The overall objective of this research program are to elucidate the molecular and cellular mechanisms of neurofilament assembly. I. Mechanism, Structural Requirements, and Regulation of Neurofilament Assembly: The specific structural requirement for in vitro assembly and exchange will be examined by a combination of protein biochemical and molecular biological approaches. Specifically, isolated domains of the core protein, chemical modification of selected residues, and the effects of synthetic peptides corresponding to subdomains will be examined on the kinetics of assembly and exchange. The construction and expression of genetically altered NF-L core protein with subdomain deletions, site- specific mutations, or chimeras constructed between neurofilaments and glial filaments will be tested for assembly and exchange in vitro to identify those structural domains responsible for neurofilament assembly. The role of phosphorylation in neurofilament assembly and interactions will be examined by fluorescence photobleach recovery and deep freeze etch electron microscopy. Further detailed studies on the molecular characterization of the neurofilament specific protein kinase will be continued and antibodies to the kinase will be used for immunocytochemistry in both normal and Alzheimer's brain where abnormal phosphorylation of neurofilaments has been observed. In addition, suicide inhibitory peptides based on the phosphorylation sequence will be used to assess the role of phosphorylation in axonal growth and stability. II. Cellular Dynamics of Neurofilaments: Fluorescently labeled neurofilament proteins and subfragments will be microinjected into cultured neurons to determine whether in vivo there is a dynamic equilibrium of NF-L between assembled and unassembled states and what portions of NF-L are responsible for assembly and exchange in vivo. In addition, models of axonal transport of the axoplasmic matix, desposition of newly synthesized neurofilament proteins during neurite outgrowth, turnover of subunits in mature axons, and the reorganization neurofilaments in regenerating axons will be directly tested at the single cell level by fluorescence photobleach recovery and low light video microscopy.
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