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MMP-mediated modulation of ECM environment during axonal growth

and NMJ development

Zora Chui-Kuen Chan, Marilyn Janice Oentaryo, Chi Wai Lee

PII: S0304-3940(20)30092-6
DOI: https://doi.org/10.1016/j.neulet.2020.134822
Reference: NSL 134822

To appear in: Neuroscience Letters

Received Date: 27 October 2019

Revised Date: 31 January 2020
Accepted Date: 4 February 2020

Please cite this article as: Chui-Kuen Chan Z, Oentaryo MJ, Lee CW, MMP-mediated
modulation of ECM environment during axonal growth and NMJ development, Neuroscience
Letters (2020), doi: https://doi.org/10.1016/j.neulet.2020.134822

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© 2020 Published by Elsevier.

MMP-mediated modulation of ECM environment during axonal growth and
NMJ development
Zora Chui-Kuen Chan*, Marilyn Janice Oentaryo*, and Chi Wai Lee

School of Biomedical Sciences, Li Ka Shing Faculty of Medicine, The University of Hong Kong,
Hong Kong
* Equal contribution

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Correspondence: Chi Wai Lee, School of Biomedical Sciences, The University of Hong Kong
L4-63, Lab Block, 21 Sassoon Road, Pokfulam, Hong Kong.
Email: chiwai.lee@hku.hk

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Highlights
 The composition and structure of ECM proteins at the basal lamina modulate NMJ
formation and maintenance.
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 Both secreted and membrane-type MMPs modulate ubiquitously expressed and/or
synapse-specific ECM proteins at developing NMJ.
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 Increased levels of MMPs in MG patients highlight the implication of studying the

MMP-mediated ECM modulation in NMJ development together with its clinical
relevance.
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Abstract
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Motor neurons, skeletal muscles, and perisynaptic Schwann cells interact with
extracellular matrix (ECM) to form the tetrapartite synapse in the peripheral nervous system.
Dynamic remodeling of ECM composition is essential to diversify its functions for distinct
cellular processes during neuromuscular junction (NMJ) development. In this review, we give
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an overview of the proteolytic regulation of ECM proteins, particularly by secreted and

membrane-type matrix metalloproteinases (MMPs), in axonal growth and NMJ development. It
is suggested that MMP-2, MMP-9, and membrane type 1-MMP (MT1-MMP) promote axonal
outgrowth and regeneration upon injury by clearing the glial scars at the lesion site. In addition,
these MMPs also play roles in neuromuscular synaptogenesis, ranging from spontaneous
formation of synaptic specializations to activity-dependent synaptic elimination, via proteolytic
cleavage or degradation of growth factors, neurotrophic factors, and ECM molecules. For
instance, secreted MMP-3 has been known to cleave agrin, the main postsynaptic differentiation
inducer, further highlighting the importance of MMPs in NMJ formation and maintenance.

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Furthermore, the increased level of several MMPs in myasthenia gravis (MG) patient suggest the
pathophysiological mechanisms of MMP-mediated proteolytic degradation in MG pathogenesis.
Finally, we speculate on potential major future directions for studying the regulatory functions of
MMP-mediated ECM remodeling in axonal growth and NMJ development.

Keywords: matrix metalloproteinases, extracellular matrix, axonal outgrowth, axonal pathfinding,

neuromuscular junction, synaptogenesis

Chemical synapses are specialized cell-to-cell junctions where nerve impulse is

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transmitted unidirectionally from one neuron to either another neuron or a non-neuronal target
cell, which allows the nervous system to perform complex computations and to regulate other
systems of the body. Structurally, they are asymmetric synapses composed of presynaptic and

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postsynaptic compartments, a synaptic cleft, and surrounding glial cells. Among different
chemical synapses, the neuromuscular junction (NMJ) has perhaps been studied most extensively
because of its simplicity and accessibility, and these NMJ studies have provided a framework for

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investigating synapses in the central nervous system (CNS) [66]. A novel concept of the
tetrapartite synapse has emerged in this field, according to which the synaptic structure and
function are regulated not only by the presynaptic neurons, postsynaptic muscles and glial cells,
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but also by extracellular matrix (ECM) and its associated molecules enriched in the synaptic
basal lamina (BL) [16, 64]. In this review, we summarize recent studies on the modulation of
ECM proteins by matrix metalloproteinases (MMPs) during distinct processes in neuromuscular
development, including axonal growth, synaptic formation, elimination and maintenance.
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Roles of ECM proteins during axonal growth and NMJ development

