Janet Cheung

Cdk5 is involved in neuregulin-induced AChR expression at the neuromuscular junction

Here we describe an important involvement of Cdk5/p35 in regulating the gene expression of acetylcholine receptor (AChR) at the neuromuscular synapse. Cdk5 and p35 were prominently expressed in embryonic muscle, and concentrated at the neuromuscular junction in adulthood. Neuregulin increased the p35-associated Cdk5 kinase activity in the membrane fraction of cultured C2C12 myotubes. Co-immunoprecipitation studies revealed the association between Cdk5, p35 and ErbB receptors in muscle and cultured myotubes. Inhibition of Cdk5 activity not only blocked the NRG-induced AChR transcription, but also attenuated ErbB activation in cultured myotubes. In light of our finding that overexpression of p35 alone led to an increase in AChR promoter activity in muscle, Cdk5 activation is sufficient to mediate the up-regulation of AChR gene expression. Taken together, these results reveal the unexpected involvement of Cdk5/p35 in neuregulin signaling at the neuromuscular synapse.

Expression of Eph receptors in skeletal muscle and their localization at the neuromuscular junction

The participation of ephrins and Eph receptors in guiding motor axons during muscle innervation has been well documented, but little is known about their expression and functional significance in muscle at later developmental stages. Our present study investigates the expression and localization of Eph receptors and ephrins in skeletal muscle. Prominent expression of EphA4, EphA7, and ephrin-A ligands was detected in muscle during embryonic development. More importantly, both EphA4 and EphA7, as well as ephrin-A2, were localized at the neuromuscular junction (NMJ) of adult muscle. Despite their relative abundance, they were not localized at the synapses during embryonic stages. The concentration of EphA4, EphA7, and ephrin-A2 at the NMJ was observed at postnatal stages and the synaptic localization became prominent at later developmental stages. In addition, expression of Eph receptors was increased by neuregulin and after nerve injury. Furthermore, we demonstrated that overexpression of EphA4 led to tyrosine phosphorylation of the actin-binding protein cortactin and that EphA4 was coimmunoprecipitated with cortactin in muscle. Taken together, our findings indicate that EphA4 is associated with the actin cytoskeleton. Since actin cytoskeleton is critical to the formation and stability of NMJ, the present findings raise the intriguing possibility that Eph receptors may have a novel role in NMJ formation and/or maintenance.

The expression profiles of neurotrophins and their receptors in rat and chicken tissues during development

Neurotrophic factors are target-derived proteins that promote the survival and differentiation of the innervating neurons. Increasing evidence indicate the involvement of these factors and receptors during the formation and maturation of the neuromuscular junction. To gain further insight on the expression pattern of these factors and receptors in developing spinal cord and skeletal muscle during the critical stages of synapse formation, a systematic study was performed with chicken and rat tissues using Northern blot analysis. The expression of all the neurotrophins was detected in skeletal muscle early in development, coincidental with the appearance of their corresponding receptors in the spinal cord. Taken together, the similar regulatory patterns observed in both rat and chicken tissues suggest that the potential roles of neurotrophins at the neuromuscular synapse are conserved throughout evolution.

Overexpression of muscle specific kinase increases the transcription and aggregation of acetylcholine receptors in Xenopus embryos

Muscle specific kinase (MuSK) mediates agrin-induced acetylcholine receptor (AChR) aggregation on muscle membrane at the neuromuscular junction (NMJ). To examine whether MuSK enhances NMJ formation during embryonic development in vivo, the level of expression of MuSK was manipulated in Xenopus embryos and the functional consequence at the NMJ was assessed. We found that overexpression of MuSK enhanced the formation of NMJ by increasing the aggregation of AChRs at innervated regions in developing embryos. The area of AChR aggregation increased by approximately 2-fold in MuSK injected embryos during the critical stages of NMJ formation. Interestingly, overexpression of MuSK in Xenopus embryos was found to induce the level of AChR transcript. Deletion of the Kringle domain in the MuSK construct did not attenuate the observed induction of AChR transcription and aggregation. Taken together, our findings provide the first demonstration that increased level of MuSK expression in vivo significantly elevate the aggregation and transcription of AChR at the NMJ in developing Xenopus embryos.