Molecular Mechanisms Underlying Maturation and Maintenance of The

Review

Feature Review

Molecular mechanisms underlying

maturation and maintenance of the vertebrate neuromuscular junction

1,2,3 1,2,3 1,2,3

Lei Shi , Amy K.Y. Fu and Nancy Y. Ip

Division of Life Science, The Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong, China

State Key Laboratory of Molecular Neuroscience, The Hong Kong University of Science and Technology, Clear Water Bay,

Hong Kong, China

JNU-HKUST Joint Laboratory for Neuroscience and Innovative Drug Research, Jinan University, 601 Huang-Pu Avenue West,

Guangzhou 510632, Guangdong, China

The vertebrate neuromuscular junction (NMJ), a periph- stability of the NMJ increases on maturation, and the

eral synapse formed between motoneuron and skeletal synaptic structure, once mature, persists throughout post-

muscle, is characterized by a protracted postnatal period natal life, thus enabling life-long effective motor perfor-

of maturation and life-long maintenance. In neuromus- mance [2,3]. This is in contrast to the rapid turnover of

cular disorders such as congenital myasthenic syn- central synapses; for example, mature dendritic spines

dromes (CMSs), disruptions of NMJ maturation and/ (where excitatory synapses reside) can be rapidly elimi-

or maintenance are frequently observed. In particular, nated and replaced by newly formed ones when neural

defective neuromuscular transmission associated with circuits are reconstructed [1].

structural and molecular abnormalities at the pre- and Abnormalities in NMJ maturation or maintenance are

postsynaptic membranes, as well as at the synaptic cleft, among the most common pathological causes of congenital

has been reported in these patients. Here, we review myasthenic syndromes (CMSs), a heterogeneous group of

recent advances in the understanding of molecular and hereditary neuromuscular diseases characterized by mus-

cellular events that mediate NMJ maturation and main- cle weakness and a reduced safety margin for synaptic

tenance. The underlying regulatory mechanisms, includ- transmission [4]. Thus, delineation of the molecular mech-

ing key molecular regulators at the presynaptic nerve anisms underlying NMJ maturation and maintenance is

terminal, synaptic cleft, and postsynaptic muscle mem- important for an understanding of the pathogenesis of

brane, are discussed. CMSs and to provide signiﬁcant insights for potential

therapeutic approaches. Here we summarize the molecular

Introduction and cellular events mediating NMJ maturation and main-

Structural and functional maturation of established syn- tenance, and review current knowledge on the regulation

apses during development controls the consolidation and of these processes.

reﬁnement of neural circuits. For example, experience-

dependent neuronal plasticity in the brain requires matu- Structural and molecular changes during NMJ

ration and maintenance of a subset of newly formed syn- maturation and maintenance

apses, while others are eliminated [1]. The peripheral Remarkable changes occur at the postsynaptic membrane

synapse neuromuscular junction (NMJ) has long been used during postnatal maturation of the NMJ [2,3]. First,

as a model system for studying the general principles of alterations in AChR clusters take place at the levels of

synapse development owing to its large size and experi- morphology, subunit composition and stability. The neo-

mental accessibility [2]. The vertebrate NMJ utilizes ace- natal plaque-like AChR clusters with uniform receptor

tylcholine (ACh) as its neurotransmitter, which binds to density are transformed into multi-perforated elaborate

ion-channel ACh receptors (AChRs) highly concentrated on branches that display a pretzel-like shape (Figure 1a and

the postsynaptic membrane. Notably, the NMJ goes Box 1) [5–7]. The embryonic-speciﬁc g-subunit of AChR is

through a unique series of maturation processes that differ replaced by the e-subunit during early postnatal stage,

from those of the central synapse [2,3]. First, whereas leading to increased calcium conductance of the receptor

maturation of the central synapse normally occurs within [8,9]. The half-life of the synaptic AChRs increases on

hours, the NMJ takes weeks to reﬁne its molecular and maturation, and insertion of newly synthesized AChRs

structural organization to achieve its mature form, which and recycling of internalized AChRs contribute to mainte-

exhibits efﬁcient neurotransmission. Furthermore, the nance of the high density of synaptic receptors [10–12].

Second, remodeling of AChR clusters is accompanied by a

Corresponding author: Ip, N.Y. ([email protected]).

change in the topological organization of the postsynaptic

Keywords: synapse; acetylcholine receptor; neuromuscular disorder; congenital

myasthenic syndrome; agrin; MuSK. membrane, which forms invaginations called junctional

Review Trends in Neurosciences July 2012, Vol. 35, No. 7

polyinnervated NMJs transform into singly innervated

(a) P5 P7 P10 P14 P18 Ad

ones through axon withdrawal during early postnatal

weeks (Figure 1b) [13]. The axon terminals subsequently

differentiate and become perfectly aligned with the AChR

clusters, and synaptic vesicles loaded with readily releas-

able neurotransmitters are concentrated at the active

(b) P5 P10 P14 Ad

zone. Furthermore, the molecular constituents of the ex-

tracellular matrix (ECM; the basal lamina) become spe-

* cialized at the synaptic cleft during NMJ maturation [14].

Terminal Schwann cells, which cap the nerve terminals,

* retract from the synaptic cleft after NMJ formation and

contribute to growth and long-term maintenance of the

TRENDS in Neurosciences synapse [15].

Figure 1. Morphological changes in the mouse NMJ during postnatal development.

(a) The morphology of early postnatal AChR clusters appears as an oval-like plaque. Presynaptic regulation during NMJ maturation and

Within the first three postnatal weeks, AChR plaque is transformed into a multi- stabilization

perforated array of branches that has a pretzel-like shape. P, postnatal day; Ad, adult.

Agrin: a master organizer of the NMJ

(b) During the first two postnatal weeks, elimination of axonal inputs occurs at the

initial poly-innervated endplates (asterisks), transforming them into singly- Before the arrival of approaching motoneuron terminals,

innervated (the retraction bulb of an eliminated axon is indicated by an

AChR clusters and postsynaptic-like structures are spon-

arrowhead). Furthermore, the axon terminals differentiate to perfectly align with

taneously formed at the center of muscle ﬁbers, a process

the AChR cluster branches. Photomicrographs show examples of NMJs at the tibialis

anterior muscle from ICR mice during the postnatal stages indicated. Postsynaptic known as muscle prepatterning (Box 3) [16,17]. Although

AChR clusters and presynaptic nerve terminals are labeled by a-bungarotoxin (BTX;

the initial formation of prepatterned structures is indepen-

red) and synaptophysin/neurofilament (green), respectively. Scale bars, 10 mm.

Readers are referred to [5,6] for more details. dent of nerve terminals, subsequent postsynaptic differen-

tiation requires trans-synaptic activity. Nerve-derived

folds (Box 2). Finally, the postsynaptic molecular composi- agrin, a large heparin sulfate proteoglycan synthesized

tion becomes specialized. A distinct set of postsynaptic and released from the nerve terminal, is a master organiz-

proteins and signaling molecules is selectively transcribed er that governs postsynaptic differentiation and stabiliza-

in the subsynaptic myoﬁber nuclei and aggregates at high tion [18]. Functional NMJ is absent in mice deﬁcient in

density in the postsynaptic membrane, accompanied by neural agrin [19]. The action of agrin on postsynaptic

reorganization of the cytoskeleton at the postsynaptic assembly and stabilization is primarily through activation

submembrane. of the muscle-speciﬁc kinase (MuSK) receptor on the mus-

Concurrent with the postsynaptic maturation, pro- cle membrane [20]. Agrin also mediates MuSK-indepen-

nounced alterations also occur at motor nerve terminals, dent signaling through binding to dystroglycan, a

the synaptic cleft and terminal Schwann cells. Newborn transmembrane protein important for NMJ maturation

and maintenance [21–24]. Furthermore, it has been sug-

gested that proteolytic cleavage-mediated downregulation

Box 1. Topological transformation of AChR clusters during of agrin inﬂuences the development of junctional folds and

NMJ maturation the topological transformation of AChR clusters [25].

Three morphological changes occur at AChR clusters during

postsynaptic maturation of the NMJ: The presynaptic ubiquitin system and integrity regulate

postsynaptic maturation

(i) Selective regions within the initial oval-like AChR plaques are

Ubiquitin proteasome-dependent degradation of proteins

disassembled to generate multiple perforations, which then

is essential for controlling precise synaptic function

develop into a complex array of branches (pretzel-like appear-

ance) [5–7]. through targeted protein degradation and clearance.

(ii) The size of AChR clusters continues to increase as muscle fibers Two ubiquitin-regulating proteins, ubiquitin speciﬁc pep-

grow. Two patterns of AChR addition have been found during

tidase 14 (Usp14) and ubiquitin carboxyl-terminal hydro-

NMJ maturation. During the plaque-to-pretzel transition, new

lase L1 (UCH-L1), regulate the development of presynaptic

receptors are preferentially added to peripheral regions of the

terminals and postsynaptic maturation of the NMJ [26,27].

clusters, suggesting circumferential growth of AChR clusters

[115]. Once the pretzel shape is obtained, AChR cluster expansion Presynaptic deﬁcits, including nerve terminal swelling and

changes to an intercalar pattern whereby the number of AChRs

accumulation of phosphorylated neuroﬁlament, and post-

increases at a constant density, whereas the number or arrange-

synaptic abnormalities, including defective plaque-to-

ment of the receptor branches remains unchanged [2,138].

pretzel transition of AChR clusters and imprecise synaptic

(iii) The stability of AChR clusters increases on maturation. Mature

clusters have a significantly longer half-life than plaque-shaped apposition, are observed in mice lacking Usp14 or UCH-L1

clusters and they are more resistant to disassembly [10,139]. [26,27]. Thus, maintenance of a proper ubiquitin pool in

motoneurons is crucial for the development of presynaptic

Signaling pathways governing assembly, stabilization and dis-

terminals and postsynaptic maturation of the NMJ.

persal of synaptic receptors work in concert to drive the maturation

The effect of presynaptic function on NMJ maturation is

of AChR clusters (Figure 2). Notably, the topological maturation of

AChR clusters can occur to a remarkable extent in the absence of also evident in molecular pathological studies of spinal

presynaptic input [115]. Thus, myotubes aneurally cultured on muscular atrophy (SMA), a motor neuron disease caused

essential basal lamina components are a useful model for studying

by mutations of the survival motor neuron 1 (SMN1) gene.

the molecular mechanism underlying AChR maturation in vitro.

