Combination of agrin and laminin increase acetylcholine receptor clustering and enhance functional neuromuscular junction formation In vitro.

Clustering of acetylcholine receptors (AChR) at the postsynaptic membrane is a crucial step in the development of neuromuscular junctions (NMJ). During development and after denervation, aneural AChR clusters form on the sarcolemma. Recent studies suggest that these receptors are critical for guiding and initiating synaptogenesis. The aim of this study is to investigate the effect of agrin and laminin-1; agents with known AChR clustering activity; on NMJ formation and muscle maturation. Primary myoblasts were differentiated in vitro on collagen, laminin or collagen and laminin-coated surfaces in the presence or absence of agrin and laminin. The pretreated cells were then subject to innervation by PC12 cells. The number of neuromuscular junctions was assessed by immunocytochemical co-localization of AChR clusters and the presynaptic marker synaptophysin. Functional neuromuscular junctions were quantitated by analysis of the level of spontaneous as well as neuromuscular blocker responsive contractile activity and muscle maturation was assessed by the degree of myotube striation. Agrin alone did not prime muscle for innervation while a combination of agrin and laminin pretreatment increased the number of neuromuscular junctions formed and enhanced acetylcholine based neurotransmission and myotube striation. This study has direct clinical relevance for treatment of denervation injuries and creating functional neuromuscular constructs for muscle tissue repair.

Agrin requires specific proteins to selectively activate γ-aminobutyric acid neurons for pain suppression.

Agrin, a heparan sulfate proteoglycan functioning as a neuro-muscular junction inducer, has been shown to inhibit neuropathic pain in sciatic nerve injury rat models, via phosphorylation of N-Methyl-d-aspartate receptor NR1 subunits in gamma-aminobutyric acid neurons. However, its effects on spinal cord injury-induced neuropathic pain, a debilitating syndrome frequently encountered after various spine traumas, are unknown. In the present investigation, we studied the 50kDa agrin isoform effects in a quisqualic acid dorsal horn injection rat model mimicking spinal cord injury-induced neuropathic pain. Our results indicate that 50kDa agrin decreased only in the dorsal horn of neuropathic animals and increased 50kDa agrin expression in the dorsal horn, via intra-spinal injection of adeno-associated virus serum type two, suppressed spinal cord injury-induced neuropathic pain. Also, the reason why 50kDa agrin only activates the N-Methyl-d-aspartate receptor NR1 subunits in the GABA neurons, but not in sensory neurons, is unknown. Using immunoprecipitation and Western-blot analysis, two dimensional gel separation, and mass spectrometry, we identified several specific proteins in the reaction protein complex, such as neurofilament 200 and mitofusin 2, that are required for the activation of the NR1 subunits of gamma-aminobutyric acid inhibitory neurons by 50kDa agrin. These findings indicate that 50kDa agrin is a promising agent for neuropathic pain treatment.

Injection of a soluble fragment of neural agrin (NT-1654) considerably improves the muscle pathology caused by the disassembly of the neuromuscular junction.

Treatment of neuromuscular diseases is still an unsolved problem. Evidence over the last years strongly indicates the involvement of malformation and dysfunction of neuromuscular junctions in the development of such medical conditions. Stabilization of NMJs thus seems to be a promising approach to attenuate the disease progression of muscle wasting diseases. An important pathway for the formation and maintenance of NMJs is the agrin/Lrp4/MuSK pathway. Here we demonstrate that the agrin biologic NT-1654 is capable of activating the agrin/Lrp4/MuSK system in vivo, leading to an almost full reversal of the sarcopenia-like phenotype in neurotrypsin-overexpressing (SARCO) mice. We also show that injection of NT-1654 accelerates muscle re-innervation after nerve crush. This report demonstrates that a systemically administered agrin fragment has the potential to counteract the symptoms of neuromuscular disorders.

Local induction of acetylcholine receptor clustering in myotube cultures using microfluidic application of agrin.

During neuromuscular synaptogenesis, the exchange of spatially localized signals between nerve and muscle initiates the coordinated focal accumulation of the acetylcholine (ACh) release machinery and the ACh receptors (AChRs). One of the key first steps is the release of the proteoglycan agrin focalized at the axon tip, which induces the clustering of AChRs on the postsynaptic membrane at the neuromuscular junction. The lack of a suitable method for focal application of agrin in myotube cultures has limited the majority of in vitro studies to the application of agrin baths. We used a microfluidic device and surface microengineering to focally stimulate muscle cells with agrin at a small portion of their membrane and at a time and position chosen by the user. The device is used to verify the hypothesis that focal application of agrin to the muscle cell membrane induces local aggregation of AChRs in differentiated C2C12 myotubes.

A single pulse of agrin triggers a pathway that acts to cluster acetylcholine receptors.

