Low dystrophin levels are insufficient to normalize the neuromuscular synaptic abnormalities of mdx mice.

Dystrophin is a sub-sarcolemmal component of skeletal muscle fibres and is enriched at the postsynaptic membrane of the neuromuscular junction (NMJ). In the mdx mouse, dystrophin absence not only causes muscle damage but also mild synaptic dysfunctions and clear morphological aberrations at NMJs. In particular, reduction of postsynaptic sensitivity for the neurotransmitter acetylcholine and extra exhaustion of presynaptic acetylcholine release during intense synaptic activity exists. Current experimental therapeutic approaches in Duchenne muscular dystrophy aim to restore dystrophin expression. An important question is what dystrophin levels are needed to improve muscle function. Recent experimental and clinical studies suggested that levels as low as a few percent of normal can be beneficial. Similarly, it is of interest to know how dystrophin levels relate to NMJ function and morphology. We investigated NMJs of a series of mdx-XistΔhs mice, which expressed dystrophin between ~2% and 19% of normal. Most functional and morphological NMJ parameters of these mice remained comparable to mdx. On the other hand, mdx+/- mice (expressing ~50% dystrophin) showed normal NMJ features. Thus, the minimal dystrophin level required for normal NMJ function and morphology lies between 19% and 50% of normal when expression of dystrophin is not uniform.

van der Pijl EM ，van Putten M ，Niks EH ，Verschuuren JJGM ，Aartsma-Rus A ，Plomp JJ ... -  《-》

Characterization of neuromuscular synapse function abnormalities in multiple Duchenne muscular dystrophy mouse models.

Duchenne muscular dystrophy (DMD) is an X-linked myopathy caused by dystrophin deficiency. Dystrophin is present intracellularly at the sarcolemma, connecting actin to the dystrophin-associated glycoprotein complex. Interestingly, it is enriched postsynaptically at the neuromuscular junction (NMJ), but its synaptic function is largely unknown. Utrophin, a dystrophin homologue, is also concentrated at the NMJ, and upregulated in DMD. It is possible that the absence of dystrophin at NMJs in DMD causes neuromuscular transmission defects that aggravate muscle weakness. We studied NMJ function in mdx mice (lacking dystrophin) and wild type mice. In addition, mdx/utrn(+/-) and mdx/utrn(-/-) mice (lacking utrophin) were used to investigate influences of utrophin levels. The three Duchenne mouse models showed muscle weakness when comparatively tested in vivo, with mdx/utrn(-/-) mice being weakest. Ex vivo muscle contraction and electrophysiological studies showed a reduced safety factor of neuromuscular transmission in all models. NMJs had ~ 40% smaller miniature endplate potential amplitudes compared with wild type, indicating postsynaptic sensitivity loss for the neurotransmitter acetylcholine. However, nerve stimulation-evoked endplate potential amplitudes were unchanged. Consequently, quantal content (i.e. the number of acetylcholine quanta released per nerve impulse) was considerably increased. Such a homeostatic compensatory increase in neurotransmitter release is also found at NMJs in myasthenia gravis, where autoantibodies reduce acetylcholine receptors. However, high-rate nerve stimulation induced exaggerated endplate potential rundown. Study of NMJ morphology showed that fragmentation of acetylcholine receptor clusters occurred in all models, being most severe in mdx/utrn(-/-) mice. Overall, we showed mild 'myasthenia-like' neuromuscular synaptic dysfunction in several Duchenne mouse models, which possibly affects muscle weakness and degeneration.

van der Pijl EM ，van Putten M ，Niks EH ，Verschuuren JJ ，Aartsma-Rus A ，Plomp JJ ... -  《-》

Acetylcholine receptors in innervated muscles of dystrophic mdx mice degrade as after denervation.

Xu R ，Salpeter MM 《-》

Dystrophin is required for organizing large acetylcholine receptor aggregates.

Kong J ，Anderson JE 《-》

Defects in neuromuscular junction structure in dystrophic muscle are corrected by expression of a NOS transgene in dystrophin-deficient muscles, but not in muscles lacking alpha- and beta1-syntrophins.

Muscular dystrophies that arise from mutations of genes that encode proteins in the dystrophin-glycoprotein complex (DGC) frequently involve defects in the structure of neuromuscular junctions (NMJs). DGC mutations that cause NMJ defects typically cause a secondary loss of neuronal nitric oxide synthase (nNOS) from the post-synaptic membrane. We tested the hypothesis that reduction of muscle-derived NO production causes NMJ defects in DGC mutants by analyzing the effect of modulating muscle NO production on NMJ structure in mutant and wild-type muscles. We found that nNOS null mutants, dystrophin-deficient mdx mice and alpha-syntrophin null mutants showed reductions in the concentration of acetylcholine receptors (AChRs) at the post-synaptic membrane. Also, expression of a muscle-specific NOS transgene increased AChR concentration, which reflected an increase in both AChR expression and clustering. NOS transgene expression also increased the size of NMJs, and partially corrected defects in normal NMJ architecture that were observed in mdx and alpha-syntrophin null muscles. In addition, stimulation of AChR clustering in vitro by application of laminin or VVA B4 lectin induced a 3-4-fold increase in NOS activity and increased AChR clustering that could be prevented by NOS inhibition. However, the partial rescue of NMJ structure by expression of a NOS transgene required the expression of alpha- or beta1-syntrophin at the NMJ; partial NMJ rescue was seen in the muscles of alpha-syntrophin mutants that expressed beta1-syntrophin, but no rescue was observed in muscles of alpha-syntrophin mutants that also lacked beta1-syntrophin. These findings show that NO promotes AChR expression and clustering in vivo and contributes to normal NMJ architecture. The results suggest that defects in NMJ structure that occur in some DGC mutants can result from the secondary loss of NOS from muscle.

Shiao T ，Fond A ，Deng B ，Wehling-Henricks M ，Adams ME ，Froehner SC ，Tidball JG ... -  《HUMAN MOLECULAR GENETICS》