Permanent paralysis after human spinal cord injuries or other CNS trauma results from the failure of most long CNS axons to regenerate in adults. This project examines the hypothesis that regeneration in the mammalian CNS is substantially limited by the failure of most injured neurons to induce expression of a small set of genes whose protein products are necessary for axon elongation. Previous work has shown that successful axon regeneration is strongly correlated with elevated synthesis of certain axonal proteins, including one of the most abundant protein components of growth cone membranes, GAP-43. Expression of these """"""""growth-associated proteins"""""""" is low in the majority of adult CNS neurons and remains low after the most common types of CNS injury. The present proposal is to determine whether failure to induce GAP expression after axotomy actually limits the ability of adult mammalian neurons to regenerate their axons. Taking GAP-43 as an accessible example of this class of proteins, the proposed studies ask whether neurons that ordinarily would extend long axons show a reduced proclivity for axon growth after experimental disruption of GAP expression. Expression of GAP-43 will be interrupted in axotomized adult neurons in tissue culture by microinjecting antibodies targeted against specific sites on the GAP-43 molecules necessary for its processing and transport to growth cone membranes, or by injecting oligonucleotides complementary to GAP-43 mRNA. The cells then will be monitored for their ability to extend axons on a series of cellular and extracellular matrix substrata previously shown to support long axonal outgrowth. If GAP-43 proves to be necessary for some steps in axon growth, then effective regeneration of injured axons will require not only induction of GAP-43 synthesis in neuron cell bodies, but also delivery of the protein to axonal growth cones and appropriate regulation of the protein's activity in response to specific cues in the extracellular environment. Intracellular routing of GAP-43 and its interaction with growth cone membranes appears to be regulated physiologically by post-translational addition and removal of covalently linked fatty acid chains to the protein. Preliminary pharmacological evidence suggests that inhibition of the acylating enzyme leads to a rapid disruption of growth cone activity and retraction of neurites in vitro. This application therefore proposes to continue investigating post-translational modification of GAP-43 in response to specific cues in the PNS and CNS environments, with particular emphasis on the biochemical regulation of the protein-acylating reaction in CNS growth cones and its functional role in axon regeneration. Finally, to continue the search for additional growth-associated proteins, a recently isolated monoclonal antibody will be used to characterize a new protein antigen expressed on growth cones, but not on mature axon terminals. Metabolic labeling, immunoprecipitation, and protein microsequencing will be used to determine whether the strongly developmentally regulated appearance of this antigen represents growth=associated expression of a new GAP gene.
| Patterson, S I; Skene, J H (1999) A shift in protein S-palmitoylation, with persistence of growth-associated substrates, marks a critical period for synaptic plasticity in developing brain. J Neurobiol 39:423-37 |
| Hess, D T; Smith, D S; Patterson, S I et al. (1999) Rapid arrest of axon elongation by brefeldin A: a role for the small GTP-binding protein ARF in neuronal growth cones. J Neurobiol 38:105-15 |
| Smith, D S; Skene, J H (1997) A transcription-dependent switch controls competence of adult neurons for distinct modes of axon growth. J Neurosci 17:646-58 |
| Herdegen, T; Skene, P; Bahr, M (1997) The c-Jun transcription factor--bipotential mediator of neuronal death, survival and regeneration. Trends Neurosci 20:227-31 |
