Search

Agrin-deficient mice die at birth because of aberrant development of the neuromuscular junctions. Here, we examined the role of agrin at brain synapses. We show that agrin is associated with excitatory but not inhibitory synapses in the cerebral cortex. Most importantly, we examined the brains of agrin-deficient mice whose perinatal death was prevented by the selective expression of agrin in motor neurons. We find that the number of presynaptic and postsynaptic specializations is strongly reduced in the cortex of 5- to 7-week-old mice. Consistent with a reduction in the number of synapses, the frequency of miniature postsynaptic currents was greatly decreased. In accordance with the synaptic localization of agrin to excitatory synapses, changes in the frequency were only detected for excitatory but not inhibitory synapses. Moreover, we find that the muscle-specific receptor tyrosine kinase MuSK, which is known to be an essential component of agrin-induced signaling at the neuromuscular junction, is also localized to a subset of excitatory synapses. Finally, some components of the mitogen-activated protein (MAP) kinase pathway, which has been shown to be activated by agrin in cultured neurons, are deregulated in agrin-deficient mice. In summary, our results provide strong evidence that agrin plays an important role in the formation and/or the maintenance of excitatory synapses in the brain, and we provide evidence that this function involves MAP kinase signaling.

The heparan sulphate proteoglycan agrin is expressed as several isoforms in various tissues. Agrin is best known as a crucial organizer of postsynaptic differentiation at the neuromuscular junction, but it has recently also been implicated in the formation of the immunological synapse, the organization of the cytoskeleton and the amelioration of function in diseased muscle. So the activities of agrin might be of broader significance than previously anticipated.

Neuromuscular junctions (NMJs) normally form in the central region of developing muscle. In this process, agrin released from motor neurons has been considered to initiate the formation of synaptic acetylcholine receptor (AChR) clusters (neurocentric model). However, in muscle developing in the absence of nerves and thus of agrin, AChR clusters still form in the muscle center. This raises the possibility that the region of NMJ formation is determined by muscle-derived cues that spatially restrict the nerve to form synapses from aneural AChR clusters, e.g., by patterned expression of the agrin receptor MuSK (muscle-specific kinase) (myocentric model). Here we examine at initial stages of synaptogenesis whether the responsiveness of myotubes to agrin is spatially restricted, whether the regions of NMJ formation in wild-type muscle and of aneural AChR cluster formation in agrin-deficient animals correlate, and whether AChR cluster growth depends on the presence of agrin. We show that primary myotubes form AChR clusters in response to exogenous agrin in their central region only, a pattern that can spatially restrict NMJ formation. However, the nerve also makes synapses in regions in which aneural AChR clusters do not form, and agrin promotes synaptic cluster growth from the first stages of neuromuscular contact formation. These data indicate that aneural AChR clusters per se are not required for NMJ formation. A model is proposed that explains either the neurocentric or the myocentric mode of NMJ formation depending on a balance between the levels of MuSK expression and the availability of nerve-released agrin. [ABSTRACT FROM AUTHOR]

The formation of the vertebrate neuromuscular junction (NMJ) requires the receptor tyrosine kinase MuSK and the adaptor molecule rapsyn. Here, we report that the phenotypes of mice deficient in these two molecules can be reproduced by RNA interference (RNAi) in rat muscle in vivo. Specifically, doublestranded RNA (dsRNA) targeting MuSK and rapsyn inhibited the formation of the NMJ in rat muscle fibres in vivo, while dsRNA targeting nonessential proteins did not have any effect. Moreover, plasmids that trigger RNAi to MuSK induced the disassembly of existing NMJs. These results thus demonstrate for the first time the functionality of dsRNA in silencing endogenous genes in adult mammalian muscle in vivo. Moreover, they show that MuSK is also required for the maintenance of the NMJ, offering a mechanistic explanation for the myasthenia gravis caused by auto-antibodies to MuSK. [ABSTRACT FROM AUTHOR]

Focuses on the influence of releasing the molecule agrin on understanding the sequence of steps involved in establishing synapses. Derivation of the synapse action from studies of the vertebrate neuromuscular junction (NMJ); Assessment of the structure and binding sites of the agrin; Control of gene transcription.

Agrin is a large, multidomain heparan sulfate proteoglycan that is associated with basement membranes of several tissues. Particular splice variants of agrin are essential for the formation of synaptic structures at the neuromuscular junction. The binding of agrin to laminin appears to be required for its localization to synaptic basal lamina and other basement membranes. Here, electron microscopy was used to determine the structure of agrin and to localize its binding site in laminin-1. Agrin appears as an ∼95 nm long particle that consists of a globular, N-terminal laminin-binding domain, a central rod predominantly formed by the follistatin-like domains and three globular, C-terminal laminin G-like domains. In a few cases, heparan sulfate glycosaminoglycan chains were seen emerging from the central portion of the core protein. Moreover, we show that agrin binds to the central region of the three-stranded, coiled-coil oligomerization domain in the long arm of laminin-1, which mediates subunit assembly of the native laminin molecule. In summary, our data show for the first time a protein­protein interaction of the extracellular matrix that involves a coiled-coil domain, and they assign a novel role to this domain of laminin-1. Based on this, we propose that agrin associates with basal lamina in a polarized way. [ABSTRACT FROM AUTHOR]