Neuromuscular Junction Formation-A great game for Agrin/LrP4/Musk signalling

AbstractOne of the greatest virtues of high vertebrates and mammals lies in their capability of performing complex and sophisticated tasks rendered by their behavioural changes according to environmental alterations. A well-orchestrated nervous system and locomotion system are two keys to this feature. The synapses, which are the fundamental function units of nervous system, have empowered the nervous system a speed of communication by electrical signals that is incomparable to anything on the planet. Neuromuscular junctions (NMJs) are special synapses formed between motoneurons and muscle fibres, making them the bridging structures between central nervous system (CNS) and muscular system. For this reason, the formation of NMJs has been an important research interest. Furthermore, abnormal NMJ formation encompasses a broad spectrum of diseases affecting the neuromotor system, making it an attractive therapeutic target. This review will focus on the underlying molecular mechanism and associated diseases of NMJ formation.

IntroductionSynapses are fundamental units of neuronal circuitry, which is the building block of nerve network of nerve network. A typical synapse comprises three elements. Namely, the pre-synaptic apparatus, post-synaptic apparatus and the intermediate synaptic cleft (Figure 1). In a neuromuscular junction (NMJ), which is a special type of synapse formed between motoneuron (the pre-synaptic apparatus) and muscle fibre (the post-synaptic apparatus), neurotransmitters, which are hormones of the nervous system, are synthesised and released from the pre-synaptic nerve terminals into the synaptic cleft where neurotransmitters diffuse and ultimately bind to specific receptors on post-synaptic fibre, causing cellular changes (i.e. transcriptional changes, opening of ion channels etc.) that subsequently lead to contraction (excitatory synapses) or relaxation (inhibitory synapses) of target muscle. The appearance of NMJs is believed to begins between embryonic development stage of day 12 to day 13.5 (1,2). At this stage, motoneurons that aggregate in the spinal cord stretch out and form growing axons in an effort to guide themselves to target muscle fibres. While nerve terminals play an active role in the path-exploring and guidance process, it is now being increasingly appreciated that post-synaptic muscle fibres and extra-synaptic glial cells (e.g. Schwann cells in peripheral nervous system, PNS) play indispensable roles in synapse formation and the subsequent events, including synapse maturation and synaptic plasticity is post-neonatal life. In addition to the interplay between those three elements (i.e. nerve terminal, muscle fibre and glial cell), the molecular interactions are regarded causative and deserve serious investigations. Owing to their large size and abundance, NMJs have fulfilled neurobiologist’s desire for a research model that renders them the possibility to study NMJ formation and the associated diseases (i.e. congenital myasthenia syndrome and myasthenia gravis) extensively. Indeed, research in the past decades has significantly extended our understandings. In particular, the discovery of Agrin/Lrp4/Musk signalling in NMJ formation has been exciting and

inspiring novel research that tries to identify therapeutic targets for neuromotor system diseases.

This review will begin with an assertion of NMJ formation at cellular and molecular level followed by an explanation of how are Wnt and other molecules involved. The associated diseases (i.e. congenital myasthenia syndrome and myasthenia gravis) and the potential therapeutic strategies will be discussed.

NMJ formation at Cellular levelThe formation of NMJs at cellular level is largely attributable to interactions between the growing nerve terminal, muscle fibre and glial cell. At the very stage, motoneurones are arranged into different groups according to their specific locations in the spinal cord (3,4). Those neurones will innervate different target muscle. When innervation process is triggered, motoneurones start growing out trajectories from their growth zones, which are guided to take up the path of travelling to specific muscle fibres. The guidance process is believed to engage both diffusible and in-diffusible molecules, including extracellular matrix (ECM) molecules (e.g. netrins) and adhesion molecules (e.g. cadheirns and immunoglobulins) (5,6), both of which can either attract or repel the growth zone, resulting in the formation of a characteristic physical path by which an axon migrates to its target muscle/cell. There are four physical forces generated in such a process. Namely, chemo-attraction/repulsion, and contact attraction/repulsion (7). It is worth noting that majority of those molecules are bifunctional with few exceptions, meaning many of them can be either attractants or repellents, depending on the specific receptors expressed on growth zone (7,8).

