Neural agrin controls acetylcholine receptor stability in skeletal muscle fibers

Abstract

At mammalian neuromuscular junctions (NMJs), innervation induces and maintains the metabolic stability of acetylcholine receptors (AChRs). To explore whether neural agrin may cause similar receptor stabilization, we injected neural agrin cDNA of increasing transfection efficiencies into denervated adult rat soleus (SOL) muscles. As the efficiency increased, the amount of recombinant neural agrin expressed in the muscles also increased. This agrin aggregated AChRs on muscle fibers, whose half-life increased in a dose-dependent way from 1 to 10 days. Electrical muscle stimulation enhanced the stability of AChRs with short half-lives. Therefore, neural agrin can stabilize aggregated AChRs in a concentration- and activity-dependent way. However, there was no effect of stimulation on AChRs with a long half-life (10 days). Thus, at sufficiently high concentrations, neural agrin alone can stabilize AChRs to levels characteristic of innervated NMJs.

Figure 1

Neural agrin mRNA, recombinant protein, and positive fibers in SOL muscles injected with neural agrin cDNA. SOL muscles were examined 8 and 16 days after injections of full-length (a) or GFP-tagged (b) rat neural agrin cDNA of low, medium, or high transfection efficiency, as indicated. Note the increase in the amount of recombinant neural agrin mRNA (by PCR) and protein (by Western blots) in a and the number of GFP-positive fibers in b (±SD, fibers from two muscles) for each increase in transfection efficiency. Amount of mRNA for muscle creatine kinase (MCK), a “housekeeping” gene, was essentially unaffected. Results in a are representative of results from three (PCR) and two (Western) muscles.

Figure 2

Recombinant neural agrin and accompanying AChR aggregates in transfected SOL muscles. SOL muscles were denervated and injected with solutions of GFP-tagged neural agrin cDNA of low, medium, or high transfection efficiencies, as indicated. Eight days later, the muscles were removed, teased into bundles, incubated with Rh-BuTx, and examined with confocal microscope. Note the increase in the amount of recombinant GFP-tagged neural agrin (green fluorescence) and the number of AChR aggregates (red) on transfected (see overlay) and adjacent muscle fibers for each increase in transfection efficiency. (Scale bar, 50 μm.)

Figure 3

AChRs at neural agrin-induced aggregates turn over at different rates. SOL muscles were denervated and injected with solutions of neural agrin (nAgrin) cDNA that resulted in expression of low or high amounts of recombinant nAgrin as indicated. Eight days later Rh-BuTx was injected into all muscles and electrical stimulation started for some of the muscles. At different times thereafter (+2 to +9 days), the muscles were removed, fixed, and labeled with Fl-BuTx to visualize AChRs inserted after day 8. In muscles expressing low amounts of nAgrin, AChR aggregates were induced whose Rh-BuTx labeling essentially disappeared in 2 days in unstimulated muscles (b) but was still present after 6 days in stimulated muscles (d). In muscles expressing high amounts of nAgrin, AChR aggregates displayed Rh-BuTx labeling as late as 9 days after Rh-BuTx injection in both unstimulated (b′) and stimulated (d′) muscle. Shown in a and a′ are nAgrin-induced AChR aggregates examined 1 h after injection of Rh-BuTx. Note also that labeling by Rh- and Fl-BuTx overlap where Rh-BuTx is still present. (Scale bar, 10 μm.)

Figure 4

Neural agrin and electrical muscle stimulation affect AChR stability. SOL muscles were injected with Rh-BuTx 8 days after denervation and injection of solutions of nAgrin cDNA that resulted in low, medium, and high amounts of recombinant nAgrin in the muscles as indicated. Relative fluorescence at nAgrin-induced AChR aggregates (ordinate, see Materials and Methods) declined at fast, intermediate, and slow rates in muscles expressing low, medium, or high amounts of recombinant protein (■). Electrical muscle stimulation starting on the day of Rh-BuTx injection decreased the degradation rate at AChR aggregates induced in muscles expressing low or medium but not high amounts of recombinant nAgrin (○). For each time point, 13–64 measurements were done in at least three different animals.

Figure 5

γ and ɛ AChR subunits at nAgrin-induced AChR aggregates. SOL muscles were denervated and injected with solutions of nAgrin cDNA that resulted in the expression of low, intermediate, and high amounts of recombinant nAgrin as indicated. Eight days later, Rh-BuTx was injected into the muscles and the muscles removed 1 h later. The muscles were then incubated with antibodies against γ- or ɛ-subunits. Note the increase in ɛ-subunit labeling at AChR aggregates for each increase in the amount of recombinant neural agrin in the muscles (a4, b4, and c4). (Scale bar, 10 μm.)