Building, Breaking, and Repairing Neuromuscular Synapses.

A coordinated and complex interplay of signals between motor neurons, skeletal muscle cells, and Schwann cells controls the formation and maintenance of neuromuscular synapses. Deficits in the signaling pathway for building synapses, caused by mutations in critical genes or autoantibodies against key proteins, are responsible for several neuromuscular diseases, which cause muscle weakness and fatigue. Here, we describe the role that four key genes, Agrin, Lrp4, MuSK, and Dok7, play in this signaling pathway, how an understanding of their mechanisms of action has led to an understanding of several neuromuscular diseases, and how this knowledge has contributed to emerging therapies for treating neuromuscular diseases.

Deficiency of skeletal muscle Agrin contributes to the pathogenesis of age-related sarcopenia in mice.

Sarcopenia, a progressive and prevalent neuromuscular disorder, is characterized by age-related muscle wasting and weakening. Despite its widespread occurrence, the molecular underpinnings of this disease remain poorly understood. Herein, we report that levels of Agrin, an extracellular matrix (ECM) protein critical for neuromuscular formation, were decreased with age in the skeletal muscles of mice. The conditional loss of Agrin in myogenic progenitors and satellite cells (SCs) (Pax7 Cre:: Agrin flox/flox) causes premature muscle aging, manifesting a distinct sarcopenic phenotype in mice. Conversely, the elevation of a miniaturized form of Agrin in skeletal muscle through adenovirus-mediated gene transfer induces enhanced muscle capacity in aged mice. Mechanistic investigations suggest that Agrin-mediated improvement in muscle function occurs through the stimulation of Yap signaling and the concurrent upregulation of dystroglycan expression. Collectively, our findings underscore the pivotal role of Agrin in the aging process of skeletal muscles and propose Agrin as a potential therapeutic target for addressing sarcopenia.

Agrin Inhibition in Enteric Neural Stem Cells Enhances Their Migration Following Colonic Transplantation.

Regenerative cell therapy to replenish the missing neurons and glia in the aganglionic segment of Hirschsprung disease represents a promising treatment option. However, the success of cell therapies for this condition are hindered by poor migration of the transplanted cells. This limitation is in part due to a markedly less permissive extracellular environment in the postnatal gut than that of the embryo. Coordinated interactions between enteric neural crest-derived cells (ENCDCs) and their local environment drive migration along the embryonic gut during development of the enteric nervous system. Modifying transplanted cells, or the postnatal extracellular environment, to better recapitulate embryonic ENCDC migration could be leveraged to improve the engraftment and coverage of stem cell transplants. We compared the transcriptomes of ENCDCs from the embryonic intestine to that of postnatal-derived neurospheres and identified 89 extracellular matrix (ECM)-associated genes that are differentially expressed. Agrin, a heparin sulfate proteoglycan with a known inhibitory effect on ENCDC migration, was highly over-expressed by postnatal-derived neurospheres. Using a function-blocking antibody and a shRNA-expressing lentivirus, we show that inhibiting agrin promotes ENCDC migration in vitro and following cell transplantation ex vivo and in vivo. This enhanced migration is associated with an increased proportion of GFAP + cells, whose migration is especially enhanced.

Perlecan (HSPG2) promotes structural, contractile, and metabolic development of human cardiomyocytes.

Perlecan (HSPG2), a heparan sulfate proteoglycan similar to agrin, is key for extracellular matrix (ECM) maturation and stabilization. Although crucial for cardiac development, its role remains elusive. We show that perlecan expression increases as cardiomyocytes mature in vivo and during human pluripotent stem cell differentiation to cardiomyocytes (hPSC-CMs). Perlecan-haploinsuffient hPSCs (HSPG2+/-) differentiate efficiently, but late-stage CMs have structural, contractile, metabolic, and ECM gene dysregulation. In keeping with this, late-stage HSPG2+/- hPSC-CMs have immature features, including reduced ⍺-actinin expression and increased glycolytic metabolism and proliferation. Moreover, perlecan-haploinsuffient engineered heart tissues have reduced tissue thickness and force generation. Conversely, hPSC-CMs grown on a perlecan-peptide substrate are enlarged and display increased nucleation, typical of hypertrophic growth. Together, perlecan appears to play the opposite role of agrin, promoting cellular maturation rather than hyperplasia and proliferation. Perlecan signaling is likely mediated via its binding to the dystroglycan complex. Targeting perlecan-dependent signaling may help reverse the phenotypic switch common to heart failure.