In mouse models of SMA, impaired structure and function

442

Review Trends in Neurosciences July 2012, Vol. 35, No. 7

Box 2. Junctional fold: a unique postsynaptic structure of vertebrate NMJ

As the NMJ matures during the postnatal stage, the postsynaptic voltage-gated Na channels are concentrated in the depths of the

membrane sinks to form a gutter (the primary fold), and the gutter folds. It has been suggested that this mutually exclusive arrange-

membrane invaginates to form a number of secondary folds (the ment of neurotransmitter receptors and Na channels amplifies the

junctional folds, Figure Ia) [2,3]. The junctional folds run parallel to synaptic current by causing a voltage drop across the electrical

each other, spaced at regular intervals, and are oriented orthogonal to resistance of the interfolds, and thus lowers the threshold for action

the long axis of the muscle fiber (Figure Ib) [7]. Abnormalities of the potential generation [141].

fold architecture are the most common phenotypes associated with Different subdomains of the junctional folds serve as platforms for

neuromuscular diseases, highlighting the junctional fold as a spatial segregation of postsynaptic receptors and membrane-

structural determinant for efficient neuromuscular transmission [140]. associated proteins (Figure Ib,c) [142–144]. Furthermore, the

Here, we summarize several functional properties of junctional constituents of the synaptic basal lamina (BL) are also segregated.

folds that are important for governing synaptic transmission and Laminin a2, a5 and b2 are present at both the synaptic cleft and fold

orchestrating postsynaptic signaling: BL, whereas a4 only shows high levels between folds at the

AChRs are highly enriched (10 000/mm ) at the crests of junctional synaptic cleft BL [36].

folds, where ACh-releasing sites are in close proximity, whereas

(a) P0 P5 P14 Ad

(b)

Schwann Nerve Schwann cell terminal cell

Synaptic Synaptic vesicles Extrasynaptic basal lamina basal lamina

Active zones

Junctional folds

Muscle

Agrin, neuregulin, laminin α 2/α5/β2 , nidogen-2, collagen IV (α3- α6)/XIII, ColQ, perlecan, AChE

AChRs, MuSK, LRP4, rapsyn, integrin, utrophin, α/β-dystroglycan, sarcoglycan, α-syntrophin, α-dystrobrevin-1

Na+ channels, ErbB2/B4, ankyrin, dystrophin, α/β2-syntrophin, α-dystrobrevin-2, nNOS

TRENDS in Neurosciences

Figure I. Schematic illustrating development of the vertebrate NMJ, with a particular emphasis on junctional folds. (a) Formation of synaptic gutters and junctional folds

during postnatal maturation of NMJ. P, postnatal day; Ad, adult. (b) An adult vertebrate NMJ. (c) Molecules expressed at the tops and troughs of adult junctional folds,

as well as those expressed at the synaptic basal lamina. Such molecules include glycoproteins (e.g., laminins), receptors (e.g., MuSK and ErbB receptors), and

scaffolding proteins (e.g., dystrophin, syntrophins, and utrophin).

443

Review Trends in Neurosciences July 2012, Vol. 35, No. 7

Box 3. Prepatterning of the postsynaptic apparatus and its maturation. First, synaptic laminins regulate the matura-

role in NMJ development tion of presynaptic terminals. Laminin b2 controls the

proper distribution of active zones and synaptic vesicles

In the early phase of synaptogenesis of the NMJ, intrinsically

through binding to voltage-gated calcium channels at the

programmed muscle aggregation of AChR clusters and postsynap-

tic proteins occurs in the central regions of myofibers [16,17]. This nerve terminal membrane [31–33]. Laminins also regulate

prepatterned process is innervation-independent and involves differentiation of nerve terminals by inhibiting excessive

assembly of the MuSK–LRP4–Dok-7–rapsyn complex and locally

outgrowth of terminal branches [30,34,35]. Second, synaptic

activated transcription of genes encoding AChR subunits, MuSK,

laminins guide the development of junctional folds and the

and AChE. MuSK activity, the embryonic AChR g-subunit and the

2+ topological maturation of AChR clusters. Laminin-b2-null

muscle L-type Ca channel dihydropyridine receptor are important

for the generation of muscle prepatterning [145–147]. It has been mice display markedly reduced junctional folds [35,36].

reported that MuSK activation in the absence of agrin is through its Studies on individual laminin heterotrimers show that

binding to Wnt ligands, which play regulatory roles in both muscle

laminin 421 and 521 are important for the plaque-to-pretzel

prepatterning and synapse formation [148–152].

transition of AChR clusters [36,37]. Third, synaptic lami-

Why is it necessary to form a prepatterned postsynaptic apparatus

before nerve innervation? Several lines of evidence suggest that nins ensure the precise apposition of pre- and postsynaptic

prepatterning is required to guide the growth of motoneurons and structures of the NMJ by preventing the intrusion of

thus restrict the sites of synaptogenesis within a narrow band in the

Schwann cell processes into the synaptic cleft [35]. Further-

central regions of muscle cells [145,147]. Moreover, when myotubes

more, laminin 421 is speciﬁcally required for the precise

in the initial stage of synaptogenesis are cultured in vitro, AChR

apposition of presynaptic active zones and openings of post-

clusters only form in the central regions in response to exogenous

agrin application [153]. This suggests that prepatterning determines synaptic junctional folds [36]. In line with the morphological

the sites that are recognizable by agrin, which may spatially restrict defects observed, electrophysiological studies revealed that

synapse formation. However, the initial synaptic contact is not

laminin b2 mutant mice exhibit dramatically reduced

determined by aneural AChR clusters, because synapses can be

evoked and spontaneous neuromuscular transmission from

formed either by incorporating the preexisting postsynaptic appa-

the ﬁrst postnatal week onwards, providing direct evidence

ratus or by assembling new postsynaptic structures in regions

where aneural AChR clusters are absent [153,154]. that synaptic laminins are crucial for NMJ maturation at

On innervation, aneural AChR clusters that are not directly opposed the functional level [38]. It is interesting to note that deletion

to motoneuron terminals are rapidly dispersed by the neurotransmit-

of individual synaptic laminin a chains results in relatively

ter ACh [155,156]. Cdk5, a serine/threonine kinase concentrated at the

subtle but non-overlapping NMJ defects compared to the

postsynaptic NMJ, is activated by ACh and is required for ACh-

severe NMJ defects exhibited by laminin-b2-deﬁcient mice.

induced removal of prepatterned AChR clusters that are not stabilized

by agrin [155–157]. It has been shown that Cdk5 mediates ACh- Indeed, different laminin a chains have distinct distribution

dependent dispersal of AChR clusters through two molecular patterns at the synaptic basal lamina, suggesting that the

mechanisms, upregulation of calpain activity and the interaction of

three laminin heterotrimers act collaboratively to coordi-

Cdk5 with the intermediate filament protein nestin [67,158,159].

nate the process of NMJ maturation [36].

Collagens comprise another group of core constituents of

of the NMJ are among the initial pathological signs that synaptic basal lamina. Collagen IV chains (a3–a6) accu-

become evident during the ﬁrst postnatal week [28,29]. mulate at the mature NMJ and are important for mainte-

Presynaptic abnormalities include decreased synaptic ves- nance of adult motoneuron terminals [32]. Collagen XIII, a

icle release and swollen nerve terminals with accumula- transmembrane collagen concentrated at the postnatal

tion of neuroﬁlament. Postsynaptic maturation is also NMJ, regulates topological maturation of AChR clusters

impaired in these SMA mouse models, as indicated by and precise apposition of active zones and junctional folds

the aberrant pretzel transition of AChR clusters and [39]. Moreover, collagen Q (ColQ) is a synaptic collagen

delayed switch of the fetal AChR g-subunit to the adult that binds to the ACh-hydrolyzing enzyme acetylcholines-

e-subunit. This ﬁnding provides evidence that the integrity terase (AChE) and regulates ACh accumulation at the

of presynaptic structure and function is a determining synaptic basal lamina [40]. AChE deﬁciency due to ColQ

factor for postsynaptic maturation. mutations is commonly associated with CMSs [4]. The

synaptic anchorage of AChE by ColQ is partly dependent

ECM proteins at the synaptic cleft control both pre- and on the association of ColQ with MuSK receptor [40].

postsynaptic maturation Reciprocally, ColQ inﬂuences MuSK-dependent AChR

The synaptic basal lamina, composed of a distinct set of clustering [41]. Perlecan, a synaptic heparan sulfate pro-

ECM proteins, extends through the synaptic cleft into the teoglycan linked to both ColQ and dystroglycan, is also

junctional folds. The synaptic basal lamina comprise four important for synaptic recruitment of AChE [42].

main types of ECM proteins: heparan sulfate proteogly- Nidogen-2, the synaptic isoform of nidogen, is the third

cans (e.g., the neural agrin), laminins, collagens and nido- main group of synaptic ECM proteins. Nidogen-2 becomes

gens [14]. As described above, neural agrin is an concentrated at the NMJ from the second postnatal week,

indispensible organizer for postsynaptic assembly. The and is essential for topological maturation of AChR clus-

other three core species, synthesized and deposited by ters and long-term postsynaptic maintenance [43].

muscle ﬁbers, play important roles in the maturation

and maintenance of both pre- and postsynaptic structures. Postsynaptic receptors and associated signaling

Three laminin heterotrimers, laminin 221 (a2b2g1), 421 pathways during NMJ maturation and maintenance

(a4b2g1) and 521 (a5b2g1), are present at the synaptic cleft MuSK-mediated postsynaptic signaling pathways

