Search

The c-ret protooncogene encodes Ret, the functional tyrosine kinase receptor for glial cell line-derived neurotrophic factor (GDNF). K-252b, a known protein tyrosine kinase inhibitor, has been shown earlier to inhibit the trophic activity of brain-derived neurotrophic factor on dopaminergic (DAergic) neurons and nerve growth factor on basal forebrain cholinergic neurons while potentiating neurotrophin-3 activity on central cholinergic and peripheral sensory neurons and PC12 cells. We tested whether K-252b would modulate GDNF-induced differentiation in DAergic neuron cultures. Exposure to 1 ng/ml GDNF increased dopamine (DA) uptake 80% above control, whereas treatment with 5 microM K-252b decreased the efficacy of GDNF by 60%. Concentrations of GDNF of100 pg/ml were completely inhibited, whereas concentrations of100 pg/ml were moderately active, between 10 and 20% above control. In addition, K-252b shifted the ED50 from 20 to 200 pg/ml. GDNF treatment increased soma size and neurite outgrowth in tyrosine hydroxylase-immunoreactive neurons. K-252b inhibited differentiation of these morphological parameters induced by GDNF. Furthermore, GDNF stimulated Ret autophosphorylation at maximal levels, whereas the inhibition of DA uptake and morphological differentiation by K-252b correlated with a significantly decreased level of Ret autophosphorylation. Therefore, K-252b is able to inhibit intracellular activities induced by GDNF on mesencephalic DAergic neurons.

Here we report the generation and characterization of two distinct monoclonal antibodies, G-90 and B-1531, specific to glial cell line-derived neurotrophic factor (GDNF). ELISA results confirmed that G-90 and B-1531 both recognize GDNF. Western blots showed that G-90 recognized only the GDNF dimer, whereas B-1531 recognized both the monomer and dimer. Peptide competition ELISA (PCE) and BIAcore data suggested that G-90 and B-1531 recognize different epitopes: PCE confirmed that B-1531 binds to NH2-terminal peptides between amino acids 18 and 37, whereas G-90 does not; BIAcore data showed that B-1531 binds to the NH2 terminus of GDNF, whereas G-90 does not. G-90, in a concentration-dependent manner, completely neutralized the GDNF-induced increases of choline acetyltransferase in cultured motoneuron and of dopamine uptake and morphological differentiation in dopaminergic neuron cultures. B-1531 had no neutralizing effects. GDNF-induced Ret autophosphorylation in NGR-38 cells was completely neutralized by G-90, whereas B-1531 had a moderate effect. These data show that G-90 and B-1531 are specific antibodies to GDNF. The data also suggest that the NH2 terminus of GDNF is not critical for activity. Partial inhibition of Ret phosphorylation is insufficient to downregulate GDNF-induced biological activity.

Phospholipase C gamma 1 (PLC-gamma 1) is involved at an early step in signal transduction of many hormones and growth factors and catalyzes the hydrolysis of phosphatidylinositol (PI) 4,5-bisphosphate to diacylglycerol and inositol trisphosphate, two potent intracellular second messenger molecules. The transformation of PC12 cells into neuron-like cells induced by nerve growth factor is preceded by a rapid stimulation of PLC-gamma 1 phosphorylation and PI hydrolysis. The present study analyzed the effects of brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) on phosphorylation of PLC-gamma 1 in primary cultures of embryonic rat brain cells. BDNF and NT-3 stimulated the phosphorylation of PLC-gamma 1, followed by hydrolysis of PI. The stimulation of PLC-gamma 1 phosphorylation occurred within 20 s after addition of BDNF or NT-3 and lasted up to 30 min, with a peak after 4 min. ED50 values were similar for BDNF and NT-3, with approximately 25 ng/ml. Phosphorylation of PLC-gamma 1 by BDNF and NT-3 was found in cultures from all major brain areas. K-252b, a compound known to inhibit selectively neutrophin actions by interfering with the phosphorylation of trk-type neutrophin receptors, prevented the BDNF- and NT-3-stimulated phosphorylation of PLC-gamma 1. Receptors of the trk type were coprecipitated with anti-PLC-gamma 1 antibodies. The presence of trkB mRNA in the cultures was substantiated by northern blot analysis. The action of BDNF and NT-3 seems to be neuron specific because no phosphorylation of PLC-gamma 1 was observed in cultures of nonneuronal brain cells. The results provide evidence that developing neurons of the cerebral cortex and other brain areas are responsive to BDNF and NT-3, and they indicate that the transduction mechanism of BDNF and NT-3 in the brain involves rapid phosphorylation of PLC-gamma 1 followed by PI hydrolysis.

