This proposal is intended to meet the aims of the Latin American Initiative from the Fogarty International Research Collaboration program by seeking support for the continuation of a collaboration begun in the U.S. and currently continuing in Argentina. The collaborative work which forms the basis of this proposal developed the novel technique of antisense oligonucleotides to suppress the expression of the microtubule-associated protein (MAP), tau. Subsequently this work has been widely cited for making an important technical contribution and for demonstrating a cell biological function of a neuronal MAP in neurite outgrowth. Since then, collaborative work has continued in which the use of antisense techniques have been expanded to address the important area of motor proteins. The long distances over which neurons must deliver molecules along microtubule tracks makes these proteins an essential aspect of neurite growth in both neural development and in regeneration. Cerebellar macroneurons and hippocampal pyramidal cells in culture will be used to study the molecular basis of neurite outgrowth. Focusing upon the MAPs, which have a role in the sequential development of neuronal morphological features, we will use antisense oligonucleotides to create null phenotypes in post-mitotic neurons. The specificity of the antisense oligonucleotides permits the selective suppression of a single mRNA and its translation product. We plan to analyze additional MAPs, particularly MAP1B and the microtubule-based motor protein, kinesin. By combining direct video-microscopic analyses of antisense-treated null cells with morphometric, immunocytochemical, and ultrastructural images, we hope to assign discrete functions to these proteins during neuritogenesis.
