Targeting of the ETS factor GABPalpha disrupts neuromuscular junction synaptic function.

The GA-binding protein (GABP) transcription factor has been shown in vitro to regulate the expression of the neuromuscular proteins utrophin, acetylcholine esterase, and acetylcholine receptor subunits delta and epsilon through the N-box promoter motif (5'-CCGGAA-3'), but its in vivo function remains unknown. A single point mutation within the N-box of the gene encoding the acetylcholine receptor epsilon subunit has been identified in several patients suffering from postsynaptic congenital myasthenic syndrome, implicating the GA-binding protein in neuromuscular function and disease. Since conventional gene targeting results in an embryonic-lethal phenotype, we used conditional targeting to investigate the role of GABPalpha in neuromuscular junction and skeletal muscle development. The diaphragm and soleus muscles from mutant mice display alterations in morphology and distribution of acetylcholine receptor clusters at the neuromuscular junction and neurotransmission properties consistent with reduced receptor function. Furthermore, we confirmed decreased expression of the acetylcholine receptor epsilon subunit and increased expression of the gamma subunit in skeletal muscle tissues. Therefore, the GABP transcription factor aids in the structural formation and function of neuromuscular junctions by regulating the expression of postsynaptic genes.

Functional analysis of neurotransmission at beta2-laminin deficient terminals.

beta2-Laminin is important for the formation of neuromuscular junctions in vertebrates. Previously, we have inactivated the gene that encodes for beta2-laminin in mice and observed predominantly prejunctional structural defects. In this study, we have used both intra- and extracellular recording methods to investigate evoked neurotransmission in beta2-laminin-deficient mice, from postnatal day 8 (P8) through to day 18 (P18). Our results confirmed that there was a decrease in the frequency of spontaneous release, but no change in the postjunctional response to such release. Analysis of evoked neurotransmission showed an increase in the frequency of stimuli that failed to elicit an evoked postjunctional response in the mutants compared to litter mate controls, resulting in a 50 % reduction in mean quantal content at mutant terminals. Compared to littermate controls, beta2-laminin-deficient terminals showed greater synaptic depression when subjected to high frequency stimulation. Furthermore, the paired pulse ratio of the first two stimuli was significantly lower in beta2-laminin mutant terminals. Statistical analysis of the binomial parameters of release showed that the decrease in quantal content was due to a decrease in the number of release sites without any significant change in the average probability of release. This suggestion was supported by the observation of fewer synaptic vesicle protein 2 (SV2)-positive varicosities in beta2-laminin-deficient terminals and by ultrastructural observations showing smaller terminal profiles and increased Schwann cell invasion in beta2-laminin mutants; the differences between beta2-laminin mutants and wild-type mice were the same at both P8 and P18. From these results we conclude that beta2-laminin plays a role in the early structural development of the neuromuscular junction. We also suggest that transmitter release activity may act as a deterrent to Schwann cell invasion in the absence of beta2-laminin.

Neuromuscular synapses mediate motor axon branching and motoneuron survival during the embryonic period of programmed cell death.

The embryonic period of motoneuron programmed cell death (PCD) is marked by transient motor axon branching, but the role of neuromuscular synapses in regulating motoneuron number and axonal branching is not known. Here, we test whether neuromuscular synapses are required for the quantitative association between reduced skeletal muscle contraction, increased motor neurite branching, and increased motoneuron survival. We achieved this by comparing agrin and rapsyn mutant mice that lack acetylcholine receptor (AChR) clusters. There were significant reductions in nerve-evoked skeletal muscle contraction, increases in intramuscular axonal branching, and increases in spinal motoneuron survival in agrin and rapsyn mutant mice compared with their wild-type littermates at embryonic day 18.5 (E18.5). The maximum nerve-evoked skeletal muscle contraction was reduced a further 17% in agrin mutants than in rapsyn mutants. This correlated to an increase in motor axon branch extension and number that was 38% more in agrin mutants than in rapsyn mutants. This suggests that specializations of the neuromuscular synapse that ensure efficient synaptic transmission and muscle contraction are also vital mediators of motor axon branching. However, these increases in motor axon branching did not correlate with increases in motoneuron survival when comparing agrin and rapsyn mutants. Thus, agrin-induced synaptic specializations are required for skeletal muscle to effectively control motoneuron numbers during embryonic development.