[30]. Genetic deletion studies of different laminin chains Agrin-induced activation of the MuSK receptor is the most

revealed that synaptic laminins play multiple roles in NMJ extensively studied signaling pathway in the assembly and

444

Review Trends in Neurosciences July 2012, Vol. 35, No. 7

maintenance of the postsynaptic apparatus. Agrin acti- agrin-dependent and -independent manners [48,62].

vates MuSK through binding to the MuSK co-receptor Multiple mutations of Dok-7 have been reported in a

low-density lipoprotein receptor-related protein 4 (LRP4) major form of CMS that affects proximal muscle strength

[44,45]. Structural analysis has revealed that a tetrameric [63]. Smaller NMJ size and simpliﬁed junctional folds are

complex formed by two agrin–LRP4 heterodimers medi- two major abnormalities identiﬁed in these patients,

ates MuSK dimerization and activation [46,47]. Activation although signiﬁcant clinical heterogeneity is associated

of MuSK also requires its interaction with the cytoplasmic with Dok-7 mutations [4]. Dok-7 comprises an N-termi-

adaptor docking protein-7 (Dok-7) [48]. Like agrin-deﬁcient nal pleckstrin homology (PH) domain, a phosphotyrosine-

mice, mice lacking either MuSK receptor, LRP4, or Dok-7 binding (PTB) domain and a C-terminal region contain-

show a complete absence of postsynaptic NMJ structure ing several tyrosine sites. Whereas Dok-7 binds to the

and die perinatally because of respiratory failure [48–51]. juxtamembrane region of MuSK through its PTB domain,

The MuSK–LRP4–Dok-7 complex is also required for pre- it self-dimerizes through its PH-PTB domain, and thus

patterning of AChR clusters (Box 3). It is noteworthy that promotes dimerization and trans-autophosphorylation of

CMSs caused by mutations in components of MuSK sig- MuSK [48,64]. Notably, mutations that truncate the C-

naling can manifest after birth or later during postnatal terminal region of Dok-7 are the most common Dok-7

development [4]. Thus, insufﬁciency or misregulation of mutations found in CMSs, suggesting that the intact C-

MuSK signaling may exert a minimal effect on initial NMJ terminal domain is required for precise NMJ function in

assembly, but mainly impacts subsequent synapse matu- vivo. Agrin stimulation triggers phosphorylation of Dok-7

ration or maintenance. at two C-terminal tyrosine sites, leading to subsequent

The role of MuSK in NMJ maintenance has been char- recruitment of adaptor proteins Crk and Crk-L, which

acterized in mice with conditional inactivation of MuSK are required for proper NMJ formation [65]. The C

during early postnatal stages [52]. AChR clusters disperse terminus of Dok-7 is also required for agrin-independent

on postnatal MuSK inactivation, and the mutant mice transformation of AChR clusters into a pretzel shape on

show severe muscle weakness and signiﬁcantly shortened maturation [63]. The mechanism underlying the require-

lifespan. Several mutations of MuSK that lead to reduced ment of Dok-7 in this agrin-independent regulation

MuSK expression and impaired interaction with Dok7 awaits further characterization.

have been identiﬁed in CMS patients [53–55]. Reduced MuSK recruits a plethora of signaling molecules to

AChR clusters and simpliﬁed junctional folds are found in guide both the aggregation and stabilization of AChR

these patients, indicative of impaired NMJ maturation. A clusters [20]. Rapsyn, a membrane-associated cytoplasmic

mouse model of CMS with mutations in MuSK (a missense protein that directly interacts with AChRs, is indispensible

mutation on one allele and deletion of the kinase domain on for MuSK-induced AChR clustering and stabilization [49].

the other allele) shows progressive neuromuscular impair- On agrin stimulation, the AChR–rapsyn interaction is

ments during postnatal development, including fragmen- strengthened, thereby enhancing the linkage of AChRs

tation of AChR clusters with reduced receptor density, to the cytoskeleton [66]. Several rapsyn-interacting mole-

aberrant plaque-to-pretzel transition, simpliﬁed junctional cules, including heat shock protein 90 (HSP90), a-actinin

folds, and defective neuromuscular transmission [54]. It and calpain, are important for stabilizing AChR clusters

has been suggested that both the kinase activity and the [66–68]. Rapsyn is also involved in agrin-stimulated tar-

integrity of the extracellular domain of MuSK are impor- geting of AChRs to lipid rafts, which are membrane micro-

tant for the laminin-induced formation of topologically domains serving as a platform for AChR clustering [69,70].

complex AChR clusters [56]. Moreover, MuSK activation leads to reorganization of the

Several mechanisms that modulate the extent of MuSK AChR-associated actin cytoskeleton through modulation of

activation are important for postsynaptic maintenance. Rho family GTPases or their downstream molecules [71–

First, association of MuSK with the adapter protein 74]. Caveolin-3, a component of caveolae lipid rafts, reg-

Dok-7 directly leads to activation of the receptor (see ulates MuSK-induced Rac1 activation and AChR cluster-

below). Tid1 (tumorous imaginal disc 1), a heat shock ing [75]. Interestingly, several components of the Wnt

protein that binds to MuSK, promotes the MuSK–Dok-7 signaling pathway participate in MuSK-dependent AChR

interaction and is important for AChR clustering [57]. clustering, including b-catenin, dishevelled, and adenoma-

Second, surface expression of MuSK is regulated by the tous polyposis coli (APC) [71,76,77].

postsynaptic ubiquitin–proteasome system or endocytic A subset of molecules downstream of MuSK is

pathway. E3 ubiquitin ligase PDZ-domain-containing speciﬁcally important for AChR stabilization. The non-

RING ﬁnger 3 (PDZRN3), which binds to MuSK and receptor tyrosine kinase Src-family kinases (SFKs) phos-

promotes its ubiquitination or degradation, is implicated phorylate the AChR b-subunit in response to agrin,

in NMJ growth and maturation [58]. Furthermore, endo- which facilitates cytoskeletal anchorage and stabiliza-

cytosis of MuSK by N-ethylmaleimide-sensitive factor tion of AChR clusters [78]. Interestingly, SFK activity is

(NSF) is required for mediation of the effect of agrin–MuSK not important for NMJ formation, but is required for

signaling [59]. Finally, MuSK activity can be modulated postsynaptic maintenance and stabilization of AChR

via its phosphorylation by cytoplasmic kinases, including clusters [79,80]. It has been reported that SFKs enhance

serine/threonine kinase casein kinase 2 (CK2) and tyrosine the association of postsynaptic components with choles-

kinase Abl [60,61]. terol-rich lipid rafts of the postsynaptic membrane,

Dok-7, a muscle adaptor protein associated with which in turn maintain the phosphorylation of these

MuSK, acts as a co-activator of MuSK in both components by SFKs [81].

445

Review Trends in Neurosciences July 2012, Vol. 35, No. 7

Muscle erythroblastic leukemia viral oncogene homolog regulates the integrity of AChR morphology and junc-

B (ErbB) receptors modulate AChR clustering and tional folds [94,95]. Third, AChR clusters are important

synapse maintenance for the accumulation and maintenance of other synaptic

ErbB receptors, a group of receptor tyrosine kinases, are regulators, including rapsyn and actin skeletal-associat-

expressed at the postsynaptic muscle membrane and the ing proteins [96,97].

surface of terminal Schwann cells. Neuregulins, the

ligands for ErbB receptors, can be synthesized and re- Molecules that regulate the postsynaptic cytoskeleton

leased into the basal lamina by both motoneurons and The dystrophin–glycoprotein complex controls

muscles. Whereas Schwann cell ErbB receptors are postsynaptic maturation and maintenance

essential for the survival and maintenance of Schwann The dystrophin–glycoprotein complex (DGC) is a multi-

cells [82], muscle ErbB receptors play modulatory roles molecular complex linking AChR clusters to both the

in postsynaptic gene transcription and AChR clustering. extracellular basal lamina and the intracellular cytoskele-

Although early studies reported that neuregulin-activat- ton [98]. The main components of the DGC include a

ed ErbB signaling promotes the transcription of synaptic transmembrane module containing a- and b-dystroglycan,

genes using in vitro cultured myotubes, later in vivo the associated cytoplasmic actin-binding proteins dystro-

evidence conﬁrmed that muscle ErbB receptors are dis- phin and utrophin, and two groups of cytoplasmic proteins,

pensable for synaptic gene expression and synapse a-dystrobrevins and syntrophins [98]. Some of the compo-

formation [83]. Nevertheless, muscle ErbB receptors nents are expressed along the entire muscle ﬁber, whereas

are associated with the MuSK receptor via the adaptor others have synaptic isoforms that are present exclusively

protein Erbin, and it has been suggested that they or concentrated at the NMJ. Whereas extrasynaptic DGC

potentiate MuSK-dependent AChR clustering [84,85]. provides structural support for the integrity and stability

Furthermore, muscle ErbB receptors regulate AChR sta- of muscle ﬁbers [99], synaptic DGC mainly contributes to

bilization and efﬁcient neuromuscular transmission via postsynaptic maturation and long-term maintenance of

phosphorylation of a-dystrobrevin-1, a cytoplasmic pro- the NMJ.

tein that is important for NMJ maturation and mainte- The dystroglycan module, containing extracellular a-

nance [86]. dystroglycan, which binds to agrin and laminins, and its

associated transmembrane b-dystroglycan, which binds to

The neurotrophin receptors rapsyn and dystrophin/utrophin, is a key component that

The neurotrophins and their cognate receptors, which play links other DGC proteins to the synaptic basal lamina and

multi-functional roles in synapse development, regulate to AChR clusters. Dystroglycan is aggregated by synaptic

postsynaptic maturation and maintenance of the NMJ. laminins and acts as a laminin receptor to mediate lami-

Tropomyosin receptor kinase B (TrkB)-mediated postsyn- nin-dependent clustering and topological maturation of

aptic signaling is important for long-term stabilization of AChRs [37,100]. Moreover, dystroglycan participates in