The Matrisome Project has characterized and organized the ensemble of genes encoding
the “matrisome” that includes ECM proteins accounting for 4% of the mammalian proteome
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[31]. The ECM is a complex meshwork of highly cross-linked proteins featuring a unique
composition in different tissues. One form of the ECM, the interstitial matrix, is present as a
hydrated, porous three-dimensional lattice located in the space surrounding cells. Another
specialized form of the ECM, the basement membrane (BM), appears as a thin, sheet-like layer
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in the basal surface of cells forming connective tissues. The BM is ubiquitously found in the
peripheral nervous system (PNS), and in skeletal muscle. The BM comprises two layers: the
external layer is a reticular lamina composed predominantly of collagenous fibrils that contribute
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to the elasticity and strength of muscle cells, and the internal layer composed of collagen, non-
collagenous glycoproteins, and proteoglycans that directly link to the plasma membrane of
muscle cells [58].
The ECM was initially considered to function only in providing architectural support and
anchorage for cells. However, based on the proteomic studies of different ECM-associated
proteins in the BL of the NMJ, the ECM is now believed to perform complex and diverse
functions [50]. Apart from serving as the structural scaffold, the spatiotemporal remodeling of
ECM organization and composition can contribute to various cellular functions as follows,
1. Acts as a physical barrier to define tissue boundaries and maintain tissue integrity;

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 2. Activates cell surface receptors to modulate different physiological processes initiated
by extracellular mechanical signals;
3. Sequesters and stores different ECM-associated growth factors; and
4. Controls the distribution of bioactive ECM-bound molecules spatiotemporally by
context-specific stimuli.
Before synaptogenesis, the growth cone, a motile structure at the tip of growing axons,
probes the environment along different extracellular guidance cues to transmit the signals that
elicit intracellular changes in axonal growth and pathfinding [12]. The motility and turning of
growth cones are aided by the assembly and disassembly of filopodial and lamellipodial
protrusions in the leading edge of growth cones. These protrusions harbor membrane receptors
for various guidance cues that initiate distinct signaling mechanisms asymmetrically across the
growth cone [49], allowing the growth cones to move away from or toward the guidance cues.

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The cytoskeletal framework of growth cones and the roles of actin-binding proteins in axonal
growth and guidance have been well documented [13, 24, 73]. Particularly, the actin-rich
filopodia and lamellipodia are stabilized by ECM molecules, aiding the adhesion of growth

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cones to the substratum [62]. These ECM molecules include proteoglycans and fibrous proteins
that activate receptors located on the surface of growth cones [22]. Because integrins function as
receptors for several ECM molecules, different types of integrins and their trafficking and
activation mechanisms have been extensively investigated [8, 28]. Collectively, many functional

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studies have been conducted to demonstrate the importance of ECM and its associated proteins
in various developmental stages of axonal growth and NMJ development, which are summarized
in Table 1.
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Table 1. A summary on the functional roles of ECM and its associated proteins in axonal growth and/or NMJ development.

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ECM and its
Subunit Localization1 Related functional studies in axonal growth and/or NMJ development
associated proteins

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IV(α1)2(α2) E • Col4a1 and Col4a2 mutant: Myopathy [36]
IV(α3)(α4)(α5) S • Col4a3-/- and Col4a6-/-: No defects at NMJ [21]

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IV(α5)2(α6) S • Col4a5-/-: Fragmented synaptic structures; retraction of axons [21]
Collagen

Collagen
XIII S • Col13a1-/-: Incomplete adhesion of pre- and post-synaptic partners [37]

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XVIII E • Col18a1 knockdown: Pronounced effect on motor axon migration in zebrafish [69]
XIX(α1) E • Regulates axonal guidance at intermediate targets in zebrafish [26]
ColQ2 • Col13a1-/-: Lack of asymmetric acetylcholinesterase [18]
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al S
• Promotes neurite outgrowth [54]
111 E
• Laminin α1 transgene reduces muscular dystrophy in laminin α2-/- mice [23]
Non-collagenous glycoprotein

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211 E+S • Laminin α2-/-: Congenital muscular dystrophy type 1A [17]
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• Serves as a stop signal for growing neurites [57]

Laminin 221 S
• Laminin β2-/-: Aberrant NMJ structures [48]
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411 E+S
• Laminin α4-/-: Fewer active zones or junctional folds [53]
421 S
511 E+S
• Laminin α5-/-: Delayed topological maturation of AChR clusters [47]
521 S
Perlecan - E+S • Perlecan mutant: Reduces endplate AChE levels; muscle stiffness [71]

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 Agrin Z+ S • Agrin-/-: Excessive axon growth; broader end-plate region [40, 83]
Proteoglycan