LRP4-related signalling pathways and their regulatory role in neurological diseases.

The mechanism of action of low-density lipoprotein receptor related protein 4 (LRP4) is mediated largely via the Agrin-LRP4-MuSK signalling pathway in the nervous system. LRP4 contributes to the development of synapses in the peripheral nervous system (PNS). It interacts with signalling molecules such as the amyloid beta-protein precursor (APP) and the wingless type protein (Wnt). Its mechanisms of action are complex and mediated via interaction between the pre-synaptic motor neuron and post-synaptic muscle cell in the PNS, which enhances the development of the neuromuscular junction (NMJ). LRP4 may function differently in the central nervous system (CNS) than in the PNS, where it regulates ATP and glutamate release via astrocytes. It mayaffect the growth and development of the CNS by controlling the energy metabolism. LRP4 interacts with Agrin to maintain dendrite growth and density in the CNS. The goal of this article is to review the current studies involving relevant LRP4 signaling pathways in the nervous system. The review also discusses the clinical and etiological roles of LRP4 in neurological illnesses, such as myasthenia gravis, Alzheimer's disease and epilepsy. In this review, we provide a theoretical foundation for the pathogenesis and therapeutic application of LRP4 in neurologic diseases.

Synergistically Acting on Myostatin and Agrin Pathways Increases Neuromuscular Junction Stability and Endurance in Old Mice.

Sarcopenia is the primary cause of impaired motor performance in the elderly. The current prevailing approach to counteract such condition is increasing the muscle mass through inhibition of the myostatin system: however, this strategy only moderately improves muscular strength, not being able to sustain the innervation of the hypertrophic muscle per se, leading to a progressive worsening of motor performances. Thus, we proposed the administration of ActR-Fc-nLG3, a protein that combines the soluble activin receptor, a strong myostatin inhibitor, with the C-terminal agrin nLG3 domain. This compound has the potential of reinforcing neuro-muscular stability to the hypertrophic muscle. We previously demonstrated an enhancement of motor endurance and ACh receptor aggregation in young mice after ActR-Fc-nLG3 administration. Now we extended these observations by demonstrating that also in aged (2 years-old) mice, long-term administration of ActR-Fc-nLG3 increases in a sustained way both motor endurance and muscle strength, compared with ActR-Fc, a myostatin inhibitor, alone. Histological data demonstrate that the administration of this biological improves neuromuscular stability and fiber innervation maintenance, preventing muscle fiber atrophy and inducing only moderate hypertrophy. Moreover, at the postsynaptic site we observe an increased folding in the soleplate, a likely anatomical substrate for improved neurotransmission efficiency in the NMJ, that may lead to enhanced motor endurance. We suggest that ActR-Fc-nLG3 may become a valid option for treating sarcopenia and possibly other disorders of striatal muscles.

Bioinformatics-based analysis of the roles of basement membrane-related gene AGRN in systemic lupus erythematosus and pan-cancer development.