The destination of every individual motoneuron is a muscle fibre, which, according to in vivo and in vitro experiments, shows acetylcholine

receptor (AchR) clusters prior to the arrival of nerve terminal (9,10). This peculiar phenomenon is referred to as pre-patterning, describing the presence of aneural AchRs and challenging the conventional notion of nerve terminal’s leading role in post-synaptic AchR clustering. For example, previous research on embryonic diaphragm innervation has shown that centralisation of post-synaptic AchRs is a biological event that takes place posterior to innervation (11), indicating the importance of nerve terminal in pre-patterning. However, an in vivo study shows that AchR clusters are present before nerve terminal arrives (9,10) . This notion is further backed-up by the same observations made in mutant mice lacking phrenic or motor nerves (12), suggesting autonomy of post-synaptic muscle fibre in pre-patterning. Further research show that both motoneuron and muscle fibre play important roles in pre-patterning and that whichever predominates depends on specific species (11,13–15) . Nevertheless, there are some issues I would like to address. First, those experiments were done using different muscle types and in different system (i.e. in vivo and in vitro), putting doubts on the credential. Second, in the mutant mice lacking phrenic and motor nerve experiments, a absence of innervation does not necessitate a mutual absence of synaptogenic factors, which could have been produced from the neurones or glial cells and have spatially-restricted impacts on the aggregation of AchRs even before the arrival of nerve terminals (see more in next section).

The formation of NMJs is subsequently followed by synapses maturation, a process that involves changes in both pre-synaptic and post-synaptic structures. Specifically, the maturation of synaptic apparatus is characterised by changes in the quantity, stability and distribution of post-synaptic AchRs (16). The exact underlying molecular mechanisms are well elucidated and will be discussed in the next section. The maturation of pre-synaptic apparatus is largely attributable to increases in nerve ramification, active zone and the distribution and type of calcium channels expressed (to facilitate neurotransmitter release) (16). Moreover, the

number of nerve terminal that forms NMJs with muscle fibre decreases significantly. This process is necessary and important for increasing the specificity and strengthening the power of inter-neuronal communication. At early stage of innervation, a given nerve terminal contacts multiple muscle fibres (17–19). This kind of NMJ is unspecific and is unable to induce muscle contraction. By the end of synapse maturation, a given motoneuron terminal establishes only one NMJ with a given muscle fibre (note that a muscle fibre can receive innervation from several motoneurones) (20). Unlike post-synaptic maturation, the molecular mechanisms that underline pre-synaptic maturation remains largely elusive. Nevertheless, there has been research showing that laminins, which are muscle-derived heterotrimeric glycoproteins comprising three different subunits, including alpha, beta and gamma subunit (21,22). Laminins are believed to be able to interact with different types of calcium ion channels at different development stages to modulate the release of neurotransmitters (23–25). This notion is further backed-up by research carried out on laminin mutant mice which show reduction in synaptic activity (26–28).

Mature synapses (NMJs) do not stay in a standstill state. Rather, they are subjected to a dynamic environment which requires constant modulation at both transcriptional and molecular level. This scenario is called synaptic plasticity. Although it is true that nerve terminal and muscle fibre are the two responsible elements of synaptic plasticity, extrasynaptic glial cells also play an active role. First of all, there are solid evidence demonstrating the bilateral interactions between Schwann cell (glial cell in PNS) and motoneuron, which produces Nrg4, the motoneuron-derived synaptogenic factor. Nrg4 binds to Erbb4, the receptor expressed on Schwann cell, favouring its proliferation and survival. Secondly, Schwann cells produce diffusible factors, including beta-transforming growth factor (beta-TGF), axotactin and Wnt (Wingless in Drosophila), all of them act in a retrograde manner to facilitate the assembly of NMJs (11,29–32). An interesting novel observation has been made on glial cell in NMJ

formation. Acknowledging its phagocytic nature, glial cell has been shown to be able to facilitate NMJ formation by engulfing fragmented synapses generated during synapse maturation (33–35).

