Although developing motor neurons express low-affinity nerve growth factor (NGF) receptors, there is no known biological effect of NGF on developing or adult motor neurons. In this study, we found that, unlike NGF, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4/5 (NT-4/5) stimulated cholinergic phenotype by increasing choline acetyltransferase (CAT) activity in cultures enriched with embryonic rat motor neurons. Ciliary neurotrophic factor (CNTF) also stimulated CAT activity. The effects of BDNF and NT-4/5 on CAT activity appeared to be synergistic with that of CNTF. Cotreatment with BDNF and NT-3 resulted in an additive effect, suggesting that signal transduction was mediated through different high-affinity receptors tyrosine kinases B and C (Trk B and Trk C). However, cotreatment with BDNF and NT-4/5 did not result in an increase in CAT activity greater than that of either BDNF or NT-4/5 alone, suggesting that their effects were mediated via the same receptor Trk B. Supporting our findings that spinal cholinergic neurons are responsive to trophic actions of members of the neurotrophin family, motor neuron-enriched cultures were found to express mRNA for Trk B and Trk C, which have been identified as high-affinity receptors for BDNF and NT-4/5, and NT-3, respectively.

Hepatocyte growth factor (HGF) has been shown to function as a potent mitogen for a variety of cells, transducing its signal through the c-met tyrosine kinase receptor. Ciliary neurotrophic factor (CNTF) is a cytokine that has been shown to promote survival of motor neurons. We show here that c-met mRNA is present in the embryonic rat spinal cord. Peak expression of c-met (at E14) coincides with the period of naturally occurring cell death in motor neurons, suggesting a possible role of HGF in the regulation of this process. Utilizing a neuron-enriched culture system, we established that HGF, like CNTF, stimulates choline acetyltransferase (CAT) activity in motor neurons. When co-administered to motor neuron cultures, saturating concentrations of HGF and CNTF produced a synergistic increase in CAT levels. We show that this synergy reflects enhanced motor neuron survival. Exposure of motor neuron cultures to the cytostatic agent vincristine markedly decreased CAT levels; co-treatment with HGF and CNTF (but not either factor alone) restored CAT activity to control levels. Our findings indicate that HGF is a survival factor for motor neurons, that it acts synergistically with CNTF, and that HGF and CNTF can together be neuroprotective in the face of vincristine toxicity.

Ciliary neurotrophic factor (CNTF) is a cytokine whose actions are largely restricted to the nervous system because of the predominant neuronal distribution of its receptor, CNTFR alpha. In this study, we sought to define the binding characteristics of CNTF to cultured sympathetic neurons and cell lines of neuronal origin. We report that 125I-CNTF binds to cultured sympathetic neurons, MAH, PC12, and EW-1 cells via high and low affinity receptors that can be distinguished on the basis of their dissociation constants (KD1 approximately 10(-12) M and KD2 approximately 10(-9) M). Competition experiments showed that the IC50 for rat and human CNTF were, respectively, 65 pM and 5 nM for sympathetic neurons and 75 pM and 1.2 nM for EW-1 cells. Interestingly, leukemia inhibitory factor (LIF) did not compete for CNTF binding even at 100 nM concentration. The binding of 125I-CNTF to sympathetic neurons involved all three components of the CNTF receptor complex, namely CNTFR alpha, LIFR, and gp130, as shown by cross-linking experiments. CNTF and LIF treatments down-regulated CNTF binding to sympathetic neurons and EW-1 cells, suggesting that heterologous ligands can regulate CNTF receptor levels, which may in turn modulate the efficacy of CNTF in vitro and in vivo.