Botulinum neurotoxin (BoNT) has become a powerful therapeutic tool, and is extensively used in aesthetic medicine and in the treatment of neurological disorders. However, its duration of effect is limited, mainly owing to nerve sprouting. Inhibition of nerve sprouting to prolong the effective duration of BoNT is therefore of great clinical interest. However, appropriate interventional strategies to accomplish this are currently unavailable. In this study, we determined the role of the neurogenic regulator agrin in BoNT type A (BoNT/A)-induced nerve sprouting in a rat model. We then determined whether agrin could be used as an interventional target for prolonging the duration of effect of BoNT/A, and made a preliminary study of the upstream and downstream regulatory mechanisms by which agrin could influence the effective duration of BoNT/A. Our results showed that agrin was involved in the regulation of BoNT/A-induced nerve sprouting, and blocking of agrin function with anti-agrin antibody temporarily could delay muscle strength recovery and prolong the duration of BoNT/A effect. Moreover, agrin influenced the duration of BoNT/A effect by regulating downstream myogenic muscle-specific receptor tyrosine kinase (MuSK), and was simultaneously regulated by upstream miR-144. In conclusion, agrin could regulate BoNT/A-induced nerve sprouting through miR-144-agrin-MuSK signaling; it influences the effective duration of BoNT/A, and could find clinical application as an interventional target for prolonging the effect of BoNT/A.

The C-terminal 95 kDa fragment of some isoforms of vertebrate agrins is sufficient to induce clustering of acetylcholine receptors but despite two decades of intense agrin research very little is known about the function of the other isoforms and the function of the larger, N-terminal part of agrins that is common to all isoforms. Since the N-terminal part of agrins contains several follistatin-domains, a domain type that is frequently implicated in binding TGFbetas, we have explored the interaction of the N-terminal part of rat agrin (Agrin-Nterm) with members of the TGFbeta family using surface plasmon resonance spectroscopy and reporter assays. Here we show that agrin binds BMP2, BMP4 and TGFbeta1 with relatively high affinity, the K(D) values of the interactions calculated from SPR experiments fall in the 10(-8) M-10(-7) M range. In reporter assays Agrin-Nterm inhibited the activities of BMP2 and BMP4, half maximal inhibition being achieved at approximately 5x10(-7) M. Paradoxically, in the case of TGFbeta1 Agrin N-term caused a slight increase in activity in reporter assays. Our finding that agrin binds members of the TGFbeta family may have important implications for the role of these growth factors in the regulation of synaptogenesis as well as for the role of agrin isoforms that are unable to induce clustering of acetylcholine receptors. We suggest that binding of these TGFbeta family members to agrin may have a dual function: agrin may serve as a reservoir for these growth factors and may also inhibit their growth promoting activity. Based on analysis of the evolutionary history of agrin we suggest that agrin's growth factor binding function is more ancient than its involvement in acetylcholine receptor clustering.

Agrin is a heparan sulfate proteoglycan that induces aggregation of acetylcholine receptors (AChRs) at the neuromuscular synapse. This aggregating activity is modulated by alternative splicing. Here, we compared binding of agrin isoforms to heparin, alpha-dystroglycan, and cultured myotubes. We find that the alternatively spliced 4 amino acids insert (KSRK) is required for heparin binding. The binding affinity of agrin isoforms to alpha-dystroglycan correlates neither with binding to heparin nor with their AChR-aggregating activities. Moreover, the minimal fragment sufficient to induce AChR aggregation does not bind to alpha-dystroglycan. Nevertheless, this fragment still binds to cultured muscle cells. Its binding is completed only by agrin isoforms that are active in AChR aggregation, and therefore this binding site is likely to represent the receptor that initiates AChR clustering.