NMJ formation at molecular level---Agrin/Lrp4/Musk signalling The identification of Agrin/Lrp4/Musk signalling pathway is considered as a prominent breakthrough in NMJ research because it enables neurobiologists, for the first time, to understand and elucidate NMJ assembly at molecular level. Agrins are motoneuron-derived glycoproteins that were originally identified for its ability to induce AchR clustering (36). Agrin’s role in NMJ assembly is further confirmed by the observation of mice carrying mutations in gene-coding agrin have defects in NMJ complicated by a lack of post-synaptic AchRs (37). Furthermore, ectopic expression of agrin is able to induce AchR cluster formation in murine muscle tissues (38). An intriguing observation has also been made in agrin mutant mice which surprisingly is able to assemble aneural AchR prior to nerve terminal arrival, indicating that agrin is neither involved in nor is important for pre-patterning (9,10). The exact mechanism by which agrin promotes AchR clustering has been shown to via counteracting the inhibitory effects of electrical activity and released acetylcholine (Ach). It has been shown that increased electrical activities in muscle fibres accompanied by Ach release causes reduction in Ach subunits expression and increase in AchR degradation, indicating a negative role of Ach in AchR clustering and therefore in NMJ assembly (11,39).

The introduction of Musk (muscle-specific kinase), a muscle-derived receptor tyrosine kinase, was originally made due to its profuseness in synapse-rich organ (40). Agrin is not able to induce post-synaptic AchR clustering in Musk-/- mice (41), indicating a connection between those two molecules that probably suggest an “agonist-receptor relationship”. Further research confirmed this plausible relationship by showing that

muscle fibre was able to restore agrin responsiveness when exogenous wild-type Musk was expressed (42,43). In addition, ectopically expressed Musk is capable of inducing synapse formation in the absence of agrins (44), suggesting the presence of other agonists or rather, a more sophisticated mechanism of activation. Crystal structure studies revealed agrin does not directly interact with Musk and that there is a mediating co-receptor (45,46), low-density lipoprotein receptor 4 (Lrp4) that serves as a connector. Lrp4 belongs to Lrp receptor family (47) and is required for agrin-induced Musk activation.

The protein structure of Lrp4 characterised by a large extracellular domain, a single transmembrane domain and a small intracellular domain that has no known kinase-associated activity (48–50). Lrp4-/- mice die at birth because of respiratory retardation that resemble those of Musk-/- mice, indicating an important role for Lrp4 in embryonic NMJ assembly (51). Interestingly, it has been shown that the extracellular domain of Lrp4 is able to self-associate and then interact with the extracellular domain of Musk (44,47), causing basal Musk activation that is relatively less potent than what is induced by agrin. This may explain why Musk induces synaptogenesis in the absence of agrin. Given the intrinsic biochemical property of Musk as a receptor tyrosine kinase, it is not surprising to suggest that Musk mediated downstream signalling cascade involves many kinase-induced protein phosphorylation. Indeed, the downstream effector molecules include downstream-of-tyrosine-kinase-7 (Dok7), which is essential for Musk’s interaction with downstream molecules (52), and rapsyn, which is a scaffold molecule that interacts with AchR and is important for its trafficking (53–56). Others are summarised in somewhere else (11).

How does exactly Musk activation leads to AchR clustering? Research has suggested that this process involves rearrangement of cytoskeleton. Specifically, activated Musk interacts with tyrosine kinase Abl (57) and metalloenzyme geranylgeranyl transferase I (GGT I) (58), both of which

ultimately contribute to Rho GTPase activation (59,60). Rho GTPase has several downstream target molecules, including the serine/threonine kinase Pak1, which stabilises actin by phosphorylating cortactin (61), a substrate of Src kinase enriched at sites where heavy actin assembly occurs (62,63). Moreover, Pak1 counteracts the effect of actin depolymerising factors (ADFs) (64).

Wnt-signalling as a novel player

Recent proceedings in NMJ assembly research has revealed a novel player, the Wnt signalling pathway.

Wnt signalling pathway is probably one of the most investigated signalling pathway in biology owing to its pivotal role in a broad range of developmental biology events, including body axis patterning, limb development and somitogenesis (65–71). Its role in NMJ assembly was originally discovered for its participation in regulation of synaptogenesis of developing rodent cerebellar granular neuron that uses Wnt7a as a retrograde signal to influence the growth of axon (72). Subsequent research using invertebrates (i.e. Drosophila) as animal models has shown that Wingless (the Drosophila orthologue of Wnt) mutation causes a reduction in synapse button number (73). Furthermore, other Wnt isoforms shown positive effects in promoting pre-and post-synaptic apparatus differentiation (74,75). The role that Wnt plays in vertebrate NMJ formation is largely attributable to two aspects. First, a solid body of evidence has been laid down showing that the cysteine-rich domain (CRD) in Musk, the receptor for agrin, shows homology with the Wnt-binding domain of Frizzled’s (76,77). Second, previous research has also shown that beta-catenin, which is the key signal molecule of Wnt signalling cascade, participates in NMJ assembly by regulating actin polymerisation