AChR clusters during postnatal life. Disruption of TrkB- agrin-regulated AChR stabilization in a MuSK-indepen-

mediated signaling in muscle in either early postnatal dent manner. Dystroglycan-deﬁcient myotubes fail to form

stages or adulthood by overexpression of a dominant-neg- stable AChR clusters in response to agrin, although the

ative TrkB mutant causes disassembly of AChR clusters detailed mechanism of how agrin modulates the function of

[87]. Furthermore, a precocious age-like decline in neuro- DGC is not clear [101,102].

muscular function including decreased muscle strength is Dystrophin and its synaptic homolog utrophin consti-

+/–

evident in trkB mice, which is associated with fragmen- tute a group of actin-binding proteins that interact with b-

tation of postsynaptic specializations in these mutants dystroglycan. Studies on mice with single or double mu-

[88]. TrkB-dependent regulation of postsynaptic mainte- tation of dystrophin or utrophin have revealed that the two

nance is probably mediated, at least in part, by its muscle- proteins play similar but non-overlapping roles in regu-

released ligand neurotrophin-4 (NT-4) in an autocrine lating AChR maintenance and junctional fold formation

fashion [89]. NT-4 and brain-derived neurotrophic factor [102–105]. Deﬁciency of both dystrophin and utrophin

(BDNF), another cognate ligand of TrkB, are also involved leads to failed synaptic localization of two cytoplasmic

in regulating the maturation of presynaptic nerve term- DGC components, a-dystrobrevins and syntrophins,

inals [90]. which are crucial downstream molecules in DGC signal-

ing. a-Dystrobrevin-null mice exhibit fragmentation of

AChR clusters themselves are required for postsynaptic AChR clusters and abnormal distribution of receptors to

maintenance the troughs of junctional folds [102]. Two isoforms of a-

A high synaptic density of AChRs is the hallmark of dystrobrevin, a-dystrobrevin-1 and a-dystrobrevin-2, are

mature NMJs. Interestingly, AChRs themselves also present at the NMJ, whereas only a-dystrobrevin-1 is

play crucial roles in guiding synapse maturation and important for the synaptic function of a-dystrobrevins

maintenance. First, replacement of the embryonic AChR [106]. Tyrosine phosphorylation of a-dystrobrevin-1 at

g-subunit by the postnatal e-subunit not only increases its C terminus by ErbB receptors is a regulatory mecha-

the calcium permeability of the receptor but is also nism for dystrobrevin-dependent stabilization of AChR

important for synaptic maintenance of AChR clusters clusters [86].

and the development of junctional folds [91–93]. Second, Two syntrophins, a- and b2-syntrophin, are present at

phosphorylation of the AChR b-subunit by SFKs the NMJ [107]. a-Syntrophin is indispensible for the mat-

enhances the association of AChRs with rapsyn and uration and stabilization of AChR clusters, as well as

446

Review Trends in Neurosciences July 2012, Vol. 35, No. 7

junctional fold development [108,109]. b2-Syntrophin Protein kinases and other synaptic regulators

plays additional roles in synaptic maintenance, because implicated in NMJ maturation and maintenance

deﬁciency of both syntrophins results in more severe post- Two serine/threonine kinases, protein kinase C (PKC) and

synaptic abnormalities than those observed in mice with a Ca /calmodulin-dependent protein kinase II (CaMKII),

single a-syntrophin mutation [110]. Detailed analysis of are involved in NMJ maturation and maintenance. PKC

AChR dynamics further revealed an increased turnover activity is important for the plaque-to-pretzel transition of

rate and a decreased recycling rate for receptors in a- AChRs and for motor axon withdrawal during synapse

syntrophin-null mice, leading to a reduction in synaptic elimination [122]. CaMKII promotes the insertion of

AChR density [108]. Interestingly, this a-syntrophin-de- recycled AChRs into synaptic sites, and thus contributes

pendent regulation of AChR stability is mediated in part to the maintenance of synaptic receptor density [123]. PKC

through the recruitment of a-dystrobrevin-1 [108]. Neuro- and CaMKII are also implicated in suppression of the

nal nitric oxide synthase (nNOS), another molecule extrasynaptic expression of AChR subunits [124,125].

recruited by syntrophin, is also important for syntro- Neural cell adhesion molecule (NCAM) is a group of

phin-mediated signaling in synaptic maintenance membrane-associated adhesion molecules concentrated at

[111,112]. both pre- and postsynaptic sites and terminal Schwann

cells. Whereas NMJ formation is normal in mice lacking

Cytoplasmic regulators for the actin cytoskeleton the three synaptic isoforms of NCAM, presynaptic matu-

modulate plaque-to-pretzel transformation ration, including synaptic vesicle cycling and neurotrans-

The aggregation, movement, stabilization and disassembly mitter release, is disrupted in these mutants [126–128].

of AChR clusters are tightly controlled by the local dynam- Moreover, delayed formation of junctional folds is found at

ics of the postsynaptic membrane-associated cytoskeleton the postsynaptic side of the mutant NMJ [129].

[113,114]. The use of aneurally cultured myotubes as an in The P2X2 receptors are ligand-gated ion channels acti-

vitro model to study postsynaptic NMJ maturation (Box 1) vated by extracellular ATP released by the nerve terminal.

has begun to unravel the molecular mechanisms underly- Mice lacking the P2X2 receptor subunit exhibit delayed

ing cytoskeletal regulation during the plaque-to-pretzel plaque-to-pretzel transition, AChR fragmentation and re-

transition of AChR clusters [115]. Concentration of F-actin duced junctional folds [130].

with elevated turnover rate occurs at sites where perfora-

tions of AChRs subsequently occur, suggesting that elevat- Synaptic transcription during NMJ maturation and

ed actin cytoskeletal dynamics guides the loss of AChRs maintenance

[116,117]. Locally increased endocytosis may also play a Synapse-speciﬁc genes are locally transcribed at special-

role in the removal of AChRs [115–117]. Moreover, AChRs ized nuclei located beneath motoneuron terminals (known

that are newly added to perforations are rapidly incorpo- as subsynaptic nuclei). A number of synaptic genes, includ-

rated into existing AChR branches, indicating that AChR ing those encoding AChR subunits, utrophin and AChE,

redistribution is another mechanism for maintaining pre- contain an Ets-binding promoter sequence (the N-box ele-

viously formed perforations [117]. A group of actin-regu- ment) to target their synaptic transcription through bind-

lating proteins are present at perforations, among which ing to Ets transcription factors. A mutation of the N-box of

actin-depolymerizing factor (ADF)/coﬁlin appears to be the gene encoding AChRe is associated with CMSs, sug-

important for AChR dynamics [117]. Moreover, perfora- gesting that N-box-directed subsynaptic transcription is

tion-associated proteins form a podosome-like structure (a important for NMJ development [131]. Erm (Ets-related

dynamic actin-rich, adhesive organelle) that is capable of molecule), an Ets transcription factor enriched postsynap-

remodeling ECM localization [116]. LL5b, a phosphatidy- tically, is important for the N-box-dependent subsynaptic

linositol-binding protein associated with podosomes, is transcription and maintenance of AChR clusters [132].

implicated in the regulation of AChR clustering in cultured Another Ets transcription factor, growth-associated bind-

myotubes [116,118]. Thus, it would be interesting to fur- ing protein (GABP), is implicated in NMJ maturation,

ther explore the in vivo function of podosomes in NMJ although it remains unclear whether GABP regulates

maturation. subsynaptic transcription in vivo [133–136].

Ephexin1, a guanine nucleotide exchange factor (GEF) N-box-dependent subsynaptic transcription is activated

of RhoA GTPase, has recently been identiﬁed as an by MuSK receptors through a Rac/Cdc42-mediated JNK

essential actin regulator for both structural and func- pathway [137]. Interestingly, this MuSK–Rac/Cdc42–JNK

tional maturation of the NMJ [119,120]. Whereas NMJ pathway also upregulates the transcription of genes with-

formation is normal in ephexin1-null mice, both the out the N-box element, such as musk [137]. Which tran-

plaque-to-pretzel transition of AChR clusters and junc- scription factors are involved in this N-box-independent

tional fold development are impaired when ephexin1 is transcription awaits further investigation. The neuregu-

absent. Ephexin1 destabilizes AChR clusters through lin–ErbB pathway, although not directly acting on sub-

activation of RhoA, which is important for receptor loss synaptic transcription, may have modulatory roles in

during AChR perforation. Given the upregulation of MuSK-regulated synaptic gene expression.

ephexin1 activity by EphA receptors and cyclin-depen-

dent kinase 5 (Cdk5) at central synapses [121], it would Concluding remarks

be of interest to investigate whether a similar mecha- Studies on the prototypic synapse NMJ have shed light on

nism underlies ephexin1-dependent postsynaptic matu- the general principles of synapse assembly. Nonetheless,

ration in the NMJ. the molecular mechanisms underlying NMJ maturation

447

Review Trends in Neurosciences July 2012, Vol. 35, No. 7

Table 1. Molecules involved in NMJ maturation and maintenance

Name Roles during NMJ maturation and maintenance Related Function and regulation Refs neuromuscular

disease

AChR cluster JFD Other synaptic development development

Presynaptic molecules

Agrin Aggregation, + CMS MuSK activation; binding [19,25]

maintenance, PPT to dystroglycan;

proteolytic cleavage by

motoneuron proteases

NCAM Maintenance + Presynaptic maturation [126–129]

SMN PPT, AChR g- to + Presynaptic function SMA [28,29]

e-subunit switch and maintenance

UCH-L1 Presynaptic function Regulation of the ubiquitin [27]

and maintenance system in motoneurons

Usp14 PPT Presynaptic differentiation; Regulation of the ubiquitin [26]

synaptic apposition system in motoneurons

Molecules at the synaptic basal lamina

Laminins (a2b2g1, PPT + Distribution of active CMS Binding to and [30–38]

a4b2g1, a5b2g1) zones and synaptic aggregating dystroglycan

vesicles; synaptic

apposition

Collagens (IV, XIII) PPT Presynaptic maturation [32,39]

and maintenance;

synaptic apposition

Nidogen-2 PPT, maintenance [43]

