The p75 neurotrophin receptor influences NT-3 responsiveness of sympathetic neurons in vivo.

To determine the role of the p75 neurotrophin receptor (p75NTR) in sympathetic neuron development, we crossed transgenic mice with mutations in p75NTR, nerve growth factor (NGF) and neurotrophin-3 (NT-3). Neuron number is normal in sympathetic ganglia of adult p75NTR-/- mice. Mice heterozygous for a NGF deletion (NGF+/-) have 50% fewer sympathetic neurons. In the absence of p75NTR (p75NTR-/- NGF+/-), however, neuron number is restored to wild-type levels. When NT-3 levels are reduced (p75NTR-/- NGF+/- NT3 +/-), neuron number decreases compared to p75NTR-/- NGF+/- NT3+/+. Thus, without p75NTR, NT3 substitutes for NGF, suggesting that p75 alters the neurotrophin specificity of TrkA in vivo.

Src-class kinases act within the agrin/MuSK pathway to regulate acetylcholine receptor phosphorylation, cytoskeletal anchoring, and clustering.

Synaptogenesis at the neuromuscular junction requires agrin-induced stable localization of acetylcholine receptors (AChRs) at the endplate. The effects of agrin are transduced by the muscle-specific receptor tyrosine kinase (MuSK). This study provides evidence that Src-class protein tyrosine kinases mediate the effects of agrin-activated MuSK to regulate clustering and anchoring of AChRs in skeletal muscle. MuSK was complexed with both Src and Fyn in the C2 mouse muscle cell line. These associations were enhanced by agrin and by increasing protein tyrosine phosphorylation with pervanadate. Coupling between MuSK and the Src-class kinases in vivo appeared to be caused by a phosphotyrosine-SH2 domain interaction because binding of MuSK to the SH2 domains of Fyn and Src in vitro was specific, enhanced by phosphorylation, and dependent on MuSK autophosphorylation. In addition, Src and Fyn phosphorylated MuSK. AChR phosphorylation, stimulated by agrin or pervanadate, was inhibited by blocking Src-class kinases with PP1. Furthermore, agrin-induced clustering and cytoskeletal anchoring of AChRs was dependent on Src-family kinases. These data support the conclusion that Fyn and Src act downstream of MuSK to regulate the stable localization of AChRs at the neuromuscular endplate during agrin-induced synaptogenesis.

NT-3, like NGF, is required for survival of sympathetic neurons, but not their precursors.

Superior cervical ganglia of postnatal mice with a targeted disruption of the gene for neurotrophin-3 have 50% fewer neurons than those of wild-type mice. In culture, neurotrophin-3 increases the survival of proliferating sympathetic precursors. Both precursor death (W. ElShamy et al., 1996, Development 122, 491-500) and, more recently, neuronal death (S. Wyatt et al., 1997, EMBO J. 16, 3115-3123) have been described in mice lacking NT-3. Consistent with the second report, we found that, in vivo, neurogenesis and precursor survival were unaffected by the absence of neurotrophin-3 but neuronal survival was compromised so that only 50% of the normal number of neurons survived to birth. At the time of neuron loss, neurotrophin-3 expression, assayed with a lacZ reporter, was detected in sympathetic target tissues and blood vessels, including those along which sympathetic axons grow, suggesting it may act as a retrograde neurotrophic factor, similar to nerve growth factor. To explore this possibility, we compared neuron loss in neurotrophin-3-deficient mice with that in nerve growth factor-deficient mice and found that neuronal losses occurred at approximately the same time in both mutants, but were less severe in mice lacking neurotrophin-3. Eliminating one or both neurotrophin-3 alleles in mice that lack nerve growth factor does not further reduce sympathetic neuron number in the superior cervical ganglion at E17.5 but does alter axon outgrowth and decrease salivary gland innervation. Taken together these results suggest that neurotrophin-3 is required for survival of some sympathetic neurons that also require nerve growth factor.