Assembly and Maintenance of the Axonal Cytoskeleton in Cultured Neurons

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NB2a/dl neuroblastoma cells provide a model system for the study of axonal cytoskeletal dynamics. Following differentiation with dbcAMP, NB2a/dl cells express and phosphorylate all three neurofilament protein subunit (NFPs) and assemble them into neurofilaments in a sequence of events closely paralleling that observed in vivo. Axonal microtubules of these cells contain acetylated alpha.tubulin, a post.translational modification of tubulin that confers stability to microtubules. NB2a/dl cells also express calpain, the calcium.activated protease that preferentially degrades cytoskeletal proteins and its regulatory molecule, calpastatin. In this project, the sequence of events of NFP and alpha.tubulin synthesis, modification, assembly and degradation, and the synthesis and activity of calpain and calpastatin will be defined during differentiation by: 1. immunoblot analysis of steady.state levels present in cytoskeletal and soluble fractions utilizing antisera specific for unmodified subunits: 2. immunoprecipitation of radiolabeled . J H.subunits; and 3. proteolysis of isolated subunits by endogenous calpain. The hypothesis that alterations in one or more of the events is responsible for axonal atrophy will be tested by withdrawal of dbcAMP (which induces resorption of axons) followed by analysis of cultures by the above methods. Direct introduction into cells of calpain inhibitory molecules will be utilized to further examine the role of proteolysis in axonal atrophy.