Early appearance of and neuronal contribution to agrin-like molecules at embryonic frog nerve-muscle synapses formed in culture

Abstract

Antibodies against chicken and Torpedo agrin were used for immunofluorescent staining in order to assess the spatial distribution and temporal appearance of agrin-like molecules at newly formed synaptic contacts in cultures of embryonic Xenopus nerve and muscle cells. The antibodies stained Xenopus neuromuscular junctions and removed ACh receptor (AChR)-aggregating activity from extracts of Xenopus brain. Immunofluorescence was observed at almost all nerve- induced AChR aggregates, even at microaggregates in cocultures as young as 7.5 hr and at nerve-muscle contacts less than 2 hr old. Microdeposits of immunofluorescence extended as far distally as, or farther than, the microaggregates of AChRs along young nerve-muscle contacts. They also occurred along portions of growing neurites that were not in contact with muscle. By contrast, immunofluorescence was rarely observed at the nonsynaptic aggregates of AChRs that form on noninnervated muscle cells. These results raise the possibility that neuronally derived microaggregates of agrin-like molecules may be primary sites of nerve-induced clustering of AChRs, and they indicate that these molecules are present at embryonic nerve-muscle synapses from the very onset of AChR aggregation. The cellular origin of the agrin-like molecules at synapses was examined in cross-species cocultures in which the neurons and muscle cells were obtained from embryos of Xenopus laevis and Rana pipiens. Immunofluorescent staining with anti-agrin antibodies reactive at both Rana and Xenopus neuromuscular junctions revealed immunofluorescence at AChR aggregates along nerve-muscle contacts involving both cross-species combinations. Immunofluorescent staining with an anti-agrin antibody reactive at Rana but not at Xenopus neuromuscular junctions was positive only at cross- species nerve-muscle contacts involving Rana neurons. These results provide the first demonstration that embryonic neurons supply agrin- like molecules to the synapses they form with embryonic muscle cells.

Cerebellar granule cells express a specific isoform of agrin that lacks the acetylcholine receptor aggregating activity

“…Agrin, a synapse-organizing protein, was implicated to be important in directing the formation of these postsynaptic specializations in the developing and regenerating neuromuscular junctions [2~,]. During development, agrin is expressed by motor neurons of the spinal cord [5]; it is released at the sites of contact with developing muscle fibers [6,7] and thus, induces the aggregation of AChRs and other components at the neuromuscular junctions [8]. Anti-agrin antibodies block nerve-induced AChR aggregation in motor neuron-myotube co-cultures [9].…”

Acetylcholine receptor-aggregating activity of agrin isoforms and mapping of the active site.

“…On cultured myotubes, agrin induces aggregation of several molecules that are concentrated at the NMJ including AChRs (Nitkin et al, 1987;Wallace, 1989). Motor neurons synthesize agrin, transport it to the nerve terminal from where it is released to induce AChR aggregation McMahan, 1988, 1990;Reist et al, 1992;Cohen and Godfrey, 1992).…”

Agrin induced morphological and structural changes in growth cones of cultured hippocampal neurons

“…Together, they provide further insights into the molecular mechanisms underlying agrin effects on neurite elongation in central neurons. A potential role for this extracellular matrix protein during neurite outgrowth was first suggested based on its predominant expression during the early stages of development and its subcellular localization in growth cones (Cohen and Godfrey, 1992;Mann and Kroger, 1996;Ferreira, 1999;Serpinskaya et al, 1999;Neuhuber and Daniels, 2003). Functional studies provided additional evidence for a regulatory role of agrin on the rate of axonal and dendritic elongation in central neurons (Chang et al, 1997;Gautman et al, 1996;Ferreira, 1999;Mantych and Ferreira, 2001).…”

“…This multi-domain glycoprotein is encoded by a single gene that is alternatively spliced to give rise to several isoforms (Deyst et al, 1998;Glass et al, 1996Glass et al, & 1997Godfrey, 1991;Godfrey et al, 1988;Hoch et al, 1994;McMahan, 1990;Ruegg et al, 1992Ruegg et al, & 1998Rupp, et al & 1992Sanes, 1997;Stone and Nikolics, 1995). The neuron-specific isoform was initially detected in motor neurons (Cohen and Godfrey, 1992;Magill-Solc and McMahan, 1988). Later, it was shown that this isoform was also expressed in the brain (Bowe and Fallon, 1995;Hoch et al, 1993;Kroger et al, 1996;Mann and Kroger, 1996;Mantych and Ferreira, 2001;O'Toole et al, 1996;Rupp et al, 1991Rupp et al, & 1992Stone and Nikolics, 1995).…”