Introduction: Systemic lupus erythematosus (SLE) is an autoimmune disease involving many systems and organs, and individuals with SLE exhibit unique cancer risk characteristics. The significance of the basement membrane (BM) in the occurrence and progression of human autoimmune diseases and tumors has been established through research. However, the roles of BM-related genes and their protein expression mechanisms in the pathogenesis of SLE and pan-cancer development has not been elucidated. Methods: In this study, we applied bioinformatics methods to perform differential expression analysis of BM-related genes in datasets from SLE patients. We utilized LASSO logistic regression, SVM-RFE, and RandomForest to screen for feature genes and construct a diagnosis model for SLE. In order to attain a comprehensive comprehension of the biological functionalities of the feature genes, we conducted GSEA analysis, ROC analysis, and computed levels of immune cell infiltration. Finally, we sourced pan-cancer expression profiles from the TCGA and GTEx databases and performed pan-cancer analysis. Results: We screened six feature genes (AGRN, PHF13, SPOCK2, TGFBI, COL4A3, and COLQ) to construct an SLE diagnostic model. Immune infiltration analysis showed a significant correlation between AGRN and immune cell functions such as parainflammation and type I IFN response. After further gene expression validation, we finally selected AGRN for pan-cancer analysis. The results showed that AGRN's expression level varied according to distinct tumor types and was closely correlated with some tumor patients' prognosis, immune cell infiltration, and other indicators. Discussion: In conclusion, BM-related genes play a pivotal role in the pathogenesis of SLE, and AGRN shows immense promise as a target in SLE and the progression of multiple tumors.

Plasticity changes in neuromuscular junction morphology and related regulatory proteins in the hibernating ground squirrel.

Skeletal muscle disuse atrophy can cause degenerative changes in neuromuscular junction morphology. Although Daurian ground squirrels (Spermophilus dauricus) are a natural anti-disuse animal model for studying muscle atrophy during hibernation, little is known about the morphological and regulatory mechanisms of their neuromuscular junctions. Here, we found that morphological indices of the soleus muscle were significantly lower during hibernation (torpor and interbout arousal) compared with pre-hibernation but recovered during post-hibernation. In the extensor digitorum longus muscle, neuromuscular junction morphology did not change significantly during hibernation. Agrin-Lrp4-MuSK is a key pathway for the formation and maintenance of the neuromuscular junction. Our results showed that low-density lipoprotein receptor-associated protein 4 (Lrp4) expression in the soleus (slow muscle) decreased by 46.2% in the interbout arousal group compared with the pre-hibernation group (P = 0.019), with recovery in the post-hibernation group. Compared with the pre-hibernation group, agrin expression in the extensor digitorum longus (fast muscle) increased by 67.0% in the interbout arousal group (P = 0.016). In conclusion, periodic up-regulation in agrin expression during interbout arousal may be involved in the maintenance of neuromuscular junction morphology in the extensor digitorum longus muscle during hibernation. The degenerative changes in neuromuscular junction morphology and the periodic decrease in Lrp4 protein expression in the soleus during hibernation, these changes recovered to the pre-hibernation levels in the post-hibernation group, exhibiting significant plasticity. This plasticity may be one of the important mechanisms for resisting disuse atrophy in hibernating animals.NEW & NOTEWORTHY This study is the first to explore the neuromuscular junction morphology of slow- and fast-twitch muscles in Daurian ground squirrels during different periods of hibernation. Results showed that the neuromuscular junction maintained stable morphology in the extensor digitorum longus muscle. The degenerative changes in neuromuscular junction morphology and the periodic decrease in Lrp4 protein expression in the soleus muscle during hibernation recovered in post-hibernation, exhibiting significant plasticity.

AGRN promotes lung adenocarcinoma progression by activating Notch signaling pathway and acts as a therapeutic target.

