PBL3 NMJ/MG Symposium 2 Speaker 4 The Agrin-Musk system and acetylcholine signaling are said to regulate postsynaptic AChR

R clustering. What is the main evidence for this idea? Agrin Promotes Synaptic Differentiation by Counteracting an Inhibitory Effect of Neurotransmitter (Misgeld et al 2005)

The premise: Synaptic development is regulated by synaptic organizing molecules (e.g. Musk, agrin) and neurotransmission (i.e. the exocytosis of ACh vesicles). Agrin is synthesized and released by motor neurons at the NMJ promotes AChR aggregation and initiates postsynaptic differentiation The experiments dealt with the instability in the formation of new postsynaptic sites caused by ACh and that a major role of agrin is to counteract this influence. The experiments also assessed the interdependence of these two elements during synaptic development. 3 mutant mice were used in the experiments: agrin deficient mice (agrn -/-), ACh deficient mice (chat -/-) [by means of mutation in the ACh synthetic enzyme, Acetyl Transferase] and double knockout mice (dko, agrn -/-, chat -/-) To test other parameters they also used myoblast cell lines and chick myotubes. agrn -/- mice are born paralyzed due to extensive synaptic defects. chat -/- mice also die at birth but has extensive embryologic NMJ development/differentiation. The first experiment revealed that neuromuscular junctions (NMJs) develop extensively in a deficiency of agrin when ACh is also absent. They mated agrn deficient mice with ACh deficient mice thus producing double knockouts. It was found that the dkos imperfectly rescued AChR clustering (comparing them to agrn -/- mice). An interesting result of this was that in dko mice there was 90% motor axon AchR apposition and the presence of vesicle markers such as synaptophysin and VS2 is noted. There was also a comparable level of colocalisation of postsynaptic membrane proteins, such as MuSK and rapsyn (compared to control) Embryological studies revealed that in agrn -/- mice, after ~15 days there is AChR aggregate dispersal (or dismantling). In dkos, premature development is observed. To test the effect of ACh on AChR dispersal they used a non-hydrolyzable cholinergic agonist, CCh, on myoblasts. Incubation with the CCh dispersed the spontaneously formed AChR aggregates. The use of curare revealed that its ability to disperse the aggregates relied on its binding to the receptor. Moreover, they postulated that it took more than ligand induced conformational changes in the AChR to cause disaggregation; the subsequent ion flux was also required. In assessing the ability of agrin to safeguard against cholinergic dispersal of AChR clusters they cocultured myotubes with heterologous Chinese hamster ovary cells which were engineered to express a membrane anchored agrin isoform (Z+ isoform). Incubation with CCh had no effect on the number, size, or receptor density of agrin-associated aggregates. The Z+ isoform (contains inserts at splice site Z; selectively expressed by motorneurons) was compared to the Z- (lacks the Z exon; expressed by both neurons and myotubes). Only Z+ agrin associated clusters were cholinergic resistant. ACh and agrin have a balanced antagonistic relationship in NMJ formation