Beyond their deep theoretical interest, the molecular events controlling axon growth compel attention because axon regeneration fails so miserably in the human brain and spinal cord. Where axon regeneration is successful, it is typically accompanied by large increases in the synthesis of a small set of """"""""growth-associated proteins"""""""" (GAPs). Injured, non-regenerating mammalian CNS neurons fail to elevate GAP synthesis. The protein most consistently correlated with axon growth is one designated GAP-43. The proposed work moves from correlations to direct tests of the hypothesis that expression of GAPs may regulate some aspects of axon growth. The first part of this work asks how changes in GAP-43 expression affect neurite growth in cultured cells. GAP-43 expression will be manipulated by transfecting a neuron-like cell line (PC12) with plasmids expressing GAP-43 mRNA or the complementary (anti-sense) RNA, and by microinjection of the purified protein. Cells over- or under-expressing GAP-43 will be assayed for neurite growth in response to nerve growth factor (NGF), laminin-containing extracellular matrix, and activation of cellular kinases by cyclic AMP or phorbol esters. The ability of cells to extend very long processes through explanted peripheral nerves will also be measured as a function of GAP-43 expression. These studies will be augmented by analysis of the GAP-43 sequence to identify functionally characterized domains or possible homologies with known proteins, as clues to the protein's functions. Phosphorylation of GAP-43 has been correlated with axon growth and synaptic plasticity. Is phosphorylation, or other modification, of GAP-43 deficient in non-regenerating axons? Antibodies against the relevant kinases will be used to determine whether the abundance of these kinases is similar in successfully regenerating and non-regenerating axons. Monoclonal antibodies selective for GAP-43 from growth cones (the presumed """"""""active form"""""""" of the protein) will be used to assess whether growth- associated modifications of GAP-43 are inhibited when regenerating axons encounter the non-permissive environment of the mammalian CNS. In parallel with detailed studies of GAP-43, the search for other GAPs continues. Differential hybridization is being used to identify cDNA clones that hybridize preferentially with cDNA derived from developing and regenerating neurons, compared to mature controls.
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