Postsynaptic molecules

MuSK Aggregation, + CMS, myasthenia Activation of multiple [20,50,53–56]

maintenance, PPT gravis signaling pathways

Dok-7 Aggregation, + CMS MuSK [48,62–65]

maintenance, PPT

Rapsyn Aggregation, + CMS Linking AChRs to [49,69]

maintenance, PPT cytoskeleton and

membrane lipid rafts

SFKs Maintenance Tyr phosphorylation of [78–80]

AChR b

AChR b Maintenance + CMS Tyr phosphorylated by [94,95]

SFKs; anchored to the

cytoskeleton via binding

to rapsyn

AChR e Maintenance + AChR deﬁciency [9,91–93]

syndrome, CMS

TrkB Maintenance Presynaptic maturation [87–90]

ErbB Maintenance + Tyr phosphorylation of [86]

a-dystrobrevin

P2X2 nucleotide Maintenance + Synaptic apposition [130] receptor

Synaptic DGC Maintenance, PPT + Linking AChRs to the [102–106,

synaptic basal lamina and 108–110,112]

cytoskeleton

CaMKII Synaptic recycling Suppression of [123,124]

extrasynaptic gene

transcription

Ephexin1 PPT, destabilization + Synaptic apposition RhoA activation [119]

ADF/coﬁlin PPT, vesicular trafﬁcking Depolymerization of F- [117]

actin; regulated by 14-3-3z

PKC PPT Synaptic elimination; [122,125]

Suppression of

extrasynaptic gene

transcription

Erm PPT, maintenance AChR positioning at Activation of subsynaptic [132]

the muscle central transcription

band

GABP PPT + [133,134]

Abbreviations: JFD, junctional fold development; PPT, plaque-to-pretzel transition.

NCAM is expressed at nerve terminals, muscle membranes, and Schwann cells. Modulation of presynaptic maturation is more consistent with regulation by presynaptic

NCAM [126–128].

Components of synaptic DGC include a- and b-dystroglycan, dystrophin/utrophin, a-dystrobrevin, a- and b2-syntrophin, and nNOS.

448

Review Trends in Neurosciences July 2012, Vol. 35, No. 7

Laminin Synaptic basal lamina

Wnt EphrEphrinAinA AChE AgAgrinrin ACh AgrinAgrin ColQColQ PerlecanPerlecan AgAgrinrin ActivityActivity α-DG-DG NRGNRG NT-4NT-4 AATPTP AChRAChR NRGRG AChRAChR LRP4 LRP4 β-DG LipidLipid rafts ErbBrbB ErbBErbB TrkBTrkB EphAEphA P P P P CCaveolin-3aveolin-3 ErbinErbin RRapsynapsyn RRapsynapsyn RapsRapsynyn RapsynRapsyn α APC AAblbl -act-actinin Hsp9Hsp90 β Calpai MMuSKuSK PPDZRN3DZRN3 P2X 2+ Utrophin/Utrophin/ -catenin CK2 2 Ca inin SFKSFKss receptorreceptor dystrophindystrophin 0β n CaCa2+2+ NNSFSF P DDvlvl Ephexin1Ephexin1 α GGGTGT Tid1 PKCPKC -DB CalpainCalpain Dok-7Dok-7 SyntrophiSyntrophinn CaMKIICaMKII PTPsPTPs nNOnNOSS Crk/Crk-LCrk/Crk-L RhoA p35 p25 F-acF-actintin 14-3-314-3-3ζ Rac1/Cdc42Rac1/Cdc42 JJNKNK Nestin Cdk5Cdk5 ADFADF/cofilin/cofilin

SubsynapticSubsynaptic nucleus Erm, GABP, etc GGeneene transcriptiontranscription

Addition or stabilization of AAChRsChRs RemovalRemoval or destabilization of AChRAChRs

PostsynapticPostsynaptic maturation Muscle

TRENDS in Neurosciences

Figure 2. Postsynaptic maturation is regulated by positive and negative signaling pathways for AChR stabilization. Positive signaling pathways – including those activated

by the MuSK receptor, the dystroglycan–glyoprotein complex, and ErbB and TrkB receptors – act on the assembly and maintenance of AChR clusters. CaMKII, a serine/

threonine kinase activated by electrical activity, promotes insertion of AChRs into synaptic sites. Synapse-specific gene transcription, such as that mediated by the Ets

transcription factor Erm, is important for AChR maintenance. By contrast, ephexin1-mediated upregulation of RhoA and activation of PKC and the actin modulator ADF/

cofilin regulate AChR removal during plaque-to-pretzel transition. Cdk5-dependent dispersal of AChR clusters, enclosed in a dotted square, is a signaling pathway that has

only been demonstrated in embryonic stages. In addition, dashed arrows indicate regulatory pathways established in vitro or in systems other than the NMJ. Abbreviations:

Abl, v-abl Abelson murine leukemia viral oncogene homolog; ACh, acetylcholine; AChE, acetylcholinesterase; AChR, acetylcholine receptor; ADF, actin depolymerizing

factor; APC, adenomatous polyposis coli; CaMKII, Ca /calmodulin-dependent kinase II; Cdk5, cyclin-dependent kinase 5; CK2, casein kinase 2; ColQ, collagen Q; DB,

dystrobrevin; DG, dystroglycan; Dvl, dishevelled; Eph, erythropoietin-producing human hepatocellular carcinoma; ErbB, erythroblastic leukemia viral oncogene homolog;

Erm, Ets-related molecule; GABP, growth-associated binding protein; GGT, geranylgeranyltransferase; JNK, c-Jun N-terminal kinase; LRP4, low-density lipoprotein

receptor-related protein 4; MuSK, muscle-specific kinase; nNOS, neuronal nitric oxide synthase; NRG, neuregulin; NT-4, neurotrophin-4; NSF, N-ethylmaleimide sensitive

factor; PDZRN, PDZ-domain-containing RING finger 3; PKC, protein kinase C; PTP, protein tyrosine phosphatase; SFKs, Src-family kinases; Tid1, tumorous imaginal disc 1;

Trk, tropomyosin receptor kinase.

Box 4. Outstanding questions

What signaling pathways control the plaque-to-pretzel transition What are the molecular mechanisms that underlie junctional fold

of AChR clusters? formation?

How are signals transduced from laminins to AChR maturation? The The structural integrity of the folds is tightly associated with the

DGC transmembrane protein dystroglycan may act as a laminin synaptic basal lamina and cytoskeleton network in subsynaptic

receptor to regulate this process. However, most of the components areas, although how these molecules are regulated is not fully

of the DGC have more direct roles in the maintenance of synaptic understood. The presynaptic input seems essential for the forma-

receptors, and not in the plaque-to-pretzel transformation per se. tion of junctional folds, because denervated muscles and aneurally

The MuSK receptor is required for the formation of pretzel clusters cultured myotubes fail to develop folds [7,115]. Notably, forced

induced by laminin-containing substrates [46,115]. It would be of expression of neural agrin in the extrasynaptic regions of inner-

interest to dissect whether laminin directly activates MuSK or vated muscle fibers is sufficient to induce junctional fold-like

indirectly activates MuSK via the regulation of other factors, such as membrane invaginations [160]. It will thus be important to elucidate

Wnt or LRP4. the signaling pathways regulated by agrin or other trans-synaptic

What signals initiate receptor loss within AChR plaques? An inputs that control junctional fold development.

enigmatic issue is how selective regions of AChRs clusters are

dispersed during receptor maturation. Cytoplasmic actin regula- What roles does muscle electrical activity play in NMJ develop-

tors, such as ephexin1 and ADF/cofilin, act as AChR-dispersing ment?

factors during pretzel formation. However, how these factors are Neurally evoked muscle electrical activity has been implicated in

activated is not clear. Another important question is whether AChR subunit switch, in suppression of extrasynaptic gene

AChR-dispersing factors and stabilizing factors have spatially transcription and in muscle development. However, the mole-

differentiated regulation within AChR plaques. cular events mediated by muscle activity remain elusive. More-

What is the role of presynaptic input in the plaque-to-pretzel over, because numerous molecules regulate NMJ development,

transition of AChR clusters? Although topologically complex it is important to verify whether the roles of these molecules

AChR clusters can form independent of nerve innervation, agrin in NMJ development involve primary effects on NMJ structure

and normal presynaptic development influence AChR maturation and/or function, or secondary effects via modulation of muscle

physiologically. Their roles are worthy of further investigation. activity.

449

Review Trends in Neurosciences July 2012, Vol. 35, No. 7

16 Lin, W. et al. (2001) Distinct roles of nerve and muscle in

and maintenance are only beginning to be understood. We

postsynaptic differentiation of the neuromuscular synapse. Nature

have reviewed molecular regulators at pre- and postsyn-

410, 1057–1064

aptic sites and the synaptic cleft, discussed the regulation

17 Yang, X. et al. (2001) Patterning of muscle acetylcholine receptor gene

of postsynaptic maturation and maintenance (Table 1 and expression in the absence of motor innervation. Neuron 30, 399–410

Figure 2), and summarized some outstanding questions 18 Bezakova, G. and Ruegg, M.A. (2003) New insights into the roles of

agrin. Nat. Rev. Mol. Cell Biol. 4, 295–308

that remain to be addressed (Box 4). It is noteworthy that

19 Gautam, M. et al. (1996) Defective neuromuscular synaptogenesis in

there are other important molecular events during NMJ

agrin-deﬁcient mutant mice. Cell 85, 525–535

maturation and maintenance. These include the molecular

20 Wu, H. et al. (2010) To build a synapse: signaling pathways in

mechanism by which synapse elimination is regulated [13], neuromuscular junction assembly. Development 137, 1017–1033

retrograde signals for presynaptic development [20], and 21 Bowe, M.A. et al. (1994) Identiﬁcation and puriﬁcation of an agrin

receptor from Torpedo postsynaptic membranes: a heteromeric

the roles of terminal Schwann cells in NMJ maintenance

complex related to the dystroglycans. Neuron 12, 1173–1180

[15]. A thorough understanding of these molecular mecha-

22 Campanelli, J.T. et al. (1994) A role for dystrophin-associated

nisms will potentially contribute to the development of

glycoproteins and utrophin in agrin-induced AChR clustering. Cell

targeted therapeutic approaches for CMSs. 77, 663–674

23 Gee, S.H. et al. (1994) Dystroglycan-alpha, a dystrophin-associated

Acknowledgments glycoprotein, is a functional agrin receptor. Cell 77, 675–686

We apologize to authors whose work could not be discussed or cited 24 Sugiyama, J. et al. (1994) Dystroglycan binds nerve and muscle agrin.

because of space limitations. This study was supported in part by the Neuron 13, 103–115

Research Grants Council of Hong Kong (6421/05 M, 661007, 661109, 25 Bolliger, M.F. et al. (2010) Speciﬁc proteolytic cleavage of agrin

660810 and HKUST6/CRF/08), the Area of Excellence Scheme of the regulates maturation of the neuromuscular junction. J. Cell Sci.