Lung cancer is the main reason for cancer-associated death globally, and lung adenocarcinoma (LUAD) is the most prevalent subtype of lung cancer. Recently, AGRN is considered playing an vital role in the development of some cancers. However, the regulatory effects and mechanisms of AGRN in LUAD remain elusive. In this study, we clarified the significant upregulation of AGRN expression in LUAD by single-cell RNA sequencing combined with immunohistochemistry. Besides, we confirmed that LUAD patients with high AGRN expression are more susceptible to lymph node metastases and have a worse prognosis by a retrospective study of 120 LUAD patients. Next, we demonstrated that AGRN directly interact with NOTCH1, which results in the release of the intracellular structural domain of NOTCH1 and the subsequent activation of the NOTCH pathway. Moreover, we also found that AGRN promotes proliferation, migration, invasion, EMT and tumorigenesis of LUAD cells in vitro and in vivo, and that these effects are reversed by blocking the NOTCH pathway. Furthermore, we prepared several antibodies targeting AGRN, and clarify that Anti-AGRN antibody treatment could significantly inhibit proliferation and promote apoptosis of tumor cells. Our study highlights the important role and regulatory mechanism of AGRN in LUAD development and progression, and suggests that antibodies targeting AGRN have therapeutic potential for LUAD. We also provide theoretical and experimental evidence for further development of monoclonal antibodies targeting AGRN.

The collagen ColQ binds to LRP4 and regulates the activation of the Muscle-Specific Kinase-LRP4 receptor complex by agrin at the neuromuscular junction.

Collagen Q (ColQ) is a nonfibrillar collagen that plays a crucial role at the vertebrate neuromuscular junction (NMJ) by anchoring acetylcholinesterase to the synapse. ColQ also functions in signaling, as it regulates acetylcholine receptor clustering and synaptic gene expression, in a manner dependent on muscle-specific kinase (MuSK), a key protein in NMJ formation and maintenance. MuSK forms a complex with low-density lipoprotein receptor-related protein 4 (LRP4), its coreceptor for the proteoglycan agrin at the NMJ. Previous studies suggested that ColQ also interacts with MuSK. However, the molecular mechanisms underlying ColQ functions and ColQ-MuSK interaction have not been fully elucidated. Here, we investigated whether ColQ binds directly to MuSK and/or LRP4 and whether it modulates agrin-mediated MuSK-LRP4 activation. Using coimmunoprecipitation, pull-down, plate-binding assays, and surface plasmon resonance, we show that ColQ binds directly to LRP4 but not to MuSK and that ColQ interacts indirectly with MuSK through LRP4. In addition, we show that the LRP4 N-terminal region, which contains the agrin-binding sites, is also crucial for ColQ binding to LRP4. Moreover, ColQ-LRP4 interaction was reduced in the presence of agrin, suggesting that agrin and ColQ compete for binding to LRP4. Strikingly, we reveal ColQ has two opposing effects on agrin-induced MuSK-LRP4 signaling: it constitutively reduces MuSK phosphorylation levels in agrin-stimulated myotubes but concomitantly increases MuSK accumulation at the muscle cell surface. Our results identify LRP4 as a major receptor of ColQ and provide new insights into mechanisms of ColQ signaling and acetylcholinesterase anchoring at the NMJ.

Embryonic Ethanol but Not Cannabinoid Exposure Affects Zebrafish Cardiac Development via Agrin and Sonic Hedgehog Interaction.

Recent studies demonstrate the adverse effects of cannabinoids on development, including via pathways shared with ethanol exposure. Our laboratory has shown that both the nervous system and cardiac development are dependent on agrin modulation of sonic hedgehog (shh) and fibroblast growth factor (Fgf) signaling pathways. As both ethanol and cannabinoids impact these signaling molecules, we examined their role on zebrafish heart development. Zebrafish embryos were exposed to a range of ethanol and/or cannabinoid receptor 1 and 2 agonist concentrations in the absence or presence of morpholino oligonucleotides that disrupt agrin or shh expression. In situ hybridization was employed to analyze cardiac marker gene expression. Exposure to cannabinoid receptor agonists disrupted midbrain-hindbrain boundary development, but had no effect on heart development, as assessed by the presence of cardiac edema or the altered expression of cardiac marker genes. In contrast, exposure to 1.5% ethanol induced cardiac edema and the altered expression of cardiac marker genes. Combined exposure to agrin or shh morpholino and 0.5% ethanol disrupted the cmlc2 gene expression pattern, with the restoration of the normal expression following shh mRNA overexpression. These studies provide evidence that signaling pathways critical to heart development are sensitive to ethanol exposure, but not cannabinoids, during early zebrafish embryogenesis.

