric restriction (CR), have been shown to
inhibit (green T-bars) some of those challenges. For some
of the affected systems, inhibitors are not known (indicated
with green question mark). See main text for details.
autophagosome
exercise
CR
mitochondria
exercise
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The essential role of neuromuscular transmission for skeletal muscle contraction (e.g., respiration) warrants a highly
reliable and redundant system to build and maintain the
NMJ. In the past two decades, we have witnessed the rapid
progress in the understanding of the molecular basis of
NMJ development. These findings are largely corroborated
by genetic evidence showing that many of the key molecules
involved in NMJ development are also the cause of CMS or
are targets of sporadic MG. Because of the vital function of
neuromuscular transmission, these diseases are, however,
very rare.
TINTIGNAC ET AL.
vidual motor neurons. Longitudinal studies indicate that
there is an up to 50% loss of motor neurons at high age
(427). This process leaves muscle fibers transiently denervated (FIGURE 7) and evokes sprouting by the surviving
motor neurons, which increases the size of the motor units.
As sarcopenic patients indeed show an increase in motor
unit size (119), motor neuron loss may also contribute to
the loss of muscle mass although reinnervation of muscle by
the surviving motor neurons may delay this process. In addition to this presynaptic effect, there is plenty of evidence
that changes in the function and the metabolism of skeletal
muscle contribute to sarcopenia (FIGURE 7). Thus current
evidence strongly indicates that sarcopenia is caused by
pathological processes in both pre- and postsynaptic cells.
ACKNOWLEDGMENTS
We thank Drs. C. Slater, A. Punga, and P. Castets for helpful comments on the manuscript. We thank Drs. Iwona
Ksiazek and Shuo Lin for the pictures shown in FIGURES 1C
AND 7A, respectively.
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Address for reprint requests and other correspondence:
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Another exciting new avenue is the observation that pathways and behavioral paradigms involved in longevity, such
as CR and endurance exercise, also show antisarcopenic
effects in animal studies and affect NMJ structure. Moreover, there is also evidence that the molecular pathways
mediating the positive effects of CR or endurance training
also affect sarcopenia and NMJ structure. These observations raise the exciting possibility that identifying the molecular signatures in skeletal muscle that affect whole-body
metabolism may allow identifying pathways that are also
beneficial to the neuromuscular system. Finally, recent heterochronic parabiosis experiments, where the circulation of
two animals of different age is joined, provide evidence that
factors circulating in the blood may counteract aspects of
aging in muscle (100).
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