University Grants Committee (AoE/B-15/01-II), the Hong Kong Jockey 123, 3944–3955

Club, the National Natural Science Foundation of China (81101015), and 26 Chen, P.C. et al. (2009) The proteasome-associated deubiquitinating

the Bureau of Science and Information Technology of Guangzhou enzyme Usp14 is essential for the maintenance of synaptic ubiquitin

Municipality (2011J2200048). N.Y.I. was a recipient of the Croucher levels and the development of neuromuscular junctions. J. Neurosci.

Foundation Senior Research Fellowship. 29, 10909–10919

27 Chen, F. et al. (2010) Ubiquitin carboxyl-terminal hydrolase L1 is

References required for maintaining the structure and function of the

neuromuscular junction. Proc. Natl. Acad. Sci. U.S.A. 107, 1636–1641

1 Holtmaat, A. and Svoboda, K. (2009) Experience-dependent

28 Kariya, S. et al. (2008) Reduced SMN, protein impairs maturation of

structural synaptic plasticity in the mammalian brain. Nat. Rev.

the neuromuscular junctions in mouse models of spinal muscular

Neurosci. 10, 647–658

atrophy. Hum. Mol. Genet. 17, 2552–2569

2 Sanes, J.R. and Lichtman, J.W. (2001) Induction, assembly,

29 Kong, L. et al. (2009) Impaired synaptic vesicle release and

maturation and maintenance of a postsynaptic apparatus. Nat.

immaturity of neuromuscular junctions in spinal muscular atrophy

Rev. Neurosci. 2, 791–805

mice. J. Neurosci. 29, 842–851

3 Sanes, J.R. and Lichtman, J.W. (1999) Development of the vertebrate

30 Patton, B.L. et al. (1997) Distribution and function of laminins in the

neuromuscular junction. Annu. Rev. Neurosci. 22, 389–442

neuromuscular system of developing, adult, and mutant mice. J. Cell

4 Engel, A.G. et al. (2010) What have we learned from the congenital

Biol. 139, 1507–1521

myasthenic syndromes. J. Mol. Neurosci. 40, 143–153

31 Nishimune, H. et al. (2004) A synaptic laminin–calcium channel

5 Slater, C.R. (1982) Postnatal maturation of nerve–muscle junctions in

interaction organizes active zones in motor nerve terminals. Nature

hindlimb muscles of the mouse. Dev. Biol. 94, 11–22

432, 580–587

6 Balice-Gordon, R.J. and Lichtman, J.W. (1993) In vivo observations of

32 Fox, M.A. et al. (2007) Distinct target-derived signals organize

pre- and postsynaptic changes during the transition from multiple to

formation, maturation, and maintenance of motor nerve terminals.

single innervation at developing neuromuscular junctions. J.

Cell 129, 179–193

Neurosci. 13, 834–855

33 Chen, J. et al. (2011) Calcium channels link the muscle-derived

7 Marques, M.J. et al. (2000) From plaque to pretzel: fold formation and

synapse organizer laminin beta2 to Bassoon and CAST/Erc2 to

acetylcholine receptor loss at the developing neuromuscular junction.

organize presynaptic active zones. J. Neurosci. 31, 512–525

J. Neurosci. 20, 3663–3675

34 Noakes, P.G. et al. (1995) Aberrant differentiation of neuromuscular

8 Villarroel, A. and Sakmann, B. (1996) Calcium permeability increase

junctions in mice lacking s-laminin/laminin beta 2. Nature 374,

of endplate channels in rat muscle during postnatal development. J.

258–262

Physiol. 496, 331–338

35 Patton, B.L. et al. (1998) Synaptic laminin prevents glial entry into

9 Missias, A.C. et al. (1996) Maturation of the acetylcholine receptor in

the synaptic cleft. Nature 393, 698–701

skeletal muscle: regulation of the AChR gamma-to-epsilon switch.

36 Patton, B.L. et al. (2001) Properly formed but improperly localized

Dev. Biol. 179, 223–238

synaptic specializations in the absence of laminin alpha4. Nat.

10 Akaaboune, M. et al. (1999) Rapid and reversible effects of activity on

Neurosci. 4, 597–604

acetylcholine receptor density at the neuromuscular junction in vivo.

37 Nishimune, H. et al. (2008) Laminins promote postsynaptic

Science 286, 503–507

maturation by an autocrine mechanism at the neuromuscular

11 Bruneau, E. et al. (2005) Identiﬁcation of nicotinic acetylcholine

junction. J. Cell Biol. 182, 1201–1215

receptor recycling and its role in maintaining receptor density at

38 Knight, D. et al. (2003) Functional analysis of neurotransmission at

the neuromuscular junction in vivo. J. Neurosci. 25, 9949–9959

beta2-laminin deﬁcient terminals. J. Physiol. 546, 789–800

12 Bruneau, E.G. and Akaaboune, M. (2006) The dynamics of recycled

39 Latvanlehto, A. et al. (2010) Muscle-derived collagen XIII regulates

acetylcholine receptors at the neuromuscular junction in vivo.

maturation of the skeletal neuromuscular junction. J. Neurosci. 30,

Development 133, 4485–4493

12230–12241

13 Wyatt, R.M. and Balice-Gordon, R.J. (2003) Activity-dependent

40 Cartaud, A. et al. (2004) MuSK is required for anchoring

elimination of neuromuscular synapses. J. Neurocytol. 32, 777–

794 acetylcholinesterase at the neuromuscular junction. J. Cell Biol.

165, 505–515

14 Singhal, N. and Martin, P.T. (2011) Role of extracellular matrix

41 Sigoillot, S.M. et al. (2010) ColQ controls postsynaptic differentiation

proteins and their receptors in the development of the vertebrate

at the neuromuscular junction. J. Neurosci. 30, 13–23

neuromuscular junction. Dev. Neurobiol. 71, 982–1005

42 Arikawa-Hirasawa, E. et al. (2002) Absence of acetylcholinesterase at

15 Feng, Z. and Ko, C.P. (2008) The role of glial cells in the formation and

the neuromuscular junctions of perlecan-null mice. Nat. Neurosci. 5,

maintenance of the neuromuscular junction. Ann. N. Y. Acad. Sci. 119–123 1132, 19–28

450

Review Trends in Neurosciences July 2012, Vol. 35, No. 7

43 Fox, M.A. et al. (2008) A synaptic nidogen: developmental regulation 72 Luo, Z.G. et al. (2003) Implication of geranylgeranyltransferase I in

and role of nidogen-2 at the neuromuscular junction. Neural Dev. 3, 24 synapse formation. Neuron 40, 703–717

44 Zhang, B. et al. (2008) LRP4 serves as a coreceptor of agrin. Neuron 60, 73 Weston, C. et al. (2000) Agrin-induced acetylcholine receptor

285–297 clustering is mediated by the small guanosine triphosphatases Rac

45 Kim, N. et al. (2008) Lrp4 is a receptor for agrin and forms a complex and Cdc42. J. Cell Biol. 150, 205–212

with MuSK. Cell 135, 334–342 74 Weston, C. et al. (2003) Cooperative regulation by Rac and Rho of

46 Gomez, A.M. and Burden, S.J. (2011) The extracellular region of Lrp4 agrin-induced acetylcholine receptor clustering in muscle cells. J.

is sufﬁcient to mediate neuromuscular synapse formation. Dev. Dyn. Biol. Chem. 278, 6450–6455

240, 2626–2633 75 Hezel, M. et al. (2010) Caveolin-3 promotes nicotinic acetylcholine

47 Zong, Y. et al. (2012) Structural basis of agrin–LRP4–MuSK signaling. receptor clustering and regulates neuromuscular junction activity.

Genes Dev. 26, 247–258 Mol. Biol. Cell 21, 302–310

48 Okada, K. et al. (2006) The muscle protein Dok-7 is essential for 76 Wang, J. et al. (2003) Regulation of acetylcholine receptor clustering

neuromuscular synaptogenesis. Science 312, 1802–1805 by the tumor suppressor APC. Nat. Neurosci. 6, 1017–1018

49 Gautam, M. et al. (1995) Failure of postsynaptic specialization to 77 Zhang, B. et al. (2007) b-Catenin regulates acetylcholine receptor

develop at neuromuscular junctions of rapsyn-deﬁcient mice. Nature clustering in muscle cells through interaction with rapsyn. J.

