Agrin, a synaptically located protein, is a key player during initial formation and maintenance of NMJs where it induces acetylcholine receptor (AChR) assembly and aggregation by binding to its receptor LRP4 followed by signaling through MuSK / DOK-7. During development, nerve cells approach muscle fibers and establish synaptic contacts defined as NMJ. Initially, the NMJ is small and weak, but if the contact is successful the connection is maintained and reinforced. Agrin induces and stabilizes clusters of AChRs at the NMJ, promotes synaptic maturation, and maintains the mature state of the NMJ.
Agrin is specifically cleaved by neurotrypsin, a serine protease, leading to a soluble C-terminal Agrin Fragment (22 kDa named CAF or CAF22; present in blood) thereby inactivating the synapse promoting activity and counteracting the effect of agrin. The molecular structure and the cleavage mechanism of serine proteases have been studied extensively and are well known. Both agrin and neurotrypsin are located in the synaptic cleft, the space between the pre-synaptic (signal-emitting) and the post-synaptic (signal-receiving) cell.
Agrin, LRP4, MuSK and DOC7 knock-out mutations in transgenic mice lead to the same phenotype seen with neurotrypsin overexpression indicating that disturbance of this signaling pathway leads to irregular NMJ formation.
The agrin / neurotrypsin system is well known for its pivotal role in the formation and maintenance of NMJs, the cholinergic connections between nerves and muscles. More recently, a similar role of this system in central synaptogenesis and in other organs has been discovered.
Weakening of neuromuscular junctions in an animal model (using slight overexpression of neurotrypsin) has resulted in the reproduction of many hallmarks of neuromuscular diseases such as reduced muscle strength, loss of coordination, and premature death. Other hallmarks are the pathology such as highly fragmented NMJs, loss of muscle mass, muscle fiber type changes or reduced mitochondrial function (SARCO mice; Bütikofer et al, 2011; Hettwer et al, 2014).
Concomitantly with disturbed NMJ formation, reduced mitochondrial function is observed. Addition of exogenous agrin corrected the defect suggesting neuromuscular junctions and mitochondrial viability is linked and that restoration of NMJs also revives mitochondria. About half of amyotrophic lateral sclerosis (ALS) patients appear to have reduced COX levels.
Manipulation of the agrin signaling path may therefore be a promising way to correct neuromuscular defects. Elevation of MuSK signaling has improved motor performance and delayed denervation in ALS mice (Perez-Garcia and Burden, 2012).
Neurotune’s therapeutic agrin fragment (agrin biologic) is able to bind to LRP4 and stimulate NMJ. Key features of the agrin biologic are its excellent solubility allowing subcutaneous administration and its resistance to neurotrypsin cleavage allowing prolonged signaling. It has proven its effectiveness in various animal models including models of SMA, ALS, nerve injury, MG and sarcopenia.