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 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 microM 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.
Pong
K
K
Department of Neuroscience, Amgen, Inc., Thousand Oaks, California 91320, USA.
Xu
R Y
RY
Baron
W F
WF
Louis
J C
JC
Beck
K D
KD
D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D002454:Cell Differentiation / Q000502:physiology; D002867:Chromones / Q000494:pharmacology; D004298:Dopamine / Q000378:metabolism*; D029721:Drosophila Proteins; D004791:Enzyme Inhibitors / Q000494:pharmacology; D051100:Glial Cell Line-Derived Neurotrophic Factor; D051097:Glial Cell Line-Derived Neurotrophic Factor Receptors; D009025:Morpholines / Q000494:pharmacology; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins / Q000494:pharmacology*; D009474:Neurons / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation / Q000187:drug effects; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051096:Proto-Oncogene Proteins c-ret; D051381:Rats / Q000196:embryology; D017207:Rats, Sprague-Dawley; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism; D000077191:Wortmannin
D000730:Androstadienes; D002867:Chromones; D029721:Drosophila Proteins; D004791:Enzyme Inhibitors; C495041:Gdnf protein, rat; D051100:Glial Cell Line-Derived Neurotrophic Factor; D051097:Glial Cell Line-Derived Neurotrophic Factor Receptors; D009025:Morpholines; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011518:Proto-Oncogene Proteins; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D014446:Tyrosine 3-Monooxygenase; D051096:Proto-Oncogene Proteins c-ret; D020794:Receptor Protein-Tyrosine Kinases; C416649:Ret protein, Drosophila; C495001:Ret protein, rat; D004298:Dopamine; D000077191:Wortmannin