Lead-induced attenuation in the aggregation of acetylcholine receptors during the neuromuscular junction formation

Lead (Pb2+) toxicity is more common in children and is associated with cognitive deficits, which may reflect lead-induced changes in central synaptic development and function. Aside from neurotoxicity, lead exposure may also impact mature neuromuscular junction (NMJ) and cause muscle weakness. NMJ is known as a peripheral cholinergic synapse and its signaling cascades responsible for development are similar to those for the central synapses. However, the effect of lead exposure on the formation of NMJ in mammals is unclear. In the present study, a NG108-15/C2C12 coculture model was used to measure the acetylcholine receptor (AM) aggregates formed on the myotubes which was an early hallmark for the NMJ formation. AChR aggregates were identified by alpha-bungarotoxin under fluorescent microscope. Single dose of lead acetate with final concentrations at 10(-3), 10(-1), or 10 mu M was applied to dishes at the beginning of coculturing. Following 3-day exposure, although NG108-15 cells could extend long neurites to nearby myotubes, obvious dose-dependent attenuation in AChR aggregation was shown. The averaged area of an AChR aggregate, the averaged number of AChR aggregates per myotube, and the total area of AChR aggregates per myotube were all significantly decreased. In addition, the distribution percentages of various sizes of AM aggregates showed that almost half of the AChR aggregates were formed with a size of 2-5 mu m(2) regardless of lead exposure. After treating 10 mu M of lead acetate, significantly more AChR aggregates ranged from 2 to 20 mu m(2) were formed and significantly less AChR aggregates larger than 20 mu m(2) were formed. These results indicated that lead exposure reduced the extent of AChR aggregation concerning both the size and number of AChR aggregates and large AChR aggregates could hardly be formed after acute high-level lead exposure. No significant change was found in the total amount of AChRs on the myotubes after lead exposure, which indicated that the attenuation of AChR aggregation was not caused by reducing the synthesis of AChRs but by remaining dispersed pattern of AChRs on the myotubes. These data suggest that lead exposure exerts detrimental effects on the formation of NMJ. (c) 2005 Elsevier Ireland Ltd. All rights reserved.