Although physiological data suggest that neuromuscular junction (NMJ) dysfunction is a principal mechanism underpinning sarcopenia, genetic studies have implicated few genes involved in NMJ function. Accordingly, we explored whether genes encoding agrin (AGRN) and neurotrypsin (PRSS12) were associated with sarcopenia phenotypes: muscle mass, strength and plasma C-terminal agrin fragment (CAF). PhenoScanner was used to determine if AGRN and/or PRSS12 variants had previously been implicated with sarcopenia phenotypes. For replication, we combined genotype from whole genome sequencing with phenotypic data from 6715 GenoFit participants aged 18-83 years. Dual energy X-ray absorptiometry assessed whole body lean mass (WBLM) and appendicular lean mass (ALM), hand dynamometry determined grip strength and ELISA measured plasma CAF in a subgroup (n = 260). Follow-up analyses included eQTL analyses, carrier analyses, single-variant and gene-burden tests. rs2710873 (AGRN) and rs71608359 (PRSS12) associate with muscle mass and strength phenotypes, respectively, in the UKBB (p = 8.9 × 10-6 and p = 8.4 × 10-6) and GenoFit cohort (p = 0.019 and p = 0.014). rs2710873 and rs71608359 are eQTLs for AGRN and PRSS12, respectively, in ≥ three tissues. Compared to non-carriers, carriers of rs2710873 had 4.0% higher WBLM and ALM (both p < 0.001), and 9.5% lower CAF concentrations (p < 0.001), while carriers of rs71608359 had 2.3% lower grip strength (p = 0.034). AGRN and PRSS12 are associated with muscle strength and mass in single-variant analyses, while PRSS12 has further associations with muscle strength in gene-burden tests. Our findings provide novel evidence of the relevance of AGRN and PRSS12 to sarcopenia phenotypes and support existing physiological data illustrating the importance of the NMJ in maintaining muscle health during ageing.

Filopodia sense the extracellular environment and direct movement in many cell types, including neurons. Recent reports suggest that the transmembrane form of the widely expressed proteoglycan agrin (TM-agrin) regulates formation and stability of neuronal filopodia. In order to elucidate the mechanism by which TM-agrin regulates filopodia, we investigated the role of agrin's glycosaminoglycan (GAG) chains in the induction of filopodia formation by TM-agrin over-expression in hippocampal neurons, and in the induction of filopodia-like processes in COS7 cells. Deletion of the GAG chains of TM-agrin sharply reduced formation of filopodia-like branched retraction fibers (BRFs) in COS7 cells, with deletion of the heparan sulfate GAG chains being most effective, and eliminated filopodia induction in hippocampal neurons. GAG chain deletion also reduced the activation of Cdc42 and Rac1 resulting from TM-agrin over-expression. Moreover, dominant-negative Cdc42 and Rac1 inhibited BRF formation. Lastly, over-expression of TM-agrin increased the adhesiveness of COS7 cells and this increase was reduced by deletion of the GAG chains. Our results suggest that TM-agrin regulates actin-based protrusions in large part through interaction of its GAG chains with extracellular or transmembrane proteins, leading to the activation of Cdc42 and Rac1.

Formation of th neuromuscular junction depends upon reciprocal inductive interactions between the developing nerve and muscle, resulting in the precise juxtaposition of a differentiated nerve terminal with a highly specialized patch on the muscle membrane, termed the motor endplate. Agrin is a nerve-derived factor that can induced molecular reorganizations at the motor endplate, but the mechanism of action of agrin remains poorly understood. MuSK is a receptor tyrosine kinase localized to the motor endplate, seemingly well positioned to receive a key nerve-derived signal. Mice lacking either agrin or MuSK have recently been generated and exhibit similarly profound defects in their neuromuscular junctions. Here we demonstrate that agrin acts via a receptor complex that includes MuSK as well as a myotube-specific accessory component.

Recently, the extracellular matrix protein agrin has been reported to promote tumor angiogenesis that supports tumorigenesis and metastasis; however, there is a lack of in vivo genetic evidence to prove whether agrin derived from the tumors or endothelial cells (ECs) systemically should be the therapeutic target. To date, the physiological role of endothelial agrin has also not been investigated. In the EC-specific agrin knockout mice, we observed normal endothelial and haematopoietic cell development during embryogenesis. Moreover, these mice develop normal vascular barrier integrity and vasoreactivity at the adult stage. Importantly, the growth of localized or metastatic cancer cells was not affected after implantation into endothelial agrin depleted mice. Mechanistically, agrin did not regulate endothelial ERK1/2, YAP or p53 activation in vivo that is central to support endothelial proliferation, survival and invasion. Cumulatively, our findings may suggest that agrin could play a redundant role in endothelial development during physiological and tumor angiogenesis. Targeting the endothelial derived agrin might not be effective in inhibiting tumor angiogenesis.

