Glial cell line-derived neurotrophic factor and target-dependent regulation of large-conductance KCa channels in developing chick lumbar motoneurons

Abstract

The functional expression of large-conductance Ca2+-activated K+ (K(Ca)) channels in lumbar motoneurons (LMNs) of the developing chick embryo is regulated in part by interactions with striated muscle target tissues. Here we show that the functional expression of K(Ca) channels in LMNs developing in vitro can be stimulated by application of a skeletal muscle extract (MEX) or by coculture with hindlimb myotubes. A similar stimulation of K(Ca) channels in vitro can be produced by the trophic factors glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor but not by neurotrophin (NT)-3 or NT-4. The actions of MEX and hindlimb myotubes are blocked by a GDNF-neutralizing antiserum. Moreover, injection of this same antiserum into the embryonic hindlimb reduced the functional expression of K(Ca) channels in vivo to levels seen in LMNs deprived of interactions with the hindlimb. The effects of GDNF on K(Ca) channel expression in LMNs require 24 hr of continuous exposure to reach maximum and are blocked by the translation inhibitor anisomycin, indicating the need for synthesis of new proteins. GDNF actions are also blocked by the farnesyl transferase inhibitor manumycin, suggesting a role for Ras in the actions of GDNF. Finally, the actions of GDNF are inhibited by PP2, an inhibitor of Src family tyrosine kinases, and by LY29003, an inhibitor of phosphatidylinositol 3 kinases, but not by PD98059, an inhibitor of the Erk signaling cascade. None of these treatments alter expression of voltage-activated Ca2+ channels. Thus, the actions of GDNF on LMN K(Ca) channel expression appear to use a transduction pathway similar to that used for regulation of apoptosis.

Fig. 1.

Effect of MEX and growth factors on the expression of macroscopic K_Ca current in LMNs in vitro. A, Representative traces of outward currents evoked in E8 LMNs and after 72 hr in the presence of MEX (50 μg/ml). Outward currents were evoked in control and Ca²⁺-free salines (left traces) by 25 msec depolarizing pulses to +30 mV from a holding potential of −40 mV (shown at bottomleft). Net macroscopic K_Ca was obtained after digital subtraction of raw traces (right trace). B, Summary of the effects of MEX and trophic factors on the functional expression of K_Ca in cultured LMNs. Motoneurons were dissociated on E8 and maintained in culture for 72 hr in the presence of 50 μg/ml MEX or 10 ng/ml of the trophic factors BDNF, NT-3, NT-4, and GDNF. All of these factors are capable of promoting LMN survival in vitro. E8 represents control neurons examined 3 hr after dissociation. Note robust stimulation of K_Ca by MEX, GDNF, and BDNF. C, Time course of GDNF stimulation of K_Ca channel expression. Maximal expression of K_Ca channel occurred after 24 hr exposure to GDNF (10 ng/ml). The effect of GDNF on K_Ca channel expression is independent of electrical activity because TTX did not prevent the functional expression of K_Ca channels. Note that 24 hr incubation with NT-4 fails to increase K_Ca channel expression. In these experiments, LMNs were dissociated on E8 and maintained in culture for 3, 12, or 24 hr in the presence of GDNF as indicated. Control represents E8 LMNs examined 3 hr after dissociation. In this and subsequent figures, error bars indicate SEM, the number of cells recorded is given above each bar, andasterisks denote p < 0.05 from control as determined by one-way ANOVA followed by Tukey's honest significant difference test for unequal n.

Fig. 2.

Effect of trophic factor-neutralizing antisera on K_Ca channel expression in developing LMNs.A, Coculture of E8 LMNs with hindlimb myotubes for 24 hr allowed for a significant developmental increase in the functional expression of K_Ca channels. The stimulatory effect of hindlimb myotubes on K_Ca channel expression was reduced by incubation with neutralizing antisera specific for GDNF but not NT-4.B, GDNF-neutralizing antiserum did not affect the density of Ca²⁺ currents in LMNs cocultured for 24 hr with hindlimb myotubes. C, The stimulatory effect of 24 hr treatment with MEX on K_Ca channel expression in E8 LMNs was also reduced by overnight incubation of MEX with neutralizing antisera specific for GDNF but not NT-4. Neutralizing antisera were used at 10 μg/ml. D, Reduction in the functional expression of K_Ca channels in LMNs developing in ovo after hindlimb injections of the microtubule inhibitor colchicine or GDNF-neutralizing antiserum. Both of these treatments evoked a comparable reduction in K_Ca expression.

Fig. 3.

Effect of protein synthesis inhibition on the GDNF-induced expression of K_Ca channels. A, Inhibition of protein synthesis with anisomysin (25 μm) blocks GDNF stimulation of K_Ca channels. B, Protein synthesis inhibition does not change the expression of voltage-dependent Ca²⁺ currents. LMNs were dissociated on E8 and maintained in culture for 24 hr in the presence of GDNF with or without anisomycin.

Fig. 4.

Effect of transduction cascade inhibitors on GDNF stimulation of K_Ca channels. In these experiments, LMNs were dissociated on E8 and maintained in culture for 24 hr in the presence of GDNF (10 ng/ml). NT-4 (10 ng/ml) was also included in the cultured medium to prevent motoneuron cell death. Control neurons were cultured for 24 hr in the presence of GDNF and NT-4 and in the absence of inhibitors. A, Role of Erk, PI3 kinase, and Ras in the stimulation of K_Ca channels by GDNF. Incubation of LMNs in the presence of LY 29400 and manumycin, but not PD98059, significantly reduced the GDNF-induced expression of K_Cacurrents. B, Role of src-family kinases in GDNF actions. GDNF actions were inhibited by the active src-kinase inhibitor PP2 but not by the inactive structural congener PP3. C, No role for CaM kinase-II in GDNF actions. Stimulatory effects of GDNF were not different in cultures containing the CaM kinase-II inhibitor KN93 and its inactive congener KN92.