ABSTRACT
How a neuron becomes polarized remains largely unknown. Results obtained with a function-blocking antibody and an siRNA targeting the insulin-like growth factor-1 (IGF-1) receptor suggest that an essential step in the establishment of hippocampal neuronal polarity and the initiation of axonal outgrowth is the activation of the phosphatidylinositol 3-kinase (PI3k)-Cdc42 pathway by the IGF-1 receptor, but not by the TrkA or TrkB receptors.
Subject(s)
Cell Polarity , Hippocampus/cytology , Neurons/cytology , Receptor, IGF Type 1/metabolism , Animals , Cells, Cultured , Neurons/metabolism , Phosphatidylinositol 3-Kinases/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Rats , Receptor, IGF Type 1/genetics , Receptor, trkA/metabolism , Receptor, trkB/metabolism , cdc42 GTP-Binding Protein/metabolismABSTRACT
Exocytotic incorporation of plasmalemmal precursor vesicles (PPVs) into the cell surface is necessary for axonal outgrowth and is known to occur mainly at the nerve growth cone. We have demonstrated recently that plasmalemmal expansion is regulated at the growth cone by IGF-1, but not by BDNF, in a manner that is quasi independent of the neuron's perikaryon. To begin elucidating the signaling pathway by which exocytosis of the plasmalemmal precursor is regulated, we studied activation of the IRS/PI3K/Akt pathway in isolated growth cones and hippocampal neurons in culture stimulated with IGF-1 or BDNF. Our results show that IGF-1, but not BDNF, significantly and rapidly stimulates IRS/PI3K/Akt and membrane expansion. Inhibition of PI3K with Wortmannin or LY294002 blocked IGF-1-stimulated plasmalemmal expansion at the growth cones of cultured neurons. Finally, our results show that, upon stimulation with IGF-1, most active PI3K becomes associated with distal microtubules in the proximal or central domain of the growth cone. Taken together, our results suggest a critical role for IGF-1 and the IRS/PI3K/Akt pathway in the process of membrane assembly at the axonal growth cone.
Subject(s)
Cell Membrane/metabolism , Central Nervous System/embryology , Exocytosis/physiology , Growth Cones/metabolism , Insulin-Like Growth Factor I/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Animals , Cell Differentiation/drug effects , Cell Differentiation/physiology , Cell Membrane/drug effects , Cells, Cultured , Central Nervous System/cytology , Central Nervous System/growth & development , Enzyme Activation/drug effects , Enzyme Activation/physiology , Enzyme Inhibitors , Exocytosis/drug effects , Growth Cones/drug effects , Growth Cones/ultrastructure , Hippocampus/cytology , Hippocampus/embryology , Hippocampus/growth & development , Insulin-Like Growth Factor I/pharmacology , Membrane Fusion/drug effects , Membrane Fusion/physiology , Microtubules/drug effects , Microtubules/metabolism , Phosphoinositide-3 Kinase Inhibitors , Protein Transport/drug effects , Protein Transport/physiology , Rats , Signal Transduction/physiology , Transport Vesicles/drug effects , Transport Vesicles/metabolism , Transport Vesicles/ultrastructureABSTRACT
Exocytotic incorporation of plasmalemmal precursor vesicles (PPVs) into the cell surface is necessary for neurite extension and is known to occur mainly at the growth cone. This report examines whether this is a regulated event controlled by growth factors. The Golgi complex and nascent PPVs of hippocampal neurons in culture were pulse-labeled with fluorescent ceramide. We studied the dynamics of labeled PPVs upon arrival at the axonal growth cone. In controls and cultures stimulated with brain-derived neurotrophic factor (BDNF), PPV clusters persisted in growth cones with a half-life (t(1/2)) of >14 minutes. Upon challenge with IGF-1, however, fluorescent elements cleared from the growth cones with a t(1/2) of only 6 minutes. Plasmalemmal expansion was measured directly as externalization of membrane glycoconjugates in resealed growth cone particles (GCPs) isolated from fetal forebrain. These assays demonstrated that membrane expansion could be stimulated by IGF-1 in a dose-dependent manner but not by BDNF, even though intact, functional BDNF receptor was present on GCPs. Because both BDNF and IGF-1 are known to enhance neurite growth, but BDNF did not stimulate membrane expansion at the growth cone, we studied the effect of BDNF on the IGF-1 receptor. BDNF was found to cause the translocation of the growth-cone-specific IGF-1 receptor subunit beta(gc) to the distal axon, in a KIF2-dependent manner. We conclude that IGF-1 stimulates axonal assembly at the growth cone, and that this occurs via regulated exocytosis of PPVs. This mechanism is affected by BDNF only indirectly, by regulation of the beta(gc) level at the growth cone.