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Biglycan - E+S • Biglycan-/-: Abnormal NMJs; focal misalignment of AChE and AChRs [1]
• NCAM-/-: Smaller NMJ, delayed synaptic elimination [60]

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NCAM3 - S
• Maintains normal synaptic function at reinnervated NMJs [9]
• Stabilizes postsynaptic apparatus via phosphorylation of α-dystrobrevin [68]
Neuregulin - S
Growth factors

• Regulates Schwann cell-mediated developmental synaptic elimination [38]

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• Inhibits nerve-induced AChR clustering by suppressing agrin synthesis [55]
BDNF - E • Differentially regulates synaptic competition by BDNF precursor (proBDNF) and
mature BDNF (mBDNF) [32]

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TGF-β1 - E • Regulates agrin synthesis and NMJ formation [20]

α E+S • α-/β-Dystroglycan-/-: Muscular dystrophy [11]

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receptors

• Muscle-specific α-/β-Dystroglycan-/-: Perturbation of satellite cell function leading

ECM

Dystroglycan
β S to insufficient repair of skeletal muscle cells [10]
Integrin α8β1 E+S • Interacts with tenascin-C to promote neurite outgrowth [78]
MMP-2, 9 -
al ND • Regulates conversion of proBDNF to mBDNF during synaptic competition [32]
MMPs

MMP-3 - ND • MMP-3-/-: Increased AChRs, junctional folds, and agrin immunoreactivity [75, 77]
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Notes:
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1
E: Extrasynaptic; S: Synaptic; ND: Not yet determined
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ColQ: Acetylcholinesterase collagenic tail peptide
3
NCAM: Neural cell adhesion molecule
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MMPs function as major proteases for ECM remodeling
Reciprocal interaction between the ECM and cells is crucial for the regulation of
diverse physiological processes [30]. ECM remodeling through synthesis, degradation,
reassembly, and chemical modification produces changes in the composition and structure of
ECM environment to affect cellular behaviors [42]. These processes are all tightly and
precisely regulated to maintain microenvironmental homeostasis and to promote repair after
injury. ECM cleavage and processing are regulated by various proteases and their regulators,
including MMPs, adamalysins, meprins, and tissue inhibitors of metalloproteinases (TIMPs)
[4].
MMPs were first described in 1962 by Gross and Lapiere, who found that MMPs are
involved in tadpole tail morphogenesis [25], and, to date, 23 human MMPs have been
discovered. The conserved protease domain shared by mammalian MMPs consists of both a
catalytic domain and an autoinhibitory pro-domain [50, 70]. The catalytic domain contains
the zinc ion, a linker peptide, and a hemopexin domain, which is critical for MMP binding to
its substrate and to TIMPs [5]. The pro-domain maintains MMPs in an inactive state through

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the interaction of a cysteine thiol group with the catalytic zinc atom in the active site. MMPs
are activated when this cysteine-zinc pairing is disrupted through conformational change
and/or proteolysis. Most MMPs are in the soluble form but some MMPs are localized to the

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cell surface through a transmembrane segment or a glycosylphosphatidylinositol anchor, and
they are referred to as membrane-type MMPs (MT-MMP) [29]. MT-MMPs are activated by
a pro-protein convertase such as furin within the exocytic pathway. Conversely, soluble
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MMPs, such as MMP-2 and MMP-9, are generally secreted as pro-enzymes, known as
zymogens, which can be proteolytically activated in the extracellular environment. To affect
different cellular behaviors, MMPs act spatiotemporally to control targeted degradation or
proteolytic processing of ECM molecules, growth factors, cell adhesion molecules, and other
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surface proteins [29]. Specifically, MMPs have been implicated in modulating ECM
microenvironment through the following mechanisms [50]:
1. Creating space for cell migration;
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2. Regulating tissue architecture by modulating the ECM and intercellular junctions;

3. Releasing ECM-bound growth factors; and
4. Producing substrate-cleavage fragments with differential biological activities.
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MMP-mediated modulation of ECM proteins during axonal growth and guidance

The mechanisms that regulate the localization and/or activity of ECM proteins,
growth factors, and guidance cues play critical roles in the control of axonal outgrowth and
guidance. Active remodeling of the ECM can expose or disrupt specific integrin-binding
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sites, as frequently observed in cancer cells [86], where degradative proteases promote the
migration of invasive cancer cells into tissues [56]. The actin-rich protrusions responsible for
the secretion and activation of matrix proteases in cancer cells are called invadopodia.
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Similar structures, known as podosomes, have also been identified in normal and healthy
cells [41, 59], which suggest a common mechanism underlying MMP-mediated modulation
of ECM proteins in both invasive and normal cells. Given the resemblance between
invadopodia and podosomes, these structures are commonly referred to as invadosomes [14,
41]. Invadosomes are similar to filopodia in the way that both structures contain a dense
filamentous actin core associated with various actin-associated proteins [51]. However, one
of the distinctive features of invadosomes is that they are long-lived protrusions typically
emerged from the basal surface at the leading edge of migratory cells. Invadosomes secrete
and activate matrix proteases such as MMPs and thereby degrade the ECM that allow cells to
migrate between layers in a three-dimensional space. Similarly, neuronal growth cones

