During neural development, a population of morphologically homogenous, mitotically active precursor cells gives rise to all the neuronal and glial cells of the adult cerebral cortex. Postmitotic neurons extend processes by which they migrate to their adult positions and begin the elaboration of axonal and dendritic arbors. To understand the molecular events that permit a cell to assume neuronal properties, we previously used 2- dimensional gel electrophoresis to identify 15 proteins that are differentially regulated over the course of corticogenesis. One of these, TOAD-64, is a 64,000 dalton protein that is up regulated nearly 7-fold over the course of late embryonic cortical development and is subsequently down regulated during postnatal life. The sequence of a full length cDNA of TOAD-64 is homologous to the unc-33 gene from C. elegans, mutations in which lead to defective patterns of axon outgrowth. Several lines of evidence suggest that TOAD-64 may, similarly, play a role in axonogenesis in the rodent. First, TOAD-64 is re-expressed in adult motor neurons as they extend axons following a peripheral nerve lesion. Second, TOAD-64 expression increases coincident with neuritogenesis in two cell lines, pC12 and p19. Finally, TOAD-64 protein is present at the most peripheral portions of the growth cone, in lamellipodia and filopodia. Together, these data support the possibility that TOAD-64 is part of the intracellular machinery by which growing neurons elaborate axons. The experiments proposed here are designed to further characterize TOAD-64 and will also address the possible functions of this new protein in neurite outgrowth.
The first aim i s to further characterize TOAD-64. A possible role for TOAD-64 in neuronal remodeling in the adult will be investigated. A new panel of antibodies to primate TOAD-64 will be generated to permit studies of early corticogenesis in the monkey. For studies of the mechanisms by which the TOAD-64 gene is regulated, a genomic clone will be isolated for future experiments to identify its regulatory sequences.
The second aim i s to determine the function of TOAD-64. We will explore the role TOAD-64 may play in axonogenesis by changing levels of protein expression in primary neuronal cultures and in two cell lines. To test whether TOAD-64 is required for a cell to elaborate neurites, antisense oligonucleotides will be used to reduce TOAD-64 expression in neurons in primary culture. We will also determine whether a reduction in TOAD-64 can alter the response of growth cones and neurites to repulsive substrates. To determine whether TOAD-64 is critical for the elaboration of neurites in PC12 and P19 cells, expression of TOAD-64 will be reduced or increased using stable transfection.
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