Structural insights into the assembly of the agrin/LRP4/MuSK signaling complex.

MuSK is a receptor tyrosine kinase (RTK) that plays essential roles in the formation and maintenance of the neuromuscular junction. Distinct from most members of RTK family, MuSK activation requires not only its cognate ligand agrin but also its coreceptors LRP4. However, how agrin and LRP4 coactivate MuSK remains unclear. Here, we report the cryo-EM structure of the extracellular ternary complex of agrin/LRP4/MuSK in a stoichiometry of 1:1:1. This structure reveals that arc-shaped LRP4 simultaneously recruits both agrin and MuSK to its central cavity, thereby promoting a direct interaction between agrin and MuSK. Our cryo-EM analyses therefore uncover the assembly mechanism of agrin/LRP4/MuSK signaling complex and reveal how MuSK receptor is activated by concurrent binding of agrin and LRP4.

Neural Isoforms of Agrin Are Generated by Reduced PTBP1-RNA Interaction Network Spanning the Neuron-Specific Splicing Regions in AGRN.

Agrin is a heparan sulfate proteoglycan essential for the clustering of acetylcholine receptors at the neuromuscular junction. Neuron-specific isoforms of agrin are generated by alternative inclusion of three exons, called Y, Z8, and Z11 exons, although their processing mechanisms remain elusive. We found, by inspection of splicing cis-elements into the human AGRN gene, that binding sites for polypyrimidine tract binding protein 1 (PTBP1) were extensively enriched around Y and Z exons. PTBP1-silencing enhanced the coordinated inclusion of Y and Z exons in human SH-SY5Y neuronal cells, even though three constitutive exons are flanked by these alternative exons. Deletion analysis using minigenes identified five PTBP1-binding sites with remarkable splicing repression activities around Y and Z exons. Furthermore, artificial tethering experiments indicated that binding of a single PTBP1 molecule to any of these sites represses nearby Y or Z exons as well as the other distal exons. The RRM4 domain of PTBP1, which is required for looping out a target RNA segment, was likely to play a crucial role in the repression. Neuronal differentiation downregulates PTBP1 expression and promotes the coordinated inclusion of Y and Z exons. We propose that the reduction in the PTPB1-RNA network spanning these alternative exons is essential for the generation of the neuron-specific agrin isoforms.

Expression of Basement Membrane Molecules by Wharton Jelly Stem Cells (WJSC) in Full-Term Human Umbilical Cords, Cell Cultures and Microtissues.

Wharton's jelly stem cells (WJSC) from the human umbilical cord (UC) are one of the most promising mesenchymal stem cells (MSC) in tissue engineering (TE) and advanced therapies. The cell niche is a key element for both, MSC and fully differentiated tissues, to preserve their unique features. The basement membrane (BM) is an essential structure during embryonic development and in adult tissues. Epithelial BMs are well-known, but similar structures are present in other histological structures, such as in peripheral nerve fibers, myocytes or chondrocytes. Previous studies suggest the expression of some BM molecules within the Wharton's Jelly (WJ) of UC, but the distribution pattern and full expression profile of these molecules have not been yet elucidated. In this sense, the aim of this histological study was to evaluate the expression of main BM molecules within the WJ, cultured WJSC and during WJSC microtissue (WJSC-MT) formation process. Results confirmed the presence of a pericellular matrix composed by the main BM molecules-collagens (IV, VII), HSPG2, agrin, laminin and nidogen-around the WJSC within UC. Additionally, ex vivo studies demonstrated the synthesis of these BM molecules, except agrin, especially during WJSC-MT formation process. The WJSC capability to synthesize main BM molecules could offer new alternatives for the generation of biomimetic-engineered substitutes where these molecules are particularly needed.

Acetylcholine receptors of the neuromuscular junctions present normal distribution after peripheral nerve injury and repair through nerve guidance associated with fibrin biopolymer.

