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.

Cloning and characterization of muscle-specific kinase in chicken.

Muscle-specific kinase (MuSK) is part of the receptor complex that is involved in the agrin-induced formation of the neuromuscular junction. In the rodent, prominent mRNA expression of MuSK is restricted to skeletal muscle while the expression of agrin can also be detected in brain and certain nonneuronal tissues. The recent identification of Xenopus MuSK reveals that MuSK can be detected in tissues other than skeletal muscle, such as the neural tube, eye vesicles, and spleen. In this study, we describe the cloning and characterization of the chicken ortholog of MuSK and demonstrate that the regulation of MuSK expression in muscle is conserved from avian to rodent. Abundant mRNA expression of MuSK can be detected in early embryonic chick muscle and is up-regulated after nerve injury. More importantly, we also demonstrate that, in the chicken, MuSK mRNA is expressed during development in brain and liver, suggesting possible novel functions for MuSK. Furthermore, the regulatory profile of MuSK expression in chick muscle closely parallels that observed for acetylcholine receptor, in terms of both mRNA expression and protein localization. Finally, studies with paralyzed chicken muscle as well as with cultured chick myotubes demonstrate the dependence of MuSK on both electrical activity and trophic factors.

Identification and characterization of splice variants of ephrin-A3 and ephrin-A5.

Ephrins and Eph receptors have been implicated to play important roles in axon guidance. A variable spacer region exists that differs significantly among distinct ephrins. An ephrin-A5 isoform has previously been isolated which lacks 27 amino acids within the spacer region. The expression and biological activities of this isoform, as well as the existence of isoforms for other ephrins that show variation within the spacer region, remain unknown. We report here a novel alternatively spliced isoform of ephrin-A3 which lacks the corresponding variable region. When compared to the longer isoforms, the shorter isoforms of both ephrin-A3 and ephrin-A5 remained less prominent in the brain during development, though their expression increased at postnatal stages. In addition, they could inhibit neurite outgrowth of dorsal root ganglia (DRG) neurons, suggesting that the corresponding variable regions were not essential for their axon guidance activities.

Induction of NG108-15 cells differentiation by human bone marrow stromal cells.

The survival and differentiation of neuronal cells is dependent on factors such as neurotrophins, cytokines and components of extracellular matrix. Bone marrow stromal cells have been shown to support the growth and differentiation of neuroblastoma cells. In an attempt to study the effects of bone marrow stromal cells on neuronal differentiation, we have co-cultured neuroblastoma x glioma hybrid NG108-15 cells with human bone marrow stromal cells. After co-culturing, clones exhibiting morphological differentiated phenotype and high level of neurofilament expression were isolated. Interestingly, these clones maintain their ability to proliferate in contrast to differentiated NG108-15 cells induced by dibutyryladenosine 3',5'-cyclic monophosphate. These results suggested that bone marrow stromal cells can induce partial differentiation of NG108-15 cells.

Cloning and expression of a novel neurotrophin, NT-7, from carp.

Neurotrophins have been demonstrated to play important roles in the development and functioning of the nervous system. This family of proteins consists of four homologous members in mammals: NGF, BDNF, NT-3, and NT-4/5. A new member, called NT-6, was recently cloned from the platyfish Xiphophorus maculatus. This protein shares closer structural relationship to NGF than the other neurotrophins, but contains a characteristic insertion of 22 amino acids that constituted the heparin-binding domain. Here we report the cloning of a novel neurotrophin from the fish Cyprinus carpio (carp), which shared about 66% amino acid identity to Xiphophorus NGF and NT-6. The neurotrophin, designated NT-7, possesses structural characteristics common to all known neurotrophins, such as the presence of six conserved cysteine residues and the flanking conserved sequences. In addition, there is an insertion of 15 amino acids at the position corresponding to that observed for NT-6. The neurotrophic activity of NT-7 was demonstrated by its ability to promote neurite outgrowth and neuronal survival of chick dorsal root ganglia. Phosphorylation assay of various Trk receptors overexpressed in fibroblasts suggested that NT-7 could activate TrkA but not TrkB or TrkC. Northern blot analysis revealed that NT-7 was predominantly expressed in peripheral tissues, though weak expression was also detected in the brain. Like NT-6, this novel neurotrophin might represent yet another NGF-like neurotrophin in lower vertebrates.

Muscle-derived neurotrophin-3 increases the aggregation of acetylcholine receptors in neuron-muscle co-cultures.

Neurotrophins, a group of protein ligands that are structurally related to the prototype nerve growth factor (NGF), are prominently expressed in the skeletal muscle during the critical period of synapse formation. In the present study, we utilized a co-culture system of NG108-15 cells expressing the Trk receptors and C2C12 myotubes expressing the individual neurotrophins to examine whether these factors can act in a target-derived manner to influence the postsynaptic specializations. Our findings demonstrated that muscle-derived neurotrophin-3 (NT-3) has the unique ability to enhance the aggregation of acetylcholine receptors (AChRs) on the myotubes following co-culture with NG108-15 cells expressing TrkC. Taken together, our findings suggest that NT-3 can act as a retrograde factor to modulate the postsynaptic specializations.

Structural determinants of Trk receptor specificities using BDNF-based neurotrophin chimeras.

Neurotrophins play very important roles in the development and maintenance of the vertebrate nervous system. In mammals, there are four members of the family: NGF, BDNF, NT-3 and NT-4/5. Members of the neurotrophin family activate different receptors that belong to a class of receptor tyrosine kinases known as "Trks." For example, NGF is the specific ligand of TrkA, while BDNF activates TrkB. To elucidate which regions of the two neurotrophins determine the receptor specificities, chimeric neurotrophins were constructed using BDNF as the backbone, with various regions being substituted by the corresponding regions of NGF. The activity of the chimeras on the Trk receptors was assayed in transfected fibroblasts ectopically expressing the Trk receptors. Our findings revealed that, although BDNF is absolutely conserved in mammals, substitution of several small variable regions from NGF into the BDNF backbone did not lead to significant loss in TrkB activity or gain in TrkA activity. Moreover, important determinants of TrkB activation might be located in the carboxy-terminal half of BDNF. On the other hand, critical elements for TrkA activation might be located within the amino-terminal half of the mature NGF molecule.