In recent years, the presence of several novel neuronal intermediate filament species has been documented. Dr. Shea has shown that the "66kDa neuronal intermediate filament," also known as alpha-internexin, is constitutively expressed by NB2a/d1 cells, and displays a unique distribution within the neuritic base and axon hillock during differentiation. Dr. Shea will modulate this protein in cells at various stages of differentiation by two methods already in use for the study of axonal cytoskeletal dynamics: 1) inhibiting the translocation, assembly, and interaction of this neuronal intermediate filament species with other cytoskeletal components, and 2) inhibiting the synthesis of this protein by treating cells with antisense oligonucleotides. Cells will be examined at hourly intervals by phase-contrast microscopy for alterations in neurite outgrowth, neurite retraction, compromise of stability, and immunocytochemistry for intracellular localization of the 66kDa neuronal intermediate filament/alpha-internexin as well as other axonal cytoskeletal antigens. These analyses will help determine whether or not NF-66 is required for neurite outgrowth, continued elongation, or stabilization. Comparison of results obtained following down-regulation of the 66kDa neuronal intermediate filament/alpha-internexin with H-200 will provide information regarding the role played by this novel neuronal intermediate filament species in axonal outgrowth and maturation.