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secrete MMPs to degrade the ECM that allows extension and pathfinding of axons [45, 84]
(Figure 1A). Intriguingly, stable F-actin puncta in invadosomes have recently been
identified in growth cones of cultured Xenopus spinal neurons [67], and the dominant-
negative mutant of an actin regulatory protein, Tks5, causes a reduction of ECM degradation
and inhibits the extension of motor neurons in vitro. This study further corroborates that
Tks5 is a critical scaffold protein for the formation of invadosomes [15], however the
mechanistic regulation of invadosome assembly and disassembly in axonal growth remains to
be further investigated.

MMP-mediated modulation of BL proteins during NMJ formation and maintenance

When axonal growth cones of motor neurons contact myotubes, synaptic transmission
between the presynaptic and postsynaptic cells can be detected within minutes after their
contact [34]. Interestingly, neuronal growth cones are capable of releasing neurotransmitters
spontaneously before contacting their synaptic target [85], and AChRs are aggregated in the
central region of muscle fibers prior to innervation [40, 83]. These suggest that nerve-muscle

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contacts appear to recruit the spontaneously formed presynaptic and postsynaptic components
for further development and maturation of synaptic specializations during NMJ formation.
Although it remains debatable whether the pre-patterned AChR clusters play an active role in

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guiding axonal growth of motor neurons in vivo, the requirement of the ECM for aneural
AChR clustering and the contribution of surface AChRs to synaptic assembly in cultured
muscle cells have been widely recognized [46, 65, 72]. In vitro studies have shown that
aneural AChR clusters, induced by laminin in cultured myotubes, morphologically resembles
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synaptic AChR clusters observed at the NMJ in vivo [35]. The formation of laminin-induced
AChR clusters is likely mediated through interactions with dystroglycan [46, 72], although it
is dispensable for NMJ formation in vivo. The identification of podosome-like structures
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(PLSs) at perforated regions of aneural AChR clusters has suggested that AChR cluster
remodeling could be regulated by PLSs [59]. Our unpublished data suggest that PLSs can
direct the vesicular trafficking and surface insertion of MT1-MMP, which functions to
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regulate AChR clustering via focal degradation of ubiquitously expressed ECM proteins
(Figure 1B), further indicating the requirement of MMP activity in NMJ formation.
Considerable effort has been devoted toward pinpointing the critical molecules
involved in AChR clustering at the synaptic region. Among the identified molecules, agrin
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has been most extensively studied for its role in the induction of postsynaptic differentiation
at the NMJ. Agrin is a heparan sulfate proteoglycan that is secreted by motor neurons,
skeletal muscles, and Schwann cells. Alternative splicing produces distinct agrin isoforms,
among which neural agrin potently induces AChR clustering. The signaling pathways
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initiated by agrin have been well elucidated, which involves Lrp4 (low-density lipoprotein
receptor-related protein 4), and MuSK (muscle-specific kinase) as the co-receptors [81].
MMP-3 null mice exhibited increased agrin deposition and AChR aggregation at NMJs, and
the purified MMP-3 protein was able to directly cleave agrin [75]. Therefore, the proteolytic
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activity of MMP-3 is essential for the removal of agrin from the synaptic basal lamina [77],
which subsequently changes AChR density and NMJ structures. Moreover, synaptic activity
also influences MMP-3 activity that further alters the ECM environment surrounding the
synapse [76]. A recent proteomic study has provided a list of other possible MMPs for agrin
processing, including MMP-1, -2, -7, and MT1-MMP, by which MMP-mediated cleavage of
agrin may also result in the disruption of laminin-agrin complex formation and AChR
clustering ability [52].
Endogenous expression of agrin in neurons can be regulated by a Schwann cell-
derived factor, transforming growth factor (TGF)-β1 [19, 55]. A mixture of neurotrophins