Peripheral nerve injuries (PNI) lead to alterations in the Agrin-LRP4-MuSK pathway. This results in disaggregation of AChRs and change from epsilon (mature, innervated) to gamma (immature, denervated) subunit. Tubulization technique has been shown to be effective for PNI repair and it also allows the use of adjuvants, such as fibrin biopolymer (FB). This study evaluated the effect of the association of tubulization with FB after PNI on AChRs and associated proteins. Fifty-two adults male Wistar rats were used, distributed in 4 experimental groups: Sham Control (S), Denervated Control (D); Tubulization (TB) and Tubulization + Fibrin Biopolymer (TB+FB). Catwalk was performed every 15 days. Ninety days after surgery the right soleus muscles and ischiatic nerves were submitted to the following analyses: (a) morphological and morphometric analysis of AChRs by confocal microscopy; (b) morphological and morphometric analysis of the ischiatic nerve; (c) protein quantification of AChRs: alpha, gama, and epsilon, of Schwann cells, agrin, LRP4, MuSK, rapsyn, MMP3, MyoD, myogenin, MURF1 and atrogin-1. The main results were about the NMJs that in the TB+FB group presented morphological and morphometric approximation (compactness index; area of the AChRs and motor plate) to the S group. In addition, there were also an increase of S100 and AChRε protein expression and a decrease of MyoD. These positive association resulted in AChRs stabilization that potentiate the neuromuscular regeneration, which strengthens the use of TB for severe injuries repair and the beneficial effect of FB, along with tubulization technique.

DOK7 Promotes NMJ Regeneration After Nerve Injury.

Motor function recovery from injury requires the regeneration of not only muscle fibers, but also the neuromuscular junction-the synapse between motor nerve terminals and muscle fibers. However, unlike muscle regeneration which has been extensively studied, little is known about the molecular mechanisms of NMJ regeneration. Recognizing the critical role of agrin-LRP4-MuSK signaling in NMJ formation and maintenance, we investigated whether increasing MuSK activity promotes NMJ regeneration. To this end, we evaluated the effect of DOK7, a protein that stimulates MuSK, on NMJ regeneration. Reinnervation, AChR cluster density, and endplate area were improved, and fragmentation was reduced in the AAV9-DOK7-GFP-injected muscles compared with muscles injected with AAV9-GFP. These results demonstrated expedited NMJ regeneration associated with increased DOK7 expression and support the hypothesis that increasing agrin signaling benefits motor function recovery after injury. Our findings propose a potentially new therapeutic strategy for functional recovery after muscle and nerve injury, i.e., promoting NMJ regeneration by increasing agrin signaling.

A link between agrin signalling and Cav3.2 at the neuromuscular junction in spinal muscular atrophy.

SMN protein deficiency causes motoneuron disease spinal muscular atrophy (SMA). SMN-based therapies improve patient motor symptoms to variable degrees. An early hallmark of SMA is the perturbation of the neuromuscular junction (NMJ), a synapse between a motoneuron and muscle cell. NMJ formation depends on acetylcholine receptor (AChR) clustering triggered by agrin and its co-receptors lipoprotein receptor-related protein 4 (LRP4) and transmembrane muscle-specific kinase (MuSK) signalling pathway. We have previously shown that flunarizine improves NMJs in SMA model mice, but the mechanisms remain elusive. We show here that flunarizine promotes AChR clustering in cell-autonomous, dose- and agrin-dependent manners in C2C12 myotubes. This is associated with an increase in protein levels of LRP4, integrin-beta-1 and alpha-dystroglycan, three agrin co-receptors. Furthermore, flunarizine enhances MuSK interaction with integrin-beta-1 and phosphotyrosines. Moreover, the drug acts on the expression and splicing of Agrn and Cacna1h genes in a muscle-specific manner. We reveal that the Cacna1h encoded protein Cav3.2 closely associates in vitro with the agrin co-receptor LRP4. In vivo, it is enriched nearby NMJs during neonatal development and the drug increases this immunolabelling in SMA muscles. Thus, flunarizine modulates key players of the NMJ and identifies Cav3.2 as a new protein involved in the NMJ biology.