To determine if patients with myasthenia gravis (MG) have antibodies to agrin, a proteoglycan released by motor neurons and is critical for neuromuscular junction (NMJ) formation, we collected serum samples from 93 patients with MG with known status of antibodies to acetylcholine receptor (AChR), muscle specific kinase (MuSK) and lipoprotein-related 4 (LRP4) and samples from control subjects (healthy individuals and individuals with other diseases). Sera were assayed for antibodies to agrin. We found antibodies to agrin in 7 serum samples of MG patients. None of the 25 healthy controls and none of the 55 control neurological patients had agrin antibodies. Two of the four triple negative MG patients (i.e., no detectable AChR, MuSK or LRP4 antibodies, AChR-/MuSK-/LRP4-) had antibodies against agrin. In addition, agrin antibodies were detected in 5 out of 83 AChR+/MuSK-/LRP4- patients but were not found in the 6 patients with MuSK antibodies (AChR-/MuSK+/LRP4-). Sera from MG patients with agrin antibodies were able to recognize recombinant agrin in conditioned media and in transfected HEK293 cells. These sera also inhibited the agrin-induced MuSK phosphorylation and AChR clustering in muscle cells. Together, these observations indicate that agrin is another autoantigen in patients with MG and agrin autoantibodies may be pathogenic through inhibition of agrin/LRP4/MuSK signaling at the NMJ.

Astrocytic endfeet membranes are studded with aquaporin-4 (AQP4) containing orthogonal arrays of particles (OAP) which can be visualized exclusively by the freeze-fracturing method. They are predominantly expressed where the astroglial membrane is in contact with the superficial and perivascular basal lamina. This polarity seems to be essential for the integrity of the blood-brain barrier (BBB). The basal lamina containing many extracellular matrix (ECM) components such as collagen, laminin and heparansulfate proteoglycans like agrin is thought to influence this OAP-related polarity of astrocytes. Recently, we have shown that agrin, in particular the neuronal isoform A4B8, is capable of influencing the formation of OAPs in astrocytes when cultured in the presence of agrin-conditioned media. In this paper we wanted to investigate whether coating with exogenous agrin compared to coating with other ECM components would induce OAP formation in astrocytes of the agrin-null mouse. For this purpose, we cultured astrocytes from agrin-null and wild-type mice on agrin- or ECM-coated surfaces. Immunofluorescent cytochemical staining of AQP4 indicated a higher AQP4 expression level in cultures with agrin- or ECM-coated than in cultures with uncoated surfaces, whereas western blot analyses and PCR showed no differences. α-Dystroglycan is thought to be a potential receptor of agrin and was immunostained in wild-type as well as in agrin-null astrocytes. In freeze-fracture replicas, we observed an increase in OAP density in astrocytes when growing on agrin- and ECM-coatings. These results concurred with other experiments in which changes in volume were measured following hypotonic stress, which supported the positive influence of exogenous agrin on AQP4 insertion into the membrane, on OAP formation and on water transport.

Agrin is a heparan sulfate proteoglycan that is required for the formation and maintenance of neuromuscular junctions. During development, agrin is secreted from motor neurons to trigger the local aggregation of acetylcholine receptors (AChRs) and other proteins in the muscle fiber, which together compose the postsynaptic apparatus. After release from the motor neuron, agrin binds to the developing muscle basal lamina and remains associated with the synaptic portion throughout adulthood. We have recently shown that full-length chick agrin binds to a basement membrane-like preparation called Matrigel. The first 130 amino acids from the NH2 terminus are necessary for the binding, and they are the reason why, on cultured chick myotubes, AChR clusters induced by full-length agrin are small. In the current report we show that an NH2-terminal fragment of agrin containing these 130 amino acids is sufficient to bind to Matrigel and that the binding to this preparation is mediated by laminin-1. The fragment also binds to laminin-2 and -4, the predominant laminin isoforms of the muscle fiber basal lamina. On cultured myotubes, it colocalizes with laminin and is enriched in AChR aggregates. In addition, we show that the effect of full-length agrin on the size of AChR clusters is reversed in the presence of the NH2-terminal agrin fragment. These data strongly suggest that binding of agrin to laminin provides the basis of its localization to synaptic basal lamina and other basement membranes.

Humans are exposed to nitrate predominantly through diet with peak plasma concentrations within an hour after ingestion, but additional exposure is obtained from the environment, and minimally through de novo synthesis. Higher nitrate consumption has been associated with methemoglobinemia, spontaneous abortions, atherosclerosis, myocardial ischemia, septic and distressed lung, inflammatory bowel disease, amyotrophic lateral sclerosis, and neural tube defects. However, skeletal muscle development has not been examined.