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was found to inhibit agrin deposition and nerve-induced AChR clustering, and these
inhibitory effects could be reversed following the addition of Schwann cell-derived factors
[55], particularly TGF-β1 [19]. TGF-β1 can exist in a latent form, in which active TGF-β1 is
enwrapped by latency-associated peptide (LAP) [61]. LAP links to the ECM by interacting
with latent TGF-β binding protein-1, and this results in TGF-β1 being embedded in the ECM
as an inactive state. TGF-β1 activation involves cell contractile forces transmitted by
integrins [27] and MMP-mediated mechanisms. Thus, MMPs might release ECM-bound
TGF-β1, which in turn increase agrin expression in motor neurons and facilitate AChR
clustering at the synaptic region (Figure 1B). Moreover, loss-of-function studies
demonstrated that the structural integrity of NMJs depends on postsynaptic Lrp4 in muscles
[2, 80]. Specifically, conditional Lrp4 knockout mice showed fragmented AChR clusters,
together with a reduction in junctional folds and synaptic activity, which led to diminished
muscle strength. These phenotypes indicate that neuromuscular transmission is impaired
when Lrp4 is absent, consistent with the requirement of agrin in NMJ maintenance [63].
Interestingly, pharmacological studies indicated that MMPs are able to cleave Lrp4 and then

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release its ectodomain [80]. The soluble ecto-Lrp4 fragments bind to secreted agrin in
forming a binary complex to promote MuSK phosphorylation and AChR clustering [80, 87].
However, which exact MMP isoform acts to cleave Lrp4 remains to be determined.

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During the maintenance of NMJs, collagen IV and laminin in the BL have been
shown to be critical for maintaining the integrity of NMJs [3]. If the BL is digested by
protease treatment, this causes detachment and subsequent retraction of nerve terminals from
the endplate. In early postnatal animals, the transient poly-innervated muscle fibers by
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multiple motor axons are progressively converted to singly-innervated through the activity-
dependent synaptic elimination process [39]. Although the precise mechanisms underlying
synaptic elimination remain unclear, electrical activity, retrograde signals, and microtubule
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dynamics have been suggested to facilitate this process [6, 39, 82]. In particularly, the
precursor and mature forms of BDNF have been shown to differentially affect the elimination
and stabilization of presynaptic nerve terminals, respectively [32, 33]. While proBDNF-
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p75NTR signaling promotes synapse elimination, mBDNF-Trk B (tropomyosin receptor kinase

B) signaling stabilizes the synapse. Intriguingly, it is believed that proBDNF can be
proteolytically converted to mBDNF by MMPs. Pharmacological inhibition of MMP-2 and
MMP-9 activity increased synaptic elimination mediated by proBDNF-p75NTR signaling,
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suggesting that MMPs serve as the activity-dependent regulators to proteolytically process

the same neurotrophic factor in generating differential biological activities (Figure 1B).

Significance and Future Perspectives

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Understanding the complexity of the ECM environment and its proteolytic regulation
by MMPs in both axonal growth and NMJ development may shed insight on their
dysregulation in various neurodegenerative and neuromuscular diseases. For example,
dysregulations of MMPs and TIMPs have been associated with Alzheimer’s disease and
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amyotrophic lateral sclerosis [7, 44, 79]. Another disease specific to the NMJ is myasthenia
gravis (MG), an autoimmune disease in which patients harbor autoantibodies against AChR,
Lrp4, MuSK, agrin, or other NMJ-associated proteins. Recently, collagen XIII, a regulator
involved in NMJ maturation, has been reported to be a target of autoantibodies in some MG
patients [74]. In addition, MG patients have been found to exhibit elevated levels of MMP-3
[43]. This may result in increased agrin degradation and altered NMJ structure that
contribute to the pathophysiological features in MG patients. Our future studies aim to
further investigate the detailed roles of MMPs and ECM proteins, and to elucidate their
signaling pathways, underlying the pathogenesis of motor neuron and neuromuscular
diseases.

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Acknowledgements: This work was partly supported by the Early Career Grant (27102316) and
General Research Fund (17100718 and 17100219) from Research Grants Council of Hong Kong, the
Health and Medical Research Fund (04151086) from Food and Health Bureau of Hong Kong to
CWL.

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Figure Legend

Figure 1. Schematic diagram showing MMP-mediated ECM modulation during axonal

growth and NMJ development.
(A) Neuronal growth cones secrete MMPs, particularly MMP-2 and MMP-9, to degrade the
surrounding ECM proteins required for axonal outgrowth and pathfinding.
(B) MT1-MMP localized in the postsynaptic membrane degrades ECM proteins necessary for
the re-distribution and clustering of AChRs during neuromuscular synaptogenesis.
Additionally, MMP-mediated proteolytic activation of latent TGF-β1 and proBDNF is
suggested to regulate agrin expression and synapse competition, respectively. TSC: terminal
Schwann cell.

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