Neuronal Agrin Promotes Proliferation of Primary Human Myoblasts in an Age-Dependent Manner.

Neuronal agrin, a heparan sulphate proteoglycan secreted by the α-motor neurons, promotes the formation and maintenance of the neuromuscular junction by binding to Lrp4 and activating muscle-specific kinase (MuSK). Neuronal agrin also promotes myogenesis by enhancing differentiation and maturation of myotubes, but its effect on proliferating human myoblasts, which are often considered to be unresponsive to agrin, remains unclear. Using primary human myoblasts, we determined that neuronal agrin induced transient dephosphorylation of ERK1/2, while c-Abl, STAT3, and focal adhesion kinase were unresponsive. Gene silencing of Lrp4 and MuSK markedly reduced the BrdU incorporation, suggesting the functional importance of the Lrp4/MuSK complex for myoblast proliferation. Acute and chronic treatments with neuronal agrin increased the proliferation of human myoblasts in old donors, but they did not affect the proliferation of myoblasts in young donors. The C-terminal fragment of agrin which lacks the Lrp4-binding site and cannot activate MuSK had a similar age-dependent effect, indicating that the age-dependent signalling pathways activated by neuronal agrin involve the Lrp4/MuSK receptor complex as well as an Lrp4/MuSK-independent pathway which remained unknown. Collectively, our results highlight an age-dependent role for neuronal agrin in promoting the proliferation of human myoblasts.

SMN controls neuromuscular junction integrity through U7 snRNP.

The neuromuscular junction (NMJ) is an essential synapse whose loss is a key hallmark of the neurodegenerative disease spinal muscular atrophy (SMA). Here, we show that activity of the SMA-determining SMN protein in the assembly of U7 small nuclear ribonucleoprotein (snRNP)-which functions in the 3'-end processing of replication-dependent histone mRNAs-is required for NMJ integrity. Co-expression of U7-specific Lsm10 and Lsm11 proteins selectively enhances U7 snRNP assembly, corrects histone mRNA processing defects, and rescues key structural and functional abnormalities of neuromuscular pathology in SMA mice-including NMJ denervation, decreased synaptic transmission, and skeletal muscle atrophy. Furthermore, U7 snRNP dysfunction drives selective loss of the synaptic organizing protein Agrin at NMJs innervating vulnerable muscles of SMA mice. These findings reveal a direct contribution of U7 snRNP dysfunction to neuromuscular pathology in SMA and suggest a role for histone gene regulation in maintaining functional synaptic connections between motor neurons and muscles.

Impaired signaling for neuromuscular synaptic maintenance is a feature of Motor Neuron Disease.

A central event in the pathogenesis of motor neuron disease (MND) is the loss of neuromuscular junctions (NMJs), yet the mechanisms that lead to this event in MND remain to be fully elucidated. Maintenance of the NMJ relies upon neural agrin (n-agrin) which, when released from the nerve terminal, activates the postsynaptic Muscle Specific Kinase (MuSK) signaling complex to stabilize clusters of acetylcholine receptors. Here, we report that muscle from MND patients has an increased proportion of slow fibers and muscle fibers with smaller diameter. Muscle cells cultured from MND biopsies failed to form large clusters of acetylcholine receptors in response to either non-MND human motor axons or n-agrin. Furthermore, levels of expression of MuSK, and MuSK-complex components: LRP4, Caveolin-3, and Dok7 differed between muscle cells cultured from MND patients compared to those from non-MND controls. To our knowledge, this is the first time a fault in the n-agrin-LRP4-MuSK signaling pathway has been identified in muscle from MND patients. Our results highlight the n-agrin-LRP4-MuSK signaling pathway as a potential therapeutic target to prolong muscle function in MND.