INHIBITION OF PHOSPHATIDYLINOSITOL 3-KINASE ACTIVITY BLOCKS CELLULAR-DIFFERENTIATION MEDIATED BY GLIAL-CELL LINE-DERIVED NEUROTROPHIC FACTOR IN DOPAMINERGIC-NEURONS

Glial cell line-derived neurotrophic factor (GDNF) is a potent surviva l factor for midbrain dopaminergic neurons. To begin to understand the intracellular signaling pathways used by GDNF, we investigated the ro le of phosphatidylinositol 3-kinase activity in GDNF-stimulated cellul ar 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 blo t analysis and lipid kinase assays. Treatment with 1 mu M 2-(4-morphol inyl)-8-phenylchromone (LY294002) or 100 nM wortmannin, two distinct a nd potent inhibitors of phosphatidylinositol 3-kinase activity, comple tely inhibited GDNF-induced phosphatidylinositol 3-kinase activation, but did not affect Ret phosphorylation. Furthermore, we examined speci fic biological functions of dopaminergic neurons: dopamine uptake acti vity and morphological differentiation of tyrosine hydroxylase-immunor eactive neurons. GDNF significantly increased dopamine uptake activity and promoted robust morphological differentiation. Treatment with LY2 94002 completely abolished the GDNF-induced increases of dopamine upta ke and morphological differentiation of tyrosine hydroxylase-immunorea ctive neurons. Our findings show that GDNF-induced differentiation of dopaminergic neurons requires phosphatidylinositol 3-kinase activation .