Thrombin reduces MuSK and acetylcholine receptor expression along with neuromuscular contact size in vitro

Abstract

In the course of studies on thrombin and its inhibitor(s) in synaptic plasticity, we addressed the question of their roles in the formation of neuromuscular junctions (NMJ) and used a model of rat neuron-myotube cocultures. We report that the size of acetylcholinesterase (AChE) patches used as a marker of neuromuscular contacts was decreased in the presence of either thrombin or SFLLRN, the agonist peptide of the thrombin receptor PAR-1, whereas it was increased with hirudin, a specific thrombin inhibitor. In an attempt to relate these neuromuscular contact size variations to molecular changes, we studied muscle-specific tyrosine kinase receptor (MuSK), acetylcholine receptor (AChR) and rapsyn expression in the presence of thrombin. We showed that thrombin did not change rapsyn gene and protein expression. However, the expression of MuSK and surface AChR proteins was diminished in both myotube cultures and neuron-myotube cocultures. These reductions in protein expression were associated with a decrease in MuSK and AChR alpha-subunit gene expression in myotube cultures but not in neuron-myotube cocultures. Moreover, the expression of the AChR epsilon-subunit gene, specifically enhanced by neuron-released factors, was not modified by thrombin in neuron-myotube cocultures. This suggests that thrombin did not affect the expression of synaptic AChRs enhanced by neuron-released factors but rather reduced the level of extrasynaptic AChRs. Taken together, these results indicate that thrombin in balance with its inhibitor(s) could modulate the formation of neuromuscular contacts in vitro by affecting the expression of two essential molecules in NMJ postsynaptic differentiation, MuSK and AChR.

Concentration-Dependent Dual Role of Thrombin in Protection of Cultured Rat Cortical Neurons

“…Additionally, thrombin has shown some potential therapeutic value via preconditioning (34). Our demonstration of a non-toxic neuroprotection mediated by low-concentration thrombin (1 nM) aligns well with studies on the peripheral nervous system, which showed important effects of lowconcentration thrombin in neuromuscular junction development (35) and in promoting motoneuron regeneration after injury (32). An inverted U effect of thrombin concentration on brain cells experiencing hypoglycemic stress has been reported in the past (3); however our study is the first demonstration of neuroprotective effects of thrombin on viability and microarchitecture in primary cortical neurons (glial-reduced cultures) in response to excitotoxic damage.…”

Plastic‐embedded semithin cross‐sections as a tool for high‐resolution immunofluorescence analysis of the neuromuscular junction molecules: Specific cellular location of protease‐activated receptor‐1

“…We had evidence that normal AChR cluster maturation in neonatal rat muscle may be mediated through activation of PAR‐1 (Lanuza et al, 2003) and that thrombin and its inhibitor hirudin affect the stability of the AChR aggregates in aneural and innervated myotubes in vitro (Davenport et al, 2000; Lanuza et al, 2000). Moreover, it has been suggested that thrombin, through PAR‐1 and in balance with its inhibitor(s), could modulate the formation of neuromuscular contacts in vitro by affecting the expression of two essential molecules in NMJ postsynaptic differentiation, MuSK and AChR (Faraut et al, 2004). Our demonstration of the postsynaptic presence of PAR‐1 near the AChRs may confirm the previously suggested role for thrombin and its receptor in the maintenance of the postsynaptic apparatus and in adult synaptic plasticity (Lanuza et al, 2001, 2002, 2003; Nelson et al, 2003).…”

‘Body-on-a-Chip’ Technology and Supporting Microfluidics

“…Measurement of functional neuromuscular transmission in vitro was rst reported in 1970 using chick muscle cells and motoneurons. 135 Since then, the ability to promote NMJ formation in vitro has been demonstrated using both rodent [136][137][138][139][140] and human 9,141 cell sources. Motoneurons maintained in coculture with skeletal muscle myotubes display normal responses to neurotransmitter application, 140 as well as characteristic ionic currents and action potential ring.…”