ABSTRACT
Neurotrophins and cell adhesion molecules regulate axon guidance, but their potential coordinate interactions are not well defined. In particular, it has been difficult to define the role of signaling from different surface molecules in neurotrophin-induced axon growth because of the strong dependence of embryonic neurons on this class of molecules for survival. We have addressed this issue using Bax deficient neurons, which do not require neurotrophins for survival. The L1 neural cell adhesion molecule and laminin each supported NGF-independent axon growth of cultured sensory neurons from dorsal root ganglia of embryonic Bax(-/-) mice. However, nerve growth factor (NGF) stimulated additional axon growth of sensory neurons on laminin but not on L1 substrates. Inhibition of the small GTPase RhoA by the dominant-negative mutant RhoA(T19N) restored NGF responsiveness of axon growth on L1 to Bax(-/-) neurons. Constitutively activated RhoA(Q63L) did not affect axon growth on L1 but inhibited NGF-stimulated axon growth on laminin. Consistent with the concept that RhoA was downregulated by NGF in neurons on laminin but not L1, the RhoA inhibitor C2IN-C3 toxin stimulated axon growth on L1 in wild-type DRG neurons in NGF. These results demonstrate a novel substrate-dependent regulation of NGF-induced growth of embryonic sensory axons mediated by RhoA GTPase.
Subject(s)
Botulinum Toxins , Cell Differentiation/physiology , Ganglia, Spinal/embryology , Growth Cones/metabolism , Laminin/metabolism , Membrane Glycoproteins/metabolism , Nerve Growth Factor/metabolism , Neural Cell Adhesion Molecules/metabolism , Neurons, Afferent/metabolism , Proto-Oncogene Proteins c-bcl-2 , ADP Ribose Transferases/pharmacology , Animals , Cell Differentiation/drug effects , Cells, Cultured , Drug Interactions/physiology , Female , Fetus , Ganglia, Spinal/cytology , Ganglia, Spinal/metabolism , Growth Cones/drug effects , Growth Cones/ultrastructure , Immunohistochemistry , Laminin/pharmacology , Leukocyte L1 Antigen Complex , Male , Membrane Glycoproteins/pharmacology , Mice , Mice, Knockout , Nerve Growth Factor/pharmacology , Neural Cell Adhesion Molecules/pharmacology , Neurons, Afferent/cytology , Neurons, Afferent/drug effects , Proto-Oncogene Proteins/deficiency , Proto-Oncogene Proteins/genetics , Receptor, trkC/drug effects , Receptor, trkC/metabolism , bcl-2-Associated X Protein , rhoA GTP-Binding Protein/agonists , rhoA GTP-Binding Protein/antagonists & inhibitors , rhoA GTP-Binding Protein/metabolismABSTRACT
The L1 adhesion molecule regulates axon growth and is mutated in the X-linked mental retardation syndrome CRASH (acronym for corpus callosum agenesis, retardation, aphasia, spastic paraplegia, hydrocephalus). A novel role for L1 as a potentiator of neuronal cell migration to extracellular matrix proteins through beta1 integrins and intracellular signaling to mitogen-activated protein (MAP) kinase was identified. L1 potentiated haptotactic migration of B35 neuroblastoma cells toward fibronectin, vitronectin, and laminin through the signaling intermediates c-Src, phosphatidylinositol-3 kinase, and MAP kinase. L1 potentiated migration toward fibronectin through alpha5beta1 integrin in human embryonic kidney 293 cells and depended on determinants of L1 endocytosis: dynamin I, c-Src, and the AP2/clathrin binding site (Arg-Ser-Leu-Glu) in the neuronal splice form of L1. L1 clustering on the cell surface enhanced the internalization of activated beta1 integrins and L1 into distinct endocytic vesicles. L1-potentiated migration, enhancement of beta1 integrin endocytosis, and activation of MAP kinase were coordinately inhibited by mutation of an RGD sequence in the sixth immunoglobulin-like domain of L1. Moreover, three CRASH mutations in the L1 cytoplasmic domain (1194L, S1224L, Y1229H), two of which interfere with ankyrin association, inhibited L1-potentiated migration and MAP kinase activation. Function-blocking antibodies to L1 and beta1 integrin retarded the migration of 5-bromo-2'-deoxyuridine-labeled mouse cerebellar granule cells in slice cultures, underscoring the potential physiological relevance of these findings. These studies suggest that L1 functionally interacts with beta1 integrins to potentiate neuronal migration toward extracellular matrix proteins through endocytosis and MAP kinase signaling, and that impairment of this function by L1 cytoplasmic domain mutations may contribute to neurological deficits in CRASH.
