Induction versus Stabilization of Postsynaptic Differentiation by Agrin

In mice with mutations that result in a complete absence of motor innervation to a muscle, such as HB9 or topoisomerase2fi mutations, a pattern of postsynaptic differentiation and AChR clustering appears in the embryonic muscle at about the same time a motor neuron would normally be making contact (E12-13 in the mouse)28,29. This pattern persists until birth, although it is not as well defined spatially or as robust as in wild-type mice. However, it is dramatically better than in agrin-knockout mice. Therefore, the complete absence of a nerve leads to more persistent postsynaptic differentiation in muscle than one sees in the presence of a nerve lacking agrin. Interestingly, in double-knockout experiments, this postsynaptic differentiation was shown to still be dependent on MuSK, but to be completely independent of agrin11,30. There are a number of conclusions to be drawn from this work. First, muscle appears to have an intrinsic program of differentiation that is not dependent on cues from the nerve. Second, this program of differentiation does depend on MuSK, either through activation by a non-agrin, non-neuronal ligand, or through autoactivation of the receptor, as suggested by the strong inhibitory effects of genetic dosage seen in MuSK heterozygous animals that also lacked innervation. Third, the presence of a nerve that lacks agrin is in some way able to disperse the sites of postsynaptic differentiation assembled by the muscle.

Acetylcholine itself has emerged as at least one possible signal for the dispersal of muscle-derived sites of innervation. In mice lacking choline acetyltransferase (ChAT), the enzyme that synthesizes ACh, innervated NMJs form even in the absence of cholinergic transmission31,32. However, abnormalities are present, including a large number of noninnervated AChR clusters. In agrin /ChAT double-knockout mice, postsynaptic differentiation is significantly restored compared to agrin knockouts 33,34. However, the pattern is still not as extensive as in muscle that completely lacks innervation, suggesting additional signals may be involved. The intracellular kinase CDK5 appears to be a part of the mechanism by which muscle fibers disperse aneural receptor clusters, and the conclusions in vivo are supported by in vitro analyses, in which agrin-induced AChR clusters were more stable in the face of ACh application than spontaneously arising clusters.

Thus, agrin's precise role in NMJ formation may be to stabilize muscle-derived sites of postsynaptic differentiation against dispersal activities of cholinergic activity, and possibly other signals, rather than inducing them de novo upon nerve contact. However, the fact that recombinant Z+ isoforms agrin are capable of inducing postsynaptic differentiation, both in vitro and when expressed in muscle in vivo, cannot be ignored. The final answer may therefore be that agrin from the motor neuron has both activities: stabilizing a muscle-derived site of AChR accumulation if it is encountered, and inducing a new site if a pre-existing site is not available.

Recent studies in zebrafish using time-lapse visualization of both postsynaptic receptor clusters and presynaptic motor neurons support this idea35. In early-forming synapses, motor neurons found and stabilized sites of AChR clustering already initiated by the muscle. However, in later-forming synapses, AChR clusters appeared at points of nerve contact, presumably induced by neuronally derived agrin.

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