(78). This is indicative of the possibility of Wnt as a ligand for Musk. Indeed, the CRD of zebrafish Mush interacts with Wnt11r, contributing to formation of aneural AchR clusters. Wnt presumably binds to Musk expressed in muscle fibres prior to the arrival of nerve terminal, thereby pre-patterning the post-synaptic apparatus. Notably, it would be worth and interesting to investigate whether ligand Wnt family interacts with the low-density lipoprotein receptor family. And, if so, how? Potential approaches include protein co-immunoprecipitation.

Neuromuscular DiseasesMyasthenia Gravis (MG) is a collection of neuromuscular disease characterised by a variety of pathophysiological background, genetic pre-deposition, and responses to treatment. Generally speaking, the clinical manifestation of MG begins with muscle weakness in the face, neck and jaw which then becomes generalised involving skeletal muscles of the whole body, especially that of upper and lower limbs. MG is classified into three groups depending on their respective pathophysiological causes. Namely, the AchR MG, Musk MG and congenital myasthenia syndrome (CMS). The former two groups are autoimmune diseases caused by auto-destruction of neuromuscular junctions (NMJs) mediated by autoantibodies. CMS is a rare inherited condition and is largely attributable to genetic pre-depositions (although postnatal environmental exposure can’t be excluded). Next, I will compare AchR and Musk MG as an example so as to demonstrate how and why should them be evaluated and treated differently. First of all, AchR and Musk MGs have very similar symptoms except that Musk MG rarely affects the ocular muscles (muscles in the eye) (79–81). Secondly, different types of autoantibodies are present. In AchR MG, the antibodies are mostly seen as IgG1 and IgG3 subclasses, whereas in Musk MG, it is IgG4 that is most commonly detected (82,83). Furthermore, it is believed that AchR type of MG has deeper involvement with CD4+ T cells as supported by the observation that thymectomy (removal of the entire thymus) actually is beneficial

(84). In contrast, Musk type of MG shows more of B cell. An increased frequency of B10 cells (85), which are major cells responsible for immune-tolerance (86–88) has been observed in Musk MG patients. In addition to B-cell and T-cell, those two types of MG differ also in genetic background (84,85).

Owing to their different pathophysiological causes, Musk and AchR MG respond differently to treatments. For example, Musk MG patients have moderate responses (89) to inhibitors of acetylcholinesterase, which is an enzyme responsible for acetylcholine degradation (90). However, Musk MG patients respond well to immunosuppressant corticosteroid, plasma exchange (filtering the autoantibodies out) and pharmaceutical drug rituximab (monoclonal antibody originally used for Non-Hodgkin Lymphoma treatment) (84,91,92). It works by binding to Fc-receptor on immune cell, thereby activating complement system, leading to the death of targeted immune cells. Musk MG patient show less sign of thymic hyperplasia, thymectomy is therefore less effective than it is in AchR type of MG (81).

ConclusionNeuromuscular junctions (NMJs) are specialised synapses formed between motor neuronal terminals and muscle fibres. They are essential coordinators of the nervous system and skeletal system, conferring high vertebrates the ability to perform complex tasks and great adaptability. Because of their abundancy and large size, NMJs have served as a fabulous model for neurobiologists to explore the molecular mechanisms underlying NMJ assembly. Research in the past decades have been proved fruitful, especially with the identification of agrin/Lrp4/Musk signalling cascade. New excitement has also been brought into this field with the appreciation of Wnt signalling pathway as a novel player in NMJ assembly. However, there are many more issues to be addressed. First of all, research investigating pre-patterning has shown contradictory results (9–12). This is probably because different experimental systems (in vivo and in vitro) and different animal models were used (vertebrates and invertebrates). Secondly, how is Wnt signalling pathway cooperated into Lrp4/Musk signalling? Does it interact with Lrp4 or other members of low-density lipoprotein receptor family? If it does, does it activate the canonical or non-canonical signalling pathway? Owing to its indispensable role in the motility of physical body, NMJ assembly retardation could lead to serious diseases known as neuromuscular diseases (represented by myasthenia gravis). Fortunately, research results have yield a great deal of clinical implications. Targeted molecular therapeutic strategies are now available for neuromuscular treatment with minimised side effects.

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