Toward understanding topographically specific branching of retinal axons in their target area, we have studied the interaction between neurotrophin receptors and members of the Eph family. TrkB and its ligand BDNF are uniformly expressed in the retina and tectum, respectively, and exert a branch-promoting activity, whereas EphAs and ephrinAs are expressed in gradients in retina and tectum and can mediate a suppression of axonal branching. We have identified a novel cis interaction between ephrinA5 and TrkB on retinal ganglion cell axons. TrkB interacts with ephrinA5 via its second cysteine-rich domain (CC2), which is necessary and sufficient for binding to ephrinA5. Their functional interaction is twofold: ephrinA5 augments BDNF-promoted retinal axon branching in the absence of its activator EphA7-Fc, whereas EphA7-Fc application abolishes branching in a local and concentration-dependent manner. The importance of TrkB in this process is shown by the fact that overexpression of an isolated TrkB-CC2 domain interfering with the ephrinA/TrkB interaction abolishes this regulatory interplay, whereas knockdown of TrkB via RNA interference diminishes the ephrinA5-evoked increase in branching. The ephrinA/Trk interaction is neurotrophin induced and specifically augments the PI-3 kinase/Akt pathway generally known to be involved in the promotion of branching. In addition, ephrinAs/TrkB modulate axon branching and also synapse formation of hippocampal neurons. Our findings uncover molecular mechanisms of how spatially restricted axon branching can be achieved by linking globally expressed branch-promoting with differentially expressed branch-suppressing activities. In addition, our data suggest that growth factors and the EphA-ephrinA system interact in a way that affects axon branching and synapse development.

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An acute intravenous administration of loo&kg body weight of recombinant tumour necrosis factor-a (TNF) resulted in a time-dependent increase in the levels of both free and conjugated ubiquitin m rat skeletal muscle. The effects of the cytokine were more pronounced in the red muscle soleus than in the white muscle EDL. In the former muscle type. TNF-treatment also resulted in a time-dependent increase in the percentage of free ubiquitin. The results suggest that the ubiquitm system for non-lysosomal protein degradation could have a very important role in the mechanism triggered by TNF which is responsible for enhanced muscle proteolysis in sepsis and other pathological states.

transduces signals mediated by NGF, BDNF, NT3 and NT4. Markedly, AmphiTrk is able to activate survival and differentiation pathways, but fails to activate the PLCγ pathway, which is involved in synaptic plasticity in higher vertebrates. AmphiTrk is expressed during amphioxus embryogenesis in sensory neural precursors in the epidermis, which possesses single migratory cells. We propose that the duplication and divergence of the Nt/Trk system, in tandem with recruitment of the PLCγ pathway, may have provided the genetic basis for a key aspect of vertebrate evolution: the complexity of the nervous system.

Rats bearmg the fast-growmg AH-l 30 Yoslnda ascItes hepatoma showed a marked cachectlc response which has been previously reported [Tessltore et al (1987) Blochem J 241, 153-1591 Thus tumour-bearmg ammals showed slgmficant decreases m body and muscle weight (soleus and gastrocnemms) as compared to both pair-fed and ad hbltum-fed animals These decreases were related to an enhanced proteolytlc rate m the muscles of the tumour-bearmg ammals as measured by the tyrosme released m m vitro assays In an attempt to elucidate which proteolytlc system IS directly responsible for the decrease m muscle mass, we have studied both lysosomal and non-lysosomal (ATP-dependent) proteolytlc systems m this animal model While the enzymatic activities of the mam cathepsm (B and B + L.) systems were actually decreased m gastrocnemms muscles of tumourbearmg rats, thus mdicatmg that lysosomal proteolysls was not involved, the ublqmtm pools (both free and conjugated) were markedly altered as a result of tumour burden These were associated with an increased ublqmtm gene expression m muscle of tumour-beanng rats, over 500% m relation to non-twnour bearers, thus suggesting that the ATP-dependent proteolytlc system may be responsible for the muscle proteolysls and wastage observed m this animal tumour model The fact that we have previously shown that TNF enhances the ublqultmlzation of muscle proteins FEBS Lett 323, 21 l-2141, together with the high clrculatmg levels of TNF detected m rats bearmg the Yoshlda hepatoma allows us to suggest that the cytokme may be responsible, most probably mdlrectly, for the activation of the referred proteolytlc system m tumour-bearing rats

Tyrosine phosphorylation of β-catenin, a component of adhesion complexes and of the Wnt pathway, affects cell adhesion, migration and gene transcription. By reducing β-catenin availability using shRNA-mediated gene silencing or expression of intracellular N-cadherin, we show that β-catenin is required for axon growth downstream of brain-derived neurotrophic factor (BDNF) signalling and hepatocyte growth factor (HGF) signalling. We demonstrate that the receptor tyrosine kinases (RTKs) Trk and Met interact with and phosphorylate β-catenin. Stimulation of Trk receptors by neurotrophins (NTs) results in phosphorylation of β-catenin at residue Y654, and increased axon growth and branching. Conversely, pharmacological inhibition of Trk or expression of a Y654F mutant blocks these effects. β-catenin phosphorylated at Y654 colocalizes with the cytoskeleton at growth cones. However, HGF, which also increases axon growth and branching, induces β-catenin phosphorylation at Y142 and a nuclear localization. Interestingly, dominant-negative ΔN-TCF4 abolishes the effects of HGF in axon growth and branching, but not that of NTs. We conclude that NT- and HGF-signalling differentially phosphorylate β-catenin, targeting this protein to distinct compartments to regulate axon morphogenesis by TCF4-transcription-dependent and -independent mechanisms. These results place β-catenin downstream of growth-factor–RTK signalling in axon differentiation.

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