Filopodia mediate axon guidance, neurite branching and synapse formation, but the membrane molecules that regulate neuronal filopodia in response to extracellular cues are largely unknown. The transmembrane isoform of the proteoglycan agrin, expressed predominantly in the CNS, may regulate neurite outgrowth, synapse formation and excitatory signaling. Here we demonstrate that agrin positively regulates neuronal filopodia. Over-expression of TM-agrin caused the formation of excess filopodia on neurites of hippocampal neurons cultured 1-6 days. Conversely, suppression of agrin expression by siRNA reduced the number of filopodia. Time lapse analysis indicated that endogenous TM-agrin regulates filopodia by increasing their stability and initiation. The N-terminal half of agrin was necessary for induction of filopodia, and over-expression of TM-agrin in a neuronal cell line increased Cdc42 activation, suggesting a role for Cdc42 downstream of agrin. By positively regulating filopodia in developing neurons, TM-agrin may influence the pattern of neurite outgrowth and synapse formation.

As synapses form and mature the synaptic partners produce organizing molecules that regulate each other's differentiation and ensure precise apposition of pre- and post-synaptic specializations. At the skeletal neuromuscular junction (NMJ), these molecules include agrin, a nerve-derived organizer of postsynaptic differentiation, and synaptic laminins, muscle-derived organizers of presynaptic differentiation. Both become concentrated in the synaptic cleft as the NMJ develops and are retained in adulthood. Here, we used mutant mice to ask whether these organizers are also required for synaptic maintenance. Deletion of agrin from a subset of adult motor neurons resulted in the loss of acetylcholine receptors and other components of the postsynaptic apparatus and synaptic cleft. Nerve terminals also atrophied and eventually withdrew from muscle fibers. On the other hand, mice lacking the presynaptic organizer laminin-α4 retained most of the synaptic cleft components but exhibited synaptic alterations reminiscent of those observed in aged animals. Although we detected no marked decrease in laminin or agrin levels at aged NMJs, we observed alterations in the distribution and organization of these synaptic cleft components suggesting that such changes could contribute to age-related synaptic disassembly. Together, these results demonstrate that pre- and post-synaptic organizers actively function to maintain the structure and function of adult NMJs.

Congenital myasthenic syndrome caused by mutations in AGRN, a gene encoding a protein with a crucial function at the neuromuscular junction, is a rare disorder. There are few studies in this area. We here present two cases with novel mutations of AGRN of which we further investigated possible pathogenesis.

Adult neurogenesis in the hippocampus may represent a form of plasticity in brain functions including mood, learning and memory. However, mechanisms underlying neural stem/progenitor cells (NSPCs) proliferation are not well understood. We found that Agrin, a factor critical for neuromuscular junction formation, is elevated in the hippocampus of mice that are stimulated by enriched environment (EE). Genetic deletion of the Agrn gene in excitatory neurons decreases NSPCs proliferation and increases depressive-like behavior. Low-density lipoprotein receptor-related protein 4 (Lrp4), a receptor for Agrin, is expressed in hippocampal NSPCs and its mutation blocked basal as well as EE-induced NSPCs proliferation and maturation of newborn neurons. Finally, we show that Lrp4 interacts with and activates receptor tyrosine kinase-like orphan receptor 2 (Ror2); and Ror2 mutation impairs NSPCs proliferation. Together, these observations identify a role of Agrin-Lrp4-Ror2 signaling for adult neurogenesis, uncovering previously unexpected functions of Agrin and Lrp4 in the brain.

Agrin is a nerve-derived factor that directs neuromuscular synapse formation, however its role in regulating interneuronal synaptogenesis is less clear. Here, we examine agrin's role in synapse formation between cholinergic preganglionic axons and sympathetic neurons in the superior cervical ganglion (SCG) using agrin-deficient mice. In dissociated cultures of SCG neurons, we found a significant decrease in the number of synapses with aggregates of presynaptic synaptophysin and postsynaptic neuronal acetylcholine receptor among agrin-deficient neurons as compared to wild-type neurons. Moreover, the levels of pre- and postsynaptic markers at the residual synapses in agrin-deficient SCG cultures were also reduced, and these defects were rescued by adding recombinant neural agrin to the cultures. Similarly, we observed a decreased matching of pre- and postsynaptic markers in SCG of agrin-deficient embryos, reflecting a decrease in the number of differentiated synapses in vivo. Finally, in electrophysiological experiments, we found that paired-pulse depression was more pronounced and posttetanic potentiation was significantly greater in agrin-deficient ganglia, indicating that synaptic transmission is also defective. Together, these findings indicate that neural agrin plays an organizing role in the formation and/or differentiation of interneuronal, cholinergic synapses.