377, 232–236 Neurosci. 27, 3968–3973

50 DeChiara, T.M. et al. (1996) The receptor tyrosine kinase MuSK is 78 Mohamed, A.S. et al. (2001) Src-class kinases act within the agrin/

required for neuromuscular junction formation in vivo. Cell 85, 501–512 MuSK pathway to regulate acetylcholine receptor phosphorylation,

51 Weatherbee, S.D. et al. (2006) LDL-receptor-related protein 4 is cytoskeletal anchoring, and clustering. J. Neurosci. 21, 3806–3818

crucial for formation of the neuromuscular junction. Development 79 Smith, C.L. et al. (2001) Src, Fyn, and Yes are not required for

133, 4993–5000 neuromuscular synapse formation but are necessary for

52 Hesser, B.A. et al. (2006) Synapse disassembly and formation of new stabilization of agrin-induced clusters of acetylcholine receptors. J.

synapses in postnatal muscle upon conditional inactivation of MuSK. Neurosci. 21, 3151–3160

Mol. Cell. Neurosci. 31, 470–480 80 Sadasivam, G. et al. (2005) Src-family kinases stabilize the

53 Chevessier, F. et al. (2004) MUSK, a new target for mutations causing neuromuscular synapse in vivo via protein interactions,

congenital myasthenic syndrome. Hum. Mol. Genet. 13, 3229–3240 phosphorylation, and cytoskeletal linkage of acetylcholine

54 Chevessier, F. et al. (2008) A mouse model for congenital myasthenic receptors. J. Neurosci. 25, 10479–10493

syndrome due to MuSK mutations reveals defects in structure and 81 Willmann, R. et al. (2006) Cholesterol and lipid microdomains

function of neuromuscular junctions. Hum. Mol. Genet. 17, 3577–3595 stabilize the postsynapse at the neuromuscular junction. EMBO J.

55 Maselli, R.A. et al. (2010) Mutations in MUSK causing congenital 25, 4050–4060

myasthenic syndrome impair MuSK–Dok-7 interaction. Hum. Mol. 82 Lin, W. et al. (2000) Aberrant development of motor axons and

Genet. 19, 2370–2379 neuromuscular synapses in erbB2-deﬁcient mice. Proc. Natl. Acad.

56 Mazhar, S. and Herbst, R. (2012) The formation of complex Sci. U.S.A. 97, 1299–1304

acetylcholine receptor clusters requires MuSK kinase activity and 83 Escher, P. et al. (2005) Synapses form in skeletal muscles lacking

structural information from the MuSK extracellular domain. Mol. neuregulin receptors. Science 308, 1920–1923

Cell. Neurosci. 49, 475–486 84 Simeone, L. et al. (2010) Identiﬁcation of Erbin interlinking MuSK

57 Linnoila, J. et al. (2008) A mammalian homolog of Drosophila and ErbB2 and its impact on acetylcholine receptor aggregation at the

tumorous imaginal discs, Tid1, mediates agrin signaling at the neuromuscular junction. J. Neurosci. 30, 6620–6634

neuromuscular junction. Neuron 60, 625–641 85 Ngo, S.T. et al. (2012) Neuregulin-1 potentiates agrin-induced

58 Lu, Z. et al. (2007) Regulation of synaptic growth and maturation by a acetylcholine receptor clustering via muscle speciﬁc kinase

synapse-associated E3 ubiquitin ligase at the neuromuscular phosphorylation. J. Cell Sci. 125, 1531–1543

junction. J. Cell Biol. 177, 1077–1089 86 Schmidt, N. et al. (2011) Neuregulin/ErbB regulate neuromuscular

59 Zhu, D. et al. (2008) Muscle-speciﬁc receptor tyrosine kinase junction development by phosphorylation of alpha-dystrobrevin. J.

endocytosis in acetylcholine receptor clustering in response to Cell Biol. 195, 1171–1184

agrin. J. Neurosci. 28, 1688–1696 87 Gonzalez, M. et al. (1999) Disruption of TrkB-mediated signaling

60 Cheusova, T. et al. (2006) Casein kinase 2-dependent serine induces disassembly of postsynaptic receptor clusters at

phosphorylation of MuSK regulates acetylcholine receptor neuromuscular junctions. Neuron 24, 567–583

aggregation at the neuromuscular junction. Genes Dev. 20, 1800–1816 88 Kulakowski, S.A. et al. (2011) Reduced TrkB expression results in

61 Finn, A.J. et al. (2003) Postsynaptic requirement for Abl kinases in precocious age-like changes in neuromuscular structure,

assembly of the neuromuscular junction. Nat. Neurosci. 6, 717–723 neurotransmission, and muscle function. J. Appl. Physiol. 111,

62 Inoue, A. et al. (2009) Dok-7 activates the muscle receptor kinase 844–852

MuSK and shapes synapse formation. Sci. Signal. 2, ra7 89 Belluardo, N. et al. (2001) Neuromuscular junction disassembly and

63 Beeson, D. et al. (2006) Dok-7 mutations underlie a neuromuscular muscle fatigue in mice lacking neurotrophin-4. Mol. Cell. Neurosci. 18,

junction synaptopathy. Science 313, 1975–1978 56–67

64 Bergamin, E. et al. (2010) The cytoplasmic adaptor protein Dok7 90 Wang, T. et al. (1995) Neurotrophins promote maturation of

activates the receptor tyrosine kinase MuSK via dimerization. Mol. developing neuromuscular synapses. J. Neurosci. 15, 4796–4805

Cell 39, 100–109 91 Witzemann, V. et al. (1996) Acetylcholine receptor epsilon-subunit

65 Hallock, P.T. et al. (2010) Dok-7 regulates neuromuscular synapse deletion causes muscle weakness and atrophy in juvenile and adult

formation by recruiting Crk and Crk-L. Genes Dev. 24, 2451–2461 mice. Proc. Natl. Acad. Sci. U.S.A. 93, 13286–13291

66 Dobbins, G.C. et al. (2008) a-Actinin interacts with rapsyn in agrin- 92 Missias, A.C. et al. (1997) Deﬁcient development and maintenance of

stimulated AChR clustering. Mol. Brain 1, 18 postsynaptic specializations in mutant mice lacking an ‘adult’

67 Chen, F. et al. (2007) Rapsyn interaction with calpain stabilizes AChR acetylcholine receptor subunit. Development 124, 5075–5086

clusters at the neuromuscular junction. Neuron 55, 247–260 93 Schwarz, H. et al. (2000) Different functions of fetal and adult AChR

68 Luo, S. et al. (2008) HSP90 beta regulates rapsyn turnover and subtypes for the formation and maintenance of neuromuscular

subsequent AChR cluster formation and maintenance. Neuron 60, synapses revealed in epsilon-subunit-deﬁcient mice. Eur. J.

97–110 Neurosci. 12, 3107–3116

69 Marchand, S. et al. (2002) Rapsyn escorts the nicotinic acetylcholine 94 Friese, M.B. et al. (2007) Synaptic differentiation is defective in mice

receptor along the exocytic pathway via association with lipid rafts. J. lacking acetylcholine receptor beta-subunit tyrosine phosphorylation.

Neurosci. 22, 8891–8901 Development 134, 4167–4176

70 Zhu, D. et al. (2006) Lipid rafts serve as a signaling platform for 95 Borges, L.S. et al. (2008) Identiﬁcation of a motif in the acetylcholine

nicotinic acetylcholine receptor clustering. J. Neurosci. 26, 4841–4851 receptor beta subunit whose phosphorylation regulates rapsyn

71 Luo, Z.G. et al. (2002) Regulation of AChR clustering by Dishevelled association and postsynaptic receptor localization. J. Neurosci. 28,

interacting with MuSK and PAK1. Neuron 35, 489–505 11468–11476

451

Review Trends in Neurosciences July 2012, Vol. 35, No. 7

96 Marangi, P.A. et al. (2001) Acetylcholine receptors are required 121 Fu, W.Y. et al. (2007) Cdk5 regulates EphA4-mediated dendritic spine

for agrin-induced clustering of postsynaptic proteins. EMBO J. 20, retraction through an ephexin1-dependent mechanism. Nat.

7060–7073 Neurosci. 10, 67–76

97 Bruneau, E.G. et al. (2008) Acetylcholine receptor clustering is 122 Lanuza, M.A. et al. (2002) Pre- and postsynaptic maturation of the

required for the accumulation and maintenance of scaffolding neuromuscular junction during neonatal synapse elimination

proteins. Curr. Biol. 18, 109–115 depends on protein kinase C. J. Neurosci. Res. 67, 607–617

98 Banks, G.B. et al. (2003) The postsynaptic submembrane machinery 123 Martinez-Pena and Valenzuela, I. et al. (2010) Calcium/calmodulin

at the neuromuscular junction: requirement for rapsyn and the kinase II-dependent acetylcholine receptor cycling at the mammalian

utrophin/dystrophin-associated complex. J. Neurocytol. 32, 709–726 neuromuscular junction in vivo. J. Neurosci. 30, 12455–12465

99 Straub, V. and Campbell, K.P. (1997) Muscular dystrophies and the 124 Tang, H. et al. (2001) CaM kinase II-dependent suppression of

dystrophin–glycoprotein complex. Curr. Opin. Neurol. 10, 168–175 nicotinic acetylcholine receptor delta-subunit promoter activity. J.