Agrin triggers signaling mechanisms of high temporal and spatial specificity to achieve phosphorylation, clustering, and stabilization of postsynaptic acetylcholine receptors (AChRs). Agrin transiently activates the kinase MuSK; MuSK activation has largely vanished when AChR clusters appear. Thus, a tyrosine kinase cascade acts downstream from MuSK, as illustrated by the agrin-evoked long-lasting activation of Src family kinases (SFKs) and their requirement for AChR cluster stabilization. We have investigated this cascade and report that pharmacological inhibition of SFKs reduces early but not later agrin-induced phosphorylation of MuSK and AChRs, while inhibition of Abl kinases reduces late phosphorylation. Interestingly, SFK inhibition applied selectively during agrin-induced AChR cluster formation caused rapid cluster dispersal later upon agrin withdrawal. We also report that a single 5-min agrin pulse, followed by extensive washing, triggered long-lasting MuSK and AChR phosphorylation and efficient AChR clustering. Following the pulse, MuSK phosphorylation increased and, beyond a certain level, caused maximal clustering. These data reveal novel temporal aspects of tyrosine kinase action in agrin signaling. First, during AChR cluster formation, SFKs initiate early phosphorylation and an AChR stabilization program that acts much later. Second, a kinase mechanism rapidly activated by agrin acts thereafter autonomously in agrin's absence to further increase MuSK phosphorylation and cluster AChRs.

ATP potentiates agrin-induced AChR aggregation in cultured myotubes: activation of RhoA in P2Y1 nucleotide receptor signaling at vertebrate neuromuscular junctions.

At vertebrate neuromuscular junctions, ATP is known to stabilize acetylcholine in the synaptic vesicles and to be co-released with it. We have shown previously that a nucleotide receptor, P2Y(1) receptor, is localized at the nmjs, and we propose that this mediates a trophic role for synaptic ATP there. In cultured myotubes, the activation of P2Y(1) receptors modulated agrin-induced acetylcholine receptor (AChR) aggregation in a potentiation manner. This potentiation effect in agrin-induced AChR aggregation was reduced by antagonizing the P2Y(1) receptors. The guanosine triphosphatase RhoA was shown to be responsible for this P2Y(1)-potentiated effect. The localization of RhoA in rat and chicken skeletal muscles was restricted at the neuromuscular junctions. Application of P2Y(1) agonists in cultured myotubes induced RhoA activation, which showed an additive effect with agrin-induced RhoA activation. Over-expression of dominant-negative mutant of RhoA in cultured myotubes diminished the agrin-induced AChR aggregation, as well as the potentiation effect of P2Y(1)-specific agonist. Application of UTP in the cultures also triggered similar responses as did 2-methylthioadenosine 5'-diphosphate, suggesting the involvement of other subtypes of P2Y receptors. These results demonstrate that RhoA could serve as a downstream mediator of signaling mediated by P2Y(1) receptor and agrin, which therefore synergizes the effects of the two neuron-derived trophic factors in modulating the formation and/or maintenance of post-synaptic apparatus at the neuromuscular junctions.

Muscle activity and muscle agrin regulate the organization of cytoskeletal proteins and attached acetylcholine receptor (AchR) aggregates in skeletal muscle fibers.

In innervated skeletal muscle fibers, dystrophin and beta-dystroglycan form rib-like structures (costameres) that appear as predominantly transverse stripes over Z and M lines. Here, we show that the orientation of these stripes becomes longitudinal in denervated muscles and transverse again in denervated electrically stimulated muscles. Skeletal muscle fibers express nonneural (muscle) agrin whose function is not well understood. In this work, a single application of > or = 10 nM purified recombinant muscle agrin into denervated muscles preserved the transverse orientation of costameric proteins that is typical for innervated muscles, as did a single application of > or = 1 microM neural agrin. At lower concentration, neural agrin induced acetylcholine receptor aggregates, which colocalized with longitudinally oriented beta-dystroglycan, dystrophin, utrophin, syntrophin, rapsyn, and beta 2-laminin in denervated unstimulated fibers and with the same but transversely oriented proteins in innervated or denervated stimulated fibers. The results indicate that costameres are plastic structures whose organization depends on electrical muscle activity and/or muscle agrin.

Effects of purified recombinant neural and muscle agrin on skeletal muscle fibers in vivo.

Aggregation of acetylcholine receptors (AChRs) in muscle fibers by nerve-derived agrin plays a key role in the formation of neuromuscular junctions. So far, the effects of agrin on muscle fibers have been studied in culture systems, transgenic animals, and in animals injected with agrin--cDNA constructs. We have applied purified recombinant chick neural and muscle agrin to rat soleus muscle in vivo and obtained the following results. Both neural and muscle agrin bind uniformly to the surface of innervated and denervated muscle fibers along their entire length. Neural agrin causes a dose-dependent appearance of AChR aggregates, which persist > or = 7 wk after a single application. Muscle agrin does not cluster AChRs and at 10 times the concentration of neural agrin does not reduce binding or AChR-aggregating activity of neural agrin. Electrical muscle activity affects the stability of agrin binding and the number, size, and spatial distribution of the neural agrin--induced AChR aggregates. Injected agrin is recovered from the muscles together with laminin and both proteins coimmunoprecipitate, indicating that agrin binds to laminin in vivo. Thus, the present approach provides a novel, simple, and efficient method for studying the effects of agrin on muscle under controlled conditions in vivo.