K-252b, a protein kinase inhibitor, has been shown earlier to inhibit nerve growth factor actions on cholinergic neurons of the basal forebrain. In the present study, K-252b was found to prevent trophic actions of two other neurotrophins, brain-derived neurotrophic factor, and neurotrophin-3, on central cholinergic and dopaminergic neurons, peripheral sensory neurons, and PC12 pheochromocytoma cells, when used at greater than 2 microM concentration. Comparable actions of nonneurotrophin growth factors were not affected. Surprisingly, at 0.1-100 nM, K-252b selectively enhanced the trophic action of neurotrophin-3 on central cholinergic neurons, peripheral sensory neurons, and PC12 cells. In PC12 cells, K-252b potentiated the neurotrophin-3-induced tyrosine phosphorylation of trk, a protein kinase responsible for transmitting neurotrophin signals. Of the three structurally related nerve growth factor inhibitors, K-252a, K-252b, and staurosporine, only the first two also mediated neurotrophin-3 potentiation. These findings indicate that K-252b generally and selectively potentiates the neurotrophic action of neurotrophin-3 and suggest that this action involves trk-type neurotrophin receptors.

To enable us to study expression of tyrosine hydroxylase [TH; tyrosine 3-monooxygenase; L-tyrosine tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating); EC 1.14.16.2] as a measure of dopaminergic neuron function in future experiments, methods were developed to quantify TH mRNA levels in cultures of dopaminergic mesencephalic cells. The model of selective dopaminergic toxicity of 1-methyl-4-phenylpyridinium (MPP+) was used to verify the specificity of our methods. Fetal (embryonic day 15) rat ventral mesencephalic cell cultures were treated with 15 microM MPP+ for 48 h, conditions previously shown to reduce the number of TH-immunoreactive neurons, TH activity, and dopamine uptake to 5-10% of control values. This treatment decreased the number of neurons labeled by TH in situ hybridization to 9% of untreated controls and caused a strong reduction of the abundance of TH mRNA in Northern blots. Our findings establish TH mRNA expression as a parameter for future studies of toxic and trophic effects on cultured dopaminergic neurons, and they support the view that MPP+ destroys dopaminergic neurons.

Glial cell line-derived neurotrophic factor (GDNF) is a potent survival factor for midbrain dopaminergic neurons. To begin to understand the intracellular signaling pathways used by GDNF, we investigated the role of phosphatidylinositol 3-kinase activity in GDNF-stimulated cellular function and differentiation of dopaminergic neurons. We found that treatment of dopaminergic neuron cultures with 10 ng/ml GDNF induced maximal levels of Ret phosphorylation and produced a profound increase in phosphatidylinositol 3-kinase activity, as measured by western blot analysis and lipid kinase assays. Treatment with 1 µM 2-(4-morpholinyl)-8-phenylchromone (LY294002) or 100 nM wortmannin, two distinct and potent inhibitors of phosphatidylinositol 3-kinase activity, completely inhibited GDNF-induced phosphatidylinositol 3-kinase activation, but did not affect Ret phosphorylation. Furthermore, we examined specific biological functions of dopaminergic neurons: dopamine uptake activity and morphological differentiation of tyrosine hydroxylase-immunoreactive neurons. GDNF significantly increased dopamine uptake activity and promoted robust morphological differentiation. Treatment with LY294002 completely abolished the GDNF-induced increases of dopamine uptake and morphological differentiation of tyrosine hydroxylase-immunoreactive neurons. Our findings show that GDNF-induced differentiation of dopaminergic neurons requires phosphatidylinositol 3-kinase activation.