Congenital myasthenic syndromes (CMS) are predominantly characterized by muscle weakness and fatigability and can be caused by a variety of mutations in genes required for neuromuscular junction formation and maintenance. Among them, AGRN encodes agrin, an essential synaptic protein secreted by motoneurons. We have identified severe CMS patients with uncharacterized p.R1671Q, p.R1698P and p.L1664P mutations in the LG2 domain of agrin. Overexpression in primary motoneurons cultures in vitro and in chick spinal motoneurons in vivo revealed that the mutations modified agrin trafficking, leading to its accumulation in the soma and/or in the axon. Expression of mutant agrins in cultured cells demonstrated accumulation of agrin in the endoplasmic reticulum associated with induction of unfolded protein response (UPR) and impaired secretion in the culture medium. Interestingly, evaluation of the specific activity of individual agrins on AChR cluster formation indicated that when secreted, mutant agrins retained a normal capacity to trigger the formation of AChR clusters. To confirm agrin accumulation and secretion defect, iPS cells were derived from a patient and differentiated into motoneurons. Patient iPS-derived motoneurons accumulated mutant agrin in the soma and increased XBP1 mRNA splicing, suggesting UPR activation. Moreover, co-cultures of patient iPS-derived motoneurons with myotubes confirmed the deficit in agrin secretion and revealed a reduction in motoneuron survival. Altogether, we report the first mutations in AGRN gene that specifically affect agrin secretion by motoneurons. Interestingly, the three patients carrying these mutations were initially suspected of spinal muscular atrophy (SMA). Therefore, in the presence of patients with a clinical presentation of SMA but without mutation in the SMN1 gene, it can be worth to look for mutations in AGRN.

Oral squamous cell carcinoma is the most common type of cancer in the oral cavity, representing more than 90% of all oral cancers. The characterization of altered molecules in oral cancer is essential to understand molecular mechanisms underlying tumor progression as well as to contribute to cancer biomarker and therapeutic target discovery. Proteoglycans are key molecular effectors of cell surface and pericellular microenvironments, performing multiple functions in cancer. Two of the major basement membrane proteoglycans, agrin and perlecan, were investigated in this study regarding their role in oral cancer. Using real time quantitative PCR (qRT-PCR), we showed that agrin and perlecan are highly expressed in oral squamous cell carcinoma. Interestingly, cell lines originated from distinct sites showed different expression of agrin and perlecan. Enzymatically targeting chondroitin sulfate modification by chondroitinase, oral squamous carcinoma cell line had a reduced ability to adhere to extracellular matrix proteins and increased sensibility to cisplatin. Additionally, knockdown of agrin and perlecan promoted a decrease on cell migration and adhesion, and on resistance of cells to cisplatin. Our study showed, for the first time, a negative regulation on oral cancer-associated events by either targeting chondroitin sulfate content or agrin and perlecan levels.

Recent studies from our laboratory have begun to elucidate the role of agrin in zebrafish development. One agrin morphant phenotype that results from agrin knockdown is microphthalmia (reduced eye size). To begin to understand the mechanisms underlying the role of agrin in eye development, we have analyzed retina development in agrin morphants. Retinal differentiation is impaired in agrin morphants, with retinal lamination being disrupted following agrin morpholino treatment. Pax 6.1 and Mbx1 gene expression, markers of eye development, are markedly reduced in agrin morphants. Formation of the optic fiber layer of the zebrafish retina is also impaired, exhibited as both reduced size of the optic fiber layer, and disruption of retinal ganglion cell axon growth to the optic tectum. The retinotectal topographic projection to the optic tectum is perturbed in agrin morphants in association with a marked loss of heparan sulfate expression in the retinotectal pathway, with this phenotype resembling retinotectal phenotypes observed in mutant zebrafish lacking enzymes for heparan sulfate synthesis. Treatment of agrin morphants with a fibroblast growth factor (Fgf) receptor inhibitor, rescue of the retinal lamination phenotype by transplantation of Fgf8-coated beads, and disruption of both the expression of Fgf-dependent genes and activation of ERK in agrin morphants provides evidence that agrin modulation of Fgf function contributes to retina development. Collectively, these agrin morphant phenotypes provide support for a crucial role of agrin in retina development and formation of an ordered retinotectal topographic map in the optic tectum of zebrafish.

The extracellular matrix modulates the hallmarks of cancer. Here we examined the role of agrin-a member of this matrix-in progression of oral squamous cell carcinoma (OSCC).