Neurofilaments (NF) are characteristic structures in neurons, but they become abnormally prominent and distributed in certain neurological disorders and toxin-induced neuropathies. Their precise role in normal neurons is unknown. NF are the only component of the neuronal cytoskeleton whose major components 1) are different proteins from those constituting the analogous structure in other kinds of cells and 2) are not all present in neuronal precursor cells, but whose synthesis is first initiated at the time that postmitotic neurons are generated. This suggests a specific role of NF in neuronal structure. The purpose of the proposed research is to examine aspects of the synthesis of NF proteins in relation to the growth of embryonic neurites and the integrity of mature neurons. Biochemical and cytological approaches will be used to examine the regulation of NF synthesis, posttranslational modification and intracellular distribution, during differentiation in vivo and in vitro as well as in mature neurons maintained in culture. The effects of certain exogenous hormones and growth factors, of agents interfering with cytoskeletal organization, and of neurotoxins, on the synthesis and processing of NF proteins will also be examined. Synthesis of NF proteins will be followed by incorporation of labeled amino acids. This will be compared with the synthesis of other neuronal cytoskeletal proteins, tubulin and actin, and of another neuron-specific (but not structural) protein, neuron-specific enolase. The types of posttranslational modifications to be examined include phosphorylation and proteolytic processing. The possibility of other types of modifications will be explored. Distribution of NF and other elements of the cytoskeleton will be examined by immunohistochemistry, using antibodies specific to individual NF proteins, as well as by electron microscopy. The results of these experiments will provide insight into the functional significance of NF in normal neurons and their relationship to altered neuronal properties in pathological conditions.
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