100 Montanaro, F. et al. (1998) Laminin and alpha-dystroglycan mediate Biol. Chem. 276, 26057–26065

acetylcholine receptor aggregation via a MuSK-independent 125 Klarsfeld, A. et al. (1989) Regulation of muscle AChR alpha subunit

pathway. J. Neurosci. 18, 1250–1260 gene expression by electrical activity: involvement of protein kinase C

101 Jacobson, C. et al. (2001) The dystroglycan complex is necessary for and Ca . Neuron 2, 1229–1236

stabilization of acetylcholine receptor clusters at neuromuscular 126 Rafuse, V.F. et al. (2000) Structural and functional alterations of

junctions and formation of the synaptic basement membrane. J. neuromuscular junctions in NCAM-deﬁcient mice. J. Neurosci. 20,

Cell Biol. 152, 435–450 6529–6539

102 Grady, R.M. et al. (2000) Maturation and maintenance of the 127 Polo-Parada, L. et al. (2001) Alterations in transmission, vesicle

neuromuscular synapse: genetic evidence for roles of the dynamics, and transmitter release machinery at NCAM-deﬁcient

dystrophin–glycoprotein complex. Neuron 25, 279–293 neuromuscular junctions. Neuron 32, 815–828

103 Grady, R.M. et al. (1997) Subtle neuromuscular defects in utrophin- 128 Polo-Parada, L. et al. (2004) Distinct roles of different neural cell

deﬁcient mice. J. Cell Biol. 136, 871–882 adhesion molecule (NCAM) isoforms in synaptic maturation revealed

104 Deconinck, A.E. et al. (1997) Postsynaptic abnormalities at the by analysis of NCAM 180 kDa isoform-deﬁcient mice. J. Neurosci. 24,

neuromuscular junctions of utrophin-deﬁcient mice. J. Cell Biol. 1852–1864

136, 883–894 129 Moscoso, L.M. et al. (1998) Organization and reorganization of

105 Banks, G.B. et al. (2009) Truncated dystrophins can inﬂuence neuromuscular junctions in mice lacking neural cell adhesion

neuromuscular synapse structure. Mol. Cell. Neurosci. 40, 433–441 molecule, tenascin-C, or ﬁbroblast growth factor-5. J. Neurosci. 18,

106 Grady, R.M. et al. (2003) Tyrosine-phosphorylated and 1465–1477

nonphosphorylated isoforms of alpha-dystrobrevin: roles in skeletal 130 Ryten, M. et al. (2007) Abnormalities in neuromuscular junction

muscle and its neuromuscular and myotendinous junctions. J. Cell structure and skeletal muscle function in mice lacking the P2X2

Biol. 160, 741–752 nucleotide receptor. Neuroscience 148, 700–711

107 Kramarcy, N.R. and Sealock, R. (2000) Syntrophin isoforms at the 131 Ohno, K. et al. (1999) Congenital myasthenic syndrome caused by a

neuromuscular junction: developmental time course and differential mutation in the Ets-binding site of the promoter region of the

localization. Mol. Cell. Neurosci. 15, 262–274 acetylcholine receptor epsilon subunit gene. Neuromuscul. Disord.

108 Martinez-Pena and Valenzuela, I. et al. (2011) Nicotinic acetylcholine 9, 131–135

receptor stability at the NMJ deﬁcient in alpha-syntrophin in vivo. J. 132 Hippenmeyer, S. et al. (2007) ETS transcription factor Erm controls

Neurosci. 31, 15586–15596 subsynaptic gene expression in skeletal muscles. Neuron 55, 726–740

109 Adams, M.E. et al. (2000) Absence of alpha-syntrophin leads to 133 O’Leary, D.A. et al. (2007) Targeting of the ETS factor GABPalpha

structurally aberrant neuromuscular synapses deﬁcient in disrupts neuromuscular junction synaptic function. Mol. Cell. Biol.

utrophin. J. Cell Biol. 150, 1385–1398 27, 3470–3480

110 Adams, M.E. et al. (2004) Structural abnormalities at neuromuscular 134 de Kerchove D’Exaerde, A. et al. (2002) Expression of mutant Ets

synapses lacking multiple syntrophin isoforms. J. Neurosci. 24, protein at the neuromuscular synapse causes alterations in

10302–10309 morphology and gene expression. EMBO Rep. 3, 1075–1081

111 Adams, M.E. et al. (2010) The alpha-syntrophin PH and PDZ domains 135 Briguet, A. and Ruegg, M.A. (2000) The Ets transcription factor GABP

scaffold acetylcholine receptors, utrophin, and neuronal nitric oxide is required for postsynaptic differentiation in vivo. J. Neurosci. 20,

synthase at the neuromuscular junction. J. Neurosci. 30, 11004– 5989–5996

11010 136 Jaworski, A. et al. (2007) GA-binding protein is dispensable for

112 Shiao, T. et al. (2004) Defects in neuromuscular junction structure in neuromuscular synapse formation and synapse-speciﬁc gene

dystrophic muscle are corrected by expression of a NOS transgene in expression. Mol. Cell. Biol. 27, 5040–5046

dystrophin-deﬁcient muscles, but not in muscles lacking alpha- and 137 Lacazette, E. et al. (2003) A novel pathway for MuSK to induce key

beta1-syntrophins. Hum. Mol. Genet. 13, 1873–1884 genes in neuromuscular synapse formation. J. Cell Biol. 161, 727–736

113 Dai, Z. et al. (2000) The actin-driven movement and formation of 138 Balice-Gordon, R.J. and Lichtman, J.W. (1990) In vivo visualization of

acetylcholine receptor clusters. J. Cell Biol. 150, 1321–1334 the growth of pre- and postsynaptic elements of neuromuscular

114 Dobbins, G.C. et al. (2006) The role of the cytoskeleton in junctions in the mouse. J. Neurosci. 10, 894–908

neuromuscular junction formation. J. Mol. Neurosci. 30, 115–118 139 Andreose, J.S. et al. (1995) Number of junctional acetylcholine

115 Kummer, T.T. et al. (2004) Nerve-independent formation of a receptors: control by neural and muscular inﬂuences in the rat. J.

topologically complex postsynaptic apparatus. J. Cell Biol. 164, Physiol. 483, 397–406

1077–1087 140 Slater, C.R. (2008) Structural factors inﬂuencing the efﬁcacy of

116 Proszynski, T.J. et al. (2009) Podosomes are present in a postsynaptic neuromuscular transmission. Ann. N. Y. Acad. Sci. 1132, 1–12

apparatus and participate in its maturation. Proc. Natl. Acad. Sci. 141 Martin, A.R. (1994) Ampliﬁcation of neuromuscular transmission by

U.S.A. 106, 18373–18378 postjunctional folds. Proc. Biol. Sci. 258, 321–326

117 Lee, C.W. et al. (2009) Regulation of acetylcholine receptor clustering 142 Flucher, B.E. and Daniels, M.P. (1989) Distribution of Na channels

by ADF/coﬁlin-directed vesicular trafﬁcking. Nat. Neurosci. 12, and ankyrin in neuromuscular junctions is complementary to

848–856 that of acetylcholine receptors and the 43 kd protein. Neuron 3,

118 Kishi, M. et al. (2005) LL5beta: a regulator of postsynaptic 163–175

differentiation identiﬁed in a screen for synaptically enriched 143 Bewick, G.S. et al. (1996) Spatial relationships of utrophin,

transcripts at the neuromuscular junction. J. Cell Biol. 169, 355–366 dystrophin, beta-dystroglycan and beta-spectrin to acetylcholine

119 Shi, L. et al. (2010) Ephexin1 is required for structural maturation receptor clusters during postnatal maturation of the rat

and neurotransmission at the neuromuscular junction. Neuron 65, neuromuscular junction. J. Neurocytol. 25, 367–379

204–216 144 Trinidad, J.C. et al. (2000) The Agrin/MuSK signaling pathway is

120 Shi, L. et al. (2010) Multiple roles of the Rho GEF ephexin1 in synapse spatially segregated from the neuregulin/ErbB receptor signaling

remodeling. Commun. Integr. Biol. 3, 622–624 pathway at the neuromuscular junction. J. Neurosci. 20, 8762–8770

452

Review Trends in Neurosciences July 2012, Vol. 35, No. 7

145 Kim, N. and Burden, S.J. (2008) MuSK controls where motor axons 154 Flanagan-Steet, H. et al. (2005) Neuromuscular synapses can form in

grow and form synapses. Nat. Neurosci. 11, 19–27 vivo by incorporation of initially aneural postsynaptic specializations.

146 Liu, Y. et al. (2008) Essential roles of the acetylcholine receptor gamma- Development 132, 4471–4481

subunit in neuromuscular synaptic patterning. Development 135, 155 Misgeld, T. et al. (2005) Agrin promotes synaptic differentiation by

1957–1967 counteracting an inhibitory effect of neurotransmitter. Proc. Natl.

147 Chen, F. et al. (2011) Neuromuscular synaptic patterning requires the Acad. Sci. U.S.A. 102, 11088–11093

function of skeletal muscle dihydropyridine receptors. Nat. Neurosci. 156 Lin, W. et al. (2005) Neurotransmitter acetylcholine negatively

14, 570–577 regulates neuromuscular synapse formation by a Cdk5-dependent

148 Jing, L. et al. (2009) Wnt signals organize synaptic prepattern and mechanism. Neuron 46, 569–579

axon guidance through the zebraﬁsh unplugged/MuSK receptor. 157 Fu, A.K. et al. (2005) Aberrant motor axon projection, acetylcholine

Neuron 61, 721–733 receptor clustering, and neurotransmission in cyclin-dependent

149 Strochlic, L. et al. (2012) Wnt4 participates in the formation of kinase 5 null mice. Proc. Natl. Acad. Sci. U.S.A. 102, 15224–

vertebrate neuromuscular junction. PLoS ONE 7, e29976 15229

150 Zhang, B. et al. (2012) Wnt proteins regulate acetylcholine receptor 158 Yang, J. et al. (2011) Nestin negatively regulates postsynaptic

clustering in muscle cells. Mol. Brain 5, 7 differentiation of the neuromuscular synapse. Nat. Neurosci. 14,

151 Gordon, L.R. et al. (2012) Initiation of synapse formation by Wnt- 324–330

induced MuSK endocytosis. Development 139, 1023–1033 159 Mohseni, P. et al. (2011) Nestin is not essential for development of

152 Henriquez, J.P. et al. (2008) Wnt signaling promotes AChR the CNS but required for dispersion of acetylcholine receptor

aggregation at the neuromuscular synapse in collaboration with clusters at the area of neuromuscular junctions. J. Neurosci. 31,

agrin. Proc. Natl. Acad. Sci. U.S.A. 105, 18812–18817 11547–11552

153 Lin, S. et al. (2008) The role of nerve- versus muscle-derived factors in 160 Meier, T. et al. (1997) Neural agrin induces ectopic postsynaptic

mammalian neuromuscular junction formation. J. Neurosci. 28, specializations in innervated muscle ﬁbers. J. Neurosci. 17, 3333–3340 6534–6544

453