Neural Agrin Induces Ectopic Postsynaptic Specializations in Innervated Muscle Fibers

Abstract

Neural agrin, in the absence of a nerve terminal, can induce the activity-resistant expression of acetylcholine receptor (AChR) subunit genes and the clustering of synapse-specific adult-type AChR channels in nonsynaptic regions of adult skeletal muscle fibers. Here we show that, when expression plasmids for neural agrin are injected into the extrasynaptic region of innervated muscle fibers, the following components of the postsynaptic apparatus are aggregated and colocalized with ectopic agrininduced AChR clusters: laminin-␤2, MuSK, phosphotyrosinecontaining proteins, ␤-dystroglycan, utrophin, and rapsyn. These components have been implicated to play a role in the differentiation of neuromuscular junctions. Furthermore, ErbB2 and ErbB3, which are thought to be involved in the regulation of neurally induced AChR subunit gene expression, were colocalized with agrin-induced AChR aggregates at ectopic nerve-free sites. The postsynaptic muscle membrane also contained a high concentration of voltage-gated Na ϩ channels as well as deep, basal lamina-containing invaginations comparable to the secondary synaptic folds of normal endplates. The ability to induce AChR aggregation in vivo was not observed in experiments with a muscle-specific agrin isoform. Thus, a motor neuron-specific agrin isoform is sufficient to induce a full ectopic postsynaptic apparatus in muscle fibers kept electrically active at their original endplate sites.

Restoration of synapse formation inMuskmutant mice expressing a Musk/Trk chimeric receptor

“…Analysis of rapsyn mutant mice indicates that Musk may activate a signaling pathway that directly stimulates synapse-specific transcription (Gautam et al, 1995). Expression of ectopic agrin or activated Musk in adult myofibers, however, stimulates AChR transcription in an ErbBdependent manner, suggesting that synapse-specific expression may require the Musk-dependent recruitment of a Nrg-1/ErbB signaling complex to synaptic sites (Jones et al, 1999;Meier et al, 1997;Moore et al, 2001;Rimer et al, 1998). Our experiments indicate that the Musk juxtamembrane region contains sequences that are crucial for synapse-specific transcription, but they do not shed light on whether these sequences act in a manner that is independent or dependent on Nrg-1/ErbB signaling.…”

“…Herbst, E. Avetisova and S. J. Burden MMT chimera contains sequences that are sufficient for clustering postsynaptic proteins Agrin/Musk signaling is necessary and sufficient to cluster several muscle-derived proteins in addition to AChRs (DeChiara et al, 1996;Gautam et al, 1996;Meier et al, 1997;Rimer et al, 1998). We therefore examined whether the MMT chimera contains sequences sufficient for clustering these proteins at synaptic sites.…”

Traumatic Axonal Injury Induces Proteolytic Cleavage of the Voltage-Gated Sodium Channels Modulated by Tetrodotoxin and Protease Inhibitors

“…We performed immunocytochemistry to detect the identity of the processes traversing the cell-free 2 mm gap in the wells, evaluating immunostaining for microtubuleassociated protein 2 (MAP2), a specific marker for the dendrites and neuronal somata, and NaCh protein, which stains the axons in addition to the dendrites and neuronal somata. Cultures were fixed in 4% paraformaldehyde and 0.1 M PBS for 20 min, permeabilized with 0.1% Triton X-100 (PBST) for 20 min at room temperature (RT), and double labeled with monoclonal mouse anti-MAP2 antibody (Ab) (AP20; 1:500; Sigma) (Binder et al, 1986) and polyclonal rabbit anti-pan NaCh Ab (06 -811; 1:80; Upstate Biotechnology, Lake Placid, NY) specific for the intracellular III-IV loop of the NaCh ␣-subunit [ amino acid (aa) 1491-1508 of full length, aa 1-2005; P04775), a highly conserved segment of the intracellular III-IV loop (Miller et al, 1983;Dugandzija-Novakovic et al, 1995;Vabnick et al, 1996;Meier et al, 1997;Rasband et al, 1999) at 4°C overnight. After rinsing with PBS, the cultures were incubated with Alexa 594-conjugated anti-mouse IgG (Molecular Probes, Eugene, OR) and Alexa 488-conjugated anti-rabbit IgG (Molecular Probes) for 60 min at RT.…”

Basement Membranes: Cell Scaffoldings and Signaling Platforms

“…The LG domains of agrin were also found to bind strongly to a-dystroglycan and to sulfatides (Gesemann et al 1998). In the case of the neural agrin splice variant released by the peripheral nerve terminus, the interaction likely helps retain agrin within the basement membrane, enhancing its activity in neuromuscular junction assembly and/or stabilization (Meier et al 1997;Lin et al 2008). In the case of the more widely expressed nonneural ("muscle") agrin splice variants, the interactions appear to allow agrin to serve as a stabilizing collateral link among laminin and the cell surface and underlying cytoskeleton (Moll et al 2001;McKee et al 2009).…”